Immunoconjugates Comprising Kallikrein-Related Peptidase 2 Antigen-Binding Domains and Uses Thereof

Abstract

Provided herein is an immunoconjugate, such as a radioimmunoconjugate, that comprises a therapeutic moiety conjugated to an antibody or antigen-binding domain that has binding specificity for hK2. In certain embodiments, the hK2-specific immunoconjugate exhibits a short half-life. Also provided herein is a method of using the immunoconjugate to selectively target cancer cells and treat diseases such as prostate cancer.

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 303,083, filed January 26, 2022, which is hereby incorporated by reference in its entirety for all purposes.

[0002] (Reference to electronically submitted sequence listing) This application contains a computer readable sequence listing that has been submitted herewith in XML file format, the entire contents of which are incorporated herein by reference in their entirety. The sequence listing XML file submitted herewith is entitled "JBI6697WOPCT1_SL.xml", was created on November 7, 2022, and is 747,970 bytes in size.

[0003] (Technical field) The present invention provides immunoconjugates, eg, radioconjugates, comprising an antigen-binding domain that binds to the kallikrein-related peptidase 2 (hK2) protein, and methods for making and using them.

[0004] BACKGROUND OF THE INVENTION Prostate cancer is the second most frequently diagnosed cancer and the sixth leading cause of cancer death in men, accounting for approximately 14% of all new cancer cases and 6% of cancer deaths worldwide. The course of prostate cancer from diagnosis to death is best classified as a series of clinical stages based on the extent of disease, hormonal status, and the presence or absence of detectable metastases: localized disease, rising prostate-specific antigen (PSA) levels without detectable metastases after radiation therapy or surgery, and clinical metastases in the noncastrated or castrated stages. While surgery, radiation, or a combination of both can be curative for patients with localized disease, a significant proportion of these patients will have recurrent disease as evidenced by rising PSA levels, which may lead to the development of metastases, i.e., progression to terminal disease, particularly in high-risk groups.

[0005] Androgen depletion therapy (ADT) is the standard of care, with a generally predictable outcome of a decline in PSA, a stable phase in which the tumor does not grow, followed by a rise in PSA and regrowth as castration-resistant disease. For many years, ADT was the standard of care for patients with metastatic prostate cancer.

[0006] Kallikrein-related peptidase 2 (hK2, HK2) is a trypsin-like enzyme with androgen receptor (AR)-driven expression specific to prostate tissue and prostate cancer. While hK2 expression is restricted to prostate and prostate cancer tissue, it has recently been demonstrated that hK2 is detectable in breast cancer lines and primary patient samples after appropriate activation of the AR pathway by steroid hormones (U.S. Patent Application Publication No. 2018 / 0326102). When the highly structured tissue of the prostate is compromised during hypertrophy or malignant transformation, catalytically inactive hK2 is retrogradely released into the bloodstream.

[0007] There remains a need for next generation hK2 targeted therapies for therapeutic and diagnostic purposes.

[0008] (Summary of the Invention) Embodiments of the present invention relate to anti-hK2 radioconjugates comprising an antigen-binding domain conjugated to a chelator that binds to a radiometal for therapeutic applications or imaging. According to certain embodiments, the anti-hK2 radioconjugate comprising the antigen-binding domain has a shorter half-life compared to an anti-hK2 radioconjugate comprising a full-length antibody.

[0009] The circulating half-life of immunoglobulin G (IgG) in humans is typically approximately 10–21 days. The Fc domain in intact IgG can bind to the neonatal Fc receptor (FcRn), resulting in antibody recycling and minimal endosomal degradation. FcRn plays a critical role in serum IgG homeostasis and placental transfer of IgG molecules from mother to fetus. Following pinocytosis, the acidic environment of the early endosome allows IgG (as well as albumin) to bind to FcRn, protecting it from degradation and facilitating its transport into the extracellular environment, where it dissociates upon exposure to physiological pH and returns to the circulation.

[0010] The circulating half-life of an antigen-binding domain such as a Fab tends to be much shorter than that of an IgG. Because the Fab fragment lacks the Fc domain, it lacks the FcRn-mediated half-life extension mechanism, and therefore the Fab alone has a shorter half-life (e.g., less than 24 hours, or less than 12 hours, or in some cases about 2-3 hours).

[0011] One embodiment of the present invention provides an immunoconjugate comprising a therapeutic moiety conjugated to an antigen-binding domain that has binding specificity for kallikrein-related peptidase 2 (hK2).

[0012] According to certain embodiments, the therapeutic moiety is a cytotoxic drug.

[0013] According to certain embodiments, the therapeutic moiety is a contrast agent.

[0014] According to certain embodiments, the therapeutic moiety comprises a radioactive metal. Non-limiting examples of suitable radioactive metals include: 225 Ac, 177 Lu, 32 P, 47 Sc, 67 Cu, 77 As, 89 Sr, 90 Y, 99 Tc, 105 Rh, 109 Pd, 111 Ag, 131 I, 149 Tb, 152 Tb, 155 Tb, 153 Sm, 159 Gd, 165 Dy, 166 Ho, 169 Er, 186 Re, 188 Re, 194 Ir, 198 Au, 199 Au, 211 At, 212 Pb, 212 Bi, 213 Bi, 223 Ra, 255 Fm, 227 Th, 177 Lu, 62 Cu, 64 Cu, 67 Ga, 68 Ga, 86 Y, 89 Zr, and 111 In is one example.

[0015] According to certain embodiments, the therapeutic moiety is: 225 It is a cytotoxic drug containing Ac.

[0016] According to certain embodiments, the therapeutic moiety is: 111 In or 64 It is a contrast agent containing Cu.

[0017] According to certain embodiments, the therapeutic moiety comprises a radiometal complex, the radiometal complex comprising a radiometal bound to a chelator, the chelator being conjugated to an antigen-binding domain having binding specificity for kallikrein-related peptidase 2 (hK2).

[0018] According to certain embodiments, the chelating agent is 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), S-2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), 1,4,8,11-tetraazacyclododecane-1,4,8,11-tetraacetic acid (TETA), 3,6,9,15-tetraazabicyclo[9.3.1]-pentadeca-1(15),11,13-triene-4-(S)-(4-isothiocyanatobenzyl)-3,6,9-triacetic acid (PCTA), 5-S-(4-aminobenzyl)-1-oxa-4,7,10-triazacyclododecane-4,7,10-tris(acetic acid) (DO3A) or a derivative thereof.

[0019] According to a particular embodiment, the chelating agent is DOTA.

[0020] According to certain embodiments, the chelating agent is H2bp18c6 or an H2bp18c6 derivative.

[0021] According to certain embodiments, the radiometal complex is a radiocomplex of formula (Im), or formula (II-m), or formula (III-m) as described herein, wherein R 11 comprises an antigen-binding domain with binding specificity for kallikrein-related peptidase 2 (hK2), and M is a radiometal.

[0022] According to certain embodiments, the radiometal complex is a radiometal complex of Formula (IV-m), or Formula (Vm), or Formula (VI-m) described herein, where R4 comprises an antigen-binding domain having binding specificity for kallikrein-related peptidase 2 (hK2), and M+ is a radiometal.

[0023] According to certain embodiments, the therapeutic moiety is an auristatin derivative, such as MMAE (monomethylauristatin E) or MMAF (monomethylauristatin F).

[0024] According to certain embodiments, the antigen-binding domain that binds to hK2 is an scFv, (scFv)2, Fv, Fab, F(ab')2, Fd, dAb, or VHH.

[0025] According to a particular embodiment, the antigen-binding domain with binding specificity for hK2 is a Fab.

[0026] According to certain embodiments, the antigen binding domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NO: 170 (SYYWS), SEQ ID NO: 171 (YIYYSGSTNYNPSLKS), SEQ ID NO: 172 (TTIFGVVTPNFYYGMDV), SEQ ID NO: 173 (RASQGISSYLA), SEQ ID NO: 174 (AASTLQS) and SEQ ID NO: 175 (QQLNSYPLT), respectively.

[0027] According to certain embodiments, the antigen-binding domain that binds to hK2 has a sequence identity that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 100%) to the VH of SEQ ID NO: 162 (QVQLQESGPGLVKPSETLSLTCTVSGGSISSYYWSWIRQPPGKGLEWIGYIYYSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAGTTIFGVVTPNFYYGMDVWGQGTTVTVSS). and a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99% or 100%) identical to the VL of SEQ ID NO: 163 (DIQMTQSPSFLSASVGDRVTITCRASQGISSYLAWYQQKPGKAPKFLIYAASTLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQLNSYPLTFGGGTKVEIK).

[0028] According to a particular embodiment, the antigen binding domain that binds to hK2 comprises a VH of SEQ ID NO:162 and a VL of SEQ ID NO:163.

[0029] According to certain embodiments, the antigen-binding domain is a Fab comprising A) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 170, 171, 172, 173, 174 and 175, respectively, and / or B) a VH of SEQ ID NO: 162 and a VL of SEQ ID NO: 163.

[0030] According to certain embodiments, the immunoconjugate is a short half-life immunoconjugate.

[0031] According to certain embodiments, a method of treating an hK2-expressing cancer in a subject comprises administering to the subject a therapeutically effective amount of an immunoconjugate according to any of the preceding embodiments.

[0032] According to certain embodiments, a method of reducing the amount of hK2-expressing tumor cells in a subject comprises administering to the subject a therapeutically effective amount of an immunoconjugate according to any of the preceding embodiments.

[0033] According to certain embodiments, a method of treating prostate cancer in a subject comprises administering to the subject a therapeutically effective amount of an immunoconjugate according to any of the preceding embodiments.

[0034] In some embodiments, the prostate cancer is recurrent, refractory, aggressive, or castration-resistant prostate cancer, or any combination thereof.

[0035] According to certain embodiments, the prostate cancer is metastatic castration-resistant prostate cancer.

[0036] According to certain embodiments, a method of detecting the presence of prostate cancer in a subject comprises administering an immunoconjugate according to any of the preceding embodiments to a subject suspected of having prostate cancer, and visualizing the biological structure to which the conjugate is bound (e.g., by computed tomography or positron emission tomography), thereby detecting the presence of prostate cancer, wherein the immunoconjugate preferably comprises an imaging agent such as 111-In or 64-Cu. According to certain embodiments, the method comprises conjugating a therapeutic moiety to an antigen-binding domain with binding specificity for kallikrein-related peptidase 2 (hK2).

[0037] According to certain embodiments, the method for making the radioimmunoconjugates described herein comprises binding a radiometal to a chelator conjugated to an antigen-binding domain having binding specificity for kallikrein-related peptidase 2 (hK2).

[0038] According to certain embodiments, the short half-life radioimmunoconjugate comprises a radioactive metal complex, the radioactive metal complex being linked to a chelator. 225 and Ac, and the chelator is conjugated to a Fab with binding specificity for hK2, wherein the Fab comprises the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 170, 171, 172, 173, 174, and 175, respectively. In a specific embodiment, the Fab comprises a VH of SEQ ID NO: 162 and a VL of SEQ ID NO: 163. [Brief explanation of the drawings]

[0039] The foregoing Summary of the Invention and the following Detailed Description of the Invention will be better understood when read in conjunction with the accompanying drawings. It should be understood that the invention is not limited to the precise embodiments shown in the drawings.

[0040] Of the drawings: [Figure 1]FIG. 1 shows cell binding and internalization of an immunoconjugate of the invention comprising KL2B30 Fab (identified as KL2B997) conjugated to MMAF (monomethyl auristatin F) in hK2-expressing VCaP cells. [Figure 2] The amino acid sequences (heavy and light chain sequences) of KL2B997 and KL2B1251 are shown. KL2B997 has a His-tag and a sortase tag (underlined in Figure 2) on the heavy chain, while KL2B1251 has no tags. [Figure 3A] FIG. 3A shows the cell binding of KL2B30 Fab (KL2B1251) and KL2B1251 conjugated to a NOTA derivative (NODA-GA) in hK2-expressing VCaP cells, and FIG. 3B shows the lack of binding in hK2-negative DU145 cells. [Figure 3B] FIG. 3A shows the cell binding of KL2B30 Fab (KL2B1251) and KL2B1251 conjugated to a NOTA derivative (NODA-GA) in hK2-expressing VCaP cells, and FIG. 3B shows the lack of binding in hK2-negative DU145 cells. [Figure 4] 1 shows a representative structure of NODA-GA-Fab according to one embodiment of the present invention. [Figure 5] Shown are the individual body weights and administered doses of male cynomolgus monkeys given a single IV dose of KL2B1251-NOTA (in Group 1) and KL2B1251-TOPA (in Group 2). [Figure 6] Individual and mean (SD) serum pharmacodynamic estimates of KL2B1251-NOTA (Group 1) and KL2B1251-TOPA (Group 2) following a single IV dose of 1 mg / kg in male cynomolgus monkeys are shown. [Figure 7] 1 shows the mean (SD) serum concentrations of KL2B1251-NOTA and KL2B1251-TOPA over time following a single IV dose of 1 mg / kg in male cynomolgus monkeys.

[0041] DETAILED DESCRIPTION OF THE INVENTION In the "Background" section and throughout this specification, various publications, articles, and patents are cited or described, and each of these references is incorporated herein by reference in its entirety. The discussion of documents, operations, materials, devices, articles and the like which is included in the specification is for the purpose of providing a context for the present invention. Such discussion is not an admission that any or all of these items constitute part of the prior art to any invention disclosed or claimed.

[0042] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Unless otherwise defined, certain terms cited herein have the meanings set forth herein. All patents, published patent applications, and publications cited herein are incorporated by reference in their entirety as if set forth herein.

[0043] As used herein, the connective term "and / or" between multiple listed elements is understood to encompass both individual and combined alternatives. For example, when two elements are connected by "and / or," the first alternative refers to the first element being applicable without the second element. The second alternative refers to the second element being applicable without the first element. The third alternative refers to the first and second elements being applicable together. Any one of these alternatives is understood to be within the meaning and, therefore, meets the requirements of the term "and / or" as used herein. The simultaneous applicability of two or more of the alternatives is also understood to be within the meaning and, therefore, meets the requirements of the term "and / or."

[0044] To assist the reader of this application, the description of the specification is divided into various paragraphs or sections or directed to various embodiments of this application. These separations should not be considered as separating a paragraph or section or embodiment entity from another paragraph or section or embodiment entity. To the contrary, those skilled in the art will understand that the present description has broad applicability and encompasses all combinations of the various sections, paragraphs, and sentences that may be contemplated. The discussion of any embodiment is intended to be merely illustrative and is not intended to suggest that the scope of the present disclosure, including the claims, is limited to these examples.

[0045] As used herein, the use of numerical ranges expressly includes all possible subranges, including integers and fractions of values ​​within that range, and all individual numerical values ​​within that range, unless the context clearly dictates otherwise.

[0046] Where lists are presented, unless otherwise stated, it is to be understood that each individual element of that list and every combination of that list is a separate embodiment. For example, a list of embodiments presented as "A, B, or C" should be interpreted to include the embodiments "A," "B," "C," "A or B," "A or C," "B or C," or "A, B, or C."

[0047] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a cell" includes a combination of two or more cells, and the like.

[0048] The transitional phrases "comprising," "consisting essentially of," and "consisting of" are intended to connote their generally accepted meanings in patent language, i.e., (i) "comprising" is synonymous with "comprising," "containing," or "characterized by" and is inclusive or open-ended and does not exclude other unrecited elements or method steps; (ii) "consisting of" excludes any element, step, or ingredient not specified in the claim; and (iii) "consisting essentially of" limits the claim to the specified materials or steps and those that do not materially affect the basic and novel characteristics" of the claimed invention. Embodiments described with the phrase "comprising" (or its equivalents) also provide as embodiments embodiments described independently with "consisting of" and "consisting essentially of."

[0049] "About" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system.

[0050] "Antibody-dependent cellular cytotoxicity," "antibody-dependent cell-mediated cytotoxicity," or "ADCC" refers to a mechanism of cell death induction that depends on the interaction of antibody-coated target cells with lytic effector cells, such as natural killer cells (NK), monocytes, macrophages, and neutrophils, via Fc gamma receptors (FcγR) expressed on the effector cells.

[0051] "Antibody-dependent cellular phagocytosis" or "ADCP" refers to a mechanism by which antibody-coated target cells are eliminated by uptake by phagocytic cells such as macrophages or dendritic cells.

[0052] "Antigen" refers to any molecule (e.g., a protein, peptide, polysaccharide, glycoprotein, glycolipid, nucleic acid, portion thereof, or combination thereof) that can be bound by an antigen-binding domain or T-cell receptor that can mediate an immune response. Exemplary immune responses include antibody production and activation of immune cells such as T cells, B cells, or NK cells. Antigens can be expressed by genes from, synthesized from, or purified from a biological sample such as a tissue sample, tumor sample, cell, or other biologically-containing fluid, organism, protein / antigen subunit, killed or inactivated whole cell, or lysate.

[0053] An "antigen-binding fragment" or "antigen-binding domain" refers to a portion of an isolated protein that binds to an antigen. An antigen-binding fragment may be a synthetic, enzymatically obtainable, or genetically engineered polypeptide, and includes antigen-binding portions of immunoglobulins, such as VH, VL, VH and VL, Fab, Fab', F(ab')2, Fd, and Fv fragments, domain antibodies (dAbs) consisting of one VH or one VL region, shark variable IgNAR regions, camelized VH regions, VHH regions, minimal recognition units consisting of amino acid residues mimicking the CDRs of an antibody, such as the FR3-CDR3-FR4 portion, HCDR1, HCDR2, and / or HCDR3, and LCDR1, LCDR2, and / or LCDR3, alternative scaffolds that bind to the antigen, and multispecific proteins comprising antigen-binding fragments. Antigen-binding fragments (such as VH and VL) may be linked together via synthetic linkers to form various types of single-chain antibody designs in which, when the VH and VL domains are expressed as separate single chains, the VH / VL domains can pair intramolecularly or intermolecularly to form monovalent antigen-binding domains, such as single-chain Fvs (scFvs) or diabodies. As used herein, "antigen-binding fragment" or "antigen-binding domain" does not refer to a full-length antibody having an Fc region.

[0054] The term "antibody" has a broad meaning and encompasses immunoglobulin molecules, including murine, human, humanized, and chimeric monoclonal antibodies, antigen-binding fragments, bispecific, trispecific, tetraspecific, multispecific antibodies such as dimeric, tetrameric, or multimeric antibodies, single-chain antibodies, domain antibodies, and any other modified configuration of an immunoglobulin molecule containing an antigen-binding site of the required specificity. A "full-length antibody" is composed of two heavy chains (HC) and two light chains (LC), inter-connected by disulfide bonds, and multimers thereof (e.g., IgM). Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region (consisting of domains CH1, hinge, CH2, and CH3). Each light chain is composed of a light chain variable region (VL) and a light chain constant region (CL). The VH and VL regions can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs), interspersed with framework regions (FRs). Each VH and VL is composed of three CDR and four FR segments, arranged from amino- to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. Immunoglobulins can be assigned to five major classes, namely, IgA, IgD, IgE, IgG, and IgM, depending on the amino acid sequence of the heavy-chain constant domain. IgA and IgG are further subdivided into isotypes, IgA1, IgA2, IgG1, IgG2, IgG3, and IgG4. Antibody light chains of any vertebrate species can be assigned to one of two clearly distinct types, kappa (κ) and lambda (λ), based on the amino acid sequence of their constant domains.

[0055] "Cancer" refers to a broad group of diverse diseases characterized by the uncontrolled growth of abnormal cells in the body. Uncontrolled cell division and growth leads to the formation of malignant tumors that invade adjacent tissues and may metastasize to distant parts of the body via the lymphatic system or bloodstream. "Cancer" or "cancerous tissue" can include tumors.

[0056] The "complementarity-determining region (CDR)" is the region of an antibody that binds to an antigen. VH has three CDRs (HCDR1, HCDR2, and HCDR3), and VL has three CDRs (LCDR1, LCDR2, and LCDR3). CDRs may be defined using various descriptions, such as Kabat (Wu et al. (1970) J Exp Med 132:211-50; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), Chothia (Chothia et al. (1987) J Mol Biol 196:901-17), IMGT (Lefranc et al. (2003) Dev Comp Immunol 27:55-77), and AbM (Martin and Thornton J Bmol Biol 263:800-15, 1996). The correspondence between various descriptions and the numbering of variable regions has been described (see, for example, Lefranc et al. (2003) Dev Comp Immunol 27:55-77; Honegger and Pluckthun, J Mol Biol (2001) 309:657-70; the International ImMunoGeneTics (IMGT) database; web resource, http: / / www_imgt_org). CDRs can be described using available programs such as abYsis by UCL Business PLC. As used herein, the terms "CDR," "HCDR1," "HCDR2," "HCDR3," "LCDR1," "LCDR2," and "LCDR3" include CDRs defined by any of the Kabat, Chothia, IMGT, or AbM methods described above, unless otherwise expressly stated in the specification.

[0057] "Decrease," "lower," "lower," "reducing," or "alleviating" generally refers to the ability of a test molecule to mediate a diminished response (i.e., a downstream effect) when compared to a control or vehicle-mediated response. Exemplary responses are T cell expansion, T cell activation, or T cell-mediated tumor cell killing, or binding of a protein to its antigen or receptor, enhanced Fcγ binding, or enhanced Fc effector function such as enhanced ADCC, CDC, and / or ADCP. The decrease may be a statistically significant difference in the measured response between the test molecule and the control (or vehicle), or an increase in the measured response, such as an increase of about 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 30 fold or more, e.g., 500, 600, 700, 800, 900, or 1000 fold or more (including all integers above 1 and decimal points therebetween, e.g., 1.5, 1.6, 1.7, 1.8, etc.).

[0058] "Differentiation" refers to the process of decreasing the potency or proliferation of a cell or moving it toward a more developmentally restricted state.

[0059] "Encode" or "encoding" refers to the inherent property of a specific sequence of nucleotides in a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for the synthesis of other polymers and macromolecules in biological processes, having either a defined sequence of nucleotides (e.g., rRNA, tRNA, and mRNA) or a defined sequence of amino acids, and the biological properties that result therefrom. Thus, in a cell or other biological system, a gene, cDNA, or RNA encodes a protein when transcription and translation of mRNA corresponding to that gene produces the protein. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence, and the non-coding strand, which is used as a template for transcription of the gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

[0060] The terms "enhance," "promote," "increase," "expand," or "improve" generally refer to the ability of a test molecule to mediate a greater response (i.e., a downstream effect) when compared to a control or vehicle-mediated response. Exemplary responses are T-cell expansion, T-cell activation, or T-cell-mediated tumor cell killing, or binding of a protein to its antigen or receptor, enhanced Fcγ binding, or enhanced Fc effector function such as enhanced ADCC, CDC, and / or ADCP. An enhancement may be a statistically significant difference in the measured response between the test molecule and the control (or vehicle), or an increase in the measured response, such as an increase of about 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 30 fold or more, e.g., 500, 600, 700, 800, 900, or 1000 fold or more (including all integers above 1 and decimal points therebetween, e.g., 1.5, 1.6, 1.7, 1.8, etc.).

[0061] "Epitope" refers to the portion of an antigen to which an antibody specifically binds. Epitopes typically consist of surface groupings of chemically active (e.g., polar, non-polar, or hydrophobic) moieties, such as amino acids or polysaccharide side chains, and may have specific three-dimensional structural characteristics as well as specific charge characteristics. Epitopes can be composed of contiguous and / or discontinuous amino acids that form a conformational spatial unit. In discontinuous epitopes, amino acids from different parts of the linear sequence of the antigen are brought into close proximity in three-dimensional space due to folding of the protein molecule. Antibody "epitopes" vary depending on the methodology used to identify the epitope.

[0062] "Expansion" refers to the result of cell division and cell death.

[0063] "Express" and "expression" refer to the well-known transcription and translation that occurs within a cell or in vitro. Thus, an expression product, e.g., a protein, is expressed by a cell or in vitro and may be an intracellular protein, an extracellular protein, or a transmembrane protein.

[0064] An "expression vector" refers to a vector that can be utilized in a biological system or reconstituted biological system to direct the translation of a polypeptide encoded by a polynucleotide sequence present in the expression vector.

[0065] A "dAb" or "dAb fragment" refers to an antibody fragment composed of a VH domain (Ward et al., Nature 341:544 546 (1989)).

[0066] "Fab" or "Fab fragment" or "Fab region" refers to the region of an antibody that binds to an antigen. Conventional IgGs typically contain two Fab regions, each in one of the two arms of the Y-shaped IgG structure. Each Fab region typically consists of one variable region and one constant region from each of the heavy and light chains. More specifically, the variable and constant regions of the heavy chain in the Fab region are the VH and CH1 regions, and the variable and constant regions of the light chain in the Fab region are the VL and CL regions. The VH, CH1, VL, and CL regions in the Fab region can be arranged in various ways to confer antigen-binding capability according to the present disclosure. For example, the VH and CH1 regions can be on one polypeptide, while the VL and CL regions can be on separate polypeptides, as in the Fab region of a conventional IgG. Alternatively, the VH, CH1, VL, and CL regions can all be on the same polypeptide and oriented in various orders.

[0067] "F(ab')2" or "F(ab')2 fragment" refers to an antibody fragment comprising two Fab fragments connected by a disulfide bridge in the hinge region.

[0068] "Fd" or "Fd fragment" refers to an antibody fragment composed of the VH and CH1 domains.

[0069] "Fv" or "Fv fragment" refers to an antibody fragment composed of the VH and VL domains from a single arm of an antibody. Fv fragments lack the constant regions of the Fab (CH1 and CL) regions. The VH and VL in an Fv fragment are held together by non-covalent interactions.

[0070] The "Fc" polypeptide of a dimeric Fc refers to one of the two polypeptides that form the dimeric Fc domain (e.g., the Fc polypeptide of a dimeric IgG Fc comprises IgG CH2 and IgG CH3 constant domain sequences).

[0071] A "full-length antibody" is composed of two heavy chains (HC) and two light chains (LC) interconnected by disulfide bonds, and multimers thereof (e.g., IgM). Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region, the heavy chain constant region being composed of subregions CH1, hinge, CH2, and CH3. Each light chain is composed of a light chain variable region (VL) and a light chain constant region (CL). VH and VL can be further subdivided into regions of hypervariability called complementarity-determining regions (CDRs), which are interspersed with framework regions (FRs). Each VH and VL is composed of three CDR and four FR segments, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0072] A "host cell" refers to any cell that contains heterologous nucleic acid. An exemplary heterologous nucleic acid is a vector (e.g., an expression vector).

[0073] A "human antibody" refers to an antibody optimized to elicit a minimal immune response when administered to a human subject. The variable regions of a human antibody are derived from human immunoglobulin sequences. If a human antibody contains a constant region or a portion of a constant region, the constant region is also derived from a human immunoglobulin sequence. A human antibody contains heavy and light chain variable regions "derived" from sequences of human origin when the variable regions of the human antibody are obtained from a system using human germline immunoglobulins or rearranged immunoglobulin genes. Exemplary such systems are phage-displayed human immunoglobulin gene libraries and transgenic nonhuman animals, such as mice or rats, carrying human immunoglobulin loci. A "human antibody" typically contains amino acid differences compared to immunoglobulins expressed in humans due to differences in the systems used to obtain human antibodies and human immunoglobulin loci, the introduction of somatic mutations or intentional substitutions into frameworks or CDRs, or both. Typically, a "human antibody" is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical in amino acid sequence to the amino acid sequence encoded by a human germline immunoglobulin or rearranged immunoglobulin gene. Optionally, a "human antibody" may contain consensus framework sequences derived from human framework sequence analysis, e.g., as described in Knappik et al., (2000) J Mol Biol 296:57-86, or a synthetic HCDR3 incorporated into a phage-displayed human immunoglobulin gene library, e.g., as described in Shi et al., (2010) J Mol Biol 397:385-96 and WO 2009 / 085462. Antibodies in which at least one CDR is derived from a non-human species are not included in the definition of "human antibody."

[0074] A "humanized antibody" refers to an antibody in which at least one CDR is derived from a non-human species and at least one framework is derived from a human immunoglobulin sequence. Humanized antibodies can contain substitutions in the framework, so that the framework may not be an exact copy of an expressed human immunoglobulin or human immunoglobulin germline gene sequence.

[0075] "In combination with" means that two or more therapeutic agents are administered to a subject together in an admixture, simultaneously as single agents, or sequentially as single agents in any order.

[0076] "Isolated" refers to a homogenous population of molecules (e.g., synthetic polynucleotides or polypeptides) that have been substantially separated and / or purified away from other components of the system in which they are produced, such as in a recombinant cell, and to proteins that have been subjected to at least one purification or isolation step. "Isolated" refers to molecules that are substantially free of other cellular material and / or chemicals and includes molecules isolated to greater degrees of purity, e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% purity.

[0077] "Kallikrein-related peptidase 2," or "hK2," (also referred to herein as KLK2) refers to a known protein also known as kallikrein-2, granular kallikrein 2, or HK2. hK2 is produced as a preproprotein and is cleaved during proteolysis to generate the active protease. All hK2 isoforms and variants are encompassed by "hK2." The amino acid sequences of various isoforms can be found in GenBank accession numbers NP_005542.1, NP_001002231.1, and NP_001243009. The amino acid sequence of full-length hK2 is set forth in SEQ ID NO: 62. The sequence includes the signal peptide (residues 1-18) and propeptide region (residues 19-24).

[0078] SEQ ID NO: 62 MWDLVLSIALSVGCTGAVPLIQSRIVGGWECEKHSQPWQVAVYSHGWAHCGGVLVHPQWV LTAAHCLKKNSQVWLGRHNLFEPEDTGQRVPVSHSFPHPLYNMSLLKHQSLRPDEDSSHD LMLLRLSEPAKITDVVKVLGLPTQEPALGTTCYASGWGSIEPEEFLRPRSLQCVSLHLLS NDMCARAYSEKVTEFMLCAGLWTGGKDTCGGDSGGPLVCNGVLQGITSWGPEPCALPEKP AVYTKVVHYRKWIKDTIAANP

[0079] "Modulate" refers to either an enhanced or decreased ability of the test molecule to mediate a greater or lesser response (i.e., a downstream effect) when compared to a control or vehicle-mediated response.

[0080] "Monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibody molecules (i.e., the individual antibodies comprising the population are identical except for possible, well-known alterations, such as removal of the C-terminal lysine from the antibody heavy chain, or post-translational modifications such as amino acid isomerization or deamidation, methionine oxidation, or asparagine or glutamine deamidation). Monoclonal antibodies typically bind to one antigenic epitope. Bispecific monoclonal antibodies bind to two different antigenic epitopes. Monoclonal antibodies may have heterogeneous glycosylation within the antibody population. Monoclonal antibodies may be monospecific or multispecific, such as bispecific, and may be monovalent, bivalent, or multivalent.

[0081] "Operably linked" and similar phrases, when used with respect to nucleic acids or amino acids, refer to the operative linkage of nucleic acid or amino acid sequences, respectively, placed in a functional relationship with each other. For example, operably linked promoters, enhancer elements, open reading frames, 5' and 3' UTRs, and terminator sequences result in the correct production of a nucleic acid molecule (e.g., RNA), and, in some cases, the production of a polypeptide (i.e., expression of the open reading frame). An operably linked peptide refers to a peptide in which the functional domains of the peptide are positioned at an appropriate distance from each other to confer the intended function of each domain.

[0082] The term "paratope" refers to the area or region of an antibody molecule that is involved in antigen binding and contains the residues that interact with the antigen. A paratope can be composed of contiguous and / or discontinuous amino acids that form a conformational spatial unit. The paratope of a given antibody can be defined and characterized at various levels of detail using a variety of experimental and computational methods. Experimental methods include hydrogen / deuterium exchange mass spectrometry (HX-MS). Paratopes are defined differently depending on the mapping method used.

[0083] A "pharmaceutical combination" refers to a combination of two or more active ingredients, administered together or separately.

[0084] A "pharmaceutical composition" refers to a composition obtained by combining active ingredients with a pharmaceutically acceptable carrier.

[0085] A "pharmaceutically acceptable carrier" or "excipient" refers to an ingredient in a pharmaceutical composition, other than an active ingredient, that is not toxic to a subject. Exemplary pharmaceutically acceptable carriers are buffers, stabilizers, or preservatives.

[0086] "Polynucleotide" or "nucleic acid" refers to a synthetic molecule comprising a chain of nucleotides covalently linked by a sugar-phosphate backbone or other equivalent covalent chemistry. cDNA is a typical example of a polynucleotide. A polynucleotide can be a DNA or RNA molecule.

[0087] "Preventing," "preventing," "prevention," or "prophylaxis" of a disease or disorder means barring the disorder from occurring in a subject.

[0088] "Proliferation" refers to an increase in cell division, either symmetric or asymmetric division of cells.

[0089] A "promoter" refers to a minimal sequence necessary to initiate transcription. A promoter may also contain enhancer or repressor elements that enhance or repress transcription, respectively.

[0090] As used interchangeably herein, "protein" or "polypeptide" refers to a molecule comprising one or more polypeptides, each composed of at least two amino acid residues linked by a peptide bond. A protein may be a monomer or a protein complex of two or more subunits, which may be the same or different. Small polypeptides consisting of less than 50 amino acids may be referred to as "peptides." A protein may be a heterologous fusion protein, a glycoprotein, or a protein modified by post-translational modifications such as phosphorylation, acetylation, myristoylation, palmitoylation, glycosylation, oxidation, formylation, amidation, citrullination, polyglutamylation, ADP-ribosylation, pegylation, or biotinylation. A protein may be recombinantly expressed.

[0091] "Recombinant" refers to polynucleotides, polypeptides, vectors, viruses, and other macromolecules that are prepared, expressed, produced, or isolated by recombinant means.

[0092] "Regulatory element" refers to any cis- or trans-acting genetic element that controls some aspect of the expression of nucleic acid sequences.

[0093] "Relapse" refers to the return of a disease or signs and symptoms of a disease after a period of improvement following prior treatment with a therapeutic agent.

[0094] "Refractory" refers to a disease that does not respond to treatment. A refractory disease may be resistant to treatment before or at the start of treatment, or a refractory disease may become resistant during treatment.

[0095] "Single-chain Fv" or "scFv" refers to a fusion protein comprising at least one antibody fragment comprising a light chain variable region (VL) and at least one antibody fragment comprising a heavy chain variable region (VH), wherein the VL and VH are contiguously linked via a polypeptide linker and can be expressed as a single-chain polypeptide. Unless otherwise specified, as used herein, an scFv can have the VL and VH variable regions in either order, e.g., relative to the N- and C-termini of the polypeptide, and can comprise either a VL-linker-VH or a VH-linker-VL.

[0096] "(scFv)2" or "tandem scFv" or "bis-scFv" fragment refers to a fusion protein comprising two light chain variable regions (VL) and two heavy chain variable regions (VH), where the two VL regions and two VH regions are contiguously linked via a polypeptide linker and can be expressed as a single polypeptide chain. The two VL and two VH are fused by a peptide linker to form a bivalent molecule, VL. A -Linker-VH A -Linker-VL B -Linker-VH B to form two binding sites that can simultaneously bind two different antigens or epitopes.

[0097] "Specifically binds," "specific binding," "specifically binding," or "binds" refers to a proteinaceous molecule binding to an antigen or an epitope within the antigen with higher affinity than its affinity for other antigens. Typically, a proteinaceous molecule binds to an antigen with an affinity of about 1 x 10 -7 M or less, e.g., about 5 × 10 -8 M or less, approximately 1×10 -8 M or less, approximately 1×10 -9 M or less, approximately 1×10 -1 0M or less, approximately 1×10 -11 M or less, or about 1 x 10 -12 The equilibrium dissociation constant (K D ) binds to an antigen or an epitope within an antigen, typically D is the K for binding to nonspecific antigens (e.g., BSA, casein) D is at least 100-fold less than the wild-type protein of which the prostate neoantigen is a variant. In the context of the prostate neoantigens described herein, "specific binding" refers to a proteinaceous molecule binding to a prostate neoantigen without detectably binding to the wild-type protein of which the prostate neoantigen is a variant. As used herein, an antibody or antigen-binding domain "having binding specificity for hK2" refers to an antibody or antigen-binding domain, respectively, that specifically binds to hK2.

[0098] A "subject" includes any human or non-human animal. A "non-human animal" includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc. The terms "subject" and "patient" may be used interchangeably herein.

[0099] "T cells" and "T lymphocytes" are interchangeable and are used synonymously herein. T cells include thymocytes, naive T lymphocytes, memory T cells, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes. T cells can be T helper (Th) cells, e.g., T helper 1 (Th1) or T helper 2 (Th2) cells. T cells can be T helper cells (HT1, CD4 +T cells), CD4 + T cells, cytotoxic T cells (CTL, CD8 + T cells), tumor-infiltrating cytotoxic T cells (TIL, CD8 + T cells), CD4 + CD8 + They may be T cells or any other subset of T cells. Also included are "NKT cells," which refer to a specialized population of T cells that not only express the semi-invariant αβ T cell receptor, but also express various molecular markers typically associated with NK cells, such as NK1.1. NKT cells include NK1.1 + and NK1.1 - , and CD4 + , CD4 - , CD8 + , and CD8 - NKT cells include NKT cells. The TCR of NKT cells is unique in that it recognizes glycolipid antigens presented by the MHC I-like molecule CD Id. NKT cells can have either protective or harmful effects due to their ability to produce cytokines that promote either inflammation or immune tolerance. Also included are "gamma delta T cells (γδ T cells)," which refers to a specialized population of a small subset of T cells that have distinct TCRs on their surface. Unlike the majority of T cells, whose TCRs are composed of two glycoprotein chains designated α and β-TCR chains, the TCR in γδ T cells is composed of γ and δ chains. γδ T cells can play a role in immune surveillance and immune regulation, are an important source of IL-17, and have robust CD8 + It has been found that they induce cytotoxic T cell responses. Also included are "regulatory T cells" or "Tregs," which refer to T cells that suppress abnormal or excessive immune responses and play a role in immune tolerance. Tregs are typically CD4+ T cells that express the transcription factor Foxp3. + T cells and IL-10-producing CD4 + It may also include transcription factor Foxp3-negative regulatory T cells, which are T cells.

[0100] As used interchangeably herein, a "therapeutically effective amount" or "effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result. A therapeutically effective amount may vary depending on factors such as the individual's medical condition, age, sex, and weight, as well as the ability of the therapeutic agent or combination of therapeutic agents to elicit a desired response in the individual. Exemplary indicators of an effective therapeutic agent or combination of therapeutic agents include, for example, an improvement in the patient's health, a reduction in tumor burden, a halt or slowing of tumor growth, and / or the absence of metastasis of cancer cells to other locations in the body.

[0101] "Transduction" refers to the introduction of foreign nucleic acid into a cell using a viral vector.

[0102] "Treating," "treating," or "treatment" of a disease or disorder, such as cancer, hereinafter refers to achieving one or more of the following: reducing the severity and / or duration of the disorder; inhibiting the worsening of symptoms characteristic of the disorder being treated; limiting or preventing the recurrence of the disorder in a subject who previously had the disorder; or limiting or preventing the recurrence of symptoms in a subject who was previously symptomatic for the disorder.

[0103] "Tumor cells" or "cancer cells" refer to cancerous, precancerous, or transformed cells that have undergone spontaneous or induced phenotypic changes, either in vivo, ex vivo, or in tissue culture. These changes do not necessarily involve the incorporation of new genetic material. Transformation can occur through infection with a transforming virus and the incorporation of new genomic nucleic acid, through the incorporation of exogenous nucleic acid, or can occur spontaneously or after exposure to a carcinogen, resulting in mutation of an endogenous gene. Transformation / cancer is exemplified by morphological changes, cellular immortalization, aberrant growth control, formation of lesions, proliferation, malignant lesions, modulation of tumor-specific marker levels, invasiveness, and tumor growth in suitable animal hosts, such as nude mice, in vitro, in vivo, and ex vivo.

[0104] "Variant," "mutant," or "altered" refers to a polypeptide or polynucleotide that differs from a reference polypeptide or polynucleotide by one or more modifications, e.g., one or more substitutions, insertions, or deletions.

[0105] Throughout this specification, the numbering of amino acid residues in antibody constant regions is according to the EU index as set forth in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991), unless otherwise expressly stated herein.

[0106] Ig constant region mutations are designated as follows: L351Y_F405A_Y407V refers to the L351Y, F405A, and Y407V mutations in one immunoglobulin constant region, and L351Y_F405A_Y407V / T394W refers to the L351Y, F405A, and Y407V mutations in one Ig constant region and the T394W mutation in a second Ig constant region present in one multimeric protein.

[0107] "VHH" refers to a single domain antibody or nanobody composed exclusively of the antigen-binding domain of the heavy chain. VHH single domain antibodies lack the CH1 domains of the light and heavy chains of a conventional Fab region.

[0108] chemical nomenclature In general, a reference to a particular element, such as hydrogen or H, is meant to include all isotopes of that element. For example, if an R group is defined to include hydrogen or H, it also includes deuterium and tritium. Thus, tritium, C 14 , P 32 and S 35 Compounds containing radioisotopes such as are within the scope of the present technology. Procedures for incorporating such labels into compounds of the present technology will be readily apparent to those of skill in the art based on the disclosure herein.

[0109] The term "substituted" means that at least one hydrogen atom has been replaced with a non-hydrogen group, provided that all normal valences are maintained and the substitution results in a stable compound. When a particular group is "substituted," the group can have one or more substituents, preferably 1 to 5 substituents, more preferably 1 to 3 substituents, and most preferably 1 to 2 substituents, independently selected from a list of substituents. For example, "substituted" refers to an organic group (e.g., an alkyl group) as defined below, in which one or more bonds to a hydrogen atom contained therein are replaced with a bond to a non-hydrogen or non-carbon atom. Substituted groups also include groups in which one or more bonds to a carbon atom(s) or hydrogen atom(s) are replaced with a heteroatom with one or more bonds, including double or triple bonds. Thus, unless otherwise specified, a substituted group is substituted with one or more substituents. In some embodiments, a substituted group is substituted with 1, 2, 3, 4, 5, or 6 substituents. Examples of substituents include halogens (i.e., F, Cl, Br, and I), hydroxyl, alkoxy, alkenoxy, aryloxy, aralkyloxy, heterocyclyl, heterocyclylalkyl, heterocyclyloxy, and heterocyclylalkoxy groups, carbonyl (oxo), carboxylate, ester, urethane, oxime, hydroxylamine, alkoxyamine, aralkoxyamine, thiol, sulfide, sulfoxide, sulfone, sulfonyl, pentafluorosulfanyl (i.e., SF), sulfonamide, amine, N-oxide, hydrazine, hydrazide, hydrazone, azide, amide, urea, amidine, guanidine, enamine, imide, isocyanate, isothiocyanate, cyanate, thiocyanate, imine, nitro group, nitrile (i.e., CN), and the like. The term "independently," when used with reference to substituents, means that when more than one of such substituents is possible, such substituents may be the same or different from each other.

[0110] Substituted ring groups, such as substituted cycloalkyl, aryl, heterocyclyl, and heteroaryl groups, also include rings and ring systems in which a bond to a hydrogen atom is replaced with a bond to a carbon atom. Thus, substituted cycloalkyl, aryl, heterocyclyl, and heteroaryl groups can also be substituted with substituted or unsubstituted alkyl, alkenyl, and alkynyl groups as defined below.

[0111] As used herein, C1-C 11 Cm-Cn, such as C1-C8, or C1-C6, when used before a group, refers to a group containing m to n carbon atoms.

[0112] Alkyl groups include straight-chain and branched-chain alkyl groups having 1 to 12 carbon atoms, typically 1 to 10 carbon atoms, or in some embodiments, 1 to 8, 1 to 6, or 1 to 4 carbon atoms. Examples of straight-chain alkyl groups include groups such as methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl, and n-octyl. Examples of branched-chain alkyl groups include, but are not limited to, isopropyl, iso-butyl, sec-butyl, tert-butyl, neopentyl, isopentyl, and 2,2-dimethylpropyl. Alkyl groups may be substituted or unsubstituted. Representative substituted alkyl groups may be substituted one or more times with substituents such as those listed above, including, but not limited to, haloalkyl (e.g., trifluoromethyl), hydroxyalkyl, thioalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, alkoxyalkyl, carboxyalkyl, and the like.

[0113] Cycloalkyl groups include monocyclic, bicyclic, or tricyclic alkyl groups having 3 to 12 carbon atoms in the ring(s), or in some embodiments, 3 to 10, 3 to 8, or 3 to 4, 5, or 6 carbon atoms. Exemplary monocyclic cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl groups. In some embodiments, cycloalkyl groups have 3 to 8 ring members, while in other embodiments, the number of ring carbon atoms ranges from 3 to 5, 3 to 6, or 3 to 7. Bicyclic and tricyclic ring systems include both bridged and fused rings, including, but not limited to, bicyclo[2.1.1]hexane, adamantyl, decalinyl, and the like. Cycloalkyl groups can be substituted or unsubstituted. Substituted cycloalkyl groups can be substituted one or more times with non-hydrogen and non-carbon groups, as defined above. However, substituted cycloalkyl groups also include rings substituted with straight or branched chain alkyl groups as defined above. Representative substituted cycloalkyl groups may be mono-substituted or more than twice substituted, such as, but not limited to, 2,2-, 2,3-, 2,4-2,5-, or 2,6-disubstituted cyclohexyl groups, which may be substituted with substituents such as those listed above.

[0114] A cycloalkylalkyl group is an alkyl group, as defined above, in which a hydrogen or carbon bond of the alkyl group is replaced with a bond to a cycloalkyl group, as defined above. In some embodiments, the cycloalkylalkyl group has 4 to 16 carbon atoms, 4 to 12 carbon atoms, and typically 4 to 10 carbon atoms. The cycloalkylalkyl group can be substituted or unsubstituted. Substituted cycloalkylalkyl groups can be substituted at the alkyl portion, the cycloalkyl portion, or both the alkyl and cycloalkyl portions of the group. Representative substituted cycloalkylalkyl groups can be mono-substituted or substituted more than once, for example, mono-, di-, or tri-substituted with the substituents listed above, but are not limited to these.

[0115] Alkenyl groups include straight-chain and branched-chain alkyl groups, as defined above, except that at least one double bond exists between two carbon atoms. Alkenyl groups have 2 to 12 carbon atoms, typically 2 to 10 carbon atoms, or in some embodiments, 2 to 8, 2 to 6, or 2 to 4 carbon atoms. In some embodiments, an alkenyl can have one carbon-carbon double bond or multiple carbon-carbon double bonds, such as 2, 3, 4, or more carbon-carbon double bonds. Examples of alkenyl groups include, but are not limited to, methenyl, ethenyl, propenyl, butenyl, and the like. Alkenyl groups can be substituted or unsubstituted. Representative substituted alkenyl groups may be mono-substituted or substituted more than once, for example, mono-, di-, or tri-substituted with the substituents listed above, but are not limited to these.

[0116] Cycloalkenyl groups include cycloalkyl groups, as defined above, having at least one double bond between two carbon atoms. Cycloalkenyl groups can be monocyclic or polycyclic alkyl groups having 3 to 12, more preferably 3 to 8, carbon atoms in the ring(s) and containing at least one double bond between two carbon atoms. Cycloalkenyl groups can be substituted or unsubstituted. In some embodiments, cycloalkenyl groups can have one, two, or three double bonds, or multiple carbon-carbon double bonds, such as two, three, four, or more carbon-carbon double bonds, but do not include aromatic compounds. Cycloalkenyl groups have 3 to 14 carbon atoms, or in some embodiments, 5 to 14 carbon atoms, 5 to 10 carbon atoms, or even 5, 6, 7, or 8 carbon atoms. Examples of cycloalkenyl groups include cyclohexenyl, cyclopentenyl, cyclohexadienyl, cyclobutadienyl, and cyclopentadienyl.

[0117] A cycloalkenylalkyl group is an alkyl group as defined above in which a hydrogen or carbon bond of the alkyl group is replaced with a bond to a cycloalkenyl group as defined above. The cycloalkenylalkyl group may be substituted or unsubstituted. Substituted cycloalkenylalkyl groups may be substituted at the alkyl portion, the cycloalkenyl portion, or both the alkyl and cycloalkenyl portions of the group. Representative substituted cycloalkenylalkyl groups may be substituted one or more times with substituents such as those listed above.

[0118] Alkynyl groups include straight-chain and branched-chain alkyl groups, as defined above, except that at least one triple bond exists between two carbon atoms. Alkynyl groups have 2 to 12 carbon atoms, typically 2 to 10 carbon atoms, or in some embodiments, 2 to 8, 2 to 6, or 2 to 4 carbon atoms. In some embodiments, alkynyl groups have one, two, or three carbon-carbon triple bonds. Examples include, but are not limited to, -C=CH, -C=CCH3, -CH2C=CCH3, and -C=CCH2CH(CH2CH3)2, among others. Alkynyl groups can be substituted or unsubstituted. A terminal alkyne has at least one hydrogen atom attached to a triple-bonded carbon atom. Representative substituted alkynyl groups can be mono-substituted or substituted more than once, for example, but not limited to, mono-, di-, or tri-substituted with the substituents listed above. A "cyclic alkyne" or "cycloalkynyl" is a cycloalkyl ring containing at least one triple bond between two carbon atoms. Examples of cyclic alkyne or cycloalkynyl groups include, but are not limited to, cyclooctyne, bicyclononyne (BCN), difluorinated cyclooctyne (DIFO), dibenzocyclooctyne (DIBO), keto-DIBO, biarylazacyclooctynone (BARAC), dibenzoazacyclooctyne (DIBAC), dimethoxyazacyclooctyne (DIMAC), difluorobenzocyclooctyne (DIFBO), monobenzocyclooctyne (MOBO), and tetramethoxyDIBO (TMDIBO).

[0119] An aryl group is a cyclic aromatic hydrocarbon that does not contain heteroatoms. Aryl groups herein include monocyclic, bicyclic, and tricyclic ring systems. Thus, aryl groups include, but are not limited to, phenyl, azulenyl, heptalenyl, biphenyl, fluorenyl, phenanthrenyl, anthracenyl, indenyl, indanyl, pentalenyl, and naphthyl groups. In some embodiments, aryl groups contain 6 to 14 carbons, and in other cases, 6 to 12, or even 6 to 10 carbon atoms in the ring portion of the group. In some embodiments, an aryl group is phenyl or naphthyl. Aryl groups can be substituted or unsubstituted. The phrase "aryl groups" includes groups containing fused rings, such as fused aromatic-aliphatic ring systems (e.g., indanyl, tetrahydronaphthyl, etc.). Representative substituted aryl groups can be monosubstituted or substituted more than once. For example, monosubstituted aryl groups include, but are not limited to, di-, tri-, tetra-, penta-, or hexa-substituted phenyl or naphthyl groups, which may be substituted with substituents such as those listed above. Aryl moieties are well known and are described, for example, in Lewis, RJ, ed., Hawley's Condensed Chemical Dictionary, 13 th Edition, John Wiley & Sons, Inc., New York (1997). Aryl groups can be a single ring structure (i.e., monocyclic) or can contain multiple ring structures (i.e., polycyclic) that are fused ring structures. Preferably, the aryl group is a monocyclic aryl group.

[0120] An alkoxy group is a hydroxyl group (—OH) in which the bond to the hydrogen atom is replaced by a bond to a carbon atom of a substituted or unsubstituted alkyl group as defined above. Examples of straight-chain alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy, butoxy, pentoxy, hexoxy, and the like. Examples of branched-chain alkoxy groups include, but are not limited to, isopropoxy, sec-butoxy, tert-butoxy, isopentoxy, isohexoxy, and the like. Examples of cycloalkoxy groups include, but are not limited to, cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, cyclohexyloxy, and the like. An alkoxy group may be substituted or unsubstituted. Representative substituted alkoxy groups may be substituted one or more times with substituents such as those listed above.

[0121] Similarly, alkylthio or thioalkoxy refers to the group -SR, where R is an alkyl attached to the parent molecule through a sulfur bridge, e.g., -S-methyl, -S-ethyl, etc. Representative examples of alkylthio include, but are not limited to, -SCH, -SCHCH, etc.

[0122] As used herein, the term "halogen" refers to bromine, chlorine, fluorine, or iodine. Correspondingly, the term "halo" refers to fluoro, chloro, bromo, or iodo. In some embodiments, the halogen is fluorine. In other embodiments, the halogen is chlorine or bromine.

[0123] The terms "hydroxy" and "hydroxyl" may be used interchangeably and refer to --OH.

[0124] The term "carboxy" refers to --COOH.

[0125] The term "cyano" refers to -CN.

[0126] The term "nitro" refers to -NO2.

[0127] The term "isothiocyanate" refers to -N=C=S.

[0128] The term "isocyanate" refers to -N=C=O.

[0129] The term "azido" refers to -N3.

[0130] The term "amino" refers to -NH. The term "alkylamino" refers to an amino group in which one or both of the hydrogen atoms bonded to the nitrogen are replaced with an alkyl group. An alkylamine group may be represented as -NR, where each R is independently hydrogen or an alkyl group. For example, alkylamines include methylamine (-NHCH), dimethylamine (-N(CH), -NHCHCH, and the like. As used herein, the term "aminoalkyl" is intended to include both branched and straight-chain saturated aliphatic hydrocarbon groups substituted with one or more amino groups. Representative examples of aminoalkyl groups include, but are not limited to, -CHNH, -CHCHNH, and -CHCH(NH)CH.

[0131] As used herein, "amide" refers to -C(O)N(R), where each R is independently an alkyl group or hydrogen. Examples of amides include, but are not limited to, -C(O)NH, -C(O)NHCH, and -C(O)N(CH).

[0132] The terms "hydroxylalkyl" and "hydroxyalkyl" are used interchangeably and refer to an alkyl group substituted with one or more hydroxyl groups. The alkyl can be a branched or straight chain aliphatic hydrocarbon. Examples of hydroxylalkyl include, but are not limited to, hydroxylmethyl (-CHOH), hydroxylethyl (-CHCHOH), and the like.

[0133] As used herein, the term "heterocyclyl" includes stable monocyclic and polycyclic hydrocarbons containing at least one heteroatom ring member, such as sulfur, oxygen, or nitrogen. As used herein, the term "heteroaryl" includes stable monocyclic and polycyclic aromatic hydrocarbons containing at least one heteroatom ring member, such as sulfur, oxygen, or nitrogen. Heteroaryls can be monocyclic or polycyclic, e.g., bicyclic or tricyclic. Each ring of a heteroatom-containing heterocyclyl or heteroaryl group can contain one or two oxygen or sulfur atoms and / or one to four nitrogen atoms, provided that the total number of heteroatoms in each ring is four or less, and each ring has at least one carbon atom. Heteroaryl groups that are polycyclic, e.g., bicyclic or tricyclic, must contain at least one fully aromatic ring, but any other fused rings or rings can be aromatic or non-aromatic. A heterocyclyl or heteroaryl group may be attached to any available nitrogen or carbon atom of any ring of the heterocyclyl or heteroaryl group. Preferably, the term "heteroaryl" refers to 5- or 6-membered monocyclic and 9- or 10-membered bicyclic groups having at least one heteroatom (O, S, or N) in at least one of the rings, with the heteroatom-containing ring preferably having 1, 2, or 3 heteroatoms, more preferably 1 or 2 heteroatoms selected from O, S, and / or N. The nitrogen heteroatom(s) of a heteroaryl may be substituted or unsubstituted. Additionally, the nitrogen and sulfur heteroatom(s) of a heteroaryl may be optionally oxidized (i.e., N→O and S(O)).r where r is 0, 1, or 2).

[0134] The term "ester" refers to -C(O)R, where R is alkyl.

[0135] The term "carbamate" refers to -OC(O)NR2, where each R is independently alkyl or hydrogen.

[0136] The term "aldehyde" refers to --C(O)H.

[0137] The term "carbonate" refers to -OC(O)OR, where R is alkyl.

[0138] The term "maleimide" refers to a group having the chemical formula H2C2(CO)2NH. The term "maleimido" refers to a maleimide group covalently bonded to another group or molecule. Preferably, the maleimide group is N-bonded, for example:

[0139] [ka]

[0140] The term "acyl halide" refers to -C(O)X, where X is halo (e.g., Br, Cl). Exemplary acyl halides include acyl chloride (-C(O)Cl) and acyl bromide (-C(O)Br).

[0141] According to the convention used in the art,

[0142] [ka] is used in structural formulas herein to indicate the bond that is the point of attachment of a moiety, functional group, or substituent to a core, parent, or scaffold structure, such as an antigen binding domain of the invention.

[0143] When any variable occurs more than one time in any constituent or formula for a compound, its definition at each occurrence is independent of its definition at every other occurrence. Thus, for example, if a group is shown to be substituted with 0 to 3 R groups, that group may be optionally substituted with up to 3 R groups, and at each occurrence R is independently selected from the definitions of R.

[0144] When a bond to a substituent is shown to cross a bond connecting two atoms in a ring, then such substituent may be bonded to any atom on the ring.

[0145] As used herein, the terms "radioactive metal ion" or "radioactive metal ion" or "radioisotope" or "radiometal" refer to one or more isotopes of an element that emit particles and / or photons. Any radioactive metal ion known to those of skill in the art in light of the present disclosure may be used in the present invention. Other radioisotopes that may be used for therapeutic applications include, for example, beta emitters or alpha emitters, such as: 225 Ac, 177 Lu, 32 P, 47 Sc, 67 Cu, 77 As, 89 Sr, 90 Y, 99 Tc, 105 Rh, 109 Pd, 111 Ag, 131 I, 149 Tb, 152 Tb, 155 Tb, 153 Sm, 159 Gd, 165 Dy, 166 Ho, 169 Er, 186 Re, 188 Re, 194 Ir, 198Au, 199 Au, 211 At, 212 Pb, 212 Bi, 213 Bi, 223 Ra, 255 Fm and 227 Other non-limiting examples of radioisotopes that may be used as imaging agents according to the present invention include, for example: 177 Lu, 62 Cu, 64 Cu, 67 Ga, 68 Ga, 86 Y, 89 Zr, and 111 and gamma-emitting radioisotopes such as In. In certain embodiments, the radioactive metal ion is a "therapeutic emitter," meaning a radioactive metal ion useful as a therapeutic agent, e.g., as a cytotoxic agent that can reduce or inhibit the growth of, or especially kill, cancer cells, such as prostate cancer cells. Examples of therapeutic emitters include beta-emitters or alpha-emitters, e.g., 132 La, 135 La, 134 Ce, 144 Nd, 149 Tb, 152 Tb, 155 Tb, 153 Sm, 159 Gd, 165 Dy, 166 Ho, 169 Er, 177 Lu, 186 Re, 188 Re, 194 Ir, 198 Au, 199 Au, 211 At, 212 Pb, 212 Bi, 213 Bi, 223 Ra, 225 Ac, 255 Fm and 227 Th, 226 Th, 230 Preferably, the radioactive metal ion used in the present invention is an alpha-emitting radioactive metal ion, such as actinium-225 (225 Ac).

[0146] As used herein, a "radiometal complex" refers to a complex comprising a radioactive metal ion associated with a chelating agent that is a macrocycle. Typically, the radioactive metal ion is bound or coordinated to the macrocycle via a coordinate bond. Heteroatoms of the macrocycle may participate in the coordinate bonding of the radioactive metal ion to the macrocycle. The macrocycle may be substituted with one or more substituents, which may also participate in the coordinate bonding of the radioactive metal ion to the macrocycle in addition to or instead of the heteroatoms of the macrocycle.

[0147] Immunoconjugates Embodiments of the present invention relate to compositions and methods for targeting hK2 with short half-life Fab-based radioconjugates to achieve effective tumor cell death in prostate cancer patients; preferably, such radioconjugates comprise a Fab (instead of a full-length antibody) and exhibit an improved safety profile (e.g., as measured by bone marrow toxicity) compared to full-length antibody-based radioconjugates. Embodiments of the Fab-based radioconjugates of the present invention target hK2-expressing prostate cancer cells and have a short half-life.

[0148] As used herein, "immunoconjugate" refers to an antibody or antigen-binding domain conjugated (covalently bound, e.g., attached to) a second molecule, such as a toxin, a drug, a radioactive metal ion, a chelator, a radioactive metal complex, etc. A "radioimmunoconjugate" (also referred to herein as a "radioconjugate") is, inter alia, an immunoconjugate in which the antibody or antigen-binding domain is labeled with a radioactive metal or conjugated to a radioactive metal complex.

[0149] According to embodiments of the present invention, an immunoconjugate comprises a therapeutic moiety conjugated to an antigen-binding domain of the present invention having binding specificity for hK2. As used herein, a "therapeutic moiety" forming part of an immunoconjugate may be useful in therapeutic and / or imaging applications, i.e., as a therapeutic agent (e.g., a cytotoxic agent) and / or an imaging agent, respectively. For example, a therapeutic moiety of the present invention may comprise a radiometal. Certain radiometals are useful in combination with therapeutic agents (e.g., 、225 Ac) and / or contrast agents (e.g., 111 It should be noted that suitable therapeutic agents can be used as therapeutic agents (e.g., immunoglobulins ...

[0150] According to certain embodiments, immunoconjugates containing an imaging agent such as Cu-64 can be used to detect prostate cancer cells in a subject. According to certain embodiments, a method for detecting the presence of prostate cancer in a subject comprises administering an immunoconjugate of the present invention to a subject suspected of having prostate cancer, and visualizing the biological structure to which the conjugate is bound (e.g., by computed tomography or positron emission tomography), thereby detecting the presence of prostate cancer. According to another embodiment, a method for detecting the progress of cancer treatment in a subject (e.g., after the subject has begun treatment for prostate cancer) comprises administering an immunoconjugate of the present invention to the subject, and visualizing the biological structure to which the immunoconjugate is bound (e.g., by computed tomography or positron emission tomography), thereby detecting the progress of prostate cancer treatment in the subject (e.g., detecting whether prostate cancer cells have decreased after prostate cancer treatment). According to one embodiment, the method can further comprise administering an anti-cancer therapeutic agent (e.g., an anti-hK2 therapeutic agent targeting hK2-expressing cancer cells) to the subject if prostate cancer is detected in the subject.

[0151] According to preferred embodiments, the present invention relates to short half-life immunoconjugates (e.g., short half-life radioimmunoconjugates). As used herein, "short half-life immunoconjugate" refers to an immunoconjugate comprising an antigen-binding domain (e.g., a Fab), where the immunoconjugate has an in vivo half-life that is shorter than the in vivo half-life of a comparable immunoconjugate, which is identical to the short half-life immunoconjugate except that the antigen-binding domain of the comparable immunoconjugate is replaced with a full-length antibody comprising an antigen-binding domain (e.g., a full-length IgG comprising an Fc region and an antigen-binding domain). In certain embodiments, the short half-life immunoconjugates of the present invention have a half-life of 36 hours or less, or 24 hours or less, or 12 hours or less, or 6 hours or less, or 3 hours or less. For example, a short half-life immunoconjugate of the invention may have a half-life of about 1 hour to about 36 hours, or about 1 hour to about 24 hours, or about 1 hour to about 12 hours, or about 1 hour to about 6 hours, or about 1 hour to about 3 hours, or about 2 hours to about 3 hours.

[0152] Actinium 225( 225 Ac) is an alpha-emitting radioisotope of particular interest for medical applications. Another radioisotope of interest for medical applications is lutetium-177 ( 177 Other radioisotopes that can be used for therapeutic purposes include, for example, beta-emitters or alpha-emitters, such as 225 Ac, 177 Lu, 32 P, 47 Sc, 67 Cu, 77 As, 89 Sr, 90 Y, 99 Tc, 105 Rh, 109 Pd, 111 Ag, 131 I, 149 Tb, 152 Tb, 155 Tb, 153 Sm,159 Gd, 165 Dy, 166 Ho, 169 Er, 186 Re, 188 Re, 194 Ir, 198 Au, 199 Au, 211 At, 212 Pb, 212 Bi, 213 Bi, 223 Ra, 255 Fm and 227 Other non-limiting examples of radioisotopes that may be used as imaging agents according to the present invention include, for example: 177 Lu, 62 Cu, 64 Cu, 67 Ga, 68 Ga, 86 Y, 89 Zr, and 111 Examples include gamma-ray emitting isotopes such as In.

[0153] In certain embodiments, the therapeutic moiety is a cytotoxic drug that is an auristatin derivative, such as MMAE (monomethylauristatin E) or MMAF (monomethylauristatin F). For example, the auristatin derivative may be attached to an antibody or antigen-binding domain of the invention via the N-terminus or C-terminus of the peptidic drug moiety (WO 02 / 088172), or via any cysteine ​​engineered into the antibody or antigen-binding domain.

[0154] Anti-hK2 antibodies and antigen-binding domains As described herein, embodiments of the present invention relate to immunoconjugates comprising a therapeutic moiety conjugated to an antigen-binding domain having binding specificity for kallikrein-related peptidase 2 (hK2). According to certain embodiments, the antigen-binding domain is an scFv, (scFv)2, aFv, Fab, F(ab')2, Fd, dAb, or VHH. According to a preferred embodiment, the antigen-binding domain with binding specificity for hK2 is a Fab.

[0155] According to certain embodiments, the antigen binding domain that binds to hK2 (e.g., Fab) comprises the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 170, 171, 172, 173, 174, and 175, respectively.

[0156] According to certain embodiments, an antigen-binding domain that binds hK2 (e.g., a Fab) comprises a VH that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to the VH of SEQ ID NO: 162, and a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to the VL of SEQ ID NO: 163. For example, an antigen-binding domain (e.g., a Fab) that binds hK2 comprises a VH that is at least 95% identical to the VH of SEQ ID NO: 162, and a VL that is at least 95% identical to the VL of SEQ ID NO: 163. According to certain embodiments, an antigen-binding domain (e.g., a Fab) that binds hK2 comprises a VH of SEQ ID NO: 162 and a VL of SEQ ID NO: 163.

[0157] According to certain embodiments, the antigen-binding domain is a "KL2B30 Fab", also referred to as "Fab of KL2B30" (a) a Fab comprising the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 170, 171, 172, 173, 174 and 175, respectively, and / or (b) a VH at least 95% or 100% identical to SEQ ID NO: 162 and a VL at least 95% or 100% identical to SEQ ID NO: 163.

[0158] Non-limiting examples of KL2B30 Fabs include KL2B997 and KL2B1251, which are described in the Examples section below. The heavy and light chain sequences of KL2B997 (SEQ ID NOs: 472 and 473, respectively) and KL2B1251 (SEQ ID NOs: 474 and 475, respectively) are shown in Figure 2. KL2B997 has a His-tag and a sortase tag (underlined in Figure 2) on the heavy chain, while KL2B1251 has no tags.

[0159] According to certain embodiments, the antigen-binding domain that binds to hK2 comprises a heavy chain that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to SEQ ID NO: 474, and a light chain that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to SEQ ID NO: 475. In embodiments, the antigen-binding domain is a Fab comprising a heavy chain that is at least 95%, at least 99%, or 100% identical to SEQ ID NO: 474 and a light chain that is at least 95%, at least 99%, or 100% identical to SEQ ID NO: 475.

[0160] According to certain embodiments, the antigen-binding domain that binds to hK2 comprises a heavy chain that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to SEQ ID NO: 472, and a light chain that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to SEQ ID NO: 473. In embodiments, the antigen-binding domain is a Fab comprising a heavy chain that is at least 95%, at least 99%, or 100% identical to SEQ ID NO: 472 and a light chain that is at least 95%, at least 99%, or 100% identical to SEQ ID NO: 473.

[0161] In some embodiments, an immunoconjugate of the invention comprises an antigen-binding domain comprising a VL, VH, or CDR having the amino acid sequence of a specific antibody as set forth below, selected from the group consisting of m11B6, hu11B6, HCF3-LCD6, HCG5-LCB7, KL2B357, KL2B358, KL2B359, KL2B360, KL2B413, KL2B30, KL2B53, KL2B242, KL2B467, and KL2B494. The foregoing antibodies and antigen-binding domains, as well as methods for making them, are described in PCT / IB 2020 / 056972, which is incorporated herein by reference.

[0162] An embodiment of the present invention provides a radioimmunoconjugate having the following structure (not showing the lysine residue of the Fab linked to the phenylthiourea moiety):

[0163] [ka] (also known as TOPA-[C7]-phenylthiourea-Fab), In the formula, M + is a radioactive isotope, e.g., actinium 225 ( 225 Ac), wherein the Fab has binding specificity for hK2, e.g., KL2B30 Fab (e.g., KL2B997 or KL2B1251). The Fab preferably comprises (a) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 170, 171, 172, 173, 174, and 175, respectively, and / or (b) a VH that is at least 95% or 100% identical to SEQ ID NO: 162 and a VL that is at least 95% or 100% identical to SEQ ID NO: 163.

[0164] An embodiment of the present invention provides a radioimmunoconjugate having the following structure (not showing the lysine residue of the Fab linked to the phenylthiourea moiety):

[0165] [ka] wherein the Fab has binding specificity for hK2, e.g., KL2B30 Fab (e.g., KL2B997 or KL2B1251). The Fab preferably comprises (a) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 170, 171, 172, 173, 174, and 175, respectively, and / or (b) a VH that is at least 95% or 100% identical to SEQ ID NO: 162 and a VL that is at least 95% or 100% identical to SEQ ID NO: 163.

[0166] Embodiments of the present invention provide a Fab conjugated to NOTA or a derivative of NOTA, such as NODA or NODA-GA. Embodiments of the present invention provide a NODA-GA-Fab, which can be represented by the structure of Figure 4 (not showing any lysine chains of the Fab connected to the chelator-linker), where M + is Cu-64( 64 and the Fab has binding specificity for hK2, such as KL2B30 Fab (e.g., KL2B997 or KL2B1251). The Fab preferably comprises (a) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 170, 171, 172, 173, 174, and 175, respectively, and / or (b) a VH at least 95% or 100% identical to SEQ ID NO: 162 and a VL at least 95% or 100% identical to SEQ ID NO: 163.

[0167] An embodiment of the present invention is + is Cu-64( 64Cu), and wherein the Fab is KL2B30 Fab (e.g., KL2B997 or KL2B1251), and the Fab is KL2B30 Fab (e.g., KL2B997 or KL2B1251), and the Fab comprises (a) the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 170, 171, 172, 173, 174, and 175, respectively; and / or (b) a VH that is at least 95% or 100% identical to SEQ ID NO: 162 and a VL that is at least 95% or 100% identical to SEQ ID NO: 163. A method for detecting hK2-expressing prostate cancer cells in a subject includes administering NODA-GA-KL2B30 Fab to a subject suspected of having prostate cancer, visualizing (preferably by positron emission tomography) the biological structure to which NODA-GA-KL2B30 Fab is bound, thereby detecting hK2-expressing prostate cancer cells.

[0168] Specific Enumerated Embodiments Exemplary numbered embodiments of the present invention are set forth below. 1. An immunoconjugate comprising a therapeutic moiety conjugated to an antigen-binding domain that has binding specificity for kallikrein-related peptidase 2 (hK2). 2. The immunoconjugate of embodiment 1, wherein the therapeutic moiety is a cytotoxic drug. 3. The immunoconjugate of embodiment 1, wherein the therapeutic moiety is an imaging agent. 4. The immunoconjugate of any of embodiments 1-3, wherein the therapeutic moiety comprises a radioactive metal. 5. Radioactive metals 225 Ac, 177 Lu, 32 P, 47 Sc, 67 Cu, 77 As, 89 Sr, 90 Y, 99 Tc, 105 Rh, 109 Pd, 111 Ag, 131 I, 149 Tb, 152 Tb,155 Tb, 153 Sm, 159 Gd, 165 Dy, 166 Ho, 169 Er, 186 Re, 188 Re, 194 Ir, 198 Au, 199 Au, 211 At, 212 Pb, 212 Bi, 213 Bi, 223 Ra, 255 Fm, 227 Th, 177 Lu, 62 Cu, 64 Cu, 67 Ga, 68 Ga, 86 Y, 89 Zr, and 111 The immunoconjugate of embodiment 4, selected from the group consisting of In. 6. The treatment area is 225 2. The immunoconjugate of embodiment 1, wherein the cytotoxic agent comprises Ac. 7. The treatment area is 111 In or 64 2. The immunoconjugate of embodiment 1, wherein the immunoconjugate is a Cu-containing imaging agent. 8. The immunoconjugate of any of embodiments 4-7, wherein the therapeutic moiety comprises a radiometal complex, the radiometal complex comprising a radiometal bound to a chelator, and the chelator is conjugated to an antigen-binding domain having binding specificity for kallikrein-related peptidase 2 (hK2). 9. The immunoconjugate of embodiment 8, wherein the chelating agent is 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), S-2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), 1,4,8,11-tetraazacyclododecane-1,4,8,11-tetraacetic acid (TETA), 3,6,9,15-tetraazabicyclo[9.3.1]-pentadeca-1(15),11,13-triene-4-(S)-(4-isothiocyanatobenzyl)-3,6,9-triacetic acid (PCTA), 5-S-(4-aminobenzyl)-1-oxa-4,7,10-triazacyclododecane-4,7,10-tris(acetic acid) (DO3A), or a derivative thereof. 10. The immunoconjugate of embodiment 8, wherein the chelating agent is DOTA or NOTA or a derivative thereof. 11. The immunoconjugate of embodiment 8, wherein the chelating agent is H2bp18c6 or an H2bp18c6 derivative. 12. The radioactive metal complex is a radioactive complex of formula (Im), or formula (II-m), or formula (III-m) described herein, wherein R 11 9. The immunoconjugate of embodiment 8, wherein X comprises an antigen-binding domain having binding specificity for kallikrein-related peptidase 2 (hK2); and M is a radioactive metal. 13. The immunoconjugate of embodiment 8, wherein the radiometal complex is a radiometal complex of Formula (IV-m), or Formula (Vm), or Formula (VI-m), as described herein, wherein R4 comprises an antigen-binding domain having binding specificity for kallikrein-related peptidase 2 (hK2), and M+ is a radiometal. 14. The immunoconjugate of embodiment 1, wherein the therapeutic moiety is an auristatin derivative. 15. The immunoconjugate of embodiment 14, wherein the therapeutic moiety is MMAE (monomethylauristatin E). 16. The immunoconjugate of embodiment 14, wherein the therapeutic moiety is MMAF (monomethyl auristatin F). 17. The immunoconjugate of any of embodiments 1 to 16 or 51 to 56, wherein the antigen-binding domain that binds to hK2 is an scFv, (scFv)2, Fv, Fab, F(ab')2, Fd, dAb, or VHH. 18. The immunoconjugate of any of embodiments 1 to 16 or 51 to 56, wherein the antigen-binding domain with binding specificity for hK2 is a Fab. 19. The immunoconjugate of any of embodiments 1-18 or 51-56, wherein the antigen-binding domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NO: 170 (SYYWS), SEQ ID NO: 171 (YIYYSGSTNYNPSLKS), SEQ ID NO: 172 (TTIFGVVTPNFYYGMDV), SEQ ID NO: 173 (RASQGISSYLA), SEQ ID NO: 174 (AASTLQS) and SEQ ID NO: 175 (QQLNSYPLT), respectively. 20. A VH in which the antigen-binding domain that binds to hK2 is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to the VH of SEQ ID NO: 162 (QVQLQESGPGLVKPSETLSLTCTVSGGSISSYYWSWIRQPPGKGLEWIGYIYYSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAGTTIFGVVTPNFYYGMDVWGQGTTVTVSS); and a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99% or 100%) identical to the VL of SEQ ID NO: 163 (DIQMTQSPSFLSASVGDRVTITCRASQGISSYLAWYQQKPGKAPKFLIYAASTLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQLNSYPLTFGGGTKVEIK). 21. The immunoconjugate of any of embodiments 1 to 19 or 51 to 56, wherein the antigen-binding domain that binds to hK2 comprises a VH of SEQ ID NO: 162 and a VL of SEQ ID NO: 163. 22. The antigen-binding domain is a. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 170, 171, 172, 173, 174 and 175, respectively; and / or b. VH of SEQ ID NO: 162 and VL of SEQ ID NO: 163, and / or c. An immunoconjugate of any of embodiments 1-18 or 51-56, comprising a heavy chain that is at least 95%, at least 99%, or 100% identical to SEQ ID NO: 474, and a light chain that is at least 95%, at least 99%, or 100% identical to SEQ ID NO: 475. 23. The immunoconjugate of any of embodiments 1-22 or 51-56, which is a short half-life immunoconjugate. 24. A method of treating an hK2-expressing cancer in a subject, comprising administering to the subject a therapeutically effective amount of an immunoconjugate of any of embodiments 1-23 or 51-56. 25. A method for reducing the amount of hK2-expressing tumor cells in a subject, comprising administering to the subject a therapeutically effective amount of the immunoconjugate of any of embodiments 1-23 or 51-56. 26. A method of treating prostate cancer in a subject, comprising administering to the subject a therapeutically effective amount of the immunoconjugate of any of claims 1-23 or 51-56. 27. The method of embodiment 26, wherein the prostate cancer is recurrent, refractory, aggressive, or castration-resistant prostate cancer, or any combination thereof. 28. The method of embodiment 26, wherein the prostate cancer is metastatic castration-resistant prostate cancer. 29. A method for detecting the presence of prostate cancer in a subject, comprising administering an immunoconjugate of any of embodiments 1-23 or 51-56 to a subject suspected of having prostate cancer, and visualizing the biological structure to which the conjugate is bound (e.g., by computed tomography or positron emission tomography), thereby detecting the presence of prostate cancer, wherein the immunoconjugate preferably comprises an imaging agent such as 111-In or 64-Cu. 30. A method of making the immunoconjugate of any one of embodiments 1-23 or 51-56, comprising conjugating a therapeutic moiety to an antigen-binding domain that has binding specificity for kallikrein-related peptidase 2 (hK2). 31. A method for making a radioimmunoconjugate, comprising binding a radiometal to a chelator conjugated to an antigen-binding domain having binding specificity for kallikrein-related peptidase 2 (hK2). 32. The method of embodiment 31, wherein the chelating agent is DOTA or NOTA or a derivative thereof. 33. The method of embodiment 31, wherein the chelating agent is H2bp18c6 or an H2bp18c6 derivative. 34. The chelating agent is selected from the group consisting of chelating agents of formula (I), formula (II), and formula (III) described herein, wherein R 11 32. The method of embodiment 31, wherein said antibody comprises an antigen-binding domain that has binding specificity for kallikrein-related peptidase 2 (hK2). 35. The method of embodiment 31, wherein the chelator is selected from the group consisting of chelators of formula (IV), formula (V), and formula (VI) described herein, wherein R4 comprises an antigen-binding domain that has binding specificity for kallikrein-related peptidase 2 (hK2). 36. The method of any one of embodiments 31 to 35, wherein the antigen-binding domain that binds to hK2 is scFv, (scFv)2, Fv, Fab, F(ab')2, Fd, dAb, or VHH. 37. The method of any one of embodiments 31 to 35, wherein the antigen-binding domain that binds to hK2 is a Fab. 38. The method of any one of claims 31 to 37, wherein the antigen-binding domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 170, 171, 172, 173, 174 and 175, respectively. 39. The method of any one of embodiments 31 to 38, wherein the antigen-binding domain that binds to hK2 comprises a VH that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99% or 100%) identical to the VH of SEQ ID NO: 162, and a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99% or 100%) identical to the VL of SEQ ID NO: 163. 40. The method of any one of embodiments 31 to 38, wherein the antigen-binding domain that binds to hK2 comprises a VH of SEQ ID NO: 162 and a VL of SEQ ID NO: 163. 41. The antigen-binding domain is a. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 170, 171, 172, 173, 174 and 175, respectively; and / or b. A method according to any one of embodiments 31 to 37, wherein the Fab comprises a VH of SEQ ID NO: 162 and a VL of SEQ ID NO: 163. 42. A radioactive metal complex is included, and the radioactive metal complex is bound to a chelating agent. 225 A short half-life radioimmunoconjugate comprising Ac, wherein the chelator is conjugated to a Fab having binding specificity for hK2, the Fab comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 170, 171, 172, 173, 174 and 175, respectively. 43. A short half-life radioimmunoconjugate according to embodiment 42, wherein the Fab comprises a VH of SEQ ID NO: 162 and a VL of SEQ ID NO: 163. 44. A short half-life radioimmunoconjugate according to embodiment 42 or 43, wherein the chelating agent is DOTA or NOTA or a derivative thereof. 45. The short half-life radioimmunoconjugate of embodiment 42 or 43, wherein the chelating agent is H2bp18c6 or an H2bp18c6 derivative. 46. ​​The radioactive metal complex is selected from the group consisting of chelators of formula (Im), formula (II-m), and formula (III-m) described herein, and R 1144. The short half-life radioimmunoconjugate of embodiment 42 or 43, wherein said Fab has binding specificity for hK2. 47. A short half-life radioimmunoconjugate according to embodiment 42 or 43, wherein the radiometal complex is selected from the group consisting of chelators of formula (IV-m), formula (Vm) and formula (VI-m) described herein, and R4 comprises a Fab having binding specificity for hK2. 48. The method of any of embodiments 31-37, further comprising conjugating the chelator to an antigen-binding domain having binding specificity for kallikrein-related peptidase 2 (hK2) prior to conjugating the radiometal to the chelator. 49. Before conjugating the chelator to the antigen-binding domain,

[0169] [ka] 49. The method of embodiment 48, having the structure: 50. Before conjugating the chelator to the antigen-binding domain,

[0170] [ka] 49. The method of embodiment 48, having the structure: 51. The radioactive metal complex is a radioactive metal complex of the formula (Im): The radiometal complex of formula (Im) has the following structure:

[0171] [ka] During the ceremony, M is a radioactive metal, preferably an alpha-emitting radioactive metal ion, more preferably actinium 225 ( 225 Ac), Each of ring A and ring B is independently a 6- to 10-membered aryl or a 5- to 10-membered heteroaryl, and each of ring A and ring B is independently halo, alkyl, alkenyl, cycloalkyl, cycloalkenyl, aryl, heterocyclyl, heteroaryl, -OR 13 , -SR 13 , -(CH2) p COOR 13 , -OC(O)R 13 , -N(R 13 )2, -CON(R 13 )2, -NO2, -CN-OC(O)N(R 13 ) 2, and X, Each of Z1 and Z2 independently represents (C(R 12 )2) m - or -(CH2) n -C(R 12 )(X)-(CH2) n - and Each X is independently -L1-R 11 and each n is independently 0, 1, 2, 3, 4, or 5; each m is independently 1, 2, 3, 4, or 5; each p is independently 0 or 1; L1 is absent or a linker, R 11 comprises an antigen-binding domain that has binding specificity for kallikrein-related peptidase 2 (hK2), Each R 12 are independently hydrogen, alkyl, cycloalkyl, aryl, heterocyclyl, or heteroaryl; Each R 13 are independently hydrogen or alkyl; R 14 , R 15 , R 16 and R 17 each independently is hydrogen, alkyl, or X; Alternatively, R 14 and R 15 and / or R 16 and R 17together with the carbon atoms to which they are attached form a 5- or 6-membered cycloalkyl ring optionally substituted with X, provided that when the radiometal complex contains at least one X and X is present on ring A or ring B, L is a linker or R 12 and R 14 ~R 17 The immunoconjugate of embodiment 8, wherein at least one of is not hydrogen. 52. The radioactive metal complex is a radioactive metal complex of formula (II-m): The radiometal complex of formula (II-m) has the following structure:

[0172] [ka] During the ceremony, M is a radioactive metal, preferably an alpha-emitting radioactive metal ion, more preferably actinium 225 ( 225 Ac), A1 is N or CR1 or absent; A2 is N or CR2; A3 is N or CR3; A4 is N or CR4; A5 is N or CR5; A6 is N or CR6 or absent; A7 is N or CR7; A8 is N or CR8; A9 is N or CR9, A 10 is N or CR 10 and However, three or less of A1, A2, A3, A4, and A5 are N, and A6, A7, A8, A9, and A 10 Not more than three of R1, R2, R3, R4, R5, R6, R7, R8, R9, and R 10 each independently represents hydrogen, halo, alkyl, alkenyl, cycloalkyl, cycloalkenyl, aryl, heterocyclyl, heteroaryl, OR 13 , -SR13 , -(CH2) p COOR 13 , -OC(O)R 13 , -N(R 13 )2, -CON(R 13 )2, -NO2, -CN-OC(O)N(R 13 )2, and -X; Alternatively, any two immediately adjacent R1, R2, R3, R4, R5, R6, R7, R8, R9, and R 10 together with the atoms to which they are attached form a 5- or 6-membered substituted or unsubstituted carbocyclic or nitrogen-containing ring; Each of Z1 and Z2 independently represents (C(R 12 )2) m - or -(CH2) n -C(R 12 )(X)-(CH2) n - and Each X is independently -L1-R 11 and each n is independently 0, 1, 2, 3, 4, or 5; each m is independently 1, 2, 3, 4, or 5; each p is independently 0 or 1; L1 is absent or a linker, R 11 comprises an antigen-binding domain that has binding specificity for kallikrein-related peptidase 2 (hK2), Each R 12 are independently hydrogen, alkyl, cycloalkyl, aryl, heterocyclyl, or heteroaryl; Each R 13 are independently hydrogen or alkyl; R 14 , R 15 , R 16 and R 17 each independently is hydrogen, alkyl, or X; Alternatively, R 14 and R 15 and / or R 16 and R 17together with the carbon atoms to which they are attached form a 5- or 6-membered cycloalkyl ring which may be substituted by X; provided that the radiometal complex contains at least one X and R1, R2, R3, R4, R5, R6, R7, R8, R9, and R 10 is X, then L1 is a linker, or R 12 and R 14 ~R 17 The immunoconjugate of embodiment 8, wherein at least one of is not hydrogen. 53. The radioactive metal complex is a radioactive metal complex of formula (III-m): The radiometal complex of formula (III-m) has the following structure:

[0173] [ka] During the ceremony, M is a radioactive metal, preferably an alpha-emitting radioactive metal ion, more preferably actinium 225 ( 225 Ac), Each A 11 are independently O, S, NMe, or NH; Each of Z1 and Z2 independently represents (C(R 12 )2) m - or -(CH2) n -C(R 12 )(X)-(CH2) n - and Each X is independently -L1-R 11 and each n is independently 0, 1, 2, 3, 4, or 5; each m is independently 1, 2, 3, 4, or 5; each p is independently 0 or 1; L1 is absent or a linker, R 11 comprises an antigen-binding domain that has binding specificity for kallikrein-related peptidase 2 (hK2), Each R 12are independently hydrogen, alkyl, cycloalkyl, aryl, heterocyclyl, or heteroaryl; Each R 13 are independently hydrogen or alkyl; R 14 , R 15 , R 16 and R 17 each independently is hydrogen, alkyl, or X; Alternatively, R 14 and R 15 and / or R 16 and R 17 together with the carbon atoms to which they are attached form a 5- or 6-membered cycloalkyl ring which may be substituted by X; Each R 18 are independently hydrogen, halo, alkyl, alkenyl, cycloalkyl, cycloalkenyl, aryl, heterocyclyl, heteroaryl, OR 13 , -SR 13 , -(CH2) p COOR 13 , -OC(O)R 13 , -N(R 13 )2, -CON(R 13 )2, -NO2, -CN-OC(O)N(R 13 )2, and -X; However, the radioactive metal complex contains at least one X and R 18 is X, L1 is a linker, or R 12 and R 14 ~R 17 The immunoconjugate of embodiment 8, wherein at least one of is not hydrogen. 54. The radioactive metal complex is a radioactive metal complex of formula (IV-m): The radiometal complex of formula (IV-m) has the following structure:

[0174] [ka] [In the formula, M + is a radioactive metal, preferably actinium-225 ( 225Ac), radium-223( 233 Ra), Bismuth-213( 213 Bi), lead-212( 212 Pb(II) and / or 212 Pb(IV)), terbium-149( 149 Tb), terbium-152( 152 Tb), terbium-155( 155 Tb), Fermium-255( 255 Fm), thorium-227( 227 Th), thorium-226( 226 Th 4+ ), astatine-211( 211 At), cerium-134( 134 Ce), neodymium 144( 144 Nd), Lanthanum-132( 132 La), lanthanum-135( 135 La) and uranium-230 ( 230 U) R1 is hydrogen and R2 is -L1-R4; Alternatively, R1 is -L1-R4 and R2 is hydrogen; R3 is hydrogen; or R2 and R3 together with the carbon atom to which they are attached form a 5- or 6-membered cycloalkyl, which 5- or 6-membered cycloalkyl is optionally substituted with -L1-R4; L1 is absent or a linker, R4 comprises an antigen-binding domain that has binding specificity for kallikrein-related peptidase 2 (hK2), or a pharmaceutically acceptable salt thereof. 55. The radioactive metal complex is a radioactive metal complex of the formula (Vm): The radiometal complex of formula (Vm) has the following structure:

[0175] [ka] [In the formula, M +is a radioactive metal, preferably actinium-225 ( 225 Ac), radium-223( 233 Ra), Bismuth-213( 213 Bi), lead-212( 212 Pb(II) and / or 212 Pb(IV)), terbium-149( 149 Tb), terbium-152( 152 Tb), terbium-155( 155 Tb), Fermium-255( 255 Fm), thorium-227( 227 Th), thorium-226( 226 Th 4+ ), astatine-211( 211 At), cerium-134( 134 Ce), neodymium 144( 144 Nd), Lanthanum-132( 132 La), lanthanum-135( 135 La) and uranium-230 ( 230 U) L1 is absent or a linker, R4 comprises an antigen-binding domain that has binding specificity for kallikrein-related peptidase 2 (hK2), or a pharmaceutically acceptable salt thereof. 56. The radioactive metal complex is a radioactive metal complex of formula (VI-m): The radiometal complex of formula (VI-m) has the following structure:

[0176] [ka] [In the formula, M + is a radioactive metal, preferably actinium-225 ( 225 Ac), radium-223( 233 Ra), Bismuth-213( 213 Bi), lead-212( 212 Pb(II) and / or 212 Pb(IV)), terbium-149( 149Tb), terbium-152( 152 Tb), terbium-155( 155 Tb), Fermium-255( 255 Fm), thorium-227( 227 Th), thorium-226( 226 Th 4+ ), astatine-211( 211 At), cerium-134( 134 Ce), neodymium 144( 144 Nd), Lanthanum-132( 132 La), lanthanum-135( 135 La) and uranium-230 ( 230 U) L1 is absent or a linker, R4 comprises an antigen-binding domain that has binding specificity for kallikrein-related peptidase 2 (hK2), or a pharmaceutically acceptable salt thereof. 57. An immunoconjugate comprising a radioactive metal complex conjugated to an antigen-binding domain having binding specificity for kallikrein-related peptidase 2 (hK2), wherein the radioactive metal complex is conjugated to a chelator. 64 An immunoconjugate comprising Cu, wherein a chelator is conjugated to an antigen-binding domain having binding specificity for kallikrein-related peptidase 2 (hK2). 58. The chelating agent is 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), S-2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), 1,4,8,11-tetraazacyclododecane-1,4,8,11-tetraacetic acid (TETA), 3,6,9,15-tetraazabicyclohexyl 58. The immunoconjugate of embodiment 57, which is 5-[9.3.1]-pentadeca-1(15),11,13-triene-4-(S)-(4-isothiocyanatobenzyl)-3,6,9-triacetic acid (PCTA), 5-S-(4-aminobenzyl)-1-oxa-4,7,10-triazacyclododecane-4,7,10-tris(acetic acid) (DO3A), or derivatives thereof. 59. The immunoconjugate of embodiment 57, wherein the chelating agent is NOTA or a derivative thereof, such as NODA-GA, NODA-GA(t-butyl)3, di-t-butyl-NOTA, NOTA-thiosemicarbazide, NODA-MPAA, or NODA-MPAEM, preferably NODA-GA. 60. The immunoconjugate of any of embodiments 57 to 59, wherein the antigen-binding domain with binding specificity for hK2 is a Fab. 61. The immunoconjugate of any of embodiments 57-60, wherein the antigen-binding domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NO: 170 (SYYWS), SEQ ID NO: 171 (YIYYSGSTNYNPSLKS), SEQ ID NO: 172 (TTIFGVVTPNFYYGMDV), SEQ ID NO: 173 (RASQGISSYLA), SEQ ID NO: 174 (AASTLQS) and SEQ ID NO: 175 (QQLNSYPLT), respectively. 62. The antigen-binding domain that binds to hK2 is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to the VH of SEQ ID NO: 162 (QVQLQESGPGLVKPSETLSLTCTVSGGSISSYYWSWIRQPPGKGLEWIGYIYYSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAGTTIFGVVTPNFYYGMDVWGQGTTVTVSS). 62. The immunoconjugate of any of embodiments 57-61, comprising a VH and a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99% or 100%) identical to the VL of SEQ ID NO: 163 (DIQMTQSPSFLSASVGDRVTITCRASQGISSYLAWYQQKPGKAPKFLIYAASTLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQLNSYPLTFGGGTKVEIK). 63. The method of any one of embodiments 57 to 62, wherein the antigen-binding domain that binds to hK2 comprises a VH of SEQ ID NO: 162 and a VL of SEQ ID NO: 163. 64. The antigen-binding domain is a. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 170, 171, 172, 173, 174 and 175, respectively; and / or b. An immunoconjugate according to any of embodiments 571 to 60, which is a Fab comprising a VH of SEQ ID NO: 162 and a VL of SEQ ID NO: 163. 65. The immunoconjugate of any of embodiments 57-64, which is a short half-life immunoconjugate. 66. A radioactive metal complex conjugated to an antigen-binding domain having binding specificity for kallikrein-related peptidase 2 (hK2), The radiometal complex is bound to a chelator that is NOTA or a derivative thereof, such as NODA-GA, NODA-GA(t-butyl)3, di-t-butyl-NOTA, NOTA-thiosemicarbazide, NODA-MPAA, or NODA-MPAEM, preferably NODA-GA. 64 Contains Cu, a chelator conjugated to an antigen-binding domain that has binding specificity for kallikrein-related peptidase 2 (hK2); The antigen-binding domain is (a) comprising the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NO: 170 (SYYWS), SEQ ID NO: 171 (YIYYSGSTNYNPSLKS), SEQ ID NO: 172 (TTIFGVVTPNFYYGMDV), SEQ ID NO: 173 (RASQGISSYLA), SEQ ID NO: 174 (AASTLQS) and SEQ ID NO: 175 (QQLNSYPLT), respectively; (b) an immunoconjugate comprising a VH that is at least 95%, at least 99% or 100% identical to the VH of SEQ ID NO: 162, and a VL that is at least 95%, at least 99% or 100% identical to the VL of SEQ ID NO: 163. 67. A method for detecting the presence of prostate cancer in a subject, comprising administering to a subject suspected of having prostate cancer an immunoconjugate of any of embodiments 57-66 and detecting the presence of prostate cancer by visualizing (e.g., by computed tomography or positron emission tomography) the biological structure to which the conjugate is bound. 68. A method for detecting the progress of cancer treatment in a subject, comprising administering to the subject an immunoconjugate described in any of embodiments 57-66, and visualizing (e.g., by computed tomography or positron emission tomography) the biological structure to which the immunoconjugate is bound, thereby detecting the progress of prostate cancer treatment in the subject. 69. The method of embodiment 67 or embodiment 68, comprising using positron emission tomography (PET) imaging to visualize the biological structure to which the conjugate is bound. 70. The method of any of embodiments 67-69, further comprising administering an anti-cancer therapeutic agent (e.g., an anti-hK2 therapeutic agent) to the subject if prostate cancer is detected in the subject. 71. Use of the immunoconjugate of any of embodiments 57 to 66 for diagnosing a subject with prostate cancer. 72. Use of an immunoconjugate according to any of embodiments 57 to 66 for visualizing prostate cancer in a patient. 73. Use of the immunoconjugate of any of embodiments 57-66 for determining the stage of prostate cancer in a subject.

[0177] chelating agents In certain embodiments, the present invention relates to immunoconjugates, e.g., radioimmunoconjugates, comprising a chelating agent, preferably a chelating agent capable of chelating a radiometal via a coordinate bond. In certain embodiments, the chelating agent of the present invention refers to a chelating agent capable of complexing a metal, preferably a radiometal, to form a radiometal complex. Preferably, the chelating agent is a macrocyclic compound. In certain embodiments, the chelating agent comprises a macrocycle or macrocyclic ring containing one or more heteroatoms, such as oxygen and / or nitrogen, as ring atoms.

[0178] According to certain embodiments, the chelator comprises a macrocyclic chelating moiety. Examples of macrocyclic chelating moieties include, but are not limited to, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), S-2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), 1,4,8,11-tetraazacyclododecane-1,4,8,11-tetraacetic acid (TETA), 3,6,9,15-tetraazabicyclo[9.3.1]-pentadeca-1(15),11,13-triene-4-(S)-(4-isothiocyanatobenzyl)-3,6,9-triacetic acid (PCTA), 5-S-(4-aminobenzyl)-1-oxa-4,7,10-triazacyclododecane-4,7,10-tris(acetic acid) (DO3A) or derivatives thereof. In some embodiments, the chelating agent is 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA). In other embodiments, the chelating agent is S-2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA). In further embodiments, the chelating agent is 1,4,8,11-tetraazacyclododecane-1,4,8,11-tetraacetic acid (TETA). In still other embodiments, the chelating agent is 3,6,9,15-tetraazabicyclo[9.3.1]-pentadeca-1(15),11,13-triene-4-(S)-(4-isothiocyanatobenzyl)-3,6,9-triacetic acid (PCTA). In yet further embodiments, the chelating agent is 5-S-(4-aminobenzyl)-1-oxa-4,7,10-triazacyclododecane-4,7,10-tris(acetic acid) (DO3A). In other embodiments, the chelating agent is DOTA, DFO, DTPA, NOTA, or TETA.

[0179] In certain embodiments, the chelator comprises NOTA or a derivative thereof. Non-limiting examples of NOTA derivatives include NODA, NODA-GA, NODA-GA(t-butyl)3, di-t-butyl-NOTA, NOTA-thiosemicarbazide, NODA-MPAA, and NODA-MPAEM. In one embodiment, a conjugable version of the chelator-linker can be referred to as NODA-GA-NHS, or 2,2',2"-(1,4,7-triazacyclononane-1,4,7-triyl)diacetic acid-glutamic acid, as shown below:

[0180] [ka]

[0181] In certain embodiments, the chelating agent comprises a macrocycle that is a derivative of 4,13-diaza-18-crown-6. 4,13-diaza-18-crown-6 may be prepared by various methods (see, for example, Gatto et al., Org. Synth. 1990, 68, 227; doi:10.15227 / orgsyn.068.0227). According to further embodiments of the present invention, the chelating agent is H2bp18c6 or an H2bp18c6 derivative, such as those described in WO 2020 / 229974. H2bp18c6 refers to N,N'-bis[(6-carboxy-2-pyridyl)methyl]-4,13-diaza-18-crown-6 as described herein. H2bp18c6 and H2bp18c6 derivatives are also described, for example, in Thiele et al., "An Eighteen-Membered Macrocyclic Ligand for Actinium-225 Targeted Alpha Therapy," Angew. Chem. Int. Ed. (2017) 56, 14712-14717, and Roca-Sabio et al., "Macrocyclic Receptor Exhibiting Unprecedented Selectivity for Light Lanthanides," J. Am. Chem. Soc. (2009) 131, 3331-3341, all of which are incorporated herein by reference. Additional chelators suitable for use according to the present invention are described in WO 2018 / 183906 and WO 2020 / 106886, which are incorporated herein by reference. As used herein, the term "TOPA" refers to the macrocyclic compound known in the art as H2bp18c6, or which may alternatively be referred to as N,N'-bis[(6-carboxy-2-pyridyl)methyl]-4,13diaza-18-crown-6 (see, e.g., Roca-Sabio et al.).

[0182] Chelating agents of formula (I), (II) and (III) Additional chelating agents suitable for use according to the present invention are described in WO 2020 / 229974, which is incorporated herein by reference. According to certain embodiments, for example, as described in WO 2020 / 229974, the chelating agent has the structure of Formula (I):

[0183] [ka] During the ceremony, Each of ring A and ring B is independently a 6- to 10-membered aryl or a 5- to 10-membered heteroaryl, and each of ring A and ring B is independently halo, alkyl, alkenyl, cycloalkyl, cycloalkenyl, aryl, heterocyclyl, heteroaryl, -OR 13 , -SR 13 , -(CH2) p COOR 13 , -OC(O)R 13 , -N(R 13 )2, -CON(R 13 )2, -NO2, -CN-OC(O)N(R 13 ) 2, and X, Each of Z1 and Z2 independently represents (C(R 12 )2) m - or -(CH2) n -C(R 12 )(X)-(CH2) n - and Each X is independently -L1-R 11 and each n is independently 0, 1, 2, 3, 4, or 5; each m is independently 1, 2, 3, 4, or 5; each p is independently 0 or 1; L1 is absent or a linker, R 11 is a nucleophilic or electrophilic moiety, or R 11 comprises an antigen-binding domain, Each R 12are independently hydrogen, alkyl, cycloalkyl, aryl, heterocyclyl, or heteroaryl; Each R 13 are independently hydrogen or alkyl; R 14 , R 15 , R 16 and R 17 each independently is hydrogen, alkyl, or X; Alternatively, R 14 and R 15 and / or R 16 and R 17 together with the carbon atoms to which they are attached form a 5- or 6-membered cycloalkyl ring which may be substituted by X; provided that the chelating agent contains at least one X, and when X is present on ring A or ring B, L1 is a linker or R 12 and R 14 ~R 17 At least one of them is not hydrogen.

[0184] According to an embodiment of the present invention, the chelating agent comprises at least one X group, where X is -L-R 11 L1 is absent or a linker, and R 11 is an electrophilic or nucleophilic moiety, or R 11 contains the antigen-binding domain. 11 When R is a nucleophilic or electrophilic moiety, such a moiety can be used to attach a chelator to the antigen-binding domain, either directly or indirectly via a linker. 11 comprises an antibody or antigen-binding domain with binding specificity for hK2, e.g., the Fab of KL2B30.

[0185] In certain embodiments, the chelator comprises one X group, preferably L1 of the X group is a linker.

[0186] In the chelating agent of the present invention, when ring A or ring B contains an X group, L1 is a linker or R 12 and R 14-R 17 Substitutions at carbon atoms of the macrocyclic ring, at the Z1 or Z2 positions, or at X on ring A or ring B, provided that at least one of is not hydrogen (i.e., at least one of the carbon atoms of Z1, Z2 and / or the macrocyclic ring carbons is substituted with an alkyl group such as, for example, methyl or ethyl). Preferably, substitutions at such positions are made to provide a radioactive metal ion, especially 225 The substitution does not affect the chelation efficiency of the chelator towards Ac, and in some embodiments, can increase the chelation efficiency.

[0187] In some embodiments, L is absent. When L is absent, R 11 is directly attached (eg, via a covalent bond) to the chelator.

[0188] In some embodiments, L1 is a linker. As used herein, the term "linker" refers to a chemical moiety that connects a chelator to a nucleophilic moiety, an electrophilic moiety, or an antigen-binding domain. In light of the present disclosure, any suitable linker known to those of skill in the art may be used in the present invention. Linkers can include, for example, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl moieties, substituted or unsubstituted aryl or heteroaryl, polyethylene glycol (PEG) linkers, peptide linkers, sugar-based linkers, or cleavable linkers, such as disulfide bonds or protease cleavage sites, e.g., valine-citrulline-p-aminobenzyl (PAB). Exemplary linker structures suitable for use in the present invention include:

[0189] [ka] In the formula, n is an integer of 0 to 10, preferably an integer of 1 to 4; and m is an integer of 0 to 12, preferably an integer of 0 to 6.

[0190] In some embodiments, R11 is a nucleophilic moiety or an electrophilic moiety. A "nucleophilic moiety" or "nucleophilic group" refers to a functional group that donates an electron pair to form a covalent bond in a chemical reaction. An "electrophilic moiety" or "electrophilic group" refers to a functional group that accepts an electron pair to form a covalent bond in a chemical reaction. A nucleophilic group reacts with an electrophilic group to form a new covalent bond in a chemical reaction, and vice versa. Reaction of a nucleophilic or electrophilic group of a chelating agent of the invention with an antigen-binding domain or other chemical moiety (e.g., a linker) comprising a corresponding reaction partner allows for the covalent attachment of the antigen-binding domain or chemical moiety to the chelating agent of the invention.

[0191] Illustrative examples of nucleophilic groups include, but are not limited to, azides, amines, and thiols. Illustrative examples of electrophilic groups include, but are not limited to, amine-reactive groups, thiol-reactive groups, alkynyls, and cycloalkynyls. Amine-reactive groups preferably react with primary amines, including those present at the N-terminus of each polypeptide chain and in the side chains of lysine residues. Examples of amine-reactive groups suitable for use in the present invention include, but are not limited to, N-hydroxysuccinimide (NHS), substituted NHS (such as sulfo-NHS), isothiocyanate (-NCS), isocyanate (-NCO), esters, carboxylic acids, acyl halides, amides, alkylamides, and tetrafluorophenyl esters and perfluorophenyl esters. Thiol-reactive groups react with thiols or sulfhydryls, preferably with thiols present in the side chains of cysteine ​​residues in polypeptides. Examples of thiol-reactive groups suitable for use in the present invention include, but are not limited to, Michael acceptors (e.g., maleimides), haloacetyls, acyl halides, activated disulfides, and phenyloxadiazole sulfones.

[0192] In certain embodiments, R 11is -NH2, -NCS (isothiocyanate), -NCO (isocyanate), -N3 (azide), alkynyl, cycloalkynyl, carboxylic acid, ester, amide, alkylamide, maleimide, acyl halide, tetrazine, or trans-cyclooctene, more particularly -NCS, -NCO, -N3, alkynyl, cycloalkynyl, -C(O)R 13 , -COOR 13 , -CON(R 13 ) 2, maleimide, acyl halide (e.g., —C(O)Cl, —C(O)Br), tetrazine, or trans-cyclooctene, and each R 13 are independently hydrogen or alkyl.

[0193] In some embodiments, R 11 is an alkynyl, cycloalkynyl, or azido group, thus enabling attachment of the chelator to an antigen-binding domain or other chemical moiety (e.g., a linker) using click chemistry. In such embodiments, a click chemistry reaction that may be performed is a Huisgen cycloaddition or a 1,3-dipolar cycloaddition between azide (-N3) and an alkynyl or cycloalkynyl group to form a 1,2,4-triazole linker or moiety. In one embodiment, the chelator comprises an alkynyl or cycloalkynyl group, and the antigen-binding domain or other chemical moiety comprises an azide group. In another embodiment, the chelator comprises an azide group, and the antigen-binding domain or other chemical moiety comprises an alkynyl or cycloalkynyl group.

[0194] In certain embodiments, R 11is an alkynyl group, more preferably a terminal alkynyl group or a cycloalkynyl group, which is particularly reactive with azide groups via strain-promoted azide-alkyne cyclocycloaddition (SPAAC). Examples of cycloalkynyl groups that can react with azide groups via SPAAC include, but are not limited to, cyclooctynyl or bicyclononynyl (BCN), difluorinated cyclooctynyl (DIFO), dibenzocyclooctynyl (DIBO), keto-DIBO, biarylazacyclooctynyl (BARAC), dibenzoazacyclooctynyl (DIBAC, DBCO, ADIBO), dimethoxyazacyclooctynyl (DIMAC), difluorobenzocyclooctynyl (DIFBO), monobenzocyclooctynyl (MOBO), and tetramethoxydibenzocyclooctynyl (TMDIBO).

[0195] In certain embodiments, R 11 is a dibenzoazacyclooctynyl (DIBAC, DBCO, ADIBO) having the following structure:

[0196] [ka] R 11 In embodiments where is a DBCO, the DBCO can be covalently attached to the compound directly or indirectly via a linker, and is preferably attached to the compound indirectly via a linker.

[0197] In some embodiments, R 11 comprises an antigen-binding domain. The antigen-binding domain can be linked to the chelator directly via a covalent bond or indirectly via a linker. In a preferred embodiment, the antigen-binding domain is an antibody or an antigen-binding fragment thereof. According to a preferred embodiment, R 11 comprises an antigen-binding domain with binding specificity for hK2, e.g., the Fab of KL2B30.

[0198] According to an embodiment of the present invention, each of Ring A and Ring B is independently a 6-10 membered aryl or a 5-10 membered heteroaryl. In an alternative embodiment, each of Ring A and Ring B is contemplated to be an optionally substituted heterocyclyl ring, such as oxazoline. Each of Ring A and Ring B can be selected from halo, alkyl, alkenyl, cycloalkyl, cycloalkenyl, aryl, heterocyclyl, heteroaryl, -OR 13 , -SR 13 , -(CH2) p COOR 13 , -OC(O)R 13 , -N(R 13 )2, -CON(R 13 )2, -NO2, -CN-OC(O)N(R 13 )2 and X. Examples of 6- to 10-membered aryl groups suitable for this purpose include, but are not limited to, phenyl and naphthyl. Examples of 5- to 10-membered heteroaryl groups suitable for this purpose include, but are not limited to, pyridinyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, and imidazolyl. Examples of suitable substituents for 5- to 10-membered heteroaryl groups and 6- to 10-membered aryl groups include, but are not limited to, -COOH, tetrazolyl, and -CHCOOH. In a preferred embodiment, the substituent is -COOH or tetrazolyl, which is an isostere of -COOH.

[0199] In certain embodiments, each of Ring A and Ring B is independently optionally substituted with one or more carboxyl groups, including, but not limited to, -COOH and -CH2COOH.

[0200] In certain embodiments, each of ring A and ring B is independently and optionally substituted with tetrazolyl.

[0201] In one embodiment, ring A and ring B are the same, e.g., both ring A and ring B are pyridinyl. In another embodiment, ring A and ring B are different, e.g., ring A and one of ring A and ring B is pyridinyl and the other is phenyl.

[0202] In certain embodiments, both ring A and ring B are pyridinyl substituted with -COOH.

[0203] In certain embodiments, both Ring A and Ring B are pyridinyl substituted with tetrazolyl.

[0204] In another particular embodiment, both Ring A and Ring B are picolinic acid groups having the following structure:

[0205] [ka]

[0206] According to an embodiment of the present invention, each of Z1 and Z2 independently represents -(C(R 12 )2) m or -(CH2) n -C(R 12 )(X)-(CH2) n each X is independently -L1-R 11 and each R 12 is independently hydrogen, alkyl, cycloalkyl, aryl, heterocyclyl, or heteroaryl; each n is independently 0, 1, 2, 3, 4, or 5, and each m is independently 1, 2, 3, 4, or 5.

[0207] In some embodiments, each R 12 are independently hydrogen or alkyl, more preferably hydrogen, —CH 3 , or —CH 2 CH 3 .

[0208] In some embodiments, each R 12 is hydrogen.

[0209] In some embodiments, Z and Z are both -(CH). m - and each m is preferably 1. In such embodiments, a carbon atom of the macrocycle, ring A or ring B is substituted with an X group.

[0210] In some embodiments, one of Z1 and Z2 is -(CH2) n -C(R 12 )(X)-(CH2) n - and the other is -(CH2) m -It is.

[0211] In some embodiments, Z and Z—(CH) n -C(R 12 )(X)-(CH2) n - and the other is -(CH2) m -; each n is 0; m is 1; X is -L1-R 11 and L1 is a linker.

[0212] In some embodiments, both Z and Z are —(CH) m -; each m is independently 0, 1, 2, 3, 4, or 5, preferably each m is 1; R 14 , R 15 , R 16 and R 17 One of the is X and the other is R 14 , R 15 , R 16 and R 17 The remainder of each is hydrogen.

[0213] In some embodiments, R 14 and R 15 together with the carbon atoms to which they are attached form a 5- or 6-membered cycloalkyl ring (i.e., cyclopentyl or cyclohexyl). Such 5- or 6-membered cycloalkyl rings can be substituted with an X group.

[0214] In some embodiments, R 16 and R 17together with the carbon atoms to which they are attached form a 5- or 6-membered cycloalkyl ring (i.e., cyclopentyl or cyclohexyl). Such 5- or 6-membered cycloalkyl rings can be substituted with an X group.

[0215] In certain embodiments, the chelator has the structure of formula (II):

[0216] [ka] During the ceremony, A1 is N or CR1 or absent; A2 is N or CR2; A3 is N or CR3; A4 is N or CR4; A5 is N or CR5; A6 is N or CR6 or absent; A7 is N or CR7; A8 is N or CR8; A9 is N or CR9, A 10 is N or CR 10 and However, three or less of A1, A2, A3, A4, and A5 are N, and A6, A7, A8, A9, and A 10 Not more than three of R1, R2, R3, R4, R5, R6, R7, R8, R9, and R 10 each independently represents hydrogen, halo, alkyl, alkenyl, cycloalkyl, cycloalkenyl, aryl, heterocyclyl, heteroaryl, OR 13 , -SR 13 , -(CH2) p COOR 13 , -OC(O)R 13 , -N(R 13 )2, -CON(R 13 )2, -NO2, -CN-OC(O)N(R 13 )2, and -X; Alternatively, any two immediately adjacent R1, R2, R3, R4, R5, R6, R7, R8, R9, and R 10 together with the atoms to which they are attached form a 5- or 6-membered substituted or unsubstituted carbocyclic or nitrogen-containing ring; Z1, Z2, X, n, m, p, L1, and R 11 ~R 17 is as described above for formula (I), provided that the chelating agent contains at least one X and R1, R2, R3, R4, R5, R6, R7, R8, R9 and R 10 is X, then L1 is a linker, or R 12 and R 14 ~R 17 At least one of these is not hydrogen.

[0217] In some embodiments, any two immediately adjacent R, R, R, R, R, R, R, R, R, R, R, and R 10 are taken together with the atoms to which they are attached to form a 5- or 6-membered substituted or unsubstituted carbocyclic ring or nitrogen-containing ring. Examples of such carbocyclic rings that can be formed include, but are not limited to, naphthyl. Examples of such nitrogen-containing rings that can be formed include, but are not limited to, quinolinyl. The carbocyclic or nitrogen-containing ring can be unsubstituted or substituted with one or more suitable substituents, such as -COOH, -CHCOOH, tetrazolyl, etc.

[0218] In some embodiments, L is absent. When L is absent, R 11 is directly attached (eg, via a covalent bond) to the chelator.

[0219] In some embodiments, L1 is a linker. Any suitable linker known to those of skill in the art in light of the present disclosure, such as those described above, can be used in the present invention.

[0220] In some embodiments, one of A1, A2, A3, A4, and A5 is nitrogen, one of A1, A2, A3, A4, and A5 is a carbon substituted with -COOH, and the remainder are CH, i.e., forming a pyridinyl ring substituted with a carboxylic acid.

[0221] In some embodiments, A6, A7, A8, A9 and A 10 One of them is nitrogen, and A6, A7, A8, A9 and A 10 One of the carbons is substituted with -COOH and the remaining are CH, i.e., forming a pyridinyl ring substituted with a carboxylic acid.

[0222] In one embodiment, at least one of R1, R2, R3, R4, and R5 is -COOH. In one embodiment, at least one of R6, R7, R8, R9, and R 10 In another embodiment, at least one of R1, R2, R3, R4, and R5 is -COOH, and at least one of R6, R7, R8, R9, and R 10 At least one of is -COOH.

[0223] In some embodiments, A and A 10 each is nitrogen; A2 is CR2 and R2 is -COOH; A9 is CR9, R9 is -COOH; each of A3 to A8 is CR2, CR3, CR4, CR5, CR6, CR7, and CR8, respectively, and each of R3 to R8 is hydrogen.

[0224] In some embodiments, one of A1, A2, A3, A4, and A5 is nitrogen, one of A1, A2, A3, A4, and A5 is carbon substituted with tetrazolyl, and the remainder are CH.

[0225] In some embodiments, A6, A7, A8, A9, and A 10 One of the is nitrogen, and A6, A7, A8, A9, and A 10 One of the carbons is substituted with tetrazolyl, and the rest are CH.

[0226] In one embodiment, at least one of R1, R2, R3, R4, and R5 is tetrazolyl. In one embodiment, at least one of R6, R7, R8, R9, and R 10 In another embodiment, at least one of R1, R2, R3, R4, and R5 is tetrazolyl; 10 At least one of is tetrazolyl.

[0227] In some embodiments, each R 12 is hydrogen.

[0228] In some embodiments, R 11 is an alkynyl or cycloalkynyl group, preferably cyclooctynyl or a cyclooctynyl derivative, such as DBCO.

[0229] In certain embodiments of the chelating agent of formula (II), A1 and A 10 each of which is nitrogen; A2 is CR2 and R2 is -COOH; A9 is CR9 and R9 is -COOH; A3 to A8 are CR2, CR3, CR4, CR5, CR6, CR7, and CR8, respectively; Each of R3 to R8 is hydrogen; One of Z1 and Z2 is -(CH2) m and the other of Z1 and Z2 is -(CH2) n -C(R 12 )(X)-(CH2) n - and R 12 is hydrogen, m is 1, Each n is 0, X is -L1-R 11 L1 is a linker, and -R 11 is an electrophilic group, for example, cyclooctynyl or a cyclooctynyl derivative such as DBCO; R 14 and R17 each is hydrogen, or R 16 and R 17 together with the carbon atom to which they are attached form a 5- or 6-membered cycloalkyl.

[0230] In certain embodiments, the chelator has the structure of formula (III):

[0231] [ka] During the ceremony, Each A 11 are independently O, S, NMe, or NH; Each R 18 are independently hydrogen, halo, alkyl, alkenyl, cycloalkyl, cycloalkenyl, aryl, heterocyclyl, heteroaryl, -OR 13 , -SR 13 , -COOR 13 , -OC(O)R 13 , -N(R 13 )2, -CON(R 13 )2, -NO2, -CN-OC(O)N(R 13 )2, and -X; Z1, Z2, X, n, m, L1, R 11 ~R 17 is as described above for formula (I), However, the chelating agent contains at least one X and R 18 is X, L1 is a linker, or R 12 and R 14 ~R 17 At least one of these is not hydrogen.

[0232] In some embodiments, each A 11 is the same, and each A 11 is O, S, NMe, or NH. For example, each A 11 can be S. In other embodiments, each A 11 are different and each is independently selected from O, S, NMe, and NH.

[0233] In some embodiments, each R 18 are independently -(CH2) p -COOR 13 or tetrazolyl, and R 13 is hydrogen, and each p is independently 0 or 1.

[0234] In some embodiments, each R 18 is -COOH.

[0235] In some embodiments, each R 18 is -CH2COOH.

[0236] In some embodiments, each R 18 is tetrazolyl.

[0237] In certain embodiments of the chelating agent of formula (III), Each R 18 is COOH, One of Z1 and Z2 is -(CH2) m and the other of Z1 and Z2 is -(CH2) n -C(R 12 )(X)-(CH2) n - and R 12 is hydrogen, m is 1; each n is 0, X is -L1-R 11 L1 is a linker, and -R 11 is an electrophilic group, for example, cyclooctynyl or a cyclooctynyl derivative such as DBCO or BCN; R 14 and R 17 each is hydrogen, or R 16 and R 17 together with the carbon atom to which they are attached form a 5- or 6-membered cycloalkyl.

[0238] A particular embodiment of the present invention is a chelating agent selected from the group consisting of:

[0239] [ka] During the ceremony, L1 is absent or a linker, R 11 is a nucleophilic or electrophilic moiety, or R 11 comprises an antigen-binding domain (e.g., the Fab of KL2B30), and Each R 12 are independently hydrogen, -CH3, or -CH2CH3, provided that at least one R 12 is -CH3 or -CH2CH3.

[0240] In some embodiments, R 11 -NH2, -NCS, -NCO, -N3, alkynyl, cycloalkynyl, -C(O)R 13 , -COOR 13 , -CON(R 13 ) 2, maleimide, acyl halide, tetrazine, or trans-cyclooctene.

[0241] In certain embodiments, R 11 is cyclooctynyl or a cyclooctynyl derivative selected from the group consisting of bicyclononynyl (BCN), difluorinated cyclooctynyl (DIFO), dibenzocyclooctynyl (DIBO), keto-DIBO, biarylazacyclooctynyl (BARAC), dibenzoazacyclooctynyl (DIBAC, DBCO, ADIBO), dimethoxyazacyclooctynyl (DIMAC), difluorobenzocyclooctynyl (DIFBO), monobenzocyclooctynyl (MOBO), and tetramethoxydibenzocyclooctynyl (TMDIBO).

[0242] Preferably, R 11 is an alkynyl group or a cycloalkynyl group, more preferably a cycloalkynyl group, such as DBCO or BCN.

[0243] Exemplary chelating agents of the present invention include:

[0244] [ka]

[0245] [ka] These include, but are not limited to:

[0246] Such chelators can be covalently attached to an antigen-binding domain to form an immunoconjugate or radioimmunoconjugate by reacting the chelator with an azide-labeled antigen-binding domain via click chemistry to form a 1,2,3 triazole linker, as described in WO 2020 / 229974.

[0247] The chelating agents of the present invention can be prepared by any method known in the art in light of the present disclosure, for example, the pendant aromatic / heteroaromatic group can be attached to the macrocyclic ring moiety by methods known in the art, such as those illustrated and described in WO 2020 / 229974.

[0248] Chelating agents of formula (IV), (V) and (VI) In one embodiment, the chelating agent has formula (IV):

[0249] [ka] or a pharmaceutically acceptable salt thereof, wherein R1 is hydrogen and R2 is -L1-R4; Alternatively, R1 is -L1-R4 and R2 is hydrogen; R3 is hydrogen; or R2 and R3 together with the carbon atom to which they are attached form a 5- or 6-membered cycloalkyl, which 5- or 6-membered cycloalkyl is optionally substituted with -L1-R4; L1 is absent or a linker, R4 is a nucleophilic moiety, an electrophilic moiety, or an antigen-binding domain (e.g., the Fab of KL2B30).

[0250] In some embodiments, L is absent. When L is absent, R is directly bonded to the compound (e.g., via a covalent bond).

[0251] In some embodiments, L1 is a linker. As used herein, the term "linker" refers to a chemical moiety that connects a compound of the present invention to a nucleophilic moiety, an electrophilic moiety, or an antigen-binding domain. In view of the present disclosure, any suitable linker known to one of skill in the art may be used in the present invention. The linker may have, for example, a substituted or unsubstituted alkyl, a substituted or unsubstituted heteroalkyl moiety, a substituted or unsubstituted aryl or heteroaryl, a polyethylene glycol (PEG) linker, a peptide linker, a sugar-based linker, or a cleavable linker, such as a disulfide bond or a protease cleavage site, e.g., valine-citrulline-p-aminobenzyl (PAB). Exemplary linker structures suitable for use in the present invention include:

[0252] [ka] In the formula, m is an integer of 0 to 12, but is not limited to these.

[0253] In some embodiments, R4 is a nucleophilic moiety or an electrophilic moiety. A "nucleophilic moiety" or "nucleophilic group" refers to a functional group that donates an electron pair to form a covalent bond in a chemical reaction. An "electrophilic moiety" or "electrophilic group" refers to a functional group that accepts an electron pair to form a covalent bond in a chemical reaction. A nucleophilic group reacts with an electrophilic group to form a new covalent bond in a chemical reaction, and vice versa. Reaction of a nucleophilic or electrophilic group of a compound of the invention with an antigen-binding domain or other chemical moiety (e.g., a linker) comprising a corresponding reaction partner allows the antigen-binding domain or chemical moiety to be covalently attached to the compound of the invention.

[0254] Illustrative examples of nucleophilic groups include, but are not limited to, azides, amines, and thiols. Illustrative examples of electrophilic groups include, but are not limited to, amine-reactive groups, thiol-reactive groups, alkynyls, and cycloalkynyls. Amine-reactive groups preferably react with primary amines, including those present at the N-terminus of each polypeptide chain and in the side chains of lysine residues. Examples of amine-reactive groups suitable for use in the present invention include, but are not limited to, N-hydroxysuccinimide (NHS), substituted NHS (such as sulfo-NHS), isothiocyanate (-NCS), isocyanate (-NCO), esters, carboxylic acids, acyl halides, amides, alkylamides, and tetrafluorophenyl esters and perfluorophenyl esters. Thiol-reactive groups react with thiols or sulfhydryls, preferably with thiols present in the side chains of cysteine ​​residues in polypeptides. Examples of thiol-reactive groups suitable for use in the present invention include, but are not limited to, Michael acceptors (e.g., maleimides), haloacetyls, acyl halides, activated disulfides, and phenyloxadiazole sulfones.

[0255] In certain embodiments, R4 is -NH2, -NCS (isothiocyanate), -NCO (isocyanate), -N3 (azide), alkynyl, cycloalkynyl, carboxylic acid, ester, amide, alkylamide, maleimide, acyl halide, tetrazine, or trans-cyclooctene, more particularly -NCS, -NCO, -N3, alkynyl, cycloalkynyl, -C(O)R 13 , -COOR 13 , -CON(R 13 )2, maleimide, acyl halide (e.g., —C(O)Cl, —C(O)Br), tetrazine, or trans-cyclooctene, where each R 13 is independently hydrogen or alkyl.

[0256] In some embodiments, R4 is an alkynyl, cycloalkynyl, or azido group, thus enabling attachment of the compounds of the invention to antigen-binding domains or other chemical moieties (e.g., linkers) using click chemistry. In such embodiments, a click chemistry reaction that can be performed is a Huisgen cycloaddition or a 1,3-dipolar cycloaddition between an azide (-N3) and an alkynyl or cycloalkynyl group to form a 1,2,4-triazole linker or moiety. In one embodiment, the compounds of the invention comprise an alkynyl or cycloalkynyl group, and the antigen-binding domain or other chemical moiety comprises an azido group. In another embodiment, the compounds of the invention comprise an azido group, and the antigen-binding domain or other chemical moiety comprises an alkynyl or cycloalkynyl group.

[0257] In certain embodiments, R4 is an alkynyl group, more preferably a terminal alkynyl group or a cycloalkynyl group, which is particularly reactive with azide groups via strain-promoted azide-alkyne cyclocycloaddition (SPAAC). Examples of cycloalkynyl groups that can react with azide groups via SPAAC include, but are not limited to, cyclooctynyl or bicyclononynyl (BCN), difluorinated cyclooctynyl (DIFO), dibenzocyclooctynyl (DIBO), keto-DIBO, biarylazacyclooctynyl (BARAC), dibenzoazacyclooctynyl (DIBAC, DBCO, ADIBO), dimethoxyazacyclooctynyl (DIMAC), difluorobenzocyclooctynyl (DIFBO), monobenzocyclooctynyl (MOBO), and tetramethoxydibenzocyclooctynyl (TMDIBO).

[0258] In certain embodiments, R4 has the following structure:

[0259] [ka] In embodiments where R4 is DBCO, the DBCO may be covalently bonded to the compound directly or indirectly via a linker, but is preferably bonded to the compound indirectly via a linker.

[0260] In certain embodiments, R4 comprises an antigen-binding domain. The antigen-binding domain can be directly bound to the compound via a covalent bond or indirectly bound to the compound via a linker. In a preferred embodiment, the antigen-binding domain has binding specificity for hK2, such as the Fab of KL2B30.

[0261] In another embodiment, the chelating agent has formula (V):

[0262] [ka] or a pharmaceutically acceptable salt thereof, wherein L1 is absent or a linker, R4 is a nucleophilic moiety, an electrophilic moiety, or an antigen-binding domain.

[0263] In another embodiment, the chelating agent has formula (VI):

[0264] [ka] or a pharmaceutically acceptable salt thereof, wherein L1 is absent or a linker, R4 is a nucleophilic moiety, an electrophilic moiety, or an antigen-binding domain (e.g., the Fab of KL2B30).

[0265] In another embodiment, the invention provides a compound, wherein R1 is -L1-R4, R2 and R3 together with the carbon atom to which they are attached form a 5- or 6-membered cycloalkyl, L1 is absent or is a linker, and R4 is a nucleophilic moiety, an electrophilic moiety, or an antigen-binding domain; or a pharmaceutically acceptable salt thereof.

[0266] In a further embodiment, the invention provides a compound wherein R1 is H, and R2 and R3, together with the carbon atom to which they are attached, form a 5- or 6-membered cycloalkyl substituted with -L1-R4, where L1 is absent or is a linker, and R4 is a nucleophilic moiety, an electrophilic moiety, or an antigen-binding domain; or a pharmaceutically acceptable salt thereof.

[0267] Further embodiments include those in which R4 is an antigen-binding domain. According to a preferred embodiment, R4 comprises an antigen-binding domain with binding specificity for hK2, such as the Fab of KL2B30.

[0268] In one embodiment, the compound of the invention is:

[0269] [ka]

[0270] [ka] wherein n is 1 to 10.

[0271] The chelating agent can be covalently attached to an antigen-binding domain (e.g., the Fab of KL2B30) to form an immunoconjugate or radioimmunoconjugate by reacting the compound with an azide-labeled antigen-binding domain to form a 1,2,3-triazole linker, e.g., via click chemistry as described in WO 2020 / 229974.

[0272] The chelators, radiometal complexes and radioimmunoconjugates of the invention can be prepared by any method known in the art in light of the present disclosure, for example, the pendant aromatic / heteroaromatic group can be attached to the macrocyclic ring moiety by methods known in the art, such as those illustrated and described in WO 2020 / 229974.

[0273] Radioactive metal complexes In another general aspect, the present invention relates to a radiometal complex comprising a radioactive metal ion coordinated to a chelating agent of the present invention by a coordinate bond. Any of the chelating agents of the present invention described herein can comprise a radioactive metal ion. Preferably, the radioactive metal ion is an alpha-emitting radioactive metal ion, more preferably an alpha-emitting radioactive metal ion. 225 The chelating agent of the present invention can chelate radioactive metal ions, especially radioactive metal ions, at any specific activity, regardless of metal impurities. 225 and therefore have high chelation stability in vivo and in vitro, and form stable radiometal complexes with challenge agents such as diethylenetriaminepentaacetic acid (DTPA).

[0274] According to an embodiment of the present invention, the radiometal complex has the structure of formula (Im):

[0275] [ka] wherein the variables are as defined above in the chelating agents of the invention, e.g., the chelating agents of Formula (I), and M is a radioactive metal ion. The radioactive metal ion, M, is bound to the chelating agent by a coordinate bond to form a radioactive metal complex. The heteroatoms of the macrocyclic ring of the chelating agent as well as any functional groups of the pendant arms (i.e., -Z1 ring A and / or -Z2 ring B) can participate in the coordinate bonding of the radioactive metal ion.

[0276] Any of the chelating agents of formula (I) above can be used to form a radiometal complex of formula (Im).

[0277] In certain embodiments, the radioactive metal ion M is an alpha-emitting radioactive metal ion. Preferably, the alpha-emitting radioactive metal ion is 225 It is Ac.

[0278] According to an embodiment of the present invention, the radiometal complex comprises at least one X group, where X is -L-R 11 L1 is absent or a linker, and R 11 is an electrophilic or nucleophilic moiety, or R 11 R contains an antigen-binding domain (e.g., the Fab of KL2B30). 11 When is a nucleophilic or electrophilic moiety, such a moiety can be used to attach a radiometal complex to an antigen binding domain directly or indirectly via a linker.

[0279] In certain embodiments, the radiometal comprises one X group, preferably L1 of the X group is a linker.

[0280] In certain embodiments, R 11is -NH2, -NCS (isothiocyanate), -NCO (isocyanate), -N3 (azide), alkynyl, cycloalkynyl, carboxylic acid, ester, amide, alkylamide, maleimide, acyl halide, tetrazine, or trans-cyclooctene, and more specifically, -NCS, -NCO, -N3, alkynyl, cycloalkynyl, -C(O)R 13 , -COOR 13 , -CON(R 13 )2, maleimide, acyl halide (e.g., —C(O)Cl, —C(O)Br), tetrazine, or trans-cyclooctene, where each R 13 is independently hydrogen or alkyl.

[0281] In some embodiments, R 11 is an alkynyl, cycloalkynyl, or azido group, thus allowing attachment of the chelator to an antigen-binding domain or other chemical moiety (e.g., a linker) using click chemistry.

[0282] In certain embodiments, R 11 is an alkynyl group, more preferably a terminal alkynyl group or a cycloalkynyl group that is particularly reactive with an azide group via strain-promoted azide-alkyne cyclocycloaddition (SPAAC). Examples of cycloalkynyl groups that can react with an azide group via SPAAC include, but are not limited to, cyclooctynyl or a cyclooctynyl derivative selected from the group consisting of bicyclononynyl (BCN), difluorinated cyclooctynyl (DIFO), dibenzocyclooctynyl (DIBO), keto-DIBO, biarylazacyclooctynyl (BARAC), dibenzoazacyclooctynyl (DIBAC, DBCO, ADIBO), dimethoxyazacyclooctynyl (DIMAC), difluorobenzocyclooctynyl (DIFBO), monobenzocyclooctynyl (MOBO), and tetramethoxydibenzocyclooctynyl (TMDIBO).

[0283] In certain embodiments, R 11is dibenzoazacyclooctynyl (DIBAC, DBCO, ADIBO) having the following structure:

[0284] [ka]

[0285] R 11 In such embodiments where is a DBCO, the DBCO can be covalently attached to the chelator directly or indirectly via a linker, preferably indirectly via a linker.

[0286] In another particular embodiment, R 11 is bicyclononynyl (BCN).

[0287] According to an embodiment of the present invention, each of Ring A and Ring B is independently a 6-10 membered aryl or a 5-10 membered heteroaryl. In an alternative embodiment, each of Ring A and Ring B is contemplated to be an optionally substituted heterocyclyl ring, such as oxazoline. Each of Ring A and Ring B can be selected from halo, alkyl, alkenyl, cycloalkyl, cycloalkenyl, aryl, heterocyclyl, heteroaryl, -OR 13 , -SR 13 , -(CH2) p COOR 13 , -OC(O)R 13 , -N(R 13 )2, -CON(R 13 )2, -NO2, -CN-OC(O)N(R 13)2 and X. Examples of 6- to 10-membered aryl groups suitable for this purpose include, but are not limited to, phenyl and naphthyl. Examples of 5- to 10-membered heteroaryl groups suitable for this purpose include, but are not limited to, pyridinyl, isothiazolyl, isoxazolyl, and imidazolyl. Examples of suitable substituents for 5- to 10-membered heteroaryl and 6- to 10-membered aryl groups include, but are not limited to, -COOH, tetrazolyl, and -CHCOOH.

[0288] In certain embodiments, each of Ring A and Ring B is independently optionally substituted with one or more carboxyl groups, including, but not limited to, -COOH and -CH2COOH.

[0289] In one embodiment, ring A and ring B are the same, e.g., both ring A and ring B are pyridinyl. In another embodiment, ring A and ring B are different, e.g., ring A and one of ring A and ring B is pyridinyl and the other is phenyl.

[0290] In certain embodiments, both ring A and ring B are pyridinyl substituted with -COOH.

[0291] In certain embodiments, both Ring A and Ring B are pyridinyl substituted with tetrazolyl.

[0292] In another particular embodiment, both Ring A and Ring B are picolinic acid groups having the following structure:

[0293] [ka]

[0294] According to an embodiment of the present invention, each of Z1 and Z2 independently represents -(C(R 12 )2) m or -(CH2) n -C(R 12)(X)-(CH2) n each X is independently -L1-R 11 wherein each n is independently 0, 1, 2, 3, 4, or 5; and each m is independently 1, 2, 3, 4, or 5.

[0295] In some embodiments, Z and Z are both -(CH). m - and each m is preferably 1. In such embodiments, a carbon atom of the macrocycle, ring A or ring B is substituted with an X group.

[0296] In some embodiments, one of Z1 and Z2 is -(CH2) n -C(R 12 )(X)-(CH2) n - and the other is -(CH2) m -It is.

[0297] In some embodiments, Z and Z—(CH) n -C(R 12 )(X)-(CH2) n - and the other is -(CH2) m -; each n is 0; m is 1; X is -L1-R 11 and L1 is a linker.

[0298] In some embodiments, both Z and Z are —(CH) m -; each m is independently 0, 1, 2, 3, 4, or 5, preferably each m is 1; R 14 , R 15 , R 16 and R 17 One of the is X and the other is R 14 , R 15 , R 16 and R 17 The remainder of each is hydrogen.

[0299] In some embodiments, R 14 and R 15together with the carbon atoms to which they are attached form a 5- or 6-membered cycloalkyl ring (e.g., cyclopentyl or cyclohexyl). Such 5- or 6-membered cycloalkyl rings can be substituted with an X group.

[0300] In some embodiments, R 16 and R 17 together with the carbon atoms to which they are attached form a 5- or 6-membered cycloalkyl ring (e.g., cyclopentyl or cyclohexyl). Such 5- or 6-membered cycloalkyl rings can be substituted with an X group.

[0301] In certain embodiments, the radiometal complex has the structure of Formula (II-m):

[0302] [ka] wherein the variables are as defined above in the chelating agents of the invention, e.g., the chelating agents of formula (II), and M is a radioactive metal ion, preferably an alpha-emitting radioactive metal ion, more preferably 225 It is Ac.

[0303] Any of the chelating agents of formula (II) above can be used to form a radiometal complex of formula (II-m).

[0304] In some embodiments, one of A1, A2, A3, A4, and A5 is nitrogen, one of A1, A2, A3, A4, and A5 is a carbon substituted with -COOH, and the remainder are CH, i.e., forming a pyridinyl ring substituted with a carboxylic acid.

[0305] In some embodiments, A6, A7, A8, A9 and A 10 One of them is nitrogen, and A6, A7, A8, A9 and A 10 One of the carbons is substituted with -COOH and the remaining are CH, i.e., forming a pyridinyl ring substituted with a carboxylic acid.

[0306] In one embodiment, at least one of R1, R2, R3, R4, and R5 is -COOH. In one embodiment, at least one of R6, R7, R8, R9, and R 10 In another embodiment, at least one of R1, R2, R3, R4, and R5 is -COOH; 10 At least one of is -COOH.

[0307] In some embodiments, A and A 10 each is nitrogen; A2 is CR2 and R2 is -COOH; A9 is CR9 and R9 is -COOH; each of A3 to A8 is CR2, CR3, CR4, CR5, CR6, CR7, and CR8, respectively; and each of R3 to R8 is hydrogen.

[0308] In one embodiment, at least one of R1, R2, R3, R4, and R5 is tetrazolyl. In one embodiment, at least one of R6, R7, R8, R9, and R 10 In another embodiment, at least one of R1, R2, R3, R4, and R5 is tetrazolyl; 10 At least one of is tetrazolyl.

[0309] In some embodiments, each R 12 is hydrogen.

[0310] In some embodiments, R 11 is an alkynyl or cycloalkynyl group, preferably cyclooctynyl or a cyclooctynyl derivative, such as DBCO.

[0311] In certain embodiments of the radiometal complex of formula (II-m), M is 225 Ac; A1 and A 10 each of which is nitrogen; A2 is CR2 and R2 is -COOH; A9 is CR9, R9 is -COOH; A3 to A8 are CR2, CR3, CR4, CR5, CR6, CR7, and CR8, respectively; Each of R3 to R8 is hydrogen; One of Z1 and Z2 is -(CH2) m and the other of Z1 and Z2 is -(CH2) n -C(R 12 )(X)-(CH2) n - and; R 12 is hydrogen; m is 1; Each n is 0; X is -L1-R 11 L1 is a linker, and -R 11 is an electrophilic group, for example, cyclooctynyl or a cyclooctynyl derivative such as DBCO; R 14 and R 17 each is hydrogen, or R 16 and R 17 together with the carbon atom to which they are attached form a 5- or 6-membered cycloalkyl.

[0312] In certain embodiments, the radiometal complex has the structure of Formula (III-m):

[0313] [ka] wherein the variables are as defined above in the chelating agents of the invention, e.g., the chelating agents of formula (III), and M is a radioactive metal ion, preferably an alpha-emitting radioactive metal ion, more preferably 225 It is Ac.

[0314] Any of the chelating agents of formula (III) above can be used to form a radiometal complex of formula (III-m).

[0315] In some embodiments, each A 11 is the same, and each A 11is O, S, NMe, or NH. For example, each A 11 can be S. In other embodiments, each A 11 are different and each is independently selected from O, S, NMe, and NH.

[0316] In some embodiments, each R 18 are independently -(CH2) p -COOR 13 and R 13 is hydrogen, and each p is independently 0 or 1.

[0317] In some embodiments, each R 18 is -COOH.

[0318] In some embodiments, each R 18 is -CH2COOH.

[0319] In some embodiments, each R 18 is tetrazolyl.

[0320] In certain embodiments of the radiometal complex of formula (III-m), Each R 18 is COOH; One of Z1 and Z2 is -(CH2) m and the other of Z1 and Z2 is -(CH2) n -C(R 12 )(X)-(CH2) n - and; R 12 is hydrogen; m is 1; each n is 0; X is -L1-R 11 L1 is a linker, and -R 11 is an electrophilic group, for example, cyclooctynyl or a cyclooctynyl derivative such as DBCO; R 14 and R 17 each is hydrogen, or R 16 and R 17together with the carbon atom to which they are attached form a 5- or 6-membered cycloalkyl.

[0321] In certain embodiments of the invention, the radiometal complex has one of the following structures:

[0322] [ka] During the ceremony, M is actinium-225 ( 225 Ac), and L1 is absent or a linker; R 11 is a nucleophilic or electrophilic moiety, or R 11 comprises an antigen-binding domain (e.g., the Fab of KL2B30); and Each R 12 are independently hydrogen, -CH3, or -CH2CH3, provided that at least one R 12 is -CH3 or -CH2CH3.

[0323] In certain embodiments, the present invention provides a radiometal complex structure of formula (IV-m):

[0324] [ka] or a pharmaceutically acceptable salt thereof, wherein: M + is a radioactive metal ion, and M + is actinium 225 ( 225 Ac), Radium 223 ( 233 Ra), Bismuth 213 ( 213 Bi), lead 212( 212 Pb(II) and / or 212 Pb(IV)), terbium-149( 149 Tb), terbium-152( 152 Tb), terbium-155( 155 Tb), Fermium-255 ( 255 Fm), Thorium-227 ( 227 Th), Thorium-226 ( 226Th 4+ ), Astatine 211( 211 At), Cerium-134 ( 134 Ce), neodymium 144( 144 Nd), Lanthanum 132( 132 La), Lanthanum 135 ( 135 La), and uranium-230 ( 230 U) R1 is hydrogen and R2 is -L1-R4; Alternatively, R1 is -L1-R4 and R2 is hydrogen; R3 is hydrogen; or R2 and R3 together with the carbon atom to which they are attached form a 5- or 6-membered cycloalkyl, which 5- or 6-membered cycloalkyl is optionally substituted with -L1-R4; L1 is absent or a linker, R4 is a nucleophilic moiety, an electrophilic moiety, or an antigen-binding domain (e.g., the Fab of KL2B30).

[0325] In another embodiment, the present invention provides a radiometal complex of formula (Vm):

[0326] [ka] or a pharmaceutically acceptable salt thereof, wherein: M + is a radioactive metal ion, and M + is actinium 225 ( 225 Ac), Radium 223 ( 233 Ra), Bismuth 213 ( 213 Bi), lead 212( 212 Pb(II) and / or 212 Pb(IV)), terbium-149( 149 Tb), terbium-152( 152 Tb), terbium-155( 155 Tb), Fermium-255 ( 255 Fm), Thorium-227 ( 227 Th), Thorium-226 ( 226Th 4+ ), Astatine 211( 211 At), Cerium-134 ( 134 Ce), neodymium 144( 144 Nd), Lanthanum 132( 132 La), Lanthanum 135 ( 135 La), and uranium-230 ( 230 U) L1 is absent or a linker, R4 is a nucleophilic moiety, an electrophilic moiety, or an antigen-binding domain (e.g., the Fab of KL2B30).

[0327] In another embodiment, the present invention provides a compound of formula (VI-m):

[0328] [ka] or a pharmaceutically acceptable salt thereof, wherein: M + is a radioactive metal ion, and M + is actinium 225 ( 225 Ac), Radium 223 ( 233 Ra), Bismuth 213 ( 213 Bi), lead 212( 212 Pb(II) and / or 212 Pb(IV)), terbium-149( 149 Tb), terbium-152( 152 Tb), terbium-155( 155 Tb), Fermium-255 ( 255 Fm), Thorium-227 ( 227 Th), Thorium-226 ( 226 Th 4+ ), Astatine 211( 211 At), Cerium-134 ( 134 Ce), neodymium 144( 144 Nd), Lanthanum 132( 132 La), Lanthanum 135 ( 135 La), and uranium-230 ( 230 U), and L1 is absent or a linker; R4 is a nucleophilic moiety, an electrophilic moiety, or an antigen-binding domain (e.g., the Fab of KL2B30).

[0329] In another embodiment, the present invention provides a radiometal complex, wherein: M + is a radioactive metal ion, and M + is actinium 225 ( 225 Ac), Radium 223 ( 233 Ra), Bismuth 213 ( 213 Bi), lead 212( 212 Pb(II) and / or 212 Pb(IV)), terbium-149( 149 Tb), terbium-152( 152 Tb), terbium-155( 155 Tb), Fermium-255 ( 255 Fm), Thorium-227 ( 227 Th), Thorium-226 ( 226 Th 4+ ), Astatine 211( 211 At), Cerium-134 ( 134 Ce), neodymium 144( 144 Nd), Lanthanum 132( 132 La), Lanthanum 135 ( 135 La), and uranium-230 ( 230 U), wherein R1 is -L1-R4; R2 and R3 together with the carbon atom to which they are attached form a 5- or 6-membered cycloalkyl; L1 is absent or a linker, R4 is a nucleophilic moiety, an electrophilic moiety, or an antigen-binding domain (e.g., the Fab of KL2B30). or a pharmaceutically acceptable salt thereof.

[0330] In a further embodiment, the present invention provides a radiometal complex, wherein: M + is a radioactive metal ion, and M + is actinium 225 ( 225 Ac), Radium 223 ( 233Ra), Bismuth 213 ( 213 Bi), lead 212( 212 Pb(II) and / or 212 Pb(IV)), terbium-149( 149 Tb), terbium-152( 152 Tb), terbium-155( 155 Tb), Fermium-255 ( 255 Fm), Thorium-227 ( 227 Th), Thorium-226 ( 226 Th 4+ ), Astatine 211( 211 At), Cerium-134 ( 134 Ce), neodymium 144( 144 Nd), Lanthanum 132( 132 La), Lanthanum 135 ( 135 La), and uranium-230 ( 230 U), wherein R1 is H; R2 and R3 together with the carbon atom to which they are attached form a 5- or 6-membered cycloalkyl substituted with -L1-R4; L1 is absent or a linker, R4 is a nucleophilic moiety, an electrophilic moiety, or an antigen-binding domain (e.g., the Fab of KL2B30). or a pharmaceutically acceptable salt thereof.

[0331] In certain embodiments, the present invention provides a method for producing a pharmaceutical composition comprising:

[0332] [ka]

[0333] [ka] Any one or more radioactive metal complexes selected from the group consisting of: In the formula, n is 1 to 10, and M + is a radioactive metal ion, and M + is actinium 225 ( 225 Ac), Radium 223 (233 Ra), Bismuth 213 ( 213 Bi), lead 212( 212 Pb(II) and / or 212 Pb(IV)), terbium-149( 149 Tb), terbium-152( 152 Tb), terbium-155( 155 Tb), Fermium-255 ( 255 Fm), Thorium-227 ( 227 Th), Thorium-226 ( 226 Th 4+ ), Astatine 211( 211 At), Cerium-134 ( 134 Ce), neodymium 144( 144 Nd), Lanthanum 132( 132 La), Lanthanum 135 ( 135 La), and uranium-230 ( 230 U).

[0334] The radiometal complexes can be produced by any method known in the art in view of the present disclosure. For example, a chelator of the present invention can be mixed with a radiometal ion and the mixture can be incubated to form the radiometal complex. In an exemplary embodiment, the compound can be 225 It was mixed with a solution of Ac(NO3)3 and bound to the chelator via a coordinate bond. 225 As noted above, the chelating agents of the present invention are useful for the formation of radioactive complexes containing radiometals, particularly 225 Thus, in certain embodiments, the chelators of the present invention are used in a ratio of 1:1000, 1:500, 1:400, 1:300, 1:200, 1:100, 1:50, 1:10, or 1:5, preferably 1:5 to 1:200, more preferably 1:5 to 1:100, of the chelators of the present invention. 225 The concentration ratio with Ac ions is 225 The chelating agent of the present invention can then be mixed with a solution of Ac ions to form a radiometal complex. 225 The ratio of Ac to other known 225This is much lower than the ratio achievable with Ac chelators (e.g., DOTA). This radioactive complex can be characterized by instant thin-layer chromatography (e.g., iTLC-SG), HPLC, LC-MS, etc. Exemplary methods are described, for example, in WO 2020 / 229974.

[0335] Additional Embodiments of Immunoconjugates and Radioimmunoconjugates As described herein, the chelators and radioactive metal complexes of the invention can be conjugated (i.e., covalently attached) to antigen-binding domains, such as immunoagents, to generate immunoconjugates and / or radioimmunoconjugates suitable for pharmaceutical use in subjects, e.g., humans, such as for targeted radiotherapy. The chelators and radioactive metal complexes of the invention can be used to site-specifically label antigen-binding domains, particularly antibodies or antigen-binding fragments thereof, capable of specifically binding to a target of interest (such as cancer cells), with radioactive metal ions to generate radioimmunoconjugates. In particular, the chelators and / or radioactive metal complexes of the invention can be used to site-specifically label radioactive metal ions, particularly antibodies or antigen-binding fragments thereof, capable of specifically binding to a target of interest (such as cancer cells), with radioactive metal ions to generate radioimmunoconjugates. 225 Radioimmunoconjugates can be produced with high-yield chelation of Ac and desirable chelator-to-antibody ratios (CARs). According to certain embodiments, the methods of the invention provide average CARs of less than 10, less than 8, less than 6, or less than 4, or CARs of about 2 to about 8, or about 2 to about 6, or about 2 to about 4, or about 2 to about 3, or CARs of about 2, or about 3, or about 4, or about 5, or about 6, or about 7, or about 8.

[0336] According to embodiments of the invention, an immunoconjugate comprises a chelator of the invention, e.g., a chelator of Formula (I), Formula (II), or Formula (III) described herein, covalently attached, preferably via a linker, to an antibody or antigen-binding fragment thereof (e.g., the Fab of KL2B30). Many conjugation configurations with different linkages between the chelator and the antibody or antigen-binding fragment thereof are possible depending on the reactive functional groups (i.e., nucleophilic and electrophilic) on the chelator and the antibody or antigen-binding fragment thereof.

[0337] According to an embodiment of the present invention, a radioimmunoconjugate comprises a radiometal complex of the present invention, e.g., a radiometal complex of Formula (Im), Formula (II-m), or Formula (III-m) as described herein, covalently attached, preferably via a linker, to an antibody or antigen-binding fragment thereof (e.g., a Fab of KL2B30).

[0338] Any of the chelators or radiometal complexes of the invention, such as those described herein, can be used to produce immunoconjugates or radioimmunoconjugates of the invention.

[0339] In some embodiments, the radiometal complexes of the radioimmunoconjugates of the present invention comprise an α-emitting radiometal ion coordinated to the chelating moiety of the radiocomplex. Preferably, the α-emitting radiometal ion is 225 It is Ac.

[0340] In a specific embodiment, the antibody or antigen-binding fragment thereof is conjugated to the radioconjugate via a triazole moiety to form a radioimmunoconjugate of the invention.

[0341] In certain embodiments, the antibody or antigen-binding fragment in the immunoconjugate or radioimmunoconjugate of the present application is capable of specifically binding to a tumor antigen. Preferably, the antibody or antigen-binding fragment specifically binds to hK2.

[0342] The immunoconjugates and radioimmunoconjugates of the present invention can be prepared by any method known in the art in light of the present disclosure for conjugating a ligand, e.g., an antibody, to a chelator, including chemical and / or enzymatic methods. For example, immunoconjugates and radioimmunoconjugates can be prepared by coupling reactions, including, but not limited to, the formation of esters, thioesters, or amides from activated acids or acyl halides; nucleophilic substitution reactions (e.g., nucleophilic substitution of a ring halide or ring opening of a ring system with strain); azide-alkyne Huisgen cycloadditions (e.g., 1,3 dipolar cycloadditions between azides and alkynes to form 1,2,3-triazole linkers); thiophosphorus addition reactions; imine formation; Diels-Alder reactions between tetrazines and trans-cycloctene (TCO); and Michael additions (e.g., maleimide additions). Many other addition modes with different linkages are possible, depending on the reactive functional groups used. The binding of the ligand can be to a chelating agent that is coordinated to a radioactive metal ion or to a chelating agent that is not coordinated to a radioactive metal ion.

[0343] According to embodiments, radioimmunoconjugates can be produced by covalently attaching a radiometal complex of the present invention to an antibody or antigen-binding fragment thereof, e.g., by click chemistry. Alternatively, radioimmunoconjugates can be produced by first preparing an immunoconjugate of the present invention by covalently attaching a chelating agent of the present invention to an antibody or antigen-binding fragment thereof, e.g., by click chemistry, and then labeling the immunoconjugate with a radioactive metal ion to produce a radioimmunoconjugate (referred to as "one-step direct radiolabeling"). Both residue-specific and site-specific conjugation methods can be used to produce the immunoconjugates and radioimmunoconjugates of the present invention. Such methods are described, for example, in WO 2020 / 229974.

[0344] According to an embodiment of the present invention, a method for producing a radioimmunoconjugate comprises the steps of:11 is a nucleophilic or electrophilic moiety with an antibody or antigen-binding fragment thereof (e.g., the Fab of KL2B30), or a modified antibody or antigen-binding fragment thereof containing a nucleophilic or electrophilic moiety.

[0345] In one embodiment, a method comprises reacting a chelating agent of the invention with an antibody or antigen-binding fragment thereof, or a modified antibody or antigen-binding fragment thereof comprising a nucleophilic or electrophilic functional group to form an immunoconjugate having a covalent bond between the chelating agent and the antibody or antigen-binding fragment thereof, or the modified antibody or antigen-binding fragment, and reacting the immunoconjugate with a radioactive metal ion such that the radioactive metal ion is bound to the chelating agent of the immunoconjugate by a coordinate bond, thereby forming a radioimmunoconjugate. This embodiment is sometimes referred to as a "one-step direct radiolabeling" method because there is only one chemical reaction step involving the radiometal.

[0346] In another embodiment, the method comprises reacting a radioconjugate of the present invention with an antibody or antigen-binding fragment thereof, or a modified antibody or antigen-binding fragment thereof comprising a nucleophilic or electrophilic functional group, thereby forming a radioimmunoconjugate. This embodiment may be referred to as the "click radiolabeling" method. The modified antibody or antigen-binding fragment thereof may be produced by any method known in the art in light of the present disclosure, for example, by labeling an antibody with an orthogonally reactive functional group at a specific residue using one or more of the methods described above, or by site-specifically incorporating an unnatural amino acid (e.g., an azido amino acid or an alkynyl amino acid) into an antibody using one or more of the methods described above. The degree of labeling (DOL), sometimes referred to as the degree of substitution (DOS), is a particularly useful parameter for characterizing and optimizing bioconjugates, such as antibodies modified with unnatural amino acids. It may be expressed as the average number of unnatural amino acids attached to a protein molecule (such as an antibody) or as a molar ratio of label / protein. The DOL can be determined from the absorbance spectrum of the labeled antibody by any method known in the art.

[0347] In certain embodiments, as described herein, immunoconjugates and radioimmunoconjugates of the present invention are prepared using click chemistry reactions. For example, radioimmunoconjugates of the present invention can be prepared using click chemistry reactions referred to as "click radiolabeling." Click radiolabeling uses click chemistry reaction partners, preferably azides and alkynes (e.g., cyclooctyne or cyclooctyne derivatives), to form a triazole covalent bond between a radiocomplex (a radioactive metal ion bound to a chelator) and an antibody or antigen-binding fragment thereof. Methods for click radiolabeling of antibodies are described, for example, in International Patent Application No. US 18 / 65913, entitled "Radiolabeling of Polypeptides," the relevant disclosure of which is incorporated herein by reference. In other embodiments, referred to as "one-step direct radiolabeling," an immunoconjugate is prepared using click chemistry reactions between an antibody or antigen-binding fragment thereof and a chelator, and the immunoconjugate is then contacted with a radioactive metal ion to form a radioimmunoconjugate.

[0348] According to one embodiment, a method for preparing a radioimmunoconjugate comprises binding (eg, by coordination) a radiometal ion to a chelator of the present invention.

[0349] One embodiment of the "one-step direct radiolabeling" method may be described as a method of preparing a radioimmunoconjugate, comprising contacting an immunoconjugate (i.e., a polypeptide-chelator complex) with a radioactive metal ion, thereby forming a radioimmunoconjugate, wherein the immunoconjugate comprises a chelator of the present invention. According to certain embodiments, the immunoconjugate is formed by a click chemistry reaction between a chelator of the present invention and a polypeptide. According to certain embodiments, the radioimmunoconjugate is formed without metal-free conditions (e.g., without any step of removing or actively eliminating common metal impurities from the reaction mixture). This is in contrast to certain conventional methods that require radiolabeling antibodies under strict metal-free conditions to avoid competitive (non-productive) chelation of common metals such as iron, zinc, and copper, which poses significant challenges to the manufacturing process.

[0350] In a specific embodiment, the method for preparing a radioimmunoconjugate of the present invention comprises: (i) providing a polypeptide (e.g., an antibody or antigen-binding fragment thereof) covalently attached to a first click reaction partner (e.g., an azide group); (ii) providing a chelator complex comprising a chelator of the invention covalently bonded to a second Click reaction partner (e.g., an alkynyl or cycloalkynyl group); (iii) contacting the modified polypeptide with a chelator complex under conditions that allow the first Click reaction partner (e.g., an azide group) to react with a second Click reaction partner (e.g., an alkynyl or cycloalkynyl group) to form a polypeptide-chelator complex (i.e., an immunoconjugate); and (iv) contacting the polypeptide-chelator complex with a radioactive metal ion, thereby preparing a radioimmunoconjugate (the radioimmunoconjugate comprises a radioactive metal ion-labeled polypeptide, e.g., a modified antibody or antigen-binding fragment thereof, labeled with an alpha-emitting radioactive metal ion, which is coordinatively linked to a chelator).

[0351] According to a particular embodiment, step (iv) is carried out under metal-free conditions.

[0352] In another embodiment, the method for preparing a radioimmunoconjugate comprises: (i) providing a modified antibody or antigen-binding fragment thereof, comprising an antibody or antigen-binding fragment thereof covalently bound to an azide group; (ii) providing a radioactive complex comprising an alpha-emitting radioactive metal ion bound by a coordinate bond to a chelating agent, wherein the chelating agent is covalently bound to an alkynyl or cycloalkynyl group; (iii) contacting the modified antibody or antigen-binding fragment thereof with a radioactive complex under conditions that allow the azido group to react with the alkynyl or cycloalkynyl group, thereby preparing a radioimmunoconjugate.

[0353] Conditions for carrying out click chemistry reactions are well known in the art, and any conditions for carrying out click chemistry reactions known to one of skill in the art in light of the present disclosure can be used in the present invention. Exemplary conditions include, but are not limited to, incubating the modified polypeptide and radioactive complex in a ratio of 1:1 to 1000:1 at a pH of 4 to 10 and a temperature of 20°C to 70°C.

[0354] The click radiolabeling method described above maximizes the rate of radiometal ion chelation under low or high pH and / or high temperature conditions, and this can be achieved without the risk of inactivating the alkyne reaction partner. Efficient chelation between azide-labeled antibodies or antigen-binding fragments thereof and radioactive complexes and efficient SPAAC reactions allow for the production of radioimmunoconjugates in high radiochemical yields even at low azide:antibody ratios. The only step that requires the exclusion of trace metals is the radiometal ion chelation to the chelating moiety; antibody production, purification, and conjugation steps do not need to be performed under metal-free conditions.

[0355] The chelating agents and radiometal complexes of the present invention can also be used to generate site-specific radiolabeled polypeptides (e.g., antibodies). The click radiolabeling method described herein facilitates the site-specific generation of radioimmunoconjugates by utilizing established methods for site-specifically introducing azide groups onto antibodies (Li, X. et al., Preparation of well-defined antibody-drug conjugates through glycan remodeling and strain-promoted azide-alkyne cycloadditions. Angew Chem Int Ed Engl, 2014. 53(28): pp. 7179-82; Xiao, H. et al., Genetic incorporation of multiple unnatural amino acids into proteins in mammalian cells. Angew Chem Int Ed Engl, 2013. 52(52): pp. 14080-3). Methods for attaching molecules to proteins or antibodies in a site-specific manner are well known in the art, and any method for site-specifically labeling antibodies known to those of skill in the art may be used in the present invention in light of the present disclosure. Examples of methods for site-specifically modifying antibodies suitable for use in the present invention include, but are not limited to, the incorporation of modified cysteine ​​residues (e.g., THIOMAB™), the use of unnatural amino acids or glycans (e.g., selenocysteine, p-AcPhe, formylglycine-generating enzyme (FGE, SMARTag™), etc.), and enzymatic methods (e.g., glycotransferases, endoglycosidases, microbial or bacterial transglutaminases (MTG or BTG), sortase A, etc.).

[0356] In some embodiments, modified antibodies or antigen-binding fragments thereof for use in generating immunoconjugates or radioimmunoconjugates of the invention can be obtained by trimming an antibody or antigen-binding fragment thereof with a bacterial endoglycosidase specific for β-1,4 linkages between core GlcNAc residues in the Fc-glycosylation site of the antibody, such as GlycINATOR (Genovis), which leaves the innermost GlcNAc on the Fc intact and allows site-specific incorporation of an azido sugar at that site. The truncated antibody or antigen-binding fragment thereof can then be reacted with an azido-labeled sugar, such as UDP-N-azidoacetylgalactosamine (UDP-GalNAz) or UDP-6-azido6-deoxyGalNAc, in the presence of a glycosyltransferase, such as GalT galactosyltransferase or GalNAc transferase, thereby obtaining the modified antibody or antigen-binding fragment thereof.

[0357] In other embodiments, modified antibodies or antigen-binding fragments thereof for use in producing immunoconjugates or radioimmunoconjugates of the present invention can be obtained by deglycosylating the antibody or antigen-binding fragment thereof with an amidase. The resulting deglycosylated antibody or antigen-binding fragment thereof can then be reacted with an azidoamine, preferably 3-azidopropylamine, 6-azidohexylamine, or any azido linker amine or any azidoalkyl / heteroalkylamine, such as azido-polyethylene glycol (PEG)-amine, e.g., O-(2-aminoethyl)-O'-(2-azidoethyl)tetraethylene glycol, O-(2-aminoethyl)-O'-(2-azidoethyl)pentaethylene glycol, O-(2-aminoethyl)-O'-(2-azidoethyl)triethylene glycol, or in the presence of microbial transglutaminase, to obtain the modified antibody or antigen-binding fragment thereof.

[0358] Any of the radiometal complexes described herein can be used to generate the radioimmunoconjugates of the invention. In certain embodiments, the radiometal complex has a structure of Formula (Im), Formula (II-m), or Formula (III-m). In certain embodiments, the radiometal complex has a structure selected from the group consisting of:

[0359] [ka] In the formula, M is a radioactive metal ion, preferably an alpha-emitting radioactive metal ion, more preferably actinium-225 ( 225 Ac) and R 11 is cyclooctynyl or a cyclooctynyl derivative selected from the group consisting of bicyclononynyl (BCN), difluorinated cyclooctynyl (DIFO), dibenzocyclooctynyl (DIBO), keto-DIBO, biarylazacyclooctynyl (BARAC), dibenzoazacyclooctynyl (DIBAC, DBCO, ADIBO), dimethoxyazacyclooctynyl (DIMAC), difluorobenzocyclooctynyl (DIFBO), monobenzocyclooctynyl (MOBO) and tetramethoxydibenzocyclooctynyl (TMDIBO).

[0360] In some embodiments, the antibody or antigen-binding fragment thereof is covalently attached to the azide group using any method for chemical or enzymatic modification of antibodies and polypeptides known to those of skill in the art in light of the present disclosure. The azide-labeled antibody or antigen-binding fragment thereof is reacted with a chelator or radiometal complex of the invention comprising an alkynyl or cycloalkynyl group, preferably a cyclooctynyl group, more preferably DBCO, under conditions sufficient to subject the azide and alkynyl or cycloalkynyl group to a click chemistry reaction to form a 1,2,3-triazole moiety.

[0361] In certain embodiments, the radioimmunoconjugates of the present application include:

[0362] [ka]

[0363] [ka] where "mAb" is an antibody or antigen-binding domain (e.g., the Fab of KL2B30); L1 is absent or a linker, preferably a linker; and each R 12 are independently hydrogen, CH3, or CH2CH3, provided that at least one R 12 is -CH3 or -CH2CH3; M is an alpha-emitting radionuclide, preferably 225 Ac).

[0364] Examples of radioimmunoconjugates of the present application include:

[0365] [ka] (wherein "mAb" preferably refers to an antibody or antigen-binding domain having binding specificity for hK2, e.g., the Fab of KL2B30, or an antibody or antigen-binding domain as otherwise described herein), but is not limited to these.

[0366] In certain embodiments, the radioimmunoconjugate comprises:

[0367] [ka] (In the formula, M + is a radioactive metal ion, and M + is actinium 225 ( 225 Ac), Radium 223 ( 233 Ra), Bismuth 213 ( 213 Bi), lead 212( 212 Pb(II) and / or 212 Pb(IV)), terbium-149( 149 Tb), terbium-152( 152Tb), terbium-155( 155 Tb), Fermium-255 ( 255 Fm), Thorium-227 ( 227 Th), Thorium-226 ( 226 Th 4+ ), Astatine 211( 211 At), Cerium-134 ( 134 Ce), neodymium 144( 144 Nd), Lanthanum 132( 132 La), Lanthanum 135 ( 135 La), and uranium-230 ( 230 U) L1 is absent or a linker, In the formula, "mAb" preferably refers to any one or more structures independently selected from the group consisting of an antibody or antigen-binding domain having binding specificity for hK2, e.g., the Fab of KL2B30, or an antibody or antigen-binding domain as otherwise described herein.

[0368] In another embodiment, the radioimmunoconjugate comprises:

[0369] [ka] (wherein "mAb" preferably refers to an antibody or antigen-binding domain having binding specificity for hK2, e.g., the Fab of KL2B30, or an antibody or antigen-binding domain described elsewhere herein).

[0370] Radioimmunoconjugates produced by the methods described herein can be analyzed using methods well known to those of skill in the art in light of the present disclosure. For example, LC / MS analysis can be used to determine the ratio of chelator to labeled polypeptide, e.g., antibody or antigen-binding fragment thereof, analytical size exclusion chromatography can be used to determine the oligomeric state of polypeptides and polypeptide conjugates, e.g., antibodies and antibody conjugates, radiochemical yield can be determined by instant thin layer chromatography (e.g., iTLC-SG), and radiochemical purity can be determined by size exclusion HPLC.

[0371] Pharmaceutical Compositions and Methods of Use In another general aspect, the invention relates to a pharmaceutical composition comprising a chelator, radiometal complex, immunoconjugate, or radioimmunoconjugate of the invention and a pharmaceutically acceptable carrier. The pharmaceutical composition may also include one or more pharmaceutically acceptable carriers.

[0372] In one embodiment, the pharmaceutical composition comprises a radiometal complex of the present invention and a pharmaceutically acceptable carrier.

[0373] In another embodiment, the pharmaceutical composition comprises a radioimmunoconjugate of the present invention and a pharmaceutically acceptable carrier.

[0374] As used herein, the term "carrier" refers to any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, oil, lipid, lipid-containing vesicle, microsphere, liposomal encapsulate, or other material known in the art for use in pharmaceutical formulations. It will be understood that the characteristics of the carrier, excipient, or diluent will depend on the route of administration for a particular application. As used herein, the term "pharmaceutically acceptable carrier" refers to a non-toxic material that does not interfere with the efficacy or biological activity of the compositions of the present invention. According to certain embodiments, any pharmaceutically acceptable carrier suitable for use in antibody-based or radioconjugate-based pharmaceutical compositions in light of the present disclosure may be used in the present invention.

[0375] According to certain embodiments, the compositions described herein are formulated to be suitable for the intended route of administration to a subject. For example, the compositions described herein may be formulated to be suitable for parenteral administration, e.g., intravenous, subcutaneous, intramuscular, or intratumoral administration.

[0376] In another general aspect, the present invention relates to radiotherapy and methods of selectively targeting neoplastic cells to treat neoplastic diseases or disorders. Any of the radiocomplexes or radioimmunoconjugates described herein and pharmaceutical compositions thereof can be used in the methods of the invention.

[0377] A "neoplasm" is an abnormal mass of tissue that occurs when cells divide more than necessary or do not die when they should. Tumors can be benign (not cancerous) or malignant (cancer). Neoplasm is also called a tumor. A neoplastic disease or disorder is a disease or disorder associated with a tumor, such as cancer. Examples of neoplastic diseases or disorders include, but are not limited to, disseminated cancer and solid tumor cancer.

[0378] According to embodiments, a method of treating prostate cancer (e.g., metastatic prostate cancer or metastatic castration-resistant prostate cancer) in a subject in need thereof comprises administering to the subject a therapeutically effective amount of a radioimmunoconjugate described herein, wherein the radioimmunoconjugate comprises a radiometal complex described herein conjugated to an antigen-binding domain having binding specificity for hK2, such as KL2B30Fab.

[0379] In one embodiment of the present invention, a method for selectively targeting neoplastic cells for radiation therapy comprises administering to a subject a radioimmunoconjugate or pharmaceutical composition of the present invention.

[0380] In one embodiment of the present invention, a method for treating a neoplastic disease or disorder comprises administering to a subject in need thereof a radioimmunoconjugate or pharmaceutical composition of the present invention to the subject.

[0381] In one embodiment of the present invention, a method of treating cancer in a subject in need thereof comprises administering to the subject a radioimmunoconjugate or pharmaceutical composition of the present invention.

[0382] The radioimmunoconjugate delivers radiation directly to, for example, a cell targeted by the antigen-binding domain. Preferably, the radioimmunoconjugate comprises: 225 When targeted, the alpha-emitting radioactive metal ions, e.g., Ac, are carried. 225 Alpha particles from Ac and its daughters are delivered to targeted cells, causing a cytotoxic effect on the cells, thereby selectively targeting tumor cells for radiation therapy and / or treatment of neoplastic diseases or disorders.

[0383] A pre-targeting approach for selectively targeting neoplastic cells for radiation therapy and to treat neoplastic diseases or disorders is also contemplated by the present invention. According to the pre-targeting approach, an azide-labeled antibody or antigen-binding fragment thereof is administered, binds to cells bearing the antibody's target antigen, and is removed from the circulation over time or with a removal agent. Subsequently, a radioactive complex of the present invention, preferably a radioactive complex comprising a cyclooctyne or cyclooctyne derivative (e.g., DBCO), is administered and undergoes a SPAAC reaction with the azide-labeled antibody bound to the target site, and the remaining unbound radioactive complex is rapidly removed from the circulation. This pre-targeting technique provides a method for enhancing the localization of radioactive metal ions at target sites in a subject.

[0384] In other embodiments, the modified polypeptide, e.g., an azido-labeled antibody or antigen-binding fragment thereof, and the radioconjugate of the invention are administered in the same composition or in different compositions to a subject in need of targeted radiation therapy or treatment of a neoplastic disease or disorder.

[0385] In some embodiments, a therapeutically effective amount of a radioimmunoconjugate or pharmaceutical composition of the present invention is administered to a subject to treat a neoplastic disease or disorder in the subject, such as cancer.

[0386] In other embodiments of the present invention, the radioimmunoconjugates and pharmaceutical compositions of the present invention may be used in combination with other agents effective in the treatment of neoplastic diseases or disorders.

[0387] Also provided are the radioimmunoconjugates and pharmaceutical compositions described herein for use in selectively targeting neoplastic cells for radiotherapy and / or treatment of a neoplastic disease or disorder and / or for diagnosis of a neoplastic disease or disorder, and the use of a radioimmunoconjugate or pharmaceutical composition described herein in the manufacture of a medicament for selectively treating neoplastic cells for radiotherapy and / or treatment of a neoplastic disease or disorder. [Example]

[0388] The following examples of the present invention are presented to further illustrate the principles of the present invention, but it should be understood that the following examples do not limit the present invention, the scope of which is defined by the appended claims.

[0389] Synthesis of Chelating Agents and Immunoconjugates (Examples 1-21) Methods of synthesis of embodiments of the chelating agents described herein are provided, for example, in WO 2020 / 229974, which is incorporated herein by reference. Further synthesis methods are provided in PCT / IB2021 / 060350 (incorporated herein by reference) and in Examples 1-21 below.

[0390] In Examples 1-21, some synthetic products are listed as being isolated as a residue. Those skilled in the art will understand that the term "residue" is not intended to limit the physical state from which the product is isolated, and may include, for example, solids, oils, foams, gums, syrups, etc.

[0391] Abbreviations used herein, particularly in the Schemes and Examples, are as listed in Table A below.

[0392] [Table 1-1]

[0393] [Table 1-2]

[0394] As used herein, unless otherwise specified, the term "isolated form" shall mean that the compound exists in a form separated from any solid mixture with other compound(s), solvent system, or biological environment. In one embodiment of the present invention, any of the compounds described herein exists in isolated form.

[0395] As used herein, unless otherwise specified, the term "substantially pure form" shall mean that the mole percent of impurities in the isolated compound is less than about 5 mole percent, preferably less than about 2 mole percent, more preferably less than about 0.5 mole percent, and most preferably less than about 0.1 mole percent. In one embodiment of the present invention, the compound of formula (I) is present in substantially pure form.

[0396] As used herein, unless otherwise specified, the term "substantially free of a corresponding salt form(s)," when used to describe a compound of formula (I), means that the mole percent of the corresponding salt form(s) in the isolated base of formula (I) is less than about 5 mole percent, preferably less than about 2 mole percent, more preferably less than about 0.5 mole percent, and most preferably less than about 0.1 mole percent. In one embodiment of the present invention, the compound of formula (I) is present in a form that is substantially free of the corresponding salt form(s).

[0397] Example 1 4-((6-(methoxycarbonyl)pyridin-2-yl)(16-((6-(methoxycarbonyl)pyridin-2-yl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-7-yl)methyl)benzoic acid (TOPA-[C-7]-phenyl-carboxylic acid)

[0398] [ka]

[0399] Scheme 1

[0400] [ka]

[0401] Step 1: In a 500 mL three-neck round-bottom flask under nitrogen at −78 °C, to a mixture of methyl 6-formylpicolinate (4.00 g, 24.2 mmol), (4-(tert-butoxycarbonyl)phenyl)boronic acid (10.7 g, 48.5 mmol), PdCl (0.21 g, 1.2 mmol), tri(naphthalen-1-yl)phosphine (0.50 g, 1.2 mmol), and potassium carbonate (10.0 g, 72.7 mmol) was added tetrahydrofuran (100 mL) in one portion. The mixture was purged with nitrogen and stirred at room temperature for 30 minutes, then heated at 65 °C for 24 hours. The reaction mixture was cooled to room temperature, filtered through a pad of Celite, and the filtrate was concentrated to dryness. The crude product was purified by silica gel chromatography (0-50% EtOAc / petroleum ether) to give methyl 6-((4-(tert-butoxycarbonyl)phenyl)(hydroxy)methyl)picolinate as a yellow oil (2.5 g, 30% yield).

[0402] Step 2: A stir bar, methyl 6-((4-(tert-butoxycarbonyl)phenyl)(hydroxy)methyl)picolinate (2.50 g, 7.30 mmol), PPh3 (3.43 g, 13.1 mmol), N-bromosuccinimide (2.13 g, 12.0 mmol), and dichloromethane (30 mL) were added to a 250 mL three-necked round-bottom flask at room temperature under a nitrogen atmosphere and stirred for 1 h. The reaction solution was loaded onto a silica gel column and purified by chromatography (0-30% EtOAc / petroleum ether) to give the compound methyl 6-(bromo(4-(tert-butoxycarbonyl)phenyl)methyl)picolinate as a yellow oil (1.65 g, 56% yield).

[0403] Step 3: A stir bar, methyl 6-(bromo(4-(tert-butoxycarbonyl)phenyl)methyl)picolinate (1.52 g, 3.69 mmol), methyl 6-((1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-7-yl)methyl)picolinate (1.50 g, 3.69 mmol), NaCO (1.17 g, 11.1 mmol), and acetonitrile (30 mL) were added to a 250 mL three-neck round-bottom flask, and the resulting heterogeneous mixture was heated at 90 °C under a nitrogen atmosphere for 16 h. The reaction mixture was then cooled to room temperature, filtered through a Celite pad, and concentrated to dryness in vacuo to give the crude product. The crude product was purified by silica gel chromatography (0-10% MeOH / dichloromethane) to give methyl 6-((4-(tert-butoxycarbonyl)phenyl)(16-((6-(methoxycarbonyl)pyridin-2-yl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-7-yl)methyl)picolinate as a brown oil (1.2 g, 44%).

[0404] Step 4: A stirring bar, methyl 6-((4-(tert-butoxycarbonyl)phenyl)(16-((6-(methoxycarbonyl)pyridin-2-yl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-7-yl)methyl)picolinate (1.2 g, 1.6 mmol), TFA (0.62 mL, 8.1 mmol), and DCM (20 mL) were added to a 100 mL three-necked round-bottom flask at room temperature and stirred for 1 h. The reaction mixture was concentrated to dryness, and the resulting crude product was purified by preparative HPLC (XBRIDGE C18 (19 × 150 mm) 5.0 μm column, 0.1% aqueous TFA / ACN, flow rate: 15.0 mL / min) to give TOPA-[C-7]-phenyl-carboxylic acid as a brown oil (0.8 g, 72%). LC-MS APCI:C 35 H 44 N4O 10 Calculated value 680.31, observed value m / z [M+H] +681.5. Purity by LC-MS: 99.87%. Purity by HPLC: 97.14% (97.01% at 210 nm, 97.20% at 254 nm, and 97.21% at 280 nm). Column: Atlantis dC18 (250 × 4.6 mm), 5 μm, Mobile phase A: 0.1% TFA in water, Mobile phase B: acetonitrile, Flow rate: 1.0 mL / min. 1 H NMR(400MHz,DMSO-d6):δ 8.12-8.07(m,4H),8.00-7.98(m,2H),7.75-7.73(m,4H),6.10(s,1H),4.6 7(s,2H),3.96(s,3H),3.91(s,3H),3.82(s,8H),3.56(s,8H),3.52(s,8H).

[0405] Example 2 6-((16-((6-carboxypyridin-2-yl)(4-((2-(2-(2-isothiocyanatoethoxy)ethoxy)ethyl)carbamoyl)phenyl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-7-yl)methyl)picolinic acid

[0406] [ka]

[0407] Scheme 2

[0408] [ka]

[0409] Step 1: A stir bar, 4-((6-(methoxycarbonyl)pyridin-2-yl)(16-((6-(methoxycarbonyl)pyridin-2-yl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-7-yl)methyl)benzoic acid (0.40 g, 0.60 mmol), tert-butyl (2-(2-(2-aminoethoxy)ethoxy)ethyl)carbamate (0.15 g, 0.60 mmol), triethylamine (0.18 g, 0.76 mmol), HATU (0.33 g, 0.90 mmol), and DCM (4.0 mL) were added to a 25 mL three-neck round-bottom flask under a nitrogen atmosphere at 0° C. The mixture was stirred at room temperature overnight. The reaction was treated with water (10 mL) and extracted with dichloromethane (10 mL × 3). The combined extracts were washed with 10% aqueous NaHCO (10 mL), brine (10 mL), dried over anhydrous NaSO, filtered, and concentrated to dryness to give an oil, which was purified by silica gel chromatography (0–10% MeOH / DCM) to give methyl 6-((4-((2,2-dimethyl-4-oxo-3,8,11-trioxa-5-azatridecan-13-yl)carbamoyl)phenyl)(16-((6-(methoxycarbonyl)pyridin-2-yl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-7-yl)methyl)picolinate (0.18 g).

[0410] Step 2: A stir bar, methyl 6-((4-((2,2-dimethyl-4-oxo-3,8,11-trioxa-5-azatridecan-13-yl)carbamoyl)phenyl)(16-((6-(methoxycarbonyl)pyridin-2-yl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-7-yl)methyl)picolinate (0.18 g, 0.20 mmol), MeOH (1.8 mL), and HCl in methanol (4 M, 1.0 mL, 4.0 mmol) were added to a 10 mL single-neck round-bottom flask at 0° C., then warmed to room temperature and stirred for 2 h. The volatiles were removed in vacuo to give methyl 6-((4-((2-(2-(2-aminoethoxy)ethoxy)ethyl)carbamoyl)phenyl)(16-((6-(methoxycarbonyl)pyridin-2-yl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-7-yl)methyl)picolinate (0.15 g), which was used without purification.

[0411] Step 3: A stir bar, methyl 6-((4-((2-(2-(2-aminoethoxy)ethoxy)ethyl)carbamoyl)phenyl)(16-((6-(methoxycarbonyl)pyridin-2-yl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-7-yl)methyl)picolinate (0.10 g, 0.12 mmol), triethylamine (37 mg, 0.37 mmol), dry DCM (2 mL), and carbon disulfide (14 mg, 0.18 mmol) were added to a pressure vial at room temperature under a nitrogen atmosphere. The vial was irradiated in a microwave oven at 90° C. for 30 minutes (power 150 W). The vial was then cooled to room temperature, and the reaction mixture was diluted with dichloromethane (10 mL), then washed sequentially with water (5 mL), 1 M HCl (5 mL), and water (5 mL), dried over anhydrous NaSO, filtered, and concentrated to dryness to give methyl 6-((4-((2-(2-(2-isothiocyanatoethoxy)ethoxy)ethyl)carbamoyl)phenyl)(16-((6-(methoxycarbonyl)pyridin-2-yl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-7-yl)methyl)picolinate (100 mg), which was used without purification.

[0412] Step 4: A stir bar, methyl 6-((4-((2-(2-(2-isothiocyanatoethoxy)ethoxy)ethyl)carbamoyl)phenyl)(16-((6-(methoxycarbonyl)pyridin-2-yl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-7-yl)methyl)picolinate (0.10 g, 0.12 mmol), and aqueous HCl (6 N, 0.4 mL, 2.34 mmol) were added to a 10 mL single-neck round-bottom flask and stirred at 50° C. for 3 hours. The reaction mixture was cooled to room temperature and concentrated to dryness under vacuum to give an oil, which was purified by preparative HPLC (column: XBRIDGE C18 19 x 150 mm, 5.0 μm, mobile phase: 0.1% aqueous TFA / acetonitrile, flow rate: 15.0 mL / min) to give 6-((16-((6-carboxypyridin-2-yl)(4-((2-(2-(2-isothiocyanatoethoxy)ethoxy)ethyl)carbamoyl)phenyl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-7-yl)methyl)picolinic acid (5.0 mg). LC-MS APCI:C 40 H 52 NO 11 Calculated S: 824.34, observed m / z [M+H] + 824.8. 1 H NMR(400MHz,CD3OD):δ 8.22-8.20(m,2H),8.14-8.05(m,2H),7.94(d,J=8.00Hz,2H),7.79(d,J=8.00Hz,2H),7.73-7. 67(m,2H),6.16(s,1H),4.77(s,2H),3.93-4.00(m,8H),3.59-3.70(m,27H),3.47-3.44(m,2H).

[0413] Example 3 6-((4-((6-aminohexyl)carbamoyl)phenyl)(16-((6-carboxypyridin-2-yl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-7-yl)methyl)picolinic acid and

[0414] Example 4 6-((16-((6-carboxypyridin-2-yl)(4-((6-isothiocyanatohexyl)carbamoyl)phenyl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-7-yl)methyl)picolinic acid

[0415] [ka]

[0416] Scheme 3

[0417] [ka]

[0418] Step 1: A stir bar, 4-((6-(methoxycarbonyl)pyridin-2-yl)(16-((6-(methoxycarbonyl)pyridin-2-yl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-7-yl)methyl)benzoic acid (0.12 g, 0.18 mmol), tert-butyl(6-aminohexyl)carbamate (38 mg, 0.18 mmol), triethylamine (54 mg, 0.54 mmol), HATU (0.10 g, 0.27 mmol), and DCM (4.0 mL) were added to a 25 mL three-neck round-bottom flask under a nitrogen atmosphere at 0° C. The reaction mixture was then warmed to room temperature and stirred overnight. The reaction mixture was then treated with water (10 mL) and extracted with dichloromethane (10 mL × 3). The combined extracts were washed with 10% aqueous NaHCO (10 mL), brine (10 mL), dried over anhydrous NaSO, filtered, and concentrated to dryness to give an oil that was purified by silica gel chromatography (0–10% MeOH / DCM) to give methyl 6-((4-((6-((tert-butoxycarbonyl)amino)hexyl)carbamoyl)phenyl)(16-((6-(methoxycarbonyl)pyridin-2-yl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-7-yl)methyl)picolinate (70 mg) as a gummy oil.

[0419] Step 2: A stir bar, methyl 6-((4-((6-((tert-butoxycarbonyl)amino)hexyl)carbamoyl)phenyl)(16-((6-(methoxycarbonyl)pyridin-2-yl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-7-yl)methyl)picolinate (70 mg, 0.080 mmol), MeOH (1.5 mL), and HCl in methanol (4 M, 0.4 mL, 1.6 mmol) were added to a 25 mL round-bottom flask at 0° C., followed by bringing to room temperature and stirring for 2 h. The volatiles were removed in vacuo to give methyl 6-((4-((6-aminohexyl)carbamoyl)phenyl)(16-((6-(methoxycarbonyl)pyridin-2-yl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-7-yl)methyl)picolinate (30 mg), which was used without purification.

[0420] Step 3: A stir bar, methyl 6-((4-((6-aminohexyl)carbamoyl)phenyl)(16-((6-(methoxycarbonyl)pyridin-2-yl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-7-yl)methyl)picolinate (30 mg, 0.038 mmol), aqueous LiOH (1.1 mL, 0.1 N, 0.11 mmol), and MeOH (1.0 mL) were added to an 8 mL reaction vial and stirred at room temperature overnight. The reaction mixture was then treated with acetic acid to a pH of approximately 6.5 and subsequently concentrated to dryness under vacuum at room temperature. The resulting product was purified by preparative HPLC (column: XBRIDGE C18 19 x 150 mm, 5.0 μm, mobile phase: 10 mM aqueous ammonium acetate / ACN, flow rate: 15.0 mL / min) to give Example 3: 6-((4-((6-aminohexyl)carbamoyl)phenyl)(16-((6-carboxypyridin-2-yl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-7-yl)methyl)picolinic acid (10 mg). LC-MS APCI:C 39 H 54Calculated for N6O9, No. 750.40; Observed m / z [M+H] + 751.3. 1 H NMR(400MHz,CD3OD):δ 8.22(d,J=1.60Hz,2H),8.21-8.06(m,2H),7.92(d,J=8.40Hz,2H),7.80(d,J=8.40Hz,2H),7.75-7.69(m,2H),6.20(s,1H),4.70 (s,2H),4.02-3.92(m,8H),3.76-3.62(m,14H),3.51-3.32(m,4H),2.93(t,J=8.00Hz,2H),1.67-1.64(m,4H),1.46-1.45(m,4H).

[0421] Step 4: A stir bar, methyl 6-((4-((6-aminohexyl)carbamoyl)phenyl)(16-((6-(methoxycarbonyl)pyridin-2-yl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-7-yl)methyl)picolinate (0.10 g, 0.13 mmol), triethylamine (39 mg, 0.38 mmol), dry DCM (2 mL), and carbon disulfide (15 mg, 0.19 mmol) were added to a pressure vial at room temperature under a nitrogen atmosphere. The vial was microwaved at 90° C. for 30 minutes (power 150 W). The vial was then cooled to room temperature, and the reaction mixture was diluted with dichloromethane (10 mL), washed with water (5 mL), 1 M HCl (5 mL), and water (5 mL), dried over anhydrous NaSO, filtered, and concentrated to dryness to give methyl 6-((4-((6-isothiocyanatohexyl)carbamoyl)phenyl)(16-((6-(methoxycarbonyl)pyridin-2-yl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-7-yl)methyl)picolinate (0.1 g), which was used without purification.

[0422] Step 5: A stir bar, methyl 6-((4-((6-isothiocyanatohexyl)carbamoyl)phenyl)(16-((6-(methoxycarbonyl)pyridin-2-yl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-7-yl)methyl)picolinate (0.10 g, 0.12 mmol), and aqueous HCl (6 N, 0.4 mL, 2.4 mmol) were added to a 10 mL round-bottom flask and then stirred at 50° C. for 3 hours. The reaction mixture was then cooled to room temperature and concentrated to dryness under vacuum to give a residue, which was purified by preparative HPLC (column: XBRIDGE C18 19 x 150 mm, 3.5 μm, mobile phase: 0.1% aqueous TFA / acetonitrile, flow rate: 2.0 mL / min) to give Example 4: 6-((16-((6-carboxypyridin-2-yl)(4-((6-isothiocyanatohexyl)carbamoyl)phenyl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-7-yl)methyl)picolinic acid (15 mg). LC-MS APCI:C 40 H 52 Calculated for N6O9S: 792.35, observed m / z [M+H] + 792.8. 1 H NMR(400MHz,CD3OD):δ 8.23-8.20(m,2H),8.15-8.06(m,2H),7.92(d,J=8.40Hz,2H),7.79(d,J=8.40Hz,2H),7.74-7.68(m,2H),6.17(s, 1H),4.77(s,2H),4.01-3.93(m,8H),3.75-3.56(m,16H),3.42-3.33(m,5H),1.74-1.64(m,4H),1.50-1.44(m,4H).

[0423] Example 5 6-((16-((6-carboxypyridin-2-yl)(4-((4-isothiocyanatophenethyl)carbamoyl)phenyl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-7-yl)methyl)picolinic acid

[0424] [ka]

[0425] Scheme 4

[0426] [ka]

[0427] Step 1: A stir bar, 4-((6-(methoxycarbonyl)pyridin-2-yl)(16-((6-(methoxycarbonyl)pyridin-2-yl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-7-yl)methyl)benzoic acid (0.25 g, 0.37 mmol), 4-(2-aminoethyl)aniline (60 mg, 0.37 mmol), TEA (0.11 g, 0.15 mL, 1.1 mmol), HATU (0.21 g, 0.55 mmol), and DCM (5 mL) were added to a 25 mL three-neck round-bottom flask under a nitrogen atmosphere at 0° C. After stirring overnight at room temperature, the reaction mixture was diluted with water (1 The mixture was treated with 10 mL of 10% aqueous NaHCO (10 mL) and brine (10 mL), dried over anhydrous NaSO, filtered, and concentrated to dryness to give the product, which was purified by silica gel chromatography (0-10% MeOH / DCM) to give methyl 6-((4-((4-aminophenethyl)carbamoyl)phenyl)(16-((6-(methoxycarbonyl)pyridin-2-yl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-7-yl)methyl)picolinate (0.12 g).

[0428] Step 2: A stir bar, methyl 6-((4-((4-aminophenethyl)carbamoyl)phenyl)(16-((6-(methoxycarbonyl)pyridin-2-yl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-7-yl)methyl)picolinate (0.12 g, 0.15 mmol), TEA (45 mg, 65 μL, 0.45 mmol), DCM (3 mL), and CS (17 mg, 0.23 mmol) were added to a 10 mL microwave pressure vial at room temperature under a nitrogen atmosphere. The reaction mixture was irradiated in a microwave (power 150 W) at 90° C. for 30 minutes. The reaction mixture was then cooled to room temperature, diluted with dichloromethane (10 mL), washed successively with water (5 mL), 1 M HCl (5 mL), and water (5 mL), dried over anhydrous NaSO, and concentrated to dryness to give methyl 6-((4-((4-isothiocyanatophenethyl)carbamoyl)phenyl)(16-((6-(methoxycarbonyl)pyridin-2-yl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-7-yl)methyl)picolinate (0.12 g), which was used without purification.

[0429] Step 3: A stir bar, methyl 6-((4-((4-isothiocyanatophenethyl)carbamoyl)phenyl)(16-((6-(methoxycarbonyl)pyridin-2-yl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-7-yl)methyl)picolinate (0.12 g, 0.14 mmol), and aqueous HCl (0.50 mL, 6 N, 2.8 mmol) were added to a 10 mL single-neck round-bottom flask and stirred at 50° C. for 3 hours. The reaction mixture was cooled to room temperature and concentrated to dryness under reduced pressure, and the crude product was purified by preparative HPLC (column: XBRIDGE C18 19 x 150 mm, 5.0 μm; mobile phase: 0.1% aqueous TFA / acetonitrile, flow rate: 15.0 mL / min) to give 6-((16-((6-carboxypyridin-2-yl)(4-((4-isothiocyanatophenethyl)carbamoyl)phenyl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-7-yl)methyl)picolinic acid (30 mg). LC-MS APCI:C42 H 48 N5O 10 Calculated S, No. 812.32; Observed m / z [M+H] + 812.9. 1 H NMR(400MHz,CD3OD):δ 8.22(d,J=0.80Hz,2H),8.06-8.21(m,2H),7.85(d,J=8.40Hz,2H),7.68-7.78(m,4H),7.31(d,J=8.40Hz,2H),7.21(d, J=2.00Hz,2H),6.18(s,1H),4.77(s,2H),3.70-4.00(m,7H),3.60-3.67(m,16H),3.44-3.49(m,2H),2.90-3.10(m,3H).

[0430] Example 6 (S-6,6'-((2-(((2-isothiocyanatoethyl)thio)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diyl)bis(methylene))dipicolinic acid

[0431] [ka]

[0432] Scheme 5

[0433] [ka]

[0434] Step 1: A stir bar, 1(methyl 6-((4-((tert-butoxycarbonyl)amino)phenyl)(16-((6-(methoxycarbonyl)pyridin-2-yl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-7-yl)methyl)picolinate) (0.10 g, 0.15 mmol), MeOH (0.5 mL) and HCl in methanol (4 M, 0.6 mL, 4.0 mmol) were added to a 25 mL single-neck round-bottom flask at 0° C., then brought to room temperature and stirred for 2 h. The volatiles were removed in vacuo to give dimethyl 6,6'-((2-(((2-aminoethyl)thio)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diyl)bis(methylene))(S)-dipicolinate (55 mg), which was used in the next step without purification.

[0435] Step 2: A stir bar, dimethyl 6,6'-((2-(((2-aminoethyl)thio)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diyl)bis(methylene))(S)-dipicolinate (50 mg, 0.10 mmol), triethylamine (24 mg, 0.24 mmol), DCM (2 mL), and carbon disulfide (12 mg, 0.16 mmol) were added to a microwave vial under a nitrogen atmosphere at room temperature. The vial was irradiated in a microwave oven at 90 °C for 30 minutes (power 150 W). The vial was then cooled to room temperature, and the reaction mixture was diluted with dichloromethane (10 mL), washed sequentially with water (5 mL), 1 M HCl (5 mL), and water (5 mL), dried over anhydrous NaSO, concentrated to dryness, and subjected to silica gel chromatography (0–10% MeOH / DCM) to give dimethyl 6,6′-((2-(((2-isothiocyanatoethyl)thio)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diyl)bis(methylene))(S)-dipicolinate as a yellow solid (20 mg).

[0436] Step 3: A stir bar, dimethyl 6,6'-((2-(((2-isothiocyanatoethyl)thio)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diyl)bis(methylene))(S)-dipicolinate (20 mg, 0.030 mmol) and aqueous HCl (6 N, 0.1 mL, 0.6 mmol) were added to a 10 mL single-neck round-bottom flask and stirred at room temperature overnight. The reaction mixture was concentrated to dryness in vacuo, and the resulting residue was purified by preparative HPLC (column: XBRIDGE C18 (19 × 150 mm) 5.0 μm, mobile phase: 0.1% TFA aqueous solution / acetonitrile, flow rate: 15.0 mL / min) to give (S)-6,6'-((2-(((2-isothiocyanatoethyl)thio)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diyl)bis(methylene))dipicolinic acid (6 mg). LC-MS APCI:C 30 H 41 Calculated for N5O8S2: 663.24, observed m / z [M+H] + 664.2. 1 H NMR(400MHz,DMSO-d6):δ 9.78(s,1H),8.10(s,4H),7.78(d,J=6.00Hz,2H),4.69(s,4H),3.96-3.52(m,23H),2.85(t,J=6.40Hz,2H),2.70(t,J=8.00Hz,2H).

[0437] Example 7 (S)-6,6'-((2-(((5-isothiocyanatopentyl)thio)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diyl)bis(methylene))dipicolinic acid

[0438] [ka]

[0439] Scheme 6

[0440] [ka]

[0441] Step 1: A stir bar, dimethyl 6,6'-((2-(((5-((tert-butoxycarbonyl)amino)pentyl)thio)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diyl)bis(methylene))(S)-dipicolinate (0.12 g, 0.15 mmol), MeOH (0.5 mL), and HCl in methanol (4 M, 0.6 mL, 4.0 mmol) were added to a 25 mL single-neck round-bottom flask at 0°C, warmed to room temperature, and stirred for 2 hours. The volatiles were then removed in vacuo to give dimethyl 6,6'-((2-(((5-aminopentyl)thio)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diyl)bis(methylene))(S)-dipicolinate (70 mg), which was used without purification.

[0442] Step 2: A stir bar, dimethyl 6,6'-((2-(((5-aminopentyl)thio)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diyl)bis(methylene))(S)-dipicolinate (70 mg, 0.10 mmol), triethylamine (20 mg, 0.20 mmol), dry DCM (2 mL), and carbon disulfide (15 mg, 0.20 mmol) were added to a microwave vial at room temperature under a nitrogen atmosphere. The reaction mixture was irradiated in a microwave (power 150 W) at 90 °C for 30 minutes. The vial was allowed to reach room temperature, and the reaction mixture was diluted with dichloromethane (10 mL), washed successively with water (5 mL), 1 M HCl (5 mL), and water (5 mL), dried over anhydrous sodium sulfate (NaSO), filtered, and concentrated to dryness to give a residue. The residue was subjected to silica gel chromatography (0-10% MeOH / DCM) to give dimethyl 6,6'-((2-(((5-isothiocyanatopentyl)thio)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diyl)bis(methylene))(S)-dipicolinate as a yellow solid (30 mg).

[0443] Step 3: A stir bar, dimethyl 6,6'-((2-(((5-isothiocyanatopentyl)thio)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diyl)bis(methylene))(S)-dipicolinate (30 mg, 0.040 mmol), and aqueous HCl (6 N, 0.2 mL, 0.8 mmol) were added to a 10 mL single-neck round-bottom flask and stirred at room temperature overnight. The reaction mixture was concentrated to dryness under vacuum, and the concentrate was purified by HPLC (column: XBRIDGE C18 19 x 150 mm, 5.0 μm, mobile phase: 0.1% aqueous TFA / acetonitrile, flow rate: 15.0 mL / min) to give (S)-6,6'-((2-(((5-isothiocyanatopentyl)thio)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diyl)bis(methylene))dipicolinic acid (12 mg). LC-MS APCI:C 33 H 47 Calculated for N5O8S2: 705.29, observed m / z [M+H] + 706.2. 1 H NMR(400MHz,DMSO-d6):δ 13.40(s,1H),9.90(s,1H),8.17-8.09(m,4H),7.78(d,J=6.80Hz,2H),4.70(s,4H),3.93- 3.17(m,27H),2.68-2.67(m,2H),1.64-1.60(m,2H),1.53-1.49(m,2H),1.40-1.38(m,2H).

[0444] Example 8 (S)-6,6'-((2-(((2-(2-(4-isothiocyanatophenoxy)ethoxy)ethyl)thio)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diyl)bis(methylene))dipicolinic acid

[0445] [ka]

[0446] Scheme 7

[0447] [ka]

[0448] Scheme 7a

[0449] [ka]

[0450] Scheme 7a, Step 1a: A stir bar, tert-butyl (4-hydroxyphenyl)carbamate (4.5 g, 22 mmol), 1-bromo-2-(2-bromoethoxy)ethane (5.0 g, 22 mmol), KCO (4.6 g, 43 mmol), and ACN (45 mL) were added to a 250 mL three-neck round-bottom flask under a nitrogen atmosphere, and the resulting reaction mixture was heated at 80 °C under a nitrogen atmosphere for 16 h. The reaction mixture was cooled to room temperature, filtered through Celite®, and concentrated to dryness in vacuo to give a concentrate that was purified by silica gel chromatography (0-20% EtOAc / petroleum ether) to give the product tert-butyl (4-(2-(2-bromoethoxy)ethoxy)phenyl)carbamate (2.0 g).

[0451] Step 2a: A stir bar, tert-butyl (4-(2-(2-bromoethoxy)ethoxy)phenyl)carbamate (2.0 g, 5.6 mmol), ethanethio S-acid (0.42 g, 5.6 mmol), K2CO3 (1.5 g, 11 mmol), and ACN (50 mL) were added to a 250 mL three-neck round-bottom flask under a nitrogen atmosphere. The reaction mixture was stirred at 80 °C for 2 h, then cooled to room temperature, filtered through Celite®, and concentrated to dryness in vacuo. The concentrate was purified using neutral alumina chromatography (0 to 50% EtOAc / petroleum ether) to give S-(2-(2-(4-((tert-butoxycarbonyl)amino)phenoxy)ethoxy)ethyl)ethanethioate (1.8 g).

[0452] Step 3a: A stir bar, S-(2-(2-(4-((tert-butoxycarbonyl)amino)phenoxy)ethoxy)ethyl)ethanethioate (1.8 g, 5.1 mmol), ethanol (20 mL), and hydrazine monohydrate (0.24 g, 0.24 mL, 7.6 mmol) were added to a 250 mL single-neck round-bottom flask under nitrogen and stirred at 80° C. for 1 h. The reaction mixture was then cooled to room temperature and concentrated to dryness in vacuo to give a concentrate that was purified by silica gel chromatography (5-10% EtOAc / petroleum ether) to give tert-butyl (4-(2-(2-mercaptoethoxy)ethoxy)phenyl)carbamate (0.5 g) as a colorless oil.

[0453] Scheme 7, Step 1: A solution of tert-butyl (4-(2-(2-mercaptoethoxy)ethoxy)phenyl)carbamate (0.40 g, 1.0 mmol) and DMF (3.0 mL) was added dropwise over 5 minutes to a 50 mL three-neck round-bottom flask containing a suspension of sodium hydride (0.060 g, 60% in mineral oil, 1.5 mmol) in DMF (3.0 mL) under a nitrogen atmosphere at 0° C. After the addition was complete, the reaction mixture was warmed to room temperature and stirred for 15 minutes. The mixture was recooled to 0 °C, and a solution of dimethyl 6,6'-((2-(((methylsulfonyl)oxy)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diyl)bis(methylene))(S)-dipicolinate (0.5 g, 0.7 mmol) and DMF (3.0 mL) was added dropwise. After the addition was complete, the reaction mixture was allowed to warm slowly to room temperature and stirred for 1.5 h. The reaction was treated slowly with saturated NH4Cl (0.2 mL) and then concentrated to dryness to give an oil. The oil was purified by preparative HPLC (column: XBRIDGE C18 19 × 150 mm 5.0 μm, mobile phase: 0.1% aqueous TFA / acetonitrile, flow rate: 15.0 mL / min) to give dimethyl 6,6′-((2-(((2-(2-(4-((tert-butoxycarbonyl)amino)phenoxyl)ethoxy)ethyl)thio)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diyl)bis(methylene))(S)-dipicolinate as a brown oil (0.15 g).

[0454] Step 2: A stir bar, dimethyl 6,6'-((2-((2-(2-(4-((tert-butoxycarbonyl)amino)phenoxy)ethoxy)ethyl)thio)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diyl)bis(methylene))(S)-dipicolinate (0.15 g, 0.16 mmol), MeOH (1.0 mL), and HCl in methanol (4 M, 0.80 mL, 3.2 mmol) were added to a 25 mL single-neck round-bottom flask at 0°C, then brought to room temperature and stirred for 3 h. The volatiles were removed under reduced pressure to give dimethyl 6,6'-((2-(((2-(2-(4-aminophenoxy)ethoxy)ethyl)thio)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diyl)bis(methylene))(S)-dipicolinate (0.12 g), which was used without purification.

[0455] Step 3: A stir bar, dimethyl 6,6'-((2-(((2-(2-(4-aminophenoxy)ethoxy)ethyl)thio)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diyl)bis(methylene))(S)-dipicolinate (0.12 g, 0.15 mmol), triethylamine (46 mg, 0.46 mmol), dry DCM (3 mL), and carbon disulfide (17 mg, 0.22 mmol) were added to a pressure vial under a nitrogen atmosphere at room temperature. The reaction mixture was irradiated in a microwave oven (power 150 W) at 90°C for 30 minutes. The reaction mixture was cooled to room temperature, diluted with dichloromethane (10 mL), washed successively with water (5 mL), 1 M HCl (5 mL), and water (5 mL), dried over anhydrous NaSO, and concentrated to dryness to give dimethyl 6,6'-((2-(((2-(2-(4-isothiocyanatophenoxy)ethoxy)ethyl)thio)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diyl)bis(methylene))(S)-dipicolinate (0.12 g), which was used without purification.

[0456] Step 4: A stir bar, dimethyl 6,6'-((2-(((2-(2-(4-isothiocyanatophenoxy)ethoxy)ethyl)thio)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diyl)bis(methylene))(S)-dipicolinate (0.12 g, 0.15 mmol) and aqueous HCl (6 N, 0.51 mL, 3.1 mmol) were added to a 10 mL single-neck round-bottom flask and stirred at 50 °C for 3 hours. The reaction mixture was cooled to room temperature and concentrated to dryness under reduced pressure, and the concentrate was purified by preparative HPLC (column: XBRIDGE C18 19 x 150 mm 5.0 μm; mobile phase: 0.1% aqueous TFA / acetonitrile, flow rate: 15.0 mL / min) to give (S)-6,6'-((2-(((2-(2-(4-isothiocyanatophenoxy)ethoxy)ethyl)thio)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diyl)bis(methylene))dipicolinic acid (40 mg, 37%). LC-MS APCI:C 38 H 49 N5O 10 S2 calculated value: 799.29, observed m / z [M+H] + 799.9. 1 H NMR(400MHz,CD3OD):δ 8.23-8.20(m,2H),8.15-8.09(m,2H),7.74-7.71(m,2H),7.19(d,J=8.80Hz,2H),6.92(d,J=9.20Hz,2H),4.81(s,2H) ),4.77(s,2H),4.09-4.11(m,4H),3.92-3.95(m,6H),3.79(t,J=4.00Hz,3H),3.66-3.71(m,16H),2.70-2.76(m,4H).

[0457] Example 9 (S)-6,6'-((2-(((2-(2-(2-(4-isothiocyanatophenoxy)ethoxy)ethoxy)ethyl)thio)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diyl)bis(methylene))dipicolinic acid

[0458] [ka]

[0459] Scheme 8

[0460] [ka]

[0461] Scheme 8a

[0462] [ka]

[0463] Scheme 8a, Step 1a: A stir bar, tert-butyl (4-hydroxyphenyl)carbamate (3.5 g, 17 mmol), 1,2-bis(2-bromoethoxy)ethane (4.6 g, 17 mmol), KCO (4.6 g, 33 mmol), and ACN (40 mL) were placed in a 250 mL three-necked round-bottom flask and stirred at 80 °C under a nitrogen atmosphere for 48 h. The reaction mixture was cooled to room temperature, filtered through Celite®, and concentrated to dryness in vacuo to give a concentrate that was purified by silica gel chromatography (0-10% MeOH / DCM) to give tert-butyl (4-(2-(2-(2-bromoethoxy)ethoxy)ethoxy)phenyl)carbamate (4.0 g) as a brown oil.

[0464] Step 2a: A stir bar, tert-butyl (4-(2-(2-(2-bromoethoxy)ethoxy)ethoxy)phenyl)carbamate (4.0 g, 9.9 mmol), ethanethio S-acid (0.75 g, 9.9 mmol), KCO (2.7 g, 20 mmol), and ACN (50 mL) were added to a 250 mL three-neck round-bottom flask under a nitrogen atmosphere, and the reaction mixture was heated at 60 °C under a nitrogen atmosphere for 2 hours. The reaction mixture was cooled to room temperature, filtered through Celite®, concentrated to dryness in vacuo, and the concentrate purified by alumina chromatography (0 to 50% EtOAc / petroleum ether) to give S-(2-(2-(2-(4-((tert-butoxycarbonyl)amino)phenoxy)ethoxy)ethoxy)ethyl)ethanethioate (3.0 g) as a brown oil.

[0465] Step 3a: A stir bar, S-(2-(2-(2-(4-((tert-butoxycarbonyl)amino)phenoxy)ethoxy)ethoxy)ethyl)ethanethioate (3.0 g, 7.5 mmol), ethanol (50 mL), and hydrazine monohydrate (0.36 g, 0.36 mL, 11 mmol) were added to a 250 mL single-neck round-bottom flask under nitrogen and stirred at 80° C. for 1 h. The reaction mixture was cooled to room temperature, concentrated to dryness in vacuo, and the concentrate was purified by silica gel chromatography (5-10% EtOAc / petroleum ether) to give tert-butyl (4-(2-(2-(2-mercaptoethoxy)ethoxy)ethoxy)phenyl)carbamate (1.0 g) as a colorless oil.

[0466] Scheme 7, Step 1: A solution of tert-butyl (4-(2-(2-(2-mercaptoethoxy)ethoxy)ethoxy)phenyl)carbamate (0.40 g, 1.0 mmol) and DMF (3.0 mL) was added dropwise over 5 minutes to a 50 mL three-neck round-bottom flask containing a suspension of sodium hydride (0.060 g, 60% in mineral oil, 1.5 mmol) in DMF (3.0 mL) under a nitrogen atmosphere at 0° C. After the addition was complete, the reaction mixture was allowed to reach room temperature and stirred continuously for 15 minutes. The mixture was recooled to 0 °C, and a solution of dimethyl 6,6'-((2-(((methylsulfonyl)oxy)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diyl)bis(methylene))(S)-dipicolinate (0.5 g, 0.7 mmol) and DMF (3.0 mL) was added dropwise over 10 min. After the addition was complete, the reaction mixture was allowed to warm slowly to room temperature and stirred for 1.5 h. The reaction mixture was then slowly treated with saturated aqueous NH4Cl (0.2 mL) and concentrated to dryness to give an oil. The oil was purified by preparative HPLC (column: XBRIDGE C18 (19 × 150 mm) 5.0 μm, mobile phase: 0.1% TFA aqueous solution / acetonitrile, flow rate: 15.0 mL / min) to give dimethyl 6,6′-((2-(((2-(2-(2-(4-((tert-butoxycarbonyl)amino)phenoxy)ethoxy)ethoxy)ethyl)thio)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diyl)bis(methylene))(S)-dipicolinate as a brown oil (0.15 g, 21%).

[0467] Step 2: A stir bar, dimethyl 6,6'-((2-(((2-(2-(2-(4-((tert-butoxycarbonyl)amino)phenoxy)ethoxy)ethoxy)ethyl)thio)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diyl)bis(methylene))(S)-dipicolinate (0.15 mg, 0.16 mmol), MeOH (1.0 mL), and HCl in methanol (4 M, 0.80 mL, 3.2 mmol) were added to a 25 mL single-neck round-bottom flask at 0° C. The reaction mixture was warmed to room temperature and stirred for 3 h. The volatiles were removed in vacuo to give dimethyl 6,6'-((2-(((2-(2-(2-(4-aminophenoxy)ethoxy)ethoxy)ethyl)thio)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diyl)bis(methylene))(S)-dipicolinate (0.12 g), which was used in the next step without purification.

[0468] Step 3: A stir bar, dimethyl 6,6'-((2-(((2-(2-(2-(4-aminophenoxy)ethoxy)ethoxy)ethyl)thio)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diyl)bis(methylene))(S)-dipicolinate (0.12 g, 0.14 mmol), triethylamine (44 mg, 0.43 mmol), dry DCM (5 mL), and carbon disulfide (17 mg, 0.22 mmol) were added to a microwave vial under a nitrogen atmosphere at room temperature. The reaction mixture was irradiated in a microwave oven at 90°C for 30 minutes (power 150 W). The reaction mixture was then cooled to room temperature, diluted with dichloromethane (10 mL), washed successively with water (5 mL), 1 M HCl (5 mL), and water (5 mL), dried over anhydrous NaSO, and concentrated to dryness to give dimethyl 6,6'-((2-(((2-(2-(2-(4-isothiocyanatophenoxy)ethoxy)ethoxy)ethyl)thio)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diyl)bis(methylene))(S)-dipicolinate (0.12 g), which was used in the next step without purification.

[0469] Step 4: A stir bar, dimethyl 6,6'-((2-(((2-(2-(2-(4-isothiocyanatophenoxy)ethoxy)ethoxy)ethyl)thio)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diyl)bis(methylene))(S)-dipicolinate (0.12 mg, 0.14 mmol) and aqueous HCl (6 N, 0.50 mL, 2.8 mmol) were added to a 10 mL single-neck round-bottom flask and stirred at 50° C. for 3 hours. The reaction mixture was cooled to room temperature and concentrated to dryness under reduced pressure to give a residue, which was purified by preparative HPLC (column: XBRIDGE C18 19 x 150 mm 5.0 μm, mobile phase: 0.1% aqueous TFA / acetonitrile, flow rate: 15.0 mL / min) to give (S)-6,6'-((2-(((2-(2-(2-(4-isothiocyanatophenoxy)ethoxy)ethoxy)ethyl)thio)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diyl)bis(methylene))dipicolinic acid (50 mg). LC-MS APCI:C 40 H 53 N5O 11 Calculated value for S2: 843.32, observed m / z [M+H] + 843.9. 1 H NMR(400MHz,CD3OD):δ 8.24-8.21(m,2H),8.21-8.11(m,2H),7.74(d,J=7.60Hz,2H),7.23-7.20(m,2H),6.97-6.95(m,2H ),4.84-4.79(m,5H),4.14-4.12(m,4H),3.97-3.94(m,6H),3.83-3.59(m,23H),2.75-2.67(m,4H).

[0470] Example 10 6-((4-((2-(2-(2-aminoethoxy)ethoxy)ethyl)carbamoyl)phenyl)(16-((6-carboxypyridin-2-yl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-7-yl)methyl)picolinic acid

[0471] [ka]

[0472] Scheme 9

[0473] [ka]

[0474] Step 1: A stirring bar, 4-((6-(methoxycarbonyl)pyridin-2-yl)(16-((6-(methoxycarbonyl)pyridin-2-yl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-7-yl)methyl)benzoic acid (0.40 g, 0.60 mmol), tert-butyl (2-(2-(2-aminoethoxy)ethoxy)ethyl)carbamate (0.15 g, 0.60 mmol), triethylamine (0.18 g, 0.76 mmol), HATU (0.33 g, 0.90 mmol), and DCM (4.0 mL) were added to a 25 mL three-neck round-bottom flask under a nitrogen atmosphere at 0° C. The mixture was stirred overnight at room temperature, diluted with water (10 mL), and extracted with dichloromethane (10 mL × 3). The combined extracts were washed with 10% aqueous NaHCO (10 mL) and brine (10 mL), dried over anhydrous NaSO, filtered, and concentrated to dryness to give a concentrate that was purified by silica gel chromatography (0–10% MeOH / DCM) to give methyl 6-((4-((2,2-dimethyl-4-oxo-3,8,11-trioxa-5-azatridecan-13-yl)carbamoyl)phenyl)(16-((6-(methoxycarbonyl)pyridin-2-yl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-7-yl)methyl)picolinate (0.18 g).

[0475] Step 2: A stir bar, methyl 6-((4-((2,2-dimethyl-4-oxo-3,8,11-trioxa-5-azatridecan-13-yl)carbamoyl)phenyl)(16-((6-(methoxycarbonyl)pyridin-2-yl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-7-yl)methyl)picolinate (0.18 g, 0.20 mmol), MeOH (1.8 mL), and HCl in methanol (4 M, 1.0 mL, 4.0 mmol) were added to a 10 mL single-neck round-bottom flask at 0° C., then brought to room temperature and stirred for 2 h. The volatiles were removed in vacuo to give methyl 6-((4-((2-(2-(2-aminoethoxy)ethoxy)ethyl)carbamoyl)phenyl)(16-((6-(methoxycarbonyl)pyridin-2-yl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-7-yl)methyl)picolinate (0.15 g), which was used without purification.

[0476] Step 3: A stir bar, methyl 6-((4-((2-(2-(2-aminoethoxy)ethoxy)ethyl)carbamoyl)phenyl)(16-((6-(methoxycarbonyl)pyridin-2-yl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-7-yl)methyl)picolinate (0.1 g, 0.1 mmol), aqueous LiOH (3 mL, 0.1 N, 0.3 mmol), and MeOH (1.0 mL) were added to an 8 mL reaction vial at room temperature and stirred overnight. The reaction mixture was adjusted to a pH of about 6.5 with acetic acid and then concentrated to dryness under vacuum at room temperature to obtain a concentrate, which was purified by preparative HPLC (column: XBRIDGE C18 19 x 150 mm, 5.0 μm, mobile phase: 0.1% aqueous TFA / ACN, flow rate: 15.0 mL / min) to obtain 6-((4-((2-(2-(2-aminoethoxy)ethoxy)ethyl)carbamoyl)phenyl)(16-((6-carboxypyridin-2-yl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-7-yl)methyl)picolinic acid (40 mg). LC-MS APCI:C 39 H 54 NO 11Calculated m / z [M+H] 782.39; observed m / z [M+H] 783.0.

[0477] Example 11 6,6'-((18-(((2-(2-aminoethoxy)ethyl)thio)methyl)tetradecahydro-4H,13H,17H-cyclopenta[b][1,4,10,13]tetraoxa[7,16]diazacyclooctadecine-4,13-diyl)bis(methylene))dipicolinic acid and

[0478] Example 12 N-Acyl-DBCO-tagged 6,6'-((18-(((2-(2-aminoethoxy)ethyl)thio)methyl)tetradecahydro-4H,13H,17H-cyclopenta[b][1,4,10,13]tetraoxa[7,16]diazacyclooctadecine-4,13-diyl)bis(methylene))dipicolinic acid

[0479] [ka]

[0480] Scheme 10

[0481] [ka]

[0482] Step 1: A stir bar, methyl cyclopent-3-ene-1-carboxylate (25.0 g, 198 mmol), THF (600 mL), methanol (12.6 g, 16.0 mL, 397 mmol), and lithium borohydride (198 mL, 2.0 M in THF, 397 mmol) were added to a 3000 mL three-neck round-bottom flask at 0° C. After the addition was complete, the reaction mixture was stirred at 70° C. for 6 hours. The reaction mixture was then cooled to room temperature, slowly treated with ice water (250 mL), further cooled to 0° C., adjusted to pH ≈2 with 1.5 N HCl (pH ≈2), and then extracted with DCM (1000 mL × 3). The combined extracts were washed with water (500 mL), dried over anhydrous NaSO, filtered, and concentrated to dryness to obtain a concentrate. This was purified by silica gel chromatography (50-80% EtOAc / petroleum ether) to give cyclopent-3-en-1-ylmethanol (13.8 g).

[0483] Step 2: A solution of cyclopent-3-en-1-ylmethanol (13.7 g, 139 mmol) and DMF (50 mL) was added dropwise over 30 minutes to a 1000 mL three-necked round-bottom flask containing a suspension of sodium hydride (6.69 g, 60% in mineral oil, 167 mmol) in DMF (50 mL) at 0 °C under a nitrogen atmosphere. After the addition was complete, the reaction mixture was slowly warmed to room temperature and stirring was continued for 30 minutes. The mixture was then recooled to 0 °C and treated dropwise over 15 minutes with a solution of benzyl bromide (19.8 g, 167 mmol) and DMF (50 mL). After the addition was complete, the reaction mixture was slowly warmed to room temperature and then stirred for 16 hours. The reaction mixture was slowly treated with saturated NH4Cl (50 mL) and then extracted with ethyl acetate (1000 mL x 3). The combined extracts were washed with water (500 mL × 3), dried over anhydrous NaSO, filtered, and concentrated to dryness to give a concentrate that was purified by silica gel chromatography (0–20% EtOAc / petroleum ether) to give ((cyclopent-3-en-1-ylmethoxy)methyl)benzene (21.0 g).

[0484] Step 3: A stir bar, NMO (38.0 g, 50 wt% in HO, 158 mmol), THF (180 mL), and osmium tetroxide (16.2 g, 3.21 mL, 2.5 wt% in t-butanol, 0.158 mmol) were added to a 1000 mL three-neck round-bottom flask at 0 °C. The reaction mixture was brought to room temperature, stirred for 10 min, and re-cooled to 0 °C. After cooling, the mixture was treated dropwise over 15 min with a solution of ((cyclopent-3-en-1-ylmethoxy)methyl)benzene (20.0 g, 158 mmol) and THF (180 mL). The reaction was brought to room temperature and stirred for 16 h before being treated with saturated aqueous NaHCO (100 mL) and extracted with DCM (1000 mL × 3). The combined extracts were washed with water (500 mL), dried over anhydrous Na2SO4, filtered, and concentrated to dryness to give a concentrate. This was purified by silica gel chromatography (0–20% EtOAc / petroleum ether) to give 4-((benzyloxy)methyl)cyclopentane-1,2-diol as a colorless oil. The isomers were separated by SFC (apparatus: PIC 100, column: Chiralpak OXH (250 × 30) mm, 5 μm, mobile phase: CO2:0.5% isopropylamine in IPA (60:40), total flow rate: 70 g / min, back pressure: 100 bar, wavelength: 220 nm, cycle time: 8.0 min), which gave both cis-1,2 isomers of 4-((benzyloxy)methyl)cyclopentane-1,2-diol: the first-eluting isomer (10 g) and the second-eluting isomer (5 g).

[0485] Step 4: A solution of the first-eluting isomer of 4-((benzyloxy)methyl)cyclopentane-1,2-diol (10.0 g, 45.0 mmol) and DMF (60 mL) was added dropwise over 1 h to a 250 mL three-neck round-bottom flask containing a suspension of sodium hydride (8.62 g, 60% in mineral oil, 225 mmol) in DMF (60 mL) at 0 °C under a nitrogen atmosphere. After the addition was complete, the reaction mixture was allowed to warm to room temperature and stirred for 30 min. The mixture was then recooled to 0 °C and treated dropwise over 15 min with a solution of 2-(2-bromoethoxy)tetrahydro-2H-pyran (47.0 g, 225 mmol) and DMF (60 mL). After the addition was complete, the reaction mixture was slowly warmed to room temperature and stirred for 2 h. The mixture was then slowly treated with saturated aqueous NH4Cl (50 mL) and then extracted with ethyl acetate (500 mL x 3). The combined extracts were washed with water (500 mL), dried over anhydrous NaSO, filtered, and concentrated to dryness to give an oil, which was purified by silica gel chromatography (0-30% EtOAc / petroleum ether) to give 2,2'-((((4-((benzyloxy)methyl)cyclopentane-1,2-diyl)bis(oxy))bis(ethane-2,1-diyl))bis(oxy))bis(tetrahydro-2H-pyran) (21.0 g).

[0486] Step 5: A stir bar, 2,2'-((((4-((benzyloxy)methyl)cyclopentane-1,2-diyl)bis(oxy))bis(ethane-2,1-diyl))bis(oxy))bis(tetrahydro-2H-pyran) (29.0 g, 61.0 mmol), MeOH (200 mL), and HCl in 1,4-dioxane (4 M, 3.0 mL, 12.0 mmol) were added to a 1000 mL three-neck round-bottom flask and then heated to reflux for 1 h. The flask was then cooled to room temperature and the volatiles were removed under vacuum to give 2,2'-((4-((benzyloxy)methyl)cyclopentane-1,2-diyl)bis(oxy))bis(ethan-1-ol) (20.0 g) as a residue, which was used without purification.

[0487] Step 6: Under a nitrogen atmosphere, a 1000 mL round-bottom flask was charged with a stir bar, 2,2'-((4-((benzyloxy)methyl)cyclopentane-1,2-diyl)bis(oxy))bis(ethan-1-ol) (20.0 g) (20.0 g, 64.4 mmol), DCM (200 mL), and triethylamine (32.6 mL, 322 mmol), and the resulting mixture was cooled to 10°C. The mixture was then treated with pTsCl (36.9 g, 193 mmol) added portionwise and then allowed to reach room temperature. After the addition was complete, the reaction mixture was stirred for 16 h, during which time a precipitate formed. The mixture was then diluted with DCM (500 mL), washed with cold aqueous HCl (1 M, 500 mL × 3) and ice-cold water (500 mL × 2), dried over anhydrous NaSO, filtered, and concentrated to dryness to give a residue which was purified by silica gel chromatography (0-30% EtOAc / petroleum ether) to give ((4-((benzyloxy)methyl)cyclopentane-1,2-diyl)bis(oxy))bis(ethane-2,1-diyl)bis(4-methylbenzenesulfonate) (26.0 g).

[0488] Step 7: A stir bar, N,N'-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))bis(4-methylbenzenesulfonamide) (21.0 g, 42.0 mmol), CsCO (41.3 g, 126 mmol), and dry DMF (250 mL) were added to a 2000 mL three-neck round-bottom flask under a nitrogen atmosphere, and the resulting heterogeneous mixture was stirred at room temperature for 1.5 h. The mixture was then treated dropwise over 2 h with a solution of ((4-((benzyloxy)methyl)cyclopentane-1,2-diyl)bis(oxy))bis(ethane-2,1-diyl)bis(4-methylbenzenesulfonate) (26.0 g, 42.0 mmol) and DMF (250 mL). Stirring was continued for 20 h, after which the mixture was concentrated to dryness in vacuo to give a pasty solid. The paste was suspended in DCM (1000 mL), stirred for 30 min, and filtered by vacuum filtration. The filtrate was concentrated to dryness under vacuum to give a concentrate that was purified by silica gel chromatography (0–40% EtOAc / petroleum ether) to give 18-((benzyloxy)methyl)-4,13-ditosyltetradecahydro-2H,11H,17H-cyclopenta[b][1,4,10,13]tetraoxa[7,16]diazacyclooctadecine (24 g).

[0489] Step 8: A solution of HBr in HOAc (50%, 112 mL, 695 mmol) was added to a 500 mL round-bottom flask containing a stir bar and 18-((benzyloxy)methyl)-4,13-ditosyltetradecahydro-2H,11H,17H-cyclopenta[b][1,4,10,13]tetraoxa[7,16]diazacyclooctadecine (24.0 g, 32.8 mmol) under a nitrogen atmosphere. The mixture was stirred at room temperature until homogeneous and then treated with phenol (16.3 g, 174 mmol). The reaction mixture was then heated at 60° C. for 6 hours, cooled to room temperature, and concentrated to dryness in vacuo to give a concentrate. The concentrate was purified by reverse-phase column chromatography (column: Revelries C18-330 g, mobile phase A: 0.1% TFA aqueous solution, mobile phase B: acetonitrile, flow rate: 60 mL / min) to give methyl (tetradecahydro-2H,11H,17H-cyclopenta[b][1,4,10,13]tetraoxa[7,16]diazacyclooctadecin-18-yl)acetate (8.0 g).

[0490] Step 9: A stir bar, (tetradecahydro-2H,11H,17H-cyclopenta[b][1,4,10,13]tetraoxa[7,16]diazacyclooctadecin-18-yl)methyl acetate (8.0 g, 21 mmol), methyl 6-(chloromethyl)picolinate (12.2 g, 53.2 mmol), NaCO (11.1 g, 106 mmol), and acetonitrile (100 mL) were added to a 500 mL three-neck round-bottom flask under a nitrogen atmosphere, and the resulting heterogeneous mixture was heated at 90° C. for 16 hours under a nitrogen atmosphere. The resulting mixture was then cooled to room temperature and filtered through a pad of Celite®, and the filtrate was concentrated to dryness in vacuo to give a concentrate. The concentrate was subjected to silica gel chromatography (0–10% MeOH / DCM) to give dimethyl 6,6′-((18-(acetoxymethyl)tetradecahydro-4H,13H,17H-cyclopenta[b][1,4,10,13]tetraoxa[7,16]diazacyclooctadecine-4,13-diyl)bis(methylene))dipicolinate (5.0 g).

[0491] Step 10: To a 250 mL round-bottom flask under a nitrogen atmosphere was added a stir bar, dimethyl 6,6'-((18-(acetoxymethyl)tetradecahydro-4H,13H,17H-cyclopenta[b][1,4,10,13]tetraoxa[7,16]diazacyclooctadecine-4,13-diyl)bis(methylene))dipicolinate (5.0 g, 7.4 mmol), KCO (0.10 g, 0.74 mmol), and methanol (50 mL), and the resulting mixture was stirred at room temperature for 10 minutes. The mixture was then concentrated to dryness under vacuum, and the resulting residue was purified by silica gel chromatography (0–10% MeOH / DCM) to give 6,6′-((18-(hydroxymethyl)tetradecahydro-4H,13H,17H-cyclopenta[b][1,4,10,13]tetraoxa[7,16]diazacyclooctadecine-4,13-diyl)bis(methylene))dipicolinate (3.0 g).

[0492] Step 11: A stir bar, 6,6'-((18-(hydroxymethyl)tetradecahydro-4H,13H,17H-cyclopenta[b][1,4,10,13]tetraoxa[7,16]diazacyclooctadecine-4,13-diyl)bis(methylene))dipicolinate (2.0 g, 3.1 mmol), DCM (20 mL), and triethylamine (1.2 g, 9.5 mmol) were added to a 100 mL three-neck round-bottom flask under a nitrogen atmosphere, and the resulting mixture was cooled to 10° C. The mixture was treated portionwise with MsCl (0.48 g, 6.3 mmol), and after the addition was complete, the reaction vessel was allowed to reach room temperature and stirred for 30 minutes, during which time a precipitate formed. The heterogeneous mixture was diluted with DCM (50 mL), washed with cold aqueous HCl (1 M, 50 mL × 3) and ice-cold water (50 mL × 2), dried over anhydrous NaSO, filtered, and concentrated to dryness to give a gummy solid. The gummy solid was purified by neutral alumina column chromatography (0–10% MeOH / DCM) to give dimethyl 6,6′-((18-(((methylsulfonyl)oxy)methyl)tetradecahydro-4H,13H,17H-cyclopenta[b][1,4,10,13]tetraoxa[7,16]diazacyclooctadecine-4,13-diyl)bis(methylene))dipicolinate (1.5 g).

[0493] Step 12: A solution of dimethyl 6,6'-((18-(((methylsulfonyl)oxy)methyl)tetradecahydro-4H,13H,17H-cyclopenta[b][1,4,10,13]tetraoxa[7,16]diazacyclooctadecine-4,13-diyl)bis(methylene))dipicolinate (0.69 g, 3.2 mmol) and DMF (5 mL) was added dropwise over 5 minutes to a 25 mL three-neck round-bottom flask containing a suspension of sodium hydride (162 mg, 60% in mineral oil, 4.22 mmol) in DMF (0.5 mL) under a nitrogen atmosphere at 0°C. After the addition was complete, the reaction mixture was allowed to warm to room temperature and stirred for 15 minutes. The reaction mixture was then recooled to 0°C and treated dropwise over 5 minutes with a solution of tert-butyl (2-(2-mercaptoethoxy)ethyl)carbamate (1.50 g, 2.11 mmol) and DMF (3 mL). After the addition was complete, the reaction mixture was allowed to warm slowly to room temperature and then stirred for 1 hour. The reaction was then slowly treated with saturated NH4Cl and then extracted with ethyl acetate (3 x 10 mL). The combined extracts were washed with water (10 mL), dried over anhydrous Na2SO4, filtered, and concentrated to dryness to give an oil. The oil was purified by preparative HPLC (column: XBRIDGE C18 19 × 150 m) 5.0 μm, mobile phase: 0.1% TFA aqueous solution / acetonitrile, flow rate: 15.0 mL / min) to give cyclopenta[b][1,4,10,13]tetraoxa[7,16]diazacyclooctadecine-4,13-diyl)bis(methylene))dipicolinate (0.2 g).

[0494] Step 13: A stir bar, cyclopenta[b][1,4,10,13]tetraoxa[7,16]diazacyclooctadecine-4,13-diyl)bis(methylene))dipicolinate (0.20 g, 0.24 mmol), MeOH (1.0 mL), and HCl in methanol (4 M, 1.2 mL, 4.8 mmol) were added to a 25 mL single-neck round-bottom flask at 0° C., and the resulting mixture was allowed to warm to room temperature and stirred for 2 h. The volatiles were removed in vacuo to give dimethyl 6,6'-((18-(((2-(2-aminoethoxy)ethyl)thio)methyl)tetradecahydro-4H,13H,17H-cyclopenta[b][1,4,10,13]tetraoxa[7,16]diazacyclooctadecine-4,13-diyl)bis(methylene))dipicolinate (150 mg), which was used without purification.

[0495] Step 14: A stir bar, dimethyl 6,6'-((18-(((2-(2-aminoethoxy)ethyl)thio)methyl)tetradecahydro-4H,13H,17H-cyclopenta[b][1,4,10,13]tetraoxa[7,16]diazacyclooctadecine-4,13-diyl)bis(methylene))dipicolinate (40 mg, 0.054 mmol), aqueous LiOH (1.6 mL, 0.1 N, 0.16 mmol), and MeOH (0.5 mL) were added to an 8 mL reaction vial at room temperature, and the resulting mixture was stirred overnight. The pH of the reaction mixture was adjusted to about pH 6.5 with acetic acid, then concentrated to dryness under vacuum at room temperature, and the resulting concentrate was purified by preparative HPLC (column: XBRIDGE C18 19 x 150 mm 5.0 μm, mobile phase: 10 mM aqueous ammonium acetate / ACN, flow rate: 15.0 mL / min) to give Example 11: 6,6'-((18-(((2-(2-aminoethoxy)ethyl)thio)methyl)tetradecahydro-4H,13H,17H-cyclopenta[b][1,4,10,13]tetraoxa[7,16]diazacyclooctadecine-4,13-diyl)bis(methylene))dipicolinic acid (23 mg). LC-MS APCI:C 34 H 51 Calculated for N5O9S, No. 705.34; Observed m / z [M+H] + 706.4.

[0496] Step 15: A stir bar, dimethyl 6,6'-((18-(((2-(2-aminoethoxy)ethyl)thio)methyl)tetradecahydro-4H,13H,17H-cyclopenta[b][1,4,10,13]tetraoxa[7,16]diazacyclooctadecine-4,13-diyl)bis(methylene))dipicolinate (70 mg, 0.95 mmol), 11,12-didehydro-γ-oxodibenz[b,f]azocine-5(6H)-butyric acid (29 mg, 0.95 mmol), triethylamine (29 mg, 0.76 mmol), HATU (54 mg, 0.14 mmol), and DCM (0.5 mL) were added to a 25 mL three-neck round-bottom flask under a nitrogen atmosphere at 0° C. The resulting mixture was allowed to warm to room temperature and stirred overnight. The reaction mixture was diluted with water (10 mL) and extracted with dichloromethane (10 mL × 3). The combined extracts were washed with 10% aqueous NaHCO (10 mL), brine (10 mL), dried over anhydrous NaSO, filtered, and concentrated to dryness to give an oil. The oil was purified by silica gel chromatography (0–10% MeOH / DCM) to give N-acyl-DBCO-tagged dimethyl 6,6′-((18-(((2-(2-aminoethoxy)ethyl)thio)methyl)tetradecahydro-4H,13H,17H-cyclopenta[b][1,4,10,13]tetraoxa[7,16]diazacyclooctadecine-4,13-diyl)bis(methylene))dipicolinate (10 mg).

[0497] Step 16: A stir bar, N-acyl-DBCO-tagged dimethyl 6,6'-((18-(((2-(2-aminoethoxy)ethyl)thio)methyl)tetradecahydro-4H,13H,17H-cyclopenta[b][1,4,10,13]tetraoxa[7,16]diazacyclooctadecine-4,13-diyl)bis(methylene))dipicolinate (10 mg, 0.01 mmol), aqueous LiOH (0.3 mL, 0.1 N, 0.03 mmol), and methanol (0.25 mL) were added to an 8 mL reaction vial at room temperature, and the resulting mixture was stirred overnight. The reaction mixture was adjusted to pH 6.5 with acetic acid and concentrated to dryness under reduced pressure at room temperature. The resulting concentrate was purified by preparative HPLC (column: XBRIDGE C18 (19 × 150 mm) 5.0 μm, mobile phase: 10 mM aqueous ammonium acetate / ACN, flow rate: 15.0 mL / min).

[0498] Example 12: N-Acyl-DBCO tagged 6,6'-((18-(((2-(2-aminoethoxy)ethyl)thio)methyl)tetradecahydro-4H,13H,17H-cyclopenta[b][1,4,10,13]tetraoxa[7,16]diazacyclooctadecine-4,13-diyl)bis(methylene))dipicolinic acid (3 mg). LC-MS APCI:C 53 H 64 NO 11 Calculated S, 992.44; observed m / z [M−H] - :991.4.

[0499] Example 13 6-((16-(1-(6-carboxypyridin-2-yl)-8-isothiocyanatooctyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-7-yl)methyl)picolinic acid

[0500] [ka]

[0501] Scheme 11

[0502] [ka]

[0503] Step 1: A 500 mL three-neck round-bottom flask purged and maintained with an inert atmosphere of nitrogen was charged with a solution of 8-((tert-butoxycarbonyl)amino)octanoic acid (20.0 g, 77.1 mmol) in dichloromethane (200 mL), N,O-dimethylhydroxylamine (7.0 g, 115 mmol), and diisopropylethylamine (29.90 g, 231 mmol). Then, with stirring at 0 °C, HATU (43.9 g, 115 mmol) was added. The resulting solution was stirred at room temperature for 1 h. The reaction was then quenched by the addition of 200 mL of water. The resulting solution was extracted with dichloromethane (100 mL × 2). The combined organic layer was washed sequentially with HCl (1 M) (300 mL × 2), aqueous NH4CO3 (400 mL × 3), and brine (400 mL). After drying over anhydrous Na2SO4, concentration gave tert-butyl (8-(methoxy(methyl)amino)-8-oxooctyl)carbamate as a pale yellow oil (15.4 g, 66% yield).

[0504] Step 2: A 500 mL three-neck round-bottom flask purged and maintained with an inert atmosphere of nitrogen was charged with a solution of 2,6-dibromopyridine (23.0 g, 927 mmol) in THF (400 mL). This was cooled to −78° C., and n-BuLi (60.4 mL, 927 mmol) was added dropwise quickly. After stirring for 10 minutes, tert-butyl (8-(methoxy(methyl)amino)-8-oxooctyl)carbamate (14.0 g, 463.5 mmol) in THF (40 mL) was added dropwise with stirring at −78° C. The resulting solution was stirred at room temperature for 30 minutes. The reaction was quenched by the addition of 500 mL of water. The resulting solution was extracted with ethyl acetate (200 mL × 2). The combined organic layers were washed with brine (400 mL), dried over anhydrous sodium sulfate, and concentrated to dryness to give the crude product. Silica gel chromatography (0-10% ethyl acetate in petroleum ether) gave tert-butyl (8-(6-bromopyridin-2-yl)-8-oxooctyl)carbamate as a pale yellow solid (11.8 g, 50% yield).

[0505] Step 3: A 1 L high-pressure reaction vessel maintained under an inert nitrogen atmosphere was charged with tert-butyl (8-(6-bromopyridin-2-yl)-8-oxooctyl)carbamate (11.5 g, 28.8 mmol, 1.0 equiv.) in MeOH (500 mL), followed by Pd(dppf)Cl (2.1 g, 2.88 mmol) and TEA (8.7 g, 86.4 mmol). CO (20 atm) was then introduced. The resulting solution was stirred at 100 °C for 16 h. The reaction solution was filtered and used directly in the next step.

[0506] Step 4: The above MeOH solution was cooled to 0 °C, and NaBH (1.08 g, 28.8 mmol) was added. The resulting solution was stirred at room temperature for 1 h. The reaction was quenched by the addition of 500 mL of aqueous NH CO and extracted with ethyl acetate (300 mL × 2). The combined organic layers were washed with brine (600 mL), dried over anhydrous Na SO , and concentrated to give methyl 6-(8-((tert-butoxycarbonyl)amino)-1-hydroxyoctyl)picolinate as a brown oil (10 g).

[0507] Step 5: A 250 mL three-necked round-bottom flask purged and maintained under an inert atmosphere of nitrogen was charged with a solution of methyl 6-(8-((tert-butoxycarbonyl)amino)-1-hydroxyoctyl)picolinate (10 g) in DCM (100 mL). After cooling to 0° C., TEA (7.9 g, 78.9 mmol) and mesyl chloride (3.6 g, 31.5 mmol) were added. The resulting solution was stirred at room temperature for 1 h. The mixture was concentrated in vacuo. MeCN (100 mL) was added and concentrated in vacuo. The crude product, methyl 6-(8-((tert-butoxycarbonyl)amino)-1-((methylsulfonyl)oxy)octyl)picolinate, was used directly in the next step.

[0508] Step 6: To a solution of the above crude product, methyl 6-(8-((tert-butoxycarbonyl)amino)-1-((methylsulfonyl)oxy)octylpicolinate, in ACN (100 mL) was added NaI (4.3 g, 28.9 mmol). The resulting solution was stirred at 80° C. for 1 h. The mixture was filtered and concentrated. The crude product was purified by flash preparative HPLC (column C18, mobile phase, HO / ACN=50 / 50% to HO / ACN=20 / 80% in 30 min). 4 g of methyl 6-(8-((tert-butoxycarbonyl)amino)-1-iodooctyl)picolinate was obtained as a brown oil.

[0509] Step 7: To a solution of methyl 6-(8-((tert-butoxycarbonyl)amino)-1-iodooctyl)picolinate (3.0 g, 6.12 mmol) in DCM (200 mL) was added methyl 6-((1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-7-yl)methyl)picolinate (3.0 g, 7.34 mmol), diisopropylethylamine (3.9 g, 30.61 mmol). The resulting solution was diluted with 80 mL of HCl. ℃The mixture was stirred at rt for 16 hours. The reaction was concentrated. The crude product was purified by flash preparative HPLC (column C18, mobile phase, A: HO (0.05% TFA), B: CAN, eluting from 20% B to 40% B in 20 min), which gave 1.9 g of methyl 6-(8-((tert-butoxycarbonyl)amino)-1-(16-((6-(methoxycarbonyl)pyridin-2-yl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-7-yl)octyl)picolinate as a brown oil.

[0510] Step 8: To a stirred solution of methyl 6-(8-((tert-butoxycarbonyl)amino)-1-(16-((6-(methoxycarbonyl)pyridin-2-yl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-7-yl)octyl)picolinate (1.7 g, 2.19 mmol, 77% by LCMS) in DCM (8.5 mL) at 0 °C was added HCl / dioxane dropwise. The resulting solution was stirred at room temperature for 1 h. The reaction was quenched by the portionwise addition of aqueous NH4CO3 (20 mL × 3). The resulting solution was extracted with dichloromethane (100 mL × 2). The combined organic layers were washed with brine (400 mL), dried over anhydrous NaSO, and concentrated to give 1.3 g of methyl 6-(8-amino-1-(16-((6-(methoxycarbonyl)pyridin-2-yl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-7-yl)octyl)picolinate as a brown oil.

[0511] Step 9: To a solution of methyl 6-(8-amino-1-(16-((6-(methoxycarbonyl)pyridin-2-yl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-7-yl)octyl)picolinate (1.0 g, 1.48 mmol) in DCM (17 mL) under N was added 1,1′-thiocarbonylbis(pyridin-2(1H)-one) (0.38 g, 1.63 mmol). The resulting solution was stirred at room temperature for 1 hour. This was concentrated to give 1.6 g of methyl 6-((16-(1-(6-(methoxycarbonyl)pyridin-2-yl)-8-thiocyanatooctyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-7-yl)methyl)picolinate as a brown oil.

[0512] Step 10: To a solution of methyl 6-((16-(1-(6-(methoxycarbonyl)pyridin-2-yl)-8-thiocyanatooctyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-7-yl)methyl)picolinate (1.40 g, 1.42 mmol) in ACN (4 mL) was added HCl (6 M) (7 mL). The resulting solution was stirred in an oil bath at 50 °C for 5 h. It was diluted with 10 mL of HO. The crude product was purified by flash preparative HPLC (column, C18, mobile phase, A: HO (0.05% TFA), B: ACN, 20% B to 36% B in 20 min, detector UV @ 210 nm). The product fractions were concentrated to remove ACN. The aqueous phase was adjusted to pH 7-8 with aqueous NaHCO3. It was again purified by flash-preparative HPLC (column, C18, mobile phase, A: HO, B: ACN, 95% B to 100% B in 20 min). The product solution was concentrated to remove CAN and then lyophilized. This gave 190 mg of 6-((16-(1-(6-carboxypyridin-2-yl)-8-thiocyanatooctyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-7-yl)methyl)picolinic acid as a brown solid. 1H NMR(300MHz,D2O)δ 7.91(s,4H),7.54(s,2H),4.52(d,J=17.9Hz,3H),3.77(d,J=9.3Hz,8H), 3.56-3.41(m,18H),2.11(s,2H),1.51(s,2H),1.17(s,7H),0.97(s,1H). MS(ES,m / z):688.3(M+H + ).

[0513] Example 14 6-((16-(1-(6-carboxypyridin-2-yl)-2-(2-(2-isothiocyanatoethoxy)ethoxy)ethyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-7-yl)methyl)picolinic acid

[0514] [ka]

[0515] Scheme 12

[0516] [ka]

[0517] Step 1: Under a nitrogen atmosphere, to a stirred solution of 2,2-dimethyl-4-oxo-3,8,11-trioxa-5-azatridecan-13-oic acid (10.00 g, 37.98 mmol) and diisopropylethylamine (14.73 g, 113.94 mmol) in dichloromethane (100 mL) at 0 °C, [bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (15.16 g, 39.88 mmol) and N,O-dimethylhydroxylamine (5.55 g, 56.97 mmol) were added dropwise. The resulting mixture was stirred at room temperature for 1 h and then poured into saturated aqueous NH₄Cl. The resulting mixture was extracted with dichloromethane (100 mL × 2). The combined organic layers were washed with brine and dried over anhydrous Na₂SO₄. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by chromatography (column, C18, mobile phase A: HO:0.05% TFA, B: ACN, gradient from 20% B to 40% B in 20 min, detector: UV @ 210 nm). tert-Butyl (3-methyl-4-oxo-2,6,9-trioxa-3-azaundecan-11-yl)carbamate was obtained as a pale yellow oil (9.90 g, 85% yield).

[0518] Step 2: Under a nitrogen atmosphere, n-BuLi (28.0 mL, 56.1 mmol) was added dropwise to a solution of 2,6-dibromopyridine (13.3 g, 56.1 mmol) in THF (260 mL) in a 500 mL three-necked round-bottom flask at −78° C. The solution was stirred at −78° C. for 10 minutes. A solution of tert-butyl (3-methyl-4-oxo-2,6,9-trioxa-3-azoundecan-11-yl)carbamate (7.0 g, 28.0 mmol) in THF (30 mL) was added dropwise to the reaction mixture at −78° C. and stirred at room temperature for 30 minutes. The reaction was quenched by the addition of water / ice (200 mL) at 0° C. The aqueous layer was extracted with ethyl acetate (100 mL × 3). The combined extracts were dried over Na2SO4 and concentrated in vacuo. The residue was purified by chromatography (column, C18, mobile phase, mobile phase A: HO: 0.05% TFA, B: ACN, gradient from 38% B to 58% B in 20 min, detector: UV @ 210 nm). tert-Butyl (2-(2-(2-(6-bromopyridin-2-yl)-2-oxoethoxy)ethoxy)ethyl)carbamate was obtained as a yellow solid (4.6 g, 50% yield). MS (ES, m / z): 425, 427 (M+Na + ).

[0519] Step 3: To a 250 mL high-pressure reactor was added tert-butyl (2-(2-(2-(6-bromopyridin-2-yl)-2-oxoethoxy)ethoxy)ethyl)carbamate (4.0 g, 18.1 mmol), triethylamine (5.5 g, 54.3 mmol), Pd(dppf)Cl (1.3 g, 1.8 mmol), and MeOH (40 mL). The reaction solution was degassed and filled with N. Then, CO (10 atm) was introduced. The resulting solution was stirred at 100 °C overnight. The reaction mixture was filtered, and the filtrate was concentrated to dryness. The residue was purified by chromatography (column, C18, mobile phase A: HO:0.05% TFA, B: ACN, gradient from 38% B to 58% B in 20 min, detector: UV @ 210 nm). Methyl 6-(2,2-dimethyl-4-oxo-3,8,11-trioxa-5-azatridecan-13-oyl)picolinate was obtained as a brown oil (2.4 g, 63% yield). MS (ES, m / z): 405 (M+Na+ ).

[0520] Step 4: To a solution of methyl 6-(2,2-dimethyl-4-oxo-3,8,11-trioxa-5-azatridecan-13-yl)picolinate (2.30 g, 6.01 mmol) in MeOH (46 mL) at 0 °C under a N atmosphere was added NaBH (0.23 g, 6.01 mmol). The resulting solution was stirred at room temperature for 1 h and quenched by adding 50 mL of saturated aqueous NHHCO solution. The resulting solution was extracted with ethyl acetate (30 mL × 2). The combined organic layers were washed with brine (60 mL), dried over NaSO, and concentrated. 2.2 g of crude product, methyl 6-(13-hydroxy-2,2-dimethyl-4-oxo-3,8,11-trioxa-5-azatridecan-13-yl)picolinate, was obtained as a brown oil.

[0521] Step 5: To a solution of methyl 6-(13-hydroxy-2,2-dimethyl-4-oxo-3,8,11-trioxa-5-azatridecan-13-yl)picolinate (2.2 g, 5.72 mmol) in dichloromethane (22 mL) was added triethylamine (1.74 g, 17.16 mmol) and MsCl (0.79 g, 6.86 mmol) under a N atmosphere at 0 °C. The resulting solution was stirred at room temperature for 1 hour and quenched with HO (22 mL). The resulting mixture was extracted with dichloromethane (20 mL × 2). The combined organic layers were washed with brine (40 mL), dried over NaSO, and concentrated. 2.2 g of crude product methyl 6-(2,2-dimethyl-13-((methylsulfonyl)oxy)-4-oxo-3,8,11-trioxa-5-azatridecan-13-yl)picolinate was obtained as a brown oil.

[0522] Step 6: Under a N2 atmosphere, to a solution of methyl 6-(2,2-dimethyl-13-((methylsulfonyl)oxy)-4-oxo-3,8,11-trioxa-5-azatridecan-13-yl)picolinate (2.2 g, 4.75 mmol) in ACN (22 mL) was added NaI (0.78 g, 5.23 mmol). The resulting solution was stirred at 80 °C for 1 h. The mixture was filtered and concentrated. The crude product was purified by chromatography (column, C18, mobile phase A: HO, B: ACN, gradient from 50% B to 80% B in 30 min, detector: UV @ 210 nm). Methyl 6-(13-iodo-2,2-dimethyl-4-oxo-3,8,11-trioxa-5-azatridecan-13-yl)picolinate was obtained as a brown oil (1.2 g). MS (ES, m / z): 517 (M+Na + ), 495(M+H + ).

[0523] Step 7: A solution of methyl 6-(13-iodo-2,2-dimethyl-4-oxo-3,8,11-trioxa-5-azatridecan-13-yl)picolinate (840 mg, 1.69 mmol) and methyl 6-(1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-7-yl)methyl)picolinate (839 mg, 2.03 mmol) in ACN (16.8 mL) was stirred overnight at 80 °C under a nitrogen atmosphere. The cooled reaction mixture was filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by chromatography (C18 column, mobile phase A: HO, B: ACN, gradient from 40% B to 60% B in 20 min, detector: UV @ 210 nm). Methyl 6-((16-(13-(6-(methoxycarbonyl)pyridin-2-yl)-2,2-dimethyl-4-oxo-3,8,11-trioxa-5-azatridecan-13-yl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-7-yl)methyl)picolinate was obtained as a brown oil (450 mg). MS (ES, m / z): 700 (M+Na + ), 678(M+H + ).

[0524] Step 8: To a solution of methyl 6-((16-(13-(6-(methoxycarbonyl)pyridin-2-yl)-2,2-dimethyl-4-oxo-3,8,11-trioxa-5-azatridecan-13-yl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-7-yl)methyl)picolinate (450 mg, 579 mmol) in dichloromethane (2.5 mL) at 0 °C, HCl / dioxane (2.5 mL, 4 M) was added. The resulting solution was stirred at room temperature for 20 minutes. The reaction was quenched by adding saturated aqueous Na2CO3 solution. The aqueous layer was extracted with DCM:IPA (5:1) (30 mL × 2). The combined organic layers were dried over anhydrous NaSO and concentrated under reduced pressure to give crude product methyl 6-(2-(2-(2-aminoethoxy)ethoxy)-1-(16-((6-(methoxycarbonyl)pyridin-2-yl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-7-yl)ethyl)picolinate (330 mg). This crude product was used directly in the next step.

[0525] Step 9: A solution of methyl 6-(2-(2-(2-aminoethoxy)ethoxy)-1-(16-((6-(methoxycarbonyl)pyridin-2-yl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-7-yl)ethyl)picolinate (300 mg, 0.44 mmol) and 1-(2-oxopyridine-1-carbothioyl)pyridin-2-one (113.08 mg, 0.48 mmol) in dichloromethane (3 mL) was stirred at room temperature under a nitrogen atmosphere for 1 hour. The resulting mixture was concentrated under reduced pressure to give crude product methyl 6-(2-(2-(2-isothiocyanatoethoxy)ethoxy)-1-(16-((6-(methoxycarbonyl)pyridin-2-yl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-7-yl)ethyl)picolinate (380 mg). This crude product was used directly in the next step.

[0526] Step 10: A solution of methyl 6-(2-(2-(2-isothiocyanatoethoxy)ethoxy)-1-(16-((6-(methoxycarbonyl)pyridin-2-yl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-7-yl)ethyl)picolinate (380 mg, 0.52 mmol) and HCl (1.9 mL, 6 M) in dichloromethane (1.9 mL) was stirred at 50 °C under nitrogen atmosphere for 3 h. The resulting mixture was concentrated under reduced pressure and basified to pH 6-7 with saturated aqueous NaHCO. The residue was purified by chromatography (C18 column, mobile phase A: HO with 0.05% TFA, B: ACN, gradient from 20% B to 36% B in 20 min, detector: UV @ 210 nm). The product fractions were then concentrated under vacuum to remove MeCN. The solution was again purified by chromatography (C18 column, mobile phase A: HO, B: ACN, gradient 95% B to 100% B (20 min)). The solution was concentrated to remove most of the MeCN, and the aqueous solution was lyophilized to give 6-((16-(1-(6-carboxypyridin-2-yl)-2-(2-(2-isothiocyanatoethoxy)ethoxy)ethyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-7-yl)methyl)picolinic acid as a brown solid (130 mg). 1 H NMR(300MHz,D2O)8.03-7.84(m,2H),7.57(dd,J=22.3,7.4Hz,1H),5.00(s,0H),4.59(s,1H) ,4.20(dd,J=23.4,9.5Hz,1H),3.82(d,J=15.4Hz,4H),3.70-3.58(m,6H),3.58-3.49(m,6H). MS(ES, m / z):692.3(M+H + ).

[0527] Example 15 6,6'-(((S-2-(((5-(((((1R,8S,9r-bicyclo[6.1.0]non-4-yn-9-yl)methoxy)carbonyl)amino)pentyl)thio)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diyl)bis(methylene))dipicolinic acid

[0528] [ka]

[0529] Scheme 13

[0530] [ka]

[0531] Step 1: At 0° C. under a nitrogen atmosphere, a solution of tert-butyl (5-mercaptopentyl)carbamate (0.30 g, 1.0 mmol) and DMF (3.0 mL) was added dropwise over 5 minutes to a 50 mL three-neck round-bottom flask containing a suspension of sodium hydride (0.07 g, 60% in mineral oil, 2 mmol) in DMF (3.0 mL). After the addition was complete, the reaction mixture was allowed to reach room temperature and stirring was continued for 15 minutes. The reaction mixture was then cooled back to 0°C and treated dropwise over 10 min with a solution of dimethyl 6,6'-((2-(((methylsulfonyl)oxy)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diyl)bis(methylene))(S-dipicolinate (0.6 g, 0.9 mmol) and DMF (3.0 mL). After the addition was complete, the reaction mixture was allowed to warm slowly to room temperature and stirring was continued for 1.5 h. The reaction mixture was then carefully treated with saturated aqueous NH4Cl (1.0 mL) and concentrated to dryness to give an oil. The oil was purified by preparative HPLC (column: XBRIDGE C18 19 × 150 mm, 5.0 μm, mobile phase: 0.1% Purification with aqueous TFA / acetonitrile (flow rate: 15.0 mL / min) gave dimethyl 6,6'-((2-(((5-((tert-butoxycarbonyl)amino)pentyl)thio)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diyl)bis(methylene))(S)-dipicolinate (0.25 g).

[0532] Step 2: A stir bar, dimethyl 6,6′-((2-(((5-((tert-butoxycarbonyl)amino)pentyl)thio)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diyl)bis(methylene))(S)-dipicolinate (0.25 g, 0.32 mmol), MeOH (1.0 mL), and HCl in methanol (4 M, 1.5 mL, 6.3 mmol) were added to a 25 mL round-bottom flask at 0° C., which was then allowed to reach room temperature, and the mixture was stirred for 3 h. The volatiles were then removed under reduced pressure to give dimethyl 6,6'-((2-(((5-aminopentyl)thio)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diyl)bis(methylene))(S)-dipicolinate (0.21 g), which was used without purification.

[0533] Step 3: Stir bar, dimethyl 6,6'-((2-(((5-aminopentyl)thio)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diyl)bis(methylene))(S-dipicolinate (0.15 g, 0.22 mmol), ((1R,8S,9r)-bicyclo[6.1.0]non-4-yn-9-yl)methyl 4-nitrophenyl carbonate (68 mg, 0.22 mmol), triethylamine (66 mg, 0.65 mmol), and a mixture of DCM (2 mL) and DMF (0.1 mL). was added to a 25 mL three-neck round-bottom flask at 0° C. under a nitrogen atmosphere. The resulting solution was allowed to warm slowly to room temperature and stirred overnight. The mixture was then concentrated to dryness to give dimethyl 6,6'-(((S-2-(((5-((((1R,8S,9r)-bicyclo[6.1.0]non-4-yn-9-yl)methoxy)carbonyl)amino)pentyl)thio)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diyl)bis(methylene))dipicolinate (0.1 g), which was used without purification.

[0534] Step 4: A stir bar, dimethyl 6,6'-(((S-2-(((5-(((((1R,8S,9r)-bicyclo[6.1.0]non-4-yn-9-yl)methoxy)carbonyl)amino)pentyl)thio)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diyl)bis(methylene))dipicolinate (0.10 g, 0.12 mmol), aqueous LiOH (3.5 mL, 0.1 N, 0.35 mmol), and MeOH (0.5 mL) were added to an 8 mL reaction vial, and the resulting mixture was stirred at room temperature overnight. The reaction mixture was then treated with acetic acid until the pH reached approximately 6.5, and then concentrated to dryness in vacuo at room temperature to give an oil, which was purified by preparative HPLC (column: XBRIDGE C18 Purification using a 19 × 150 mm column, 5.0 μm column, mobile phase: 0.1% formic acid / ACN in HO, flow rate: 15.0 mL / min) gave 6,6'-(((S)-2-(((5-((((1R,8S,9r)-bicyclo[6.1.0]non-4-yn-9-yl)methoxy)carbonyl)amino)pentyl)thio)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diyl)bis(methylene))dipicolinic acid (42 mg).

[0535] Example 16 N-Acyl-DBCO-tagged 6,6'-((2-(((5-aminopentyl)thio)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diyl)bis(methylene))(S)-dipicolinic acid

[0536] [ka]

[0537] Scheme 14

[0538] [ka]

[0539] Step 1: A stir bar, dimethyl 6,6'-((2-(((5-((tert-butoxycarbonyl)amino)pentyl)thio)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diyl)bis(methylene))(S)-dipicolinate (0.12 g, 0.15 mmol), MeOH (0.5 mL), and HCl in methanol (4 M, 0.75 mL, 3.0 mmol) were added to a 25 mL round-bottom flask at 0°C, then brought to room temperature and stirred for 2 hours. The volatiles were removed in vacuo to give dimethyl 6,6'-((2-(((5-aminopentyl)thio)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diyl)bis(methylene))(S)-dipicolinate (70 mg), which was used without purification.

[0540] Step 2: A stir bar, dimethyl 6,6′-((2-(((5-aminopentyl)thio)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diyl)bis(methylene))(S)-dipicolinate (50 mg, 0.070 mmol), 11,12-didehydro-γ-oxodibenz[b,f]azocine-5(6H)-butanoic acid (20 mg, 0.070 mmol), triethylamine (21 mg, 0.21 mmol), HATU (38 mg, 0.10 mmol), and DCM (0.5 mL) were added to a 25 mL three-neck round-bottom flask under a nitrogen atmosphere at 0° C., followed by warming to room temperature and stirring overnight. The reaction mixture was treated with water (10 mL) and extracted with dichloromethane (10 mL × 3). The combined extracts were washed with 10% aqueous NaHCO (10 mL) and brine (10 mL), dried over anhydrous NaSO, filtered, and concentrated to dryness to give an oil. The oil was purified by silica gel chromatography (0–10% MeOH / DCM) to give N-acyl-DBCO-tagged dimethyl 6,6′-((2-(((5-aminopentyl)thio)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diyl)bis(methylene))(S)-dipicolinate (16 mg).

[0541] Step 3: A stir bar, N-acyl-DBCO-tagged dimethyl 6,6′-((2-(((5-aminopentyl)thio)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diyl)bis(methylene))(S)-dipicolinate (16 mg, 0.016 mmol), aqueous LiOH (0.49 mL, 0.1 N, 0.049 mmol), and MeOH (0.25 mL) were added to an 8 mL reaction vial and the mixture was stirred at room temperature overnight. The reaction mixture was then treated with acetic acid until the pH reached approximately 6.5 and concentrated to dryness under vacuum at room temperature to give a concentrate, which was purified by preparative HPLC (column: XBRIDGE C18 19 x 150 mm 5.0 μm, mobile phase: 10 mM aqueous ammonium acetate / ACN, flow rate: 15.0 mL / min) to give N-acyl-DBCO-tagged 6,6'-((2-(((5-aminopentyl)thio)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diyl)bis(methylene))(S)-dipicolinic acid as an off-white solid (5 mg). LC-MS APCI:C 51 H 62 NO 10 Calculated S, 950.42; observed m / z [M+H] + 951.4. 1 H NMR(400MHz,D2O):δ 7.81-7.75(m,4H),7.52-7.17(m,10H),4.97-4.90(m,1H),4.80(s,4H),4.14(s,3H),3.77-3.46(m,16H),3.10(s,7H) ),2.83-2.80(m,2H),2.55-2.53(m,2H),2.42-2.38(m,3H),2.11-2.08(m,3H),1.39-1.35(m,2H),1.20-1.10(m,4H).

[0542] Example 17 N-Acyl-DBCO-tagged 6-((4-((6-aminoethyl)carbamoyl)phenyl)16-((6-carboxypyridin-2-yl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-7-yl)methyl)picolinic acid (TOPA-[C7]-benzimide-DBCO)

[0543] [ka]

[0544] Scheme 15

[0545] [ka]

[0546] Step 1: In a 500 mL three-neck round-bottom flask under nitrogen at −78° C., to a mixture of methyl 6-formylpicolinate (4.00 g, 24.2 mmol), (4-(tert-butoxycarbonyl)phenyl)boronic acid (10.7 g, 48.5 mmol), PdCl (0.21 g, 1.2 mmol), tri(naphthalen-1-yl)phosphine (0.50 g, 1.2 mmol), and potassium carbonate (10.0 g, 72.7 mmol) was added tetrahydrofuran (100 mL) in one portion. The mixture was purged with nitrogen and stirred at room temperature for 30 minutes, then heated at 65° C. for 24 hours. The reaction mixture was cooled to room temperature and filtered through a pad of Celite®, and the filtrate was concentrated to dryness. The crude product was subjected to silica gel chromatography (0-50% EtOAc / petroleum ether) to afford methyl 6-((4-(tert-butoxycarbonyl)phenyl)(hydroxy)methyl)picolinate as a yellow oil (2.5 g, 30%).

[0547] Step 2: A stir bar, methyl 6-((4-(tert-butoxycarbonyl)phenyl)(hydroxy)methyl)picolinate (2.50 g, 7.30 mmol), PPh3 (3.43 g, 13.1 mmol), N-bromosuccinimide (2.13 g, 12.0 mmol), and DCM (30 mL) were placed in a 250 mL three-necked round-bottom flask at room temperature under a nitrogen atmosphere and stirred for 1 h. The reaction solution was loaded onto a silica gel column and purified using 0-30% ethyl acetate in petroleum ether to give the compound methyl 6-(bromo(4-(tert-butoxycarbonyl)phenyl)methyl)picolinate as a yellow oil (1.65 g, 56%).

[0548] Step 3: A stir bar, methyl 6-((1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-7-yl)methyl)picolinate (1.52 g, 3.69 mmol), 6-(bromo(4-(tert-butoxycarbonyl)phenyl)methyl)picolinate (1.50 g, 3.69 mmol), NaCO (1.17 g, 11.1 mmol), and acetonitrile (30 mL) were added to a 250 mL three-neck round-bottom flask and the resulting heterogeneous mixture was heated at 90° C. for 16 hours under a nitrogen atmosphere. The reaction mass was then cooled to room temperature, filtered through a pad of Celite®, and concentrated to dryness in vacuo to give the crude product. The crude product was subjected to silica gel chromatography (0-10% MeOH / DCM) to afford methyl 6-((4-(tert-butoxycarbonyl)phenyl)(16-((6-(methoxycarbonyl)pyridin-2-yl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-7-yl)methyl)picolinate as a brown oil (1.2 g, 44%).

[0549] Step 4: A stir bar, methyl 6-((4-(tert-butoxycarbonyl)phenyl)(16-((6-(methoxycarbonyl)pyridin-2-yl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-7-yl)methyl)picolinate (1.2 g, 1.6 mmol), TFA (0.62 mL, 8.1 mmol) and DCM (20 mL) were added to a 100 mL three-neck round-bottom flask at room temperature and stirred for 1 hour. The reaction mixture was concentrated to dryness, and the resulting crude product was subjected to preparative HPLC (column: XBRIDGE C18 (19 × 150 mm) 5.0 μm, mobile phase: 0.1% TFA aqueous solution / ACN, flow rate: 15.0 mL / min) to obtain 4-((6-(methoxycarbonyl)pyridin-2-yl)(16-((6-(methoxycarbonyl)pyridin-2-yl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-7-yl)methyl)benzoic acid as a brown oil (0.8 g, 72%). LC-MS APCI: C 35 H 44 N4O 10 Calculated value 680.31, observed value m / z [M+H] + 681.5. Purity by LC-MS: 99.87%. Purity by HPLC: 97.14% (97.01% at 210 nm, 97.20% at 254 nm, and 97.21% at 280 nm), Column: Atlantis dC18 (250 × 4.6 mm), 5 μm, Mobile phase A: 0.1% TFA in water, Mobile phase B: acetonitrile, Flow rate: 1.0 mL / min. 1 H NMR(400MHz,DMSO-d6):δ 8.12-8.07(m,4H),8.00-7.98(m,2H),7.75-7.73(m,4H),6.10(s,1H),4.6 7(s,2H),3.96(s,3H),3.91(s,3H),3.82(s,8H),3.56(s,8H),3.52(s,8H).

[0550] Step 5: A stir bar, 4-((6-(methoxycarbonyl)pyridin-2-yl)(16-((6-(methoxycarbonyl)pyridin-2-yl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-7-yl)methyl)benzoic acid (0.25 g, 0.37 mmol), DBCO (0.10 g, 0.37 mmol), triethylamine (0.16 mL, 1.1 mmol), HBTU (0.21 g, 0.55 mmol), and DCM (10 mL) were added to a 25 mL three-neck round-bottom flask at 0 °C under a nitrogen atmosphere at room temperature and stirred for 16 h. The reaction was quenched with water (20 mL) and extracted with DCM (3 × 20 mL). The combined extracts were washed with 10% aqueous NaHCO (20 mL), brine (20 mL), dried over anhydrous NaSO, filtered, and concentrated to dryness to give the crude product as an oil, which was subjected to silica gel chromatography (0-10% MeOH / DCM) to give TOPA dimethyl ester-[C]-phenyl-DBCO as a colorless gummy oil (0.12 g, 35%).

[0551] Step 6: A stir bar, TOPA dimethyl ester-[C7]-phenyl-DBCO (0.1 g, 0.1 mmol), aqueous LiOH·HO (3 mL, 0.1 N, 0.3 mmol), and THF / MeOH / HO (4:1:1 v / v / v, 2 mL) were added to an 8 mL reaction vial at room temperature and stirred for 2 hours. The reaction mixture was neutralized with aqueous HCl (1 N) to a pH of approximately 6.5. The reaction mixture was concentrated to dryness in vacuo at room temperature, and the resulting crude product was subjected to preparative HPLC (column: XBRIDGE C18 (19 × 150 mm) 5.0 μm, mobile phase: 10 mM aqueous ammonium acetate / ACN, flow rate: 15.0 mL / min) to give TOPA-[C7]-phenyl-DBCO as an off-white solid (20 mg, 21%). LC-MS APCI:C 51 H 54 NO 10 Calculated value 910.39, observed value m / z [MH] +909.3. Purity by LC-MS: 92.47%. Purity by HPLC: 90.68% (88.04% at 210 nm, 90.43% at 254 nm, and 93.56% at 280 nm), Column: XBRIDGE C8 (50 × 4.6 mm), 3.5 μm, Mobile phase A: 10 mM ammonium bicarbonate aqueous solution, Mobile phase B: acetonitrile, Flow rate: 1.0 mL / min. 1 H NMR(400MHz,DMSO-d6):δ 7.84-7.82(m,4H),7.60-7.29(m,12H),7.13-7.10(m,2H),5.12-5.02(m ,2H),3.97(s,2H),3.59-3.44(m,20H),2.85(s,4H),2.73-2.68(m,6H).

[0552] Example 18 TOPA-[C7]-benzimide-DBCO-triazole-PSMB-127 antibody conjugate

[0553] [ka]

[0554] Step 1. Azide modification and click reaction of mAb: PSMB127 was site-selectively modified with a 100-fold molar excess of 3-azidopropylamine and microbial transglutaminase (MTG, Activa TI) at 37 °C. The addition of two azides onto the heavy chain of the mAb was monitored by intact mass ESI-TOF LC-MS on an Agilent G224 instrument. Excess 3-azidopropylamine and MTG were removed, and the azide-modified mAb (azido-mAb) was purified using a 1 mL GE Healthcare MabSelect column. The azido-mAb was eluted from the resin using 100 mM sodium citrate (pH 3.0) and then exchanged into 20 mM Hepes, 100 mM NaCl (pH 7.5) using a 7K Zeba® desalting column. A 10-fold molar excess of TOPA-[C7]-phenyl-DBCO was reacted with site-specific azide-PSMB127 (DOL=2) for 1 hour at 37°C without shaking. Completion of the DBCO-azide click reaction was monitored by intact mass spectrometry. Excess free chelator was removed by desalting the conjugate into 20 mM Hepes, 100 mM NaCl (pH 7.5) on a Zeba® 7K desalting column, followed by three 15-fold serial dilutions and concentration into 20 mM Hepes, 100 mM NaCl (pH 7.5) using a 30K MWCO Amicon concentrator device by spinning at 3800 x g. This yielded the final site-specific TOPA-[C7]-phenyl-DBCO-PSMB127 conjugate with a CAR=2. The final conjugate was confirmed to be monomeric by analytical size exclusion chromatography using a Tosoh TSKgel G3000SWxl 7.8 mm x 30 cm, 5 u column, column temperature: room temperature, column elution with DPBS buffer (x1, calcium and magnesium free), flow rate: 0.7 mL / min, 18 min run, injection volume: 18 μL.

[0555] Step 2. Chelation: Stock solutions of the following metal salts were prepared in purified water:

[0556] [Table 2]

[0557] The final conjugate was confirmed to be monomeric by analytical size-exclusion chromatography, eluting with 10 mM sodium acetate at a 5-fold molar excess. The metal solution was added to TOPA-[C7]-phenyl-DBCO-PSMB127 (6.8 μM antibody, 34 μM metal ion) in 10 mM sodium acetate (pH 5.2) and incubated for 2 hours at 37°C. Excess metal was removed by desalting on a Zeba® column (ThermoFisher®), followed by two cycles of 10-fold dilution and concentration on a 50K MWCO Amicon concentrator (EMD Millipore®). Chelation was assessed by intact and reduced-mass LC-MS.

[0558] Step 3. Stability Determination: DTPA induction was performed to determine the stability of the chelate. 50 μL of sample (6.3 μM antibody) was combined with 50 μL of 10 mM DTPA (pH 6.5) and incubated overnight at 37°C. Chelation was assessed by intact and reduced-mass LC-MS. LC-MS was performed on an Agilent 1260 HPLC system connected to an Agilent G6224 MS-TOF mass spectrometer. LC was performed on an Agilent RP-mAb C4 column (2.1 × 50 mm, 3.5 microns) at a flow rate of 1 mL / min using mobile phases of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (Sigma-Aldrich catalog no. 34688) (B), with a gradient of 20% B (0–2 min), 20–60% B (2–3 min), and 60–80% B (3–5.5 min). The instrument was operated in positive electrospray ionization mode, scanning from m / z 600 to 6000. Mass-to-charge spectra were deconvoluted using a maximum entropy algorithm, and the relative abundances of related species were estimated by the peak heights of the deconvoluted masses. Instrument settings included a capillary voltage of 3500 V, a fragmentor voltage of 175 V, a skimmer voltage of 65 V; a gas temperature of 325 °C, a drying gas flow rate of 5.0 L / min, a nebulizer pressure of 30 psig, and an acquisition mode range of 100-7000 with a 0.42 scan rate.

[0559] A shift in MW relative to TOPA-[C7]-phenyl-DBCO-PSMB127 was observed for the cerium and neodymium samples. The intact mass of the conjugate incubated with cerium showed an increase in MW of 139 Da (20% by peak area) or 276 Da (77%), corresponding to the addition of one or two cerium ions. After DTPA induction, the masses remained similar, as did the abundances (30% and 67% for the +138 and +274 species).

[0560] Example 19 6-((16-((6-carboxypyridin-2-yl)(4-isothiocyanatophenyl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-7-yl)methyl)picolinic acid (H2bp18c6 benzyl-phenyl) (TOPA-[C7]-phenyl isothiocyanate and sodium salt forms

[0561] [ka]

[0562] Compound 2 was prepared in a manner similar to that described in the literature, see J. Org. Chem. 1987, 52, 5172.

[0563] [ka]

[0564] Compound 3 was prepared by a method similar to that described in the literature, see Chemistry - a European Journal, 2015, 21, 10179.

[0565] [ka]

[0566] Preparation of Compound 4:

[0567] [ka]

[0568] 1,4,10,13-Tetraoxa-7,16-diazacyclooctadecane (494 g, 1.88 mol, 2.5 equiv.), NaCl (44.1 g, 0.75 mol, 1.0 equiv.), HO (140 mL, 1 volume relative to compound 3), and acetonitrile (2.1 L, 15 volumes) were charged to a 10 L reactor at 15-20 °C under a N atmosphere and heated to 65 °C. To the resulting mixture was added a solution of compound 3 (140 g, 0.75 mol) in acetonitrile (280 mL, 2 volumes) dropwise over 1 h at 65 °C. The solution was aged at 65 °C for 0.5 h. LCMS analysis of the mixture indicated the reaction was complete. The mixture was allowed to cool to room temperature and concentrated under reduced pressure. Acetone (700 mL, 5 volumes) was added to the mixture, and the suspension was stirred for an additional 1 h. The mixture was filtered (the filtered solid was unreacted compound 2). The filtrate was concentrated in vacuo and then dissolved in DCM (1.4 L, 10 vol). The organic phase was washed with water (3 x 750 mL), dried over Na2SO4, and then concentrated in vacuo to give 212 g of compound 4 (63% yield, assay: 85% w / w). LCMS: (ES, m / z): 412.15 [M+H] + 1 H-NMR(300MHz,DMSO-d6,ppm):δ 7.98-7.87(m,2H),7.81(dd,J=6.4,2.6Hz,1H),3.87(s,3H),3.81(s,2H),3.61-3.38(m,16H),2.77(dt,J=19.0,5.2Hz,8H).

[0569] Preparation of Compound 7:

[0570] [ka]

[0571] Methyl 6-formylpicolinate 5 (250 g, 1.0 equiv.), (4-((tert-butoxycarbonyl)amino)phenyl)boronic acid 6 (538 g, 1.5 equiv.), and degassed THF (6.5 L, 26 volumes relative to 5) were charged to a 10 L reactor at 15–20 °C under a N atmosphere. Following this, PdCl (14.0 g, 0.05 equiv.), tri(naphthalen-1-yl)phosphane (31 g, 0.05 equiv.), and KCO (650 g, 3.1 equiv.) were added. The resulting solution was stirred at 20 °C for 0.5 h. The mixture was then heated to 65 °C and aged for 17 h. Analysis by LCMS indicated the reaction was complete. The resulting solution was cooled to room temperature and diluted with ice water (2.5 L, 10 volumes) and ethyl acetate (5 L, 20 volumes). The mixture was filtered through a Celite pad. The solution was separated and the lower aqueous layer was discarded. The organic phase was washed with water (2 x 1.5 L, 12 vol). The layers were separated and the organic layer was dried over Na2SO4 and concentrated in vacuo. The resulting residue was treated with heptane (1.25 L, 5 vol) and the resulting suspension was stirred for 0.5 h. The mixture was filtered and the filter cake was washed with n-heptane (500 mL, 2 vol) to afford 530 g (98% yield, LCAP purity: 90%) of the desired product 7 as a yellow solid, which was used directly in the next step without further purification. LCMS: (ES, m / z): 381.10 [M+Na] + 1 H-NMR(300MHz,DMSO-d6,ppm):δ 9.27(s,1H),8.03-7.85(m,2H),7.79(dd,J=7.7,1.4Hz,1H),7.39(d,J=8.4Hz,2H),7.26 (d,J=8.4Hz,2H),6.13(d,J=4.0Hz,1H),5.72(d,J=3.9Hz,1H),3.87(s,3H),1.46(s,9H).

[0572] Preparation of Compound 8:

[0573] [ka]

[0574] Methyl 6-((4-((tert-butoxycarbonyl)amino)phenyl)(hydroxy)methyl)picolinate 7 (310 g, 1.0 equiv.), triethylamine (219 g, 2.5 equiv.), and DCM (6.2 L, 20 volumes relative to 7) were charged to a 10 L reactor at 15-20 °C under a nitrogen atmosphere, and the solution was cooled to 0 °C. Methanesulfonyl chloride (99.2 g, 1.0 equiv.) was added dropwise over 30 min while maintaining the temperature at 0 °C. The cooling bath was removed, the temperature was allowed to reach ambient temperature, and the mixture was aged at this temperature for 1 h. The solution was concentrated under vacuum at 10-15 °C, and the residue was then dissolved in acetonitrile (438 mL, 2 volumes). The resulting solution was concentrated under vacuum to give 518 g (crude) of the desired product 8. The crude product was used directly in the next step without further purification.

[0575] Preparation of Compound 9:

[0576] [ka]

[0577] Methyl 6-((4-((tert-butoxycarbonyl)amino)phenyl)-((methylsulfonyl)oxy)methyl)picolinate 8 (212 g, 1.0 equiv., 85% pure by Q-NMR), NaCO (137.6 g, 3.0 equiv.), and acetonitrile (3.56 L, 20 volumes relative to 8) were charged to a 10 L reactor at room temperature under a nitrogen atmosphere, and the mixture was then heated to 65 °C and aged for 1 h. A solution of methyl 6-((1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-7-yl)methyl)picolinate 4 (377.8 g, 2.0 equiv.) in acetonitrile (3 L, 10 volumes) was added dropwise at 65 °C over 0.5 h. The mixture was aged at this temperature until HPLC analysis showed the reaction was complete. The resulting solution was cooled to room temperature, then filtered, and the filter cake was washed with MeOH (2 × 1 volume). The filtrate was concentrated under vacuum, and the resulting residue was dissolved in EA (700 mL). Silica gel (800 g, type: ZCX-2, 100-200 mesh, 2.11 w / w) was then added. The mixture was concentrated under vacuum while maintaining the temperature below 35 °C. Silica gel (9.6 kg, type: ZCX-2, 100-200 mesh, 26.3 w / w) was loaded onto a column, followed by the prepared dried silica gel containing the adsorbed crude product 9. The column was eluted with ethyl acetate, petroleum ether:dichloromethane (3:3:1) / methanol, dichloromethane (1:1) (gradient from 100:0 to 90:10, sample collection every 4 L ± 0.5 L). The fractions were analyzed by TLC (ethyl acetate:ethyl acetate:petroleum ether:dichloromethane:methanol=4:4:1:1). The product-containing fractions were combined and concentrated to give 260 g of compound 9 as a yellow solid (HPLC: 94%, QNMR: 92%). An additional 70 g of compound 9 was obtained as a yellow oil (HPLC: 75%, QNMR: 60%). LCMS (ES, m / z): 752.30 [M+H] + Observed m / z 1H-NMR (400MHz, CDCl3, ppm): δ 7.53-7.32(m,3H),7.28-7.18(m,3H),6.86(d,J=8.4Hz,2H),6.76(d,J=8.4Hz,2H),6.09(s,1H),4.63( s,1H),3.48(s,3H),3.44(bs,5H),3.17-2.92(m,16H),2.38(dq,J=25.0,7.2,6.8Hz,8H),0.97(s,9H).

[0578] Preparation of compound 10:

[0579]

change

[0580] Compound 9 (260 g, QNMR: 92%, 1.0 equiv.), N,O-bis(trimethylsilyl)acetamide (BSA, 6.0 equiv.), and acetonitrile (4 L, 15 vol.) were placed in a 10 L reaction vessel under a nitrogen atmosphere at 15–20 °C. The mixture was stirred at 20 °C for 40 min. A solution of TMSOTf (212.9 g, 3.0 equiv.) in acetonitrile (1.3 L, 5 vol.) was added dropwise over 0.5 h while maintaining the internal temperature at 15–20 °C. The resulting solution was aged at 15–20 °C for 1 h. When in-process analysis (sample preparation: 0.1 mL solution + 0.9 mL ACN + 1 drop of diisopropylethylamine) indicated complete conversion of the starting material, the mixture was quenched with diisopropylethylamine (617 g, 15.0 equiv.) while maintaining the temperature at 5–10 °C. The mixture was stirred at 5-10°C for 20 minutes, then saturated aqueous NH4Cl (2.6 L, 10 vol) was charged while maintaining the temperature at 5-10°C. The mixture was aged at this temperature for an additional 30 minutes. The aqueous phase (containing solids) was collected and extracted with 2-MeTHF (520 mL, 2 vol). The organic phases were combined, the water content was checked by KF (KF: 9.18%), and then dried over anhydrous Na2SO4 (500 g, 10.0 equiv.). The solids were removed by filtration, and the filter cake was washed with acetonitrile (2 × 520 mL, 2 vol.). The filtrate was then dried over anhydrous Na2SO4 (500 g, 10.0 equiv.). After filtration, the filter cake was washed with acetonitrile (2 × 520 mL, 2 vol.) and the water content was checked by KF (KF: 8.15%). The acetonitrile / 2-MeTHF extract 10 was used directly in the next step (the product was not stable to LCMS conditions).

[0581] Preparation of Compound 14 (Free Acid) [ka]

[0582] Methyl 6-((4-((tert-butoxycarbonyl)amino)phenyl)(16-((6-(methoxycarbonyl)pyridin-2-yl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-7-yl)methyl)picolinate (6.0 g, 1.0 equiv.), BSA (9.7 g, 6.0 equiv.), and MeCN (120 mL, 20 volumes in 9 parts by volume) were charged into a 500 mL reaction vessel at room temperature under a nitrogen atmosphere. A solution of TMSOTf (5.4 g, 2.3 equiv.) in MeCN (120 mL, 20 volumes) was added dropwise at room temperature over 30 minutes. The mixture was aged overnight at room temperature. Analysis of the mixture (sample preparation: 0.1 mL solution system + 0.9 mL ACN + 1 drop of diisopropylethylamine) indicated that the reaction had reached completion. The mixture was quenched with diisopropylethylamine (15.4 g, 15.0 equiv.) while maintaining the temperature at 0-5 °C. The mixture was stirred at 0-5 °C for 5 min, and then saturated NH4Cl solution (60 mL, 10 vol.) was added dropwise while maintaining the temperature at 0-5 °C. The aqueous phase was removed by extraction, and the organic phase was collected and used directly in the next step. The organic phase was placed in a 500 mL three-neck round-bottom bottle, and a solution of LiOH (1.15 g, 6.0 equiv.) in water (60 mL, 10 vol.) was added to the solution at room temperature. The solution was stirred at room temperature for 1 h. Analysis of the mixture (sample preparation: 0.1 mL of solution system + 0.9 mL of acetonitrile) indicated incomplete conversion. Additional LiOH (576 mg, 3.0 equiv.) was added, and the solution was stirred at room temperature for an additional 1 h. Analysis of the mixture (sample preparation: 0.1 mL of solution + 0.9 mL of acetonitrile) indicated that the reaction had reached completion. Next, TCDI (5.6 g, 3.9 equiv.) was added, and the solution was stirred at room temperature for 1 h. Analysis of the mixture (sample preparation: 0.1 mL of solution + 0.9 mL of acetonitrile) indicated incomplete conversion. Further TCDI (2.8 g, 2.0 equiv.) was added, and the solution was stirred at room temperature for another 1 h. Analysis of the mixture (sample preparation: 0.1 mL of solution + 0.9 mL of acetonitrile) indicated that the reaction had reached completion. The reaction solution was separated by reverse-phase Combi-Flash. Method: Column C18, solution A: HO (containing 0.01% formic acid), solution B: ACN. From 5% to 35% in 40 min, flow rate (100 mL / min), product between 20 and 25 min.The solution was collected. The solution was concentrated to remove the ACN and separated again by reverse-phase Combi-Flash. Method: Column C18, Solution A HO, Solution B ACN. 5% 10 min, 5% to 35% in 5 min, 95% 10 min, flow rate (100 mL / min), product 13 to 25 min. The solution was collected. The solution was concentrated under reduced pressure below 20 °C and dried by lyophilization. This gave 2.5 g (47% yield over three steps) of compound 14 as a yellow solid. Compound 14 (6-((16-((6-carboxypyridin-2-yl)(4-isothiocyanatophenyl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-7-yl)methyl)picolinic acid) needed to be stored at -80 °C. LCMS: (ES, m / z): 666.3 [M+H]. + 1 H-NMR:(400MHz,D2O,ppm):7.94-7.84(m,4H),7.56-7.40(m,4H),7.16-7.14(m,2H), 5.83(s,1H),4.56(s,2H),3.80-3.75(m,8H),3.60-3.49(m,14H),3.36-3.33(m,2H).

[0583] Preparation of Compound 11 (sodium salt):

[0584] [ka]

[0585] The prepared solution of compound 10 in ACN and 2-MeTHF was placed in a 10 L four-neck reaction vessel, and the solution was cooled to 5-10 °C. Powdered NaOH (56.9 g, 4.5 equiv.) was added while maintaining the temperature at 5-10 °C. The resulting solution was stirred at 15-20 °C for 0.5 h. Analysis of the mixture (sample preparation: 0.1 mL of solution + 0.9 mL of acetonitrile) showed no conversion. Additional powdered NaOH (25.3 g, 2.0 equiv.) was added at 5-10 °C. The solution was aged at 15-20 °C for an additional 0.5 h. A second IPC analysis showed 50% conversion. The final addition of powdered NaOH (25.3 g, 2.0 equiv.) was added at 5-10 °C. The mixture was stirred at 15-20 °C for an additional 0.5 h. Analysis showed complete conversion of starting material 10. The mixture was filtered, and the filter cake was washed with acetonitrile (2 × 520 mL, 2 vol). The final solution (approximately 7.5 L, 28.8 vol) was concentrated to 1–2 vol while maintaining a temperature of 15–20 °C. The residue was then treated with acetonitrile (2 L, 7.7 vol), and the water content was checked by KF (KF: 5.7%). The mixture was filtered, and the filter cake was washed with ACN (2 × 520 mL, 2 vol). The solution was then concentrated under vacuum at 15–20 °C to 1–2 vol. The water content was again checked by KF (KF: 5.5%). The solution was diluted with acetonitrile (390 mL, 1.5 vol) and added dropwise to MTBE (2.6 L, 10 vol) over 0.5 h, maintaining the temperature between 15–20 °C. The solvent was decanted to leave a viscous oil, which was redissolved in acetonitrile (520 mL, 2 vol) and added to MTBE (2.6 L, 10 vol). This process was repeated four more times. A viscous oil was obtained, which was finally dissolved in acetonitrile (520 mL, 2 vol), dried, and then concentrated under reduced pressure at 15-20 °C. Residual solvent was then removed by evaporation using an oil pump at 15-20 °C. After drying, 335 g of compound 11 was obtained as a yellowish solid (QNMR: 70%, 87% overall yield over two steps). LCMS (ES, m / z): 624.3 [M-TfONa-2Na+3H] + 1H-NMR(300MHz,Methanol-d4,ppm):δ 7.97(dd,J=7.8,2.1Hz,2H),7.84(t,J=7.7Hz,1H),7.75(t,J=7.8Hz,1H),7.36(dd,J=7.8,1.1Hz,1H),7.23(d,J=7.7Hz,1H ),7.11(d,J=8.5Hz,2H),6.72(d,J=8.5Hz,2H),3.96(s,1H),3.83-3.36(m,18H),3.03-2.62(m,6H),2.55(d,J=14.3Hz,2H).

[0586] Preparation of Compound 12 (TOPA-[C7]-phenylisothiocyanate sodium salt):

[0587] [ka]

[0588] TCDI (68.7 g, 1.4 equiv.) and acetonitrile (2.6 L, 8 vol.) were placed in a 10 L reaction vessel under a nitrogen atmosphere at 15-20 °C. Compound 11 (330 g, NaCl) in acetonitrile (660 mL, 2 vol.) was added while maintaining the temperature at 15-20 °C. +A solution of 2,4-dichloromethane (2,4-dichloromethane, QNMR: 70%, 1.0 equiv.) was added dropwise over 30 min. The mixture was aged at 15-20 °C for 0.5 h. Analysis of the mixture (sample preparation: 30 μL of solution + 300 μL of ACN + 1 drop of water) showed that the reaction had reached completion. The water content was checked by KF (KF: 0.19%). The solution was dried and concentrated under reduced pressure at 15-20 °C. The resulting residue was dissolved in acetonitrile (945 mL, 2.9 vol.), and the water content was measured by KF (KF: 0.34%). Isopropyl acetate (660 mL, 2 vol.) was added to the solution over 40 min at 15-20 °C. No nucleation was observed, and additional isopropyl acetate (6.6 L, 18 vol) was slowly charged dropwise over 40 min at 15-20°C, resulting in precipitation of product 12, which was collected by filtration as a yellowish solid. The solid was dissolved in acetonitrile (330 mL, 1 vol), and IPAc (6.6 L, 20 vol) was added dropwise over 40 min at 15-20°C. The mixture was filtered to give 230 g of product as a yellowish solid (LCAP: 80.99%, QNMR: 59%, 10% IPAc). The wet cake was dried under vacuum at 15-20°C for 2 h to give 224 g of crude product 12 as a yellowish solid (LCAP: 80.9%, QNMR: 60.4%, ca. 6% IPAc). The crude product 12 was redissolved in acetonitrile (330 mL, 1 vol) and isopropyl acetate (412 mL, 1.25 vol) was added dropwise over 40 min at 15–20°C. The resulting mixture was filtered, and 12 was recovered (30.5 g, HPLC = 60.9%, assay: 25.5%). The mother liquor was diluted with isopropyl acetate (6.6 L, 20 vol) added over 40 min at 15–20°C. The mixture was filtered, and the cake was dried to give 173.5 g of crude product 12 as a yellowish solid (LC / Cat. No. 85.4%, QNMR: 66%, 3.9% IPAc, RRT = 3.9%). 190 g of crude product 12 was dissolved in 760 mL of acetonitrile:isopropyl acetate (2:1), and the mixture was passed through a silica gel column (380 g, 2×).The silica was flushed with acetonitrile:isopropyl acetate (2:1, 5.7 L) and then with 12 L of acetonitrile (very little product). The product-containing fractions were concentrated to give 118 g of product 12 as a yellowish solid (LCAP: 95%). The silica pad was then rinsed with MeCN / HO (12 L, 10:1). The solvent was removed in vacuo to give an additional 60 g of crude product 12 as a yellowish solid, which was dissolved in acetonitrile (1.5 L), stirred for 30 min, and then filtered. The mother liquor was then concentrated to give 24 g of crude product 12 as a yellowish solid (LCAP = 92%). Crude product 12 (118 g) prepared as above and crude product 12 (24 g) were dissolved in acetonitrile (330 mL, 1 vol), and isopropyl acetate (6.6 L, 20 vol) was added dropwise over 40 min at 15-20 °C. The mixture was then filtered to give 133 g of product 12 as a yellowish solid of suitable purity (LCAP: 95%, QNMR: 60.8%, 7.8% IPAc). Note: Compound 12 required storage at -20 °C. LCMS: (ES, m / z): 666.61 [M-TfONa-2Na+3H]. + 1 H-NMR: (400 MHz, methanol-d4, ppm): δ 8.00(ddd,J=13.8,7.7,1.0Hz,2H),7.84(dt,J=20.4,7.7Hz,2H),7.58-7.49 (m,2H),7.40(dd,J=7.6,1.0Hz,1H),7.36-7.28(m,2H),7.28-7.20(m,1H),4. 96(hept,J=6.3Hz,1H),3.96-3.88(m,1H),3.83(d,J=15.1Hz,1H),3.70-3.52 (m,11H),3.55-3.39(m,4H),3.07-2.73(m,6H),2.62(dt,J=15.1,3.6Hz,2H).

[0589] Example 20 TOPA-[C7]-phenylthiourea-h11B6 antibody conjugate

[0590] [ka]

[0591] (In the TOPA-[C7]-phenylthiourea-h11B6 antibody conjugate above, the structure does not show the lysine residue of h11B6 linked to the phenylthiourea moiety.)

[0592] TOPA-[C7]-phenylthiourea modification of mAbs: h11b6 mAb (10.2 mg / mL) was diluted to 1 mg / mL in 10 mM sodium acetate pH 5.2 buffer. Just prior to conjugation, the pH was adjusted to pH 9 with sodium bicarbonate buffer (VWR 144-55-8). The pH was confirmed with pH paper. Next, a 10-fold molar excess of disodium salt TOPA-[C7]-phenylisothiocyanate sodium salt (50 mM stock dissolved in water) was added to h11b6 mAb, and the mixture of antibody and TOPA-[C7]-phenylisothiocyanate sodium salt was incubated at room temperature for approximately 1 hour without shaking. The addition of TOPA-[C7]-phenylisothiocyanate sodium salt was monitored by intact mass ESI-TOF LC-MS on an Agilent® G224 instrument until a CAR value of 1.5-2.0 was achieved. The mixture was immediately quenched by adding 1 M Tris pH 8.5 (Teknova T1085) to a final concentration of 100 mM. Excess free chelator was removed by desalting the reaction in 10 mM sodium acetate pH 5.2 using a 7K Zeba® desalting column. To confirm the absence of excess chelator, three sample dilutions to 15 mL were performed, followed by concentration to 1 mL using a 50,000 MWCO Amicon concentrator. The sample was then concentrated to its final concentration for radiolabeling. The final conjugate was confirmed to be monomeric by analytical size exclusion chromatography using a Tosoh TSKgel G3000SWxl 7.8 mm x 30 cm, 5 μL column, column temperature: room temperature, column elution with 0.2 M sodium phosphate (pH 6.8), flow rate: 0.8 mL / min, 18 min run, injection volume: 18 μL.

[0593] Example 21 Ac-225 labeled TOPA-[C7]-phenylthiourea-h11B6 antibody conjugate

[0594] [ka]

[0595] (In the Ac-225 labeled TOPA-[C7]-phenylthiourea-h11B6 antibody conjugate above, the structure does not show the lysine residue of h11B6 linked to the phenylthiourea moiety.)

[0596] (i) Labeling of TOPA-[C7]-phenylthiourea-h11B6 with Ac-225 in 3M NaOAc buffer: To a solution of NaOAc (3 M in HO, 60 μL) in a plastic vial, Ac-225 (10 mCi / mL in 0.1 N HCl, 15 μL) and TOPA-[C]-phenylthiourea-h11B6 (1.13 mg / mL in 10 mM NaOAc pH=5.5, 441 μL, 0.5 mg) were added sequentially. After mixing, the pH was approximately 6.5 by pH paper. The vial was left at 37°C for 2 hours.

[0597] iTLC of the labeling reaction mixture: 0.5 μL of the labeling reaction mixture was loaded onto iTLC-SG and developed with 10 mM EDTA (pH 5-6). The dried iTLC-SG was left overnight at room temperature and then scanned on a Bioscan AR-2000 radio-TLC scanner. Under the elution conditions described herein, TOPA-[C7]-phenylthiourea-h11B6-bound Ac-225 remained at the origin, while any free Ac-225 migrated with the solvent to the solvent front. iTLC scans showed that 99.9% of TOPA-[C7]-phenylthiourea-h11B6 was bound to Ac-225.

[0598] DTPA derivatization of the labeling reaction mixture: Further, 0.5 μL of the labeling reaction mixture was mixed with 10 mM DTPA (pH = 6.5, 15 μL) at 37 °C. After 30 min, 10 μL of the mixture was spotted onto iTLC-SG and developed with 10 mM EDTA. The dried iTLC-SG was left overnight at room temperature and then scanned using a Bioscan AR-2000 radio-TLC scanner. Under the elution conditions described herein, TOPA-[C7]-phenylthiourea-h11B6-chelated Ac-225 remained at the origin, while any free Ac-225 migrated with the solvent to the solvent front. iTLC scans showed that 99.7% of TOPA-[C7]-phenylthiourea-h11B6 was chelated to Ac-225.

[0599] Purification on PD10 column: The PD-10 resin was conditioned with NaOAc buffer (25 mM NaOAc, 0.04% PS-20, pH 5.5) by passing three 5 mL portions of the buffer through the column and discarding the wash solution. The entire labeling reaction mixture was applied to the column reservoir, and the eluate was collected in pre-numbered plastic tubes. The reaction vial was washed three times with 0.2 mL of NaOAc buffer (25 mM NaOAc, 0.04% PS-20, pH 5.5), and the wash solution was pipetted into the PD-10 column reservoir. The eluate was collected. Each tube contained approximately 1 mL of eluate. Continuous application of NaOAc buffer (25 mM NaOAc, 0.04% PS-20, pH 5.5) to the PD-10 column reservoir was continued until a total elution volume of 10 mL was reached. The radiochemical purity of the collected fractions was checked by iTLC. 10 μL of each collected fraction was spotted onto iTLC-SG and developed with 10 mM EDTA. The dried iTLC-SG was left at room temperature overnight and then scanned on a Bioscan AR-2000 radio-TLC scanner. Pure fractions should have no radioactive signal at the solvent front of the iTLC-SG.

[0600] Refined 225 DTPA induction of Ac-TOPA-[C7]-phenylthiourea-h11B6: 10 μL of fraction #3 collected after the PD-10 column was mixed with 15 μL of 10 mM DTPA solution (pH 6.5) and incubated for 30 minutes. 10 μL of the mixture was loaded onto iTLC-SG, which was developed with 10 mM EDTA and allowed to dry overnight. It was scanned using a Bioscan AR-2000 radio-TLC scanner. No radioactive signal was observed at the solvent front of iTLC-SG, indicating the absence of free Ac-225 in fraction #3.

[0601] Refined 225 HPLC analysis of Ac-TOPA-[C7]-phenylthiourea-h11B6: Fraction #3 collected after the PD-10 column was analyzed by HPLC. HPLC method: Tosoh TSKgel G3000SWxl 7.8 mm x 30 cm, 5 μm column; column temperature: room temperature. The column was eluted with DPBS buffer (x1, calcium and magnesium free), flow rate: 0.7 mL / min, 20 min run, injection volume: 40 μL. After HPLC, fractions were collected at 30 s or 1 min time intervals. The collected HPLC fractions were left overnight at room temperature. The radioactivity of each collected fraction was counted in a gamma counter. HPLC radioactivity traces were constructed from the radioactivity in each HPLC fraction. The HPLC radioactivity traces showed a radioactive peak corresponding to the TOPA-[C7]-phenylthiourea-h11B6 peak on the HPLC UV trace.

[0602] (ii) Labeling of higher concentrations of TOPA-[C7]-phenylthiourea-h11B6 with Ac-225 in 1.5 M NaOAc buffer: To a solution of NaOAc (1.5 M in HO, containing 0.04% PS-20, 63 μL) in a plastic vial, Ac-225 (10 mCi / mL in 0.1 N HCl, 10 μL) and TOPA-[C7]-phenylthiourea-h11B6 (9.36 mg / mL in 10 mM NaOAc pH=5.2, 0.04% PS-20, 36 μL, 337 μg) were added sequentially. After mixing, the pH was approximately 6.5 as determined by pH paper. The vial was left at 37°C for 2 hours.

[0603] iTLC of the labeling reaction mixture: Next, 0.5 μL of the labeling reaction mixture was loaded onto iTLC-SG, which was developed with 10 mM EDTA. The dried iTLC-SG was left overnight at room temperature and then scanned on a Bioscan AR-2000 radio-TLC scanner. Under the elution conditions described herein, TOPA-[C7]-phenylthiourea-h11B6-bound Ac-225 remained at the origin, while any free Ac-225 migrated with the solvent to the solvent front. The iTLC scan showed that 99.9% of TOPA-[C7]-phenylthiourea-h11B6 was bound to Ac-225.

[0604] DTPA derivatization of the labeling reaction mixture: Further, 0.5 μL of the labeling reaction mixture was mixed with 10 mM DTPA (pH = 6.5, 15 μL) at 37 °C. After 30 min, 10 μL of the mixture was spotted onto iTLC-SG...

Claims

1. 1. An immunoconjugate comprising a radiometal complex conjugated to an antigen binding domain having binding specificity for kallikrein-related peptidase 2 (hK2), wherein said radiometal complex is conjugated to a chelator. ６４ An immunoconjugate comprising Cu, wherein the chelator is conjugated to the antigen-binding domain having binding specificity for kallikrein-related peptidase 2 (hK2).

2. The chelating agent is 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), S-2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), 1,4,8,11-tetraazacyclododecane-1,4,8,11-tetraacetic acid (TETA), 3,6,9,15-tetraazabi ...

2. The immunoconjugate of claim 1, which is chloro[9.3.1]-pentadeca-1(15),11,13-triene-4-(S)-(4-isothiocyanatobenzyl)-3,6,9-triacetic acid (PCTA), 5-S-(4-aminobenzyl)-1-oxa-4,7,10-triazacyclododecane-4,7,10-tris(acetic acid) (DO3A), or a derivative thereof.

3. The chelating agent is NOTA or a derivative thereof, such as NODA-GA, NODA-GA(t-butyl) ３ , di-t-butyl-NOTA, NOTA-thiosemicarbazide, NODA-MPAA or NODA-MPAEM, preferably NODA-GA.

4. 2. The immunoconjugate of claim 1, wherein the antigen-binding domain having binding specificity for hK2 is a Fab.

5. 2. The immunoconjugate of claim 1, wherein the antigen-binding domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NO: 170 (SYYWS), SEQ ID NO: 171 (YIYYSGSTNYNPSLKS), SEQ ID NO: 172 (TTIFGVVTPNFYYGMDV), SEQ ID NO: 173 (RASQGISSYLA), SEQ ID NO: 174 (AASTLQS) and SEQ ID NO: 175 (QQLNSYPLT), respectively.

6. the antigen-binding domain that binds to hK2 shares at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) of the VH of SEQ ID NO: 162 (QVQLQESGPGLVKPSETLSLTCTVSGGSISSYYWSWIRQPPGKGLEWIGYIYYSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAGTTIFGVVTPNFYYGMDVWGQGTTVTVSS).

2. The immunoconjugate of claim 1, comprising a VH that is identical to the VL of SEQ ID NO: 163 (DIQMTQSPSFLSASVGDRVTITCRASQGISSYLAWYQQKPGKAPKFLIYAASTLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQLNSYPLTFGGGTKVEIK) and a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99% or 100%) identical to the VL of SEQ ID NO: 163 (DIQMTQSPSFLSASVGDRVTITCRASQGISSYLAWYQQKPGKAPKFLIYAASTLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQLNSYPLTFGGGTKVEIK).

7. 2. The immunoconjugate of claim 1, wherein the antigen-binding domain that binds to hK2 comprises a VH of SEQ ID NO: 162 and a VL of SEQ ID NO:

163.

8. the antigen-binding domain a. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 170, 171, 172, 173, 174, and 175, respectively; and / or b. The immunoconjugate of claim 1, which is a Fab comprising a VH of SEQ ID NO: 162 and a VL of SEQ ID NO:

163.

9. The immunoconjugate of claim 1 , wherein the immunoconjugate is a short half-life immunoconjugate.

10. An immunoconjugate comprising a radioactive metal complex conjugated to an antigen-binding domain having binding specificity for kallikrein-related peptidase 2 (hK2), The radioactive metal complex may be NOTA or a derivative thereof, such as NODA-GA, NODA-GA(t-butyl) ３ , di-t-butyl-NOTA, NOTA-thiosemicarbazide, NODA-MPAA or NODA-MPAEM, preferably NODA-GA, linked to a chelator. ６４ Contains Cu, the chelator is conjugated to the antigen-binding domain that has binding specificity for kallikrein-related peptidase 2 (hK2); The antigen-binding domain is (a) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 170 (SYYWS), SEQ ID NO: 171 (YIYYSGSTNYNPSLKS), SEQ ID NO: 172 (TTIFGVVTPNFYYGMDV), SEQ ID NO: 173 (RASQGISSYLA), SEQ ID NO: 174 (AASTLQS), and SEQ ID NO: 175 (QQLNSYPLT), respectively; and / or (b) an immunoconjugate comprising a VH that is at least 95%, at least 99%, or 100% identical to the VH of SEQ ID NO: 162, and a VL that is at least 95%, at least 99%, or 100% identical to the VL of SEQ ID NO:

163.

11. 11. A detection agent for use in a method for detecting the presence of prostate cancer in a subject, the detection agent comprising an immunoconjugate according to any one of claims 1 to 10, the method comprising administering the immunoconjugate to a subject suspected of having prostate cancer and detecting the presence of prostate cancer by visualizing (e.g., by computed tomography or positron emission tomography) a biological structure to which the conjugate is bound.

12. 11. A detection agent for use in a method for detecting the progress of cancer treatment in a subject, the detection agent comprising an immunoconjugate of any one of claims 1 to 10, the method comprising administering the immunoconjugate to the subject and visualizing (e.g., by computed tomography or positron emission tomography) a biological structure to which the conjugate is bound, thereby detecting the progress of prostate cancer treatment in the subject.

13. The detection agent described in claim 11, wherein the method includes using positron emission tomography (PET) imaging to visualize the biological structure to which the immunoconjugate is bound.

14. The detection agent described in claim 12, wherein the method includes using positron emission tomography (PET) imaging to visualize the biological structure to which the immunoconjugate is bound.

Images