Alpha-particle radionuclide-antibody conjugates for treatment of solid tumors

Abstract

Compositions comprising one or more targeting (or high affinity) radionuclide-antibody conjugates and one or more non-targeting (or low affinity) radionuclide-antibody conjugates and their use in treating solid tumors are disclosed.

Description

[0001] ALPHA-PARTICLE RADIONUCLIDE-ANTIBODY CONJUGATES FOR TREATMENT OF SOLID TUMORS

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Application No. 63 / 618,604, filed January 8, 2024, which is incorporated herein by reference in its entirety.

[0004] BACKGROUND

[0005] Advanced (i.e., vascularized), disseminated solid tumors are incurable. Radionuclide-antibody conjugates are among the leading approaches in targeted alphaparticle (a-particle) therapies for the treatment of vascularized, soft-tissue, solid tumors overexpressing the antibody-targeted cell markers. Jang et al., 2023. Importantly, a-particle radionuclide- antibody conjugates are now also being evaluated in the clinic on patients with tumors expressing only moderate levels of the targeted markers (NCT04147819), following reports on promising outcomes in preclinical studies. Jang et al., 2023.

[0006] These findings are possibly attributed to the high killing potency of a-particle radiotherapy, since the tumor absorbed doses (i.e., absorbed energy per mass of tumor) are expected to decrease with the lowering levels of antibody-targeted marker expression. Independent of expression levels of the targeted markers / receptors on cancer cells, however, the success of a-particle RadioPharmaceutical Therapy (aRPT) against soft-tissue cancer metastases has been confined to the treatment of disseminated relatively small metastases, Navarro-Teulon et al., 2013; Kratochwil et al., 2017; Sathekge et al., 2019, and, inevitably, patients progress and die from their disease. Hence, there is an urgent unmet need for a therapy that selectively and effectively kills cancer cells in large, vascularized solid tumors (at the primary site and / or the metastatic sites) resulting in prolonged survival.

[0007] SUMMARY

[0008] In some aspects, the presently disclosed subject matter provides a composition comprising a first radionuclide-antibody conjugate having a first affinity for a target and a second radionuclide-antibody conjugate having a second affinity for the same target, wherein the first affinity for the target is higher than the second affinity for the target. Tn certain aspects, the composition comprises more than one first radionuclideantibody conjugate and / or more than one second radionuclide-antibody conjugate.

[0009] In certain aspects, the composition comprises a total radioactivity having a ratio ranging about 10:90 to about 90:10 between the first radionuclide-antibody conjugate and the second radionuclide- antibody conjugate.

[0010] In certain aspects, the composition comprises a total radioactivity that is approximately equally divided between the first radionuclide-antibody conjugate and the second radionuclide-antibody conjugate.

[0011] In certain aspects, the radionuclide of the first radionuclide- antibody conjugate and the second radionuclide- antibody conjugate comprises an alpha-particle emitter.

[0012] In certain aspects, the alpha-particle emitter for the first radionuclide- antibody conjugate and the second radionuclide-antibody conjugate can be the same or different and is selected from actinium-225, astatine-211, lead-212, terbium-149, thorium-227, radium- 223, radium-224, bismuth-212, and bismuth-213. In particular aspects, the alpha-particle emitter is actinium-225 (225Ac).

[0013] In certain aspects, the first radionuclide- antibody conjugate and the second radionuclide- antibody conjugate each independently comprise an antibody selected from the group consisting of trastuzumab, cetuximab, panitumumab, rituximab, and bevacizumab, wherein the antibody of the first radionuclide-antibody conjugate and the antibody of the second radionuclide-antibody conjugate can be the same or different. In particular aspects, the antibody of the first radionuclide- antibody conjugate is selected from trastuzumab and cetuximab.

[0014] In certain aspects, the antibody of the second radionuclide-antibody conjugate comprises a non-targeting antibody. In particular aspects, the non-targeting antibody comprises rituximab.

[0015] In certain aspects, the non-targeting antibody comprises an inactive form of the antibody comprising the first radionuclide- antibody conjugate. In particular aspects, the inactive form of the antibody comprising the non-targeting antibody comprises a capped antibody having an epitope-peptide sequence bound at one or more binding sites and / or covalently modified at one or more binding sites. Tn certain aspects, the second radionuclide-antibody conjugate comprises the same antibody as the first radionuclide-antibody conjugate, wherein the antibody of the second radionuclide- antibody conjugate is modified such that its affinity for the target is decreased relative to the affinity for the target of the antibody of the first radionuclide- antibody conjugate.

[0016] In certain aspects, one or more binding sites of the antibody of the second radionuclide- antibody conjugate are sterically hindered. In particular aspects, the one or more binding sites of the antibody of the second radionuclide-antibody conjugate are sterically hindered by conjugation of one or more sterically-hindering moieties to one or more a-amino groups of N-terminal amino acids on the binding sites of the antibody via click chemistry. In such aspects, the antibody comprises one or more sterically-hindering moieties bound to one or more a-amino groups of N-terminal amino acids on the binding sites of the antibody. In particular aspects, the one or more sterically-hindering moieties are selected from a dye, a chelating moiety, and a click-reactive linker. In more particular aspects: (a) the dye is selected from a coumarin dye, a rhodamine dye, a cyanine dye, a xanthene dye, a pyrylium dye, and sulfonated, azetidine derivatives thereof; (b) the chelating moiety comprises a chelator moiety disclosed herein; and (c) the click-reactive linker comprises a linker disclosed herein.

[0017] In certain aspects, an immunoreactivity of the antibody of the second radionuclideantibody conjugate is less than an immunoreactivity of the antibody of the first radionuclideantibody conjugate. In particular aspects, the immunoreactivity of the antibody of the second radionuclide-antibody conjugate is less than the immunoreactivity of the antibody of the first radionuclide- antibody conjugate by a range of about 1% less to about 99% less.

[0018] In certain aspects, the first radionuclide- antibody conjugates and the second radionuclide- antibody conjugates each independently further comprise a linker.

[0019] In certain aspects, the linker is selected from the group consisting of isothiocyanate (SCN), isothiocyanato-benzyl (SCN-Bn), N-succinimidyl 4-(2pyridyldithiojpentanoate (SPP), N-succinimidyl 4-(2-pyridyldithio)-2-sulfopentanoate (sulfoSPP), N-succinimidyl 4- (2-pyridyldithio)butanoate (SPDB), N-succinimidyl 4-(2-pyridyldithio)2-sulfobutanoate (sulfo-SPDB), N-succinimidyl 4-(maleimidomethyl) cyclohexanecarboxylate (SMCC), N- sulfosuccinimidyl 4-(maleimidomethyl) cyclohexanecarboxylate (sulfoSMCC), N- succinimidyl-4-(iodoacetyl)-aminobenzoate (STAB), and N-succinimidyl-[(N- malcimidopropionamido-tctracthylcncglycol] ester (NHS-PEG4-malcimidc) .

[0020] In certain aspects, the first radionuclide- antibody conjugates and the second radionuclide- antibody conjugate each independently further comprise a chelating moiety.

[0021] In certain aspects, the chelating moiety is selected from the group consisting of DOTAGA (1,4,7,10-tetraazacyclododececane, l-(glutaric acid)-4,7,10-triacetic acid), DOTA (l,4,7,10-tetraazacyclododecane-l,4,7,10-tetraacetic acid), DOTASA (1,4,7,10- tetraazacyclododecane-l-(2-succinic acid)-4,7,10-triacetic acid), CB-DO2A (10- bis(carboxymethy 1)- 1 ,4,7 , 10-tetraazabicyclo [5.5.2] tetradecane), DEP A (7 -[2-(Bis- carboxymethylamino)-ethyl] -4, 10-bis-carboxymethyl- 1 ,4,7 , 10-tetraaza-cy clododec- 1 -yl- acetic acid)), 3p-C-DEPA (2-[(carboxymethyl)][5-(4-nitrophenyl-l-[4,7,10- tris(carboxymethyl)-l,4,7,10-tetraazacyclododecan-l-yl]pentan-2-yl)amino]acetic acid)), TCMC (2-(4-isothiocyanotobenzyl)- 1,4,7, 10-tetraaza- 1,4,7, 10-tetra-(2-carbamonyl methyl)- cyclododecane), oxo-DO3A (l-oxa-4,7,10-triazacyclododecane-5-S-(4- isothiocyanatobenzyl)-4,7,10-triacetic acid), p-NH2-Bn-Oxo-DO3A (l-Oxa-4,7,10- tetraazacyclododecane-5-S-(4-aminobenzyl)-4,7,10-triacetic acid), TE2A ((1,8-N,N ” -bis- (carboxymethyl)-l,4,8,l l-tetraazacyclotetradecane), MM-TE2A, DM-TE2A, CB-TE2A (4,1 l-bis(carboxymethyl)-l,4,8,l l-tetraazabicyclo[6.6.2]hexadecane), CB-TE1A1P (4,8,11- tetraazacyclotetradecane-l-(methanephosphonic acid)-8-(methanecarboxylic acid)), CB- TE2P (l,4,8,ll-tetraazacyclotetradecane-l,8-bis(methanephosphonic acid), TETA (1,4,8,11- tetraazacyclotetradecane- 1,4, 8, 11 -tetraacetic acid), NOTA (l,4,7-triazacyclononane-N,N " ,N"-triacetic acid), NODA (l,4,7-triazacyclononane-l,4-diacetate); NODAGA (1,4,7- triazacyclononane,l -glutaric acid-4, 7-acetic acid); NOTAGA (l,4,7-triazonane-l,4- diyl)diacetic acid); DFO (Desferoxamine), NETA ([4-[2-(bis-carboxymethylamino)-ethyl]- 7-carboxymethl-[l,4,7]triazonan-l-yl}-acetic acid), TACN-TM’(N”N',N", tris(2- mercaptoethyl)- 1,4,7-triazacyclononane), Diamsar (1 ,8-Diamino-3,6, 10,13, 16, 19- hexaazabicyclo(6,6,6)eicosane, 3,6,10,13,16,19-Hexaazabicyclo[6.6.6]eicosane-1,8- diamine), Sarar (l-N-(4-aminobenzyl)-3, 6,10,13,16,19-hexaazabicyclo[6.6.6] eicosane-1,8- diamine), AmBaSar (4-((8-amino-3,6,10,13,16,19-hexaazabicyclo [6.6.6] icosane-1- ylamino) methyl) benzoic acid), macropa, and BaBaSar. In particular aspects, the chelating moiety is selected from dodecane tetraacetic acid (DOTA) and dicthylcnctriamincpcntaacctic acid (DTP A).

[0022] In other aspects, the presently disclosed subject matter provides a method for treating a solid tumor in a subject in need of treatment thereof, the method comprising administering a therapeutically effective amount of the presently disclosed composition to the subject.

[0023] In particular aspects, the solid tumor comprises a cancer selected from breast cancer, pancreatic cancer, liver cancer, kidney cancer, prostate cancer, lung cancer, colorectal cancer, ovarian cancer, brain cancer, skin cancer, and combinations thereof.

[0024] In certain aspects, the first radionuclide- antibody conjugate and the second radionuclide- antibody conjugate are administered at the same time. In other aspects, the first radionuclide-antibody conjugate and the second radionuclide- antibody conjugate are administered separately with a time interval between administration of each conjugate. In particular aspects, the first radionuclide-antibody conjugate and the second radionuclideantibody conjugate are administered between about 12 hours and about 18 hours apart.

[0025] In certain aspects, the first radionuclide- antibody conjugate is administered first.

[0026] In certain aspects, a total dose of the first radionuclide-antibody conjugate and / or the second radionuclide-antibody conjugate is administered in a series of fractional doses at predetermined time intervals.

[0027] Certain aspects of the presently disclosed subject matter having been stated hereinabove, which are addressed in whole or in part by the presently disclosed subject matter, other aspects will become evident as the description proceeds when taken in connection with the accompanying Examples and Drawings as best described herein below.

[0028] BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0030] Having thus described the presently disclosed subject matter in general terms, reference will now be made to the accompanying Figures, which are not necessarily drawn to scale, and wherein: FIG. 1 A shows that alpha-particles cause lethal, difficult to repair, double strand DNA breaks (trajectory in red). Alpha-particles do not need radiosensitizers, in contrast to beta-particles, which mostly induce single strand breaks in DNA (shown in blue), that are easier for the cell to repair. Beta-particle emitters are already in the clinic (e.g., TheraSphere®: Yttrium-90 microspheres, Lutathera®: Lutetium-177 dotatate);

[0031] FIG. IB shows the range of alpha- and beta-particles in tissue (in red and blue, respectively) relative to cell size (in gray);

[0032] FIG. 1C shows that spheroids, 3D cell culture, utilized as surrogates of tumor avascular regions, are not uniformly irradiated by targeting antibodies delivering the alphaparticle emitting radionuclides: the spheroid core does not receive alpha-particle therapy, dark region, and cancer cells in this region are not killed. Zhu et al., 2017;

[0033] FIG. ID show heterogeneous distributions of the alpha-particle emitter Actinium- 225 in tumors: alpha-camera image of a tumor section, a HER2-positive BT-474 breast cancer tumor, excised from mice which were injected intravenously with the HER2- targeting radionuclide-antibody conjugates. Antibodies populated areas close to the vasculature (indicated by green-framed regions that stained more strongly, more spots, for CD31) and could not reach tumor regions of poor vascularization (yellow square). Howe et al., 2022;

[0034] FIG. 2 show florescence histograms of the extent of targeting to each of the cancer cell lines by the three fluorescently-labeled antibodies studied herein: the HER2-targeting Trastuzumab (in red); the HER 1 -targeting Cetuximab (in orange); and the CD20-targeting Rituximab, that is the non-targeting antibody conjugate in this study (in blue), (left) BT- 474; (middle) HEPG-2; (right) BxPC-3;

[0035] FIG. 3 shows binding isotherms of the targeting antibodies to each of the three cancer cell lines studied herein.i nIn-DTPA-labeled HER2-targeting Trastuzumab orU 1ln- DTPA-labeled HER 1 -targeting Cetuximab were incubated with the cancer cell lines as indicated on the plots. The immunoreactivity of the targeting antibodies for these studies is indicated on Table 3. The plotted concentration of the targeting antibody (on the x-axis) was corrected by multiplying the antibody concentration by its corresponding immunoreactivity. The y-axis was corrected for the non-specific binding of antibody. Howe et al., 2022; Salerno et al., 2022. The calculated values, from the fitted curves, for KD and the average number of targeted receptors per cell (HER2 or HER1), arc shown on each plot;

[0036] FIG. 4A shows the effect of alpha-particle therapy delivered by antibodies to HER2- overexpressing BT-474 breast cancer tumors in mice. Left column indicates the tumor growth inhibition over time for different treatment groups, and the plot on the right shows the corresponding animal survival following treatment with a single intravenous injection and same total injected radioactivity of 2.96 kBq Actinium-225 per 20 g NSG-mouse delivered by: (a) only the HER2-targeting radionuclide-Trastuzumab conjugate (red); (b) only the non-targeting radionuclide-Rituximab conjugate (blue); (c) both radionuclideantibody conjugates of same total radioactivity at equal split between the targeting and the non-targeting antibodies (purple). Non-treated animals’ tumor growth and survival are shown by the black line. Animals were sacrificed when the tumor size increased beyond the point at which the ability of mice to freely reach the food and water containers was affected. Significance in survival was calculated with one-way ANOVA (p-value<0.05);

[0037] FIG. 4B shows animal weights over time during the treatment study on the BT-474 subcutaneous mouse models, shown on FIG. 4A;

[0038] FIG. 4C shows characteristic H&E-stained sections of normal organs and of BT-474 tumors of animals being treated as stated on FIG. 4 A, acquired at the endpoint of the study. Scale bar = 200 pm;

[0039] FIG. 5A shows the effect of alpha-particle therapy delivered by antibodies to moderately HER2-expressing HEPG-2 hepatic cancer tumors in mice. Left column indicates the tumor growth inhibition over time for different treatment groups, and the plot on the right shows the corresponding animal survival following treatment with a single intravenous injection and same total injected radioactivity of 2.96 kBq Actinium-225 per 20 g NSG-mouse delivered by: (a) only the HER2-targeting radionuclide-Trastuzumab conjugate (red); (b) only the non-targeting radionuclide-Rituximab conjugate (blue); (c) both radionuclide-antibody conjugates of same total radioactivity at equal split between the targeting and the non-targeting antibodies (purple). Non-treated animals’ tumor growth and survival are shown by the black line. Animals were sacrificed when the tumor size increased beyond the point at which the ability of mice to freely reach the food and water containers was affected. Significance in survival was calculated with one-way ANOVA (p- valuc<0.05);

[0040] FIG. 5B shows animal weights over time during the treatment study on the HEPG-2 subcutaneous mouse models shown on FIG. 5A;

[0041] FIG. 5C shows characteristic H&E-stained sections of normal organs and of HEPG-

[0042] 2 tumors of animals being treated as stated on FIG. 5A, acquired at the endpoint of the study. Scale bar = 200 pm;

[0043] FIG. 6A shows the effect of alpha-particle therapy delivered by antibodies to BxPC-

[0044] 3 pancreatic cancer tumors that express low levels of HERE Left column indicates the tumor growth inhibition over time for different treatment groups, and the plot on the right shows the corresponding animal survival following treatment with a single intravenous injection and same total injected radioactivity of 2.96 kBq Actinium-225 per 20 g NSG- mouse delivered by: (a) only the HER 1 -targeting radionuclide-Cetuximab conjugate (orange); (b) only the non-targeting radionuclide-Rituximab conjugate (blue); (c) both radionuclide- antibody conjugates of same total radioactivity at equal split between the targeting and the non-targeting antibodies (green). Non-treated animals’ tumor growth and survival are shown by the black line. Animals were sacrificed when the tumor size increased beyond the point at which the ability of mice to freely reach the food and water containers was affected. Significance in survival was calculated with one-way ANOVA (p- value<0.05);

[0045] FIG. 6B shows animal weights over time during the treatment study on the BxPC-3 subcutaneous mouse models shown on FIG. 6A;

[0046] FIG. 6C shows characteristic H&E-stained sections of normal organs and of BxPC- 3 tumors of animals being treated as stated on FIG. 6 A, acquired at the endpoint of the study. Scale bar = 200 pm;

[0047] FIG. 7 shows the biodistributions of the targeting (red) and the non-targeting (blue) radionuclide- antibody conjugates injected intravenously on NSG mice bearing BT-474 subcutaneous tumors overexpressing the targeted marker (HER2). (red) the HER2-targeting111In-DTPA-SCN-antibody, Trastuzumab; (blue) the non-targetingl nIn-DTPA-SCN- antibody, Rituximab. Indium-I ll was utilized as surrogate of the parent Actinium-225. Error bars correspond to standard deviations of n=3 mice per condition per time point; FIG. 8 shows the biodistributions of the targeting (red) and the non-targeting (blue) radionuclide- antibody conjugates injected intravenously on NSG mice bearing HEPG-2 subcutaneous tumors moderately overexpressing the targeted marker (HER2). (red) the HER2-targeting111In-DTPA-SCN-antibody, Trastuzumab; (blue) the non-targetingn iIn- DTPA-SCN-antibody, Rituximab. Indium- 111 was utilized as surrogate of the parent Actinium-225. Error bars correspond to standard deviations of n=3 mice per condition per time point;

[0048] FIG. 9 shows the biodistributions of the targeting (orange) and the non-targeting (blue) radionuclide- antibody conjugates injected intravenously on NSG mice bearing BxPC3 subcutaneous tumors expressing low levels of the targeted marker (HER1). (orange) the HER 1- targetingi nIn-DTPA-SCN-antibody, Cetuximab; (blue) the non-targetingU 1ln- DTPA-SCN-antibody, Rituximab. Indium-I l l was utilized as surrogate of the parent Actinium-225. Error bars correspond to standard deviations of n=3 mice per condition per time point;

[0049] FIG. 10 shows the colony survival of different types of cancer cells following a 6- hour incubation, when in monolayers, with different radioactivity concentrations of225Ac in various forms: (a) in free form,225Ac-DOTA (grey symbols), (b) as the HER2-targeting radionuclide- antibody conjugate,225Ac-DOTA-SCN-Trastuzumab conjugate (red symbols); (c) as the HER 1 -targeting radionuclide-antibody conjugate, the HER 1 -targeting225Ac - DOTA-SCN-Cetuximab conjugate (orange circles); and (d) as the non-targeting radionuclide- antibody conjugate, the225Ac-DOTA-SCN-Rituximab conjugate, which binds to CD20 that is not expressed by any of the three cancer cell lines, and was utilized in these studies as a model “non-targeting” antibody (blue symbols). Data points indicate the mean values, and error bars the standard deviations of n = 3 independent experiments;

[0050] FIG. 11 shows the spatiotemporal microdistributions (left and middle columns) in 200-pm radius BT-474 spheroids overexpressing the targeted receptor HER2, and the time- integrated radial microdistributions (right column) of the targeting antibody (top panel, the HER2-targeting Trastuzumab, in red) and the non-targeting antibody (bottom panel, Rituximab, shown in blue), each fluorescently labeled with FITC. Data points indicate the mean values, and error bars the standard deviations of n = 3 different spheroids studied per time point; FIG. 12 shows the spatiotemporal microdistributions (left and middle columns) in 200-pm radius HEPG-2 spheroids moderately expressing the targeted receptor HER2, and the time- integrated radial microdistributions (right column) of the targeting antibody (top panel, the HER2-targeting Trastuzumab, in red) and the non-targeting antibody (bottom panel, Rituximab, shown in blue), each fluorescently labeled with FITC. Data points indicate the mean values, and error bars the standard deviations of n = 3 different spheroids studied per time point;

[0051] FIG. 13 shows HER2-overexpressing BT-474 spheroids. The extent of outgrowth / regrowth inhibition (used as an indirect surrogate of tumor recurrence) of BT- 474 spheroids of 200-pm radius (at the time of treatment). Two different radioactivity concentrations were evaluated (1.0 kBq / mL and 3.0 kBq / mL), which were divided at different ratios between the targeting radionuclide-antibody conjugates and the nontargeting radionuclide-antibody conjugates, at two different total antibody concentrations (10 pg / mL and 2 pg / mL, of each antibody conjugate, as shown on the top and lower panel, respectively). Spheroids were incubated for 24 hours with the antibody compositions. For BT-474, the targeting radionuclide-antibody conjugate was the HER2-targeting225Ac - DOTA-SCN-antibody Trastuzumab (TRA on plot); the non-targeting radionuclide- antibody conjugate was the225Ac-DOTA-SCN-antibody Rituximab (RIX on plot). Error bars correspond to the standard deviations of repeated measurements (n=18 spheroids per condition, n=3 independent spheroid and radionuclide- antibody conjugate preparations);

[0052] FIG. 14 shows moderately HER2-expressing HEPG-2 spheroids. The extent of outgrowth / regrowth inhibition (used as an indirect surrogate of tumor recurrence) of HEPG-2 spheroids of 200-pm radius (at the time of treatment). Two different radioactivity concentrations were evaluated (1.0 kBq / mL and 3.0 kBq / mL), which were divided at different ratios between the targeting radionuclide-antibody conjugates and the nontargeting radionuclide-antibody conjugates, at two different total antibody concentrations (10 pg / mL and 2 pg / mL, of each antibody conjugate, as shown on the top and lower panel, respectively). Spheroids were incubated for 24 hours with the antibody compositions. For HEPG-2, the targeting radionuclide-antibody conjugate was the HER2-targeting225Ac - DOTA-SCN-antibody Trastuzumab (TRA on plot); the non-targeting radionuclide- antibody conjugate was the225Ac-DOTA-SCN-antibody Rituximab (RIX on plot). Error bars correspond to the standard deviations of repeated measurements (n=18 spheroids per condition, n=3 independent spheroid and radionuclide- antibody conjugate preparations);

[0053] FIG. 15A, FIG. 15B, and FIG. 15C demonstrate selective steric hindrance of the binding sites of trastuzumab. FIG. 15A is an in-house Matlab-based eroding code applied to average the radial fluorescence intensities on the spheroid images, shown in the fluorescent images (in FIG. 15B), and to generate the quantitative radial distributions of antibody concentrations. Decreasing antibody reactivity was enabled by conjugation of FITC-SCN onto the antibody’s binding sites’ a-amino groups of the N-terminal amino acids. (FIG. 15B). FIG. 15C is a cartoon that frames the use of spheroids in the context of solid tumors: spheroids of different diameter are employed as surrogates of the solid tumors’ avascular regions of different size;

[0054] FIG. 16 is flow cytometry indicating extent of binding of fluorescently-labeled antibodies to HER2-expressing HEPG2 cancer cells. Gray: cells only; Blue: fluorescence shift by cells incubated with FITC-labeled “low-affinity” trastuzumab; Red: fluorescence shift by cells inculabed with FITC-labeled “high-affinity” trastuzumab.

[0055] FIG. 17 shows clonogenic cell survival of HEPG2 hepatoma cells exposed to [225Ac]Ac-DOTA (white symbols), [225Ac]Ac-DOTA-SCN-labeled “high-affinity” trastuzumab (red symbols), [225Ac]Ac-DOTA-SCN-labeled “low-affinity” trastuzumab (blue symbols), and / or the presently disclosed radionuclide-antibody conjugates with equal activity split between the two antibodies (purple symbols);

[0056] FIG. 18 shows the extent of inhibiting regrowth (used as indirect surrogate of tumor recurrence) of 400-pm-in-diameter HEPG2 spheroids treated with the same total activity of actinium-225 delivered by [225Ac]Ac-DOTA-SCN-labeled “high-affinity” trastuzumab (red bars), [225Ac]Ac-DOTA-SCN-labeled “low-affinity” trastuzumab (blue bars), and / or different activity ratios employing both antibodies as indicated on the plot. Spheroids were exposed to radioconjugates for 24 hours. Mean values ± the standard deviations of n-6 spheroids per condition (n=3 independent radioconjugate preparations) as shown. *** indicates j>-value <0.001; **<0.01; *<0.05; and

[0057] FIG. 19 shows survival plots of NSG-mice with subcutaneous HER2-expressing HEPG2 tumors treated with the same total injected radioactivity (2.96 kBq per 20 g mouse) that was split into different ratios (purple lines) between the “high-affinity” trastuzumab and the “low-affinity” trastuzumab and was compared to the treatment outcomes, at the same total injected (radio)activity, delivered by each antibody alone. The red line corresponds to the group treated with the “high-affinity” trastuzumab-radioconjugate alone (which is the current targeted alpha-particle therapy approach currently evaluated in clinical trials). Treatment was injected intravenously when tumors reached 100 mm3. The number n of animals treated in each cohort is indicated in the parentheses on the plot legend.

[0058] DETAILED DESCRIPTION

[0059] The presently disclosed subject matter now will be described more fully hereinafter with reference to the accompanying Figures, in which some, but not all embodiments of the inventions arc shown. Like numbers refer to like elements throughout. The presently disclosed subject matter may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements. Indeed, many modifications and other embodiments of the presently disclosed subject matter set forth herein will come to mind to one skilled in the art to which the presently disclosed subject matter pertains having the benefit of the teachings presented in the foregoing descriptions and the associated Figures. Therefore, it is to be understood that the presently disclosed subject matter is not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims.

[0060] A. Compositions

[0061] In some embodiments, the presently disclosed subject matter provides a composition comprising a first radionuclide-antibody conjugate having a first affinity for a target, also referred to herein as a “targeting radionuclide-antibody conjugate” or a “high-affinity radionuclide- antibody conjugate” and a second radionuclide-antibody conjugate having a second affinity for the same target, also referred to herein as a “non-targeting radionuclideantibody conjugate” or a “low(er) affinity radionuclide-antibody conjugate,” wherein the first affinity for the target is higher than the second affinity for the target. In certain embodiments, the composition comprises more than one first radionuclide- antibody conjugate and / or more than one second radionuclide-antibody conjugate. The term “antibody,” as used herein, refers to a protein that is found in blood or other bodily fluids of vertebrates, which is used by the immune system to identify and neutralize foreign objects, such as bacteria and viruses. Typically, an antibody is a protein that comprises at least one complementarity determining region (CDR). The CDRs form the “hypervariable region” of an antibody, which is responsible for antigen binding. A whole antibody typically consists of four polypeptides: two identical copies of a heavy (H) chain polypeptide and two identical copies of a light (L) chain polypeptide. Each of the heavy chains contains one N-terminal variable (VH) region and three C-terminal constant (CHI, Cm, and Cm) regions, and each light chain contains one N-terminal variable (VL) region and one C-terminal constant (CL) region. The light chains of antibodies can be assigned to one of two distinct types, either kappa (K) or lambda ( ), based upon the amino acid sequences of their constant domains. The VH and VL regions have the same general structure, with each region comprising four framework (FW or FR) regions. The term “framework region,” as used herein, refers to the relatively conserved amino acid sequences within the variable region which are located between the CDRs. In a typical antibody, each light chain is linked to a heavy chain by disulphide bonds, and the two heavy chains are linked to each other by disulphide bonds. The light chain variable region is aligned with the variable region of the heavy chain, and the light chain constant region is aligned with the first constant region of the heavy chain. The remaining constant regions of the heavy chains are aligned with each other. The variable regions of each pair of light and heavy chains form the antigen binding site of an antibody, (see, e.g., C. A. Janeway et al. (eds.), Immunobiology, 5th Ed., Garland Publishing, New York, N.Y. (2001)).

[0062] The terms “fragment of an antibody,” “antibody fragment,” and “antigen-binding fragment” of an antibody are used interchangeably herein to refer to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (see, generally, Holliger et al., Nat. Biotech., 23(9): 1126-1129 (2005)). An antibody fragment can comprise, for example, one or more CDRs, the variable region (or portions thereof), the constant region (or portions thereof), or combinations thereof. Examples of antibody fragments include, but are not limited to, (i) a Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL, and CHI domains, (ii) a F(ab’)2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region, (iii) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (iv) a Fab’ fragment, which results from breaking the disulfide bridge of an F(ab’)2 fragment using mild reducing conditions, (v) a disulfide-stabilized Fv fragment (dsFv), and (vi) a domain antibody (dAb), which is an antibody single variable region domain (VH or VL) polypeptide that specifically binds antigen.

[0063] “Binding” as used herein (e.g., with reference to a nanoparticle and / or antibody binding to cancer cells) refers to a non-covalent interaction between macromolecules (e.g., between a protein and a nucleic acid or a protein and a protein). While in a state of non- covalent interaction, the macromolecules are said to be “associated” or “interacting” or “binding” (e.g., when a molecule X is said to interact with a molecule Y, it is meant the molecule X binds to molecule Y in a non-covalent manner). Not all components of a binding interaction need be sequence-specific (e.g., contacts with phosphate residues in a DNA backbone), but some portions of a binding interaction may be sequence specific. Binding interactions are generally characterized by a dissociation constant (Kd) of less than

[0064] 106M, less than 107M, less than 10sM, less than 109M, less than 1010M, less than 10

[0065] 11M, less than 1012M, less than 1012M, less than 1014M, or less than 1015M. “Affinity” refers to the strength of binding, increased binding affinity being correlated with a lower Kd. With respect to antibodies in particular, when an antibody or other entity (e.g., antigen binding domain) “specifically recognizes” or “specifically binds” an antigen or epitope, it preferentially recognizes the antigen in a complex mixture of proteins and / or macromolecules, and binds the antigen or epitope with affinity which is substantially higher than to other entities not displaying the antigen or epitope. In this regard, “affinity which is substantially higher” means affinity that is high enough to enable detection of an antigen or epitope which is distinguished from entities using a desired assay or measurement apparatus.

[0066] More particularly, in some embodiments, the targeting radionuclide- antibody conjugate comprises an antibody that binds to a cancer- specific receptor. The terms “cancer- specific receptor,” “tumor- specific receptor,” “cancer- specific antigen,” and “tumorspecific antigen,” may be used interchangeably herein to refer to a cell surface receptor that is uniquely expressed by and / or displayed on cancer cells and is not expressed by or displayed on other cells in the body (e.g., normal healthy cells). In contrast, the terms “cancer-associated-receptor,” “tumor-associated-receptor,” “cancer-associated-antigen,” and “tumor-associated-antigen” may be used interchangeably herein to refer to a cell surface receptor that is not uniquely expressed by or displayed on a tumor cell and instead is also expressed on normal cells under certain conditions.

[0067] In some embodiments, the antibody is a monoclonal antibody. The term “monoclonal antibody,” as used herein, refers to an antibody produced by a single clone of B lymphocytes that is directed against a single epitope on an antigen. Monoclonal antibodies typically are produced using hybridoma technology, as first described in Kohler and Milstein, Eur. J. Immunol., 5: 511-519 (1976). Monoclonal antibodies may also be produced using recombinant DNA methods (see, e.g., U.S. Patent 4,816,567), isolated from phage display antibody libraries (see, e.g., Clackson et al. Nature, 352: 624-628 (1991)); and Marks et al., J. Mol. Biol., 222: 581-597 (1991)), or produced from transgenic mice carrying a fully human immunoglobulin system (see, e.g., Lonberg, Nat. Biotechnol., 23(9): 1117-25 (2005), and Lonberg, Handb. Exp. Pharmacol., 181: 69-97 (2008)). In contrast, “polyclonal” antibodies are antibodies that are secreted by different B cell lineages within an animal. Polyclonal antibodies are a collection of immunoglobulin molecules that recognize multiple epitopes on the same antigen.

[0068] Monoclonal antibodies that bind to cancer- specific receptors (referred to herein as “cancer- specific” or “tumor- specific” antibodies) typically cause selective cellular toxicity first by binding to a specific target antigen followed by cell lysis via antibody -dependent cellular cytotoxicity, complement activation, complement-dependent cytotoxicity, or by inhibition of signal transduction (e.g. the inhibition of dimerization of a receptor by receptor blocking through a monoclonal antibody) (Attarwala, H., J Nat Sci Biol Med., 7(1): 53-56 (2010)). In the context of the disclosed methods, however, the antibody in the second composition serves primarily to deliver the anti-cancer agent to target cancer cells, and not for any therapeutic effect of the antibody itself. The antibody may bind to any cancerspecific receptor known in the art, as well as cancer-specific receptors not yet identified. Exemplary cancer-specific receptors include, but are not limited to, HER2, epidermal growth factor receptor (EGFR), vascular endothelial growth factor receptor (VEGFR), interleukin-4 (IL-4), ccv(33 integrin, insulin-like growth factor receptor 1 (IGFR1), insulinlike growth factor receptor 2 (IGFR1), folate receptor, transferrin receptor, estrogen receptor, CXCR4, interleukin-6 (IL-6), transforming growth factor-beta receptor (TGF-DR), prostate specific membrane antigen (PSMA), a6[31 integrin, IGF1, EphA2, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), platelet derived growth factor receptor (PDGFR), CD20, and fibroblast growth factor receptor (FGFR). Other cancer- specific receptors are described in, e.g., Zeromski J., Arch Immunol Ther Exp (Warsz), 50(2): 105- 110 (2002); and Boonstra et al., Biomarkers in Cancer, 8: 119-133 (2016); doi:10.4137 / BIC.S38542.

[0069] A number of monoclonal antibodies that bind to cancer-specific receptors have been approved to treat a variety of different cancers, any of which may be included in the second composition. Such monoclonal antibodies include, but are not limited to, trastuzumab (HERCEPTIN®, Genentech, Inc.), cetuximab (ERBITUX®, Eli Lilly and Company), panitumumab (VECTIBIX®, Amgen, Inc.), rituximab (RITUXAN®, Genentech, Inc.), and bevacizumab (AVASTIN®, Genentech, Inc.). The disclosure is not limited to these particular antibodies, however, and any antibody that binds to a cancer- specific receptor may be included in the radionuclide-antibody conjugates.

[0070] In some embodiments, the first radionuclide-antibody conjugate and the second radionuclide- antibody conjugate each independently comprise an antibody selected from the group consisting of trastuzumab, cetuximab, panitumumab, rituximab, and bevacizumab. In certain embodiments, the first, or targeting, radionuclide-antibody conjugate comprises trastuzumab or cetuximab. In certain embodiments, the second, or non-targeting, radionuclide- antibody conjugate comprises rituximab.

[0071] In certain embodiments, the non-targeting radionuclide-antibody conjugate comprises an inactive form of the targeting antibody. For example, if the antibody comprising the targeting radionuclide- antibody is trastuzumab, then the antibody comprising non-targeting radionuclide- antibody conjugates in an inactive form of trastuzumab. In some embodiments, the inactive form of the targeting antibody used as the non-targeting antibody can be a “capped” antibody having a tightly bound epitope-peptide sequence at its binding sites and / or covalently modified at its binding sites).

[0072] In other embodiments, the second radionuclide-antibody conjugate comprises the same antibody as the first radionuclide- antibody conjugate, wherein the antibody of the second radionuclide-antibody conjugate is modified such that its affinity for the target is decreased relative to the affinity for the target of the antibody of the first radionuclideantibody conjugate.

[0073] In certain embodiments, one or more binding sites of the antibody of the second radionuclide- antibody conjugate are sterically hindered. In particular embodiments, the one or more binding sites of the antibody of the second radionuclide- antibody conjugate are sterically hindered by conjugation of one or more sterically-hindering moieties to one or more a-amino groups of N-terminal amino acids on the binding sites of the antibody via click chemistry. In such embodiments, the antibody comprises one or more sterically- hindering moieties bound to one or more a-amino groups of N-terminal amino acids on the binding sites of the antibody. In particular embodiments, the one or more sterically- hindering moieties are selected from a dye, a chelating moiety, and a click-reactive linker. In more particular embodiments: (a) the dye is selected from a coumarin dye, a rhodamine dye, a cyanine dye, a xanthene dye, a pyrylium dye, and sulfonated, azetidine derivatives thereof; (b) the chelating moiety comprises a chelator moiety disclosed herein; and (c) the click-reactive linker comprises a linker disclosed herein.

[0074] Representative dyes suitable for use with the presently disclosed radionuclideantibody conjugates include, but are not limited to:

[0075] Alexa Fluor® dyes, including Alexa Fluor® 350, 405, 430, 488, 500, 514, 532, 546, 555, 568, 594, 610, 633, 635, 647, 660, 680, 700, 750, 790, or DyLight® dyes, including DyLight® 350, 405, 488, 550, 594, 633, 650, 680, 755, and 800, in which a coumarin, a rhodamine, a cyanine dye, or a xanthene dye is sulfonated, among other modifications;

[0076] Chromeo™ dyes, including Chromeo™ 488, 494, 505, 546, and 642, which aminereactive pyrylium dyes; and

[0077] Janelia Fluor® Dyes, including but not limited to, JF503, JF525, JF549, JF585, JF635, and JF646, in which a N, N-dimethylamino group on a fluorophore is replaced with an azetidine.

[0078] Such dyes can include a alkyne, azide, an N-hydroxy succinimide (NHS) ester, or an isothiocyanato (SCN) reactive group.

[0079] In more particular embodiments, the antibody comprises one or more fluorescein isothiocyanate (FITC-SCN) moieties bound to one or more a-amino groups of N-terminal amino acids on the binding sites of the antibody. Tn certain embodiments, an immunoreactivity of the antibody of the second radionuclide- antibody conjugate is less than an immunorcactivity of the antibody of the first radionuclide- antibody conjugate. In particular embodiments, the immunoreactivity of the antibody of the second radionuclide- antibody conjugate is less than the immunoreactivity of the antibody of the first radionuclide- antibody conjugate by a range of about 1% less to about 99% less, including about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,

[0080] 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,

[0081] 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67,

[0082] 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,

[0083] 92, 93, 94, 95, 96, 97, 98, and 99% less immunoreactivity.

[0084] The antibody desirably is conjugated to a radionuclide. In some embodiments, the antibody is conjugated to the radionuclide using a linker. A linker is any chemical moiety that is capable of linking a compound, usually a drug, to a cell-binding agent such as an antibody or fragment thereof in a stable, covalent manner. Linkers can be susceptible to or be substantially resistant to acid-induced cleavage, light-induced cleavage, peptidase- induced cleavage, esterase-induced cleavage, and disulfide bond cleavage, at conditions under which the antibody remains active. Suitable linkers are well known in the art and include, for example, disulfide groups, thioether groups, acid labile groups, photolabile groups, peptidase labile groups, and esterase labile groups. Linkers also include charged linkers, and hydrophilic forms thereof as described herein and known in the art. In some embodiments, the linker may be a cleavable linker, a non-cleavable linker, a hydrophilic linker, and a dicarboxylic acid based linker. Exemplary linkers that may be used in the disclosed method include, but are not limited to isothiocyanato (SCN), isothiocyanato- benzyl (SCN-Bn), N-succinimidyl 4-(2-pyridyldithiojpentanoate (SPP); N-succinimidyl 4- (2-pyridyldithio)-2-sulfopentanoate (sulfoSPP); N-succinimidyl 4-(2- pyridyldithiojbutanoate (SPDB); N-succinimidyl 4-(2-pyridyldithio)2-sulfobutanoate (sulfo- SPDB); N-succinimidyl 4-(maleimidomethyl) cyclohexanecarboxylate (SMCC); N- sulfosuccinimidyl 4-(maleimidomethyl) cyclohexanecarboxylate (sulfoSMCC); N- succinimidyl-4-(iodoacetyl)-aminobenzoate (SIAB); and N-succinimidyl-[(N- maleimidopropionamido-tetraethyleneglycol] ester (NHS-PEG4-maleimide) .

[0085] The terms “radionuclide,” “radioisotope,” and “radioactive isotope” may be used interchangeably herein to refer to an atom that emits radiation as it undergoes radioactive decay through the emission of alpha particles (a), beta particles (P), or gamma rays (y).

[0086] Alpha-particle radiopharmaceutical therapy (aRPT) has shown promise in difficult- to-treat cancers, such as metastatic castration-resistant prostate cancer and triple-negative breast cancer. The highly efficient irradiation of a-particle emitters (1-10 MeV energy), endows a-particles with a 3- to 8-fold greater relative biological effectiveness compared to photon or P-particle radiation. Alpha particles typically cause double-strand DNA breaks, and their high killing efficacy (1-3 tracks across the nucleus result in cell death) is mostly independent of the cell-oxygenation state and cell-cycle. Hence, the complexity and level of DNA damage induced by aRPT rapidly overwhelms cellular repair mechanisms, and, if optimally delivered, aRPT is impervious to resistance irrespective of cell type or of resistance to other agents.

[0087] Actinium-225 (225Ac) is one of the most potent emitters, having a 10-day half-life and yielding 4 a-particles per physical decay to a stable element. Comparatively, the halflife of Francium-221,221Fr is about 4.9 minutes, the half-life of Astatine-217,217At is about 32 msec, and the half-life of Bismuth-213,213Bi is about 45.6 min.

[0088] The short range of a-particles in tissue (5-10 cell diameters) makes them ideal for precise cell irradiation, but presents challenges for using aRPT to treat established solid tumors. In addition, the diffusion-limited penetration depths of traditional targeted radionuclide vectors (e.g., antibodies) combined with the short range of a-particles result in only partial irradiation of solid tumors, compromising efficacy. That is, tumor regions not hit by the delivered a-particles likely are not killed.

[0089] In some embodiments, the radionuclide comprises an alpha-particle emitter. In certain embodiments, the alpha-particle emitter is selected from actinium-225, astatine-211, lead-212, terbium-149, thorium- 227, radium- 223, radium- 224, bismuth-212, and bismuth- 213. In particular embodiments, the alpha-particle emitter is actinium-225 (225Ac). The radionuclide of the first and the second radionuclide- antibody conjugate can be the same or different, and in some embodiments, is the same.

[0090] In some embodiments, the composition comprises a total radioactivity having a ratio ranging about 10:90 to about 90:10 between the first, targeting radionuclide-antibody conjugate and the second, non-targeting radionuclide- antibody conjugate, including about 10:90, about 15:85, about 20:80, about 25:75, about 30:70, about 35:65, about 40:60, about 45:55, about 50:50; about 55:45, about 60:40, about 65:35, about 70:30, about 75:25, about 80:20, about 85:15, and about 90:10 any integers or fraction thereof in between. In some embodiments, the composition comprises a total radioactivity that is approximately equally divided , e.g., 50:50, between the first radionuclide-antibody conjugate and the second radionuclide- antibody conjugate.

[0091] For example, the presently disclosed composition can have a total radioactivity in the range of about 50 to about 200 kilobecquerel (kBq) per mouse kilogram or per human kilogram; however, doses below or above this exemplary range are within the scope of the invention. The daily dose can be about 50 kBq / kg, about 55 kBq / kg, about 60 kBq / kg, about 65 kBq / kg, about 70 kBq / kg, about 75 kBq / kg, about 80 kBq / kg, about 85 kBq / kg, about 90 kBq / kg, about 95 kBq / kg, about 100 kBq / kg, about 105 kBq / kg, about 110 kBq / kg, about 115 kBq / kg, about 120 kBq / kg, about 125 kBq / kg, about 130 kBq / kg, about 135 kBq / kg, about 140 kBq / kg, about 145 kBq / kg, about 150 kBq / kg, about 155 kBq / kg, about 160 kBq / kg, about 165 kBq / kg, about 170 kBq / kg, about 175 kBq / kg, about 180 kBq / kg, about 185 about kBq / kg, about 190 kBq / kg, about 195 kBq / kg, or a range defined by any two of the foregoing values. Therapeutic or prophylactic efficacy can be monitored by periodic assessment of treated patients.

[0092] In some embodiments, the first radionuclide-antibody conjugate and the second radionuclide- antibody conjugates each independently further comprise a chelating moiety.

[0093] In certain embodiments, the chelating moiety is selected from the group consisting of DOTAGA (1,4,7,10-tetraazacyclododececane, l-(glutaric acid)-4,7,10-triacetic acid), DOTA (l,4,7,10-tetraazacyclododecane-l,4,7,10-tetraacetic acid), DOTASA (1,4,7,10- tetraazacyclododecane-l-(2-succinic acid)-4,7,10-triacetic acid), CB-DO2A (10- bis(carboxymethyl)-l,4,7,10-tetraazabicyclo[5.5.2]tetradecane), DEPA (7-[2-(Bis- carboxymethy lamino)-ethyl] -4, 10-bis-carboxymethyl- 1 ,4,7 , 10-tetraaza-cyclododec- 1 -yl- acetic acid)), 3p-C-DEPA (2-[(carboxymethyl)][5-(4-nitrophenyl-l-[4,7,10- tris(carboxymethyl)-l,4,7,10-tetraazacyclododecan-l-yl]pentan-2-yl)amino]acetic acid)), TCMC (2-(4-isothiocyanotobenzyl)- 1,4,7, 10-tetraaza- 1,4,7, 10-tetra-(2-carbamonyl methyl)- cyclododecane), oxo-DO3A (l-oxa-4,7,10-triazacyclododecane-5-S-(4- isothiocyanatobenzyl)-4,7,10-triacetic acid), p-NH2-Bn-Oxo-DO3A (l-Oxa-4,7,10- tetraazacyclododecane-5-S-(4-aminobenzyl)-4,7,10-triacetic acid), TE2A ((l,8-N,N'-bis- (carboxymcthyl)-l,4,8,l l-tctraazacyclotctradccanc), MM-TE2A, DM-TE2A, CB-TE2A (4,1 l-bis(carboxymethyl)-l,4,8,l l-tetraazabicyclo[6.6.2]hexadecane), CB-TE1A1P (4,8,11- tetraazacyclotetradecane-l-(methanephosphonic acid)-8-(methanecarboxylic acid)), CB- TE2P (l,4,8,ll-tetraazacyclotetradecane-l,8-bis(methanephosphonic acid), TETA (1,4,8,11- tetraazacyclotetradecane- 1,4, 8, 11 -tetraacetic acid), NOTA (1,4,7-triazacyclononane- N,N',N"-triacetic acid), NODA (l,4,7-triazacyclononane-l,4-diacetate); NODAGA (1,4,7- triazacyclononane, 1 -glutaric acid-4, 7-acetic acid); NOTAGA (l,4,7-triazonane-l,4- diyl)diacetic acid); DFO (Desferoxamine), NETA ([4-[2-(bis-carboxymethylamino)-ethyl]- 7-carboxymethl-[l,4,7]triazonan-l-yl}-acetic acid), TACN-TM (N,N',N", tris(2- mercaptoethyl)-l,4,7-triazacyclononane), Diamsar (1,8-Diamino-3,6,10,13,16,19- hexaazabicyclo(6,6,6)eicosane, 3,6,10,13,16,19-Hexaazabicyclo[6.6.6]eicosane-1,8- diamine), Sarar (l-N-(4-aminobenzyl)-3, 6,10,13,16,19-hexaazabicyclo[6.6.6] eicosane-1,8- diamine), AmBaSar (4-((8-amino-3,6,10,13,16,19-hexaazabicyclo [6.6.6] icosane-1- ylamino) methyl) benzoic acid), macropa, and BaBaSar.

[0094] In certain embodiments, the chelating moiety is selected from the group consisting of:

[0095]

[0096]

[0097] In particular embodiments, the chelating moiety is selected from dodecane tetraacetic acid (DOTA) and diethylenetriaminepentaacetic acid (DTPA). B. Methods for Treating a Solid Tumor

[0098] In other embodiments, the presently disclosed subject matter provides a method for treating a solid tumor in a subject in need of treatment thereof, the method comprising administering a therapeutically effective amount of a composition described herein to the subject.

[0099] The term “tumor,” as used herein, refers to an abnormal mass of tissue that results when cells divide more than they should or do not die when they should. In the context of the present disclosure, the term tumor may refer to tumor cells and tumor- associated stromal cells. Tumors may be benign and non-cancerous if they do not invade nearby tissue or spread to other pails of the organism. In contrast, the terms “malignant tumor,” “cancer,” and “cancer cells” may be used interchangeably herein to refer to a tumor comprising cells that divide uncontrollably and can invade nearby tissues. Cancer cells also can spread or “metastasize” to other parts of the body through the blood and lymph systems. The terms “primary tumor” or “primary cancer” refer to an original, or first, tumor in the body. The term “metastasis,” as used herein, refers to the process by which cancer spreads from the location at which it first arose as a primary tumor to distant locations in the body. The terms “metastatic cancer” and “metastatic tumor” refer to the cancer or tumor resulting from the spread of a primary tumor. It will be appreciated that cancer cells of a primary tumor can metastasize through the blood or lymph systems.

[0100] In certain embodiments, the solid tumor comprises a cancer selected from breast cancer, pancreatic cancer, liver cancer, kidney cancer, prostate cancer, lung cancer, and colorectal cancer, ovarian cancer, brain cancer, skin cancer, and combinations thereof.

[0101] The disclosure provides a method of inhibiting cancer cell growth, which comprises contacting cancer cells with the above-described compositions. Ideally, administration of the compositions described herein inhibits the growth of cancer cells from a primary tumor or primary cancer, such as an established solid tumor.

[0102] A primary cancer or tumor may arise in any organ or tissue. For example, the primary cancer or tumor may be a carcinoma (cancer arising from epithelial cells), a sarcoma (cancer arising from bone and soft tissues), a lymphoma (cancer arising from lymphocytes), a melanoma, or brain and spinal cord tumors. The primary tumor or cancer cells can arise in the oral cavity (e.g., the tongue and tissues of the mouth) and pharynx, the digestive system, the respiratory system, bones and joints (e.g., bony metastases), soft tissue, the skin (e.g., melanoma), breast, the genital system, the urinary system, the eye and orbit, the brain and nervous system (e.g., glioma), or the endocrine system (e.g., thyroid). More particularly, primary tumors or cancers of the digestive system can arise in the esophagus, stomach, small intestine, colon, rectum, anus, liver, gall bladder, and pancreas. Primary cancers or tumors of the respiratory system can arise in the larynx, lung, and bronchus and include, for example, non- small cell lung carcinoma. Primary cancers or tumors of the reproductive system can affect the uterine cervix, uterine corpus, ovaries, vulva, vagina, prostate, testis, and penis. Primary cancers of the urinary system can arise in the urinary bladder, kidney, renal pelvis, and ureter. Primary cancer cells also can be associated with lymphoma (e.g., Hodgkin’s disease and Non-Hodgkin’s lymphoma), multiple myeloma, or leukemia (e.g., acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, chronic myeloid leukemia, and the like). In some embodiments, the cancer cells are from a primary cancer or tumor located in the breast, pancreas, or liver.

[0103] The presently disclosed method comprises contacting cancer cells with a dose of the compositions described above. The cancer cells may be contacted with the compositions in vitro or in vivo. The term “zz? vivo” refers to a method that is conducted within living organisms in their normal, intact state, while an “zn vitro” method is conducted using components of an organism that have been isolated from its usual biological context. When cancer cells are contacted with the composition in vitro, the cell may be any suitable prokaryotic or eukaryotic cell. When cancer cells are contacted with the composition in vivo, the composition may be administered to an animal, such as a mammal, particularly a human, using standard administration techniques and routes. Suitable administration routes include, but are not limited to, oral, intravenous, intraperitoneal, subcutaneous, subcutaneous, or intramuscular administration. The compositions ideally are suitable for parenteral administration. The term “parenteral,” as used herein, includes intravenous, intramuscular, subcutaneous, rectal, vaginal, and intraperitoneal administration. In other embodiments, the compositions may be administered to a mammal using systemic delivery by intravenous, intramuscular’, intraperitoneal, or subcutaneous injection.

[0104] In some embodiments, the disclosed method promotes inhibition of cancer cell proliferation, the eradication of cancer cells, and / or a reduction in the size of at least one cancer or tumor such that the cancer or tumor is treated in a mammal (e.g., a human). By “treatment of cancer” is meant alleviation of a cancer in whole or in part. In one embodiment, the disclosed method reduces the size of a cancer or tumor by at least about 20% (e.g., at least about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%). Ideally, the cancer or tumor is completely eliminated.

[0105] For in vivo applications, the presently disclosed composition may be administered to a mammal (e.g., a human), so long as the anti-cancer agent is efficiently delivered to target cancer cells such that cancer cell growth is inhibited. To this end, the inventive method comprises administering a “therapeutically effective amount” of the anti-cancer agent. A “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result. The therapeutically effective amount may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the anti-cancer agent to elicit a desired response in the individual. For example, a therapeutically effective amount of the anti-cancer agent is an amount which is cytotoxic to cancer cells, such that the cancer or tumor is eliminated.

[0106] Alternatively, the pharmacologic and / or physiologic effect may be prophylactic, i.e., the effect completely or partially prevents cancer cell growth. In this respect, the inventive method comprises administering a “prophylactically effective amount” of the anti-cancer agent. A “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired prophylactic result (e.g., prevention of cancer or metastases).

[0107] As used herein, the term “treating” can include reversing, alleviating, inhibiting the progression of, preventing, or reducing the likelihood of the disease, disorder, or condition to which such term applies, or one or more symptoms or manifestations of such disease, disorder, or condition. Preventing refers to causing a disease, disorder, condition, or symptom or manifestation of such, or worsening of the severity of such, not to occur. Accordingly, the presently disclosed compounds can be administered prophylactically to prevent or reduce the incidence or recurrence of the disease, disorder, or condition.

[0108] The “subject” treated by the presently disclosed methods in their many embodiments is desirably a human subject, although it is to be understood that the methods described herein are effective with respect to all vertebrate species, which are intended to be included in the term “subject.” Accordingly, a “subject” can include a human subject for medical purposes, such as for the treatment of an existing condition or disease or the prophylactic treatment for preventing the onset of a condition or disease, or an animal subject for medical, veterinary purposes, or developmental purposes. Suitable animal subjects include mammals including, but not limited to, primates, e.g., humans, monkeys, apes, and the like; bovines, e.g., cattle, oxen, and the like; ovines, e.g., sheep and the like; caprines, e.g., goats and the like; porcines, e.g., pigs, hogs, and the like; equines, e.g., horses, donkeys, zebras, and the like; felines, including wild and domestic cats; canines, including dogs; lagomorphs, including rabbits, hares, and the like; and rodents, including mice, rats, and the like. An animal may be a transgenic animal. In some embodiments, the subject is a human including, but not limited to, fetal, neonatal, infant, juvenile, and adult subjects. Further, a “subject” can include a patient afflicted with or suspected of being afflicted with a condition or disease. Thus, the terms “subject” and “patient” are used interchangeably herein. The term “subject” also refers to an organism, tissue, cell, or collection of cells from a subject.

[0109] In general, the “effective amount” of an active agent or refers to the amount necessary to elicit the desired biological response. As will be appreciated by those of ordinary skill in this art, the effective amount of an agent may vary depending on such factors as the desired biological endpoint, the agent to be delivered, the makeup of the pharmaceutical composition, the drug target, and the like.

[0110] In some embodiments, the first radionuclide-antibody conjugate and the second radionuclide- antibody conjugates can be administered at the same time. In other embodiments, the first radionuclide-antibody conjugate and the second radionuclideantibody conjugate can be administered separately at different times, i.e., with a time interval between administration of each conjugate. For example, in some embodiments, the first radionuclide-antibody conjugate and the second radionuclide-antibody conjugate are injected between about 30 seconds and about 18 hours apart, including 30 seconds, 1 minute, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, and 18 hours apart. In particular embodiments, the first radionuclideantibody conjugate and the second radionuclide- antibody conjugate are administered between about 12 hours and about 18 hours apart, including 12, 13, 14, 15, 16, 17, and 18 hours apart, in vivo. In certain embodiments, the first radionuclide- antibody conjugate is administered first.

[0111] For repeated administrations over several days or longer, depending on the condition, the treatment can be repeated until a desired suppression of disease symptoms occurs. Other dosage regimens, however, may be useful and are within the scope of the presently disclosed methods. For example, in some embodiments, a total dose of the first radionuclide- antibody conjugate and / or the second radionuclide-antibody conjugates is administered in a series of fractional doses at predetermined time intervals. In such embodiments, the total dose administered via the series of fractional doses equals the total dose of the one or more targeting radionuclide-antibody conjugates and / or the one or more non-targeting radionuclide- antibody conjugates to be administered.

[0112] Additional therapies that can be used in combination with the presently disclosed compositions include surgery, radiation, including proton therapy, and chemotherapy.

[0113] The disclosed method can be performed in combination with other therapeutic methods to achieve a desired biological effect in a human patient. Ideally, the disclosed method may include, or be performed in conjunction with, one or more cancer treatments. Suitable cancer treatments that may be employed include, but are not limited, surgery, chemotherapy, radiation therapy, immunotherapy, and hormone therapy.

[0114] For example, one or more cancer treatments can include administering an “anticancer agent,” “anti-cancer drug,” “anti-cancer therapy,” and “anti-cancer therapeutic,” each of which may be used interchangeably herein to refer to any compound, molecule, substance, or procedure that partially or completely inhibits any or all aspects of cancer development and / or metastases. For example, an anti-cancer agent may inhibit the initiation, promotion, progression, metastasis, and / or neovascularization of a malignant tumor or cancer, as well as any adverse symptoms attributable to the particular cancer. Examples of anti-cancer agents include, but are not limited to, radiation therapeutic agents (e.g., radiopharmaceuticals), chemotherapeutic agents (e.g., alkylating agents, antimetabolites, plant alkaloids, antitumor antibiotics), immunotherapeutic agents (e.g., immune checkpoint inhibitors, monoclonal antibodies, CAR-T cells, cancer vaccines), targeted agents (e.g., small molecule drugs, monoclonal antibodies), and hormone therapies. In some embodiments, the anti-cancer agent is a chemotherapeutic agent, such as, for example, asparaginase, bleomycin, busulphan, cisplatin, carboplatin, carmustinc, capecitabine, chlorambucil, cytarabine, cyclophosphamide, camptothecin, dacarbazine, dactinomycin, daunorubicin, dexrazoxane, docetaxel, doxorubicin, etoposide, floxuridine, fludarabine, fluorouracil, gemcitabine, hydroxyurea, idarubicin, ifosfamide, irinotecan, lomustine, mechlorethamine, mercaptopurine, melphalan, methotrexate, mitomycin, mitotane, mitoxantrone, nitrosurea, paclitaxel, pamidronate, pentostatin, plicamycin, procarbazine, rituximab, streptozocin, teniposide, thioguanine, thiotepa, vinblastine, vincristine, vinorelbine, taxol, transplatinum, anti-vascular endothelial growth factor compounds (“anti-VEGFs”), anti-epidermal growth factor receptor compounds (“anti- EGFRs”), 5 -fluorouracil, and the like. However, any suitable anti-cancer agent may be used in the context of the present disclosure.

[0115] The term “combination” is used in its broadest sense and means that a subject is administered at least two agents, more particularly a composition as provided herein, i.e., composition comprising a first, targeting radionuclide- antibody conjugate and a second, radionuclide- antibody conjugate, in combination with a second therapeutic agent or therapy. More particularly, the term “in combination” refers to the concomitant administration of two (or more) active agents or therapies for the treatment of a single disease state. As used herein, the active agents or therapies may be combined and administered in a single dosage form, may be administered as separate dosage forms at the same time, or may be administered as separate dosage forms that are administered alternately or sequentially on the same or separate days. In one embodiment of the presently disclosed subject matter, the active agents or therapies are combined and administered in a single dosage form. In another embodiment, the active agents or therapies are administered in separate dosage forms (e.g., wherein it is desirable to vary the amount of one but not the other). The single dosage form may include additional active agents therapies for the treatment of the disease state.

[0116] Further, the presently disclosed composition in combination an additional therapeutic agent or therapy can be further administered with adjuvants that enhance stability of the agents, alone or in combination with one or more therapeutic agents, facilitate administration of pharmaceutical compositions containing them in certain embodiments, provide increased dissolution or dispersion, increase inhibitory activity, provide adjunct therapy, and the like, including other active ingredients. Advantageously, such combination therapies utilize lower dosages of the conventional therapeutics, thus avoiding possible toxicity and adverse side effects incurred when those agents are used as monotherapies.

[0117] The timing of administration of the presently disclosed composition in combination with an additional therapeutic agent or therapy can be varied so long as the beneficial effects of the combination of these agents are achieved. Accordingly, the phrase “in combination with” refers to the administration of a composition described herein and an additional therapeutic agent or therapy either simultaneously, sequentially, or a combination thereof. Therefore, a subject administered a combination of a presently disclosed composition and an additional therapeutic agent or therapy can receive a composition and additional therapeutic agent or therapy at the same time (i.e., simultaneously) or at different times (i.e., sequentially, in either order, on the same day or on different days), so long as the effect of the combination of both agents is achieved in the subject.

[0118] When administered sequentially, the agents can be administered within 1, 5, 10, 30, 60, 120, 180, 240 minutes or longer of one another. In other embodiments, agents administered sequentially, can be administered within 1, 5, 10, 15, 20 or more days of one another. Where the composition and additional agent or therapy are administered simultaneously, they can be administered to the subject as separate pharmaceutical compositions, each comprising either a presently disclosed composition or at least one additional therapeutic agent, or they can be administered to a subject as a single pharmaceutical composition comprising both agents.

[0119] When administered in combination, the effective concentration of each of the agents to elicit a particular biological response may be less than the effective concentration of each agent when administered alone, thereby allowing a reduction in the dose of one or more of the agents relative to the dose that would be needed if the agent was administered as a single agent. The effects of multiple agents may, but need not be, additive or synergistic. The agents may be administered multiple times.

[0120] In some embodiments, when administered in combination, the two or more agents can have a synergistic effect. As used herein, the terms “synergy,” “synergistic,” “synergistically” and derivations thereof, such as in a “synergistic effect” or a “synergistic combination” or a “synergistic composition” refer to circumstances under which the biological activity of a combination of a compound described herein and at least one additional therapeutic agent is greater than the sum of the biological activities of the respective agents when administered individually.

[0121] Synergy can be expressed in terms of a “Synergy Index (SI),” which generally can be determined by the method described by F. C. Kull et al., Applied Microbiology 9, 538 (1961), from the ratio determined by;

[0122] QH / QA + Qn / Qu = Synergy Index (SI) wherein:

[0123] QA is the concentration of a component A, acting alone, which produced an end point in relation to component A;

[0124] Qais the concentration of component A, in a mixture, which produced an end point;

[0125] QB is the concentration of a component B, acting alone, which produced an end point in relation to component B ; and

[0126] Qb is the concentration of component B, in a mixture, which produced an end point.

[0127] Generally, when the sum of Q / QA and Qb / Qa is greater than one, antagonism is indicated. When the sum is equal to one, additivity is indicated. When the sum is less than one, synergism is demonstrated. The lower the SI, the greater the synergy shown by that particular mixture. Thus, a “synergistic combination” has an activity higher that what can be expected based on the observed activities of the individual components when used alone. Further, a “synergistically effective amount” of a component refers to the amount of the component necessary to elicit a synergistic effect in, for example, another therapeutic agent present in the composition.

[0128] C. Pharmaceutical Formulations

[0129] The presently disclosed compositions can comprise a pharmaceutically acceptable (e.g., physiologically acceptable) carrier. Accordingly, a variety of suitable formulations are possible. Methods for preparing compositions for pharmaceutical use are known to those skilled in the art and are described in more detail in, for example, Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins; 21sted. (May 1 , 2005). The choice of carrier will be determined, in part, by the particular use of the compositions (c.g., administration to an animal) and the particular method used to administer the compositions. In some embodiments, the pharmaceutical compositions are sterile.

[0130] Suitable compositions include aqueous and non-aqueous isotonic sterile solutions, which can contain anti-oxidants, buffers, and bacteriostats, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. The compositions can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, water, immediately prior to use. Each of the nanoparticle and antibody desirably is part of a composition formulated to protect the nanoparticle, antibody, and anti-cancer agent from damage prior to administration to cells. For example, the composition can be formulated to decrease the light sensitivity and / or temperature sensitivity of the nanoparticle, antibody, and / or anti-cancer agent.

[0131] One of ordinary skill in the art will appreciate that the composition can include other therapeutic or biologically-active agents. For example, factors that control inflammation, such as ibuprofen or steroids, can be part of the composition to reduce swelling and inflammation associated with in vivo administration of the first and / or second composition.

[0132] D. Kits

[0133] In some embodiments, the presently disclosed compositions can be provided in a kit, i.e., a packaged combination of reagents in predetermined amounts with instructions for using the compositions (e.g., for administration to a human subject). As such, the disclosure provides a kit comprising the first composition and second composition described herein and instructions for use thereof. The instructions can be in paper form or computer-readable form, such as a disk, CD, DVD, etc. Ideally, the kit comprises all components, i.e., reagents, standards, buffers, diluents, etc., which are necessary to deliver the composition to cells in vitro or in vivo. The kit components may be provided as dry powders (typically lyophilized), including excipients which on dissolution will provide a reagent solution having the appropriate concentration. Following long-standing patent law convention, the terms “a,” “an,” and “the” refer to “one or more” when used in this application, including the claims. Thus, for example, reference to “a subject” includes a plurality of subjects, unless the context clearly is to the contrary (e.g., a plurality of subjects), and so forth.

[0134] Throughout this specification and the claims, the terms “comprise,” “comprises,” and “comprising” are used in a non-exclusive sense, except where the context requires otherwise. Likewise, the term “include” and its grammatical variants are intended to be non-limiting, such that recitation of items in a list is not to the exclusion of other like items that can be substituted or added to the listed items.

[0135] For the purposes of this specification and appended claims, unless otherwise indicated, all numbers expressing amounts, sizes, dimensions, proportions, shapes, formulations, parameters, percentages, quantities, characteristics, and other numerical values used in the specification and claims, are to be understood as being modified in all instances by the term “about” even though the term “about” may not expressly appear- with the value, amount, or range. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are not and need not be exact, but may be approximate and / or larger or smaller as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the ait depending on the desired properties sought to be obtained by the presently disclosed subject matter. For example, the term “about,” when referring to a value can be meant to encompass variations of, in some embodiments, ± 100% in some embodiments ± 50%, in some embodiments ± 20%, in some embodiments ± 10%, in some embodiments ± 5%, in some embodiments ±1%, in some embodiments ± 0.5%, and in some embodiments ± 0.1% from the specified amount, as such variations are appropriate to perform the disclosed methods or employ the disclosed compositions.

[0136] Further, the term “about” when used in connection with one or more numbers or numerical ranges, should be understood to refer to all such numbers, including all numbers in a range and modifies that range by extending the boundaries above and below the numerical values set forth. The recitation of numerical ranges by endpoints includes all numbers, e.g., whole integers, including fractions thereof, subsumed within that range (for example, the recitation of 1 to 5 includes 1 , 2, 3, 4, and 5, as well as fractions thereof, e.g., 1.5, 2.25, 3.75, 4.1, and the like) and any range within that range.

[0137] EXAMPLES

[0138] The following Examples have been included to provide guidance to one of ordinary skill in the art for practicing representative embodiments of the presently disclosed subject matter. In light of the present disclosure and the general level of skill in the art, those of skill can appreciate that the following Examples are intended to be exemplary only and that numerous changes, modifications, and alterations can be employed without departing from the scope of the presently disclosed subject matter. The synthetic descriptions and specific examples that follow are only intended for the purposes of illustration, and are not to be construed as limiting in any manner to make compounds of the disclosure by other methods.

[0139] EXAMPLE 1 Alpha-Particle Radionuclide- Antibody Conjugates for Treatment of Solid Tumors

[0140] 1.1 Overview

[0141] Radionuclide- antibody conjugates are among the leading approaches in targeted alpha-particle therapies for the treatment of vascularized, soft-tissue, solid tumors. We have discovered an unexpected, unique intervention that improves the therapeutic outcomes of therapies with alpha-particle radionuclide- antibody conjugates without increasing the total injected radioactivity: our intcrv ention improves the efficacy of the systemically injected targeting radionuclide-antibody conjugates by separately injecting a fraction of the total radioactivity using a different radionuclide-antibody conjugate. The choice of this second radionuclide- antibody conjugate is key: it is emphatically chosen to not bind to cancer cells. Our composition is different from previously reported compositions of alpha-particle radionuclide- antibody conjugates, where each and every one of the antibodies in the composition are chosen to bind to a receptor expressed on the surface of the same cancer cells comprising the tumor.

[0142] With our intervention, we observed significantly improved therapeutic outcomes when we split the injected radioactivity between the two types of antibodies - (a) the targeting radionuclide-antibody conjugate and (b) the non-targeting radionuclide-antibody conjugate - compared to the therapeutic outcomes of the same total radioactivity delivered only by the targeting radionuclide- antibody conjugate alone, on tumors of different origin expressing a wide range of targeted receptors. Without wishing to be bound to any one particular theory, one possible explanation for the observed improved outcomes with our unexpected intervention is the better spreading of the delivered radioactivity within solid tumors, in spite the fact that with our intervention lower absorbed doses are generally delivered at the tumors (i.e., the total absorbed energy averaged over the tumor mass) compared to the tumor absorbed doses delivered by the targeting radionuclide- antibody conjugate alone. Our in vitro investigations in 3D multicellular spheroids, that are used as surrogates of the avascular regions of solid tumors, demonstrate that the non-targeting radionuclide- antibody conjugates penetrate deeper into the avascular regions, compared to the targeting radionuclide- antibody conjugates, since they are not consumed / exhausted by their binding to / internalization by cancer cells, as is the case with the targeting radionuclideantibody conjugates. As a result, with our intervention, there is a significant population of cancer cells located far from the tumor periphery that is now irradiated by alpha-particles, delivered by the non-targeting radionuclide- antibody conjugates. It is possible that killing of this population is the main reason for the improved overall therapeutic outcomes that we observed in vivo. Better radioactivity spreading within the tumor enables more uniform irradiation of cancer cells, therefore, potentially increasing the probability of killing more of the cancer cells; since with alpha-particle radionuclidetherapy, cells that are not hit by alpha-particles will not be killed.

[0143] 1.2 Scope

[0144] High energy a-particles act like bullets and cause double-strand DNA-breaks (FIG. 1 A) that result in cell death, making aRPT impervious to resistance, if optimally delivered. A major advantage, but also a key challenge of aRPT, is the short range of a-particles in tissue (4-5 cell-diameters) (FIG. IB). Although it makes them ideal for precisely localized irradiation, which is critical in sparing neighboring healthy cells and tissues, limited tumor penetration may result in only partial tumor irradiation (FIG. 1C and FIG. ID). Importantly, tumor regions not being hit by a-particles will not be killed. We unexpectedly discovered that when we inject the same total radioactivity of an alpha-particlc emitter conjugated on two separate antibodies (a first antibody that targets surface markers on cancer cells comprising the solid tumors; and a second antibody chosen to emphatically not recognize / not target any markers on the surface of cancer cells comprising the same solid tumors), then we observe better tumor growth inhibition and longer survival of tumor-bearing mice compared to the corresponding outcomes when the same total radioactivity is injected as a radioconjugate of the targeting-antibody alone, for tumors of different origin, and with different levels of expression of the surface markers (that are targeted by the first antibody).

[0145] The newly discovered composition of radionuclide- antibody conjugates is different from compositions of radionuclide-antibody conjugates that have been reported in the previous art. On previously reported targeting-antibody compositions, each of the antibodies are chosen to bind to a receptor expressed on the surface of cancer cells comprising the solid tumor. In other words, in compositions of radionuclide- antibody conjugates known in the art, more than one type of surface markers / receptors are collectively targeted by the antibodies and all antibodies are targeting a receptor expressed by the cancer cells. Milenic et al., 2010; Blumenthal et al., 1991.

[0146] The idea behind these approaches is based on the rationale of maximizing the radioactivity delivered per each cancer cell (and / or maximizing the radioactivity delivered at the tumor (Milenic et al., 2010)): when the expression levels of one type of targeted marker is relatively low, then a second type of targeted marker is identified, and therefore, a composition of two separate targeting radionuclide-antibody conjugates is developed aiming to collectively deliver greater levels of radioactivity per cell since the collective number of targeted receptors, on cancer cells, is practically increased. Alternatively and additionally, the heterogeneous distributions of the expression levels of surface markers on cancer cells have been shown to justify compositions of separate (all being) targeting antibodies, each targeting a different receptor on same cancer cells, to again, collectively increase the radioactivity delivered per cancer cell. Pasternack et al., 2014; Howell, 2015.

[0147] As provided herein below in the Examples, a potential mechanism that could explain the unexpected finding of the presently disclosed compositions of radionuclide-antibody conjugates is that in solid tumors the targeting antibodies have limited penetration depths within the tumor avascular regions whereas, contrary, the non-targeting radionuclideantibody conjugates (which wc introduce) penetrate significantly deeper into the tumor avascular regions. The non-targeting antibodies must be chosen to be any antibody not targeting any of the markers on cancer cells and / or any other markers on the tumor microenvironment.

[0148] 1.3 Results

[0149] 1.3.1. Justification of antibody-pair choice and cancer cell characterization

[0150] The following receptors were chosen as model targets on cancer cells; the Human Epidermal growth factor Receptor-2 (HER2) and the Human Epidermal growth factor Receptor-1 (HER1). We utilized the following “targeting” antibodies: the HER2-targeting Trastuzumab, and the HER 1- targeting Cetuximab, as well as the following model “nontargeting” antibody: the CD20-targeting antibody Rituximab. Three cancer cells lines were studied, which were chosen based on their expression of the targeted surface markers: high, moderate and low, as follows: the HER2-overexpressing BT474 breast cancer cells (1.2 million HER2 copies per cell), the moderately HER2-expressing HEPG-2 human liver cancer cells line (410,000 HER2 copies per cell), and the BxPC3 pancreatic cancer cell line expressing low levels of HER1 (117,000 HER1 copies per cell). None of the three cancer cell lines expressed any detectable levels of CD20. Therefore, Rituximab was utilized as the non-targeting radionuclide- antibody conjugate in all three cancer cell lines.

[0151] The extend of targeting / binding of each of the antibodies listed above to cancer cells and / or the lack of binding was demonstrated both by flow cytometry (FIG. 2) and by measuring the binding isotherms of each of the antibodies to cancer cells (FIG. 3), with the aim to extract the expression levels of each of the targeted receptors by each cancer cell line, that are stated above, and the antibodies’ dissociation constants, KD.

[0152] Based on FIG. 2 and FIG. 3, the following antibody pairs were evaluated in our studies, that are shown on Table 1:

[0153] Table 1. Pairs of specific and non-specific antibodies evaluated as carriers of alphaparticle radiotherapy for each of the cell lines expressing different levels of the targeted receptor.

[0154] Radiolabeling of all antibodies was stable, as shown in Table 2 (for labeling with

[0155] Actinium-225) and in Table 3 (for labeling with Indium- 111, that was used for the KD and biodistributions studies). In all studies, comparable specific radioactivities were maintained for each radionuclide across all three antibodies.

[0156] Table 2. Characterization of Actinium-225-antibody conjugates.

[0157] Table 3. Characterization of Indium- 111 -antibody conjugates

[0158] 1.3.2. In vivo evaluation: tumor response, animal survival and dosimetry

[0159] In mice bearing subcutaneous tumors, formed by each of the three cancer cell lines, were injected once intravenously with each treatment when the tumor volume reached 100 mm3, and tumor volume and animal weight (and behavior) were checked and recorded daily. The study endpoint was defined by the tumor reaching a size beyond which the ability of mice to freely reach the food and water containers was affected. For each animal model, both the extent of tumor growth inhibition and animal survival were significantly improved (p-value > 0.05) when the indicated antibody pairs were employed to deliver the same total injected radioactivity (purple line in FIG. 4A and FIG. 5A; green line in FIG. 6A) compared to the tumor growth inhibition and animal survival observed when only the targeting antibody was used (red line in FIG. 4A and FIG. 5A; orange line in FIG. 6A) even at greater tumor absorbed doses (for BT-474 and HEPG-2 tumors) as shown in Table 4 and Table 5 below, for the marker-overexpressing BT-474 and moderately marker-expressing HEPG-2 tumors, respectively. On the low marker-expressing BxPC3 tumors, both the targeting and the non-targeting antibodies delivered comparable absorbed doses (Table 6), but, still, the composition of both radionuclide-antibody conjugates at equal split of the same total radioactivity demonstrated slower tumor growth and significantly longer survival.

[0160] In all studies, the injected dose was 2.96 kBq, which was 80% of the MTD (which was 3.7 kBq / 20 g mouse). All animals demonstrated weight loss less than 20% during the study (FIG. 4B, FIG. 5B, FIG. 6B). On euthanized animals, at the indicated endpoints, histopathology evaluation did not reveal off-target toxicitics (FIG. 4C, FIG. 5C, FIG. 6C).

[0161] In mice bearing subcutaneous tumors formed by each of the three cancer cell lines, the Indium- 111 -radiolabeled conjugates of each antibody studied herein were intravenously injected and were employed to measure the biodistributions of each antibody in all three animal models, as shown in FIG. 7, FIG. 8 and FIG. 9 for mice with subcutaneous BT-474, HEPG-2 and BxPC-3 tumors, respectively. Indium- 111 has been repeatedly validated as a reliable surrogate for evaluating the biodistributions of the parent Actinium- 225. Howe et al., 2022; Salerno et al., 2022; Prasad et al., 2021.

[0162] On the evaluation of dosimetry, for the HER1 and HER2 targeting antibodies, given their internalization, all emitted four alpha-particles were assumed to remain within the tumor contributing to the delivered dose, in agreement with previous reports. Howe et al., 2022; Salerno et al., 2022; Prasad et al., 2021.

[0163] For the non-targeting, augmenting, antibody approximately 50% of the emitted Bismuth-213 was assumed to clear from the tumor; this dose was added to the kidneys. Briefly, an effective diffusion coefficient of 6xl0‘12mm2 / sec was assumed for any protein, a radionuclide-carrier protein, on which the generated Bismuth-213 cation is expected to adhere on right after its generation within the tumor interstitial space, and the root mean square displacement of this radionuclide-carrier protein was calculated for the time period of 10 x [the half-life of Bismuth-213] (=10x45 minutes). Then the fraction of alpha-particles emitted from Bismuth-213 that escaped the tumor was estimated to be equal to the ratio of the volume of the outer shell of thickness equal to this root mean square displacement divided by the total tumor volume of 100 mm3, assuming spherical tumor shape.

[0164] Table 4, Table 5 and Table 6 show the dosimetry on mice with subcutaneous BT- 474, HEPG-2 and BxPC-3 tumors, respectively. In the first two animal models with high and moderate expression of the HER2-targeted receptors, the tumor delivered dose by the antibody pairs was less than the dose delivered by the targeting antibody alone. For the low- HER1 -expressing BxPC-3 tumors, either type of antibody delivered comparable doses to the tumor. Table 4. Tumor and normal organ absorbed doses (Gy) from 2.96 kBq225Ac injected intravenously and delivered by each radionuclide-antibody conjugate alone and / or by their combination (at equal injected radioactivity split ratio) on NSG mice bearing BT-474 subcutaneous tumors overexpressing the targeted marker (HER2). Reported are the mean values and standard deviations of measurements averaged over 3 mice per time point.

[0165] Table 5. Tumor and normal organ absorbed doses (Gy) from 2.96 kBq225Ac injected intravenously and delivered by each radionuclide-antibody conjugate alone and / or by their combination (at equal injected radioactivity split ratio) NSG mice bearing HEPG-2 subcutaneous tumors moderately overexpressing the targeted marker (HER2). Reported are the mean values and standard deviations of measurements averaged over 3 mice per time point.

[0166] >

[0167] Table 6. Tumor and normal organ absorbed doses (Gy) from 2.96 kBq225Ac injected intravenously and delivered by each radionuclide-antibody conjugate alone and / or by their combination (at equal injected radioactivity split ratio) on NSG mice bearing BxPC3 subcutaneous tumors expressing low levels of the targeted marker (HER1). Reported are the mean values and standard deviations of measurements averaged over 3 mice per time point.

[0168] 1.3.3 Radiosensitivity of cancer cells in monolayers

[0169] The colony survival fractions of cells exposed to Actinium-225 in different forms are shown in FIG. 10. As expected the225Ac-DOTA form and the225Ac-DOTA-SCN- Rituximab conjugate (the non-targeting radionuclide-antibody conjugate) did not associate with cells and resulted in similar and less effective colony kill compared to the targeting225Ac-labeled antibody conjugates (225Ac-DOTA-SCN-Trastuzumab for BT-474 and HEPG- 2, and225Ac-DOTA-SCN-Cetuximab for BxPC3, respectively). The antibody concentration was kept constant across all different radioactivity concentrations, at 20 pg / mL. 1.3.4 Spatiotemporal Profiles of antibodies in 3D spheroids and spheroid response to radiotherapy delivered by different antibodies

[0170] Two of the cancer cells lines, BT-474 and HEPG-2, formed spheroids, in which the spatiotemporal microdistributions of fluorescently labeled targeting and non-targeting antibodies were measured, and the time-integrated radial antibody concentrations were calculated as shown in FIG. 11 and FIG. 12, respectively. Comparison of the time-integrated profiles confirmed (as shown before (Howe et al., 2022; Salerno et al., 2022; Prasad et al., 2021.)) that the targeting antibodies exhibited heterogeneous spatiotemporal distributions, and their penetration into the deeper distances from the spheroid edge was limited. Conversely, the non-targeting (non-binding) antibody demonstrated almost uniform infiltration of spheroids. Both types of antibodies used herein, the targeting and nontargeting, were incubated with spheroids for the same period of time to match their similar blood clearance kinetics (as shown by the biodistribution studies). The decreasing concentrations of all antibodies at the edges of spheroids is an experimental artifact, as previously discussed (Kavousanakis et al., 2022): when at different time points, during incubation of spheroids with the antibodies, spheroids are removed from the incubating suspension and are quickly placed into the freezing fluid medium (before freezing and sectioning), significant mass transport occurs within the antibody-free fluid in which the spheroids are embedded. In our computational models, not shown herein, this point is being taken into account. Kavousanakis et al., 2022.

[0171] The spatiotemporal distributions in spheroids, shown in FIG. 11 and FIG. 12, should be adequate to qualitatively appreciate that by splitting the same total radioactivity between the antibody pairs, the non-targeting antibody should enable part of the radioactivity to reach the spheroid core at greater extents - although none of the radionuclides will enter the cancer cells by antibody-mediated internalization as is the case with the targeting antibodies. It is possible that the locally delivered radioactivity by the targeting antibodies on cancer cells residing within the first 50-75 pm from the spheroid edge, to be well above the lethal levels, which may not be affected by the fraction of the radioactivity concentration separately labeled on the non-targeting antibody. We are in the process of computing this ideal ratio to optimize both the locally delivered doses at the spheroid periphery (by the targeting radionuclide-antibody) and the delivered dose towards the spheroid core (by the non-targeting radionuclide- antibody conjugate).

[0172] In agreement with the above observations on the microdistributions of antibodies within spheroids are the findings shown in FIG. 13 and FIG. 14, which confirm that the best inhibition of spheroid regrowth / outgrowth was obtained by splitting the same total radioactivity concentration between the two separate antibodies, a targeting and a nontargeting, in each pair rather than delivering all the radioactivity by the targeting radionuclide- antibody conjugate alone.

[0173] 1.4 Materials and Methods

[0174] Phosphate Buffered Saline (PBS), trisodium citrate dihydrate, anhydrous citric acid, poly(2-hydroxyethyl methacrylate) (polyHEMA) and 2-Amino-2-(hydroxymethyl)-l,3- propanediol (Trizma®) were purchased from Sigma-Aldrich (Atlanta, GA, USA). Trypsin and Matrigel™ were purchased from Coming (Coming, NY, USA), Ethylenediaminetetraacetic acid (EDTA) was purchased from Fisher Scientific (Pittsburgh, PA, USA), penicillinstreptomycin was from ThermoFisher Scientific (Waltham, MA, USA), S-2-(4- Isothiocyanatobenzyl) -diethylenetriamine pentaacetic acid (DTPA-SCN) and S-2-(4- isothiocyanatobenzyl)- 1 ,4,7, 10-tetraazacyclododecane- 1 ,4,7, 10- tetraacetic acid (DOTA- SCN) were from Macrocyclics (Dallas, TX, USA). Chelex® resin, chromatography and desalting columns from Bio-Rad (Hercules, CA, USA), syringe filters (0.22 pm, Cat No. 76479-024) from VWR (Radnor, PA, USA), the Eagle’s Minimal Essential Medium (EMEM) was purchased from Quality Biological (Gaithersburg, MD,USA) and, Hybricare™ and Roswell Park Memorial Institute (RPMI) medium were from ATCC (Manassas, VA, USA), the Fetal Bovine Serum (FBS) was from Omega Scientific (Tarzana, CA, USA), and the HER 1 -binding antibody Cetuximab was purchased from Eli Lilly (Indianapolis, IN, USA) while the HER-2 binding antibody Trastuzumab was from Genentech (San Francisco, CA, USA).The non-specific antibody Rituximab was purchased from the Johns Hopkins Pharmacy (Baltimore, MD, USA). Indium-Il l (111In), indium chloride, was purchased from BWXT (Ontario, Canada). Actinium-225 (225Ac), actinium chloride, was supplied by the U.S. Department of Energy Isotope Program, managed by the Office of Science for Nuclear- Physics. 1.4.1 Cell lines

[0175] The cell lines BT-474 and HEPG-2 were cultured using Hybricarc™ Media (made using manufacturer’s guidelines) and Eagle’s Minimum Essential Medium (EMEM), respectively, each supplemented with 10% FBS, 100 units / mL Penicillin and 100 g / mL Streptomycin in an incubator at 37 °C and 5% CO2. The cell line BxPC3 was cultured in Roswell Park Memorial Institute (RPMI) media, supplemented as above.

[0176] 1.4.2 Antibody labeling and characterization

[0177] DOTA-SCN (or DTPA-SCN or FITC-SCN), was dissolved in DMF at 10 mg / mL (or dissolved in DMSO at 20 mg / mL or in DMF at 10 mg / mL), following which it was added dropwise to the antibody (Trastuzumab, Cetuximab or Rituximab) (2.5 mg / mL) in 0.1 -M sodium carbonate buffer at pH 9.0 at 40:1 (or 15:1 or 5:1) chelator (or fluorophore): antibody mole ratio. The reaction was then allowed to proceed at 4 °C overnight on a plate shaker. The unconjugated chelator (or fluorophore) was removed by passing the mixture through a 10DG column (equilibrated with 0.1-M Tris-HCl buffer at pH 9.0 for225Ac-radiolabeling, or with IM acetate buffer at pH 4.5 fori nIn-radiolabeling, or with PBS at pH 7.4 in the case of the fluorophore). The antibody concentration was measured using the BCA assay, and, for the FITC-labeled antibody, it was correlated to fluorescence to develop a calibration curve.

[0178] For antibody radiolabeling, radioactivity dissolved in 0.2-M HC1 was added to the chelator-conjugated antibody, suspended in 500 pL of Tris-HCl buffer (or acetate buffer), and the reaction mixture was incubated at 37 °C for one hour. Following these steps, the radiolabeled antibody was then purified using a 10DG column equilibrated with PBS at 1 mM, pH 7.4. Radiolabeling efficiency was calculated as the ratio of the measured radioactivity before and after the 10DG column. Radiochemical purity was evaluated using iTLC with 10-mM EDTA in water as the mobile phase. McDevitt et al., 2002.

[0179] Immunoreactivity of the (radio)labeled antibody was measured by incubating with cells on ice for 1 hour at 100:1 receptor: antibody ratio and, upon separation of non-cell- bound antibodies, by quantifying the radioactivity associated with cells relative to the total radioactivity added. The fraction bound was corrected for non-specific antibody binding to cells by evaluating, in parallel suspensions, the fraction of radiolabeled antibody bound to cells in the presence of 50x excess unlabeled antibody. The stability of the antibody radiolabeling was evaluated by adding the radiolabeled antibody to media at pH 7.4. Following 24 hours of incubation, the antibody was passed through a 10DG column equilibrated with PBS at 1 mM, pH 7.4, and the antibody fractions that were eluted from the column were collected. The stability of radiolabeling was then calculated as the ratio of the measured radioactivity associated with the antibody before incubation and after the completion of the 24-hour incubation.

[0180] 1.4.3 Colony Survival Assay

[0181] The cancer cells were plated in 6-well plates at 500,000 cells per well and were allowed to adhere overnight before being incubated with varying concentrations of radioactivity (4.63, 9.25, 18.5, 37, 74 kBq / mL) delivered either in the form of the radioactive pay load225Ac-DOTA or the radiolabeled Antibody, at pH 7.4 for a duration of 6 hours. Post completion of incubation, the cells in each of the wells were washed with PBS at pH 7.4, gently scraped and resuspended at a concentration of 10,000 cells / mL in media at pH 7.4. Following this, they were plated into tissue culture dishes at varying cell densities. Once cell colonies were observed (approximately 10 doubling times), the media was removed, dishes were washed with water, and the colonies were fixed and stained using 6% (w / v) glutaraldehyde and 0.05% (w / v) Crystal violet, respectively, following which they were counted using a colony counter pen. The number of colonies counted for each of the treatment groups was then normalized by the number of colonies from the control group to obtain the survival fraction, while accounting for the plating efficiency. Franken et al., 2006.

[0182] 1.4.4 Flow cytometry

[0183] Cells were trypsinized and a suspension of 1 million cells / mL in media was obtained. For the fluorescent antibody conditions, this suspension was incubated with FITC- labeled Antibody at 50 times excess Antibody to receptor assuming 1 million receptors / cell on ice for 1 hour. Following this, the cell suspensions were centrifuged, supernatant was taken off and the pelleted cells were resuspended in ice cold PBS. This procedure was repeated three times following which the cells were suspended in 1 mL of PBS and were analyzed on the BD FACS Canto Flow cytometer (Franklin Lakes, NJ, USA). For the cells only condition, 1 million cells / mL were incubated with a volume of ice-cold PBS equal to the volume of fluorescently labeled antibody that was added. 1.4.5 Spheroid formation, spatiotemporal distributions and treatment

[0184] Spheroids were formed by seeding 3200 BT-474 and / or 1000 HEPG-2 cells per well in a PolyHEMA-coated 96-well round bottom plate, and centrifuging it for 10 minutes at 1023 ref and 4° C. The formed spheroids were tracked for their size and were used upon reaching the desired size. The cell line BxPC3 did not form spheroids.

[0185] The spatiotemporal profiles of the specific and non-specific antibodies were evaluated by incubating 400 pm diameter spheroids with FITC-labeled specific Antibody (Trastuzumab) (0.06 pM, ex / em: 494 / 518 nm) or with the FITC-labeled non-specific Antibody (Rituximab) (0.06 pM, ex / em: 494 / 518 nm). Spheroids were harvested at various timepoints, during incubation with the carriers (uptake) and upon being transferred in fresh media (clearance), were flash frozen in cryochrome, mounted on OCT gel and sectioned at 20 pm thickness. The equatorial slices were then imaged using a confocal fluorescence microscope (Zeiss LSM 780 Confocal Microscope with filters, 10X objective, White Plains, NY). To generate the corresponding calibration curves, known concentrations of FITC- labeled antibody were measured, using the same microscope settings, in a quartz cuvette of 20 pm path-length. An in-house developed MATLAB erosion code was applied on the images of spheroid sections to calculate the average intensity / concentration within each 5 pm-wide concentric ring of the section which was then plotted vs. its radial position, and the time-integrated, radial concentrations were calculated using the trapezoidal rule.

[0186] In treatment studies, upon reaching a size of 400 pm in diameter, the spheroids were incubated with varying combinations of radiolabeled specific and non-specific antibody, for 24 hours, to roughly match their blood clearance kinetics in mice, at three different total radioactivity concentration of 1.0 and 3.0 kBq / mL. The total antibody mass for both the specific and non-specific antibodies, each, was maintained at 200 times excess of the HER2 receptors expressed by all cells comprising the spheroid. After incubation, the treated spheroids were transferred into fresh media (one spheroid per well in PolyHEMA-coated U- bottom plates), and the spheroid volume was monitored until an asymptote was reached for the volume of untreated spheroids. At that point, spheroids were transferred to adherent 96- well plates (one spheroid per well in cell culture- treated F-bottom plates), and, once the nontreated condition reached confluency, cells from each well were trypsinized and counted. The percent outgrowth / regrowth was evaluated as the number of cells counted in each treated condition normalized by the number of cells in the untreated condition.

[0187] 1.4.6 Animal study

[0188] All animal studies were performed in compliance with Institutional Animal Care and Use Committee protocol (IACUC) guidelines. Female and male NSG (NOD scid gamma) mice, 20 g in weight, four to six weeks old were purchased from JHU Breeding Facility. These mice were housed in filter top cages with sterile food and water.

[0189] For the breast cancer model, subcutaneous tumor inoculation was performed in female NSG mice, by injecting 1,000,000 BT-474 cells (per mouse) suspended in a 100 pL mixture of 50:50 v / v ratio of serum-free medium and Matrigel™. For the hepatoma and pancreatic cancer models, the NSG male mice were inoculated subcutaneously by injecting 1,500,000 HEPG-2 cells or 500,000 BxPC3 cells (per mouse) suspended in lOOpL mixture of 50:50 v / v ratio of serum-free medium and Matrigel™. The tumors were allowed to grow to a volume of approximately 100 mm3following which the animals were randomly assigned to a treatment / control group.

[0190] For treatment, the tumor bearing mice were administered lOOpL of radiotherapy at a total radioactivity of 2.96 kBq per 20 g animal (either 2.96 kBq of225Ac-DOT A-SCN- labeled specific Antibody, 2.96 kBq of225Ac-DOTA-SCN-labeled non-specific Antibody, or a combination of both at the same total radioactivity) intravenously on Day 50 (for BT-474 tumors) and Day 55 (for HEPG-2 tumors). In the study with the BxPC-3 tumors, the therapy was administered in two parts: 2.22 kBq per 20 g animal was given on Day 40 and a subsequent 0.74 kBq per 20 g animal was given on Day 50. Tumor volume measured using a digital caliper (with a resolution of 0.01 mm) and animal weights were recorded daily. The tumor volume was calculated using the ellipsoid formula (V=4*7r*a* 132 / 3, where a and 13 are the major and minor diameters, respectively).

[0191] For each of the models, the endpoint criterion was set as the point when the onset of symptoms associated with uncontrolled tumor growth was observed. The tumor growth control and animal survival advantage were monitored for each of these. Following euthanasia, animals, from all models, were dissected to recover the tumors and critical organs. Fixed tissues of harvested sections were processed, and H&E stained for histological evaluation. EXAMPLE 2

[0192] High- Affinity and Low(cr)-Affinity Alpha-Particle Radionuclide- Antibody Conjugates

[0193] 2.1 Overview

[0194] This Example provides an embodiment of the presently disclosed antibody transport conjugates, in which the same antibody-radioconjugate is employed in two distinct forms: a “high-affinity” form and a “low(er)-affinity” form, each of which targets the same tumor cell marker. More particularly, this Example demonstrates that the affinity of any targeting antibody for the target can be decreased, thereby enabling the targeting antibody to penetrate deeper into a solid tumor. When a “low(er)-affinity” form is combined with a “high affinity” form of the same antibody-radioconjugate, a composition of radiolabeled antibodies of variable affinities can be created, which collectively enables more uniform irradiation of solid tumors by alpha-particles resulting in prolonged survival without increasing the administered radioactivity. The “collective immunoreactivity ” / “collective affinity” of the antibody composition is quantifiable and can be tailored as desired.

[0195] This delivery strategy uniformly distributes a-particles within large solid tumors by simultaneously delivering the same a-particle emitter by different antibodies, each killing a different region of the tumor: (1) a “high-affinity” radiolabeled- antibody irradiating the tumor perivascular regions (where the aggressive cancer cells reside) (FIG. 15, red frame / symbols), and (2) a separately administered “low(er)-affinity” form of the same radiolabeled-antibody that upon tumor uptake penetrates the deeper parts of tumors where “high-affinity” antibodies do not reach (and where cancer recurrence originates) (FIG. 15, blue frame / symbols). The “low(er)-affinity” antibodies clear too fast from the tumor perivascular regions, since they do not strongly bind / adhere to cells and / or the tumor microenvironment to delay their clearance from the tumor.

[0196] 2.2 Representative Results

[0197] FIG. 15A demonstrates that at the core of the spheroid, which is employed as surrogate of the avascular regions of solid tumors (see FIG. 15C), the blue symbols (i.e., the “low-affinity” antibody) penetrate more; at the spheroid edge, it is the red symbols (i.e., the “high-affinity” antibody) that accumulates the most. The presently disclosed approach engages both antibody types as separate radioconjugates of the same alpha-particle emitter that (when given at the right activity ratios) deliver lethal doses at every location within the avascular tumor regions. The best activity split ratio between the two antibody- radioconjugatcs, with the constraint of keeping the total activity at a minimum, is calculated using a digital twin, Kavousanakis et al., 2024, that is experimentally informed by the spatiotemporal distributions of each agent in said spheroids, along with the measured binding affinities of antibodies for each cancer cell type.

[0198] In the following representative examples, Trastuzumab was used as the “high- affinity” antibody. Trastuzumab also was used as the “low(er)-affinity” radiolabeled- antibody after its active sites were partially sterically hindered. One way to achieve steric hindrance of an antibody is via click chemistry. In this example, fluorescein isothiocyanate (FITC-SCN) was reacted with trastuzumab at almost neutral pH to selectively target the a- amino groups of the N-terminal amino acids on the binding sites of trastuzumab, thereby decreasing the “immunoreactivity” of trastuzumab from 88% (measured for the “high- affinity” trastuzumab) to less than 17% (measured for the “low(er)-affinity” trastuzumab).

[0199] 2.2.1. Penetration of 3D spheroids

[0200] Referring once again to FIG. 15, selective steric hindrance of the binding sites of trastuzumab (producing the “low(er)-affinity” antibody, shown in blue) enabled its penetration into the deeper parts of 3D multicellular spheroids, which were utilized as surrogates of tumors’ avascular regions, where “high-affinity” trastuzumab (shown in red) cannot reach. (FIG. 15A): an in-house Matlab-based “eroding code” was applied to average the radial fluorescence intensities on the spheroid images, shown in the fluorescent images (in FIG. 15B), and to generate the quantitative radial distributions of antibody concentrations. Our lab has pioneered such quantitative approaches that employ the spatiotemporal microdistributions of carriers and of therapeutic agents in spheroids as predictors for in vivo responses of solid tumors. Kavousanakis et al., 2024; Howe et al., 2022.

[0201] In this Example, decreasing antibody reactivity was enabled by conjugation of FITC- SCN onto the antibody’s binding sites’ a- amino groups of the N-terminal amino acids. FIG. 15B shows fluorescence microscopy images of HER2-positive BT474 breast cancer spheroids’ equatorial sections after incubation for 24 hours with fluorescently labeled (a) HER2-targeting “high- affinity” trastuzumab (in red) and (b) “low-affinity” trastuzumab (in blue). FIG. 15C is a cartoon that aims to frame the use of spheroids in the context of solid tumors: spheroids of different diameter are employed as surrogates of the solid tumors’ avascular regions of different size.

[0202] 2.2.2. Engineering the antibody affinity toward HER2- expressing cancer cells

[0203] Flow cytometry confirmed (a) the greater fluorescence shift by the “high-affinity” trastuzumab (shown in red), compared to (b) the minimal shift of a the “low(er)-affinity” trastuzumab on same cells (shown in blue). Referring now to FIG. 16, flow cytometry indicates the extent of binding of fluorescently-labeled antibodies to HER2-expressing HEPG2 cancer cells. Gray: cells only; Blue: fluorescence shift by cells incubated with FITC-labeled “low-affinity” trastuzumab; Red: fluorescence shift by cells inculabed with FITC-labeled “high-affinity” trastuzumab.

[0204] 2.2.3. Killing efficacy in vitro

[0205] First, in the absence of transport limitations (FIG. 17), monolayers of HEPG2 cancer cells were exposed to actinium-225 delivered (i) by each antibody alone, and / or (ii) by their “transport conjugate compositions” with the (radio)activity concentrations equally split between the two separate antibody-radioconjugates. The clonogenic survival fractions were the least when cells were treated with the “high-affinity” antibody-radioconjugates (red, FIG. 17) and greatest (among all three antibody-radioconjugates’ treatments) when treated with the “low-affinity” antibody-radioconjugates (blue symbols). The “transport conjugate compositions” (purple symbols) resulted in intermediate extents of clonogenic survival fractions. For comparison, the free chelate ([225Ac]Ac -DOTA, white symbols) resulted in the greatest survival fractions among all forms of radioconjugates studied; this results was expected, since the free chelate does not associate with cancer cells.

[0206] In 3D spheroids that were utilized as surrogates of the tumors’ avascular regions, and contrary to the killing efficacy trends on monolayers, the greatest inhibition of spheroid regrowth was achieved when the same total activity was delivered by affinity compositions (purple and pink bars in FIG. 18), and not when the entire activity was delivered by the high-affinity antibody-radioconjugate alone (red bars).

[0207] 2.2.4. Killing efficacy in vivo

[0208] The “transport conjugate compositions” were evaluated in vivo (FIG. 19) on HEPG2 tumor-bearing NSG mice. Treatment was initiated when the subcutaneous xenografts reached 100 mm3; the “transport conjugate composition” at 70:30 activity split ratio between the two antibody-radioconjugates demonstrated longer mean survival (even though, not statistically significant) from the equally split activity ratio (22 vs 20 days, p- value = 0.2510), and was more effective in prolonging survival compared to the survival of animals that were injected the same total activity delivered by the “high affinity” antibody alone (red line, 18 days mean survival, -valucs< 0.02).

[0209] REFERENCES

[0210] All publications, patent applications, patents, and other references mentioned in the specification are indicative of the level of those skilled in the art to which the presently disclosed subject matter pertains. All publications, patent applications, patents, and other references are herein incorporated by reference to the same extent as if each individual publication, patent application, patent, and other reference was specifically and individually indicated to be incorporated by reference. It will be understood that, although a number of patent applications, patents, and other references are referred to herein, such reference does not constitute an admission that any of these documents form part of the common general knowledge in the art.

[0211] Jang A, Kendi AT, Johnson GB, Halfdanarson TR, Sartor O. Targeted AlphaParticle Therapy: A Review of Current Trials. Int J Mol Sci. (24), 2023.

[0212] Karlsson J. Abstract ND07: HER2-TTC: A Targeted thorium conjugate to treat HER2 expressing cancers with potent alpha radiation. Cancer Research. (81) ND07-ND, 2021.

[0213] Navarro-Teulon I, Lozza C, Pelegrin A, Vives E, Pouget J-P. General overview of radioimmunotherapy of solid tumors. Immunotherapy. (5) 467-87, 2013.

[0214] Kratochwil C, Bruchertseifer F, Rathke H, Bronzel M, Apostolidis C, Weichert W, Haberkom U, Giesel FL, Morgenstern A. Targeted a-Therapy of Metastatic Castration- Resistant Prostate Cancer with225Ac-PSMA-617: Dosimetry Estimate and Empiric Dose Finding. Journal of Nuclear Medicine. (58) 1624-31, 2017.

[0215] Sathekge M, Bruchertseifer F, Knoesen O, Reyneke F, Lawai I, Lengana T, Davis C, Mahapane J, Corbett C, Vorster M, Morgenstern A. (225)Ac-PSMA-617 in chemotherapy-I patients with advanced prostate cancer: a pilot study. European journal of nuclear medicine and molecular imaging. (46) 129-38, 2019.

[0216] Zhu C, Sempkowski M, Holleran T, Linz T, Bertalan T, Josefsson A, Bruchertseifer F, Morgenstern A, Sofou S. Alpha-particle radiotherapy: For large solid tumors diffusion trumps targeting. Biomaterials. (130) 67— <75, 2017.

[0217] Howe A, Bhatavdekar O, Salerno D, Josefsson A, Pacheco-Torres J, Bhujwalla ZM, Gabrielson KL, Sgouros G, Sofou S. Combination of carriers, with complementary intratumoral microdistributions of delivered a-particles, may realize the promise for Actinium-225 in large solid tumors. Journal of Nuclear Medicine. (63) 1223— >30, 2022.

[0218] Milenic DE, Brady ED, Garmestani K, Albert PS, Abdulla A, Brechbiel MW. Improved efficacy of a-particle-targeted radiation therapy. Cancer. (116) 1059-66, 2010.

[0219] Blumenthal RD, Kashi R, Stephens R, Sharkey RM, Goldenberg DM. Improved radioimmunotherapy of colorectal cancer xenografts using antibody mixtures against carcinoembryonic antigen and colon- specific antigen-p. Cancer Immunology, Immunotherapy. (32) 303-10, 1991.

[0220] Pasternack JB, Domogauer JD, Khullar A, Akudugu JM, Howell RW. The Advantage of Antibody Cocktails for Targeted Alpha Therapy Depends on Specific Activity. Journal of Nuclear Medicine. (55) 2012-9, 2014.

[0221] U.S. Patent Application Publication No. US20150030534 for Antibody Cocktails for Breast Cancer Radiommuno therapy, to Howell et al., published January 29, 2015.

[0222] Salerno D, Howe A, Bhatavdekar O, Josefsson A, Pacheco-Torres J, Bhujwalla ZM, Gabrielson KL, Sofou S. Two diverse carriers are better than one: A case study in a-particle therapy for prostate specific membrane antigen-expressing prostate cancers. Bioengineering & Translational Medicine. (7) el0266, 2022.

[0223] Prasad A, Nair R, Bhatavdekar O, Howe A, Salerno D, Sempkowski M, Josefsson A, Pacheco-Torres J, Bhujwalla ZM, Gabrielson KL, Sgouros G, Sofou S. Transport-driven engineering of liposomes for delivery of a-particle radiotherapy to solid tumors: effect on inhibition of tumor progression and onset delay of spontaneous metastases. Eur J Nucl Med Mol Imaging. (48) 4246-58, 2021. Kavousanakis M, Bhatavdekar O, Sofou S, Kevrekidis I. Transport and Reaction Modeling of Nanocarricrs for Cancer Therapeutics: Experimental and in silico approaches. IFAC-PapersOnLine. (55) 104-8, 2022.

[0224] McDevitt MR, Ma D, Simon J, Frank RK, Scheinberg DA. Design and synthesis of225Ac radioimmunopharmaceuticals. Appl Radiat Isot. (57) 841-7, 2002.

[0225] Franken NA, Rodermond HM, Stap J, Haveman J, van Bree C. Clonogenic assay of cells in vitro. Nat Protoc. (1) 2315-9, 2006.

[0226] Kavousanakis M, Bhatavdekar O, Bastiaannet R, Kevrekidis Y, Sofou S. Transport Cocktails for Cancer Therapeutics. Phys Rev Research (6) 033137, 2024. Howe A, Bhatavdekar O, Salerno D, et al. Combination of Carriers with

[0227] Complementary Intratumoral Microdistributions of Delivered ot-Particles May Realize the Promise for225Ac in Large, Solid Tumors. J. Nucl. Med. (63) 1223-1230, 2022.

[0228] Although the foregoing subject matter has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be understood by those skilled in the art that certain changes and modifications can be practiced within the scope of the appended claims.

Claims

THAT WHICH IS CLAIMED:

1. A composition comprising a first radionuclide- antibody conjugate having a first affinity for a target and a second radionuclide- antibody conjugate having a second affinity for the same target, wherein the first affinity for the target is higher than the second affinity for the target.

2. The composition of claim 1, wherein the composition comprises a total radioactivity having a ratio ranging about 10:90 to about 90:10 between the first radionuclide- antibody conjugate and the second radionuclide- antibody conjugate.

3. The composition of claim 1 or claim 2, wherein the composition comprises a total radioactivity that is approximately equally divided between the first radionuclideantibody conjugate and the second radionuclide- antibody conjugate.

4. The composition of any one of claims 1 to claim 3, wherein the radionuclide of the first radionuclide-antibody conjugate and the second radionuclide- antibody conjugate comprises an alpha-particle emitter.

5. The composition of claim 4, wherein the alpha-particle emitter for the first radionuclide- antibody conjugate and the second radionuclide- antibody conjugate can be the same or different and is selected from actinium- 225, astatine-211, lead-212, terbium- 149, thorium-227, radium-223, radium-224, bismuth-212, and bismuth-213.

6. The composition of claim 5, wherein the alpha-particle emitter is actinium- 225 (225Ac).

7. The composition of any one of claims 1 to 6, wherein the first radionuclideantibody conjugate and the second radionuclide- antibody conjugate each independently comprise an antibody selected from the group consisting of trastuzumab, cetuximab, panitumumab, rituximab, and bevacizumab, wherein the antibody of the first radionuclide-antibody conjugate and the antibody of the second radionuclide-antibody conjugate can be the same or different.

8. The composition of claim 7, wherein the antibody of the first radionuclideantibody conjugate is selected from trastuzumab and cetuximab.

9. The composition of any one of claims 1 to 8, wherein the antibody of the second radionuclide-antibody conjugate comprises a non-targeting antibody.

10. The composition of claim 9, wherein the non-targeting antibody comprises rituximab.

11. The composition of claim 9, wherein the non-targeting antibody comprises an inactive form of the antibody comprising the first radionuclide-antibody conjugate.

12. The composition of claim 11, wherein the inactive form of the antibody comprising the non-targeting antibody comprises a capped antibody having an epitope- peptide sequence bound at one or more binding sites and / or covalently modified at one or more binding sites.

13. The composition of claim 1, wherein the second radionuclide- antibody conjugate comprises the same antibody as the first radionuclide- antibody conjugate, wherein the antibody of the second radionuclide- antibody conjugate is modified such that its affinity for the target is decreased relative to the affinity for the target of the antibody of the first radionuclide- antibody conjugate.

14. The composition of claim 13, wherein one or more binding sites of the antibody of the second radionuclide- antibody conjugate are sterically hindered.

15. The composition of claim 14, wherein the one or more binding sites of the antibody of the second radionuclide- antibody conjugate are sterically hindered byconjugation of one or more sterically-hindering moieties to one or more a-amino groups of N-tcrminal amino acids on the binding sites of the antibody via click chemistry.

16. The composition of claim 15, wherein the antibody comprises one or more sterically-hindering moieties bound to one or more a-amino groups of N-terminal amino acids on the binding sites of the antibody.

17. The composition of claim 16, wherein the one or more sterically-hindering moieties are selected from a dye, a chelating moiety, and a click-reactive linker.

18. The composition of claim 17, wherein:(a) the dye is selected from a coumarin dye, a rhodamine dye, a cyanine dye, a xanthene dye, a pyrylium dye, and sulfonated, azetidine derivatives thereof;(b) the chelating moiety comprises a chelator moiety of any one of claims 23 to 25; and(c) the click-reactive linker comprises a linker of claim 21.

19. The composition of any one of claims 1 to 18, wherein an immunoreactivity of the antibody of the second radionuclide- antibody conjugate is less than an immunoreactivity of the antibody of the first radionuclide-antibody conjugate.

20. The composition of claim 19, wherein the immunoreactivity of the antibody of the second radionuclide-antibody conjugate is less than the immunoreactivity of the antibody of the first radionuclide-antibody conjugate by a range of about 1% less to about 99% less.

21. The composition of any one of claims 1 to 20, comprising more than one first radionuclide- antibody conjugate and / or more than one second radionuclide-antibody conjugate.

22. The composition of any one of claims 1 to 21, wherein the first radionuclide-antibody conjugates and the second radionuclide-antibody conjugates each independently further comprise a linker.

23. The composition of claim 22, wherein the linker is selected from the group consisting of isothiocyanato (SCN), isothiocyanato-benzyl (SCN-Bn), N-succinimidyl 4- (2pyridyldithiojpentanoate (SPP), N-succinimidyl 4-(2-pyridyldithio)-2-sulfopentanoate (sulfoSPP), N-succinimidyl 4-(2-pyridyldithio)butanoate (SPDB), N-succinimidyl 4-(2- pyridyldithio)2-sulfobutanoate (sulfo-SPDB), N-succinimidyl 4-(maleimidomethyl) cyclohexanecarboxylate (SMCC), N-sulfosuccinimidyl 4-(maleimidomethyl) cyclohexanecarboxylate (sulfoSMCC), N-succinimidyl-4-(iodoacetyl)-aminobenzoate (SIAB), and N-succinimidyl-[(N-maleimidopropionamido-tetraethyleneglycol] ester (NHS- PEG4-maleimide).

24. The composition of any one of claims 1 to 23, wherein the one or more targeting radionuclide-antibody conjugates and one or more non-targeting radionuclideantibody conjugates each independently further comprise a chelating moiety.

25. The composition of claim 24, wherein the chelating moiety is selected from the group consisting of DOTAGA (1,4,7,10-tetraazacyclododececane, l-(glutaric acid)- 4,7,10-triacetic acid), DOTA (l,4,7,10-tetraazacyclododecane-l,4,7,10-tetraacetic acid), DOTASA (1,4,7, 10-tetraazacyclododecane-l -(2- succinic acid)-4,7,10-triacetic acid), CB- D02A (10-bis(carboxymethyl)-l,4,7,10-tetraazabicyclo[5.5.2]tetradecane), DEPA (7-[2- (Bis-carboxymethylamino)-ethyl] -4, 10-bis-carboxymethyl- 1 ,4,7 , 10-tetraaza-cyclododec- 1 - yl-acetic acid)), 3p-C-DEPA (2-[(carboxymethyl)][5-(4-nitrophenyl-l-[4,7,10- tris(carboxymethyl)-l,4,7,10-tetraazacyclododecan-l-yl]pentan-2-yl)amino]acetic acid)), TCMC (2-(4-isothiocyanotobenzyl)- 1 ,4,7, 10-tetraaza- 1 ,4,7, 10-tetra-(2-carbamonyl methyl)- cyclododecane), oxo-DO3A (l-oxa-4,7, 10-triazacyclododecane-5-S-(4- isothiocyanatobenzyl)-4,7,10-triacetic acid), p-NH2-Bn-Oxo-DO3A (l-Oxa-4,7,10- tetraazacyclododecane-5-S-(4-aminobenzyl)-4,7,10-triacetic acid), TE2A ((l,8-N,N'-bis- (carboxymethyl)-l,4,8,l l-tetraazacyclotetradecane), MM-TE2A, DM-TE2A, CB-TE2A (4,l l-bis(carboxymethyl)-l,4,8,l l-tetraazabicyclo[6.6.2]hexadecane), CB-TE1A1P (4,8,11-tetraazacyclotetradecane-l -(methanephosphonic acid)-8-(methanecarboxylic acid)), CB- TE2P (l,4,8,ll-tctraazacyclotctradccanc-l,8-bis(mcthancphosphonic acid), TETA (l,4,8,l l-tetraazacyclotetradecane-l,4,8,l l-tetraacetic acid), NOTA (1,4,7- triazacyclononane-N,N',N"-triacetic acid), NODA (l,4,7-triazacyclononane-l,4-diacetate); NODAGA (l,4,7-triazacyclononane,l-glutaric acid-4,7 -acetic acid); NOTAGA (1,4,7- triazonane-l,4-diyl)diacetic acid); DFO (Desferoxamine), NETA ([4-[2-(bis- carboxymethylamino)-ethyl]-7-carboxymethl-[l,4,7]triazonan-l-yl}-acetic acid), TACN- TM’(N”N’,N", tris(2-mercaptoethyl)-l,4,7-triazacyclononane), Diamsar (1,8-Diamino- 3,6,10,13,16,19-hexaazabicyclo(6,6,6)eicosane, 3,6,10,13,16,19- Hexaazabicyclo[6.6.6]eicosane-l,8-diamine), Sarar (l-N-(4-aminobenzyl)-3, 6,10,13,16,19- hexaazabicyclo[6.6.6] eicosane- 1,8-diamine), AmBaSar (4-((8-amino-3,6,10,13,16,19- hexaazabicyclo [6.6.6] icosane-l-ylamino) methyl) benzoic acid), macropa, and BaBaSar.

26. The composition of claim 24, wherein the chelating moiety is selected from the group consisting of:

27. The composition of claim 24, wherein the chelating moiety is selected from dodecane tetraacetic acid (DOTA) and diethylenetriaminepentaacetic acid (DTPA).

28. A method for treating a solid tumor in a subject in need of treatment thereof, the method comprising administering a therapeutically effective amount of a composition of any one of claims 1 to 27 to the subject.

29. The method of claim 28, wherein the solid tumor comprises a cancer selected from breast cancer, pancreatic cancer, liver cancer, kidney cancer, prostate cancer, lung cancer, colorectal cancer, ovarian cancer, brain cancer, skin cancer, and combinations thereof.

30. The method of claim 28 or claim 29, wherein the first radionuclide- antibody conjugate and the second radionuclide- antibody conjugate are administered at the same time.

31. The method of claim 28 or 29, wherein the first radionuclide-antibody conjugate and the second radionuclide- antibody conjugate are administered separately with a time interval between administration of each conjugate.

32. The method of claim 31, wherein the first radionuclide-antibody conjugate and the second radionuclide-antibody conjugates are administered between about 12 hours and about 18 hours apart.

33. The method of any one of claims 28 to 32, wherein the first radionuclideantibody conjugate is administered first.

34. The method of any one of claims 28 to 33, wherein a total dose of the first radionuclide- antibody conjugate and / or the second radionuclide- antibody conjugate is administered in a series of fractional doses at predetermined time intervals.

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