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WO2025151464A1 - Conjugués de radionucléides-anticorps à particules alpha pour le traitement de tumeurs solides - Google Patents

Conjugués de radionucléides-anticorps à particules alpha pour le traitement de tumeurs solides

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Publication number
WO2025151464A1
WO2025151464A1 PCT/US2025/010658 US2025010658W WO2025151464A1 WO 2025151464 A1 WO2025151464 A1 WO 2025151464A1 US 2025010658 W US2025010658 W US 2025010658W WO 2025151464 A1 WO2025151464 A1 WO 2025151464A1
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antibody
radionuclide
composition
conjugate
cancer
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Stavroula Sofou
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Johns Hopkins University
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Johns Hopkins University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
    • A61K51/1045—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody against animal or human tumor cells or tumor cell determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
    • A61K51/1045—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody against animal or human tumor cells or tumor cell determinants
    • A61K51/1051—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody against animal or human tumor cells or tumor cell determinants the tumor cell being from breast, e.g. the antibody being herceptin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2887—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD20
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A61K2039/507—Comprising a combination of two or more separate antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

  • 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.
  • 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.
  • 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.
  • the composition comprises more than one first radionuclideantibody conjugate and/or more than one second radionuclide-antibody conjugate.
  • 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.
  • the composition comprises a total radioactivity that is approximately equally divided between the first radionuclide-antibody conjugate and the second radionuclide-antibody conjugate.
  • the radionuclide of the first radionuclide- antibody conjugate and the second radionuclide- antibody conjugate comprises an alpha-particle emitter.
  • 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.
  • the alpha-particle emitter is actinium-225 (225Ac).
  • 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.
  • the antibody of the first radionuclide- antibody conjugate is selected from trastuzumab and cetuximab.
  • an immunoreactivity of the antibody of the second radionuclideantibody conjugate is less than an immunoreactivity of the antibody of the first radionuclideantibody conjugate.
  • 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.
  • 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 (sulfoSM).
  • 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)
  • 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.
  • 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.
  • the first radionuclide- antibody conjugate is administered first.
  • 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-177rate);
  • FIG. IB shows the range of alpha- and beta-particles in tissue (in red and blue, respectively) relative to cell size (in gray);
  • 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);
  • 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).
  • FIG. 5B shows animal weights over time during the treatment study on the HEPG-2 subcutaneous mouse models shown on FIG. 5A;
  • FIG. 5C shows characteristic H&E-stained sections of normal organs and of HEPG-
  • FIG. 6A shows the effect of alpha-particle therapy delivered by antibodies to BxPC-
  • FIG. 6B shows animal weights over time during the treatment study on the BxPC-3 subcutaneous mouse models shown on FIG. 6A;
  • 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).
  • HER2-targeting 111 In-DTPA-SCN-antibody Trastuzumab
  • non-targeting l n In-DTPA-SCN- antibody Rituximab
  • 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).
  • HER2-targeting 111 In-DTPA-SCN-antibody Trastuzumab
  • Additional therapies that can be used in combination with the presently disclosed compositions include surgery, radiation, including proton therapy, and chemotherapy.
  • 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.
  • 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.
  • 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.
  • 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
  • hormone therapies e.g., hormone therapies.
  • 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, plic
  • 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.
  • 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.
  • 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.
  • the active agents or therapies are combined and administered in a single dosage form.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • agents administered sequentially 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.
  • 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.
  • 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.
  • the two or more agents when administered in combination, can have a synergistic effect.
  • 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.
  • 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;
  • SI Synergy Index
  • QA is the concentration of a component A, acting alone, which produced an end point in relation to component A;
  • Q a is the concentration of component A, in a mixture, which produced an end point
  • 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.
  • kit components may be provided as dry powders (typically lyophilized), including excipients which on dissolution will provide a reagent solution having the appropriate concentration.
  • dry powders typically lyophilized
  • excipients which on dissolution will provide a reagent solution having the appropriate concentration.
  • 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.
  • Radionuclide- antibody conjugates are among the leading approaches in targeted alpha-particle therapies for the treatment of vascularized, soft-tissue, solid tumors.
  • 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.
  • 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.
  • compositions of radionuclide- antibody conjugates are different from compositions of radionuclide-antibody conjugates that have been reported in the previous art.
  • each of the antibodies are chosen to bind to a receptor expressed on the surface of cancer cells comprising the solid tumor.
  • 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.
  • compositions of radionuclide-antibody conjugates are 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.
  • 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).
  • HER2 Human Epidermal growth factor Receptor-2
  • HER1 Human Epidermal growth factor Receptor-1
  • 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.
  • 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.
  • 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.
  • 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 mm 3 , assuming spherical tumor shape.
  • Table 6 Tumor and normal organ absorbed doses (Gy) from 2.96 kBq 225 Ac 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.
  • the colony survival fractions of cells exposed to Actinium-225 in different forms are shown in FIG. 10.
  • the 225 Ac-DOTA form and the 225 Ac-DOTA-SCN- Rituximab conjugate did not associate with cells and resulted in similar and less effective colony kill compared to the targeting 225 Ac-labeled antibody conjugates ( 225 Ac-DOTA-SCN-Trastuzumab for BT-474 and HEPG- 2, and 225 Ac-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
  • 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).
  • PBS Phosphate Buffered Saline
  • PBS trisodium citrate dihydrate
  • anhydrous citric acid poly(2-hydroxyethyl methacrylate)
  • polyHEMA poly(2-hydroxyethyl methacrylate)
  • Trizma® 2-Amino-2-(hydroxymethyl)-l,3- propanediol
  • Trypsin and MatrigelTM 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).
  • Indium-Il l 111 In
  • indium chloride was purchased from BWXT (Ontario, Canada).
  • Actinium-225 ( 225 Ac) 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
  • the cell lines BT-474 and HEPG-2 were cultured using HybricarcTM 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.
  • 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 for 225 Ac-radiolabeling, or with IM acetate buffer at pH 4.5 for i n In-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.
  • 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.
  • 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.
  • 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 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.
  • 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.
  • 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.
  • mice Female and male NSG 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.
  • 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 MatrigelTM.
  • 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 MatrigelTM.
  • the tumors were allowed to grow to a volume of approximately 100 mm 3 following which the animals were randomly assigned to a treatment/control group.
  • 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 of 225 Ac-DOT A-SCN- labeled specific Antibody, 2.96 kBq of 225 Ac-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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • FITC-SCN fluorescein isothiocyanate
  • 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.
  • 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.
  • 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).
  • 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.

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Abstract

L'invention concerne des compositions comprenant un ou plusieurs conjugués de radionucléides-anticorps de ciblage (ou d'affinité élevée) et un ou plusieurs conjugués de radionucléides-anticorps de non-ciblage (ou de faible affinité) et leur utilisation dans le traitement de tumeurs solides.
PCT/US2025/010658 2024-01-08 2025-01-08 Conjugués de radionucléides-anticorps à particules alpha pour le traitement de tumeurs solides Pending WO2025151464A1 (fr)

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WO2022251541A1 (fr) * 2021-05-26 2022-12-01 Cornell University Macrocycles et complexes avec des radionucléides utiles dans la radiothérapie ciblée du cancer
US20230364276A1 (en) * 2014-05-16 2023-11-16 Sloan-Kettering Institute For Cancer Research One-Step Labeling of Antibodies to High Specific Activity with Actinium-225

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US20220185803A1 (en) * 2018-11-20 2022-06-16 Cornell University Macrocyclic complexes of alpha-emitting radionuclides and their use in targeted radiotherapy of cancer
WO2022104697A1 (fr) * 2020-11-20 2022-05-27 Bliss Biopharmaceutical (Hangzhou) Co., Ltd. Anticorps egfr modifié à affinité réduite
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KONDO MISAKI, CAI ZHONGLI, CHAN CONRAD, FORKAN NUBAIRA, REILLY RAYMOND M.: "[225Ac]Ac- and [111In]In-DOTA-trastuzumab theranostic pair: cellular dosimetry and cytotoxicity in vitro and tumour and normal tissue uptake in vivo in NRG mice with HER2-positive human breast cancer xenografts", EJNMMI RADIOPHARMACY AND CHEMISTRY, SPRINGER INTERNATIONAL PUBLISHING, vol. 8, no. 1, pages 24 - 24-22, XP093337284, ISSN: 2365-421X, DOI: 10.1186/s41181-023-00208-0 *
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