Trivalent radioisotope biologically targeted radiopharmaceuticals, methods of preparation and uses

Abstract

PCTA chelation Q of formula I +3 Targeted radiopharmaceuticals are disclosed that include a targeting moiety chemically bound to a trivalent radioactive ion. 1 ~R 7 Six of the X's are H, the seventh is a reacted functional group, and Z forms a chemical bond with the targeting species T. "g" is a number having an average value of 1 to about 12. 1 , X 2 , and X 3 Q +3 An anion Y is a substituent that can coordinate to an ion and / or serve to neutralize the charge of an ion. - is optionally present to balance the ionic charge. Pharmaceutical compositions comprising a theranostically effective amount of a targeted radiopharmaceutical of Formula I in a pharma- ceutical acceptable diluent are also contemplated, as are methods for treating and / or diagnosing a mammalian host having a disease, disorder or condition characterized by unwanted angiogenesis, tumor growth and / or tumor metastasis. TIFF2024519970000022.tif7283

Description

[Background technology]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application Nos. 63 / 191,499 and 63 / 191,506, both filed May 21, 2021, the disclosures of which are incorporated by reference herein.

[0002] Sequence Listing The sequence listing associated with this application is provided in text form in lieu of a paper copy and is incorporated herein by reference. The name of the text file containing the sequence listing is ______________, said text file is _________KB, was created on ______, 2022, and is being submitted via EFS-Web together with the filing of this application.

[0003] 2. Background of the Invention Radiopharmaceuticals generally contain a radioisotope bound to a targeting moiety or carrier, which carries the radioisotope to the target where it decays. The mode of decay of the isotope determines the type of radiopharmaceutical. Generally, gamma-emitting isotopes are used to detect the fate of the construct and are used for diagnostic purposes. For therapeutic purposes, constructs with particle emitters are preferred. In the past, beta-emitters were used, but in recent years alpha-emitters have shown superior efficacy. Alpha-emitting radionuclides are effective in killing cells due in part to their short particle range and high linear energy transfer (LET). Poty et al. (J Nucl Med. 2018 Jun: 59(60):878-884) describe the use of alpha-emitters in therapeutic radiopharmaceuticals.

[0004] The relatively long half-life of the alpha-emitting radionuclide actinium-225 (Ac-225) compared to other alpha-emitters is one of the reasons why it has become popular as a therapeutic radioisotope for cancer treatment. Clinical trials with constructs using this isotope have shown excellent results. The half-life of approximately 10 days matches well with the in vivo biological half-life of monoclonal antibodies, and the four alpha emissions produced by Ac-225 and its daughter nuclides were responsible for a high rate of tumor cell killing. However, the chemistry required to attach Ac-225 to a targeting site was lacking. The Ac-225 ion has a valence of +3 and an ionic radius of 112 pm. +3 is classified as a "hard" Lewis acid according to the Hard and Soft Acids and Bases (HSAB) theory [Pearson, J Am Chem Soc 1963, 85:3533-3539] and is therefore predicted to likewise prefer "hard" non-polarizable, electronegative Lewis bases such as anionic oxygen donors. The acid-base hardness / softness properties of a particular ion can be quantified using the concept of absolute chemical hardness (h). The absolute chemical hardness (h) of an ion is given by the formula (h) = (IA) / 2, where I is the ionization energy and A is the electron affinity of the species of interest. [Parr and Pearson, J Am Chem Soc 1983, 105:7512-7516; and Pearson, Inorg Chem 1988, 27:734-740].

[0005] The Ac calculated in this way +3 and La +3 The absolute chemical hardnesses of Au and AuC are 14.4 eV and 15.4 eV, respectively. + , Ag + , and Cu + Soft ions such as Sc have absolute chemical hardness values ​​of 5.7 to 6.3 eV. +3 and Al +3 Conventional hard ions such as are characterized by absolute chemical hardness values ​​greater than 24 eV. Thiele et al., Cancer Biother Radio, 2018 33(8):336-348. The most commonly used chelates of Ac(III) are in the range of 8-12 coordination, so Ac +3 The large ionic size of actinium makes it suitable for large multidentate chelators of high densities. Actinium, similar to other actinides and rare earth elements, undergoes hydrolysis in solution to give [Ac(OH) 3-x ] x- can be formed and sub-picomolar concentrations of Ac-225 then cause the hydroxide species to form radioactive colloids that bind to surfaces such as reaction vessels.

[0006] The emission of multiple alpha particles in the decay chain of Ac-225 makes it a particularly effective isotope for killing cancer cells, but it still makes the directed delivery of the nuclide and its decay daughter nuclei difficult. Because of conservation of momentum, the emission of energetic alpha particles often imparts recoil energies of over 100 keV to the daughter nuclei, 1000 times higher than the binding energy of any chemical bond. This results in the daughter nuclei being released from the chelator of the original delivery vector. Subsequent redistribution of the alpha-emitting daughter nuclei in vivo can cause substantial harm to non-targeted healthy tissues, reducing the efficacy of the treatment. Davis et al., Nuc Med Biol 1999, 26(5):581-589, reported that there is limited information regarding the in vivo behavior of Ac-225. Preliminary studies have evaluated Ac-225 complexed with citrate for tissue uptake, biodistribution, and tumor tropism in animal models. Previous studies with Ac-225 complexed with either the polyaminocarboxylic acid chelators, ethylenediaminetetraacetic acid (EDTA), or cyclohexyldiethylenetriaminepentaacetic acid (CHX-DTPA), demonstrated variable tissue tropism and increased blood clearance compared to uncomplexed Ac-225.

[0007] Ac-225-CHX-DTPA-monoclonal antibody (Mab) complexes, used to measure biokinetic behavior in tumor-bearing nude mice, were successful in complexing in vitro but showed poor stability in vivo. Thus, Ac-225 may be useful in radiotherapy models, but there is a paucity of information on potentially effective chelators and the relative stability of such Ac-225 complexes in vivo. A recent review article on Ac-225 radiopharmaceuticals, Robertson et al., Curr Radiopharm, 2018, 11(3):156-172, points out that finding a chelating agent that binds Ac(III) with sufficient stability and also controls the release of its daughter nuclide remains a challenge. Furthermore, the limited global availability of Ac-225 and the lack of a stable alternative nuclide have limited research with this isotope to a handful of institutions worldwide that have a reliable supply of Ac-225. The authors of the above review articles, including that of Davis et al. mentioned above, indicate that the biodistribution profile of each of the purified Ac-225 complexes over an 8-day period was assessed by injecting 92 kBq (2.5 mCi) of each complex and compared with the biodistribution of Ac-225 acetate as a control.

[0008] The high liver uptake of chelated Ac-225 indicates an unstable complex in vivo, since uncomplexed Ac-225 accumulates primarily in the liver, with smaller amounts in bone, kidney, and heart. Cyclohexyldiethylenetriaminepentaacetic acid "a" isomer (CHX-A"-DTPA) and 1,4,7,10,13-pentaazacyclo-pentadecane-N,N',N",N"',N""pentaacetic acid (PEPA) reduced the liver uptake of complexed Ac-225 by more than 5.5-fold compared with Ac-225 acetate, and the data from Davis et al. suggest that -CHX-A"-DTPA was the most effective chelator complex tested in terms of in vivo stability, although the authors of the review by Robertson et al. note that "further improvements are possible to further reduce accumulation in nontarget tissues" [Robertson et al., p. 164]. Therefore, CHX-A"-DTPA chelates Ac(III) poorly. Another important finding of the initial in vivo study commented by Robertson et al. was that the maximum tolerated dose of Ac-225-CHX-A"-DTPA was less than 185 kBq (5 mCi), since at doses of 185 kBq (5 mCi) and above, severe tissue damage was observed as early as 1 hour post-injection (pi) and ultimately led to the death of the test animals, causing 100% mortality by day 8 pi.

[0009] Conjugation of actinium to targeting molecules was achieved by Sheinberg's research group (Sheinberg, Science 2001 Nov 16; 294(5546):1537-1540. doi: 10.1126 / science.1064126). The chelator chosen was a DOTA-based bifunctional molecule. However, in the Sheinberg group's report, a two-step method was used to obtain sufficient Ac-225 for the targeting moiety. Furthermore, the yield based on the Ac-225 starting material was very low, with less than 10% of the isotope incorporated into the targeting moiety. Over 90% of the isotope was wasted. The specific activity of this method ranged from about 50-70 μCi per mg of antibody. Clearly, a higher yielding one-step method would be desirable. Further investigation of potential chelators by the Scheinberg research group [McDevitt et al., App. Radiat. Isot., 2002, 57(6):841-847] showed that of six potential chelators studied, only DOTA and 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrapropionic acid (DOTMP) demonstrated complexation of Ac225 with radiochemical yields (RCYs) of >99% and 78%, respectively, after 2 hours at 37°C. However, subsequent in vitro stability assays in serum suggested that the Ac-225 DOTA complex was robust, remaining >90% intact after 10 days, whereas the Ac-225-DOMTMP complex rapidly dissociated.

[0010] A two-step labeling method was again employed, requiring first radiolabeling of the bifunctional DOTA-NCS ligand, followed by conjugation of the mAb (pH 8.7, 37°C, 52 min). Although the overall radiochemical yield was low at only 9.8±4.5%, a reasonable specific activity (4.1±2.6GBq / g, or 0.11±0.07Ci / g) was achieved, allowing preclinical therapeutic studies. The low yield was attributed to the initial Ac-225 labeling step of DOTA-NCS requiring heating, resulting in decomposition of the isothiocyanate linker and insufficient conjugation of the mAb in the next step. The Scheinberg group and coworkers [McGuire et al., J. Nucl. Med., 2014, 55(9):1492-1498] later reported a one-step method for the preparation of Ac-225-DOTA-antibody constructs. This process combines the DOTA-antibody construct and Ac-225 +3 The reaction proceeded in 2 M tetramethylammonium acetate buffer (pH 7.5) with the addition of L-ascorbic acid as a radioprotectant, with a typical final reaction pH value of 5.8. Heating for 2 h at 37 °C increased the radiochemical yield by 10-fold (80%) compared to previous two-step methods (6-12%) and allowed the preparation of bioconjugates with up to 30-fold higher specific activity (120 GBq / g versus 3.7-14.8 GBq / g). The highest non-reactive activity achieved was equivalent to one actinium per 25 antibodies.

[0011] US2004 / 0067924 A1 (Frank) teaches the use of 12-membered macrocyclic amine-based polyacetate and polyphosphonate chelators to complex Ac-225. DOTA-based chelators have been found to be useful for chelating Ac-225. Paragraph

[0082] of the patent publication states that the nitrobenzyl group of one DOTA chelant shown can be reduced to an aniline, the amine of which can then be converted to an isothiocyanate to form a bifunctional compound for linking to a targeting peptide antibody or other entity. A bifunctional analog of PCTA (below) can reportedly be prepared by attaching a linking group to one of the acetate carbons.

[0012] 3,6,9,15-tetra-azabicyclo-[9.3.1]pentadeca-1(15),11,13-triene-3,6,9-acetic acid (PCTA) based chelators were described in the body of the Flank application and actinium binding data was presented. The PCTA compounds shown and used were not compatible with linking to targeting molecules such as peptides or antibodies, other than the possibility of using one of the chelating carboxyl groups. No targeting constructs using PCTA were disclosed. Yapp et al., Mol Imaging June 2013 12(4):263-272 reported the use of PCTA, DPTA, and 1-oxa-4,7,10-triazacyclododecane-4,7,10-triacetic acid (Oxo-DO3A) for the chelation of Cu-64 [Cu(II)-64] for use in PET scan studies of tumor vasculature. This chelate was conjugated to the cyclic tetrapeptide cyclic (RGDyK) via a benzylisothiocyanate linkage to an appended lysine of the cyclic peptide. Another study reported by Bryan et al., Cancer Biol Ther June 15, 2011 11:12, 1001-1007, discussed the results of radioimmunotherapy with Cu-64 linked to internalizing and non-internalizing mAbs using 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) as a chelating agent and PET scans in the treatment of colon cancer tumors in xenografted mice. Daily tumor measurements showed that PET scans were useful and that the use of internalizing antibodies did not improve the outcome of Cu-64 radioimmunotherapy.

[0013] An earlier one-step method was disclosed in Simon, WO 2011 / 011592 A1. This patent application teaches the preparation of a protein conjugated with a chelator as a first step. After removing excess chelator, the protein chelate conjugate was reacted with an isotope. Again, a DOTA-based chelator was used in the study, and current thinking in the art still indicates that a DOTA-based chelator is optimal for Ac-225. The method disclosed by Simon required the use of high concentrations of acetate ions and high chelator:antibody ratios (CARs). Initial reactions were performed using a molar reactant ratio of 100 chelator per antibody, resulting in CAR numbers of 10-12. It is desirable to generate high specific activity Ac-225 constructs with lower CAR number conjugates. This is because as the CAR number increases, the biological targeting of the antibody decreases. Thus, even though a one-step method is taught using Ac-225 and a DOTA-type chelator, the number of CARs required was high when using a DOTA-type chelator. It is clear that a better chelator is needed for the preparation of Ac-225 constructs.

[0014] Regardless of the difficulties of using DOTA as a chelator of Ac-225 as described above, Thiele et al., Cancer Biother Radio, 2018 33(8):336-348, as recently as 2018, used the phrase “DOTA: the current gold standard” (p. 340) in a review section. The final sentence of their DOTA section states: “In summary, these shortcomings make DOTA 225 Ac-TAT[ 225 indicated that it is not ideal for use in Ac targeted alpha therapy applications. 225 These results highlight the need for a more suitable chelating scaffold for Ac." The chemistry involved in attaching Ac-225 to targeting moieties has been challenging for users and authors. Clearly, a better way to attach Ac-225 to molecules is needed. The present invention helps address that need. Surprisingly, we found that PCTA-based chelators form stable chelates with Ac-225 under mild conditions with lower CAR numbers than previously reported using the prior "gold standard" DOTA.

[0015] Bi-213 is a radioactive decay product of Ac-225, while Bi-212 is produced by the radioactive decay of lead-212 (Pb-212) following the stepwise decay of uranium-234 (U-234). The short half-lives of Bi-212 and Bi-213 may limit the application of these radionuclides in radionuclide therapy. The bismuth isotopes Bi-212 and Bi-213 are also candidates for use in radioimmunotherapy. Several preclinical studies have been published utilizing one, the other, or both isotopes. Both have a valence of +3 and therefore tend to remain complexed after actinium decay. Bi-212 has a half-life of about 1 hour and emits both alpha and beta particles in a ratio of approximately 1:2. Bi-213 has a half-life of about 45 minutes and decays almost completely by beta emission to polonium-213, which then emits an alpha particle to form lead-209, as shown in Figure 1 herein.

[0016] For example, Park et al., Blood, 2020 116(20):4231-4239, treated mice bearing Ramos lymphoma xenografts with an anti-CD20 antibody fused to streptavidin, followed by [ 213reported preclinical studies of tumor-bearing mice treated with [Bi]DOTA-biotin. Treated tumor-bearing mice showed significant growth delay and median survival was approximately four times longer than untreated controls. A review by Yong et al., AIMS Med Sci, 2021, 2(3):228-245, describes recent studies using Pb-212 / Bi-212 for targeted alpha particle therapy (TAT), including a study using the HER2-binding mAb trastuzumab linked to the chelator 2-(4-isothio-cyanatobenzyl-1,4,7,10-tetraaza-1,4,7,10-tetra(2-carbamonylmethyl)cyclododecane (TCMC). A review by Mulford et al., J Nucl Med 2005, 46(1 Suppl):199S-204S, describes several TAT therapies utilizing one or the other of the above bismuth isotopes.

[0017] As mentioned in Yong et al. above, labeling biomolecules with the precursor Pb-212 instead of Bi-212 or Bi-213 has the advantage of providing a conjugate with a half-life of 10.6 hours compared to 60 minutes for Bi-212 or 46 minutes for Bi213. Previous attempts to create a potential in vivo generator by complexing Pb-212 with a DOTA chelator failed due to reports that β-particle-induced decay of Pb-212 to form Bi-212 results in leakage of about 36% of Bi, which is not retained by DOTA. Since free bismuth ions are localized in the kidney, it may be important that the Bi-212 formed by the decay of Pb-212 remains bound to the carrier. Bartos et al., J Radioanal Nucl Chem, 2013 295:205-209.

[0018] Zirconium-89 is another useful radioisotope in that zirconium has an atomic valence and Zr-89 emits gamma rays (909 keV) and positrons of approximately 397 keV, both of which are useful for diagnostics. Zr-89 has a half-life of 3.3 days, which is similar to the circulating half-life of many monoclonal antibodies used in medicine. These isotopes have been used to radiolabel and evaluate mAbs in positron emission tomography (Immuno-PET) [Saleem et al., Sci World J 2014, Article ID 269605, page 9]. The final decay product of Zr-89 is the stable, non-radioactive isotope yttrium-89. A further isotope useful in the present invention is indium-111. Indium also has a valence of +3, and the half-life of In-111 is approximately 2.8 days. Decay of indium-111 provides gamma radiation at 0.171 MeV and 0.245 MeV, which can be used for diagnostic scans such as single photon emission computed tomography (SPECT) imaging. In-111 decays to cadmium-111, which is non-radioactive and stable.

[0019] The invention disclosed below teaches the use of a single chelator with various targeting species for both therapeutic and diagnostic (theranostic) applications, providing a single chelator-linked targeting system for both applications. Such theranostics have significant advantages in development and manufacturing, since the radioisotope labels are separate, but the manufacturing steps for the targeting species and chelator can be common. This provides several time and cost advantages in development, toxicity testing with unlabeled targeted chelators, common stability, and drug substance. Summary of the Invention

[0020] Summary of the Invention The present invention relates to the use of chelating agents containing 12-membered macrocyclic amines with surprisingly embedded pyridine rings in their structures that readily form stable metal-ligand complexes with trivalent radioisotope ions, such as Ac-225, Bi-212, Bi-213, Zr-89 and In-111, as well as with targeting species molecules to form radiotherapeutic or radiodiagnostic agents (or generally radiopharmaceuticals). These radiopharmaceuticals may also be referred to as radiotheranostics. In one embodiment of the present invention, a chelator is attached to the targeting species molecule, providing Q to that portion of the molecule. +3 The chelator is attached to a portion of the targeting molecule that does not interfere with the ability of the targeting molecule to reach its target. The targeting species allows the radiopharmaceutical to bind to a cell to be killed or whose presence, location, size, and / or shape are to be determined.

[0021] More specifically, the targeting species is a chelated trivalent radioisotope ion Q +3 to form a theranostic radiopharmaceutical having the general structural formula shown in Formula I below, and which, depending on the radioisotope being chelated, can be used therapeutically to either kill a target cell or bind to a target cell to obtain one or more signals of the presence, location, size or shape of the bound cell. [ka] In that chelator, chelation Q +3 The ion has an atomization of +3 and is R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , and R 7 Six of the X's are H and the seventh is a radioisotope that contains a reacted functional group Z that forms a chemical bond with the targeting species T. 1 , X 2 , and X 3may be coordinated to the ion and / or chelating Q such as trivalent Ac-225, Bi-213, Bi-212, Zr-89 or In-111. +3 "g" is the number of chelated PCTA-chelated trivalent radioactive ions Q per molecule of targeting species T. +3 The average value is from 1 to about 12. - can be present in an amount necessary to balance the ionic charge. First, Q +3 A chelation reaction with the ion can be performed, followed by binding to the targeting species molecule, T. This is called a two-step method, since the isotope is handled twice. Alternatively, a binding reaction (binding the chelator to the targeting species) can be performed first, followed by binding to the targeting species, Q. +3 It is also possible to insert ions, which is called a one-step method and is preferred since only one isotope is required. 225 Ac +3 is a preferred Q +3 It is an ion.

[0022] Pharmaceutical compositions are contemplated that include a therapeutically effective amount of a targeted radiopharmaceutical of Formula I dissolved or dispersed in a pharma- ceutical acceptable diluent. Preferably, the pharma- ceutical acceptable diluent is an aqueous liquid at ambient temperature and adapted for parenteral administration. In one embodiment, the pharmaceutical composition is used in a method for treating a mammalian host having a disease, disorder or condition characterized by unwanted angiogenesis, tumor growth and / or tumor metastasis comprising administering to the host a targeted cell killing (therapeutic) effective amount of the targeted radiopharmaceutical. In a further embodiment, contemplated targeted radiopharmaceuticals are used as diagnostic agents.Thus, the present invention contemplates a method for assaying a mammalian host suspected of or known to have a disease, disorder or condition characterized by unwanted angiogenesis, tumor growth and / or tumor metastasis by administering to the host a target cell binding effective amount of a targeted radiopharmaceutical and then scanning the host to detect and locate the radiation emitted by the bound targeted radiopharmaceutical. [Brief description of the drawings]

[0023] [Figure 1] FIG. 1 shows the radioactive decay scheme from 229Th through 225Ac to stable 209Bi in the development of the preparation of 213Bi, with the emission of four alpha particles (α) and four beta particles (β-) as well as the half-life of each nuclide in the decay scheme shown in boxes, as reported in Huang et al., Comput Math Method M, Vol. 2012, Article ID 153212, page 6, where the number followed by the letter indicates the half-life, d=days, h=hours, m=minutes, ms=milliseconds, and ms=microseconds. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] Detailed Description of the Invention The present invention provides a PCTA chelating Q molecule chemically linked to a targeting species. +3 Contemplated targeted radiopharmaceuticals of the present invention have the general structure shown in Formula I below. [ka] In that targeted radiopharmaceutical, Q +3 is a trivalent radioisotope ion; R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , and R 7 Six of X are H and the seventh contains a reacted linking functional group Z that forms a chemical bond with the targeting species T. 1 , X 2 , and X 3 The base is 225 Ac +3 , 212 Bi +3 , 213 Bi +3 , 89 Zr +3 or 111 In+3 Q +3 It can coordinate to ions and chelate Q +3 "g" is the number of chelated PCTA-chelated trivalent radioactive ions Q per molecule of targeting species T. +3 The average value is about 1 to about 12. - can be present in an amount necessary to balance the ionic charge.

[0025] Exemplary targeted radiopharmaceutical chelates are illustrated below in Formulas Ia, Ib, Ic, and Id, without depicting the specific targeting species, the number of chelates bound to each targeting species, or the reacted linking functional group Z. 212 Bi +3 and 213 Bi +3 ) is for convenience, 212 / 213 Bi +3 is listed together with [ka]

[0026] Actinium-225 is the preferred radiopharmaceutical isotope because it has a half-life of approximately 10 days and decays to form the stable isotope bismuth-209, emitting four alpha particles and three beta particles, as shown in Figure 1. One of the daughter decay products from Actinium-225 is Bi-213, and the final decay product is Bi-209, which is not radioactive and is stable. Bismuth-212 is a decay product of lead-212. Once obtained, bismuth-212 can be separated from lead-212 and complexed with an appropriately linked PCTA to form a chelated Q-linked chelate that is chemically bound to the targeting species. +3 ions can be formed. Bi-212 eventually decays into lead-208, which is not radioactive and is stable. Some versions of these chelating agents are called pyridine-based 12-membered tetraazamacrocyclic ligands, or PCTAs (First published April 2, 2019; chemistry-europe.onlinelibrary.wiley.com / -doi / abs / 10.1002 / ejoc.201900280).

[0027] In one embodiment, the reacted functional group Z is selected from the group consisting of one or more of a reacted Michael reaction acceptor, a reacted isocyanato group, a reacted carboxyl group, and a 1,4-disubstituted-1,2,3-triazine formed by the reaction of an azide with an alkyne. A reacted isothiocyanate [-NH-C(=S)-NH-; thiourea] is one preferred reacted functional group. The number of chelators bound per antibody molecule is an average number, since in a given composition some antibody molecules will not react and others will. When isothiocyanate groups are bound to intact antibodies, the average number of chelators bound per antibody molecule is 1 to about 12, preferably about 3 to about 12, more preferably about 8 to about 10. When the paratope-containing portion (or antigen-binding fragment) is the targeting species, the number of PCTA chelators per molecule of the targeting species tends to be small, e.g., about 1 to about 5, since in the absence of the two pairs of CH2 and CH3 portions of the heavy chain, there are fewer lysine amino groups with which the isothiocyanate groups can react.

[0028] An exemplary pre-reacted chelator reactive functional group Z can be a Michael reaction acceptor such as maleimide, allowing up to about eight chelators to be linked to the reduced intact antibody thiol group. Of course, small targeting species such as the peptidomimetic cyclic (RGDyK) described below only have one amine available for attachment to the chelator, thus limiting the number of radiopharmaceutical chelates that can be linked. Michael reaction accepting groups include ab-unsaturated carbonyl groups that can react with nucleophiles such as amines or mercaptans. Exemplary Michael reaction accepting groups include acryloyl, methacryloyl, and maleimide groups. The precursors for the formation of 1,4-disubstituted-1,2,3-triazines, azides and alkynes can be present either on the pre-reacted functional groups of the chelator or on the targeting species, one each. The coupling reaction can be catalyzed by copper(II) ions or UV irradiation.

[0029] The targeting species T is selected from the group consisting of one or more of a chemically conjugated antibody or a paratope-containing portion of an antibody, a chemically conjugated hormone, a chemically conjugated non-antibody protein, a chemically conjugated cytokine, a chemically conjugated aptamer, a chemically conjugated oligonucleotide, a chemically conjugated cytokine, a linear or cyclic oligopeptide or peptidomimetic, and a linear or branched oligosaccharide. Monoclonal antibodies (mAbs) or paratope-containing portions thereof are preferred targeting groups, with humanized monoclonal antibodies or paratope-containing portions thereof being particularly preferred.

[0030] X 1 , X 2 , and X 3 The base is Q +3 are the same or different substituents that are functional groups useful for chelating that can coordinate to ions and / or serve to neutralize the ionic charge of the targeted radiopharmaceutical. Exemplary X substituents include -(CH2) n CO2M group, phosphonic acid (-PO3M2) group and its half ester, and carboxamide -(CH2) n CONH2 and -(CH2) n CH2NR 10 R 11 Primary, secondary or tertiary amines, where R 10 and R 11 are the same or different and are H or C1-C4 alkyl. In such substituents, M is a proton (H + ), ammonium ion or alkali metal ion. 1 , X 2 , and X 3Preferably, each of the groups are the same, and more preferably, each is a -COOM group. "n" is 0 or 1, and preferably is 0, such that the X group is -CO2M. It is understood that once in an aqueous composition, such as a buffer, the cationic M will likely exchange for another cation present in the aqueous composition.

[0031] The preferred chelators are referred to in the art as PCTAs. The chemical formula of a particularly preferred form of PCTA is (4-isothiocyanato-phenyl)methyl derivative, which allows the chelator to be bifunctional, shown below in Formula II, where M is as previously described. [ka] The chelating agent of formula II is commercially available from Macrocyclics Inc. (Dallas, TX) under the name p-SCN-Bn-PCTA.

[0032] Targeted species Antibodies are a preferred class of targeting species molecules in one aspect of the invention, as they often bind to cell surface antigens on unwanted cells in the body, such as cancer cells. Once bound, the antibody and its associated chelating Q +3 The ions are taken up into the unwanted cells (the cells being treated) and then Q such as Ac-225 is absorbed into the cells. +3 ions, or 213 Bi +3 One of its daughter atoms, such as Q, can break down and release its cytotoxic α particle in unwanted cells. +3 The specific combination of ion and PCTA chelator is Q +3 It forms a particularly stable chelate product compared to that formed using ions and DOTA as a chelator, resulting in higher concentrations of the radioisotope in the target cells and lower concentrations of the radioisotope elsewhere in the recipient's body than would be achieved using a chelator such as DOTA. An exemplary list of monoclonal antibodies for use as targeting species is shown in the table below. Most of the list are human or humanized antibodies and have been approved for use in human therapy, while others are murine antibodies. It should be understood that this list is only an example of the approximately 80-90 potentially useful monoclonal antibodies awaiting approval by the U.S. FDA for use in humans. [Table 1] TIFF2024519970000007.tif244160

[0033] The mAb designated mAb MNPR-101, as illustratively used herein, is a humanized version of the murine mAb ATN-658, the hybridoma of which has ATCC Accession No. PTA-8191 and is disclosed and claimed in U.S. Patent No. 8,101,726, and the murine mAb ATN-615, also disclosed and claimed in U.S. Patent No. 8,101,726, is secreted by a hybridoma having ATCC Accession No. PTA-8192. These mAbs specifically bind (immunoreact) with the binary complex termed uPA-uPAR, i.e., urokinase plasminogen activator (uPA) and its cell surface receptor, uPAR, as well as with uPAR at loci that do not prevent the formation of the binary complex. U.S. Patent No. 8,101,726 notes that expression of uPA and uPAR has been demonstrated in many tumor types. The paratopic amino acid residue sequences (CDRs; complementarity determining regions; variable regions) of mAb MNPR-101 bind to its uPA-uPAR antigen in a manner very similar to that of mAb ATN-658 (Table 3, below). The heavy chain constant regions (CH1, CH2, and CH3) are those of a human IgG1 antibody.

[0034] Humanization of ATN-658 to prepare MNPR-101 utilized the Xoma HE™ synthetic platform, which uses human antibody amino acid residue sequences reported in Wu and Kabat, 1992 Mol. Immunol., 29(9):1141-1146 (hereinafter Kabat) combined with the variable region sequences of the antibody to be humanized to form one or more consensus sequences. The method involves several steps: (1) Human Engineer™ (HE™) of ATN-658 light and heavy chains using XOMA Corp. (Emeryville, CA) proprietary HE™ technology to generate low-risk and low-plus-moderate-risk HE™ variants; (2) Codon optimization, energy minimization, and gene synthesis of the HE™ variable (V) region sequences; (3) cloning of the four HE™ V regions into XOMA's proprietary transient expression vectors containing human gamma-1 and kappa constant region modules; (4) transient expression of HE™ mutants; (5) purification of the humanized antibodies and characterization of their purity and endotoxins; and (6) Confirmation of the affinity of the four HE™ mutants. The term "low risk" as discussed above and below relates to whether the amino acid residue change from mouse to human results in a significant reduction in therapeutic immunogenicity with little or no effect on binding affinity. The alternative term "high risk" relates to modifications at positions where the amino acid residue change from mouse to human results in reduced or lost binding activity with little or no actual reduction in therapeutic immunogenicity.

[0035] Humanization of ATN-658 to generate mAb MNPR-101 using the Xoma HE™ platform was performed according to the "low risk," "medium risk," and "high risk" substitutions suggested in the following publications, patents, and patent applications: 1) WO 93 / 11794, "Methods and materials for preparation of modified antibody variable domains and therapeutic uses thereof"; 2) U.S. Patent No. 5,766,886, "Modified antibody variable domains"; 3) U.S. Patent No. 5,770,196, "Modified antibody variable domains and therapeutic uses thereof"; 4) U.S. Patent No. 5,821,123, "Modified antibody variable domains"; 5) U.S. Patent No. 5,869,619, "Modified antibody variable domains," and 6) Studnicka et al. 1994 Protein Eng 7:805-814, the disclosures of which are all incorporated herein by reference. Further details regarding the preparation of mAb MNPR-101 are provided in the Examples below.

[0036] The term "antibody" is meant to include both intact immunoglobulin (Ig) molecules and fragments and derivatives that can be produced by proteolytic cleavage of Ig molecules or that can be genetically or chemically engineered. Paratope-containing portions or fragments include, for example, Fab, Fab', F(ab')2, and Fv, each of which can bind to an antigen. These fragments lack the Fc fragment of intact antibodies (Ab) and have the added advantage that, when used therapeutically, they are cleared from the circulation more rapidly and have less nonspecific tissue binding than intact antibodies. Papain treatment of Ig produces Fab fragments; pepsin treatment produces F(ab')2 fragments. These fragments can also be produced by genetic or protein engineering using methods well known in the art.

[0037] A Fab fragment or portion is a multimeric protein consisting of a portion of an Ig molecule that contains the immunologically active portions of a covalently linked Ig heavy (H) chain and an Ig light (L) chain and is capable of specifically binding to an antigen. Fab fragments are generally prepared by proteolytic digestion of substantially intact Ig molecules with papain, using methods well known in the art. However, Fab fragments can also be prepared by expressing the desired portions of Ig H and L chains in a suitable host cell, using methods well known in the art. F(ab')2 fragments are tetramers that contain fragments of two H and two L chains. Fv fragments consist of covalently linked IgH chain variable (V) regions (V H ) and IgL chain V region (V L Fv fragments are generally multimeric proteins consisting of the immunologically active portions of Ig V, which are capable of specifically binding to an antigen. ... H Area and V L The desired portion of the region is prepared by expressing it in a suitable host cell.

[0038] Single-chain antigen-binding proteins or single-chain Abs, also called "scFvs," areL The C-terminus of the sequence is V H IgV is a peptide linked to the N-terminus of the sequence. H Ig V linked to amino acid residue sequence L It is a polypeptide composed of an amino acid residue sequence. In a preferred embodiment, the Ab is a mouse monoclonal antibody (mAb) designated ATN-615 (Creative Biolabs, Inc., Shirley, NY) or ATN-658 (hybridoma B cell: ATCC PTA-8191; Manassas, VA), both of which are IgG1 antibodies. The Abs of the invention can be produced as single chain Abs or scFvs, instead of the usual multimeric structures. Single chain Abs contain the hypervariable region of the Ig of interest and recreate the antigen binding site of the native Ig at a fraction of the size of the intact Ig (Skerra et al., Science, 1988 240:1038-1041; Pluckthun et al. Methods Enzymol 1989 178:497-515; Winter et al., Nature 1989 349:293-299); Bird et al., Science 1988 242:423-426; Huston et al. Proc. Natl. Acad. Sci. USA 1988 85:5879-5883; Jost et al., J Biol Chem. 1994 269:26267-26273; (U.S. Patent Nos. 4,704,692, 4,853,871, 4,946,778, 5,260,203, and 5,455,030).

[0039] The DNA sequences encoding the H and L chain V regions are joined by a linker that encodes a sequence of at least about four amino acid residues (generally small neutral amino acids). The protein encoded by this fusion allows for the construction of a functional variable region that retains the specificity and affinity of the original Ab. Different types of single chain Abs are antibodies induced in camelids such as dromedaries, llamas, alpacas, and vicunas. These animals produce single heavy chain only antibodies (HcAbs) with variable and constant regions and many unique properties such as small size, good solubility, good stability, rapid clearance from blood, and deep tissue penetration. The name "nanobody" was first adopted by the Belgian company Ablynx® due to their nanometer size and molecular weight of less than about 15 kDa. Ablynx NV, an affiliate of Sanofi, is the worldwide owner of the NANOBODY® trademark.

[0040] However, the antigen-binding capacity of nanobodies remains similar to that of conventional antibodies for the following reasons. First, the complementarity determining region 3 (CDR3) of nanobodies is similar to or longer than the human VH domain (variable domain of the immunoglobulin heavy chain). The former consists of 3-28 amino acids (AA), while the latter is only 8-15 amino acids. As therapeutics, nanobodies enable targeted treatment by lesion-specific delivery of drugs and effector domains, thereby improving the specificity and efficacy of therapy [Bao et al., EJNMMI Res (2021) 11:6.]. Humanized versions of monoclonal nanobodies are generally prepared by methods similar to those used for the preparation of double-chain mAbs, except that fewer steps are generally required. The human genes encoding the constant (C) regions of the chimeric antibodies of the present invention can be derived from a human fetal liver library or any human cell, including cells that express and produce human Igs. H The region may be derived from any of the known classes or isotypes of human H chain, including gamma, mu, alpha, delta, or epsilon, and subtypes thereof, such as G1, G2, G3, and G4. The H-chain isotypes are responsible for various effector functions of Abs, so H The choice of region depends on the desired effector function, such as complement fixation, or Ab-dependent cellular cytotoxicity (ADCC) activity. HThe regions are derived from gamma 1 (IgG1), gamma 3 (IgG3), gamma 4 (IgG4), or μ (IgM). Human C L The region may be derived from human light chain isotypes kappa or gamma.

[0041] Genes encoding human IgC regions can be obtained from human cells by standard cloning techniques [Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Press, Cold Spring Harbor, NY (1989)]. Human C region genes are readily available from known clones that contain genes representing two classes of L chains, five classes of H chains, and their subclasses. In general, chimeric antibodies of the invention are prepared by cloning DNA segments encoding the heavy and light chain antigen-binding regions of a particular Ab of the invention, preferably a non-human Ab, and cloning these DNA segments into human C-terminal fragments. H and human C H These are produced by joining DNA segments encoding these regions to create a chimeric Ig-encoding gene. Thus, in a preferred embodiment, a fusion gene is made that includes a first DNA segment encoding at least an antigen-binding region of non-human origin, such as CDR1, CDR2 and CDR3 of a V region with a joining (J) segment, linked to a second DNA segment encoding at least a portion of a human C region.

[0042] Chimeric Ab fragments, such as F(ab')2 and Fab, can be prepared by designing an appropriately truncated chimeric H chain gene. For example, a chimeric gene encoding the H chain portion of an F(ab')2 fragment contains DNA sequences encoding the CH1 domain and hinge region of the H chain, followed by a translation stop codon to yield a truncated molecule. One common feature of all IgH and L chain genes and their encoded mRNAs is the J region. Although the sequences of the H and L chain J regions differ, there is a high degree of sequence homology (greater than 80%) between each group, especially near the C region. This method exploits this homology, using consensus sequences of the H and L chain J regions to design oligonucleotides for use as primers to introduce useful restriction sites into the J region for subsequent joining of the V region segments with human C region segments.

[0043] C region cDNA vectors prepared from human cells can be modified by site-directed mutagenesis to place restriction sites at the analogous positions in the human sequence. For example, the complete human kappa chain C (C κ ) region and the complete human γ-1C region (C γ-1 ) can be cloned into a bacterial system that is compatible with the C region vectors. In this case, alternative methods based on genomic C region clones as a source of C region vectors do not allow the expression of these genes in bacterial systems that lack the enzymes required for removal of the intervening sequences. The cloned V region segments are excised and ligated into L or H chain C region vectors. Alternatively, human C γ-1 The regions can also be modified to create a genetic sequence that encodes the H chain portion of a Fab molecule. The coding sequence having the combined V and C regions is then introduced into a suitable expression vehicle for expression in a suitable prokaryotic or eukaryotic host.

[0044] In another embodiment of the invention, the targeting molecule is a relatively small molecule, such as a linear or cyclic oligopeptide or peptidomimetic having a molecular weight of about 400 to about 1000 amu. One exemplary cyclic oligopeptide is a cyclic tetrapeptide called cyclic(RGDyK), which is expressed in endothelial cells of some tumors and tumor neovasculature. v Binds to the b3 receptor [Yapp et al., Mol Imaging June 2013 12(4):263-272]. A peptidomimetic is a compound whose essential elements (pharmacophores) mimic a natural peptide or protein in three-dimensional space and retain the ability to interact with a biological target to produce the same biological effect as the natural peptide or protein [Vagner et al., Curr Opin Chem Biol June 2008 12(3):292-296]. An exemplary peptidomimetic of interest here is an inhibitor of prostate-specific membrane antigen (PSMA).

[0045] PSMA is a surface type 2 integral membrane glycoprotein with folate hydrolase, carboxypeptidase, and internalization activities [Cimadamore et al., Front Oncol December 21, 2018 8:article 653]. PSMA is highly expressed on prostate cancer tumor cells as well as on blood vessels in a variety of non-prostate solid tumors. Monoclonal antibody J591 and the other three anti-PSMA monoclonals are each murine monoclonal antibodies. One or more of these mAbs are the subject matter disclosed and / or claimed in the following U.S. Patents: 6,107,090; 6,136,311; 6,649,163; 6,770,450; 7,045,605; 7,112,412; 7,163,680; 7,192,586; 7,514,078; 7,666,414; 7,666,425; and 8,951,737.

[0046] Structural and functional homology between N-acetylaspartylglutamic peptidases (N-acetylated alpha-linked acid dipeptidases; NAAALDASEs) has led to the identification of several inhibitors for NAAALDASEs. One of the most advanced peptidomimetic inhibitors is the urea-based PSMA ligand, which typically consists of three components: a binding motif (glutamic acid-urea-lysine [Glu-urea-Lys]), a linker, and a radiolabel-containing moiety (a chelator molecule for radiolabeling). A particularly useful such molecule is shown below without the chelator. [ka]

[0047] An example of a branched oligosaccharide targeting species is sialyl-Lewis a (sLe a )Sialyl-Lewis x (sLe x ) antigens, and others that may cause metastasis. Lintuzumab, an anti-CD33 mAb, binds to the sialohesin receptor CD33. Folic acid and derivatives can be used as targeting species for cancerous renal cells that overexpress folate receptors. Folate receptors are also overexpressed in brain, kidney, lung, ovarian, and breast cancers compared to lower levels in normal cells (see, for example, Sudimack et al., Adv Drug Deliv Rev 2000 41:147-162). Figliola et al., RSC Adv 2019 9:14078-14092, show synthetic routes for the preparation of folate targeting species, including the drug prodigiosene, using several α,ω-amino linking groups such as ethylenediamine, ethylene oxide, cystamine, and diamino oligo oxytheylene. Immune cells have an affinity for mannose, and several RGD-containing targeting peptides containing 4-30 amino acid residues are known, many of which are described in Beer et al., Methods Mol. Biol. 2011 680:183-200; Beer et al., Theranostics 2011 1:48-57; Morrison et al., Theranostics 2011 1:149-153; Zhou et al., Theranostics 2011 1:58-82; and Auzzas et al., Curr. Med. Chem. 2010 17:1255-1299, and Goonewardena et al., U.S. Patent No. 9,931,412.

[0048] Pharmaceutical Compositions Pharmaceutical compositions containing a therapeutically effective amount of a contemplated targeted radiopharmaceutical dissolved or dispersed in a pharma- ceutical acceptable diluent are used in the contemplated methods. In one embodiment, where the treatment is therapeutic, the therapeutically effective amount is a target cell killing effective amount. Such compositions are administered to a mammalian host animal in vivo to bind to and kill unwanted target cells, such as cancer cells and aberrant immune cells. Exemplary unwanted target cells include cells that are involved in unwanted cell migration, invasion, proliferation, immune response or angiogenesis.Examples of such cells include abnormal immune cells and cancer cells, such as lung cancer, ovarian cancer, prostate cancer, brain cancer, bladder cancer, head and neck cancer, pancreatic cancer and colon cancer.Treatment of blood cancers, such as acute myeloid leukemia that expresses CD33 marker, and breast cancer that expresses HER2 marker is also contemplated.

[0049] Targeted radiopharmaceutical Q administered therapeutically to provide an effective amount for target cell killing +3 The theranostic dose of ions usually varies depending on the patient and the severity of the disease, such as the tumor burden in the cancer situation the patient has. However, about 80 to about 120 kBq / kg body weight given every other month (every other month, about 60 days apart) for 2 to about 4 cycles generally shows good results. 225 The same dosing regimen has been reported to produce good results using AC-PSMA-617 at approximately 100 kBq / kg body weight for three cycles, leading to complete remission in some patients. See Kratochwil et al., J Nucl Med 2016 57(12):1941-1944; Langbein et al., J Nucl Med 2019 60:13S-19S; and Eder et al., Pharmaceuticals 2022 15:267. Such doses can be used to provide a dose basis for therapeutic treatment of other conditions.

[0050] For diagnostic purposes, the host is administered a theranostic amount, which is a target cell binding (diagnostic) effective amount of the targeted radiopharmaceutical. The host is then maintained for a period of about 1 hour to several days, more usually about 1 to about 4 hours, to allow the radiopharmaceutical to bind to the target cells. The maintenance time may vary depending on several factors, such as the decay rate of the trivalent isotope used and the clearance rate of the targeted radiopharmaceutical. The maintained host mammal is then scanned by a positron emission tomography (PET) scan or a gamma detector (e.g., SPECT scan) to detect and localize radiation emitted by the targeted radiopharmaceutical bound to the target cells, thereby identifying one or more of the following: 1) that the targeted cells were present within the host, 2) the location within the host body of the targeted cells, 3) the size, and in some cases 4) the shape of the cell mass bound by the targeted species. The diagnostically effective amount of the targeted radiopharmaceutical administered is generally sufficient radioisotope to provide about 0.5 to about 6 mCi for adults, and appropriately less for children. In-111 is generally used at about 111 MBq (3 mCi) to about 222 MBq (6 mCi) for intravenous administration to an average adult (70 kg). Patients can receive about 0.5 to about 2 mCi of Zr-89 intravenously for whole-body PET scans.

[0051] Since the contemplated targeted radiopharmaceutical pharmaceutical compositions are intended for parenteral administration by injection, such compositions should contain electrolytes, preferably with osmolality and pH values ​​approximately equal to the physiological osmolality and pH of the intended recipient mammalian species. The preferred concentration of monovalent electrolyte ions in the targeted radiopharmaceutical pharmaceutical composition is about 0.5 to about 1.5% (w / v), more preferably about 0.8 to about 1.2% (w / v), and most preferably about 0.9% (w / v). A concentration of about 0.9% (w / v) is particularly preferred, as it corresponds to a nearly isotonic solution for humans. In a further preferred embodiment, the electrolyte in the chemoablative pharmaceutical composition is sodium chloride. Such levels of electrolytes increase the osmolality of the targeted radiopharmaceutical pharmaceutical composition. Thus, instead of specifying a range of electrolyte concentrations, osmolality can be used to partially characterize the electrolyte levels of a composition. Preferably, the osmolality of the composition is greater than about 100 mOsm / kg and less than about 520 mOsm / kg, more preferably, the osmolality of the composition is greater than about 250 mOsm / kg, and most preferably, the osmolality is between about 300 and about 500 mOsm / kg.

[0052] The pH value of the targeted radiopharmaceutical composition is preferably from about 4 to about 9 to maximize solubility of the targeted radiopharmaceutical in the aqueous vehicle and ensure compatibility with biological tissues. Particularly preferred pH values ​​are from about 5 to about 8, more preferably from about 6 to about 7.5. The pH value of the targeted radiopharmaceutical pharmaceutical composition can be adjusted or regulated by any suitable means known to those skilled in the art. The composition can be buffered or the pH value can be adjusted by the addition of acids or bases, etc.

[0053] Since the contemplated targeted radiopharmaceutical compositions are intended for parenteral administration, they are <71> and contain negligible levels of pyrogens, such as those required for compliance with USP 1999 / 100 / 1000 / 1000 / 2000 / 1 ... <85> (limulus amebocyte assay) or USP <151> (Rabbit Pyrogen Test), or substantially equivalent requirements. <788> (i.e., NMT 3000 particles per container if the size exceeds 10 microns, and NMT 300 particles per container if the size exceeds 25 microns), or a substantially equivalent requirement. Each of these references from the USP is incorporated herein by reference.

[0054] Exemplary mammalian hosts to which contemplated targeted radiopharmaceutical compositions can be administered include primates, such as humans; apes, such as chimpanzees or gorillas; monkeys, such as cynomolgus monkeys or macaques; laboratory animals, such as rats, mice or rabbits; pet animals, such as dogs, cats, horses; or food animals, such as cows or steers, sheep, lambs, pigs, goats, llamas, etc. The contemplated pharmaceutical compositions are typically administered multiple times to a mammalian host over the course of several weeks or months. As mentioned above, a typical dosing schedule is every other month. Screening the host between doses can provide up-to-date information so that the attending physician can decide regarding further treatment. As previously mentioned, three consecutive doses of 100 kBq / kg of various Ac-225-containing targeted radiopharmaceutical compositions administered every other month (approximately 60 days apart) have resulted in complete remission in some prostate cancer patients.

[0055] Formulation of parenteral compositions is described, for example, in Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania; 1975 and Liberman, HA and Lachman, L., eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, NY, 1980. For injectable preparations, for example, sterile injectable aqueous suspensions can be formulated according to known techniques using suitable dispersing or wetting compounds and suspending materials. Sterile injectable preparations can also be sterile injectable solutions or suspensions in non-toxic parenterally acceptable diluents or solvents, for example, solutions in 1,3-butanediol. Acceptable vehicles and solvents that can be used include aqueous liquids at ambient temperature, such as water, Ringer's solution, and isotonic sodium chloride solution, phosphate-buffered saline, etc. Liquid pharmaceutical compositions include, for example, solutions suitable for parenteral administration. Sterile aqueous solutions of targeted radiopharmaceuticals or sterile solutions of targeted radiopharmaceuticals in solvents including water, ethanol, DMSO, or propylene glycol are examples of liquid compositions suitable for parenteral administration. Sterile solutions can be prepared by dissolving the targeted radiopharmaceutical component in the desired solvent system and then sterilizing the resulting solution by passing it through a membrane filter, or by dissolving the sterile compound under sterile conditions in a previously sterilized solvent. EXAMPLES

[0056] Example 1 Two bifunctional chelators were purchased from Macrocyclics, Dallas, TX. The structures of these two are shown below. Their Macrocyclics catalog names are: S-2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecanetetraacetic acid and 3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-4-S-(4-isothiocyanatobenzyl)-3,6,9-triacetic acid. They are also named p-SCN-Bn-DOTA and p-SCN-Bn-PCTA as the unreacted precursors, respectively. Once reacted with the targeting species, they are more simply called DOTA and PCTA and will be referred to as such herein. [ka]

[0057] Conjugation reactions with monoclonal antibody (MNPR-101) were performed in metal-free vials and glassware was acid washed to remove potential metal contamination. Reactions were performed using 2 mg of antibody and increasing molar reactant ratios of bifunctional chelators. Monoclonal antibody (mAb) MNPR-101 is a humanized version of murine IgG1, kappa, mAb ATN-658, ATCC Accession No. PTA-8191, disclosed and claimed in U.S. Patent No. 8,101,726. The mAb MNPR-101 paratope amino acid residue sequences (CDRs; complementarity determining regions) are identical to those of ATN-658, but the framework portions of the variable regions are humanized and the Fc portion is that of a human IgG1 antibody. For the PCTA chelator, the molar reaction ratios were 1, 3, 5, 10, and 20. For the DOTA chelator, the molar reaction ratios were 3, 10, 25, 50, and 100. The pH of the solution was adjusted to 9.2 with 1 M Na2CO3. The reaction was carried out at 37°C for 1.5 hours.

[0058] The conjugate was purified using a Bio-Rad 10DG gravity-fed column with a molecular weight cut-off of 6,000 Daltons. The column was rinsed with 15 mL of 0.1 M HEPES buffer in 0.1 M NaCl. The pH of the buffer was 7.3. The entire contents of the reaction vial were loaded onto the top of the column and collected in a 2 mL tube. Multiple 0.5 mL elutions using the same buffer were also collected in separate tubes. Protein-containing fractions were determined by measuring the UV absorbance at 280 nm of each fraction. Typically, the protein eluted in four fractions, which were combined. The protein content of the combined fractions was measured using the Pierce BCA assay kit. The concentration of the resulting protein conjugate was approximately 1 mg / mL. Analysis of each conjugate was performed by size-exclusion HPLC. The column was from IGM Tosoh (TSKgel G3000SWx1; Tosoh Bioscience LLC, King of Prussia, PA). The mobile phase was phosphate-buffered saline and the flow rate was 1 mL / min. UV detection at 280 nm was used. HPLC results showed an early eluting peak with a retention time of approximately 8 min, consistent with a highly pure conjugate. The retention time of the conjugate decreased slightly with increasing ratios of bifunctional chelator, consistent with the addition of chelator to the antibody.

[0059] Example 2 Conjugates were prepared according to the method of Example 1, except that the molar reaction ratios of chelator to protein were 12 and 25. Typical reaction yields are about 30%. Thus, the reactions are expected to have average CAR numbers of about 4 and 8. Ac-225 was obtained from ORNL. Reaction vials contained solid Ac-225, which was dissolved using 0.2M HCl. Ac-225 chelates were prepared using the same four conjugates described in the previous section. A ratio of 50 μCi of Ac-225 to 50 μg of MNPR-101-PCTA chelator conjugate was used, resulting in a specific activity of 1 mCi / mg for a 100% yield. Reactions were performed in a volume of 100 μL. This volume contained approximately 4 μL of Ac-225 in 0.2M HCl, 60 μL of 0.1M ammonium acetate buffer, and 36 μL of MNPR-101-PCTA or MNPR-101-DOTA conjugate. Reactions were incubated at pH 5.8 and 37° C. for 60 minutes.

[0060] The radiochemical yield of the reaction was determined by diluting a 50 μL aliquot of the reaction to 3 mL with buffer and passing it through a 30 kDa Amicon® filter. The small unchelated Ac-225 ion passes through the filter, while the conjugate is retained by the filter. Samples were counted after 45 minutes on a Ge detector using the first daughter of Ac-225 (Fr-221). Samples were further counted using a dose calibrator after equilibrating Ac and its daughter nuclei overnight (approximately 18 hours). Chelation results are shown below. [Table 2] The above table shows quantitative yields for both PCTA conjugates, but the yield for the DOTA conjugate is much lower, with a significant difference between the 12:1 and 25:1 conjugates. Surprisingly, even at lower CAR numbers, the PCTA conjugates show higher yields.

[0061] The specific activity of the chelate formed from PCTA was 1,000 μCi / g, whereas the specific activity of the chelates from the conjugates prepared from DOTA ranged from about 216 to 284 mCi / g. This direct comparison of DOTA and PCTA indicates the superiority of the PCTA chelator over the DOTA chelator for chelation of Ac. The purity of the above samples was determined using high performance liquid chromatography (HPLC) using a size exclusion column with phosphate buffered saline as the mobile phase. The HPLC data showed essentially the same results as the filtration method.

[0062] Example 3 The MNPR-PCTA conjugate of Example 2 was chelated with Ac-225 at a chelator to antibody starting reaction ratio of 12:1. If the reaction is quantitative, this will give a specific activity of 1 mCi / mg. The same chelation reaction was carried out with the DOTA conjugate of MNPR-101 at a molar reaction ratio of chelator to antibody of 25:1. Additionally, bovine serum albumin without added chelator was used as a negative control. The total volume of each reaction was 150 μL. The yield of each reaction was determined using the filtration method of Example 2. The percent of activity in the retentate was used as the yield of the reaction.

[0063] In a parallel study, the above reaction was run containing an additional 35 μL of 0.1 M diethylenetriaminepentaacetic acid (DTPA) and the reaction was allowed to stand at room temperature for 1 hour, again using the filtration method with counts as above to determine yield or purity. The results of both studies are shown in the table below. [Table 3] The initial yield of MNPR-PCTA (12) was 99.1%. After addition of DTPA, the chelate lost only about 1% of its activity. In contrast, the initial yield of MNPR-DOTA (25) was only 8.9%, which dropped to 5.6% after addition of DTPA. Furthermore, the control BSA showed only 3.4% of the protein-associated activity (nonspecific binding), which dropped to 1.3% after DTPA washing. This data is consistent with PCTA outperforming DOTA in its ability to chelate Ac-225 even at lower CAR ratios. Furthermore, the lack of binding to bare BSA indicates that nonspecific binding is not an issue.

[0064] Example 4 Conjugates of MNPR-101 (MNPR) and PCTA have been shown to efficiently chelate Ac-225. In a direct comparison, Ac-225 chelated much more efficiently to PCTA conjugates than to DOTA conjugates. Ac-225 was obtained from ORNL. The conjugates used in these reactions were prepared previously and are described in Examples 2 and 3 above. Bovine serum albumin (BSA) was used as a negative control protein with no chelator attached. MNPR-PCTA(12) refers to a MNPR-101 conjugate made with p-SCN-Bn-PCTA (PCTA) at a starting molar reaction ratio of PCTA to antibody of 12:1. MNPR-DOTA(25) refers to a conjugate of MNPR-101 at a starting molar reaction ratio of p-SCN-Bn-DOTA (DOTA) chelator to antibody of 25.

[0065] Reactions were targeted to produce 1 mCi / mg, assuming 100% Ac incorporation into the antibody. Reactions were performed in a volume of 150 μL and incubated at pH 5.8, 37°C for 60 min. Following reaction, a 35 μL aliquot of each reaction was mixed with 35 μL of 1 M diethylenetriaminepentaacetic acid (DTPA) and incubated at room temperature for 1 h. The solutions were tested for percent protein associated Ac-225 as a function of time (1, 24 and 72 hours) by filtration (counts) as described above. The results of the initial tests are shown in the table below as percent protein associated Ac-225 as a function of time. [Table 4]

[0066] The percentages indicate the relative amount of activity in the filter compared to the total (filter + filtrate). MNPR-PCTA (12) gave the best results with 99% and 98% binding to the antibody (on the filter) after 1 and 24 h of incubation with DTPA. After 72 h, the purity dropped to 73%. Note that no radioprotectants were added and that Ac-225 imparts a high radioactive dose to the solution. The fact that the isotope remained associated with the protein indicates a high degree of stability and little loss of bismuth decay products to the solution during the time period examined.

[0067] Both the control (BSA) and MNPR-DOTA (25) had significantly lower percentages of activity associated with the protein. High-resolution γ-spectroscopic analysis of the solutions was consistent with the filtration results in Table 1. Antibody MNPR-101 conjugated to PCTA with a starting chelator to antibody molar reaction ratio of 12:1 was shown to reproducibly chelate Ac-225 with high yields and high specific radioactivity (1,000 μCi / mg). When the material was incubated in excess of DTPA, it showed a high degree of stability even when the formulation did not contain a radioprotector. Direct comparison with the same antibody conjugated to DOTA with a starting ligand to protein molar ratio of 25:1 gave much lower yields, indicating the advantage of PCTA over DOTA for chelation of Ac-225. Naked BSA was used as a control and showed a small amount of nonspecific binding.

[0068] Example 5 Formula I (where Q +3 teeth 125 Ac +3 A targeted radiopharmaceutical containing Ac-225 chelated by PCTA bound to mAb MNPR-101, as shown by (Figure 1), was prepared as previously described. The starting molar ratio of chelator to antibody was 12:1. 50 μCi of Ac-225 was mixed with 50 μg of MNPR-PCTA conjugate, and the pH value was adjusted to 5.8 with ammonium acetate at 37°C for 60 min. The total volume of the reaction was 100 μL. A volume of 25 μL of the reaction mixture was analyzed by high performance liquid chromatography using a size exclusion column. The mobile phase was 0.1 M phosphate buffer, pH=7.4, and the flow rate was 1 mL / min. Detection was by UV absorption at 280 nm and also by a radiometric detector.

[0069] Evaluation of the UV and radiometric detectors showed radioactivity co-eluting with the protein. Size exclusion columns separate chemicals based on size. Since most of the radioactivity from the Ac-225 solution comes from its radioactive daughter nuclide, it is expected that the radiometal not bound to protein will elute later. There was no radioactive signal with a retention time consistent with a small molecule. This result is consistent with the MNPR-101-PCTA conjugate chelating radioactive Ac-225 daughter nuclides, such as Bi-213. Without being bound by theory, it is believed that the superior binding properties of the PCTA conjugates are the result of the chelator binding not only Ac-225, but also daughter nuclides, such as Bi-213, that are trivalent and / or non-radioactive.

[0070] Bismuth ions can form highly insoluble compounds that can precipitate, retaining both bismuth and actinium ions. Prevention of bismuth compound precipitation by the mAb-bound PCTA chelating functionalities provides another advantage of the present invention. Similar size-exclusion column studies using DOTA as a chelator conjugated to mAb MNPR-101 showed different results, thus showing that when DOTA is used, the on-line radiation detector shows very little protein-associated signal and that the majority of the activity is in a later-eluting peak representing radiometal not bound to protein.

[0071] Example 6 PCTA conjugates were prepared with humanized mAb MNPR-101 in parallel with two other exemplary murine monoclonal antibodies: mAb ATN-616 and mAb ATN-292. Subsequent chelation of Ac-225 was optimized using a chelator to protein molar ratio of 12:75. MNPR-101 and ATN-616 were coupled to PCTA in a molar reaction ratio of 12:1, and ATN-292 was coupled in excess of 75:1. The pH value of the solution was adjusted to 9.2 with 1 M NaH2CO3 and 0.2 M HCl. The reaction was carried out at 37°C for 1.5 hours.

[0072] The conjugates thus formed were purified using a Bio-Rad 10DG gravity-fed column (6,000 Da molecular weight cut-off) and each conjugate was eluted with 0.1 M ammonium acetate buffer pH 5.77. Elution fractions (0.5 mL) were collected in 1.5 mL metal-free tubes and UV absorbance was measured at 280 nm. Depending on the protein concentration in the eluate, three or four fractions were combined and reconcentrated using Amicon® concentrators (30 kDa). The combined fractions were analyzed using the Pierce™ BCA Assay Kit (Thermofisher; final protein concentration was approximately 2-3 mg / mL). The purity of the conjugate was analyzed using size-exclusion high performance liquid chromatography as previously described, using a phosphate buffered saline solvent and a flow rate of 1 mL / min. HPLC results revealed the expected peak at approximately 8 min observed from the naked antibody and a decrease in the retention time (Rt) of the conjugate consistent with the addition of the bifunctional chelator PCTA.

[0073] The results suggest that the increase in retention time (ΔRt) observed between the conjugates and each naked mAb is related to the ability of the conjugates to subsequently chelate Ac-225, with a larger ΔRt correlating with a higher number of chelators bound to the antibody. The difference in retention time between the three conjugates and the naked antibody is shown below. [Table 5]

[0074] Example 7 Reaction vials containing solid Ac-225 were obtained from ORNL and dissolved using 0.2M HCl. Ac-225 chelates were prepared using the three conjugates from Example 6. A ratio of 100 μCi of Ac-225 to 100 μg of mAb-PCTA chelant conjugate was used in all reactions, resulting in a specific activity of 1 mCi / mg for a 100% yield. Reactions were performed in a volume of approximately 110 μL containing approximately 10 μL of Ac-225 in 0.2M HCl, 60 μL of 0.1M ammonium acetate buffer, and 40 μL of mAb-PCTA conjugate, normalized for each protein concentration according to each protein concentration. Reactions were incubated at pH 5.7, 37° C. for 60 minutes.

[0075] A 25 μL aliquot of each chelation reaction was purified by eluting 0.5 mL fractions on a Bio-Rad 10DG gravity-fed column with 0.1 M ammonium acetate buffer. The Ac-labeled conjugate was expected to elute in 3-4 "peak fractions" which were summed with the activity remaining on the column to determine the radiochemical yield. After a minimum of 5 hours (to allow equilibration with Ac and its daughter species), the fractions and their respective columns were run on a dose calibrator (Capintec, set no. 086). The results of the gravity-fed fractions for each chelation run on the dose calibrator are shown in the table below. [Table 6]

[0076] The peak fractions from each reaction were measured at 24 and 48 hours after purification to determine the ability of the chelator to retain the Ac-225 daughter isotope. An increase in peak activity as a function of time would be evidence that the chelator did not effectively control the daughter isotope. However, if activity decreased at a rate consistent with the decomposition of Ac-225, evidence would suggest that the chelator was able to retain Ac-225 and its daughter isotopes. The results, seen in the table below, provide evidence that the three chelate systems effectively control the Ac-225 daughter isotope, as each peak activity shows no increase in activity as a function of time. [Table 7]

[0077] Example 8 A 20 μL sample of each chelation reaction was analyzed by HPLC using an isocratic method (1× PBS solvent, pH 7.4) with detection by UV absorbance at 280 nm and a radiometric detector. Fractions of 1 mL were collected every minute and, after equilibration (>5 h), were measured using a wide-window NaI detector. As observed in Example 5, HPLC results showed radioactivity co-eluting with protein from the three reactions. There were no radioactive signals with retention times consistent with small molecules, further supporting the inference that the PCTA chelator binds Ac-225 and its daughter nuclei. The yield results for each reaction are shown in the table below. [Table 8]

[0078] The peak yields for the three reactions when analyzed with the dose calibrators are 80.6%, 77.0%, and 75.5%, respectively, as described in Example 7, but these same reactions when analyzed using HPLC purification and NaI detection give reaction yields of 96.2%, 92.7%, and 97.8%, respectively. This variability is understood to result from the inability to measure the activity remaining on the size exclusion column, resulting in the more conservative yields observed from the Bio-Rad gravity-fed column and dose calibrator values. The methods and results described suggest that the subject bifunctional chelator PCTA exhibits a remarkable ability to bind Ac-225 not only when conjugated with the humanized mAb MNPR-101, but also with other antibodies, such as two murine monoclonal antibodies, mAb ATN-616 and mAb ATN-292.

[0079] Example 9 Humanization of the variable (V) region amino acid residue sequence of mouse mAb ATN-658 The light chain variable region (V L ) and the heavy chain variable region (V H The consensus amino acid sequence (single letter code) of the ATN-658 polypeptide is shown in U.S. Patent No. 8,191,726 to Parry and Mazar, which is incorporated herein by reference and will not be repeated here. cDNA from total RNA extracted from hybridomas expressing ATN-658 and the variable regions was cloned, amplified, and sequenced using standard techniques. Following the procedure set forth by Studnicka et al., supra, human Vkappa light chain subgroup 2 (VK2) and human heavy chain subgroup 1 (VH1) consensus sequences were used, and the cognate mouse signal sequence was retained.

[0080] Two sequences were prepared for each of the light and heavy chain variable regions. One sequence for each chain contained only low-risk changes, while the other sequence for the VK2 and VH1 regions contained both low- and moderate-risk changes, for a total of four sequences. Ten low-risk and one moderate-risk changes were introduced into the light chain framework sequence, and eleven low-risk and five moderate-risk changes were introduced into the heavy chain framework sequence. Changes at low-risk residue positions are solvent-exposed but do not contribute to antigen binding or antibody structure, and are likely to reduce immunogenicity with little or no effect on binding affinity. These amino acid residue sequences were sent to Blue Heron Biotech LLP (Bothell, WA) for codon (Chinese Hamster Ovary cells) and expression optimization. The optimized DNA sequences were received and sent back to Blue Heron for gene synthesis.

[0081] Construction of transient expression vectors Codon- and expression-optimized low-risk and low+moderate-risk Human Engineered™ light and heavy chains were cloned in frame into XOMA's proprietary transient antibody expression vectors containing human kappa and gamma-1 constant region modules. DNA sequences were verified to initiate expression (ELIM Biopharmaceuticals, Inc., Hayward, CA).

[0082] Production of Human Engineered™ ATN-658 Antibody Four HE™ ATN-658 mutants (designated HE™ ATN-1, HE™ ATN-2, HE™ ATN-3, and HE™ ATN-4) were produced by transient transfection in HEK293E cells. The XOMA transient transfection approach is described in detail in a poster presentation at the 2005 ASCB Annual Meeting. Briefly, light and heavy chains were co-transfected into XOMA suspension-adapted HEK293E cells grown in IS293 medium (Irvine Scientific, Irvine, CA) in 2-liter shake flasks. After 24 hours in shake flasks, 200 ml of transfected cells were centrifuged, resuspended in 40 ml of fresh medium, and transferred to Integra flasks (Wilson Wolf Manufacturing, Inc., New Brighton, MN) for production. After 7 days of incubation, the cell suspension was removed from the Integra flask, centrifuged, and the culture supernatant was retained. Antibody in the culture supernatant was purified on a Protein A spin column (Pro-Chem), dialyzed against PBS, concentrated, and sterilized by filtration.

[0083] The variable region sequences of these four antibodies are shown in Table 1 below. [Table 9]

[0084] Concentrations were determined by A280 using an extinction coefficient of 1.52. Proteins were analyzed for purity by SDS-PAGE (4-20%) and for endotoxin using the LAL assay. Purification results show that all antibody preparations had concentrations of 1mg / ml or greater, were greater than 90% pure, and contained low levels of endotoxin (<1EU / mg).

[0085] Assessment of the affinity of the Human Engineered™ ATN-658 antibody by Biacore assay Kinetic analysis of mouse monoclonal antibody ATN-658 and Human Engineered™ ATN-658 variant antibodies was performed on a Biacore 2000® surface plasmon resonance analyzer (Uppsala, Sweden) to generate sensograms based on antibody-surface interactions. Kinetic determinations were performed using a capture method. Mouse parental mAb ATN-658 was diluted to 2 mg / mL in PBS and injected over the rabbit anti-mouse capture surface. HE™ variants were diluted to 1 mg / mL and injected over the Protein A / G surface. Antibody injections were optimized to give an antibody concentration of 100-200 RU. Six 3-fold serial dilutions of soluble UPAR (suPAR) were prepared in running buffer (PBS) and each dilution was injected three times in random order at 25°C. Buffer injections were evenly distributed across runs. Sample injections were double-referenced against a blank flow cell and a buffer injection to correct for bulk shift or non-specific binding. Data were analyzed with Biacore® BiaEvaluation software. Sensorgrams were fitted using a 1:1 Langmuir model.

[0086] Humanized mAb MNPR-101 In arriving at the six CDRs of mAb MNPR-101 compared to mAb ATN-658, one residue was changed in one CDR each of the VK2 and VH1 regions of mAb MNPR-101 compared to the CDR sequences of mAb ATN-658 (CDR L1 and CDR H2). The complementarity determining regions (CDRs) of each variable region present in the paratopic region of mAb MNPR-101 are shown in Table 2 below. [Table 10]

[0087] array The sequences of the VL and VH regions and the CL and CH regions of the Fab portion of mAb MNPR-101 are shown below, as well as the low risk sequence of the variable regions of both chains (HE™ ATN-1). SEQ ID NO:1 - [mAb MNPR-101 VL] Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Ser Val Thr Ile Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu Asp Ser Asp Gly Lys Thr Tyr Leu Asn Trp Leu Leu Gln Lys Pro Gly Gln Ser Pro Gln Arg Leu Ile Tyr Leu Val Ser Lys Arg Asp Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Trp Gln Gly Thr His Phe Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys SEQ ID NO:2 - [HE™ ATN-1 VL] Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Ser Val Thr Ile Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu Asp Ser Asp Gly Lys Thr Tyr Leu Asn Trp Leu Leu Gln Lys Pro Gly Gln Ser Pro Lys Arg Leu Ile Tyr Leu Val Ser Lys Arg Asp Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Trp Gln Gly Thr His Phe Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys SEQ ID NO:3 [mAb MNPR-101 CDR L1] Arg Ser Ser Gln Ser Leu Leu Asp Ser Asp Gly Lys Thr Tyr Leu Asn SEQ ID NO:4 [mAb MNPR-101 CDR L2] Leu Val Ser Lys Arg Asp Ser SEQ ID NO:5 mAb [MNPR-101 CDR L3] Trp Gln Gly Thr His Phe Pro Leu Thr SEQ ID NO:6 [LC signal sequence] MSPAQFLFLL VLWIRETNG SEQ ID NO:7 [mAb MNPR-101 LC constant region sequence] RTVAAPSVFI FPPSDEQLKS GTASVVCLLN NFYPREAKVQ WKVDNALQSG NSQESVTEQD SKDSTYSLSS TLTLSKADYE KHKVYACEVT HQGLSSPVTK SFNRGEC SEQ ID NO:8 [mAb MNPR-101 low+medium risk-VH] Glu Val Gln Leu Val Gln Ser Gly Pro Glu Val Lys Lys Thr Gly Ala Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr Tyr Met His Trp Val Arg Gln Ala His Gly Gln Gly Leu Glu Trp Ile Gly Glu Ile Asn Pro Tyr Asn Gly Gly Ala Ser Tyr Asn Gln Lys Ile Gln Gly Arg Ala Thr Phe Thr Val Asp Thr Ser Thr Ser Thr Ala Tyr Met Glu Phe Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ser Ile Tyr Gly His Ser Val Leu Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser SEQ ID NO: 9 [HE (trademark) ATN-1 VH] Glu Val Gln Leu Val Gln Ser Gly Pro Glu Val Val Lys Thr Gly Ala Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr Tyr Met His Trp Val Lys Gln Ala His Gly Gln Gly Leu Glu Trp Ile Gly Glu Ile Asn Pro Tyr Asn Gly Gly Ala Ser Tyr Asn Gln Lys Ile Lys Gly Arg Ala Thr Phe Thr Val Asp Thr Ser Thr Arg Thr Ala Tyr Met Glu Phe Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ser Ile Tyr Gly His Ser Val Leu Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Sequence number 10 [mAb MNPR - 101 CDR H1] Gly Tyr Ser Phe Thr Ser Tyr Tyr Met His Sequence number 11 [mAb MNPR - 101 HC CDR H2] Glu Ile Asn Pro Tyr Asn Gly Gly Ala Ser Tyr Asn Gln Lys Ile Gln Gly Sequence number 12 [mAb MNPR - 101 HC CDR H3] Ser Ile Tyr Gly His Ser Val Leu Asp Tyr Sequence number 13 [mAb MNPR - 101 HC signal sequence] MGWIWIFLFL LSGTAGVHS Sequence number 14 [mAb MNPR - 101 HC constant region sequence] ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKRVEP KSCDKTHTCP PCPAPELLGG PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSREE MTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPGK To insert the encoding nucleic acid into an expression vector, a SalI restriction site was placed in frame upstream of the encoded N-terminus of each of the heavy and light chains, and an XhoI site was inserted in frame downstream of the encoded C-terminus of each chain.

[0088] Production of MNPR-101 The heavy and light chains of the monoclonal antibody candidates were packaged into a pUC19 plasmid, cDNA inserts encoding the monoclonal antibodies were cloned, and the heavy and light chains were inserted into an expression vector. After sequence verification, the DHFR-deficient CHO cell line DUX B11 was transfected with the light and heavy chain-containing vectors and a cationic liposome mixture (Lipofectamine® 2000; Invitrogen Corp., Carlsbad, Calif.). Forty-eight hours after transfection, cells were subcloned in 96-well dishes using purine-free growth medium in the presence of geneticin (G418) and 20 nM methotrexate (MTX).

[0089] After selection, all subclones were screened using a hIgG betyl ELISA kit. Three vials were frozen for each of the 12 best subclones. The top six best producing subclones were then transferred to medium supplemented with increasing amounts of methotrexate (MTX), an inhibitor of DHFR. The MTX concentration was sequentially increased from 20 to 1,000 nM and then to 1,500 nM MTX during the selection process. The expanded MTX-resistant clones were screened by ELISA. After the first round of amplification, two highest expressing population subclones were obtained in medium containing 1,000 nM MTX. These two clones were amplified to 1,500 nM MTX and then subcloned at 1,000 nM and 1,500 nM MTX. These subclones are currently being expanded into 6-well plates and will be screened by ELISA in the coming days. The top two to three best subclones will be adapted to serum-free medium and then expanded for the generation of a research cell bank.

[0090] result The ligand binding kinetics of mouse mAb ATN-658 and the Human Engineered™ ATN-658 antibody described above were measured once. The sensorgram results from each assay showed that all of the transiently expressed antibodies showed similar affinities to mAb ATN-658 and also among themselves. The results for the four combinations of two VL chains and two VH chains are shown in Table 3. [Table 11]

[0091] Example 10 Initial study of the chelating properties and stability of In-111 with the proposed PCTA-MNPR-101 chelator targeting species. Thus, freshly prepared PCTA-MNPR-101 (made at 12:1) was incubated at 37° C. for 1.5 hours in an aqueous solution (1 M NaHCO3 and HCl) with a pH value of 9.2, containing 4.0 mg / mL by protein analysis. The conjugate (220.0 mL MNPR-101-PCTA) was purified by passing through a PD10 column and eluting with 0.1 M ammonium acetate. Samples containing the conjugate were collected and concentrated using a 30 kDa Amicon® concentrator (4000 rpm, 20 min). Three aqueous chelation reactions were set up, each with approximately 200 μCi of radioactivity for a target specific activity of 10 mCi / mg. Each was mixed with In-111 chloride obtained from BWXT Medical, Ottawa, ON, Canada. All reactions were stored at 4°C and stability was measured after 24, 48, and 72 hours.

[0092] Stability in this context is the maintenance of chelation of the radioactive ion over time. Stability was determined by gravity-fed SEC column (PD10 6,000 Dalton cutoff), HPLC and TLC for comparison. Three aqueous chelation reactions were set up with approximately 200 μCi of activity each, for a target specific activity of 10 mCi / mg. These were: 1) Incubate at 37℃ for 30 minutes, then store at 4℃ for 72 hours. 2) Incubate at room temperature for 30 minutes, then store at 4°C for 24 hours. 3) Incubate at room temperature for 1.5 hours, then store at 4°C for 48 hours.

[0093] The results of this initial study are shown in the table below. [Table 12] Results of this initial study showed that a relatively high yield of chelation was obtained with a target specific activity of 10 mCi / mg. It is likely that conditions can be optimized to increase the yield. Each of the three different analytical methods indicated that the chelate was formed. Reasonable stability of the chelated In-111 was observed, given that the half-life of Indium-111 is approximately 2.8 days.

[0094] Each of the patents, patent applications, and articles cited herein is incorporated by reference. Use of the article "a" or "an" is intended to include one or more. The foregoing description and examples are intended to be illustrative and are not to be construed as limiting. Further variations within the spirit and scope of the invention are possible and will readily suggest themselves to those skilled in the art.

Claims

1. PCta Chelated Q +3 A targeted radiopharmaceutical comprising a targeting species T bound to a thiourea group with a radioisotope ion, wherein the targeted radiopharmaceutical has a general structural formula represented by the following formula I. 【Chemical 1】 (wherein Q +3 is 225 Ac +3 , 213 Bi +3 a trivalent radioisotope ion of, or a mixture thereof R 1 、R 2 、R 4 、R 5 、R 6 、and R 7 is H, and R 3 contains a reacted functional group Z that forms a thiourea bond with the targeting species T, "g" is a number with an average value of 4 to 8, which represents the average number of chelated PC1A chelated trivalent radioactive ions Q per molecule of the targeted species T. +3 and the average value is a number between 4 and 8. X 1 , X 2 , and X 3 are the same or different substituents that can coordinate to the +3 radioisotope ions of Q and / or can help neutralize the ionic charge. the targeting species T is selected from the group consisting of an antibody or a paratope-containing portion of an antibody conjugated with thiourea, a hormone conjugated with thiourea, a non-antibody protein conjugated with thiourea, a cytokine conjugated with thiourea, an aptamer conjugated with thiourea, a nucleic acid or oligonucleotide conjugated with thiourea, a linear or cyclic oligopeptide, and a linear or branched oligosaccharide, and Any anion Y - is present optionally in an amount necessary to balance the ionic charges.)

2. Said X 1 , X 2 , and X 3 each of which is -(CH 2 ), n CO 2 M or -PO 3 M 2 is a substituent, n is 0 or 1, and M is H + or an alkali metal cation, the target radiopharmaceutical according to claim 1.

3. Said X 1 , X 2 , and X 3 each is a -(CH 2 ) n CO 2 M substituent, n is 0 or 1, and M is H + or an alkali metal cation. The target radiopharmaceutical according to claim 2

4. The target radiopharmaceutical according to claim 3, wherein n is 0.

5. The target radiopharmaceutical according to claim 1, wherein the targeting species T is an antibody or a paratope-containing portion of an antibody conjugated with thiourea.

6. The target radiopharmaceutical according to claim 5, wherein the antibody or the paratope-containing portion of the antibody is a monoclonal antibody (mAb) or a paratope-containing portion thereof.

7. The target radiopharmaceutical according to claim 6, wherein the monoclonal antibody or the paratope-containing portion thereof is an mAb called ATN-658 produced by a hybridoma having the ATCC accession number PTA-8191 or a paratope-containing portion of ATN-658.

8. The target radiopharmaceutical according to claim 6, wherein the mAb is humanized.

9. A pharmaceutical composition comprising a therapeutically effective amount of the target radiopharmaceutical according to claim 1 dissolved or dispersed in a pharmaceutically acceptable diluent.

10. The pharmaceutical composition according to claim 9, wherein the pharmaceutically acceptable diluent is an aqueous liquid at ambient temperature and is suitable for parenteral administration.

11. The pharmaceutical composition according to claim 10, wherein the composition is isotonic with the blood of the intended mammalian species host recipient.

12. The pharmaceutical composition according to claim 11, wherein the intended mammalian species host recipient is a human.

13. The pharmaceutical composition according to claim 9, wherein the therapeutically effective amount is an amount effective to kill the cells of the target radiopharmaceutical.

14. The pharmaceutical composition according to claim 13, wherein the amount effective to kill the cells of the target radiopharmaceutical is an amount sufficient to administer 80 to 120 kBq / kg body weight to a mammalian host.

Images