EA044225B1 - APPLICATION OF A CLEANING AGENT BASED ON DENDRONS N-ACETYLGALACTOSAMINE IN PRE-TARGETED RADIOIMMUNOTHERAPY WITH DOTA CHELATOR - Google Patents

Description

Cross reference to related applications

The invention claims priority under U.S. Provisional Patent Application No.

62\697956, filed July 13, 2018, the contents of which are incorporated by reference in their entirety.

Field of technology

The invention generally relates to compositions comprising new scavenging agents based on N-acetylgalactosamine dendrons, and to methods for their use in pre-targeted radioimmunotherapy.

Application for state support

The invention was made with financial support from the US government under contract CA86438, awarded by the US National Institutes of Health. The government has certain rights to an invention.

State of the art

The following description of the prior art of the present invention is provided solely to assist in understanding the present invention and is not a description or summary of the prior art of the present invention.

Radiolabeling agents have been used for over 50 years as a means of delivering ionizing radiation to specific lesions (Larson SM. Cancer 67:1253-1260 (1991); Britton KE. Nucl Med Commun. 18:992-1007 (1997)). A large number of molecules have been reviewed for use in targeted radioisotope delivery, including radiolabeled antibodies, antibody fragments, alterative scaffolds, and small molecules (Tolmachev V, et al., Cancer Res. 67:2773-2782 (2007); BirchlerMT, et al., Otolaryngol Head Neck Surg 136:543-548 (2007) Reubi JC, Maecke HR J Nucl Med 49:1735-1738 (2008) The use of antibodies for targeted tumor destruction, such as in radioimmunotherapy (RIT) with targeted conjugated antibodies, is challenging, in part due to the suboptimal tumor dose and therapeutic index (TI). In addition, due to the inevitable damage to healthy tissue, increasing the dose is not possible, and therefore such therapy leads to a limited antitumor effect. Moreover, antibodies have a long half-life in the blood, which is a consequence of the low ratio of tumor absorption to background radiation. Smaller antibody fragments and other binding scaffolds are cleared more quickly from the blood, but they accumulate in large quantities in the kidneys and/or liver. Radiolabeled small molecule ligands are generally characterized by faster clearance from the blood and lower background radiation compared to antibodies and antibody fragments, but generally result in poor specificity due to relatively low affinity for the desired target.

In pretargeted radioimmunotherapy (PRIT), a non-radioactive bifunctional antibody with specificity for both a tumor antigen and a low molecular weight hapten is administered and allowed to localize to the tumor(s). After the antibody has been cleared from the blood in sufficient quantities, a small molecule with a radioactive label is administered, which is captured by the pre-targeted antibody. However, many small peptide and metal chelate haptens used in PRIT systems exhibit prolonged retention in the body, resulting in undesirable background activity that limits signal-to-background ratio values in imaging and contributes to nonspecific emission that limits the maximum tolerated dose for therapeutic applications. (Orcutt et al., Mol Imaging Biol 13:215-221 (2011)).

Thus, there is a need for new molecules that provide (a) effective pretargeted radioimmunotherapy of solid tumors in vivo and (b) rapid clearance of radiolabeled small molecules from non-tumor tissue.

Brief description of the invention

In one aspect, the present invention provides a compound of the form

- 1 044225

- 2 044225

or a pharmaceutically acceptable salt and/or solvate thereof, in which X ¹ , X ² , X ³ , X ⁴ , X ⁵ , X ⁶ , X ⁷ , X ⁸ , X ⁹ , X 10 , X ¹¹ , X ¹² , ^{X 13} , X ¹⁴ , X ¹⁵ , X ¹⁶ , X ¹⁷ , X ¹⁸ , X ¹⁹ , X ²⁰ each independently represent H or a lone pair of electrons (i.e. provide an oxygen anion); Y ¹ , Y ² , Y ³ , Y ⁴ , Y ⁵ are each independently O or S; x is 1, 2 or 3; y is 1, 2, 3 or 4.

In any embodiment, the compound may be a cleaning agent of the form

- 3 044225

- 4 044225

or a pharmaceutically acceptable salt and/or solvate thereof, in which M ¹ , M ² , M ³ , M ⁴ , M ⁵ are each independently Lu ³⁺ , Sc ³⁺ , Ga ³⁺ , Y ³⁺ , In ³⁺ , La ³⁺ , Ce ³⁺ , Eu ³⁺ , Tb ³⁺ , or Gd ³⁺ ; X ¹ , X ² , X 3, X ⁴ , X ⁵ , X ⁶ , ^{X 7 ,} X ⁸ , X 9, X ¹⁰ , X ¹¹ , X ¹² , X ¹³ , X ¹⁴ , X ¹⁵ , ^{X 16 ,} X ¹⁷ , X ¹⁸ , X ¹⁹ , X ²⁰ each independently represents H or a lone pair of electrons (ie, provides an oxygen anion); Y ¹ , Y ² , Y ³ , Y ⁴ , Y ⁵ are each independently O or S; x is 1, 2 or 3; y is 1, 2, 3 or 4.

According to any embodiment, it may turn out that M ¹ , M ² , M ³ , M ⁴ , M ⁵ are each independently not a radionuclide. In any embodiment, it may be that Y ¹ , Y ² , Y ³ , Y ⁴ , Y ⁵ are each independently S. In any embodiment, it may be that x is 1 or 2. In any embodiment it may turn out that y is equal to 2 or 3.

In a related aspect, there is provided a composition that includes one or more compounds according to any embodiment described above and a pharmaceutically acceptable carrier.

In another aspect, the present invention provides a method of sensitizing a tumor to radiation therapy in a subject diagnosed with cancer, comprising (a) administering to the subject an effective amount of an anti-DOTA bispecific antibody, wherein the anti-DOTA bispecific antibody is configured to localize to a tumor expressing a target tumor antigen; (b) administering to the subject an effective amount of a cleansing agent according to the present invention; (c) administering to the subject an effective amount of a radiolabeled DOTA hapten, wherein the DOTA hapten is configured to form a complex with an anti-DOTA bispecific antibody.

In another aspect, the present invention provides a method of treating cancer in a subject in need thereof, comprising (a) administering to the subject an effective amount of an anti-DOTA bispecific antibody, wherein the anti-DOTA bispecific antibody is configured to localize to a tumor expressing a target tumor antigen; (b) administering to the subject an effective amount of a cleansing agent according to the present invention; (c) administering to the subject an effective amount of a radiolabeled DOTA hapten, wherein the DOTA hapten is configured to form a complex with an anti-DOTA bispecific antibody. Methods of treating cancer may further include sequential, separate, or simultaneous administration to a subject of at least one chemotherapeutic agent selected from the group consisting of nitrogen mustards, ethyleneimine derivatives, alkylsulfonates, nitrosoureas, gemcitabine, triazenes, folic acid analogues, anthracyclines, taxanes, COX inhibitors, 2 (English COX-2 inhibitors), pyrimidine analogues, purine analogues, antibiotics, enzyme inhibitors, epipodophyllotoxins, platinum coordination complexes, vinca alkaloids, substituted ureas, methylhydrazine derivatives, adrenocortical suppressors, hormone antagonists, endostatin, taxols, camptothecins, SN- 38, doxorubicin, doxorubicin analogs, antimetabolites, alkylating agents, antimitotics, antiangiogenic agents, tyrosine kinase inhibitors, mTOR inhibitors, heat shock protein (HSP90) inhibitors, proteasome inhibitors, HDAC inhibitors, proapoptotic agents, methotrexate, CPT11.

Additionally or alternatively, according to some embodiments of the methods disclosed herein, the tumor antigen target is selected from the group consisting of GPA33, HER2/neu, GD2, MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE- 2, MUM-1, CDK4, Nacetylglucosaminyltransferase, p15, gp75, beta-catenin, ErbB2, cancer antigen 125 (CA-125), carcinoembryonic antigen (CEA), RAGE, MART (melanoma antigen), MUC-1, MUC-2 , MUC-3, MUC-4, MUC-5ac, MUC-16, MUC-17, tyrosinase, Pmel 17 (gp100), GnT-V subtype V intron sequences (N-acetylglucoaminyltransferase subtype V intron V sequences), prostate cancer psm , PRAME (melanoma antigen), β-catenin, EBNA (Epstein-Barr virus nuclear antigen) 16, p53, lung cancer resistance protein (LRP) Bcl-2, prostate-specific antigen (PSA), Ki-67, CEACAM6, colon specific antigen-p (CSAp), HLA-DR, CD40, CD74, CD138, EGFR, EGP-1, EGP-2, VEGF, P1GF, insulin-like growth factor (ILGF), tenascin, platelet-derived growth factor, IL-6 , CD20, CD19, PSMA, CD33, CD123, MET, DLL4, Ang-2, HER3, IGF-1R, CD30, TAG-72, SPEAP, CD45, L1-CAM, Lewis antigen Y (Ley), E-cadherin, Vcadherina, ErCAM.

Additionally or alternatively, according to some embodiments of the methods disclosed herein, the anti-DOTA bispecific antibody, scavenging agent, and/or radiolabeled DOTA hapten are administered intravenously, intramuscularly, intraarterially, intrathecally, intracapsularly, intraorbitally, intradermally, intraperitoneally, transtracheally, subcutaneously, intracerebroventricularly, orally or intranasally.

Additionally or alternatively, according to some embodiments of the methods disclosed herein, the cancer is selected from the group consisting of breast cancer, colorectal cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, hepatocellular carcinoma, cancer brain, lung cancer, gastrointestinal or stomach cancer, pancreatic cancer, thyroid cancer, kidney cancer, prostate cancer, melanoma, sarcoma, carcinoma, Wilms tumor, endometrial cancer, glioblastoma, squamous cell carcinoma, astrocytoma, salivary gland carcinoma , vulvar cancer, penile carcinoma, head and neck cancer.

Brain cancer can be a pituitary adenoma, meningioma, neuroblastoma, or craniopharyngioma.

Additionally or alternatively, according to some embodiments of the methods disclosed herein, the radiolabeled DOTA hapten comprises one or more components of Proteus-DOTA, S-2-(R-aminobenzyl)-1,4,7,10-tetraazacyclododecanetetraacetic acids (DOTA-Bn), DOTA-Bn-biotin, BAD (((S)-2-(4-(2-bromo)αcetamido)benzyl)-DOTA), NBD ((S)-2-(4nitrobenzyl)- DOTA), DOTA-RGD, DOTA-PEG-E(c(RGDyK)) ₂ , DOTA-8-AOC-BBN, p-NO ₂ -Bn-DOTA,

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DOTA-PESIN, DOTA-biotin-sarcosine (DOTA-biotin), mono 1,4,7,10-tetraazacyclododecane-1,4,7,10tetraacetic acid (N-hydroxysuccinimide ester) (DOTA-NHS) or DOTATyrLysDOTA.

The radiolabeled DOTA hapten may be labeled with a radionuclide selected from the group consisting of ²¹³ Bi, ²¹¹ At, ²²⁵ Ac, ¹⁵² Dy, ²¹² Bi, ²²³ Ra, ²¹⁹ Rn, ²¹⁵ Po, ²¹¹ Bi, ²²¹ Fr, ²¹⁷ At, ²⁵⁵ Fm, ⁸⁶ Y, ⁹⁰ Y, ⁸⁹ Sr, ¹⁶⁵ Dy, ¹⁸⁶ Re, ¹⁸⁸ Re, ¹⁷⁷ Lu, 67 Cu, ^{1n In} , ⁶⁷ Ga, ⁵¹ Cr, ⁵⁸ Co, ^99m Tc, ^103m Rh, ^195m Pt, ¹¹⁹ Sb, ¹⁶¹ Ho , ^189m Os, ¹⁹² Ir, ²⁰¹ Tl, ²⁰³ Pb, ⁶⁸ Ga, ²²⁷ Th, ⁶⁴ Cu.

Additionally or alternatively, according to some embodiments of the methods disclosed herein, the levels of radioactivity emitted by the complex of the radiolabeled DOTA hapten and the anti-DOTA bispecific antibody are detected between 4 and 24 hours after administration of the radiolabeled DOTA hapten. The levels of radioactivity emitted by a given complex can be expressed as a percentage of the administered dose per gram of tissue (% ID/g). The reference value can be calculated by measuring the levels of radioactivity present in non-tumor (healthy) tissues and calculating the average levels of radioactivity present in non-neoplastic (healthy) tissues ± standard deviation. In some embodiments, the reference value is a standard uptake value (SUV). See Thie JA, J Nucl Med. 45(9): 1431-4 (2004). The therapeutic efficacy of such a complex can be determined by calculating the ratio of the area under the pharmacokinetic curve of the tumor to the area under the pharmacokinetic curve of healthy tissue. In some embodiments, the complex provides a tumor AUC to healthy tissue AUC ratio of approximately 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9 :1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 55:1, 60:1, 65:1 , 70:1, 75:1, 80:1, 85:1, 90:1, 95:1 or 100:1. Additionally or alternatively, according to some embodiments of the methods disclosed herein, the ratio of tumor radioactivity levels to healthy tissue radioactivity levels is about 2:1, 3:1, 4:1, 5:1, 6:1, 7: 1, 8:1, 9:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 55:1, 60:1, 65:1, 70:1, 75:1, 80:1, 85:1, 90:1, 95:1 or 100:1.

The present invention also discloses kits containing components useful for treating cancer in a patient. In one aspect, the kits contain a cleaning agent according to the present invention and instructions for use. The kits of the present invention may further contain at least one anti-DOTA bispecific antibody and/or DOTA hapten, which is optionally labeled with one or more radionuclides. Examples of suitable radionuclides include ²¹³ Bi, ²¹¹ At, ²²⁵ Ac, ¹⁵² Dy, ²¹² Bi, ²²³ Ra, ²¹⁹ Rn, ²¹⁵ Po, ²¹¹ Bi, ²²¹ Fr, ²¹⁷ At, ²⁵⁵ Fm, ⁸⁶ Y, ⁹⁰ Y, ⁸⁹ Sr, ¹⁶⁵ Dy, ¹⁸⁶ Re, ¹⁸⁸ Re, ¹⁷⁷ Lu, ⁶⁷ Cu, ¹ⁿ In, ⁶⁷ Ga, ⁵¹ Cr, ⁵⁸ Co, ^99m Tc, ^103m Rh, ^195m Pt, ¹¹⁹ Sb, ¹⁶¹ Ho, ^189m Os, ¹⁹² Ir, ²⁰¹ Tl, ²⁰³ Pb, ⁶⁸ Ga, ²²⁷ Th, ⁶⁴ Cu, but not limited to them. Additionally or alternatively, in some embodiments, the at least one anti-DOTA bispecific antibody is coupled to a tumor antigen target selected from the group consisting of GPA33, HER2/neu, GD2, MAGE-1, MAGE-3, BAGE, GAGE -1, GAGE-2, MUM-1, CDK4, N-acetylglucosaminyltransferase, p15, gp75, beta-catenin, ErbB2, cancer antigen 125 (CA-125), carcinoembryonic antigen (CEA), RAGE, MART (melanoma antigen), MUC-1, MUC-2, MUC-3, MUC-4, MUC-5ac, MUC-16, MUC-17, tyrosinase, Pmel 17 (gp100), GnT-V subtype V intron sequences (Nacetylglucoaminyltransferase subtype V intron V sequences ), prostate cancer psm, PRAME (melanoma antigen), βcatenin, EBNA (Epstein-Barr virus nuclear antigen) 1-6, p53, lung cancer resistance protein (LRP) Bcl-2, prostate-specific antigen (PSA), Ki -67, CEACAM6, colon specific antigen-p (CSAp), HLA-DR, CD40, CD74, CD138, EGFR, EGP-1, EGP-2, VEGF, P1GF, insulin-like growth factor (ILGF), tenascin, platelet-derived factor growth, IL-6, CD20, CD19, PSMA, CD33, CD123, MET, DLL4, Ang-2, HER3, IGF-1R, CD30, TAG72, SPEAP, CD45, L1-CAM, Lewis Y antigen (Le ^y ), E-cadherin, V-cadherin, EpCAM.

Brief description of drawings

In fig. 1 shows the effect of the scavenging agent disclosed in the present invention on the in vivo circulation of bispecific antibody ¹³¹ I. Athymic healthy mice (without tumors) were intravenously administered at t=0 bispecific antibody ¹³¹ I (19-21 μCi; 250 μg; 1.19 nmol) followed by the introduction of either a carrier medium (saline solution) or a dendron-based cleansing agent (25 μg; 2.76 nmol) after t = 24 hours. Serial blood sampling was carried out at various time points at t = 1-28 hours. Data presented as the average value (±) root mean square error.

In fig. 2 shows the results of tumor pre-targeting obtained with the clearing agent CCA-16-DOTA-Y3+ according to the present invention.

In fig. Figure 3 shows the effect of scavenging agent CCA-16-DOTA-Y ³⁺ on tumor uptake as a function of dose. **P<0.01 compared with the 25 μg group.

In fig. 4 shows a comparison of the tumor pre-targeting results obtained using an embodiment of the scavenging agent of the present invention versus no scavenging agent (carrier medium) as well as using a 500 kDa dextran-hapten DOTA scavenging agent.

In fig. Figure 5A shows a comparison of the biodistribution of pre-targeted radioactive

- 7 044225 indicators [ ²²⁵ Ac]Proteus-DOTA (n=3) or [ ¹ⁿ In]Proteus-DOTA (n=5) in groups of nude mice with SW1222 tumor 24 hours after administration. Following intravenous (lateral tail vein) injections of huA33-C825 antibody (0.25 mg, 1.19 nmol), clearing agent, and radiolabeled DOTA haptens, animals were sacrificed 24 h later for organ harvesting and radioactivity measurements. *P<0.05;***P<0.001. Data are presented as mean (±) standard error.

In fig. 5B shows the absolute tumor uptake of pretargeted radiolabeled DOTA haptens 24 hours after administration as a function of the amount of radiotracer administered (mol); n=1-7 for each data point.

In fig. Figure 6A shows the results of tumor pre-targeting in animals administered 172 pmol/1.67 MBq [45 μCi] [ ⁿ¹ In] Proteus-DOTA (n=4) or 790 pmol/7.66 MBq [207 μCi] [ ⁿ¹ In] Proteus-DOTA (n=1).

In fig. 6B shows a statistical comparison of the effects of pretargeted [ ²²⁵ Ac]Proteus-DOTA and [ ¹¹¹ In]Proteus-DOTA with the effects of pretargeted anti-GPA33-DOTA.

In fig. 7 shows SPECT/CT images approximately 24 hours after administration of pretargeted [ ¹¹¹ In]Proteus-DOTA to human colorectal cancer tumor SW1222 in nude mice. The SW1222 xenograft is clearly visible in the lateral abdominal region. Based on image analysis of individual areas of the body such as the tumor, kidney (left), heart and liver, activity concentrations were obtained (standard error value ± 1 standard deviation; percentage of dose administered per gram) of 6.89 ± 4.68; 0.46 ± 0.47; 0.20 ± 0.24; 0.22 ± 0.27, respectively.

In fig. Figure 8 shows the structure of Proteus-DOTA (chemical formula: C ₅₀ H ₈₀ LuN ₁₁ O ₁₉ S ^3- ; exact mass: 1345.48; molecular mass: 1346.28). The framed portion of the molecule is the non-radioactive benzyl-DOTA hapten (Lu), which is recognized by the single chain variable fragment C825 of the anti-DOTA hapten antibody at Kd=10 pM. The unbound trilobal portion of the DOTA molecule can attract a variety of radiometals used in therapy and/or imaging, including ²²⁵ Ac, ⁶⁸ Ga and ⁶⁴ Cu.

In fig. Figure 9 shows the activity of ¹⁷⁷ Lu in the tumor and various healthy tissues, determined by biodistribution assessment after PRIT using huA33-C825 (0.25 mg per mouse), a dendron scavenging agent CCA-16-DOTA-Y3+ (25 μg; 2 .76 nmol) and 3.7 MBq (20 pmol) ¹⁷⁷ Luaminobenzyl DOTA ([ ¹⁷⁷ Lu]LuDOTA-Bn). Number of animals in group n=5. Data are presented as percentage of dose administered per gram (% ID/g); root mean square error: ±1 standard deviation.

In fig. Figure 10 shows the biodistribution curves of ¹⁷⁷ Lu activity, taking into account decay correction for SW1222 tumors, as well as for selected healthy tissues 1-48 hours after injection followed by PRIT using huA33-C825+ [ ¹⁷⁷ Lu]LuDOTA-Bn (3.7 MBq, 20 pmol) and dendron-based cleansing agent CCA-16-DOTA-Y ³⁺ (25 μg; 2.76 nmol). Data are presented as percentage of dose administered per gram (% ID/g); root mean square error: ±1 standard deviation.

In fig. Figure 11 shows the uptake doses of pretargeted [ ¹⁷⁷ Lu]LuDOTA-Bn and anti-GPA33-DOTA using the dendron-based scavenging agent CCA-16-DOTA-Y ³⁺ in nude mice subcutaneously injected with the GPA33(+) SW1222 tumor. The therapeutic index was determined as the calculated ratio of the dose absorbed by the tumor to the dose absorbed by healthy tissues.

Detailed Description of the Invention

It should be appreciated that certain aspects, modes, embodiments, differences and features of the present methods are described below at various levels of detail in order to provide a clear understanding of the present invention.

The present methods are practiced using many traditional techniques from molecular biology, protein biochemistry, cell biology, microbiology and recombinant DNA. For example,

- 8 044225 see Sambrook and Russell eds. (2001)

Molecular Cloning: A Laboratory Manual, 3rd edition; series by Ausubel et al. eds. (2007) Current Protocols in Molecular Biology, Methods in Enzymology series (Academic Press, Inc., N.Y.); MacPherson et al. (1991) PCR 1: A Practical Approach (IRL Press at Oxford University Press); MacPherson et al. (1995) PCR 2: A Practical Approach, Harlow and Lane eds. (1999) Antibodies, A Laboratory Manual, Freshney (2005) Culture of Animal Cells: A Manual of Basic Technique, 5th edition; Gait ed. (1984) Oligonucleotide Synthesis', US Patent No. 4683195; Hames and Higgins eds. (1984) Nucleic Acid Hybridization', Anderson (1999) Nucleic Acid Hybridization', Hames and Higgins eds. (1984) Transcription and Translation; Immobilized Cells and Enzymes (IRL Press (1986)); Perbal (1984) A Practical Guide to Molecular Cloning', Miller and Calos eds. (1987) Gene Transfer Vectors for Mammalian Cells (Cold Spring Harbor Laboratory); Makrides ed. (2003) Gene Transfer and Expression in Mammalian Cells; Mayer and Walker eds. (1987) Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); Herzenberg et al. eds (1996) Weir's Handbook of Experimental Immunology.

The DOTA-PRIT system disclosed in the present invention involves a three-step pre-targeting strategy comprising the introduction of (1) an IgG (IgG-scFv) bispecific antibody construct with a single chain variable fragment (scFv) containing antibody sequences with high affinity to the antitumor antigen antibody (part IgG) and single chain variable fragment scFv of anti-DOTA hapten (eg, C825); using (2) a scavenging agent of the present invention to rapidly clear circulating bispecific antibody after sufficient time has passed for the bispecific antibody to accumulate in the antigen-positive tumor; use of (3) a radiolabeled DOTA hapten composition (eg, ¹⁷⁷ Lu-DOTA-Bn).

The compositions of the present invention include novel N-acetylgalactosamine dendron-based scavenging agents that can be used in PRIT (eg, alpha particle radioimmunotherapy). The clearing agent compositions disclosed in the present invention demonstrate more effective clearance of radiolabeled bispecific antibody (BsAb) from the blood in DOTA-PRIT and improve the therapeutic index (TI) in DOTA-PRIT. For example, within 5 minutes after a single overdose of the dendron-based scavenging agent of the present invention (the molar ratio of administered bispecific antibody ¹³¹ I to scavenging agent is 1:2.3), the activity of ¹³¹ I in the blood decreased by 64% compared to baseline, from 6.7% ID/g to 2.4% ID/g. DOTA-PRIT studies in a colorectal cancer xenograft mouse model showed a dose-dependent scavenging agent tumor to blood uptake ratio of ¹⁷⁷ Lu-DOTA-Bn 24 h after administration of ¹⁷⁷ Lu activity (e.g., mean tumor to blood uptake ratio was 2. 9, 26, and 59 at 0 μg (carrier), 15 μg, or 20 μg for dendron-based clearing agent, respectively). A 25 μg dose of dendron-based scavenging agent resulted in an average tumor to blood uptake ratio of 76, which is almost identical to the previously optimized dosage of dextran-based scavenging agent (average tumor to blood uptake ratio of 77). Taken together, these results suggest that the dendron-based cleansing agent of the present invention is useful for increasing the therapeutic index of DOTA-HRIT.

Definitions.

Unless otherwise specified, all technical and scientific terms used in the present invention have essentially the same meaning as commonly understood by one skilled in the art to which the present invention belongs. In the present specification and the accompanying claims, the singular forms include references to the plural forms unless the content clearly indicates otherwise. For example, a reference to a cell includes a combination of two or more cells, etc. In general, the nomenclature and laboratory procedures used in the present invention in cell culture, molecular genetics, organic chemistry, analytical chemistry, nucleic acid chemistry and hybridization described below are well known and commonly used in the art.

As used herein, the term approximately, with respect to a number as a whole, includes numbers that fall within the range of 1, 5, or 10% in either direction (more or less) of the number, unless otherwise indicated or otherwise obvious from the context (unless when such a number is less than 0 or exceeds 100% of the possible value).

Pharmaceutically acceptable salts of the compounds described herein are within the scope of the present invention and include acid or base addition salts that retain the desired pharmacological activity and are not biologically undesirable (e.g., the salt is not excessively toxic, allergenic or irritant, and is bioavailable). If the connection according to the present invention with

- 9 044225 contains a basic group such as, for example, an amino group, pharmaceutically acceptable salts can be formed using inorganic acids (such as hydrochloric acid, boric acid, nitric acid, sulfuric acid and phosphoric acid), organic acids (such as alginate, formic acid, acetic acid, benzoic acid, gluconic acid, fumaric acid, oxalic acid, tartaric acid, lactic acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, naphthalenesulfonic acid and p-toluenesulfonic acid) or acidic amino acids (such as aspartic acid and glutamic acid). If the compound of the present invention contains an acidic group, such as, for example, a carboxylic acid group, it can form salts with metals such as alkali and alkaline earth metals (for example, Na ⁺ , Li ⁺ , K ⁺ , Ca ²⁺ , Mg ²⁺ , Zn ²⁺ ), ammonia or organic amines (eg dicyclohexylamine, trimethylamine, triethylamine, pyridine, picoline, ethanolamine, diethanolamine, triethanolamine) or basic amino acids (eg arginine, lysine and ornithine). Such salts may be prepared in situ during the isolation and purification of compounds or by separately reacting a purified compound in free base or free acid form with a corresponding acid or base to isolate the salt thus formed.

As used herein, the term administration of an agent or drug to a subject includes any route of administration or delivery to a subject of a compound to perform its intended function. Administration can be by any suitable route, including oral, intranasal, parenteral (intravenous, intramuscular, intraperitoneal or subcutaneous), rectal or topical. Administration includes self-administration of the drug and administration of the drug by another person.

As used herein, the term antibody collectively refers to immunoglobulins or immunoglobulin-like molecules, including by way of example, but not limited to IgA, IgD, IgE, IgG and IgM, combinations thereof, as well as similar molecules produced during an immune response in any vertebrate , for example, in mammals such as humans, goats, rabbits and mice, as well as in non-mammalian species such as shark immunoglobulins. In this context, antibodies (including intact immunoglobulins) and antigen-binding fragments specifically bind to a molecule of interest (or a group of very similar molecules of interest), substantially excluding binding to other molecules (for example, antibodies and antibody fragments that have a binding constant for the molecule of interest molecule that is about 103 M ^-1 times larger, about 10 ⁴ M ^-1 times larger, or about 10 ⁵ M ^-1 times larger than the binding constant for other molecules in the biological sample). The term antibody also includes genetically engineered forms such as chimeric antibodies (eg, humanized murine antibodies), heteroconjugated antibodies (such as bispecific antibodies). See also Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, 111.); Kuby, J., Immunology, ^3rd Ed., W. H. Freeman & Co., New York, 1997.

More specifically, an antibody refers to a polypeptide ligand comprising at least an immunoglobulin light chain variable region or an immunoglobulin heavy chain variable region that specifically recognizes and binds an epitope of an antigen. Antibodies are composed of a heavy and a light chain, each of which has a variable region called a heavy chain variable region (VH) and a light chain variable region (VL). Together, the V _H region and the V _L region are responsible for binding the antigen recognized by the antibody. Typically, immunoglobulin has heavy (H) chains and light (L) chains connected by disulfide bonds. There are two types of light chain: lambda (λ) and kappa (k). There are five main classes of heavy chains (or isotypes) that determine the functional activity of an antibody molecule: IgM, IgD, IgG, IgA and IgE. Each heavy and light chain contains a constant region and a variable region (regions are also known as domains). In combination, the heavy and light chain variable regions specifically bind antigen. The light and heavy chain variable regions contain a framework region interrupted by three hypervariable regions, also known as the complementarity determining region (CDR). The extent of the framework region and complementarity determining region has been determined (see Kabat et al., Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, 1991, incorporated herein by reference). The Kabat database is now maintained online. The framework sequences of different light or heavy chains are relatively conserved within a species. The antibody framework region, which is the combined framework regions of the light and heavy chain constituents, largely adopts a β-sheet conformation, and the complementarity determining regions form loops that connect and in some cases form part of the β-sheet structure. Thus, the framework regions act to form a scaffold that ensures the correct orientation of the complementarity determining region through interchain non-covalent interactions.

The complementarity determining region is primarily responsible for binding to the epitope of the antigen. The complementarity determining regions (CDRs) of each chain are usually designated as

- 10 044225

CDR1, CDR2 and CDR3 are numbered sequentially starting from the N-terminus and are also usually identified by the strand on which a particular CDR is located. Thus, VH CDR3 is located in the variable domain of the antibody heavy chain in which it is found, while V _L CDR1 is CDR1 from the variable domain of the light chain of the antibody in which it is found. An antibody that binds a target protein (eg, GPA33) or molecule (eg, DOTA or DOTA hapten) will have a specific V _H region and V _L region sequence and, therefore, specific CDR sequences. Antibodies with different specificities (i.e., different paratopes for different antigens) have different CDRs. Although CDRs vary from antibody to antibody, only a limited number of amino acid positions within a CDR are directly involved in antigen binding. These positions in the CDR are called specificity determining residues (SDRs). Examples of antibodies include monoclonal antibodies, polyclonal antibodies, humanized antibodies, chimeric antibodies, recombinant antibodies, multispecific antibodies, bispecific antibodies and antibody fragments. An antibody binds specifically to an antigen.

A bispecific antibody is an antibody that can be simultaneously bound to two different antigens. Bispecific antibodies (BsAb) and bispecific antibody fragments (BsFab) may have at least one arm that specifically binds, for example, to a tumor-associated antigen (eg, GPA33), and at least another arm that specifically binds to a target conjugate , carrying a therapeutic or diagnostic agent (for example, a DOTA hapten bound to a radionuclide). Many different bispecific antibody structures are known in the art. In some embodiments, each binding moiety in the bispecific antibody comprises a VH and/or _VL region from a different monoclonal antibody. In some embodiments, the bispecific antibody includes an immunoglobulin molecule comprising VH and/or VL regions that contain CDRs from the first monoclonal antibody, and an antibody fragment (e.g., Fab, F(ab'), F(ab') ₂ , Fd, Fv , dAB, scFv, etc.) containing VH and/or VL regions that contain the CDR from the second monoclonal antibody.

As used herein, the term diabodies refers to small antibody fragments with two antigen-binding regions, these fragments containing a heavy chain variable domain (V _H ) linked to a light chain variable domain (V _L ) in the same polypeptide chain (V _H V _L ). When using a linker that is too short to pair between two domains on one chain, the domains are forced to pair with complementary domains on the other chain and form two antigen-binding sites. Diabodies are described in more detail, for example, in EP 404097; WO 93/11161; 30 Hollinger et al., Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993).

As used herein, the terms single chain antibody or single chain Fv (scFv) refer to the fusion of an antibody molecule of two Fv fragment domains, V _L and V _H . Single chain antibody molecules may include a polymer with a number of individual molecules, such as a dimer, trimer, or other polymers. In addition, although the two domains of the Fv fragment, V _L and V _H are encoded by separate genes, they can be joined recombinantly using a synthetic linker that allows them to reside as a single protein chain that contains the V _L and a pair of regions V _H to form monovalent molecules (known as single-chain Fv (scFv)). Bird et al. (1988) Science 242:423-426 and Huston et al. (1988) Proc. Natl. Acad Sci. USA 85:5879-5883. Such single-chain antibodies can be produced by recombinant methods or by enzymatic or chemical digestion of intact antibodies.

As used herein, the terms intact antibody or intact immunoglobulin refer to an antibody or immunoglobulin that contains at least two heavy (H) chain polypeptides and two light (L) chain polypeptides linked by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated HCVR or VH) and a heavy chain constant region. The heavy chain constant region consists of three domains: CH1, _CH2 and CH3. Each light chain consists of a light chain variable region (abbreviated LCVR or V _L ) and a light chain constant region. The light chain constant region consists of a single C _L domain. The VH and _VL regions can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each V _H and V _L consists of three CDRs and four FRs, located from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR ₃ , CDR3, FR4. The heavy and light chain variable regions contain a binding domain that interacts with the antigen. Antibody constant regions can mediate the binding of immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system.

As used herein, the term antigen refers to a molecule to which an antibody can selectively bind. The target antigen may be a protein (eg, an antigenic peptide), carbohydrate, nucleic acid, lipid, hapten, or other naturally occurring or synthetic compound. The antigen can also be administered to an animal subject to create an immune response in the subject.

- 11 044225

As used herein, the term antigen-binding fragment refers to a fragment of the entire immunoglobulin structure that contains part of the polypeptide responsible for binding to the antigen. Examples of the antigen binding fragment used in the present invention include scFv, (scFv)2, scFvFc, Fab, Fab' and F(ab')2, diabodies; linear antibodies; single chain antibody molecules; multispecific antibodies formed from antibody fragments.

Binding affinity refers to the strength of the overall non-covalent interactions between one binding site of a molecule (for example, an antibody) and its binding partner (for example, an antigen). The affinity of a molecule X for its partner Y can usually be represented by the dissociation constant (Kd). Affinity can be measured by standard methods known in the art, including those described in the present invention. A low affinity complex contains an antibody that usually dissociates easily from the antigen, whereas a high affinity complex contains an antibody that usually remains associated with the antigen for a longer time.

As used herein, a scavenging agent is an agent that binds to excess bifunctional antibodies present in the blood compartment of a subject to facilitate rapid renal clearance. The use of a scavenging agent prior to the administration of DOTA radiotherapy promotes a better ratio of tumor uptake to background radiation in PRIT systems.

As used herein, the term control sample is an alternative sample used in an experiment for comparison purposes. The control sample can be positive or negative. For example, if the purpose of an experiment is to determine the correlation of the effectiveness of a therapeutic agent for treating a particular type of disease or condition, it is common to use a positive control sample (a compound or composition known to exhibit the desired therapeutic effect) and a negative control sample (a subject or sample that is not receiving therapy or receiving placebo).

As used herein, the term effective amount of a composition means an amount sufficient to achieve the desired prophylactic or therapeutic effect, e.g., an amount that results in a reduction in symptoms associated with the disease being treated, e.g., diseases or conditions associated with the target polypeptide (e.g. , breast cancer, colorectal cancer, brain cancer, etc.). The amount of the composition of the present invention administered to a subject will depend on the extent, type and severity of the disease and on the characteristics of the individual, such as general health, age, sex, body weight and drug tolerance. A qualified practitioner will be able to determine appropriate dosages based on these and other factors. The compositions of the present invention may also be administered in combination with one or more additional therapeutic compounds.

As used herein, the term epitope means an antigenic determinant capable of specifically binding to an antibody. Epitopes usually consist of chemically active surface moieties of molecules and usually have certain three-dimensional structural characteristics as well as certain charge characteristics.

As used herein, the term sample refers to clinical samples obtained from a subject or isolated microorganisms. In some embodiments, the sample is obtained from a biological source (ie, a biological sample), such as tissue, body fluid, or microorganisms collected from a subject. Sample sources include, but are not limited to, mucus, sputum, bronchial alveolar lavage (BAL), bronchial wash (BW), whole blood, body fluids, cerebrospinal fluid (CSF), urine, plasma, serum, or tissue.

As used herein, the term separate therapeutic administration refers to the administration of at least two active ingredients simultaneously or at substantially the same time by different routes.

As used herein, the term sequential therapeutic administration refers to the administration of at least two active ingredients at different times, the routes of administration being identical or different. More specifically, sequential administration refers to the complete administration of one of the active ingredients before administration of the other or others. Thus, it is possible to administer one of the active ingredients within a few minutes, hours or days before administering the other active ingredient or ingredients. In this case, simultaneous treatment does not occur.

As used herein, the term simultaneous therapeutic administration refers to the administration of at least two active ingredients by the same route and at the same or substantially the same time.

As used herein, the term specifically binds refers to a molecule (eg, an antibody) that recognizes and binds to another molecule (eg, an antigen) but does not substantially recognize or bind to other molecules. The terms specific binding, specifically binds to or is specific for a particular molecule (for example, antigen

- 12 044225 or an epitope on an antigen), as used in the present invention, may refer, for example, to a molecule having a K _d for the molecule to which it binds of about 10 ^-4 , 10 ^-5 , 10 ^-6 , 10 ^{- 7} , 10 ^-8 ,

9, 10' ¹⁰ , 10 ^-11 or 10'2 M.

As used herein, the terms subject, individual, or patient are used interchangeably and refer to an individual organism, vertebrate, mammal, or human. In some embodiments, the individual, patient, or subject is a human.

As used herein, the term therapeutic agent is intended to mean a compound that, when administered in an effective amount, produces a desired therapeutic effect on a subject in need thereof.

As used herein, the terms treatment or therapy include treating a disease or disorder described herein in a subject, such as a human, and includes: (i) inhibiting the disease or disorder (stopping its progression); (ii) alleviation of disease or disorder (regression); (iii) slowing the progression of the disorder; and/or (iv) inhibiting, alleviating or slowing the progression of one or more symptoms of a disease or disorder. Cancer treatment means that the symptoms caused by the cancer are, for example, alleviated, reduced, cured or put into remission.

It should also be appreciated that the various methods of treating diseases as described in the present invention are intended to represent the essential, which includes complete, but also less than complete treatment, and while achieving some biologically or medically significant result. Treatment may be continuous, long-term treatment of a chronic disease or a single or short-term administration of a drug to treat an exacerbation of the disease.

Use of clearing agents in pretargeted radioimmunotherapy (PRIT).

The therapeutic index (TI) of radioimmunotherapy (RIT) should be maximized for effective treatment of solid tumors (Larson SM et al., Nature Reviews Cancer 15: 347-60 (2015)). RIT with radiolabeled IgG antibodies has a low TI due to the slow pharmacokinetics of the IgG carrier and is therefore often ineffective at tolerated doses.

An alternative is to use a pre-targeted RIT (PRIT) approach. During PRIT, the step of slow antibody-mediated targeted delivery of the therapeutic drug to the tumor site is separated from the administration of the radioactive substance. PRIT is a multi-step process that addresses the slow clearance of tumor-targeted antibodies from the blood, which contributes to unwanted toxicity to healthy tissue such as bone marrow. During PRIT, a radionuclide or other diagnostic or therapeutic agent is coupled to a carrier that has more favorable pharmacokinetics (eg, a small molecule compound with rapid renal clearance and low healthy tissue uptake and retention (Orcutt KD et al., Molecular imaging and biology 13 : 215-21 (2011), such as the DOTA hapten), and the dose uptake into healthy tissue is minimized when targeting the therapeutic drug to the tumor site. To target the radiopharmaceutical to the tumor, the circulating therapeutic agent (for example, the DOTA hapten) is captured by a tumor-localized bispecific antibody or otherwise effectively excreted through the kidneys.

Scavenging agents can effectively reduce the concentration of circulating biomolecules by forming large complexes in the bloodstream that are recognized by the reticuloendothelial system (RES) or, using appropriate glychaptens, can be targeted to liver asialoglycoprotein receptors (ASPGR) (Rossin R et al., Journal Nuclear Medicine 54:1989-95 (2013)). The therapeutic index of DOTA-PRIT varies greatly depending on the dosage of the scavenging agent because the circulating bispecific antibody retains the ability to bind the radiolabeled DOTA hapten. To target the radiolabeled DOTA hapten to the tumor as accurately as possible, a saturating dose of the bispecific antibody is typically used to provide the highest concentration of anti-DOTA antibody domains to bind the subsequently administered radiolabeled DOTA hapten. At this stage, there is the problem of removing excess circulating unbound bispecific antibody before introducing the cytotoxic radiolabeled DOTA hapten in order to maximize the therapeutic index. Although long time intervals can be used to ensure clearance of the bispecific antibody, this may affect the binding ability of the bispecific antibody to the radiohapten in the tumor since the bispecific antibody may be degraded and/or absorbed depending on the molecular pharmacology of the bispecific antibody-antigen complex .

A 500 kDa dextran/DOTA hapten conjugate (Orcutt KD et al., Molecular Cancer Therapeutics 11: 1365-72 (2012)) has previously been used for DOTA PRIT using carcinoembryonic antigen. The dextran-based scavenging agent was designed to bind to the antiDOTA(M)-scFv domains of circulating bispecific antibody via a moiety of the DOTA(Y) molecule displayed on a dextran scaffold and clear the unbound bispecific antibody from the blood through recognition and catabolism by the reticuloendothelial system (RES). Because of him

- 13 044225 large size, the binding of the dextran-based scavenging agent to the tumor-associated bispecific antibody is limited due to poor intratumoral extravasation.

Despite its high efficiency, the use of a dextran-based cleaning agent has disadvantages. As a naturally occurring glucose polymer, the dextran scaffold is polydisperse in nature, which poses challenges associated with batch-to-batch production and in vivo application. In addition, enzymatic degradation of dextran-1,6 glucosidase by the reticuloendothelial system can lead to the introduction of its hapten fragments into the bloodstream, which can compete with radiohapten for binding to the tumor bispecific antibody. Similar problems have been observed with the use of an albumin-based scavenging agent during clinical streptavidin-biotin PRRT (Knox SJ et al., Clinical Cancer Research 6: 406-14 (2000); Breitz HB et al., Journal Nuclear Medicine 41: 131- 40 (2000)).

Compositions according to the present invention.

In one aspect, the present invention provides a compound of the form

- 14 044225

or a pharmaceutically acceptable salt and/or solvate thereof, in which X ¹ , X ² , X ³ , X ⁴ , X ⁵ , X ⁶ , X ⁷ , X ⁸ , X ⁹ , X 10 , X ¹¹ , X ¹² , ^{X 13} , X ¹⁴ , X ¹⁵ , X ¹⁶ , X ¹⁷ , X ¹⁸ , X ¹⁹ , X ²⁰ each independently represent H or a lone pair of electrons (i.e. provide an oxygen anion); Y ¹ , Y ² , Y ³ , Y ⁴ , Y ⁵ are each independently O or S; x is 1, 2 or 3; y is 1, 2, 3 or 4.

According to any embodiment of the present invention, the compound may be a cleaning agent (also referred to herein as a dendron-based cleaning agent of the present invention, a dendron-based OA of the present invention, or the like) of the form

- 15 044225

- 16 044225

or a pharmaceutically acceptable salt and/or solvate thereof, in which M ¹ , M ² , M ³ , M ⁴ , M ⁵ are each independently Lu ³⁺ , Sc ³⁺ , Ga ³⁺ , Y ³⁺ , In ³⁺ , La ³⁺ , Ce ³⁺ , Eu ³⁺ , Tb ³⁺ , or Gd ³⁺ ; X ¹ , X ² , X 3, X ⁴ , X ⁵ , X ⁶ , ^{X 7 ,} X ⁸ , X 9, X ¹⁰ , X ¹¹ , X ¹² , X ¹³ , X ¹⁴ , X ¹⁵ , ^{X 16 ,} X ¹⁷ , X ¹⁸ , X ¹⁹ , X ²⁰ each independently represents H or a lone pair of electrons (ie, provides an oxygen anion); Y ¹ , Y ² , Y ³ , Y ⁴ , Y ⁵ are each independently O or S; x is 1, 2 or 3; y is 1, 2, 3 or 4.

According to any embodiment of the present invention, it may be that M ¹ , M ² , M ³ , M ⁴ , M ⁵ are each independently not a radionuclide. In any embodiment of the present invention, it may be that Y ¹ , Y ² , Y ³ , Y ⁴ , Y ⁵ are each independently S. In any embodiment of the present invention, x may be 1 or 2. In any embodiment of the present invention, it may turn out that y is equal to 2 or 3.

In a related aspect, a composition is provided that includes one or more compounds

- 17 044225 of any embodiment of the compound as described above, and a pharmaceutically acceptable carrier (collectively, such carriers, excipients, excipients, etc. will be referred to as pharmaceutically acceptable carriers unless a more specific term is used). The compositions can be used in the methods and presentations described in the present invention. The present invention also provides pharmaceutical compositions and drugs that include one or more compounds of any embodiment as described above and a pharmaceutically acceptable carrier. Such pharmaceutical compositions may be packaged into a unit dosage form. Pharmaceutical compositions and drugs can be prepared by mixing one or more compounds of the present invention, pharmaceutically acceptable salts thereof and/or solvates thereof with pharmaceutically acceptable carriers, excipients, binders, diluents, and the like. For example, such compositions may be in the form of granules, powders, tablets, capsules, syrup, suppositories, injections, emulsions, elixirs, suspensions or solutions. The present compositions can be formulated for various routes of administration, for example, oral, parenteral or rectal administration. Parenteral or systemic administration includes, but is not limited to, subcutaneous, intravenous, intraperitoneal and intramuscular injections. The following dosage forms are provided by way of example and should not be construed as limiting the present invention.

For oral, buccal and sublingual administration, powders, suspensions, granules, tablets, pills, capsules, gelatin capsules and caplets are acceptable as solid dosage forms. They can be prepared, for example, by mixing one or more compounds of the present invention or pharmaceutically acceptable salts or tautomers thereof with at least one additive, such as starch or other additive. Acceptable additives include sucrose, lactose, cellulose sugar, mannitol, maltitol, dextran, starch, agar, alginates, chitins, chitosans, pectins, gum tragacanth, gum acacia, gelatins, collagens, casein, albumin, synthetic or semi-synthetic polymers or glycerides. Optionally, oral dosage forms may contain other ingredients to aid administration, such as an inactive diluent, or lubricants, such as magnesium stearate, or preservatives, such as paraben or sorbic acid, or antioxidants, such as ascorbic acid, tocopherol or cysteine, a disintegrant , binding agents, thickeners, buffers, sweeteners, flavoring agents or flavoring agents. Tablets and pills can be further treated with suitable coating materials known in the art.

Liquid dosage forms for oral administration may be in the form of pharmaceutically acceptable emulsions, syrups, elixirs, suspensions and solutions, which may contain an inactive diluent such as water. Pharmaceutical formulations and drugs may be prepared in the form of liquid suspensions or solutions using a sterile liquid such as, but not limited to, oil, water, alcohol, and combinations thereof. Pharmaceutically acceptable surfactants, suspending agents, emulsifying agents may be added for oral or parenteral administration.

As noted above, suspensions may include oils. Such oils include, but are not limited to, peanut oil, sesame oil, cottonseed oil, corn oil and olive oil. The suspension formulation may also contain fatty acid esters such as ethyl oleate, isopropyl myristate, fatty acid glycerides and acetylated fatty acid glycerides. Suspension formulations may include, but are not limited to, alcohols such as ethanol, isopropyl alcohol, hexadecyl alcohol, glycerin, and propylene glycol. Ethers may include, but are not limited to, polyethylene glycol, petroleum hydrocarbons such as mineral oil and petroleum jelly; water can also be used in suspension formulations.

Injectable dosage forms typically include aqueous suspensions or oily suspensions, which can be prepared using a suitable dispersant or wetting agent and suspending agent. Injectable forms can be in the solution phase or in the form of a suspension, which is prepared using a solvent or diluent. Acceptable solvents or carrier media include sterilized water, Ringer's solution, or isotonic aqueous saline. An isotonic solution should be understood as isotonic with respect to the object. Alternatively, sterile oils may be used as solvents or suspending agents. Typically the oil or fatty acid is non-volatile, including natural or synthetic oils, fatty acids, mono-, di- or triglycerides.

For injection, the pharmaceutical composition and/or drug may be in the form of a powder suitable for reconstitution with a suitable solution as described above. Examples thereof include, but are not limited to, lyophilized powders, rotary-dried or spray-dried powders, amorphous powders, granules, precipitates or particles. For injection, the formulations may optionally contain stabilizers, pH modifiers, surfactants, bioavailability modifiers, and combinations thereof.

In addition to these typical dosage forms described above, pharmaceutically acceptable

- 18 044225 excipients and carriers are generally known to those skilled in the art and are thus included in the present invention. Such excipients and carriers are described, for example, in Remingtons

Pharmaceutical Sciences Mack Pub. Co., New Jersey (1991); the contents are incorporated into the present invention by reference.

The compositions of the present invention can be formulated as short-acting, quick-release, long-acting, and slow-release as described below. Thus, pharmaceutical formulations can also be formulated as controlled release or slow release.

The present compositions may also contain, for example, micelles or liposomes or some other encapsulated form, or may be administered in a slow-release form for long-term storage and/or delivery. Therefore, pharmaceutical formulations and drugs can be compressed into pellets or cylinders and implanted intramuscularly or subcutaneously as depot injections or as implants such as stents. Such implants may use known inert materials such as silicones and biodegradable polymers.

Specific dosages may be adjusted depending on the disease state, age, body weight, general health, sex and diet of the subject, dosage intervals, routes of administration, rates of elimination, and drug combinations. Any of the above dosage forms containing effective amounts are within the scope of batch experiments and, therefore, within the scope of the present invention.

Therapeutic techniques according to the present invention.

In one aspect, the present invention provides a method of sensitizing a tumor to radiation therapy in a subject diagnosed with cancer, comprising (a) administering to the subject an effective amount of an anti-DOTA bispecific antibody, wherein the anti-DOTA bispecific antibody is configured to localize to a tumor expressing a tumor target. antigen; (b) administering to the subject an effective amount of a cleansing agent according to the present invention; (c) administering to the subject an effective amount of a radiolabeled DOTA hapten, wherein the DOTA hapten is configured to form a complex with an anti-DOTA bispecific antibody.

In another aspect, the present invention provides a method of treating cancer in a subject in need thereof, comprising (a) administering to the subject an effective amount of an anti-DOTA bispecific antibody, wherein the anti-DOTA bispecific antibody is configured to localize to a tumor expressing a target tumor antigen; (b) administering to the subject an effective amount of a cleansing agent according to the present invention; (c) administering to the subject an effective amount of a radiolabeled DOTA hapten, wherein the DOTA hapten is configured to form a complex with an anti-DOTA bispecific antibody. Methods of treating cancer may further include sequential, separate, or simultaneous administration to a subject of at least one chemotherapeutic agent selected from the group consisting of nitrogen mustards, ethyleneimine derivatives, alkylsulfonates, nitrosoureas, gemcitabine, triazenes, folic acid analogues, anthracyclines, taxanes, COX inhibitors -2, pyrimidine analogs, purine analogs, antibiotics, enzyme inhibitors, epipodophyllotoxins, platinum coordination complexes, vinca alkaloids, substituted ureas, methylhydrazine derivatives, adrenocortical suppressors, hormone antagonists, endostatin, taxols, camptothecins, SN-38, doxorubicin, doxorubicin analogs, antimetabolites, alkylating agents, antimitotics, antiangiogenic agents, tyrosine kinase inhibitors, mTOR inhibitors, heat shock protein (HSP90) inhibitors, proteasome inhibitors, HDAC inhibitors, proapoptotic agents, methotrexate, CPT-11.

Additionally or alternatively, according to some embodiments of the methods disclosed herein, the levels of radioactivity emitted by the complex of the radiolabeled DOTA hapten and the anti-DOTA bispecific antibody are detected between 4 and 24 hours after administration of the radiolabeled DOTA hapten. The levels of radioactivity emitted by the complex can be expressed as a percentage of the administered dose per gram of tissue (% ID/g). The reference value can be calculated by measuring the levels of radioactivity present in non-neoplastic (healthy) tissues and calculating the mean levels of radioactivity present in non-neoplastic (healthy) tissues ± standard deviation. In some embodiments, the reference value is a standard absorbance value (SAV). See Thie JA, J. Nucl Med. 45 (9): 1431-4 (2004). The therapeutic efficacy of such a complex can be determined by calculating the ratio of the area under the pharmacokinetic curve of the tumor to the area under the pharmacokinetic curve of healthy tissue. In some embodiments, the complex has a ratio of the area under the tumor pharmacokinetic curve to the area under the pharmacokinetic curve of healthy tissue of approximately 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9: 1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 55:1, 60:1, 65:1, 70:1, 75:1, 80:1, 85:1, 90:1, 95:1 or 100:1. Additionally or alternatively, according to some embodiments of the methods disclosed herein, the ratio of tumor radioactivity levels to healthy tissue radioactivity levels is approximately 2:1, 3:1, 4:1, 5:1, 6: 1,

7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 55: 1, 60:1, 65:1, 70:1, 75:1, 80:1, 85:1, 90:1,

95:1 or 100:1.

Additionally or alternatively, according to some embodiments of the methods disclosed herein, the target tumor antigen is selected from the group consisting of GPA33, HER2/neu, GD2, MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE -2, MUM-1, CDK4, Nacetylglucosaminyltransferase, p15, gp75, beta-catenin, ErbB2, cancer antigen 125 (CA-125), carcinoembryonic antigen (CEA), RAGE, MART (melanoma antigen), MUC-1, MUC- 2, MUC-3, MUC-4, MUC-5ac, MUC-16, MUC-17, tyrosinase, Pmel 17 (gp100), GnT-V subtype V intron sequences (N-acetylglucoaminyltransferase subtype V intron V sequences), prostate cancer psm, PRAME (melanoma antigen), β-catenin, EBNA (Epstein-Barr virus nuclear antigen) 16, p53, lung cancer resistance protein (LRP) Bcl-2, prostate-specific antigen (PSA), Ki-67, CEACAM6 , colon specific antigen-p (CSAp), HLA-DR, CD40, CD74, CD138, EGFR, EGP-1, EGP-2, VEGF, P1GF, insulin-like growth factor (ILGF), tenascin, platelet-derived growth factor, IL- 6, CD20, CD19, PSMA, CD33, CD123, MET, DLL4, Ang-2, HER3, IGF-1R, CD30, TAG-72, SPEAP, CD45, L1-CAM, Lewis antigen Y (Ley), E-cadherin , Vcadherina, ErCAM.

Additionally or alternatively, according to some embodiments of the methods disclosed herein, the anti-DOTA bispecific antibody, scavenging agent, and/or radiolabeled DOTA hapten are administered intravenously, intramuscularly, intraarterially, intrathecally, intracapsularly, intraorbitally, intradermally, intraperitoneally, transtracheally, subcutaneously, intracerebroventricularly, orally or intranasally.

Additionally or alternatively, according to some embodiments of the methods disclosed herein, the cancer is selected from the group consisting of breast cancer, colorectal cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, hepatocellular carcinoma, cancer brain, lung cancer, gastrointestinal or stomach cancer, pancreatic cancer, thyroid cancer, kidney cancer, prostate cancer, melanoma, sarcoma, carcinoma, Wilms tumor, endometrial cancer, glioblastoma, squamous cell carcinoma, astrocytoma, salivary gland carcinoma , vulvar cancer, penile carcinoma, head and neck cancer. Brain cancer can be a pituitary adenoma, meningioma, neuroblastoma, or craniopharyngioma.

Additionally or alternatively, according to some embodiments of the methods disclosed herein, the radiolabeled DOTA hapten includes one or more of Proteus-DOTA, S-2-(R-aminobenzyl)-1,4,7,10 -tetraazacyclododecanetetraacetic acid (DOTA-Bn), DOTA-Bn-biotin, BAD (((S)-2-(4-(2-bromo)acetamido)benzyl)-DOTA), NBD ((S)-2-(4nitrobenzyl )-DOTA), DOTA-RGD, DOTA-PEG-E(c(RGDyK)) ₂ , DOTA-8-AOC-BBN, p-NO ₂ -Bn-DOTA, DOTA-PESIN, DOTA-biotin-sarcosine (DOTA -biotin), mono 1,4,7,10-tetraazacyclododecane-1,4,7,10tetraacetic acid (N-hydroxysuccinimide ester) (DOTA-NHS) or DOTATyrLysDOTA. Alternatively, any radiolabeled DOTA hapten known in the art can be used in the methods disclosed in the present invention. The radiolabeled DOTA hapten may be labeled with a radionuclide selected from the group consisting of ²¹³ Bi, ²¹¹ At, ²²⁵ Ac, ¹⁵² Dy, ²¹² Bi, ²²³ Ra, ²¹⁹ Rn, ²¹⁵ Po, ²¹¹ Bi, ²²¹ Fr, ²¹⁷ At, ²⁵⁵ Fm, ⁸⁶ Y, ⁹⁰ Y, ⁸⁹ Sr, ¹⁶⁵ Dy, ¹⁸⁶ Re, ¹⁸⁸ Re, ¹⁷⁷ Lu, 67 Cu, ^{1n In} , ⁶⁷ Ga, ⁵¹ Cr, ⁵⁸ Co, ^99m Tc, ^103m Rh, ^195m Pt, ¹¹⁹ Sb, ¹⁶¹ Ho , ^189m Os, ¹⁹² Ir, ²⁰¹ Tl, ²⁰³ Pb, ⁶⁸ Ga, ²²⁷ Th, ⁶⁴ Cu.

In any of the above-described embodiments of the methods disclosed in the present invention, the subject is a human. The anti-DOTA bispecific antibody is administered under conditions and for a period of time (eg, according to a dosage regimen) sufficient to saturate the tumor cells, and any unbound anti-DOTA bispecific antibody is cleared from the bloodstream following administration of the anti-DOTA bispecific antibody. In some embodiments, the radiolabeled DOTA hapten is administered after a period of time that may be sufficient to clear the unbound anti-DOTA bispecific antibody.

Additionally or alternatively, according to some embodiments of the methods disclosed herein, the scavenging agent and radiolabeled DOTA hapten are administered without further administration of the anti-DOTA bispecific antibody. For example, in some embodiments, the anti-DOTA bispecific antibody is administered according to a regimen that includes at least one cycle of: (i) administering the anti-DOTA bispecific antibody (saturation of the appropriate tumor cells is optional); (ii) introducing a cleaning agent according to the present invention and a radiolabeled DOTA hapten; (iii) optionally additional administration of a radiolabeled DOTA hapten and/or a scavenging agent without additional administration of an anti-DOTA bispecific antibody. In some embodiments, the method may include multiple such cycles (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more cycles).

The radiolabeled DOTA hapten can be administered at any time from 1 minute to 4 days or more after administration of the anti-DOTA bispecific antibody. For example, in some embodiments, the radiolabeled DOTA hapten is administered at 1 minute, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 minutes, 1 hour,

- 20 044225

1.25, 1.5, 1.75, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,

21, 22, 23, 24, 48, 72, 96 hours or any range thereof after administration of the anti-DOTA bispecific antibody. Alternatively, radiolabeled DOTA hapten can be administered at any time 4 or more days after administration of the anti-DOTA bispecific antibody.

Additionally or alternatively, according to some embodiments of the methods disclosed herein, the radiolabeled DOTA hapten may be administered at any time from 1 minute to 4 days or more after administration of the cleansing agent. For example, in some embodiments, the radiolabeled DOTA hapten is administered at 1 minute, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 minutes, 1 hour, 1.25, 1.5, 1.75, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 48, 72, 96 hours or any range thereof after administration of the cleansing agent . Alternatively, radiolabeled DOTA hapten can be administered at any time 4 or more days after administration of the clearing agent.

Sets.

The present invention provides kits containing components useful for treating cancer in a patient. In one aspect, the kits contain a cleaning agent according to the present invention and instructions for use. The kits may further contain at least one anti-DOTA bispecific antibody. In some embodiments, at least one anti-DOTA bispecific antibody binds to a target tumor antigen selected from the group consisting of GPA33, HER2/neu, GD2, MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE- 2, MUM-1, CDK4, N-acetylglucosaminyltransferase, p15, gp75, beta-catenin, ErbB2, cancer antigen 125 (CA-125), carcinoembryonic antigen (CEA), RAGE, MART (melanoma antigen), MUC-1, MUC -2, MUC-3, MUC-4, MUC-5ac, MUC-16, MUC-17, tyrosinase, Pmel 17 (gp100), GnT-V subtype V intron sequences (N-acetylglucoaminyltransferase subtype V intron V sequences), cancer prostate psm, PRAME (melanoma antigen), β-catenin, EBNA (Epstein-Barr virus nuclear antigen) 1-6, p53, lung cancer resistance protein (LRP) Bcl-2, prostate-specific antigen (PSA), Ki- 67, CEACAM6, colon specific antigen-p (CSAp), HLA-DR, CD40, CD74, CD138, EGFR, EGP-1, EGP-2, VEGF, P1GF, insulin-like growth factor (ILGF), tenascin, platelet-derived growth factor , IL-6, CD20, CD19, PSMA, CD33, CD123, MET, DLL4, Ang-2, HER3, IGF-1R, CD30, TAG-72, SPEAP, CD45, LI-CAM, Lewis Y antigen (Ley), Ecadherin, V-cadherin, EpCAM. The at least one anti-DOTA bispecific antibody may be presented in the form of a prefilled syringe or autoinjector pen containing a sterile liquid formulation or lyophilized antibody preparation (e.g., Kivitz et al., Clin. Ther. 28:1619-29 (2006)) .

Additionally or alternatively, in some embodiments, the kits further contain a DOTA hapten, which may optionally be labeled with one or more radionuclides. Examples of DOTA haptens include compounds such as Proteus-DOTA, S-2-(Raminobenzyl)-1,4,7,10-tetraazacyclododecanetetraacetic acid (DOTA-Bn), DOTA-Bn-biotin, BAD (((S)-2 -(4-(2-bromo)-acetamido)-benzyl)-DOTA), NBD ((S)-2-(4-nitrobenzyl)-DOTA), DOTA-RGD, DOTAPEG-E(c(RGDyK)) ₂ , DOTA-8-AOC-BBN, p-NO ₂ -Bn-DOTA, DOTA-PESIN, DOTA-biotin-sarcosine (DOTA-biotin), mono 1,4,7,10-tetraazacyclododecane-1,4,7, 10-Tetraacetic acid (Nhydroxysuccinimide ester) (DOTA-NHS) or DOTATyrLysDOTA, etc., but not limited to. Additionally or alternatively, according to some embodiments of the kits of the present invention, one or more radionuclides are selected from the group consisting of ²¹³ Bi, ²¹¹ At, ²²⁵ Ac, ¹⁵² Dy, ²¹² Bi, ²²³ Ra, ²¹⁹ Rn, ²¹⁵ Po, ²¹¹ Bi , ²²¹ Fr, ²¹⁷ At, ²⁵⁵ Fm. Additionally or alternatively, in some embodiments, the one or more radionuclides are selected from the group consisting of ⁸⁶ Y, ⁹⁰ Y, ⁸⁹ Sr, ¹⁶⁵ Dy, ¹⁸⁶ Re, ¹⁸⁸ Re, ¹⁷⁷ Lu, ⁶⁷ Cu, ⁿ¹ In, ⁶⁷ Ga, ⁵¹ Cr, ⁵⁸ Co, ^99m Tc, ^103m Rh, ^195m Pt, ¹¹⁹ Sb, ¹⁶¹ Ho, ^189m Os, ¹⁹² Ir, ²⁰¹ Tl, ²⁰³ Pb, ⁶⁸ Ga, ²²⁷ Th, ⁶⁴ Cu.

If the kit components are not formulated for oral administration, a device capable of delivering the kit components by some other route may be included. Examples of such devices include syringes (for parenteral administration) or inhalation devices.

Kit components may be packaged together or divided into two or more containers. In some embodiments, the containers may be vials containing sterile lyophilized formulations of a cleaning agent disclosed herein, a DOTA hapten and/or an anti-DOTA composition and a bispecific antibody that are suitable for reconstitution. The kit may also contain one or more buffers that are suitable for reconstitution and/or dilution of other reagents. Other containers that may be used include, but are not limited to, a sachet, a cuvette, a box, a test tube, and the like. The kit components can be packaged and stored in sterile containers.

Examples

Example 1 Illustrative example of the synthesis of compounds according to the present invention.

Basic provisions. DOTA-Bn isothiocyanate (p-SCN-Bn-DOTA) was purchased from Macrocyclics, Inc. (Plano, Texas, USA); amine-PEG4-DOTA was purchased from Che

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Matech (Dijon, France). Optima™ hydrochloric acid was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Chelex-100 resin, 200-400 mesh, was purchased from Bio-Rad Laboratories (Hercules, CA, USA). PD-10 size exclusion gel filtration columns (containing 8.3 mL Sephadex™ G-25 resin per column) were purchased from GE Healthcare Life Sciences (Pittsburgh, PA, USA). All other reagents and synthetic grade chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA) and were used without further purification. All solvents used for HPLC analysis (HPLC grade) and purification of compounds were also purchased from Thermo Fisher Scientific (Waltham, MA, USA). All buffers and solutions were prepared using ultrapure water (resistivity: 18 MΩ-cm).

All liquid chromatography-mass spectrometry (LC/MS) data were acquired using a Waters Autopure system (Milford, Massachusetts, USA) containing the following instrumentation: 2767 sample management program, 2545 binary gradient module, inkjet system organizer, 2424 evaporative light scattering detector, 2998 photodiode array detector, 3100 mass detector. HPLC solvents (solvent A, 0.05% trifluoroacetic acid in water; solvent B, 0.05% trifluoroacetic acid in acetonitrile) were filtered before use. Analytical method: 5-25% solvent B, time - 10 min, flow rate - 1.2 ml/min. Analytical columns: Waters XBridge BEH300 (Milford, Massachusetts, USA), C4, 3.5 µm, 4.6x50 mm and C18, 4 µm, 4.6x50 mm. Preparative method: 5-25% solvent B, time - 30 minutes, flow rate - 20 ml/min. Preparative column: Waters XBridge Prep (Milford, Massachusetts, USA) C18, 4 µm, optimal layer density, 19x150 mm.

All NMR data were acquired using Bruker AV500 or AV600 instruments (Bruker, Billerica, MA, USA) at ambient temperature. The following abbreviations were used: singlet (s), broad singlet (bs), doublet (d), triplet (t), quartet (q), pentet (p), doublet doublet (dd), multiplet (iii).

All experiments using molecules with high metal complexing properties, such as DOTA, were carried out in glassware that was first washed with metal-free HCl, rinsed with high purity water (eg water distilled in a glass jar) and dried in an oven. Chromatography was carried out on hand-packed glass columns to avoid reaction of the complexing agent with metal leached or recovered from the metal walls of the column. Reversed phase purification was performed on clean, metal-free glass columns that were manually filled with loose C-18 silica gel. The water content of the resulting complexes was not measured.

CCA-16-methyl ether-NHBoc (illustrated below) and 5-((tert-butoxycarbonyl)amino)pentyl-2-acetamido-2-deoxy-1-thio-3,4,6-tri-O-acetyl-α-D -galactopyranoside (monoperacetyl-sugar-NHBoc) was prepared according to similar methods and protocols as described in Yoo, B. et al., N-Acetylgalactosamino Dendrons as Clearing Agents to Enhance Liver Targeting of Model Antibody-Fusion Protein, Bioconjugate Chem. 2013, 24, 2088-2103 (including related supporting information) and Cheal, S.M. et al., Evaluation of Glycodendron and Synthetically Modified Dextran Clearing Agents for Multistep Targeting of Radioisotopes for Molecular Imaging and Radioimmunotherapy, Mol. Pharmaceutics 2014, 11, 400-16 (including related supporting information), the contents of which are incorporated herein by reference. The corresponding characteristic data for CCA-16-methyl ether-NHBoc and monoperacetyl-sugar-NHBoc are given below.

CCA-16-methyl ether-NHBoc.

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Ή NMR (600 MHz, D2O) δ: 3.68, 3.66 (2s, 48H, OCH3), 3.30-3.26 (m, 32H), 3.22-3.19 (m, 32H), 2.83 (bs, NCH ₃ ), 2.35-2.25 (m, 64H), 1.69-1.51 (m, 128H), 1.44 (s, 9 H, C(CH ₃ ) ₃ ), 1.34-1.29 (m, 64H). ¹³ C NMR (125 MHz, D2O) δ: 174.10, 173.87, 172.10, 51.57, 51.48, 47.86, 47.79, 45.78, 45.70, 33.95 , 33.84, 33.83, 33.05, 32.89, 29.21, 28.90, 28.50, 27.70, 27.48, 27.46, 26.99, 26.84, 26 .55, 26.42, 25.23, 25.13, 24.68, 24.64.

Monoperacetyl-caxap-NHBoc (5-((tert-butoxycarbonyl)amino)pentyl-2-acetamido-2-deoxy-1 thio-3,4,6-tri-O-acetyl-α-O-galactopyranoside): 1H NMR (600 MHz, D2O) δ: 5.57 (d, 1H, J=7.4 Hz), 5.49 (d, 1H, J=4.4 Hz), 5.38 (d, 1H, J= 2.4 Hz), 5.05 (dd, 1H, J=2.4 Hz, J=7.4 Hz), 4.80-4.74 (m, 1H), 4.58 (bs, 1H) , 4.54 (t, 1H, J=5.4 Hz), 4.13 (dd, 1H, J=9.6 Hz, J=5.4 Hz), 4.09 (dd, 1H, J= 9.6 Hz, J=5.4 Hz), 3.14-3.08 (m, 2H), 2.662.55 (m, 2H), 2.16, 2.05, 2.01, 1.98 (4s, 12H, COCH ₃ ), 1.67-1.61 (m, 2H), 1.44 (s, 9H, C(CH ₃ ) ₃ ), 1.41-1.38 (m, 2H) . ¹³ C NMR (125 MHz, D2O) δ: 171.00, 170.39, 170.28, 170.15, 155.98, 84.93, 68.54, 67.34, 67.24, 61.83 , 48.33, 40.38, 30.97, 29.62, 29.28, 28.43, 25.94, 23.34, 20.75, 20.71.

An illustrative example of the synthesis of SCA-16 acid.

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To CCA-16-methyl ether-NHBoc (2.1 g, 0.54 mmol) contained in MeOH (60 ml), NaOH (10 N, 16 ml) and water (16 ml) were added. The resulting mixture was shaken at room temperature for 1 hour. The pH was then adjusted to 5.0 with 2.0 N. HCl and solution were loaded into a separatory funnel. Next, extraction was carried out with a mixture of dichloromethane tert-butanol (3/1, v/v), 150 mlx5, and the combined organic layers were briefly dried using Na ₂ SO ₄ . After filtration through a pad of celite, the filtrate was evaporated under reduced pressure and the residue was further dried under high vacuum for 2 hours to obtain 2.0 g of a colorless thick oil. The product was of sufficient purity for immediate use in the next step.

CCA-16-caxap-NHBoc.

CCA-16 acid (2.0 g, 0.54 mmol) was diluted in dimethylformamide (80 ml) before purification using 1-bis(dimethylamino)methylene-1H-1,2,3-triazolo-4,5-b pyridinium -3-oxide-hexafluorophosphate (HATU) (4.0 g, 10.5 mmol) and 5-amino-1-pentyl-a-thio-tetraacetylgalactosamine (5.6 g, 9.9 mmol). The resulting mixture was shaken at room temperature for 20 minutes before adding diisopropylethylamine (8.0 ml). After 1 hour, dichloromethane (200 ml) was added and the reaction mixture was washed with water (2x100 ml). The organic layer was then briefly dried using Na ₂ SO ₄ , filtered through a pad of celite, and the filtrate was concentrated in vacuo. Purification using flash column chromatography with a gradient of 0 to 15% MeOH in dichloromethane (v/v) gave CCA-16-caxapNHBoc, 4.9 g, 85% yield. 1H NMR (600 MHz, D2O) δ: 7.95 (bs, 12H, NH), 5.64-5.61 (m, 16H), 5.47-5.46 (d, 16H, J=2, 8 Hz), 5.06 (dd, 16 N, J=11.8 Hz), 4.64-4.60 (m, 32H), 4.18-4.12 (m, 32H), 3.35 (m, 32H), 3.21-3.19 (m, 36H), 3.02, 2.88 (2s, 3H, NCH ₃ ), 2.86-2.84 (bs, 4H), 2, 70-2.61 (m, 32H), 2.41-2.38 (m, 36H), 2.24 (m, 32H), 2.21, 2.06, 1.98, 1.97 (4s , 192H, COCH ₃ ), 1.69-1.64 (m, 128H, 1.59-1.53 (m, 70H), 1.48 (s, 9H, OC(CH ₃ ), 1.48- 1.46 (m, 32), 1.40-1.33 (m, 70H) ¹³ C NMR (125 MHz, D2O) δ: 174.60, 174.38, 174.30, 173.41, 172 ,23, 172.14, 170.66, 170.34, 84.07, 68.23, 67.23, 66.80, 61.78, 53.46, 45.67, 39.01, 38.88 , 35.73, 35.63, 35.58, 32.59, 32.51, 30.03, 29.06, 28.66, 28.54, 27.57, 27.08, 26.37, 26 ,28, 26.22, 26.10, 25.83, 25.45, 25.36, 25.18, 25.10, 21.26, 21.22, 19.44, 19.37, 19.24 .

CCA-16-peracetyl-a-thiogal actosamine-NH ₂ ·TFA.

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mg CCA-16-peracetyl-α-thiogalactosamine-NHBoc (1.9 μmol) was dissolved in 0.8 ml of 20% trifluoroacetic acid (TFA) and dichloromethane (v/v), then shaken at room temperature for 60 min. . Next, the mixture was evaporated under reduced pressure to remove trifluoroacetic acid and dichloromethane. The reaction progress was monitored by LCMS, which was carried out using an Autopure System (Waters) using C4 (Xbridge, Waters, 4.6x50 mm) with a gradient of 5-95% v/v acetonitrile in water containing 0 .05% trifluoroacetic acid, for 10 minutes. Detection was achieved by simultaneous operation of a mass spectrometer, a diode array, and an evaporative light scattering detector. The residue (20 mg) was further dried overnight under high vacuum and used directly in the next step.

CCA-16-peracetyl-α-thiogalactosamine-NHCSNMe-Bn-DOTA.

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Crude ammonium salt (CCA-16-peracetyl-α-thiogalactosamine-NH ₂ ·TFA) and P-SCN-Bn-DOTA (2.5 mg, 3.8 μmol, 2 eq.) were dissolved in dimethylformamide (0.3 ml ) and then triethylamine (Et ₃ N) (6 µl) was added. The resulting homogeneous mixture was shaken at room temperature for 4 hours; volatiles were removed under high vacuum at room temperature to obtain a foam of sufficient purity, which was used in the next step. LCMS evaluation was performed using the same system as the previous step, but instead used an XBridge C18 column (4.6 x 150 mm) with a gradient of 40-95% v/v acetonitrile in water containing 0.05% trifluoroacetic acid. slots.

CCA-16-α-thio-peracetylgalactosamine-DOTA-Y ³⁺ .

The raw material from the previous step CCA-16-peracetyl-α-thiogalactosamine-NHCSNMe-BnDOTA was added to a solution of YCl ₃ -6H ₂ O (6 mg) in 0.05 M HCl (0.2 ml) and 0.5 M NH ₄ OAc (0.2 ml). The mixture was shaken for 4 hours. Attempts at purification using HPLC at this stage were unsuccessful. However, analytical control showed that the reaction proceeded with acceptable purity. LCMS was performed on an XBridge C18 column with a gradient of 5-95% v/v acetonitrile in water (both containing 0.05% trifluoroacetic acid) over 10 min. The remainder was used in the next and final step. The results obtained by electrospray ionization tandem mass spectrometry (ESI-MS) (m/z) were as follows: [M+6H] ⁶⁺ cal. 1847.55, exp. 1847.86.

CCA-16-DOTA-Y ³⁺ .

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Crude CCA-16-α-thio-peracetylgalactosamine-DOTA-Y ³⁺ was dissolved in MeOH (0.5 ml) and degassed NaOH (0.2 N, 400 μl) was added to the solution. After 30 min, the reaction mixture was evaporated to dryness, dissolved in a minimum amount of water and subjected to HPLC (XBridge C18, 20-70% v/v with a gradient of acetonitrile in water (both containing 0.05% trifluoroacetic acid) for 10 min). The appropriate fractions were combined and lyophilized to obtain 22 mg of the title compound as a white foam with an overall yield of 64% based on CCA-16-methyl ester-NHBoc. 1H NMR (600 MHz, D2O) δ: 7.21-7.24 (m, 4H, Ph), 5.43 (d, 16 H, J=5.2 Hz, HChar-1), 4.27 ( dd, 16H, J=5.2 Hz, J=11.2 Hz, NSahar-2), 4.17-4.14 (m, 16 N, NSahar-5), 3.96 (d, 16 H, J=2.7 Hz, Nsahar-4), 3.75 (dd, 16H, J=2.7 Hz, J=11.2 Hz, Nsahar-3), 3.68 (d, 32H, J=6.0 Hz, Nsuhar-6), 3.23 (bs, 64H), 3.15-3.08 (m, 36H), 3.00 and 2.85 (2s, 3H, NCH3), 2.58-2, 47 (m, 34H), 2.30 (bs, 32H), 2.16-2.13 (m, 34H), 1.96 (s, 48 H, NHCOCH3), 1.55-1.30 (m , 198Н), 1.23-1.15 (m, 102Н). ¹³ C NMR (125 MHz, D2O) δ: 175.94, 174.24, 163.04, 162.81, 117.31, 115.38, 83.84, 71.62, 68.46, 67.77 , 61.07, 50.29, 48.22, 45.86, 39.19, 35.78, 32.74, 30.26, 28.77, 28.24, 27.13, 26.90, 26 ,22, 25.86, 25.80, 25.51, 25.39, 22.09. The results obtained using electrospray ionization tandem mass spectrometry (ESI-MS) (m/z) were as follows: [M+5H] ⁵⁺ cal. 1812.2, exp. 1813.1; [M+6N] ⁶⁺ calc. 1510.3, exp. 1511.2.

Example 2. Illustrative example of experimental materials and methods of biological research.

Materials. Bispecific antibody αanti-GPA33 for DOTA-PRIT (huA33-C825) was prepared according to the method previously described in WO 2016/130539. The human colorectal cancer cell line SW1222 GPA33(+) was provided by the Institute for Cancer Research. Ludwig (New York, NY, USA) and maintained in minimal culture medium supplemented with 10% heat-inactivated fetal bovine serum, 2.0 mM glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin.

Radioiodination of bispecific antibodies. The ^131I isotope was provided by Nordion (Ottawa, Ontario, Canada). Iodogen precoated tubes were provided by Thermo Fisher Scientific (Waltham, MA, USA). Prefilled Sephadex G-25 columns (PD10 columns) were provided by GE Healthcare Life Sciences (Pittsburgh, PA, USA). Aliquots of anti-GPA33 bispecific antibody were radioiodinated according to the IODO-GEN method (Salacinski PR et al., Analytical biochemistry 117: 136-46 (1981); Harlow E, Lane D. USING ANTIBODIES: A LABORATORY MANUAL. Cold Spring Harbor , NY (1999)) followed by gel filtration purification using commercially prefilled PD10 columns (Sephadex G-25 resin). The radiochemical purity was determined to be >98% using high pressure size exclusion liquid chromatography coupled with radiodetection. Briefly, to prepare ¹³¹ I-huA33-C825, 100 μg was mixed with 80 μl of 0.2 M sodium phosphate, pH 7.4, in an iodogen-precoated tube. Then 400 μCi of the isotope ¹³¹ I was added. After 5 min at room temperature, the reaction mixture was transferred to a new tube containing 50 μl of iodogen stopping buffer [10 mg/ml tyrosine (saturated), 10% glycerol, 0.1% xylene cylanol in phosphate-buffered saline] and purified using a PD10 column that had been pre-conditioned and eluted with saline supplemented with 1 w/v% bovine serum albumin.

Model animals. Female nude mice (6–8 weeks old) were provided by Harlan (Indianapolis, IN, USA) and Envigo (Huntingdon, UK). Mice were acclimatized for at least 1 week. Healthy (tumor-free) mice were used to determine the effect of the dendron-based scavenging agent CCA-16-DOTA-Y ³⁺ on the clearance of bispecific antibody ¹³¹¹ from the blood. For DOTA-PRIT biodistribution experiments, groups of mice with subcutaneously injected human colorectal cancer SW1222 xenografts were used. To introduce the SW1222 tumor, mice were inoculated with 5.0 x 106 cells in 200 μl of a cell suspension of a 1:1 mixture of basement membrane-reconstituted media (BD Matrigel, Collaborative Biomedical Products Inc., Bedford, MA, USA) into the lower abdomen by subcutaneous injection , and formed tumors (100-300 mm ³ ) were observed for 7-10 days.

Removal of bispecific antibody ¹³¹ I from the blood. Radioactive antibody tracer was prepared for injection by mixing bispecific antibody ¹³¹ I with additional antibody carrier to achieve 250 μg (1.19 nmol) per dose. All blood draws and injections were performed through the tail vein. Healthy mice (n=15) at t=0 were intravenously injected with 250 μg (1.19 nmol) of bispecific antibody ¹³¹ I, as well as either vehicle (saline; n=3) or 25 μg of clearing agent (prepared in 250 μl physiological solution; n=12) at t=24 hours. To study the carrier medium, blood samples were taken (30-40 μl; n=3 per sample) at t=1,2,3, 4, 24, 25, 26, 27 and 28 hours. To study the cleansing agent, blood samples were taken (30-40 μl; n=3 per sample) at t=1, 2, 3, 4, 24, 24.1 (5 min after administration of the cleansing agent), 24. 3 (15 min after administration of the scavenging agent), 24.5, 25, 26, 27 and 28 h. Radioactivity concentrations were determined for each sample by counting on a gamma counter (PerkinElmer Life Sciences Wallac Wizard 3). Count rates were corrected for background and attenuation, converted to activities using the isotope-specific system calibration factor, normalized to the administered activity, and expressed as percent dose/g administered (ID/g). Percentage changes in blood activity from baseline (i.e., immediately before injection of the cleansing agent; y1) at time intervals after injection of the cleansing agent (y2) were calculated using the formula ((y2-y1)/y1)x100.

Optimization of the dose of the dendron-based cleaning agent CCA-16-DOTA-Y ³⁺ in DOTA-PRIT. Groups of tumor-bearing mice (n=4 per group) were treated with bispecific antibody (250 μg, 1.19 nmol; t=28 h) followed by various amounts of CCA-16-DOTA-Y ³⁺ (0-25 μg; 0- 2.76 nmol; t=4 h). An additional group of tumor-bearing animals for comparison was administered the dendron-based scavenging agent (Y)DOTA (62.5 μg; 0.125 nmol; 7.625 nmol) instead of the dendron-based scavenging agent CCA-16-DOTA-Y ³⁺ . 4 h after administration of the scavenging agent, mice were injected with ¹⁷⁷ Lu-DOTA-Bn (150-167 µCi; 30-33 pmol). Mice were sacrificed 48 h after administration of ¹⁷⁷ Lu; organs were removed for analysis of radioactivity concentrations according to the method described above. These organs included blood, tumor, heart, liver, spleen, stomach, small intestine, large intestine, kidney, muscle, bone, and tail (injection site). Ratios of tumor uptake to non-tumor tissue uptake as a percentage of administered dose per gram were also calculated.

Obtaining [111In] Proteus-DOTA. In fig. Figure 8 shows the structure of Proteus-DOTA (chemical formula: C ₅₀ H ₈₀ LuN ₁₁ O ₁₉ S ³ '; exact mass: 1345.48; molecular mass: 1346.28). See also US Patent Application No. 18/40911, filed July 5, 2018, the contents of which are incorporated by reference in their entirety. For PRIT studies with the DOTA chelator [ ¹ⁿ In], Proteus-DOTA was prepared using the isotope [111In] indium chloride (153 MBq [4.14 mCi]) and 1 μl of Proteus-DOTA at a concentration of 10 mg/ml (10 μg; 7 .42 nmol). The [111In] Proteus-DOTA yield was 44% and the resulting specific activity (SA) was 7178 GBq/g [194 Ci/g] or 9.67 E6 GBq/mol [2.61 E5 Ci/mol]. [ ¹ⁿ In] Proteus-DOTA was purified using a Strata™ -X cartridge prior to administration to mice (Polymer Reverse Phase C-18, 33 µM, 30 mg/1 ml #8B-S1OO-TAK, Phenomenex® Inc., Torrance , California, USA), and radiochemical purity was assessed to be >98% either by in vitro binding assay with excess bispecific antibody or by analytical reverse phase HPLC coupled with radiodetection.

Statistical analysis. Differences between means were determined using unpaired parametric Student's t tests.

SPECT/CT imaging. For SPECT/CT imaging and biodistribution studies following in vivo targeting of anti-GPA33-DOTA-PRIT with [ ¹ⁿ In]Proteus-DOTA in SW1222 tumor-bearing mice that received injections of bispecific antibodies huA33-C825 and CCA-16DOTA-Y ^{3 +} ; 25 mcg; 2.76 nmol) 172 pmol/1.67 MBq [45 μCi] [ ¹ⁿ In] 1 (n=4) or 790 pmol/7.66 MBq [207 μCi] [ ¹ⁿ In] 1 (n=1) were administered. The next day, the only mouse that received a higher

- 28 044225 administered [ ¹¹¹ In]Proteus-DOTA activity was examined by SPECT/CT 20 hours after administration, and all animals were sacrificed 24 hours after administration to assess biodistribution.

Example 3 Improvement of clearance of radiolabeled bispecific antibody (BsAb) from blood in vivo using scavenging agents of the present invention.

Evaluation of the removal of bispecific antibody ¹³¹ I from the blood ( ¹³¹ I-huA33-C825). In vivo experiments were performed on healthy (tumor-free) nude mice to evaluate the effect of a single overdose of the dendron-based scavenging agent of the present invention on the clearance of bispecific antibody ¹³¹ I from the blood.

Groups of animals were initially treated with 250 μg of bispecific antibody ¹³¹ I (a dose of bispecific antibody previously optimized for PRIT with the DOTA chelator). After 24 hours, mice were administered the dendron-based clearing agent of the present invention (25 μg, 2.76 nmol) or vehicle medium. Serial blood collections were performed at various times up to 4 hours after administration of the clearing agent (or 28 hours after administration of the ^131I bispecific antibody), and the concentration of ^131I activity was determined in each sample using gamma counter analysis.

As shown in FIG. 1, initially there were no significant differences in ¹³¹ I activity in the blood at baseline (24 hours after administration) between the cleansing agent and carrier medium groups. The scavenging agent was effective in clearing circulating bispecific antibody ^131I from the blood, as the mean blood activity concentration was significantly reduced by 64% 5 min after administration from 6.7% VD/g to 2.4% VD/g. 1 hour after administration of the cleansing agent or carrier medium, the average blood activity was 5.8 (-13%) or 1.3% ID/g (-81%), respectively.

These results indicate that the scavenging agents of the present invention may be useful in methods of sensitizing a tumor to radiation therapy and/or treating cancer in a subject in need thereof.

Example 4: Applicability of the cleansing agents of the present invention in PRPT with DOTA chelator.

The tumor pre-targeting results achieved with different doses of CCA-16-DOTA-Y3+ are shown in FIG. 2. A dose of 5-25 μg of dendron-based scavenging agent resulted in an average tumor uptake of ¹⁷⁷ Lu-DOTA-Bn of 27-41%. ID/g at 24 h post-injection, ¹⁷⁷ Lu activity was comparable to the tumor uptake of ¹⁷⁷ Lu-DOTA-Bn observed with the dextran clearing agent and vehicle groups (~35% ID/g). A dose of 25 μg of dendron-based scavenging agent (CCA-16-DOTA-Y3+) produced a tumor to blood uptake ratio that was comparable to that observed with a dose of 62.5 μg of dextran-based scavenging agent. The effect of the dendron scavenging agent Nacetylgalactosamine CCA-16-DOTA-Y3+ on tumor uptake was dose dependent (see Fig. 3). In addition, in FIG. 4 shows a comparison of the tumor pretargeting results obtained using a scavenging agent according to the present invention with those obtained without the use of a scavenging agent (carrier medium), as well as with a conjugate of a dextran scavenging agent and a DOTA hapten scavenging agent in an amount 500 kDa. The tumor to healthy tissue uptake ratio values obtained using CCA-16DOTA-Y3+ were comparable to those obtained using the dextran-based clearing agent.

To demonstrate the utility of [ ^225Ac ]Proteus-DOTA in combination with PRIT with the DOTA chelator for effective tumor pre-targeting in vivo, a group of nude mice implanted with GPA33-expressing SW1222 xenografts were intravenously administered the bispecific antibody huA33-C825 (250 μg; 1. 19 nmol) 28 hours before administration of [ ²²⁵ Ac] Proteus-DOTA, and a clearing agent (62.5 μg; 0.125 nmol dextran; 7.625 nmol (Y)DOTA) was also administered intravenously 4 hours before administration of [ ²²⁵ Ac] Proteus- DOTA (182 pmol, 1.85 kBq [50 nCi]). These mice were sacrificed 24 hours after administration of [ ^225Ac ]Proteus-DOTA to assess biodistribution. This study was also repeated using [ ¹¹¹ In] Proteus-DOTA (172 pmol/1.67 MBq [45 μCi] [ ¹¹¹ In] Proteus-DOTA (n=4) or 790 pmol/7.66 MBq [207 μCi] [ ¹¹¹ In] Proteus-DOTA (n=1) using CCA-16-DOTA-Y ³⁺ (25 μg; 2.76 nmol)). The next day, the single mouse that had received the highest [ ¹¹¹ In]Proteus-DOTA activity administered was examined by SPECT/CT 20 hours after administration, and all animals were sacrificed 24 hours after administration to assess biodistribution.

As shown in FIG. 5A, for those animals that received pre-targeted radioimmunotherapy with [ ²²⁵ Ac]Proteus-DOTA, blood, tumor and kidney uptake (as a percentage of administered activity per gram of tissue; % ID/g) 24 hours after administration was 0.94 ± 0.26, 16.71 ± 2.95, and 1.08 ± 0.55, respectively, corresponding to tumor to organ activity ratios of approximately 18:1 and 16:1 for blood and kidney, respectively. The uptake of [ ¹¹¹ In]Proteus-DOTA by blood, tumor and kidney 24 h after administration was 0.76 ± 0.09, 13.18 ± 0.97 and 1.02 ± 0.06, respectively, which corresponds to the activity ratios in tumor to organ activity is approximately 17:1 and 13:1 for blood and kidneys, respectively. No significant differences between the use of [ ²²⁵ Ac]

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Proteus-DOTA and [ ^u1In ]Proteus-DOTA were not observed, except for liver uptake, which was approximately 3 times higher with [ ²²⁵ Ac]Proteus-DOTA (1.40 ± 0.47 and 0.46 ± 0. 05 with [ ²²⁵ Ac] Proteus-DOTA or [ ¹ⁿ In] Proteus-DOTA, respectively; P < 0.05) and bone absorption, which was higher with [ ¹¹¹ In] Proteus-DOTA (no data and 0.15 ± 0.01 for [ ^225Ac ]Proteus-DOTA or [ ^1nIn ]Proteus-DOTA, respectively; P<0.001). These ratios of tumor activity to organ activity are similar to previous biodistribution studies performed with anti-GPA33-DOTA using the radiotracer ¹⁷⁷ Lu-DOTA-Bn or ⁸⁶ YDOTA-Bn in the same animal model, in which average tumor uptake for both DOTA haptens were ~8% WD/g ((1.85-8.8 MBq; 10-50 pmol for any M-DOTA-Bn hapten) 24 hours after administration (see Cheal, SM et al., Eur J Nucl Med Mol Imaging 43:925–937 (2016)), suggesting that the affinity of C825 for [ ²²⁵ Ac]Proteus-DOTA was similar (see Fig. 5B).

In fig. Figure 6A shows tumor pre-targeting results obtained in a group of animals administered 172 pmol/1.67 MBq [45 μCi] [ ⁿ¹ In] Proteus-DOTA (n=4) or 790 pmol/7.66 MBq [207 μCi ] [ ¹¹¹ In] Proteus-DOTA (n=1). The ratio of tumor uptake to healthy tissue uptake was generally higher in animals receiving a higher dose of [ ¹¹¹ In]Proteus-DOTA (see Figs. 6A and 7).

As shown in FIG. 6B, PRIT results using anti-GPA33-DOTA obtained with the administration of [ ²²⁵ Ac] Proteus-DOTA (used in combination with a dextran-based scavenging agent) and [ ¹¹¹ In] Proteus-DOTA (used in combination with a dextran-based scavenging agent). dendrons CCA-16DOTA-Y3+) were comparable.

These results indicate that the scavenging agents of the present invention may be useful in methods of sensitizing a tumor to radiation therapy and/or treating cancer in a subject in need thereof.

Example 5: Use of dendron-based scavenging agent N-aietylgalactosamine in pretargeted radioimmunotherapy with DOTA chelator.

The main hypothesis was tested that anti-GPA33 PRIT with ¹⁷⁷ Lu-aminobenzyl DOTA ([ ¹⁷⁷ Lu]LuDOTA-Bn), including the dendron-based scavenging agent CCA-16-DOTA-Y3+, can be used to achieve high therapeutic indices (TI) instead of dextran-based cleaning agents. The therapeutic index is defined in this case as the calculated ratio of the dose absorbed by the tumor to the dose absorbed by healthy tissues.

Absorbed dose estimates for PRIT administered with [ ¹⁷⁷ Lu]LuDOTA-Bn, including the dendron-based scavenging agent CCA-16-DOTA-Y3+. To calculate absorbed doses for PRIT with [ ¹⁷⁷ Lu]LuDOTA-Bn, including a dendron-based scavenging agent (the scavenging agent of the present invention), groups of nude mice implanted with GPA33-expressing SW1222 xenografts (n=5 per group) were administered i.v. huA33-C825 antibody (250 μg; 1.19 nmol) 28 h before administration, and intravenous dendron-based clearing agent, CCA-16-DOTA-Y3+ (25 μg; 2.76 nmol) 4 h before administration [ ¹⁷⁷ Lu]LuDOTA-Bn (20 pmol, 3.7 MBq [100 µCi]). Mice were sacrificed 1, 4, 24, or 48 h after administration of [ ¹⁷⁷ Lu]LuDOTA-Bn for biodistribution analysis. For each tissue, activity concentration-time relationships without decay correction were fitted using Excel to a one-component, two-component, or more complex exponential function, as appropriate, and analytically integrated to calculate the accumulated concentration activity per unit of administered activity (MBq-h/g per MBq). The ¹⁷⁷ Lu equilibrium dose constant for non-penetrating radiation (8.49 g-cGy/MBq-h) was used for tumor-to-tumor estimation and selection of organ-to-organ self-absorbed doses, assuming total local absorption of only ¹⁷⁷ Lu β-rays, and ignoring the contribution y-rays and other doses. Data obtained from these studies are shown in FIG. 9.

The decay-adjusted activity-time curve 48 hours after administration of [ ¹⁷⁷ Lu]LuDOTA-Bn for tumor, blood, liver, spleen, kidney, and muscle is shown in FIG. 10. In FIG. Figure 11 shows absorbed doses (cGy/MBq) and therapeutic indices for various tissues. As shown in FIG. 11, the calculated absorbed doses of [ ¹⁷⁷ Lu]LuDOTA-Bn for blood, tumor, liver, spleen and kidney were 11.7, 468.4, 9.97, 5.49 and 13.3 cGy/MBq, respectively. The ratio of tumor absorbed dose estimates to selected healthy tissue absorbed dose estimates (ie, TI) ranged from about 40 (eg, blood and kidney) to about 550 for muscle (Fig. 11).

For optimized PRIT with administration of [ ¹⁷⁷ Lu]LuDOTA-Bn including dextran-based scavenging agent, the calculated absorbed doses (cGy/MBq) to tumor, blood, liver, spleen and kidney per cycle were 65.8, 0.9 (TI 73), 6.3 (TI 10), 6.6 (TI 10) and 5.3 (TI 12), respectively. See Cheal et al., Eur J Nucl Med Mol Imaging 43: 925 (2016). Effective treatment without any serious treatment-related toxicities (e.g., 111.0 MBq delivered as a fractionated dose has been demonstrated to induce complete remission with a 100% rate, including survival of more than 140 days in two of nine mice; absorbed doses of 7304,100 and 588 cGy by tumor, blood and kidneys). Cheal et al., Eur J Nucl Med Mol Imaging 43:925 (2016).

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Based on estimates of radiation dose tolerance for healthy human tissue obtained from clinical observations (Marks, et al., Int J Radiat Oncol Biol Phys. 76:S10-9 (2010)), the maximum tolerated dose (MTD) is 250 cGy to bone marrow , 1500 cGy for the lungs, 3000 cGy for the liver and 2000 cGy for the kidneys. Therefore, for optimized PRPT with administration of [ ^177Lu ]LuDOTA-Bn, including a dextran-based scavenging agent, the maximum tolerated pretargeting activity of [ ^177Lu ]LuDOTA-Bn is 278 MBq, with bone marrow as the dose-limiting organ. With this activity, the estimated absorbed dose delivered to the tumor is 18,292 cGy (183 Gy), of which 250 cGy is delivered to the blood (bone marrow) and 1,473 cGy is delivered to the kidneys.

For the above example of PRPT with administration of [ ^177Lu ]LuDOTA-Bn, including the dendron-based scavenging agent CCA-16-DOTA-Y ³⁺ , the maximum tolerated pre-targeting activity of [ ^177Lu ]LuDOTA-Bn is 21 MBq for the bone marrow administered as a dose-limiting organ. With this activity, the estimated absorbed dose delivered to the tumor is 9836 cGy (98 Gy), of which 246 cGy is delivered to the blood (bone marrow) and 280 cGy to the kidneys. Thus, effective and safe therapy for colorectal carcinoma is predicted in xenografts in mice, given the tumor uptake dose of ~73 Gy, which can be achieved with an administered activity of 15.6 MBq (183 cGy for blood (bone marrow) and 207 cGy for kidneys).

Ultimately, the data indicate that a dendron-based scavenging agent can be used to treat human colorectal carcinoma (eg, 70 Gy per tumor) in mouse models with less ¹⁷⁷ Lu activity administered compared to a dextran-based scavenging agent with differences in dosimetry for critical tissues (for example, with a reduced dose for the kidneys).

These results indicate that the scavenging agents of the present invention may be useful in methods of sensitizing a tumor to radiation therapy and/or treating cancer in a subject in need thereof.

Equivalents.

The present invention should not be limited to the specific embodiments described in the present invention, which are intended as separate illustrations of certain aspects of the present invention. Many modifications and variations of the present invention can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. Functionally equivalent methods and devices within the scope of the present invention, in addition to those listed herein, will be apparent to those skilled in the art from the foregoing description. Such modifications and variations are intended to be within the scope of the present invention. It should be understood that the present invention is not limited to specific methods, reagents, compounds, compositions or biological systems, which, of course, may vary. It should also be understood that the terminology used in the present invention is intended to describe specific embodiments only and is not intended to be limiting.

Moreover, if features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will understand that features or aspects of the disclosure are also described in terms of any individual member or subset of members of the Markush group.

As will be understood by one skilled in the art, for all purposes, especially from the point of view of providing a written description, all ranges disclosed in the present invention also cover any and all possible sub-ranges and combinations thereof. Any specified range can easily be characterized as sufficiently descriptive to allow the same range to be broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed in the present invention can be easily broken down into a lower third, a middle third, an upper third, etc. As will also be understood by one skilled in the art, all terms such as up to, at least more than, less than, etc. include the number indicated and refer to ranges that can subsequently be broken down into subranges as described higher. Finally, as one skilled in the art will appreciate, the range includes each individual element. Thus, for example, a group having 1-3 cells refers to groups having 1, 2 or 3 cells. Likewise, a group having 1-5 cells refers to groups having 1, 2, 3, 4 or 5 cells, etc.

All patents, patent applications, provisional applications and publications referenced or cited in the present invention are incorporated by reference in their entirety, including all figures and tables, to the extent they do not conflict with the express teachings of this specification.

Claims (11)

1. Connection selected from - 32 044225 or a pharmaceutically acceptable salt thereof, in which X ¹ , X ² , X 3, X ⁴ , X ⁵ , X ⁶ , ^{X 7 ,} X ⁸ , X 9, X ¹⁰ , X ¹¹ , X ¹² , X ¹³ , X ¹⁴ , X ¹⁵ , ^{X 16 ,} X ¹⁷ , X ¹⁸ , X ¹⁹ and X ²⁰ are each independently H; Y ¹ , Y ² , Y ³ , Y ⁴ and Y ⁵ are each independently O or S; x is 1; and y is 1. 2. A cleaning agent compound selected from - 33 044225 - 34 044225 or a pharmaceutically acceptable salt thereof, wherein M ¹ , M ² , M ³ , M ⁴ and M ⁵ are each independently Lu ³⁺ , Sc ³⁺ , Ga ³⁺ , Y ³⁺ , In ³⁺ , La ³⁺ , Ce ³⁺ , Eu ³⁺ , Tb ³⁺ or Gd ³⁺ ; X ¹ , X ² , X 3, X ⁴ , X ⁵ , X ⁶ , ^{X 7 ,} X ⁸ , X 9, X ¹⁰ , X ¹¹ , X ¹² , X ¹³ , X ¹⁴ , X ¹⁵ , ^{X 16 ,} X ¹⁷ , X ¹⁸ , X ¹⁹ and X ²⁰ are each independently H; Y ¹ , Y ² , Y ³ , Y ⁴ and Y ⁵ are each independently O or S; x is 1; yequals 1. - 35 044225 3. A compound according to any one of claims 1, 2, wherein Y ¹ , Y ² , Y ³ , Y ⁴ and Y ⁵ are each independently S. 4. A method of increasing the sensitivity of a tumor to radiation therapy in a subject diagnosed with cancer, comprising: (a) administering to the subject an effective amount of an anti-DOTA bispecific antibody, wherein the anti-DOTA bispecific antibody is configured to localize to a tumor expressing the target tumor antigen; (b) administering to the subject an effective amount of the cleansing agent compound of any one of claims 2, 3; (c) administering to the subject an effective amount of a radiolabeled DOTA hapten, wherein the DOTA hapten is configured to form a complex with an anti-DOTA bispecific antibody. 5. A method of treating cancer in a subject in need thereof, comprising: (a) administering to the subject an effective amount of an anti-DOTA bispecific antibody, wherein the anti-DOTA bispecific antibody is configured to localize to a tumor expressing the target tumor antigen; (b) administering to the subject an effective amount of the cleansing agent compound of any one of claims 2, 3; (c) administering to the subject an effective amount of a radiolabeled DOTA hapten, wherein the DOTA hapten is configured to form a complex with an anti-DOTA bispecific antibody. 6. The method of claim 5, further comprising sequential, separate or simultaneous administration to the subject of at least one chemotherapeutic agent selected from the group consisting of nitrogen mustards, ethyleneimine derivatives, alkyl sulfonates, nitrosoureas, gemcitabine, triazenes, folic acid analogues, anthracyclines, taxanes, COX-2 inhibitors, pyrimidine analogs, purine analogs, antibiotics, enzyme inhibitors, epipodophyllotoxins, platinum coordination complexes, vinca alkaloids, substituted ureas, methylhydrazine derivatives, adrenocortical suppressors, hormone antagonists, endostatin, taxols, camptothecins, SN-38, doxorubicin , doxorubicin analogs, antimetabolites, alkylating agents, antimitotics, antiangiogenic agents, tyrosine kinase inhibitors, mTOR inhibitors, heat shock protein (HSP90) inhibitors, proteasome inhibitors, HDAC inhibitors, proapoptotic agents, methotrexate and CPT-11. 7. The method according to any one of claims 4-6, in which the tumor antigen target is selected from the group consisting of GPA33, HER2/neu, GD2, MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, MUM -1, CDK4, Nacetylglucosaminyltransferase, p15, dr75, beta-catenin, ErbB2, cancer antigen 125 (CA-125), carcinoembryonic antigen (CEA), RAGE, MART (melanoma antigen), MUC-1, MUC-2, MUC- 3, MUC-4, MUC-5ac, MUC-16, MUC-17, tyrosinase, Pmel 17 (gp100), GnT-V subtype V intron sequences (N-acetylglucoaminyltransferase subtype V intron V sequences), prostate cancer psm, PRAME ( melanoma antigen), β-catenin, EBNA (Epstein-Barr virus nuclear antigen) 16, p53, lung cancer resistance protein (LRP) Bcl-2, prostate-specific antigen (PSA), Ki-67, CEACAM6, specific antigen- colon p (CSAp), HLA-DR, CD40, CD74, CD138, EGFR, EGP-1, EGP-2, VEGF, PIGF, insulin-like growth factor (ILGF), tenascin, platelet-derived growth factor, IL-6, CD20, CD19, PSMA, CD33, CD123, MET, DLL4, Ang-2, HER3, IGF-1R, CD30, TAG-72, SPEAP, CD45, L1-CAM, Lewis Y antigen (Ley), E-cadherin, Vcadherin and EpCAM . 8. The method according to any one of claims 4-7, in which the bispecific anti-DOTA antibody is administered intravenously, intramuscularly, intraarterially, intrathecally, intracapsularly, intraorbitally, intradermally, intraperitoneally, transtracheally, subcutaneously, intracerebroventricularly, orally or intranasally; and/or wherein the radiolabeled DOTA hapten and/or clearing agent is administered intravenously, intramuscularly, intraarterially, intrathecally, intracapsularly, intraorbitally, intradermally, intraperitoneally, transtracheally, subcutaneously, intracerebroventricularly, orally or intranasally. 9. The method according to any one of claims 4 to 8, wherein the cancer is selected from the group consisting of breast cancer, colorectal cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, hepatocellular carcinoma, brain cancer, cancer lung, gastrointestinal or stomach cancer, pancreatic cancer, thyroid cancer, kidney cancer, prostate cancer, melanoma, sarcoma, carcinoma, Wilms tumor, endometrial cancer, glioblastoma, squamous cell carcinoma, astrocytoma, salivary gland carcinoma, vulvar cancer , penile carcinoma, head and neck cancer, wherein the brain cancer is not necessarily a pituitary adenoma, meningioma, neuroblastoma or craniopharyngioma. 10. The method of any one of claims 4 to 9, wherein the subject exhibits a tumor to healthy tissue uptake ratio of about 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8: 1, 9:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 55:1, 60:1, 65:1, 70:1, 75:1, 80:1, 85:1, 90:1, 95:1 or 100:1. 11. The method according to any one of claims 4 to 10, in which the radiolabeled DOTA hapten is labeled with a radionuclide, - 36 044225 selected from the group consisting of ²¹³ Bi, ²¹¹ At, ²²⁵ Ac, ¹⁵² Dy, ²¹² Bi, ²²³ Ra, ²¹⁹ Rn, ²¹⁵ Po, ²¹¹ Bi, ²²¹ Fr, ²¹⁷ At, ²⁵⁵ Fm, ⁸⁶ Y, ⁹⁰ Y , ⁸⁹ Sr, ¹⁶⁵ Dy, ¹⁸⁶ Re, ¹⁸⁸ Re, ¹⁷⁷ Lu, ⁶⁷ Cu, ^1H In, ⁶⁷ Ga, ⁵¹ Cr, ⁵⁸ Co, ^99m Tc, ^103m Rh, ^195m Pt, ¹¹⁹ Sb, ¹⁶¹ Ho, ^189m Os, ¹⁹² Ir, ²⁰¹ Tl, ²⁰³ Pb, ⁶⁸ Ga, ²²⁷ Th, ⁶⁴ Cu and, optionally, wherein the radiolabeled DOTA hapten includes one or more components of Proteus-DOTA, S-2-(R-aminobenzyl)-1,4,7 ,10tetraazacyclododecanetetraacetic acid (DOTA-Bn), DOTA-Bn-biotin, BAD (((S)-2-(4-(2bromo)acetamido)benzyl)-DOTA), NBD ((S)-2-(4-nitrobenzyl )-DOTA), DOTA-RGD, DOTA-PEGE(c(RGDyK)) ₂ , DOTA-8-AOC-BBN, p-NO ₂ -Bn-DOTA, DOTA-PESIN, DOTA-biotin-sarcosine (DOTAbiotin), mono 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (Nhydroxysuccinimide ester) (DOTA-NHS) or DOTATyrLysDOTA.