Localized in VIVO prodrug activation using radioisotopes

Abstract

The invention provides targeted caged prodrugs and targeted radioemitters, pharmaceutical compositions thereof, and methods of use thereof for the purpose of killing cells.

Description

[0001] LOCALIZED IN VIVO PRODRUG ACTIVATION USING RADIOISOTOPES

[0002] BACKGROUND OF THE INVENTION

[0003] Radiopharmaceutical therapy (RPT) is becoming a critical pillar in cancer treatment, exemplified by FDA approvals of 177Lu-PSMA-617 and 177Lu-DOTATATE for managing advanced-stage prostate and neuroendocrine tumors. Encouraging Phase III trials (PSMAfore, NCT04689828, NETTER-2, NCT03972488) are poised to support extending RPT indications to treatment settings earlier in the disease course. However, current RPT agents cannot mediate complete disease control in all patients. As with other classes of therapy, RPT must be combined with other treatments for effective management of advanced disease.

[0004] Systemic chemo- and immunotherapies are being actively tested with RPT (e.g. NCT03805594, NCT02358356, NCT05247905). As standalone therapies, these modalities form the mainstay of treatment in patients with disseminated disease. While chemo- and immunotherapies are often effective for tumor control, dose-limiting toxicities to off-target sites in the body constrain therapeutic indices. In some cases, high-grade toxicities exceed 50% and are exacerbated when multiple drug modalities are used in combination. This results in dose reduction or treatment cessation, reducing treatment efficacy and potentially contributing to treatment resistance. Therefore, the ability to selectively deliver and activate potent drugs in tumors while minimizing their impact elsewhere in the body would be highly desirable.

[0005] Numerous drug delivery strategies have been developed to expand the range over which therapeutic cargoes can safely elicit effective responses. Examples include antibody-drug conjugates (ADCs) and nanomedicines designed to improve pharmacokinetics and payload delivery to tumors, prodrugs that release activated drugs in the presence of enriched enzymes or hypoxia, and strategies using externally delivered triggers like ultrasound to control drug activity. While such approaches can increase tumor target specificity and, in some cases, efficacy, unintended activity at off-target sites remains a challenge. Inevitable accumulation in clearance tissues, heterogeneity of endogenous physicochemical properties within the tumor microenvironment, and premature drug activation due to chemical instability represent challenges for existing drug delivery approaches. As a result, there is a need for better methods and compositions to deliver drugs to targeted cells and tissues while minimizing off-target delivery and toxicity.

[0006] SUMMARY OF THE INVENTION

[0007] In one aspect, the invention provides a method of killing a cell in a subject, the method including, administering to the subject a targeted caged prodrug, and administering to the subject a targeted radioemitter comprising64Cu,67Cu,67Ga,68Ga,99mTc,103Pd,1111 n,123l,125l,131l,131Cs,133Xe,153Sm,149Pm,161Tb,169Er,177Lu,201TI, or203Pb that produces ionizing radiation, wherein the ionizing radiation releases drug from the caged prodrug, and each of the targeted caged prodrug and the targeted radioemitter is configured to target the same cell type or tissue. In some embodiments, the targeted radioemitter administered to the subject includes67Ga,99mTc,1111 n, or123L

[0008] In another aspect, the invention provides a method of killing a cell in a subject, the method including, administering to the subject a targeted caged prodrug conjugated to a protein, and following from 8 hours to 48 hours after, administering to the subject a targeted radioemitter that produces ionizing radiation, wherein the ionizing radiation releases drug from the caged prodrug, and each of the targeted caged prodrug and the targeted radioemitter is configured to target the same cell type or tissue. In some embodiments, the targeted radioemitter administered to the subject, includes64Cu,67Cu,67Ga,68Ga,99mTc,103Pd,111In ,123l,125l,131l,131Cs,133Xe,153Sm,149Pm,161Tb,169Er,177Lu,201TI, or203Pb, or any other described herein.

[0009] In another aspect, the invention provides a method of killing a cell in a subject, the method including, administering to the subject a targeted radioemitter including a radiolabeled peptide pharmaceutical, radiolabeled small molecule, or a radiolabeled protein that produces ionizing radiation, and following from 4 hours to 48 hours, administering to the subject a targeted caged prodrug, wherein the ionizing radiation releases drug from the caged prodrug, and each of the targeted caged prodrug and the targeted radioemitter is configured to target the same cell type or tissue. In some embodiments, the targeted radioemitter administered to the subject includes64Cu,67Cu,67Ga,68Ga,99mTc,103Pd,111ln,123l,125l,131l,131Cs,133Xe,153Sm,149Pm,161Tb,169Er,177Lu,201TI, or203Pb, or any other described herein.

[0010] In some embodiments, the targeted radioemitter of the above-mentioned method of killing a cell in a subject includes a radiolabeled small molecule, a radiolabeled peptide pharmaceutical, or a radiolabeled protein. In some embodiments, the radiolabeled small molecules include methylene diphosphonate, phytate, meta-iodobenzylguanidine. In some embodiments, the radiolabeled peptide pharmaceuticals include one of PSMA-11 , PSMA-617, PSMA-1007, DOTA-TATE, DOTA-TOC, FAPI-04, FAPI-34, FAPI- mFS, H6F, NNS309, NeoBOMBI , RM2, BAY 86-7548, AMBA, NOTA-PRGD2, pentixafor, CCZ01048, CP04, PP-F11 N, DOTA-MG11 , and DOTA-MSH. In some embodiments, the radiolabeled protein includes one ofABY-025, MIRC213, VHH1 , 2Rs15d, ZHER2:2891 affibody, ZEGFR:2377 affibody, R3B23, CEA61 , DARPin 9_29, Adnectin CT-322, AKY 1189, CD105-CD3 BiTE nanobody, PD-L1xCD3 BiTE nanobody, Anti-CD20 x CD3 BiTE nanobody, CD3-FAP BiTE nanobody, and HER2-scFvCD3 x HER2-EGFR BiTE nanobody.

[0011] In a related aspect, the targeted caged prodrug of the above-mentioned method of killing a cell in a subject includes a peptide, small molecule, a protein, an antibody, a nanoparticle, or a polymer (e.g., polyethylene glycol (PEG), polyglucose dextran, or poly-L-glutamic acid). In some embodiments, the targeted caged prodrug is a peptide conjugate includes a targeting moiety that is a radical of a radiolabeled peptide pharmaceutical selected from PSMA-11 , PSMA-617, PSMA-1007, DOTA-TATE, DOTA-TOC, FAPI-04, FAPI-34, FAPI-mFS, H6F, NNS309, NeoBOMBI , RM2, BAY 86-7548, AMBA, NOTA-PRGD2, pentixafor, CCZ01048, CP04, PP-F11 N, DOTA-MG11 , DOTA-MSH, and MIBG. In some embodiments, the protein is an albumin. In some embodiments, the antibody is any one of amivantamab, loncastuximab, camidanlumab, rovalpituzumab, vadastuximab, trastuzumab, gemtuzumab, brentuximab, ado-trastuzumab, inotuzumab, polatuzumab, enfortumab, fam-trastuzumab, scituzumab, tisotumab, mirvetuximab belantamab, moxetumomab, cetuximab, distamab, ibritumomab, metuximab, tositumomab, rosopatamab, girentuximab, 9MW2821 , ABBV-383, AK109, AMG 160, AMG 199, AMG 211 , AMG 596, AMG 910, anbenitamab, anvatabart opadotin, apamistamab, ASKB589, atezolizumab, avelumab, AZD0901 , AZD5863, ifinatamab, GSK5764227, YL201 , BCA-101 , BL-M07D1 , BNT323, DP303c, FDA018, HLX22, HPN424, IAH0968, IBIT343, JSKN003, IMC-F106C, M108, IA0968, MDX1110, MRGO02, RO6958688, SHR-A1904, SHR-A1921 , SHR-A2009, SYSA1801 , SYS6010, TF2, TNB-383B, TORL-1-23, TQB2102, becotatug, bevacizumab, belantamab, bemarituzumab, camidanlumab, catumaxomab, caxmotabart, cetuximab, cosibelimab, datopotamab, dinutuximab, distamab, durvalumab, edrecolomab, elranatamab, enfortumab, ficlatuzumab, gemtuzumab, girentuximab, ianalumab, ibritumomab, ifinatamab, inotuzumab, izalontamab, kintuximab, labetuzumab, ligufalimab, linvoseltamab, livmoniplimab, Im-302, loncastuximab, luveltamab, margetuximab, metuximab, mirvetuximab, monalizumab, moxetumomab, naxitamab, necitumumab, olaratumab, oleclumab, onfekafusp alfa, ozekibart, panitumumab, pasotuxizumab, patritumab, panitumumab, pertuzumab, petosemtamab, polatuzumab, raludotatug, ramucirumab, acituzumab, scituzumab, sigvotatug, sugemalimab, tarlatamab, tebentafusp, telisotuzumab, tifcemalimab, tisotumab, tositumomab, solitomab, trastuzumab, uliledlimab, vadastuximab, varlilumab, xaluritamig, zanidatamab, zenocutuzumab, zilovertamab, and zolbetuximab.

[0012] In a related aspect, the targeted caged prodrug of the above-mentioned method of killing a cell in a subject is one of a cytotoxic, cytostatic, or immunomodulatory agent. In some embodiments, the targeted caged prodrug includes any one of an antitubulin agents, a DNA replication inhibitors, an alkylating agents, an antifolates, an antimetabolites, a chemotherapy sensitizers, a topoisomerase inhibitors, and a vinca alkaloids. In some embodiments, the targeted caged prodrug includes any one of pyrrolobenzodiazepine dimer (PBD dimer), exatecan, monomethyl auristatin E (MMAE), doxorubicin, and gardiquimod.

[0013] In a related aspect, the caged product, or a pharmaceutically acceptable salt thereof, of the above- mentioned method of killing a cell in a subject has the structure of Formula I:

[0014] [RSM]-Linker-Drug Moiety

[0015] Formula I wherein RSM is a radiation-sensitive moiety. In certain embodiments, the linker (L) has the structure:

[0016] Formula VIII wherein a, b, c, e, f, and g are each, independently, 0 or 1 , d is 0, 1 , 2, or 3, each of R6, R8, R10, and R12, is, independently, optionally substituted Ci-Ce alkylene, optionally substituted Ci-Ce heteroalkylene, optionally substituted C2-C6 alkenylene, optionally substituted C2-Ce alkynylene, or optionally substituted C6-C10 arylene, O, S, Se, and NR13, R7and R11are each, independently, carbonyl, thiocarbonyl, sulfonyl, or phosphoryl, wherein, if R7is phosphoryl, -(R9)d- is a bond, and e, f, and g are 0, then at least one of R6or R8is not O, and if R11is phosphoryl, -(R9)d- is a bond, and a, b, and c are 0, then at least one of R10or R12is not O, each R9is optionally substituted C1-C10 alkylene, optionally substituted C2-C10 alkenylene, optionally substituted C2-C10 alkynylene, optionally substituted C2-C10 heterocyclylene, optionally substituted C6-C12 arylene, optionally substituted C2-C100 polyethylene glycolene, or optionally substituted C1-C10 heteroalkylene, or a bond linking (R6)a-(R7)b-(R8)c to (R10)e-(R11)f-(R12)g, wherein if -(R9)d- is a bond, then at least one of a, b, c, e, f, or g is 1 , and R13is hydrogen, optionally substituted C1-C4 alkyl, optionally substituted C2-C4 alkenyl, optionally substituted C2-C4 alkynyl, optionally substituted C2-C6 heterocyclyl, optionally substituted C6-C12 aryl, or optionally substituted C1-C7 heteroalkyl. In some embodiments, the linker of Formula I includes a carbamate group. In some embodiments, the caged prodrug has the structure of Formula l-A:

[0017] Formula l-A wherein, ring A is a 5-6 member heteroaryl or a phenyl, each Ri is independently halogen, azido, or C1 - C6 alkoxy, wherein not more than one of Ri is azido, m is 2, 3, 4, or 5 each R2 is independently halogen,

[0018] C1-C6 alkyl, or C1-C6 haloalky I, n is 0, 1 , 2, 3, or 4, R3 is -NHRaor -NHRb, Ra is -H or C1-C6 alkyl, Rb is

[0019] -X, or -(PEG)t-X, R4 is a small molecule, a peptide, a protein, or an antibody, X is an electrophilic group, p, q, r, s, t, u, and v is each independently an integer from 2-20, and D is a drug moiety. In some embodiments, ring A is phenyl. In some embodiments, m is 2 or 5. In some embodiments, m is 2 and each Ri is methoxy. In some embodiments, m is 5, one Ri is azido, and the remaining Ri are each fluoro. In some embodiments, n is 0. In some embodiments, R3 is -NHRb. In some embodiments, Rb is some embodiments, Rb is embodiments, some embodiments, Rb is some embodiments, some embodiments, Rb is

[0020] O . In some embodiments, Rb is O

[0021] In some embodiments, v is 4 and caged prodrug has a structure of Formula II:

[0022] Formula II.

[0023] In some embodiments, R3 is -OH. In some embodiments, R3 is -O(C1-C6 alkyl). In some embodiments, R3 is -NHRa. In some embodiments, Rais -H. In some embodiments, Rais C1-C6 alkyl.

[0024] In a related aspect, in the method of killing a cell in a subject, the targeted caged prodrug is a conjugate including a first targeting moiety, and the targeted radioemitter is a conjugate including a second targeting moiety, wherein the first targeting moiety and the second targeting moiety target the same cell type, tissue, or receptor. In some embodiments, the first targeting moiety and the second targeting moiety are identical. In some embodiments, the first targeting moiety and the second targeting moiety are selected from small molecules and peptides and nanobodies.

[0025] In a related aspect, the method of killing a cell in a subject further includes administering to the subject a first caged prodrug including a first cytotoxic agent and administering to the subject a second caged prodrug including a second cytotoxic agent, wherein the first cytotoxic agent and the second cytotoxic agent are different. In some embodiments, the first cytotoxic agent is MMAE and the second cytotoxic agent is exatecan. In some embodiments, the cell is a cancer cell.

[0026] In another aspect, the invention provides a composition comprising a caged prodrug of Formula III:

[0027] Formula III, or a pharmaceutically acceptable salt thereof, wherein R3 is a linker bound to a targeting moiety.

[0028] In another aspect, the invention provides a composition comprising a caged prodrug of Formula IV: or a pharmaceutically acceptable salt thereof, wherein R3 is a linker bound to a targeting moiety.

[0029] In some embodiments, the targeting moiety is a peptide, small molecule, a protein, an antibody, a nanoparticle, or a polymer. In some embodiments, the targeted moiety is a is a radical of a radiolabeled peptide pharmaceutical selected from PSMA-11 , PSMA-617, PSMA-1007, DOTA-TATE, DOTA-TOC, FAPI-04, FAPI-34, FAPI-mFS, H6F, NNS309, NeoBOMBI , RM2, BAY 86-7548, AMBA, NOTA-PRGD2, pentixafor, CCZ01048, CP04, PP-F11 N, DOTA-MG11 , DOTA-MSH, and MIBG. In some embodiments, the targeting moiety includes methylene diphosphonate, phytate, or meta-iodobenzylguanidine. In some embodiments the targeting moiety is a protein and the protein is an albumin. In some embodiments, the targeting moiety is an antibody and the antibody is any one of amivantamab, loncastuximab, camidanlumab, rovalpituzumab, vadastuximab, trastuzumab, gemtuzumab, brentuximab, ado- trastuzumab, inotuzumab, polatuzumab, enfortumab, fam-trastuzumab, scituzumab, tisotumab, mirvetuximab belantamab, moxetumomab, cetuximab, distamab, ibritumomab, metuximab, tositumomab, rosopatamab, girentuximab, 9MW2821 , ABBV-383, AK109, AMG 160, AMG 199, AMG 211 , AMG 596, AMG 910, anbenitamab, anvatabart opadotin, apamistamab, ASKB589, atezolizumab, avelumab, AZD0901 , AZD5863, ifinatamab, GSK5764227, YL201 , BCA-101 , BL-M07D1 , BNT323, DP303c, FDA018, HLX22, HPN424, IAH0968, IBIT343, JSKN003, IMC-F106C, M108, IA0968, MDX1110, MRGO02, RO6958688, SHR-A1904, SHR-A1921 , SHR-A2009, SYSA1801 , SYS6010, TF2, TNB-383B, TORL-1-23, TQB2102, becotatug, bevacizumab, belantamab, bemarituzumab, camidanlumab, catumaxomab, caxmotabart, cetuximab, cosibelimab, datopotamab, dinutuximab, distamab, , durvalumab, edrecolomab, elranatamab, enfortumab, ficlatuzumab, gemtuzumab, girentuximab, ianalumab, ibritumomab, ifinatamab, inotuzumab, izalontamab, kintuximab, labetuzumab, ligufalimab, linvoseltamab, livmoniplimab, Im-302, loncastuximab, luveltamab, margetuximab, metuximab, mirvetuximab, monalizumab, moxetumomab, naxitamab, necitumumab, olaratumab, oleclumab, onfekafusp alfa, ozekibart, panitumumab, pasotuxizumab, patritumab, panitumumab, pertuzumab, petosemtamab, polatuzumab, raludotatug, ramucirumab, 7acituzumab, scituzumab, sigvotatug, sugemalimab, tarlatamab, tebentafusp, telisotuzumab, tifcemalimab, tisotumab, tositumomab, trastuzumab, uliledlimab, vadastuximab, varlilumab, xaluritamig, zanidatamab, zenocutuzumab, zilovertamab, and zolbetuximab.

[0030] In another aspect, the invention includes a method of killing a cell in a subject, the method including, contacting the cell with a radioemitter including67Ga,68Ga,99mTc,103Pd,1111 n ,123l,125l,131l,131Cs,133Xe,153Sm,149Pm,161Tb,169Er,177Lu,201TI,203Pb,211At,212Pb, or203Ac to produce ionizing radiation within the cell, and contacting the cell with cytotoxic drug, wherein the cytotoxic drug is generated in situ by exposing a caged prodrug to the ionizing radiation to produce the cytotoxic drug. In some embodiments, the radioemitter includes67Ga,99mTc,111In , or123L

[0031] In another aspect, the invention includes a method of killing a cell in a subject, the method includes, contacting the cell with a caged prodrug of a cytotoxic drug, following from 4 hours to 48 hours after, contacting the cell with a radioemitter to produce ionizing radiation within the cell, and generating the cytotoxic drug in situ by exposing the caged prodrug to the ionizing radiation to produce the cytotoxic drug. In some embodiments, the radioemitter comprises "mTc,111In,177Lu, or225Ac, or any other radioemitter described herein.

[0032] In another aspect, the invention includes a method of killing a cell in a subject, the method including, contacting the cell with a radioemitter to produce ionizing radiation within the cell, following from 4 hours to 48 hours after, contacting the cell with a caged prodrug of a cytotoxic drug, and generating the cytotoxic drug in situ by exposing the caged prodrug to the ionizing radiation to produce the cytotoxic drug. In some embodiments, the radioemitter includes64Cu,68Ga,90Y,99mTc,111In,123l,131l,177Lu,211At,212Pb,223Ra, or225Ac, or any other radioemitter described herein.

[0033] In a related aspect, the cytotoxic drug of the method of killing a cell in a subject is selected from antitubulin agents, DNA replication inhibitors, alkylating agents, antifolates, antimetabolites, chemotherapy sensitizers, topoisomerase inhibitors, and vinca alkaloids. In some embodiments, the cytotoxic drug is selected from pyrrolobenzodiazepine dimer (PBD dimer), exatecan, monomethyl auristatin E (MMAE), doxorubicin, and gardiquimod. In some embodiments, the caged prodrug has the structure of Formula l-B:

[0034] Formula l-B

[0035] Wherein, ring A is a phenyl, m is 5, one Ri is azido, and each remaining Ri is fluoro, each R2 is independently halogen, C1 -C6 alkyl, or C1 -C6 h aloalky I , n is 0, 1 , 2, 3, or 4, R3 is -NRaRb or a targeting moiety, Rais -H or C1-C6 alkyl, Rb is -H or C1-C6 alkyl, and D is a drug moiety comprising a radical of the cytotoxic drug.

[0036] DEFINITIONS

[0037] By “about” or “approximately” is meant ±10% of a recited value.

[0038] By “alkenyl” is meant an unsaturated straight or branched hydrocarbon having at least one carbon-carbon double bond, such as a straight or branched group of 2-12, 2-10, or 2-6 carbon atoms, referred to herein as C2-Ci2-alkenyl, C2-Cio-alkenyl, and C2-Ce-alkenyl, respectively.

[0039] The term “alkenylene” as used herein, represents a divalent alkenyl group derived by the removal of two hydrogen atoms, and is exemplified by ethenylene, and isopropenylene. The term “Cx-y alkenylene” represent alkenylene groups having between x and y carbons. Exemplary values for x are 2, 3, 4, 5, and 6, and exemplary values for y are 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, or 20 (e.g., C2-6, C2-10, or C2-20 alkenylene). In some embodiments, the alkenylene can be further substituted with 1 , 2, 3, or 4 substituent groups as defined herein. The term “branched alkenylene” as used herein, refers to a multivalent alkenyl group derived by the removal of more than two hydrogen atoms.

[0040] By “alkyl” is meant a straight-chain or branched alkyl radical in all of its isomeric forms, such as a straight or branched group of 1 -12, 1 -10, or 1 -6 carbon atoms, referred to herein as C1-C12 alkyl, C 1 -C 1 o-al ky I , and Ci-Ce-alkyl, respectively. Representative alkoxyl groups include methyl, ethyl, tert-butyl and the like.

[0041] The term “alkylene,” as used herein, represents a saturated divalent hydrocarbon group derived from a straight or branched chain saturated hydrocarbon by the removal of two hydrogen atoms, and is exemplified by methylene, ethylene, isopropylene, and the like. The term “Cx-Cyalkylene” represents alkylene groups having between x and y carbons. Exemplary values for x are 1 , 2, 3, 4, 5, and 6, and exemplary values for y are 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, or 20 (e.g., Ci-Ce, C1-C10, C2-C20, C2-C6, C2-C10, or C2-C20 alkylene). In some embodiments, the alkylene can be further substituted with 1 , 2, 3, or 4 substituent groups as defined herein. By “alkynyl” is meant an unsaturated straight or branched hydrocarbon having at least one carbon-carbon triple bond, such as a straight or branched group of 2-12, 2-10, or 2-6 carbon atoms, referred to herein as C2-Ci2-alkynyl, C2-Cio-alkynyl, and C2-Ce-alkynyl, respectively.

[0042] The term “alkynylene” as used herein, represents a divalent alkynyl group derived by the removal of two hydrogen atoms. The term “Cx-y alkynylene” represent alkynylene groups having between x and y carbons. Exemplary values for x are 2, 3, 4, 5, and 6, and exemplary values for y are 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, or 20 (e.g., C2-6, C2-10, or C2-20 alkynylene). In some embodiments, the alkynylene can be further substituted with 1 , 2, 3, or 4 substituent groups as defined herein. The term “branched alkynylene” as used herein, refers to a multivalent alkynyl group derived by the removal of more than two hydrogen atoms.

[0043] By “alkoxyl” or “alkoxy” is meant an alkyl group, as defined above, having an oxygen radical attached thereto. Representative alkoxyl groups include methoxy, ethoxy, tert-butoxy and the like.

[0044] By “amide” or “amido” is meant a radical of the form — R1C(O)N(R2) — , — R1C(O)N(R2)R3— , — C(O)NR2R3, or — C(O)NH2, wherein R1, R2and R3are each independently alkoxy, alkyl, alkenyl, alkynyl, amide, amino, aryl, arylalkyl, carbamate, cycloalkyl, ester, ether, formyl, halogen, haloalkyl, heteroaryl, heterocyclyl, hydrogen, hydroxyl, ketone, or nitro.

[0045] By “amine” and “amino” is meant both unsubstituted and substituted amines, wherein substituents may include, for example, alkyl, cycloalkyl, heterocyclyl, alkenyl, and aryl.

[0046] By “aryl” is meant a carbocyclic aromatic group. Representative aryl groups include phenyl, naphthyl, anthracenyl, and the like. By “aryl” includes polycyclic ring systems having two or more carbocyclic rings in which two or more carbons are common to two adjoining rings (the rings are “fused rings”) wherein at least one of the rings is aromatic and, e.g., the other ring(s) may be cycloalkyls, cycloalkenyls, cycloalkynyls, and / or aryls. Unless specified otherwise, the aromatic ring may be substituted at one or more ring positions with, for example, , alkoxy, alkyl, haloalkyl, alkenyl, alkynyl, amido, amino, aryl, carboxy, cycloalkyl, ester, halogen, haloalkyl, heteroaryl, heterocyclyl, hydroxyl, sulfonamido, sulfonyl or the like. In certain other embodiments, the aromatic ring is not substituted, e.g., it is unsubstituted. In certain embodiments, the aryl group is a 6-10 membered ring structure.

[0047] The term “arylene” as used herein, represents a divalent aryl group derived by the removal of two hydrogen atoms. The term “Cx-y arylene” represent arylene groups having between x and y carbons. Exemplary values for x are 6 and 10, and exemplary values for y are 10, 12, 14, 16, 18, or 20 (e.g., C6-10 or C6-20 arylene). In some embodiments, the arylene can be further substituted with 1 , 2, 3, or 4 substituent groups as defined herein. The term “branched arylene” as used herein, refers to a multivalent aryl group derived by the removal of more than two hydrogen atoms.

[0048] By “caged prodrug” is meant a compound which is activated by ionizing radiation by a radioemitter or an external ionizing radiation source. Caged prodrugs can be prepared by modifying functional groups present in the compounds in such a that they covalently bind to a “caging group,” which may be removed, by a source of ionizing radiation, either in routine manipulation or in vivo, to release the Drug Moiety. Caged prodrugs include compounds wherein hydroxyl, amino, sulfhydryl, or carboxyl groups are bonded to any group that, when administered to a mammalian subject and exposed to ionizing radiation, cleaves to form a free hydroxyl, amino, sulfhydryl, or carboxyl group respectively. In some embodiments, caged prodrugs are conjugated to a targeting moiety, which may be a small molecule, a radical of a radiolabeled peptide pharmaceutical, a peptide, a protein, or an antibody to form a “targeted caged prodrug.” Caged prodrugs include compounds that are caged as described in e.g., Formula II.

[0049] By “carbamate” is meant a radical of the form — R1OC(O)N(R2)— , — R1OC(O)N(R2)R3— , — OC(O)NR2R3, or — OC(O)NH2, wherein R1, R2and R3are each independently alkoxy, alkyl, alkenyl, alkynyl, amide, amino, aryl, arylalkyl, carbamate, cycloalkyl, ester, ether, formyl, halogen, haloalkyl, heteroaryl, heterocyclyl, hydrogen, hydroxyl, ketone, or nitro.

[0050] By “carboxy” is meant a radical — COOH or its corresponding salts, e.g. — COONa, etc.

[0051] By “cycloalkyl” is meant a monovalent saturated cyclic, bicyclic, or bridged cyclic (e.g., adamantyl) hydrocarbon group of 3-12, 3-8, 4-8, or 4-6 carbons, referred to herein. Unless specified otherwise, cycloalkyl groups are optionally substituted at one or more ring positions with, for example, alkoxy, alkyl, haloalkyl, alkenyl, alkynyl, amido, amino, aryl, carboxy, cycloalkyl, ester, halogen, haloalkyl, heteroaryl, heterocyclyl, hydroxyl, sulfonamido, sulfonyl. In certain embodiments, the cycloalkyl group is not substituted, e.g., it is unsubstituted.

[0052] By “Drug Moiety” is meant a radical of a drug that is covalently bonded to a cage to form a caged prodrug. The drug moiety can be for example a cytotoxic, cytostatic, or immunomodulatory drug upon release from the caged prodrug. Drug Moieties may impart their cytotoxic and cytostatic effects by mechanisms including but not limited to tubulin binding, DNA binding or intercalation, and inhibition of RNA polymerase, protein synthesis, and / or topoisomerase.

[0053] By “effective amount” is meant the amount or dose of the compound that provides the desired effect. An effective amount may be provided as a single or multiple doses to the subject. The disclosed methods may include administering an effective amount of the disclosed compounds (e.g., as present in a pharmaceutical composition) for treating a cell proliferative disease or disorder, such as a cancer.

[0054] By “ester” is meant a -R1C(O)OR2-, wherein R1and R2are each independently alkoxy, alkyl, alkenyl, alkynyl, aryl, arylalkyl, cycloalkyl, haloalkyl, heteroalkyl, heteroaryl, or heterocyclyl.

[0055] By “ether” is meant two hydrocarbons covalently linked by an oxygen. Accordingly, the substituent of an alkyl that renders that alkyl an ether is or resembles an alkoxyl, such as may be represented by one of — O-alkyl, — O-alkenyl, — O-alkynyl, and the like.

[0056] By “haloalkyl” is meant an alkyl group that is substituted with at least one halogen. For example, — CH2F, — CHF2, — CF3, — CH2CF3, — CF2CF3, and the like.

[0057] By “halogen” or “halo” is meant a fluorine (fluoro), chlorine (chloro), bromine (bromo), or iodine (iodo) radical. By “heteroalkyl” is meant an “alkyl” group in which at least one carbon atom has been replaced with a heteroatom (e.g., an O, N, or S atom). One type of heteroalkyl group is an “alkoxyl” group.

[0058] The term “heteroalkylene,” as used herein, refers to an alkylene group, as defined herein, in which one or two of the constituent carbon atoms have each been replaced by nitrogen, oxygen, or sulfur. In some embodiments, the heteroalkylene group can be further substituted with 1 , 2, 3, or 4 substituent groups as described herein.

[0059] By “heteroaryl” is meant an aromatic 3 to 10 member ring structure, alternatively 3 to 7 member rings, whose ring structures include one to four heteroatoms, such as nitrogen, oxygen, and sulfur. In certain embodiments, the heteroaryl is substituted at one or more ring positions , alkoxy, alkyl, haloalky I, alkenyl, alkynyl, amido, amino, aryl, carboxy, cycloalkyl, ester, halogen, haloalkyl, heteroaryl, heterocyclyl, hydroxyl, sulfonamido, sulfonyl, or the like. In certain other embodiments, the aromatic ring is not substituted, e.g., it is unsubstituted.

[0060] By “heterocyclyl” and “heterocyclic group” is meant a saturated, partially unsaturated, or aromatic 3- to 10-membered ring structures, alternatively 3-to 7-membered rings, whose ring structures include one to four heteroatoms, such as nitrogen, oxygen, and sulfur. The number of ring atoms in the heterocyclyl group can be specified using 5 Cx-Cx nomenclature where x is an integer specifying the number of ring atoms. For example, a C3-C7 heterocyclyl group refers to a saturated or partially unsaturated 3- to 7- membered ring structure containing one to four heteroatoms, such as nitrogen, oxygen, and sulfur. The designation “C3-C7” indicates that the heterocyclic ring contains a total of from 3 to 7 ring atoms, inclusive of any heteroatoms that occupy a ring atom position. In certain embodiments, the heterocyclyl is substituted at one or more positions with , alkoxy, alkyl, haloalkyl, alkenyl, alkynyl, amido, amino, aryl, carboxy, cycloalkyl, ester, halogen, haloalkyl, heteroaryl, heterocyclyl, hydroxyl, sulfonamido, sulfonyl, or the like. In certain other embodiments, the heterocyclyl is not substittued.

[0061] By “hydroxyl” or “hydroxy” is meant a -OH group.

[0062] The terms “include” and “including” have the same meaning as the terms “comprise” and “comprising.” The terms “comprise” and “comprising” should be interpreted as being “open” transitional terms that permit the inclusion of additional components further to those components recited in the claims. The terms “consist” and “consisting of’ should be interpreted as being “closed” transitional terms that do not permit the inclusion additional components other than the components recited in the claims. The term “consisting essentially of’ should be interpreted to be partially closed and allowing the inclusion only of additional components that do not fundamentally alter the nature of the claimed subject matter.

[0063] The term “pharmaceutically acceptable salt” refer to salts of the compounds which are substantially nontoxic to living organisms. Typical pharmaceutically acceptable salts include those salts prepared by reaction of the compounds as disclosed herein with a pharmaceutically acceptable mineral or organic acid or an organic or inorganic base. Such salts are known as acid addition and base addition salts. It will be appreciated by the skilled reader that most or all of the compounds as disclosed herein are capable of forming salts and that the salt forms of pharmaceuticals are commonly used, often because they are more readily crystallized and purified than are the free acids or bases. The particular counter-ion forming a part of any salt of a compound disclosed herein may not be critical to the activity of the compound, so long as the salt as a whole is pharmacologically acceptable and as long as the counterion does not contribute undesired qualities to the salt as a whole. Undesired qualities may include undesirably solubility or toxicity.

[0064] By “radical of a radiolabeled peptide pharmaceutical” is meant the targeting moiety of a radiolabeled peptide pharmaceutical that in the methods of the invention is repurposed to carry a caged prodrug rather than a radiolabeled peptide pharmaceutical. Examples of such targeting moieties are found in e.g. Table 1.

[0065] By “radioemitter” is meant a compound which includes a radionuclide which undergoes decay (e.g., a-, p- , p+-, or y- decay) to emit (e.g., a-, p-, positron, or y- emission) ionizing radiation in the form of a particle (e.g., a particle, positron, or electron). In some embodiments, the radioemitter is further conjugated to a targeting moiety, which may be a small molecule, a radical of a radiolabeled peptide pharmaceutical, a peptide, a protein, or an antibody to form a “targeted radioemitter.”

[0066] By “subject,” “patient,” or “individual” is meant an animal, which may be a human or non-human animal, in need of treatment. A “subject in need of treatment” may include a subject having a disease, disorder, or condition that is responsive to therapy with a sensitizing agent in combination with a DNA damaging agent. In some embodiments, the patent may be have a disease, disorder, or condition that is responsive to therapy with the RNA methyltransferase inhibitor alone or in combination with a DNA damaging agent. For example, a “subject in need of treatment” may include a subject having a cell proliferative disease, disorder, or condition such as cancer. In some embodiments, the cancer may be a breast cancer, an ovarian cancer, an esophageal cancer, a stomach cancer, a colon cancer, a lung cancer, a skin cancer, a prostate cancer, a head and neck cancer, a bone cancer, a kidney cancer, a urinary tract cancer, a bladder cancer, a pancreatic cancer, a pediatric cancer, or a blood cancer.

[0067] The term “substituted,” as used herein, refers to substituents independently selected from the group consisting of: (1) C1-6 alkoxy; (2) C1-6 alkyl; (3) amino, as defined herein (e.g., unsubstituted amino (i.e., - NH2) or a substituted amino (i.e., -N(RN1)2, where RN1is as defined for amino); (4) C6-10 aryl-Ci-6 alkoxy;

[0068] (5) azido; (6) halo; (7) (C2-9 heterocyclyl)oxy; (8) hydroxy; (9) nitro; (10) oxo; (11) C1-7 spirocyclyl; (12) C1-6 heteroalkyl; (13) thiol; (14) -CO2RA, where RAis selected from the group consisting of (a) C1-6 alkyl, (b) C2-6 alkenyl, (c) Ce-io aryl, and (d) hydrogen; and (15) -C(O)NRBRC, where each of RBand Rcis, independently, selected from the group consisting of (a) hydrogen, (b) C1-6 alkyl, (c) Ce-io aryl, and (d) Ce- io aryl C1-6 alkyl.

[0069] By “treating” or “to treat” is meant to alleviate symptoms.

[0070] BRIEF DESCRIPTION OF THE DRAWINGS

[0071] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application with color drawings will be provided by the Office upon request and payment of the necessary fee. FIG. 1 shows the RAiDER (“RAdioisotope induced Drug Engagement for Release”) concept. Targeted radioisotopes accumulate in tumor tissues, where they locally deliver radiation to chemically activate caged prodrugs. Radioisotope-mediated prodrug activation occurs through reduction of a phenyl azide caging moiety (orange), leading to linker self-immolation and release of the active drug payload (purple) from its drug delivery vehicle (blue), which in this work is long-circulating serum albumin. Consequently, locally activated therapeutic payloads combine with ionizing radiation to maximize tumor cytotoxicity while sparing off-target tissues. Created with BioRender.com.

[0072] FIG. 2 shows radioisotope-mediated drug release from caged-MMAE. (A) 10pM caged-MMAE was exposed to different radioisotopes at varying activities, with corresponding total radiation doses released as estimated using TOPAS-nBIO(39); prodrug activation was monitored by LCMS / HPLC. (B) Drug release efficiencies across different radioisotopes and external beam modalities, reported as nM activated drug per Gy. The highest release efficiency (99mTc) was compared to the other isotopes and modalities tested (mean±S.E., One-way ANOVA with Tukey’s multiple comparisons test, compared to 99mTc).

[0073] FIG. 3 shows RAiDER restores biological prodrug activities in vitro. (A) Cytotoxicity of Alb- caged-MMAE, 99mTC anc| freeMMAE on TBP3743 anaplastic thyroid cancer, measured 72h post-treatment by a resazurin-based assay (n=3, mean ±S.E.). (B) Representative images and quantification of TBP3743 colony formation (mean ±S.E., 2-way-ANOVA with Geisser-Greenhouse correction). (C) Representative images of microtubule immunofluorescence (top and apoptosis by TUNEL (bottom) 24h after treatment in TBP3743 cells (mean ±S.E., 2way-AN0VA with Geisser-Greenhouse correction, scale bar = 200pm).

[0074] FIG. 4 shows a computational modeling of radioisotope-mediated drug release using TOPAS- nBIO. (A) Conceptual mechanism to explain radioisotope-dependent RAiDER efficiencies. Red shading illustrates the spatial distribution and frequency of free-radical-generating ionization events from a given isotope within a vial. Ionization clouds interact with prodrug molecules most efficiently for isotopes emitting lower energy particles / photons (e.g. beta particles / Auger emitters, middle). (B) Correlation between observed drug release across radioisotopes, and their estimated number of electrons generated within the 1 DO- 11 OkeV energy window. (C) Comparison of drug release efficiency with dose imparted by low energy electrons (LEE) across radioisotopes Pearson correlation coefficient (r) was calculated across all data points.

[0075] FIG. 5 shows in vivo biodistribution and prodrug activation. Left: Experiment schematic of RAiDER biodistribution using B6129SF1 / J mice bearing syngeneic TBP3743 tumors. Center: Biodistribution of fluorescent Alb-caged-MMAE, measured by tissue Cy5 fluorescence and 99mTc-FAPI-34 measured by gamma scintillation counting. Right: Biodistribution of activated MMAE measured by LCMS / HPLC. Data are means ± S.E., n = 3-4 mice, two-tailed t-test shown). Created with BioRender.com

[0076] FIG. 6 shows RAiDER enhances the ability of targeted radioisotopes to block tumor growth. (A-B) Mice bearing TBP3743 tumors were treated with Alb-caged-MMAE (5mg / kg per dose) and either 99mTc-FAPI- 34 (18.5 MBq per dose, A) or ^^Lu-FAPI-34 (18.5MBq, B). Caliper measurements of tumor growth are shown over time (left) and with individual tumor sizes on the indicated day (middle, n=20 total mice, 40 total tumors, mean ± S.E). Mouse mass was measured during treatment time course (right). FIG. 7 shows estimating tumoral drug release in patients using clinical dosimetry. Lesion standardized uptake values (SUV) derived from ®^Ga-PSMA-PET and ®^Ga-DOTATATE-PET in prostate and neuroendocrine cancer patients receiving ^^Lu-PSMA-617(45) and ^^Lu- DOTATATE(46) respectively were compared to expected intratumoral active drug release (calculated based on release efficiencies in FIG. 2). Absorbed dose within each individual lesion was calculated in those previous studies using 1 ^^Lu SPECT / CT, demonstrating good correlation with PET-SUV. Red line denotes the IC50 estimated for aggressive cancers in this study (See Table 5).

[0077] FIG. 8 shows caged prodrug stability and release. (A) Caged-MMAE structure: para-azido-2, 3,5,6- tetrafluorobenzyl self-immolative linker (pATFB-SIL) conjugated to monomethyl auristatin E (MMAE, red). (B) HPLC trace of caged-MMAE. (C) Stability of Caged-MMAE and Caged- Exatecan at 4°C over 30 days. (D) Comparison of 99mTCand 177[_umediated MMAE release from caged-MMAE at room temperature and in liquid nitrogen (-196°C) over 96 h (n=3, mean±S.E.).

[0078] FIG. 9 shows an in vitro assessment of radioisotope-mediated caged-MMAE activation across multiple cell lines. (A) Cytotoxicity assay comparing Alb-caged-MMAE (with and without exposure to 99mTc) and free MMAE treatments upon different cancer cell lines, measured in a 72 h resazurin-based cytotoxicity assay. Viability compared to untreated control (n=3, mean±S.E.). (B) Representative images and (C) quantification of cancer cell line colony formation (mean±S.E., 2way-ANOVA with Geisser-Greenhouse correction, n=3 per condition).

[0079] FIG. 10 shows a 99nrrrc cytotoxicity assay. TBP3743 and HT 1080 cells were exposed to varying levels of 99mTCactivities (11.1 MBq / mL, 1.1 MBq / mL and 0.11 MBq / mL) for 72 h, followed by viability assessment (n = 3, mean±S.E). Curves fitted to the linear quadratic model (Masters et al., Invest New Drugs. 2018;36:121-135).

[0080] FIG. 11 shows identifying mechanisms for RAiDER. (A) The effect of known free radical quenchers gentisic acid (GA), ascorbic acid (AA) and hydrogen peroxide on 99mTc-mediated drug release from caged-MMAE at different concentrations. Release mediated by TCEP, tris(2- carboxyethyl)phosphine, served as a positive control (n=3, mean ±S.E., Kruskal-Wallis test). (B) Comparison of drug release and electron generated across different energy windows (0-300keV 10keV windows) for different radioisotopes, measured by Pearson correlation r. The energy window with the highest correlation between electrons generated and drug released (~100keV) was further examined in FIG. 4 (shaded).

[0081] FIG. 12 shows RAiDER is driven by the local concentration of free-radicals from LEEs and recombination effects of relatively low energy radiation. (A) Mammographic energy spectra using W-AI and W-Rh anode / filter combinations generated by SpekPy (Vorbau et al., SpekPy Web — online x-ray spectrum calculations using an interface to the SpekPy toolkit. JAppI Clin Med Phys. 2024;25:e14301 .) show an increase in low-energy photons for W-AI. SpekPy-derived spectra for 320keV x-rays compared to mammography up to 50keV and across 0-300keV (inset). Specific SpekPy parameters are noted in Table 6. (B) Experimental drug release efficiency for the different anode / filter combinations shows better W-AI efficiency. This is in keeping with this filter set having more photons emitted in the “low energy” range. Kruskall-Wallis test, n = 3. (C) Linearity of mammography-mediated caged-MMAE drug release as a function of dose delivered. Note that high-dose samples (blue) were irradiated over two days, showing linear dose-to-drug release response with this irradiation system (n=3, mean±S.E.).

[0082] FIG. 13 shows murine fibroblast activation protein expression (muFAP, green) in murine TBP3743 tumors. Scale bar = 100 pm.

[0083] FIG. 14 shows selectivity of active MMAE accumulation in tumors. (A) Tumor-to-tissue ratio of active MMAE in tissues treated with 99mTc-FAPI-34, as depicted in FIG. 5. (B) Biodistribution of 99mTc-FAPI-34 (18.5MBq) in TBP3743 tumor-bearing mice obtained 8 h post-injection (n=2, mean ±S.E).

[0084] FIG. 15 shows caged prodrug stability and release. (A) Caged-MMAE structure: para-azido-2, 3,5,6- tetrafluorobenzyl self-immolative linker (pATFB-SIL) conjugated to monomethyl auristatin E (MMAE, red). (B) HPLC trace of caged-MMAE exposed to varying amounts of 99mTc. 10pM MMAE is provided as a comparison (red) (C) Stability of Caged-MMAE and Caged-Exatecan at 4°C in PBS (pH 7.4) over 30 days. (D) Comparison of technetium-99m and lutetium-177 mediated MMAE release from caged-MMAE at room temperature and in liquid nitrogen (-196°C) over 96 h (n=3, mean±S.E.)

[0085] FIG. 16 shows drug release mediated by 177|_u-PSMA-617 in prostate cancer. RM1 .PSMA tumor- bearing mice were injected with Alb-caged-Exatecan 48 h before 18.5Mbq 177 Lu-PSMA-617. Tissues were harvested 24 h later, and exatecan release was assessed. Free exatecan was injected in separate mice for comparison (n=2-3 per treatment mean ±S.E., 2-way-ANOVA with multiple comparisons).

[0086] FIG. 17 shows blood biomarkers of toxicity during RAiDER. Blood samples were obtained from experimental animals depicted in FIG. 6A at the end of treatment, and a complete blood count (CBC) and complete metabolic panel (CMP) were obtained (mean (S.D)). The shaded box denotes normal ranges. No significant differences were noted across treatment cohorts (Kruskall-Wallis Test, n = 3 per treatment).

[0087] FIG. 18 shows representative hematoxylin and eosin staining of liver and kidney tissues across different RAiDER treatments (obtained from subjects depicted in FIG. 6A, 40x). Scale bar 100 pm.

[0088] FIG. 19 shows a computed estimation of RAiDER feasibility in patients. Estimated dosimetry from literature-derived pharmacokinetics and realistic injected doses of commonly used radiopharmaceutical therapies (Velikyan et al., Quantitative and qualitative intrapatient comparison of 68Ga-DOTATOC and 68Ga-DOTATATE: net up-take rate for accurate quantification. J Nucl Med. 2014;55:204.; Jia et al., The role of [99mTc]Tc-HFAPi SPECT / CT in patients with malignancies of digestive system: first clinical experience. Eur J Nucl Med Mol / mag / ng.2023;50:1228-1239.; Schuchardt et al., Prostate-Specific Membrane Antigen Radioligand Therapy Using (177)Lu-PSMA l&T and (177)Lu-PSMA-617 in Patients with Metastatic Castration-Resistant Prostate Cancer: Comparison of Safety, Biodistribution, and Dosimetry. J Nucl Med. 2022;63:1199-1207.). MIRCcalc was used for calculations (Behranvand et al. Cancer immunology, immunotherapy. 2022;71 :507-526). To evaluate whether the tumor absorbed dose would lead to meaningful drug release, the absorbed dose, in Gy / MBq (A), was converted to drug release concentration based on the typical injected dose of the relevant agent in patient and efficiency measures in FIG. 2B. The assumption is made that enough caged-prodrug can be delivered to the tumor, which is supported by biodistribution studies FIG. 20 shows the concept and chemical performance of bispecific Radiation-AMPlified Antibody Drug Conjugate (RAMP-ADC) therapy. A) Overview schematic of RAMP-ADC containing a caged MMAE payload (RAMP-ADCM). Systemically administered RAMP-ADCM circulates in the body, and MMAE is chemically activated in situ in irradiated tumor tissue. B-C) Concentration of active MMAE released from RAMP-ADCM following X-ray irradiation, quantified by mass spectrometry (n = 3; data are means + / - s.e.m.). C) Representative HPLC chromatogram detecting free MMAE payload following 2 Gy fractions of X-ray radiation.

[0089] FIG. 21 shows RAMP-ADC response across diverse cancer cell lines. (A) Quantification of combined EGFR and MET mRNA expression (left), and rates of copy-number-amplification, high protein levels, or high mRNA levels of EGFR and / or MET (right) as measured by clinical tumor samples in The Cancer Genome Atlas (TCGA). Bars (left) denote mean expression. (B) Relative quantification of combined EGFR and MET protein levels as measured by normalized mass spectrometry data across the Cancer Cell Line Encyclopedia (left), and the correlation of EGFR and MET protein levels across individual cell lines (right). Mean + / - s.d. are shown (left). (C) Colony formation following indicated treatments, with colony counts reported as mean + / -s.d. (n=3). (D) Cytotoxicity half-maximal inhibitory values (IC50) for cells exposed to non- irradiated or irradiated RAMP-ADCM and controls (see FIG. 35 for full data).

[0090] FIG. 22 shows cellular mechanisms of RAMP-ADC activity in vitro. (A) Representative fluorescence microscopy of RAMP-ADCM and amivantamab bound to HT1080 cells, using secondary anti-human lgG1-AF488 as control, with normalized mean fluorescence intensity of each condition. Scale bar, 40 pm. (B) 48 hr cytotoxic response to MMAE, amivantamab, RAMP-ADCM, or irradiated RAMP-ADCM on HT1080 cells measured by PrestoBlue (n = 2, mean ± s.d.). (C-D) HT1080 colony formation following treatment with radiation and / or RAMP-ADC components (C) and its quantification (D; Two-way ANOVA with Dunnett’s test for multiple comparisons, n = 3). 0.5 nM payload and 0.13 nM antibody were used. (E-H) Representative immunofluorescence of HT1080 microtubule structures following treatment (scale bar, 500 pm at 4x and 40 pm at 40x), and corresponding quantification of the mitotic index (F), total microtubule fluorescence intensity (G), and live cell count (H). Data are means ± s.d.; one-way ANOVA with Dunnett’s test for multiple comparisons, n = 5. *P<0.05, ** P<0.01 , ***P<0.001 , ****P<0.0001 .

[0091] FIG. 23 shows radiation promotes bystander effects of RAMP-ADC. (A-B) Bystander drug redistribution concept (A) and corresponding in vitro experimental schematic (B). (C-E) Representative immunofluorescence of HT1080 microtubule structure exposed to conditioned media (C; scale bar, 200 pm), and corresponding quantification of total microtubule fluorescence intensity (D), microtubule spindle abundance (E), and live cell count (F). One-way ANOVA with Dunnett’s test for multiple comparisons; n = 6, mean ± s.d. *P<0.05, ** P<0.01 , ***P<0.001 , ****P<0.0001 .

[0092] FIG. 24 shows optimizing RAMP-ADC with payload combinations. (A) Chemical structure of RAMP- ADC containing a topoisomerase 1 inhibitor payload based on exatecan (RAMP-ADCT). (B) Cytotoxicity of HT1080 cells in response to 48 hrs of irradiated or non-irradiated RAMP-ADCT or its components (n = 2). (C) Summarized cytotoxicity half-maximum inhibitory values (IC50) of free MMAE / exatecan / combination and their average across tested cell lines, along with combination index (Cl). D) Corresponding dose-response curves, in response to 48 hrs of treatment with MMAE and / or exatecan individually or at a 5:1 molar ratio (n = 2). Data are means ± s.d.

[0093] FIG. 25 shows an in vivo evaluation of RAMP-ADC. (A) Ex vivo biodistribution analysis of RAMP-ADCM antibody stably labeled with Cy5, measured by fluorescence reflectance (representative images at bottom). % I D / g , percent injected dose per gram tissue. Scale bar, 5 mm. (B-C) Biodistribution of chemically activated RAMP-ADC payloads, following intravenous co-dosing at a 5:1 molar ratio of RAMP-ADCM : RAMP-ADCT, showing individual payload concentrations (B) and combined activation yield (C). Tumors were irradiated 4 hr after intravenous RAMP-ADC injection, and 24 hr later tissues were digested for LCMS. (D-F) HT 1080 xenograft growth over time (D), with individual tumor sizes (E), humane survival Kaplan-Meier (F), and body weight measurements (G). One-way ANOVA, total n=33; data are means ± sem (D, G) and ± s.d. (E). (H-l) 24 hrs following treatment, HT1080 tumors were processed for (H) tubulin immunofluorescence and (I) apoptosis TUNEL quantification. One-way ANOVA with Dunnett’s test for multiple comparisons, n > 4 per group; data are means ± s.d. Scale bars, 100 pm. *P<0.05, **P<0.01 , ***P<0.001 , ****P<0.0001.

[0094] FIG. 26 shows a correlation of EGFR and MET levels with radiation sensitivity profiling per cell line. (A) Summed mRNA expression of EGFR and MET in each cell line relative to the radiation sensitivity (cellular survival after exposure to a RT dose-response), such that higher values denote radioresistance. Spearman rank correlation test, n = 524 cell lines. Cell lines annotated in orange are used in this manuscript. (B) Comparison of summed [EGFR+MET] mRNA expression in the 100 most radiosensitive and 100 most radioresistance cell lines, corresponding to A (two-tailed Mann-Whitney test). (C) Correlation between summed [EGFR+MET] levels and radiation sensitivity, within individual tissue-types of origin (two-tailed Spearman rank correlation test, and false discovery rate q-values using Benjamini- Hochberg correction). No statistically significant correlations were detected below a false discovery rate threshold of 0.05.

[0095] FIG. 27 shows cytotoxicity dose-response curves for multiple cell lines. (A-C) Multiple cell lines were treated with either free MMAE, amivantamab antibody, RAMP-ADCM, or RAMP-ADCM after X-ray irradiation (8 Gy), corresponding to Figure 2. (D) EGFR / MET expression and RAMP-ADC IC50values across cell lines. Spearman rank correlation test P=0.11 , r=0.89. Data are means ± s.d. (n=2).

[0096] FIG. 28 shows an Evaluation of cytotoxicity across different molar ratios of MMAE and exatecan. A-B) Cytotoxicity of varying MMAE and exatecan ratios in sarcoma (A, HT1080), anaplastic thyroid cancer (B, TBP; C, 8505c), triple-negative breast cancer (D, MDA-MB-231), and lung adenocarcinoma (E, HCC-827) cell lines. Cells were treated with either free MMAE, free exatecan, or a combination. After 72 hours, cells were analyzed using the PrestoBlue assay and data were normalized to the control wells; data correspond to Figure 3 (n=2). (F) Dose response of RAMP-ADCT payload release following X-ray exposure. Samples of RAMP-ADCT (approximate concentration: 10 pM) were X-ray irradiated, and released exatecan was measured by LCMS (n=3). Data are means ± s.d. (G) Combination index calculated from data in A-E, such that each datapoint represents an individual cell line (n=5). The 1 :5 ratio showed consistent synergy (P=0.02, two-tailed t-test), but the 5:1 ratio was consistently more cytotoxic at a given dose and was used in subsequent experiments. (H) Ratio of IC50 values comparing monotherapy with the 5:1 ratio combination, such that each datapoint represents an individual cell line (n=6). In all cell lines, the combination is more potent than the worse monotherapy, and on average is similar to the better monotherapy in IC50.

[0097] FIG. 29 shows a calibration curves for quantification. Representative calibration curves for LCMS analysis of MMAE (A) and exatecan (B), and fluorescence analysis of Cy5 for interpreting Cy5- labeled RAMP-ADC biodistribution (C).

[0098] FIG. 30 shows the effect of RAMP-ADC on individual tumor volume. Individual tumor sizes were measured by caliper during the longitudinal efficacy assessment in HT1080 tumor-bearing mice (n=16). Conditions include saline only (A), radiation only (B), RAMP-ADC (RAMP-ADCM + RAMP-ADCT) only (C), and RAMP-ADC followed by radiation (D). HT 1080 xenograft growth over time (E), with individual tumor sizes (F), and corresponding body weight measurements (G). One-way ANOVA, total n=16; data are means ± s.d.

[0099] FIG. 31 shows a RAMP toxicity assessment. Serum and blood samples from mice were collected following treatment with either RAMP-ADCM or saline control. Data are means ± SEM, n=4 mice per group. No statistically significant changes were detected according to the Mann- Whitney test.

[0100] FIG. 32 shows the concentration of uncaged Drug Moiety after incubation of the caged prodrug ADCs incubated with either99mTc or177Lu containing radioemitters for 48 hours.

[0101] FIG. 33 Shows the tumor size within the C57BL / 6 mice injected with the targeted radioemitter PSMA-617 (“Lu-177 PSMA”) and the PSMA-617 in combination with the PBD caged prodrug (“Lu-177 PSMA + prodrug”) over the course of 8 days of treatment.

[0102] FIG .34 Shows the tumor size within the nude mice injected with targeted radioemitter PSMA-617 (“Lu- 177 PSMA”), the PSMA-617 in combination with the PBD caged prodrug (“Lu-177 PSMA + prodrug”), and the control, not injected with either, over the course of 7 days of treatment.

[0103] FIG. 35 shows determination of drug-antibody ratio (DAR) and stability for RAMP conjugates. Antibody conjugates were prepared as described in the methods section, followed by incubation of an aliquot (1 mg) of conjugate with 5 units PNGase F (Promega, USA) in 100 pL PBS for 4 hours at 37 °C. These aliquots (as well as a sample of deglycosylated amivantamab) were analyzed on a Waters Acquity UPLC- MS equipped with a RDa mass detector. The mobile phase was 0.1 M ammonium formate (pH 7.4) on a XBridge Premier Protein 250 A size exclusion column (Waters, USA). Deconvoluted mass data for the native antibody (A), MMAE conjugate (B), and exatecan conjugate (C) are shown. The stability of each conjugate (D-E) was measured by incubating aliquots of each (1 pM drug concentration) at varying temperature for 72 hours. The samples were then filtered through spin filters (3 kDa MWCO, 10,000 ref for 10 min) and the flow through was analyzed by LCMS to determine the concentration of free drug released, showing <3% drug release under all unirradiated conditions. (F) As an alternative method of determining DOL, antibody and released drug concentrations were measured by Nanodrop and LC / MS, respectively (using the calibration curves in FIG.29 for LC / MS quantification). FIG. 36 shows the effect of RAMP-ADC on individual tumor volume and survival. Individual tumor sizes were measured by caliper during the longitudinal efficacy assessment in HT1080 tumorbearing mice, corresponding to FIG. 25D-E. Humane survival is shown of all experimental groups, corresponding to FIG. 25F. Experimental group assignments were uneven in the number of mice to prioritize power in detecting differences between [amivantamab+RT], [RAMP-ADC+ RT|, and [RAMP-ADCM+RT]; these three groups had n=7 mice / group..

[0104] FIG. 37 shows clonogenic dose response of HT1080 to combinations of radiation and RAMP-ADCM. 300 cell were plated per well in 6-well plates (1 mL DMEM per well), treated with RAMP-ADCM and varying doses of X-ray irradiation, then incubated for 7-10 days. Colonies were then stained with crystal violet as described in the methods section and counted. Data are means ± s.d. (n=2).

[0105] DETAILED DESCRIPTION

[0106] The invention provides targeted caged prodrugs and targeted radioemitters and methods of use thereof to treat patients suffering from a cancer that can benefit from administration of targeted caged prodrugs containing potent pharmaceuticals, which may be uncaged by ionizing radiation emitted by the targeted radioemitters, while minimizing off-target activity.

[0107] Caged Prodrugs

[0108] The present invention features caged prodrugs of Formula I - VII, or any one of the compounds of Examples I - V, or pharmaceutically acceptable salts thereof. Exemplary synthesis of the caged prodrugs is disclosed in WO 2023 / 220280 A1 , incorporated hereby reference, and further exemplary synthesis procedure for the caged prodrugs is discussed below in Examples 1 , 2, and 4.

[0109] Scaffolds

[0110] In a preferred embodiment, the caged prodrugs of the invention have the structure of Formula I:

[0111] [RSM]-Linker-Drug Moiety

[0112] Formula I, wherein the RSM is a radiation-sensitive moiety. In some embodiments, the caged prodrugs of the invention include a scaffold of Formula II:

[0113] Formula II wherein D is a drug moiety, ring A is a 5-6 member heteroaryl or phenyl, each Ri is independently halogen, azido, or C1 -C6 alkoxy, wherein not more than one of Ri is azido, m is 2, 3, 4, or 5, each R2 is independently halogen, C1 -C6 alkyl, or C1 -C6 h aloalky I , n is 0, 1 , 2, 3, or 4, and R3 is a linker bound to a targeting moiety. In some embodiments, m is 2 or 5. In some embodiments m is 2 and each Ri is methoxy. In some embodiments, m is 5, one Ri Is azido, and the remaining Ri are each fluoro. In some embodiments, ring A has the structure

[0114] Drug Moiety

[0115] In preferred embodiments, the caged prodrugs of the invention include a Drug Moiety (e.g., D of Formula II), chosen from one of a cytotoxic, cytostatic, or immunomodulatory agent. In some embodiments, the caged prodrugs of the invention include a drug moiety chosen from any one of an antitubulin agents, a DNA replication inhibitors, an alkylating agents, an antifolates, an antimetabolites, a chemotherapy sensitizers, a topoisomerase inhibitors. In some embodiments, the caged prodrugs of the invention include a Drug Moiety chosen from any one of pyrrolobenzodiazepine dimer (PBD dimer), exatecan, monomethyl auristatin E (MMAE), doxorubicin, or gardiquimod. In some embodiments, the drug moiety is the PBD dimer of Formula III, wherein X is the attachment point to the remainder of the prodrug.

[0116] In some embodiments, the drug moiety is exatecan of Formula IV, wherein X is the attachment point to the remainder of the prodrug, to form a carbamate bond.

[0117] In some embodiments, the drug moiety is MMAE of Formula V, wherein X is the attachment point to the remainder of the prodrug, to form a carbamate bond.

[0118] In some embodiments, the drug moiety is doxorubicin of Formula VI, wherein X is the attachment point to the remainder of the prodrug, to form a carbamate bond.

[0119] In some embodiments, the drug moiety is gardiquimod of Formula VII, wherein X is the attachment point to the remainder of the prodrug, to form a carbamate bond.

[0120] Formula VII

[0121] Linkers

[0122] In some embodiments, the caged prodrugs of the invention include a handle or linker (e.g., R3 of Formula II), where a targeting moiety may be covalently attached to the prodrug. Covalent conjugation of two or more components in a conjugate using a linker may be accomplished using well-known organic chemical synthesis techniques and methods. Complementary functional groups on two components may react with each other to form a covalent bond. Examples of complementary reactive functional groups include, but are not limited to, e.g., maleimide and cysteine, amine and activated carboxylic acid, thiol and maleimide, activated sulfonic acid and amine, isocyanate and amine, azide and alkyne, and alkene and tetrazine. Site-specific conjugation to a polypeptide may accomplished using techniques known in the art. Exemplary techniques for site-specific conjugation to an Fc domain are provided in Agarwall. P., et al. Bioconjugate Chem. 26:176-192 (2015). Other examples of functional groups capable of reacting with amino groups include, e.g., alkylating and acylating agents. Representative alkylating agents include: (i) an a-haloacetyl group, e.g., XCH2CO- (where X=Br, Cl, or I); (ii) a N-maleimide group, which may react with amino groups either through a Michael type reaction or through acylation by addition to the ring carbonyl group; (iii) an aryl halide, e.g., a nitrohaloaromatic group; (iv) an alkyl halide; (v) an aldehyde or ketone capable of Schiff’s base formation with amino groups; (vi) an epoxide, e.g., an epichlorohydrin and a bisoxirane, which may react with amino, sulfhydryl, or phenolic hydroxyl groups; (vii) a chlorine-containing of s-triazine, which is reactive towards nucleophiles such as amino, sufhydryl, and hydroxyl groups; (viii) an aziridine, which is reactive towards nucleophiles such as amino groups by ring opening; (ix) a squaric acid diethyl ester; and (x) an a-haloalkyl ether.

[0123] Examples of amino-reactive acylating groups include, e.g., (i) an isocyanate and an isothiocyanate; (ii) a sulfonyl chloride; (iii) an acid halide; (iv) an active ester, e.g., a nitrophenylester or N-hydroxysuccinimidyl ester; (v) an acid anhydride, e.g., a mixed, symmetrical, or N-carboxyanhydride; (vi) an acylazide; and (vii) an imidoester. Aldehydes and ketones may be reacted with amines to form Schiff’s bases, which may be stabilized through reductive amination.

[0124] It will be appreciated that certain functional groups may be converted to other functional groups prior to reaction, for example, to confer additional reactivity or selectivity. Examples of methods useful for this purpose include conversion of amines to carboxyls using reagents such as dicarboxylic anhydrides; conversion of amines to thiols using reagents such as N-acetylhomocysteine thiolactone, S- acetylmercaptosuccinic anhydride, 2-iminothiolane, or thiol-containing succinimidyl derivatives; conversion of thiols to carboxyls using reagents such as a -haloacetates; conversion of thiols to amines using reagents such as ethylenimine or 2-bromoethylamine; conversion of carboxyls to amines using reagents such as carbodiimides followed by diamines; and conversion of alcohols to thiols using reagents such as tosyl chloride followed by transesterification with thioacetate and hydrolysis to the thiol with sodium acetate.

[0125] In some embodiments, a linker of the invention, is conjugated (e.g., by any of the methods described herein) to a protein, for example the Fc portion an antibody, or the Fc portion of a fusion protein. In some embodiments of the invention, the linker is conjugated by way of: (a) a thiourea linkage (i.e., - NH(C=S)NH-) to a lysine; (b) a carbamate linkage (i.e., -NH(C=O)-O) to a lysine; (c) an amine linkage by reductive amination (i.e., -NHCH2) to a lysine; (d) an amide (i.e., -NH-(C=O)CH2) to a lysine; (e) a cysteine-maleimide conjugate between a maleimide of the linker to a cysteine; (f) an amine linkage by reductive amination (i.e., -NHCH2) between the linker and a carbohydrate (e.g., a glycosyl group of an Fc domain monomer or an Fc domain); (g) a rebridged cysteine conjugate, wherein the linker is conjugated to two cysteines; (h) an oxime linkage between the linker and a carbohydrate (e.g., a glycosyl group of an Fc domain monomer or an Fc domain); (i) an oxime linkage between the linker and an amino acid residue; (j) an azido linkage between the linker; (k) direct acylation of a linker; or (I) a thioether linkage between the linker.

[0126] In some embodiments of any of the foregoing caged prodrug conjugates, the linker (L) has the structure:

[0127] Formula VIII wherein a, b, c, e, f, and g are each, independently, 0 or 1 , d is 0, 1 , 2, or 3, each of R6, R8, R10, and R12, is, independently, optionally substituted Ci-Ce alkylene, optionally substituted Ci-Ce heteroalkylene, optionally substituted C2-C6 alkenylene, optionally substituted C2-Ce alkynylene, or optionally substituted C6-C10 arylene, O, S, Se, and NR13, R7and R11are each, independently, carbonyl, thiocarbonyl, sulfonyl, or phosphoryl, wherein, if R7is phosphoryl, -(R9)d- is a bond, and e, f, and g are 0, then at least one of R6or R8is not O, and if R11is phosphoryl, -(R9)d- is a bond, and a, b, and c are 0, then at least one of R10or R12is not O, each R9is optionally substituted C1-C10 alkylene, optionally substituted C2-C10 alkenylene, optionally substituted C2-C10 alkynylene, optionally substituted C2-C10 heterocyclylene, optionally substituted C6-C12 arylene, optionally substituted C2-C100 polyethylene glycolene, or optionally substituted C1-C10 heteroalkylene, or a bond linking (R6)a-(R7)b-(R8)c to (R10)e-(R11)f-(R12)g, wherein if -(R9)d- is a bond, then at least one of a, b, c, e, f, or g is 1 , and R13is hydrogen, optionally substituted C1-C4 alkyl, optionally substituted C2-C4 alkenyl, optionally substituted C2-C4 alky ny I, optionally substituted C2-C6 heterocyclyl, optionally substituted C6-C12 aryl, or optionally substituted C1-C7 heteroalkyl.

[0128] In some embodiments, the linker is -NHRa, or -NHRb. In some embodiments, Rais -H or C1-C6 alkyl. In , ,2s , t , p, q, r, s, t, u, and v is each independently an integer from 2-20, X is an electrophilic group, and linkers attached to R4 indicate a covalently bound linker, wherein R4 is a small molecule, a peptide, a protein, or an antibody.

[0129] In some embodiments, the handle or linker has the structure of -NHRb, wherein Rb is ated to the targeting moiety, wherein R4 is the targeting moiety which may be a small molecule, a peptide, a protein, or an antibody. In some embodiments, the linker may include - (C1-C6 alkylene)n-W or -(C1-C6 heteroalkylene)n-W, wherein the n is an integer from 1-20 and W is an attachment moiety. In some embodiments, W is chosen from one of maleimide, azide, alkyne, amine, thiol, carbonyl, and carboxyl.

[0130] Radioemitter

[0131] The disclosure provides radioemitters, pharmaceutically acceptable salt thereof, or pharmaceutical composition thereof that can be used in the methods and compositions of the invention to kill cells. Radioemitters include a radionuclide which undergoes decay (e.g., a-, p-, p+-, or y- decay) to emit (e.g., a-, p-, positron, or y- emission) ionizing radiation in the form of a particle (e.g., a particle, positron, or electron). These radioemitters have found use as radiopharmaceuticals, namely for imaging (e.g., positron emission tomography (PET) and single-photon emission computed tomography (SPECT) scans) and targeted delivery of radionuclides to targeted cells through targeting moieties (e.g., small molecules, peptides, nanobodies, and antibodies).

[0132] Moreover, the precise targeted deposition of high energy emitted by radionuclides in target cells to directly induces cell death through single- or double-strand DNA breaks has been extensively studied. In contrast to radiotherapy, which involves an external radiation source, radiopharmaceutical therapy (RPT) restricts radiation within targeted cells, by using targeted radioemitters, and exhibits few toxic effects on non-targeted cells, thereby reducing off-target cell, tissue, and organ injury. Notably, compared with conventional modalities, a small dose of targeted vectors could achieve sufficient radiation to achieve cell killing, enabling a safe and economical therapeutic modality, as well as ensuring safe and targeted delivery of ionizing radiation to a specific cell, tissue, or organ.

[0133] The radioemitters of the present invention may include radionuclides which undergo decay. In some embodiments, the radionuclides include18F,61Cu,64Cu,67Cu,67Ga,68Ga,82Rb,89Zr,89Sr, "Y,99mTc,103Pd,111ln,128l,124l,125l,131l,131Cs,133Xe,153Sm,149Pm,161Tb,166Ho,169Er,177Lu,186Re,188Re,201TI,203Pb,211At,212Pb,213Bi,223Ra,225Ac, or227Th.

[0134] In some embodiments, the radioemitters of the present invention may include radionuclides that undergo decay (e.g., a- decay, p- decay, p+- decay, or y- decay), and which may release a particle (e.g., an a particle, a positron, or an electron). In some embodiments, the radioemitter of the present invention is an a-emitter (e.g.,211At,212Pb,213Bi,223Ra,225Ac, or227Th). In some embodiments, the radioemitter of the present invention is a p-emitter (e.g.,64Cu,67Cu,89Sr, "Y,131l,166Ho,177Lu,186Re, or188Re). In some embodiments, the radioemitter of the present invention is a positron-emitter (e.g.,18F,61Cu,64Cu,66Ga,68Ga,82Rb,89Zr, or124l). In some embodiments, the radioemitter of the present invention is a y-emitter (e.g.,67Ga,68Ga,99mTc,103Pd,111ln,123l,125l,131l,131Cs,133Xe,153Sm,149Pm,161Tb,169Er,177Lu,201TI, or203Pb). In some embodiments, the radioemitter of the present invention is an Auger emitter (e.g.,67Ga,99mTc,111In,123l,or125l).

[0135] In some embodiments, the radioemitter of the present invention includes64Cu. In some embodiments, the radioemitter of the present invention includes67Ga. In some embodiments, the radioemitter of the present invention includes68Ga. In some embodiments, the radioemitter of the present invention includes "Y. In some embodiments, the radioemitter of the present invention includes99mTc. In some embodiments, the radioemitter of the present invention includes111ln. In some embodiments, the radioemitter of the present invention includes123l. In some embodiments, the radioemitter of the present invention includes1311.In some embodiments, the radioemitter of the present invention includes177Lu. In some embodiments, the radioemitter of the present invention includes223Ra. In some embodiments, the radioemitter of the present invention includes225Ac.

[0136] In some embodiments, the radionuclide of the present invention is covalently attached (e.g., a C-X bond, wherein X is a halide radionuclide) to the radioemitter. In some embodiments, the radioemitter of the present invention includes a chelator that chelates to the radionuclide. In some embodiments, the chelator is 1 ,4,7,10-tetraazacyclododecane-1 ,4,7,10-tetraacetic acid (DOTA), or a derivative thereof. In some embodiments, the chelator is 2,2',2"-(1 ,4,7-triazonane-1 ,4,7-triy l)triacetic acid (NOTA), or a derivative thereof. In some embodiments, the chelating group is (carboxyethyl)benzyl]ethylenediamine- N,N'-diacetic acid (HBED-CC), or a derivative thereof. In some embodiments, the chelating group is 6- hydrazinonicotinamide (HYNIC), or a derivative thereof. In some embodiments, the chelating group is 1 ,4,7,10-tetraazacyclododecane-1 ,4,7,10-tetramethylenephosphonate (DOTMP), or a derivative thereof. In some embodiments, the chelating group has the structure of Formula IX:

[0137] Formula IX wherein the M is the radionuclide and both R are simultaneously -OH, -L-Asn, -N-L-Glu, -N-L-Gla, or N-D- Arg.

[0138] In some embodiments, the radioemitter of the present invention is covalently bound to a targeting moiety.

[0139] In some embodiments, the radioemitter and the targeting moiety are one and the same.

[0140] Targeting Moiety

[0141] The disclosure provides targeting moieties (e.g., peptide targeting agent, antibody drug conjugate, small molecule targeting agent, or protein targeting agent), which may be used with radioemitters, pharmaceutically acceptable salt thereof, or pharmaceutical composition thereof and / or caged prodrugs, pharmaceutical acceptable salts thereof, or pharmaceutical compositions thereof, that can be used in the methods and compositions of the invention to kill cells.

[0142] In some embodiments, the targeting moieties of the present disclosure are conjugated to the caged prodrug and / or the radioemitter in order assist in targeting (e.g., delivering) the caged prodrug and / or the radioemitter to the specific cell, tissue, or organ. In some embodiments, the targeting moiety is a peptide targeting agent, an antibody used in antibody-drug conjugates (ADC), a small molecule targeting agent, or a nanobody targeting agent.

[0143] In some embodiments, the targeting moieties used for the radioemitters and caged prodrugs may be the same. In some embodiments, the targeting moieties used for the radioemitters and caged prodrugs may be different. In some embodiments, the targeting moieties used for the radioemitters and caged prodrugs may target the same cell, tissue, or organ, but be different targeting moieties. In some embodiments, identical targeting moiety may be used for the radioemitter and caged prodrug during the method of treatment of a cell, tissue, or organ of the present invention.

[0144] Peptide Targeting Agents

[0145] In some embodiments, the targeting moiety of the present invention is a peptide targeting agent. In some embodiments, the peptide targeting agent includes a peptide that is from 2 to 30 amino acids in length. In some embodiments, the peptide targeting agent is a linear peptide targeting agent. In some embodiments, the peptide targeting agent is a cyclic peptide targeting agents. In some embodiments, all of the amino acids in the peptide targeting agent are natural amino acids. In some embodiments, at least one of the amino acids in the peptide targeting agent is a non-natural (e.g., synthetic) amino acid. In some embodiments, the peptide targeting agent is further connected to a linker, through which it attaches (e.g., conjugates) to the caged prodrug or radioemitter.

[0146] In some embodiments, the peptide targeting agent is a radical of a radiolabeled peptide pharmaceutical selected from PSMA-11 , PSMA-617, PSMA-1007, DOTA-TATE, DOTA-TOC, FAPI-04, FAPI-34, FAPI- mFS, H6F, NNS309, NeoBOMBI , RM2, BAY 86-7548, AMBA, NOTA-PRGD2, pentixafor, CCZ01048, CP04, PP-F11 N, DOTA-MG11 , and DOTA-MSH. The structure (e.g., with or without the conjugated radionuclide) and specific targets of the radiolabeled peptide pharmaceuticals are provided in Table 1.

[0147] Table 1. Peptide based radiolabeled pharmaceuticals for targeted radiation delivery.

[0148]

[0149] The radiolabeled peptide pharmaceuticals shown in Table 1 contain an amide bond connecting the chelating moiety to the targeting peptide structure which, in some embodiments, which is used as a point of attachment, replacing the chelating moiety with a caged prodrug. Thus, a radical of the peptide targeting moiety may be utilized as a peptide targeting agent in conjunction with the caged prodrugs for targeting their delivery to specific tissues or cells. For example, caged prodrugs conjugated to the targeting moieties shown in Table 1 can prepared, e.g., to produce caged exatecan, caged MMAE, or caged PBD. In some embodiments, the invention features a method of killing a cell using a combination therapy of (i) a peptide based radiolabeled pharmaceutical for targeted radiation delivery, and (ii) the corresponding a peptide (as the targeting moiety) based caged exatecan, caged MMAE, and / or caged PBD.

[0150] Antibody Drug Conjugates

[0151] In some embodiments, the targeting moiety of the present invention is an antibody that is conjugated to the caged prodrug or radioemitter to form an Antibody-Drug Conjugate (ADC). Synthesis and preparation of ADCs are known in the art, and exemplary synthesis is provided in example 2 . In some embodiments, the antibodies of the present invention are monoclonal antibodies. In some embodiments, the antibodies of the present invention are bispecific antibodies. In some embodiments, the bispecific antibodies are bispecific T-cell engager (BiTE) antibodies. In some embodiments, the antibodies of the present invention are designed to bind to more than 2 (e.g., 3) targets. Exemplary antibodies, or antibody containing compounds and ADCs, which may be used to generate ADCs of the present invention are listed in Table 2. Table 2 further includes a list of non-limiting caged drug moieties which may be used to form targeted caged prodrug ADCs of the present invention.

[0152] Table 2. Exemplary antibodies, or antibody containing compounds and ADCs and their targets.

[0153] The ADCs shown in Table 2 may be utilized in the methods of the invention, where the antibody acts as the targeting moiety conjugated via a linker to a caged prodrug, where the antibody delivers the ADC to specific tissues or cells. For example, caged prodrugs conjugated to the ADCs shown in Table 2 can prepared, e.g., to produce ADCs bearing caged exatecan, caged MMAE, or caged PBD. In some embodiments, the invention features a method of killing a cell using a combination therapy of (i) a peptide, nanobody, or small molecule based radiolabeled pharmaceutical for targeted radiation delivery to a cell or tissue, and (ii) an ADC carrying caged prodrug (e.g., caged exatecan, caged MMAE, or caged PBD), where the antibody is selected to deliver the caged prodrug to the same cell or tissue as the radiopharmaceutical. Small Molecule Targeting Agents

[0154] In some embodiments, the targeting moiety of the present invention is a small molecule targeting agent. In some embodiments, the small molecule targeting agent includes methylene diphosphonate. In some embodiments, the small molecule targeting agent includes phytate. In some embodiments, the small molecule targeting agent includes meta-iodobenzylguanidine.

[0155] Protein Targeting Agents

[0156] In some embodiments, the targeting moiety of the present invention is a protein targeting agent. In some embodiments, the protein targeting agent is a native protein. In some embodiments, the protein targeting agent is a recombinant protein. In some embodiments, the protein targeting agent is conjugated to the caged prodrug and / or the radioemitter prior to administration. In some embodiments, the protein targeting agent conjugates to the caged prodrug and / or the radioemitter within the subject after administration. In some embodiments, the protein targeting agent is albumin. In some embodiments, the protein targeting agent is an affibody. In some embodiments, the protein targeting agent is a nanobody. Exemplary affibody, nanobodies, and proteins are provided in Table 3.

[0157] Table 3. Exemplary affibodies, nanobodies, and proteins.

[0158] The affibodies, nanobodies, and proteins shown in Table 3 may be utilized in the methods of the invention, e.g., to provide targeting moieties for a caged prodrug and / or a radiopharmaceutical. In some embodiments, the protein targeting agent is a nanobody-based BiTEs. In some embodiments, the nanobody-based BiTEs are a PD-L1 / CD137 BiTE, a CD3 / CD105 BiTE, CD3 / PD-L1 BiTE, CD3 / CD20 BiTE, CD3 / FAP BiTE, HER2-scFvCD3 / HER2-EGFR BiTE, or CD19 / CD20 BiTE.

[0159] In some embodiments, the protein targeting agent is a single-chain variable fragment (scFv) with affinity towards cancer markers. The scFv may be based on the structure of centuximab, trastuzumab, anti-CEA antibodies, anti-CD44 antibodies, or anti-PSMA antibodies. Pharmaceutical Compositions

[0160] The caged prodrugs (e.g., targeted caged prodrug) and radioemitters (e.g., targeted radioemitters) disclosed and utilized in the methods disclosed herein may be formulated as pharmaceutical compositions that include an effective amount of one or more targeted caged prodrugs or targeted radioemitters, as disclosed herein, and one or more pharmaceutically acceptable carriers, excipients, or diluents.

[0161] The targeted caged prodrugs for use according to the methods disclosed herein may be administered as a single targeted caged prodrug or a combination of targeted caged prodrugs. For example, a caged prodrug may be administered as a single caged prodrug or in combination with another caged prodrug that treats cancer or that has a different pharmacological activity.

[0162] The pharmaceutical composition of the caged prodrug may include the caged prodrugs in a range of about 0.1 mg to about 2000 mg. The pharmaceutical composition of the radioemitter may include the radioemitter in a range of about 0.1 mg to about 2000 mg. In some embodiments, the concentration of the radioemitter is measured by its radioactivity (e.g., MBq / mL or mCi / mL). The pharmaceutical composition may be administered to the subject to provide the caged prodrug and / or the radioemitter at a dose of about 0.01 ng / kg to about 100 mg / kg body weight. In some embodiments, after the pharmaceutical composition is provided to a subject (e.g., after about 1 , 2, 3, 4, 5, or 6 hours postadministration), the concentration of the caged prodrug and / or the radioemitter within the targeted cell, tissue, or organ is about 1 pM to about 10 pM. In some embodiments, the uncaged prodrug, after uncaging by the ionizing radiation of the targeted radioemitter is about 1 pM to about 10 pM per cell.

[0163] The caged prodrugs and / or radioemitters utilized in the methods disclosed herein may be formulated as a pharmaceutical composition that includes one or more, suspending agents, preservatives, and buffers.

[0164] The caged prodrugs and / or radioemitters employed in the compositions and methods disclosed herein may be administered as pharmaceutical compositions and, therefore, pharmaceutical compositions incorporating the caged prodrugs and / or radioemitters are considered to be embodiments of the compositions disclosed herein. Such compositions may take any physical form which is pharmaceutically acceptable; illustratively, they can be subcutaneously administered pharmaceutical compositions. Such pharmaceutical compositions contain an effective amount of a disclosed caged prodrug and / or radioemitter, which effective amount is related to the dose of the caged prodrugs and / or radioemitters to be administered. Each dosage unit may contain the dose of a given caged prodrugs and / or radioemitters or each dosage unit may contain a fraction of the dose, such as one-half or one-third of the dose. The amount of each caged prodrugs and / or radioemitters to be contained in each dosage unit can depend, in part, on the identity of the particular caged prodrugs and / or radioemitters chosen for the therapy and other factors, such as the indication for which it is given.

[0165] Subject in Need of Treatment

[0166] The caged prodrugs, or pharmaceutical compositions thereof, and radioemitters or pharmaceutical compositions thereof of the present invention may be administered to a cell in need of killing. In some embodiments, the cell in need of killing is an abnormal cell. In some embodiments, the cell in need of killing is a cancer cell. In some embodiments, the cell in need of killing is found within a subject in need of a treatment. In some embodiments, the subject in need of a treatment may include a subject having a disease, disorder, or condition. In some embodiments, the condition may be cancer. In some embodiments the cancer may be a breast cancer, an ovarian cancer, an esophageal cancer, a stomach cancer, a colon cancer, a lung cancer, a skin cancer, a prostate cancer, a head and neck cancer, a bone cancer, a kidney cancer, a urinary tract cancer, a bladder cancer, a pancreatic cancer, a pediatric cancer, or a blood cancer.

[0167] In some embodiments, the cancer may be associated with an overexpression of a specific gene or protein (e.g., targets in Table 1 , Table 2, or Table 3), through which the cancer can be identified, targeted, and / or treated. Commonly overexpressed or mutated genes, their protein products, and targeting moieties (e.g., small molecules, peptides, proteins, or antibodies) are known in the art. For example, CD20 expression may be markers and targeting agent for most B-cell leukemias and lymphomas. Thus, a practitioner skilled in the art will be able to select a targeting moiety, discussed herein, in combination with a caged prodrug and / or radioemitter to treat a subject in need of treating (e.g., killing) a specific type of cancer.

[0168] Method of Treatment

[0169] The caged prodrugs and radioemitters, and their pharmaceutical compositions described herein, may be used for methods of treatment. In particular, the targeted caged prodrugs and targeted radioemitters, and their pharmaceutical compositions may be used for methods of treatment of a cancer. In some embodiments, the method of treating a cancer include treating a breast cancer, an ovarian cancer, an esophageal cancer, a stomach cancer, a colon cancer, a lung cancer, a skin cancer, a prostate cancer, a head and neck cancer, a bone cancer, a kidney cancer, a urinary tract cancer, a bladder cancer, a pancreatic cancer, a pediatric cancer, or a blood cancer.

[0170] In some embodiments, the targeted caged prodrugs and targeted radioemitters of the present invention co-localize to the same cells, tissues, or organs which express the antigen for the targeting moieties and effectively deliver the cytotoxic drug moieties caged by the caged prodrugs, upon exposure to ionizing radiation of the radioemitters. The localization and selective release of the drug moiety from the caged prodrug ensures minimal off-target cytotoxic activity by the drug moiety, while delivering a potent cytotoxic drug in a pharmaceutically relevant concentration (e.g., at or above IC50 concentrations) to kill the target cells.

[0171] In some embodiments, the targeted radioemitter is first provided to the subject followed by the targeted caged prodrug. In some embodiments, the targeted caged prodrug is first provided to the subject followed by the targeted radioemitter. In some embodiments, the targeted radioemitter is provided to the subject at least 4 hours prior to the targeted caged prodrug. In some embodiments, the targeted caged prodrug is provided to the subject at least 4 hours prior to the targeted radioemitter.

[0172] It should be appreciated that the order of administration of the caged prodrugs and the radioemitters is important, and is determined by the toxicity of the drug moiety, the half-lives of the radionuclide within the radioemitter, and / or the clearance times of the caged prodrugs or the radioemitters. Short Half-Life Radioemitters

[0173] In one aspect of the invention, the radioemitters of the present invention will include radionuclides that have short (e.g., < 1 day) physical half-lives. In embodiments where the radioemitters have a short halflives, the treatment methods include administration of the targeted caged prodrug prior to (e.g., > 4 hours) the administration of the targeted radioemitter. This allows the targeted caged prodrug to first localize within the cells, tissue, or organs of the subject prior to being exposed to ionizing radiation from the radioemitter. Once a suitable time has passed (e.g., 4 hours to 48 hours), the targeted radioemitter with a short half-life is administered to the subject, and allowed to localize within the cells, tissue, or organs which already hold the targeted caged prodrug, whereupon the ionizing radiation released by the targeted radioemitter uncages the caged prodrug and releases the Drug Moiety from the cage.

[0174] In some embodiments, the short half-life radioemitters include99mTc or123L

[0175] In some embodiments, the targeted caged prodrug is first delivered to the subject in need of treatment. In some embodiments, the targeted caged prodrug is allowed to localize based on the targeting moiety of the targeted caged prodrug for from 4 to 48 hours. Upon completion of the localization time, the targeted caged prodrug has either localized to the target cells, tissues, or organs, or has cleared from circulation. The targeted radioemitter, which includes the targeting moiety directing the radioemitter to the same cells, tissues, or organs as the targeting moiety of the targeted caged prodrug is then administered to the subject. Upon reaching the cells, tissues, or organs with the caged prodrug, the ionizing radiation released by the radioemitters uncages the drug, restoring its cytotoxic activity, leading to the death of cells into which it was localized. In some embodiments, the short half-life of the radioemitter minimizes the amount of caged prodrug released, minimizes potential off-target release, and / or minimizes radiation related complication associated with the radioemitter.

[0176] Long Half-Life Radioemitters

[0177] In one aspect of the invention, the radioemitters of the present invention will include radionuclides that have long (e.g., > 1 day) half-lives. In embodiments where the radioemitters have a long half-lives, the treatment methods include administration of the targeted radioemitter prior to (e.g., > 24 hours) the administration of the targeted caged prodrug. The targeted radioemitters are allowed to localize within the target cells, tissues, or organs and any radioemitters that have not been localized. In some embodiments, the non-localized radioemitter clears the system prior to the administration of the targeted caged prodrug, minimizing off-target release of caged prodrugs which have yet to localized. Use of long half-life radioemitters also allows for slower and / or longer release of the caged prodrug, ensuring prolonged exposure of the targeted cells to the cytotoxic drug. Furthermore, the longer half-life radioemitters may be utilized with multiple administrations of targeted caged prodrugs, ensuring longer exposure of the cytotoxic drug, upon uncaging with ionizing radiation, with a single administration of the targeted radioemitter.

[0178] In some embodiments, the targeted radioemitters include67Ga,68Ga,103Pd,1111 n,125l,131l,131Cs,133Xe,

[0179] 153Sm,149Pm,161Tb,169Er,177Lu,201TI, or203Pb Short Clearance Time Radioemitters or Caged Prodrugs

[0180] In one aspect of the invention, the targeted radioemitters and / or the targeted caged prodrugs of the present invention will include a targeting moiety (e.g., nanobody, peptide targeting agent, etc.) which shortens the clearance time (e.g., <24 hours) of the radioemitter and / or the caged prodrug. The shortened clearance time of the radioemitter minimizes off-target radiation delivery while ensuring a long enough dwell time to still allow for localization and release of the caged prodrug. Similarly, the short clearance time of the targeted caged prodrug minimizes off-target accumulation, which may cause off-target release and toxicity if utilized with longer half-life radioemitters. In some embodiments, the short clearance time radioemitters and / or caged prodrugs may include targeting moieties which are substantially small enough to clear via the kidney’s, bypassing the liver, to further minimize potential off-target (e.g., within the liver) release of the caged prodrug.

[0181] In some embodiments, the short clearance time targeting moieties include the radical of a radiolabeled peptide pharmaceuticals selected from any one of peptide targeting agents of Table 1. In some embodiments, the short clearance time targeting moieties include the nanobodies, affibodies and proteins of Table 3.

[0182] High-Toxicity Drug Moieties

[0183] In one aspect of the invention, the drug moiety contained within the caged prodrug is highly-toxic, wherein exposure to a small concentration (e.g., pM) would lead to cell death. In such situations, the delivery of the caged prodrug and the radioemitter must be managed, to minimize off-target release and activity of the caged prodrug.

[0184] In some embodiments, the high-toxicity Drug Moiety is the PBD dimer of Formula III. In some embodiments, the high-toxicity Drug Moiety is used with a short clearance time targeting agent, to minimize circulation times and off-target release of the Drug Moiety. In some embodiments, the high- toxicity Drug Moiety is used with a long half-life radioemitters, wherein the long half-life radioemitters are first administered to the subject, allowed to localize, and clear from the non-localized radioemitters from the system prior to administering the high-toxicity Drug Moiety containing targeted prodrug. In some embodiments, short clearance time targeting agents include a radical of a radiolabeled peptide pharmaceutical of Table 1 , or the targeting agents of Table 3.

[0185] In some embodiments, the high-toxicity Drug Moiety is utilized in combination with a short clearance time radioemitter, to ensure minimal off-target exposure. In some embodiments, the fast clearance time radioemitter is a targeted radioemitter that uses a radical of a radiolabeled peptide pharmaceutical of Table 1 or is one of the radiolabeled peptides pharmaceuticals of Table 1. In some embodiments, the fast clearance time radioemitter is PSMA-617.

[0186] In some embodiments, the targeted caged prodrug with the high-toxicity Drug Moiety is administered to the subject first, followed by the targeted radioemitter. In some embodiments, the targeted radioemitter is administered to the subject at least 4 hours after the targeted caged prodrug with the high-toxicity Drug Moiety. EXAMPLES

[0187] Table of Contents

[0188] The invention will be further described by the following non-limiting examples.

[0189] Example 1 : Localized in vivo Prodrug Activation Using Radioisotopes

[0190] This study assesses the feasibility of the “Radioisotope induced Drug Engagement for Release” (RAiDER) approach (FIG. 1) for prodrug activation using a recently developed chemical strategy for long-circulating radiation-labile drug conjugates. We hypothesized that local energy delivered by molecularly targeted radioisotopes could chemically activate prodrugs at disease sites while avoiding activation in off-target sites of toxicity. As proof-of-principle, we tested whether this strategy of “Radioisotope induced Drug Engagement for Release” (RAiDER) could locally deliver combined radiation and chemotherapy to maximize tumor cytotoxicity while minimizing exposure to activated chemotherapy in off-target sites.

[0191] We screened the ability of radioisotopes to chemically activate a model radiation-activated prodrug consisting of the microtubule destabilizing monomethyl auristatin E caged by a radiation-responsive phenyl azide (“caged-MMAE”) and interpreted experimental results using the radiobiology computational simulation suite TOPAS-nBIO. RAiDER was evaluated in syngeneic mouse models of cancer using fibroblast activation protein inhibitor (FAPI) agents

[0192] 99mTc-FAPI-34 and ^^Lu-FAPI-04, the prostate-specific membrane antigen (PSMA) agent ^^Lu-

[0193] PSMA-617, combined with caged-MMAE or caged-exatecan. Biodistribution in mice, combined with clinical dosimetry, estimated the relationship between radiopharmaceutical uptake in patients and anticipated concentrations of activated prodrug using RAiDER.

[0194] RAiDER efficiency varied by 250-fold across radioisotopes yielding up to 1.22 pM prodrug activation per Gy of exposure from 99mTCResults show that radioisotopes commonly used for diagnostic and therapeutic nuclear medicine can trigger intratumoral drug release from prodrugs without significant systemic toxicity nor off-target drug activation. Computational simulations implicated low-energy electron-mediated free radical formation as driving prodrug activation. Clinically-relevant radioisotope concentrations chemically activated caged-MMAE, restored its ability to destabilize microtubules, and increased its cytotoxicity by up to 600-fold compared to non-irradiated prodrug. Mice treated with 99nT]-c_FAp|_34anc| caged-MMAE accumulated up to 3000x greater concentrations of activated MMAE in tumors compared to other tissues. RAiDER with 99mTc-FAPI-34 or ^^Lu-FAPI-04 delayed tumor growth, while monotherapies did not (P<0.03). Clinically-guided dosimetry indicates that sufficient radiation doses can be delivered to activate therapeutically meaningful levels of prodrug.

[0195] It is demonstrated that RAiDER is compatible with multiple radioisotopes commonly used in nuclear medicine and has the potential to safely improve the efficacy of radiopharmaceutical therapies to treat cancer. RAiDER provides an effective strategy to treat disseminated malignancies and broadens the capability of radiopharmaceuticals to trigger diverse biological and therapeutic responses.

[0196] RESULTS:

[0197] Synthesis and chemical characterization of long-circulating radiation-activated prodrugs

[0198] Since therapeutic radioisotopes often exhibit long radioactive half-lives (177[_utl / 2 6.7 days) and deliver radiation over multiple weeks in patients. This takes advantage of the long circulating half-life of serum albumin (human tl / 2, ~3 weeks), its preferential uptake across multiple tumor types, and consists of covalently conjugating therapeutic payloads to albumin via a radiation- labile para-azido- 2,3,5,6-tetrafluorobenzyl (pATFB) moiety and self-immolating linker. The microtubule destabilizing monomethyl auristatin E served as the model therapeutic payload using this strategy (Alb-caged- MMAE). A novel caged version of the topoisomerase inhibitor exatecan (Alb-caged-exatecan) was developed and used to assess the generalizability of the approach in some experiments (Schemes 1 - 3). Constructs were stable in PBS (pH 7.4) for 4 weeks at 4°C (FIG. 8).

[0199] Scheme 1. Prodrug synthesis. Compounds S1-1a and 2a were synthesized according to prior literature.

[0200] (Sartor et al., New England Journal of Medicine. 2021 ;385:1091 -1103; and Ouyang et al., Nature

[0201] Materials. 2020;19:1362-1371)

[0202] Scheme 2. Synthesis of pATFB-SIL-exatecan (1b). Exatecan (40 mg, 101.9 pmol) was added to a solution of pATFB-SIL-PNP (S4, 53.4 mg, 90.0 pmol) in dry DMF (2 mL) and DIPEA (25 pL, 143.5 pmol) was added. This mixture was shaken at room temperature for 18h, then loaded directly onto a reverse- phase column (C18) and purified using a gradient of 5-95% acetonitrile in water (0.1% formic acid).

[0203] Fractions containing the product were combined and evaporated to provide a brown solid (36.8 mg, 45% yield).

[0204] 1H NMR (400 MHz, CDCI3): 6 8.23 (d, J = 9.2 Hz, 1 H), 7.63-7.55 (m, 2H), 7.41 (d, J = 9.1 Hz, 2H),

[0205] 7.18 (d, J = 8.8 Hz, 1 H), 7.09 (d, J = 8.2 Hz, 1 H), 7.03 (d, J = 9.3 Hz, 1 H), 6.89 (d, J = 9.2 Hz, 1 H), 5.90 (s, 1 H), 5.62 (dd, J = 26.0, 14.4 Hz, 1 H), 5.32 (s, 1 H), 5.28-5.20 (m, 4H), 5.11 (d, J= 16.1 Hz, 1 H), 3.77

[0206] (s, 1 H), 3.75-3.70 (m, 1 H), 3.67 (s, 3H), 3.30-3.07 (m, 2H), 2.00 (s, 3H),1 .89-1 .77 (m, 2H), 1.38 (d, J = 6.7 Hz, 1 H), 1.02-0.93 (m, 3H).

[0207] 13c NMR (101 MHz, CDCI3): 6 171.7, 168.0, 166.5, 161.7, 160.8, 159.2, 155.6, 153.7, 153.4, 153.0, 150.4, 149.9, 148.6, 148.1 , 144.9, 144.2, 143.6, 142.4, 140.3, 139.6, 137.3, 136.3, 133.3,132.8, 127.7, 126.8, 126.4, 124.1 , 123.4, 123.3, 119.8, 119.4, 116.9, 116.4, 116.0, 114.5, 113.8,112.8, 108.4, 107.9,

[0208] 107.2, 96.1 , 72.7, 70.9, 64.1 , 56.1 , 52.2, 51.5, 50.7, 48.0, 45.6, 38.9, 34.7,29.6, 21.6, 18.8, 9.4, 5.9.

[0209] 19F NMR (376 MHz, CDCI3): 6 -109.6, -142.2, -151 .1 .

[0210] MS: m / z calculated for C42H33F5N7O1Q+(M+H)+890.22, found 890.42.

[0211]

[0212] Scheme 3. Synthesis of pATFB-SIL-Mal-exatecan (2b). pATFB-SIL-exatecan (36.8 mg, 24.8 umol) was dissolved in methanol (8 mL) and 0.5M LiOH (2 mL) was added. This mixture was stirred at room temperature for 30 min, then quenched with acidic resin (~1 g). This suspension was stirred for 1 minute, then filtered and washed with methanol. The solvent was removed via rotary evaporation, and the resulting residue was dissolved in DMF (1 mL). HBTU (25 mg, 65.9 umol), Mal-PEG4-amine (100 uL of a 100 mg / mL solution in THF, 31.6 umol), and DIPEA (20 uL, 1 14.8 umol) were added, and the mixture was stirred for 16h. The reaction mixture was loaded directly onto a reverse-phase column and purified using a gradient of 5-95% acetonitrile in water (0.1 % formic acid) to provide the product as a brown solid (22.1 mg, 46% yield).

[0213] 1H NMR (400 MHz, DMSO-c / 6): 6 8.26 (d, J = 8.3 Hz, 1 H), 8.01 (d, J = 8.3 Hz, 2H), 7.95 (s, 1 H), 7.40- 7.26 (m, 5H), 7.07 (dd, J = 8.4, 4.3 Hz, 1 H), 5.30-5.18 (m, 3H), 4.41 (s, 1 H), 3.06-2.99 (m, 2H), 2.92-2.77 (m, 12H), 2.73-2.67 (m, 6H), 2.39 (s, 3H), 1.89-1.78 (m, 4H), 1.17-1.13 (m, 5H), 0.85 (s, 3H).

[0214] 19F NMR (376 MHz, DMSO-c / 6): 5 -69.2, -71.1 , -73.42.

[0215] MS: m / z calculated for C55H53F5N9O15+(M+H)+1174.36, found 1174.53.

[0216] Multiple radioisotope types can efficiently mediate RAiDER.

[0217] The ability of commonly used radioisotopes in clinical nuclear medicine to induce radiation-labile prodrug activation was evaluated in vitro. Alpha, electron / positron, Auger, and gamma emitters were assessed across preclinical and clinically relevant activities (FIG. 2A, Table 4). Results show roughly linear relationships between caged-MMAE activation and total dose imparted by each isotope. However, drug release efficiency between radioisotopes was variable (FIG. 2B).

[0218] 99mTc and 177[_ushowed highest efficiencies; 99mTc was ~7-fold more efficient than external beam radiation. This was followed by other beta and alpha-emitting isotopes (64cu, 68Qa223pa) The near-pure positron-emitting isotope, wasmuch less efficient. Diverse radioisotopes thus elicit meaningful drug release using the pATFB radiation-labile caging moiety. Table 4. Properties of isotopes and in vitro experimental conditions used in this study.

[0219] In vitro cytotoxicity of isotope-released prodrugs

[0220] Cytotoxicity of the isotope-released drug was tested in vitro in a panel of aggressive murine and human cancer models spanning across anaplastic thyroid cancer (TBP3743), prostate cancer (LnCAP), colon cancer (MC38), fibrosarcoma (HT1080), and ovarian cancer (BBPNM). Many of these diseases are less responsive to conventional chemotherapy. 99rrr]-cwas used due to its high drugactivating efficiency. Alb-caged-MMAE activation by 99mTCelicited up to a 600-fold increase in drug toxicity as measured by a 72h cell proliferation assay (FIG. 3A, FIG. 9, Table 5). 99rrr]-c a|One was not cytotoxic at activities used for drug activation (FIG. 10). Clonogenic assays revealed that 99mTc- activated Alb-caged-MMAE significantly blocked colony formation (FIG. 10B). Consistent with external irradiation, 99mTc-activated Alb-caged-MMAE restored the ability of MMAE to destabilize cellular microtubules and induce apoptosis, as seen by immunofluorescence and TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling), respectively (FIG. 3C). Therefore, radioisotope-activated caged-MMAE elicits biological effects in vitro consistent with the intended chemical prodrug release.

[0221] Table 5. Estimated 50% proliferation / cytotoxicity values from viability curves generated in FIG. 3A and FIG. 10 across different cancer cell lines across Alb-caged-MMAE, free inhibition (IC50) MMAE, and 99mTc+Alb-caged-MMAE treatments. RAiDER depends on energy-specific free-radical formation

[0222] Free-radical products of water radiolysis including superoxide (’02), hydroxyl radicals (’OH), and hydrated electrons underly many radiation-dependent chemical reactions, including pATFB- uncaging and radiopharmaceutical stability. We therefore assessed whether radical scavengers could inhibit RAiDER, finding that gentisic acid, ascorbic acid, and hydrogen peroxide could all reduce caged- MMAE prodrug activation by 99rrr]-c(FIG. 11 A). These data confirm RAiDER depends on the availability of free radical species generated by ionizing radiation.

[0223] Free-radical generation from water radiolysis is effected not only by direct ionization from initial radioisotope decay events but also by secondary electron cascades with progressively lower energies along the ionization track. The spatial extent of these cascades depends on the initial energy of the electron from the original ionization event. Isotopes releasing high-energy radiation, such as would be expected to generate ionization events over a broader volume at a lower concentration than isotopes generating lower energy initial electrons. The higher concentration of ionization cascades for the latter may improve the probability of local reaction with macromolecules. These assertions (FIG. 4A) were tested using TOPAS-nBIO, a Monte- Carlo simulation platform that can examine radiobiology processes at sub-cellular scales. Simulations examining the number of electrons generated by radioisotopes across different energy windows showed the best correlation with observed drug release across all isotopes for 100-110keV electrons (r = 0.89, P<0.0001 , FIG. 4B, 11B). We additionally simulated the dose deposited by LEEs within nanoscopic volumes (~10nm to reflect the hydrodynamic radius of Alb-caged-MMAE that would enable radicals to chemically interact with the prodrug), finding that the dose delivered by LEEs at this scale correlated better with drug release than the total dose imparted across all radioisotopes (FIG. 4C, 0.91 vs. 0.55, P<0.0001).

[0224] We experimentally tested the hypothesis that lower energy radiation enables more efficient drug release using external beam irradiation. X-rays from mammographic systems are less energetic (<50 keV) than other conventionally used diagnostic and therapeutic irradiation devices. We simulated the energy spectra of two low-energy anode / filter combinations (W-AI and W-Rh) using SpekPy (Vorbau et al., SpekPy Web — online x-ray spectrum calculations using an interface to the SpekPy toolkit. J Appl Clin Med Phys. 2024;25:e14301.). Parameters are shown in Table 6. Spectra generated from W- Al and W-Rh delivering ~0.5 Gy showed that both filters generate higher fluence at lower energies (<60 keV) than irradiation at 320 keV (FIG. 12A). We observed that mammography-generated X-rays would lead to more efficient drug release (FIG. 12B, P<0.0001). Together, results indicate that RAiDER is driven by the local concentration of free-radicals from LEEs and recombination effects of relatively low energy radiation. Table 6. SpekPy parameters for simulation.

[0225] W W 32

[0226] Pfiysfcs kqp kqp qp enuation d Penelope Penelope Pene ergy bin (k 0.1 0.1 0.1 rget materia! W W W

[0227] Radioisotope-mediated drug release in vivo.

[0228] Next, we assessed the ability of RAiDER to mediate drug release in vivo, using the TBP3743 syngeneic mouse model of anaplastic thyroid cancer, an aggressive type of cancer characterized by poor clinical prognosis and traditional chemoradiotherapy resistance. Immunofluorescence indicated that TB3743 tumors express mouse fibroblast activation protein (muFAP, FIG. 13). We treated mice bearing TBP3743 tumors with fluorescent Alb-caged- MMAE (Cy5-Alb-caged-MMAE), 99mTc-FAPI-34(43) administration 48h later, and tissue harvesting 24h later to assess biodistribution and prodrug activation (FIG. 5). This showed highest accumulation and colocalization of both Cy5 fluorescence and 99mTc-FAPI-34 in tumors. Other tissues examined showed uptake that was lower and / or discordant between the two agents. Selectivity for active MMAE accumulation was dramatically enhanced in tumors, 20-3000x higher than in other tissues (FIG. 14A). Minimal caged-MMAE activation was seen without 99mTc-FAPI-34 (FIG. 5). Estimated dosimetry shows that in vivo prodrug activation by 99mTc-FAPI-34 roughly matches values observed in vitro (Table 7, FIG. 14B).

[0229] Table 7. Estimated dosimetry of 99mTc-FAPI-34 based on time-injected activity curves derived from FIG.

[0230] 5 and FIG. 15 using MIRD formalism (Behranvand et al. Cancer immunology, immunotherapy.

[0231] 2022;71 :507-526).

[0232] ★Based on 18 MBq injected Expected tumor [MMAE] based on in vitro results: 1.45E02 nM

[0233] To evaluate the generalizability of RAiDER, we performed analogous biodistribution assays using a syngeneic murine model of prostate-specific membrane antigen (PSMA)-expressing prostate cancer (RM1 .PSMA). As a proof-of-principle, we treated tumor-bearing mice with Alb- caged-exatecan and 1 7[_U-PSMA-617 (lutetium Lu 177 vipivotide tetraxetan), which is used clinically to treat PSMA+ metastatic prostate cancer. ^^Lu-PSMA-617 accumulated in tumors and triggered the local accumulation of activated exatecan more selectively in tumors compared to when free exatecan was administered as a control (FIG. 15). Taken together, biodistribution analysis indicates RAiDER efficiently and selectively releases active drugs at targeted tumor sites and is generalizable across therapeutic payloads, disease models, radioisotopes, and their targeting strategies.

[0234] RAiDER improves radioisotope efficacy to slow tumor progression.

[0235] \Ne next evaluated whether in vivo radioisotope-mediated prodrug activation could translate into detectable effects on disease progression. TBP3743 tumor-bearing mice were treated with Alb- caged-MMAE, 99mTc-FAPI-34, or their combination, and tumor growth was monitored. Tumor growth was delayed with combination treatment but not monotherapy (FIG. 6A; compared to control: 2-way ANOVA with repeated measures: P<0.001 , Day 11 : P<0.01). No noticeable toxicity was observed, as evidenced by mouse weight changes during treatment, blood biomarkers, and histology (FIG. 16, 17). Of note, equimolar doses of free MMAE are known to be toxic in mice.

[0236] We additionally tested RAiDER using 177[_U_FAP|_O4 using a single injection to account for the long half-life of 177|_uCombination treatment significantly delayed tumor growth but monotherapy did not (FIG. 6B; compared to control: 2-way ANOVA with repeated measures: P<0.02, Day 16: P<0.03), all without detectable toxicity. Together, these results demonstrate that RAiDER can safely improve radiopharmaceutical efficacy in a mouse model of aggressive cancer. Clinically realistic dosimetry is compatible with RAiDER.

[0237] Feasibility of RAiDER in patients was estimated using clinical dosimetry. Expected drug release in prostate and neuroendocrine tumors in patients treated with ^^Lu-PSMA-617 and ^^Lu- DOTATATE, respectively, was estimated based on published tumor dosimetry (Watabe et al., J Nucl Med. 2020;61 :563-569. And Violet et al., J Nucl Med. 2019;60:517-523.) and drug activation efficiencies shown in FIG. 2. Tumor accumulation of caged prodrugs was assumed not rate-limiting based on its observed biodistribution (FIG. 5) and prior pharmacokinetic modeling (Quintana et al. Cent. Sci. 2024;10:1371-1382). Dosimetric analysis showed that radiopharmaceutical uptake in all lesions examined would be sufficient to mediate drug release well above the reported in vitro IC50 values of MMAE that block cancer cell proliferation (FIGs. 3, 9). Moreover, the standardized uptake values of these lesions are well within range of those seen in typical patients receiving radiopharmaceutical therapy.

[0238] Extrapolation of these observations to other radiopharmaceuticals was assessed using MIRDcalc (Stenvall et al., EJNMMI Res. 2022;12:75.), using published pharmacokinetic data of agents currently in human use (Kesner et al. J Nucl Med. 2023;64:1117-1124; Velikyan et al., J Nucl Med.

[0239] 2014;55:204.; and Schuchardt J Nucl Med. 2022;63:1199-1207.). Higher linear energy transfer (LET) alpha-emitting isotopes are understood to deliver significantly higher tumor dose per unit of activity compared to beta- and Auger-emitting isotopes regardless of the delivery vehicle (2 -way ANOVA with Tukey’s multiple comparison correction, P<0.02). Scaling these values to clinically used activities (alpha: 20MBq, beta: 7400 MBq, Auger: 1110 MBq), followed by an estimation of drug release based on the tumor dose delivered at these activities, shows that all classes of isotopes, and especially 177Lu, are capable of mediating drug release to enact meaningful (>IC50) cytotoxicity across a range of tumor sizes and composition. This analysis indicates that RAiDER is appropriate across a range of clinical applications, including for patients with borderline RPT lesion uptake who may be resistant to RPT monotherapy.

[0240] SUMMARY

[0241] This study presents RAiDER as a method to harness ionizing radiation imparted by radioisotopes not only for their radiotoxic effects on tumor cells but also to mediate the localized release of complementary molecular therapy. Timed co-administration of caged drug payload (Alb-caged- MMAE) and its partner radiopharmaceutical enabled tumor colocalization of the two agents, enhanced tumor drug release, and reduced systemic exposure to free drug by 20-3000x in off-target tissues. Preclinical studies showed improved tumor control with RAiDER compared to RPT monotherapy without appreciable systemic toxicity.

[0242] Albumin-bound radiation-activated prodrugs were used in this proof-of-principle demonstration since serum albumin circulates for an extended period of time in the body via FcRn-mediated recycling; its 66.5kDa molecular weight makes it smaller than antibodies and nanoparticles and promotes its ability to penetrate tissues; and it accumulates in cancer cells and phagocytes across multiple tumor types facilitating translation of this approach across different malignancies. The pATFB and alternative radiation-labile chemistries have been conjugated with other macromolecules or drug delivery vehicles, including nanoparticles or antibodies, likely applicable to RAiDER. Since the phenyl azide caging moiety is less sensitive to positron- emitting 1 Bp, imaging versions of prodrugs could, in principle, be synthesized to provide a companion theranostic to guide treatment planning.

[0243] The above-described results were obtained using the following materials and methods.

[0244] MATERIALS AND METHODS

[0245] Synthesis of the radio-cleavable prodrug linker for RAiDER.

[0246] Unless stated otherwise, all materials were used as received from commercial sources. N,N- diisopropylethylamine, N,N-dimethylformamide, bis(4-nitrophenyl) carbonate, lithium hydroxide, and HBTU were purchased from Sigma Aldrich (St. Louis, MO, USA). Hydrochloric acid (HCI), methanol, dichloromethane, and acetonitrile were purchased from VWR International (Radnor, PA, USA), while Monomethyl auristatin E (MMAE) and exatecan were purchased from MedChem Express (Monmouth Junction, NJ, USA). Maleimide-PEG4-amine trifluoroacetic acid salt was purchased from BroadPharm (San Diego, CA, USA) and CDCI3 was purchased from Cambridge Isotope Laboratories (Tewksbury, MA, USA). Reaction mixtures were purified using a Biotage Star Bio C18 (300 A, 10 g) on a BUCHI C- 850 FlashPrep with a gradient composed of water (0.1 % formic acid) and acetonitrile (0.1% formic acid) for reversed-phase chromatography. ^ H, and I^C NMR spectra were recorded on a Bruker AC-400 MHz spectrometer. High-performance liquid chromatography-mass spectrometry (HPLC-MS, LCMS) analysis was performed on a Waters instrument equipped with a Waters 2424 ELS Detector, a Waters 2998 UV-Vis Diode array Detector, a Waters 2475 Multi-wavelength Fluorescence Detector, and a Waters 3100 Mass Detector. Separations employed an HPLC-grade water / acetonitrile solvent gradient. Columns: Xterra MS C18 Column, 125, 5 pm, 4.6 mm X 50 mm column. All samples were run on an Xterra MS C18 column using a gradient of 5 to 95% acetonitrile in water (0.1% formic acid) over 1 .5 minutes, followed by 95% acetonitrile for 0.5 minutes at a flow rate of 5 mL / min.

[0247] Caged-MMAE and caged-exatecan were synthesized as outlined in Scheme S1 , guided by previous examples (Sartor et al., New England Journal of Medicine. 2021 ;385:1091-1103). Briefly, the drugs were conjugated to the pATFB-SIL (para-azido-2,3,5,6- tetrafluorobenzyl self-immolative linker) via a carbamate linkage under basic conditions, followed by ester hydrolysis and amide coupling to install a maleimide anchor. The resulting prodrugs were characterized via LCMS and NMR. Synthetic details can be found below in the section following (Schemes 1-3, FIGs. 12, 18). Maleimide was reacted with the free cysteine on mouse serum albumin to yield albumin-conjugated caged prodrugs. The reactive fluorophore Cyanine5-NHS ester (Lumiprobe) was conjugated to serum albumin prodrug via amine coupling (DOL: 3.2) for biodistribution experiments (Sartor et al., New England Journal of Medicine. 2021 ;385:1091-1103). Conjugate stability was determined by treatment of the conjugates with tris(2- carboxyethyl)phosphine (TCEP, 10eq), which reduces the azide linker via a Staudinger reduction, leading to release of the free drug. Prodrug aliquots were purified at various time intervals via spin filtration through a 10kDa molecular-weight cutoff centrifugal filter (Amicon) 10,000 ref for 7 min), and the free drug was quantified via LCMS to determine the presence of intact prodrug over 30 days. Prodrug activation using radioisotopes in vitro.

[0248] Radioisotopes examined in this study, their source, and conditions used for their testing are outlined in Table 4 (above) . For in vitro studies, 10 pM of caged-MMAE was incubated with varying activities of each isotope for defined periods (Table 4) in 2 mL HPLC glass vials (Thermofisher, USA) and buffered with 10x PBS to achieve a volume of 1 mL at pH 7.4 and a final concentration of 1xPBS. Samples were kept at least 0.5 cm apart during incubation at room temperature. No appreciable cross-vial mediated drug release was found in the control vials placed in this configuration in pilot studies. Samples for which the incubation time was shorter than the physical half-life of the radioisotope were frozen and stored in a liquid nitrogen tank until further analysis could be performed. This cryopreservation prevented further drug release from the pATFB linker (FIG. 8). Chemical quenching studies were performed with gentisic acid, ascorbic acid, and hydrogen peroxide (Millipore Sigma).

[0249] Prodrug activation using external radiation.

[0250] Prodrug activation was additionally tested with external beam radiation. Samples were prepared as above, except in 96 well plates diluted to a volume of 100 pL. A total of five irradiation modalities were used:

[0251] X-ray irradiation: X-ray irradiation used an X-RAD320 cell and small animal irradiator (Strosberg et al. NETTER-1 phase III: Progression-free survival, radiographic response, and preliminary overall survival results in patients with midgut neuroendocrine tumors treated with 177-Lu-dotatate. American Society of Clinical Oncology; 2016) with a 320keV energy and dose rate of 325 ± 10cGy min"1 at room temperature (Precision X-ray).

[0252] Parameters for X-ray irradiation were 320kV, 12.5mA, and HVL ~ 1 mm Cu, 2mm Al filter.

[0253] Gamma irradiation: Irradiation was performed on a dual source 137QSGammacell 40 Exactor (Best Theratronics) with a dose rate of roughly 50 cGy min"1 .

[0254] Protons: Proton beam irradiations were performed at the Francis H. Burr Proton Therapy Center with a single field impinging vertically on the samples. The samples were placed at the center of the spread- out Bragg peak (SOBP) with a range of 13 cm and a modulation width of 7 cm, resulting in a flat dose distribution at and around the sample with a linear energy transfer (LET) ~2.3 keV prrrl and a dose rate of ~0.5 Gy s" The LET was estimated using Monte Carlo simulations with TOPAS, which was previously well-tested for this beamline (Bodei et al., Nature Reviews Clinical Oncology. 2022;19:534- 550).

[0255] MV Linac: Clinical energy (6 MV) photon irradiations were performed at the MGH Clark Center using Varian Truebeam Linacs. A solid water phantom size of 5 or 10 cm depth was used to simulate the internal scatter expected with biological tissues.

[0256] Mammography: This was performed with a Hologic 3Dimensions mammography system. Exposure dose was measured using a portable calibrated dosimeter (Raysafe X2) connected to a solid-state mammographic sensor (X2 MAM). If necessary, samples were stored at 4°C until analysis. The linearity of drug release using this approach across different doses and multiday experimental sessions was confirmed before additional experiments (FIG. 12C).

[0257] Estimation of drug release.

[0258] Samples were analyzed using LCMS (Waters instrument equipped with a Waters 2424 ELS Detector, Waters 2998 UV-Vis Diode array Detector, and a Waters 3100 Mass Detector) if the incubation time exceeded 10 half-lives of the relevant isotope or using an Agilent 1200 Series HPLC, with a multichannel-wavelength UV / Vis detector (G1365D), fluorescence detector (G1321A), and a flow- through y-detector (35900E), using PBS mobile phase at a flow rate of 0.7 mL min-'!) if the sample was radioactive at the time of analysis. On both instruments, an Xterra MS C18 Column (Waters; 124A, 5 pm, 4.6 x 50 mm) column was used, with a gradient of 5-95% acetonitrile in water with 0.1 % formic acid as the mobile phase. Drug concentration from LCMS and LC / UV samples were quantified by comparing the area under the curve from the ELSD, isolated mass chromatographs (+ESI 718.8 Da for MMAE, 436.4 Da for exatecan), or UV absorbance (210 nm for MMAE, 320 nm for exatecan) of each sample to a 7-point standard calibration curve. LCMS and UV standards were cross-calibrated to allow for direct comparison.

[0259] Cell culture

[0260] Mouse cancer cell lines underwent mouse pathogen testing (IDEXX) before use and all cells were routinely tested for mycoplasma contamination (Mycoplasma PCR test Kit, Applied Biological Materials). TBP3743 (murine anaplastic thyroid cancer), HT1080 (human fibrosarcoma), BPPNM (murine ovarian cancer), LnCAP (human prostate cancer, ATCC), RM1.PSMA (murine prostate cancer with prostate-specific membrane antigen, PSMA, constitutively expressed using a PiggyBac transposase cassette, plasmid procured from VectorBuilder with original cell line obtained from ATCC), MC38 (murine colorectal cancer), and iKras (murine pancreatic cancer) cells were prepared as previously described (Inderjeeth et al., Novel radionuclide therapy combinations in prostate cancer. TherAdv Med Oncol. 2023;15:17588359231187202). TBP3743, HT1080, RM1.PSMA, and MC38 were cultured in DMEM, while LnCAP used RPMI, all supplemented with 10% fetal bovine serum (FBS) and penicillin / streptomycin (P / S) under an atmosphere of 5% CO2- AC37 mouse cancer cells with doxycycline-inducible KrasG12D expression (iKras, a gift from Dr. Haoqiang Ying, MD Anderson Cancer Center, by way of Dr. Nabeel Bardeesy, MGH), derived from triple transgenic p48-Cre; ROSA26-LSL-rtTa-IRES- GFP;TetO-LSL-KrasG12D genetically engineered mouse model of pancreatic ductal carcinoma (Adant et al., Combination treatments to enhance peptide receptor radionuclide therapy of neuroendocrine tumours. European Journal of Nuclear Medicine and Molecular Imaging. 2019.), were maintained in DMEM / Nutrient Mixture F-12 media (DMEM / F12, Invitrogen) supplemented with 2 pg / mL doxycycline (Sigma). The BPPNM cells (Dagher et al., Cell.

[0261] 2023;186:1814-1814. E1811) were cultured in DMEM supplemented with 1 % insulin-transferrin- selenium (Thermo Fisher Scientific), epidermal growth factor (2 ng / mL), 4% heat-inactivated FBS (Thermo Fisher Scientific), and 1 % P / S. Cytotoxicity assay ofcleaved drugs.

[0262] Cytotoxicity experiments were performed by seeding 3000 cells per well overnight in a 96-well plate (Corning) before adding each drug / conjugate. MMAE or prodrug (with or without exposure to [99nr]-c]TCQ4- for at |east 60h to promote drug activation) were prepared at varying concentrations ([MMAE], 1 pM and subsequent 2-fold dilution to 1 nM) in media. Empty wells with only media or vehicle treatment served as controls. After adding the corresponding drug condition and a 72 h incubation, PrestoBlue (ThermoFisher, USA) determined the number of live cells according to the provider’s protocols.

[0263] Radiotoxicity assay

[0264] 3000 cells per well were seeded overnight in a 96-well plate (Corning) before adding [99mTc]TcO4" at varying concentrations. Empty wells with only media or vehicle treatment served as controls. After a 72h incubation, PrestoBlue (ThermoFisher, USA) determined the number of live cells.

[0265] Colony formation assay

[0266] 1 x 1C)4 cell lines were placed in each well of 96-well plates and incubated overnight. The cells were treated with either a control solution or 99mTc-activated caged-MMAE. After 48h, the cells were harvested and reseeded into individual wells of a 6-well culture plate in fresh medium at 200 cells / mL and allowed to form colonies for 1 to 3 weeks. Once colonies had developed, they were fixed with 100% methanol for 20 minutes, stained with a crystal violet staining solution (Sigma) for 20 minutes at room temperature, and then rinsed with distilled water. Plating efficiency (PE) and survival fraction were calculated as previously described: Survival fraction = (number of colonies formed after treatment) / (number of cells initially seeded x PE) x 100%, plating efficiency (PE) = (number of colonies formed for untreated cells) / (number of cells initially seeded) x 100%. The plating efficiency for non-irradiated cells was determined to be 95%.

[0267] Immunofluorescence and TUN EL assays

[0268] 1 x 1C)4 TBP3743 cells were seeded in each well of 96-well plates overnight. TBP3743 cells were then treated with vehicle, Alb-caged-MMAE (with and without 99mTc-mediated cleavage), or MMAE at a concentration of 100nM. After 6h, the cells were washed three times with PBS, replaced with fresh cell culture media, irradiated with 10 Gy X-rays, and incubated for 24h (for TUNEL marker staining) or 48 h (for a-tubulin immunofluorescence). Cells were then fixed with 4% paraformaldehyde (PFA) for 30 minutes at room temperature (RT), washed in PBS, permeabilized in 0.5% Triton X-100 in PBS for 30 minutes, and blocked with a 10% serum from the same species as the secondary antibody for 20 mins at room temperature. A-tubulin Alexa Fluor 488 mouse monoclonal antibody (DM1 A, 1 : 100, Invitrogen) was used for staining.

[0269] Apoptotic cells were determined using a DeadEnd Fluorometric TUNEL System (Promega) according to the manufacturer’s instructions. All samples were mounted with a mounting solution containing DAPI to stain the nuclei and stored until imaging. Fibroblast activating protein (FAP) expression in TBP3743 tumors was confirmed using immunofluorescence. Tumor samples were harvested and stored in 4% paraformaldehyde overnight and subsequently in sucrose 15% solution for 12h, then in sucrose 30% solution overnight, and finally stored in 1xPBS until sectioning. Intact tissue was prepared for sectioning by embedding using Optimal Cutting Temperature (OCT, Sakura) in a cryomold and frozen on dry ice. 10 pm cryosections were prepared on glass slides. Tissue sections were first blocked with blocking buffer (5% normal goat serum, 0.2% Triton X-100, 5% BSA 1 x PBS) for 2 h at RT. Samples were then incubated with a primary antibody (mouse fibroblast activation protein mFAP antibody, R&D system, monoclonal Rat lgG1 stock: 0.5 mg / ml, 1 :50 dilution in wash buffer: 10% normal serum, 0.2% Triton x-100 0.2% in PBS) overnight at 4°C in a humidified staining chamber. Samples incubated with only the secondary antibody served as negative controls. Slides were then washed with PBS 3 times, followed by staining with a secondary antibody for 1 h at RT (AF488 anti-rat lgG2b antibody, Biolegend, Mouse lgG1 : 0.5 mg / ml. Clone: MRG2b-85, 1 :200 dilution in wash buffer), and mounted with VECTASHIELD@ Antifade Mounting medium with for further analysis.

[0270] Slide-mounted samples were imaged using a fluorescent microscope (Revolve, Discover Echo). Images were analyzed using Imaged and / or CellProfiler. A-tubulin and TUNEL stains were quantified by calculating the positive signal normalized to the vehicle control.

[0271] Preparation of ^mTc and 1 Lu labeled FAPI and PSMA targeting agents

[0272] 99mTc-FAPI-34, ^^Lu-FAPI-04, and ^^Lu-PSMA-617 used in this study were prepared as guided by prior reports (8,9). Precursors were commercially obtained (MedChemExpress). Reduction of [99mTcO4]‘ to [99mTC(co)3]+wasperformed using a formulation similar to the IsoLink kit(Bailly et al., Combined cytotoxic chemotherapy and immunotherapy of cancer: modern times. NAR cancer. 2020;2:zcaa002.), consisting of 8.5 mg sodium tartrate Na2C4H4O6, 2.85 mg sodium tetraborate Na2B4O7, 7.15 mg sodium carbonate Na2CO3 and 4.5 mg sodium boranocarbonate Na2H3BCO2 constituted in 1 mL ultrapure milliQ water (Millipore, Billerica, MA, USA) in a 10mL glass vial and degassed with nitrogen for at least 15 minutes. This was subdivided into aliquots of 260 pL in microcentrifuge tubes with rubber ring-sealed screw caps (Nalgene, Rochester, NY, USA) under an anaerobic environment for further use. For labeling, up to 1 GBq [99mTcO4]‘, generator elulate (100 pL) was added to each tube and heated for 30 min at 100°C. The microcentrifuge tube was then allowed to cool to RT, and the solution neutralized with 25 pL of 1 M HCI to pH approximately 7.5, giving a total volume of 125 pL of [99mTc(CO)3]+

[0273] The radiochemical purity of the [99mTc(CO)3]+produced was checked with thin layer chromatography (TLC) plates (3 cm x 7.5 cm, Merck, Darmstadt, Germany) using a mobile phase of 1 % HCI in methanol. Plates were analyzed with a gamma ray radioTLC scanner (AR2000). [99mTc(CO)3]+was then added to a mixture of 5 pL of the individual FAPI-34 precursor (1 mM in water). The reaction was then heated to 95°C for 20 min.

[0274] Labeling completeness of all three radiopharmaceuticals was monitored by radioTLC (FIG. 20) and processed by solid-phase extraction, evaporation, and formulation with 0.9% saline before therapy or biodistribution experiments. iTLC was performed by loading each chelate solution (1 pL) onto glass microfiber chromatography paper impregnated with silica gel (iTLC-SG, Agilent), then run in a solution of 0.2 M sodium acetate (pH 6). The iTLC was then analyzed on an AR-2000 (Eckert & Ziegler) TLC scanner.

[0275] Mouse experiments.

[0276] All animal research was performed under guidelines and approval from the local Institutional Animal Care and Use Committee. Mice were housed in a pathogen-free vivarium with controlled temperature, humidity, and light / dark cycling. B6129SF1 / J (TBP) and C57BL6 / J (RM1 .PSMA) mice were purchased from the Jackson Laboratory (JAX).

[0277] Biodistribution ofcaged prodrugs and co-administered radioisotope.

[0278] TBP3743 Anaplastic Thyroid Cancer Model'. 5-12-week-old female B6129SF1 / J mice were inoculated with 5 x 1 o5 TBP3743 cells subcutaneously. Once tumor reached ~100 n roughly 10 days later, fluorescent Alb-caged-MMAE (Cy5-MSA-pATFB-MMAE) was injected intraperitoneally (13.9 mol / kg;

[0279] 10 mg / kg free MMAE equivalent). A subset of these mice was injected with 18.5MBq 99mTc-FAPI-34 intravenously via retro-orbital injection 48 h later. Tissues were harvested from the mice after an additional 24h. Tissues from additional mice obtained 8 h after 99mTc-FAPI-34 injection were also obtained for dosimetry. Biodistribution of 99mTc-FAPI-34 was performed using a gamma counter (Perkin-Elmer), calibrated with known 99mTCstock standards. Data was decay-corrected to enable cross-comparison across samples. The biodistribution of caged-MMAE (Cy5-MSA-pATFB-MMAE) was calculated by quantifying fluorescent imaging taken on the Azure Sapphire FL imaging system using stock standards.

[0280] Quantification of the images was performed using Imaged, as previously described (Quintana et al., Extended Pharmacokinetics Improve Site-Specific Prodrug Activation Using Radiation. ACS Cent Sci. 2024;10:1371 -1382.). Tissues were then cryopreserved until extraction of MMAE from tissue to estimate drug release was performed.

[0281] RM1.PSMA Prostate Cancer Model: 5-12 week-old male C57BL / 6J mice were inoculated with 2.5 x 10^ RM1 .PSMA cells subcutaneously. Roughly 7 days later, once tumors reached approximately -100 mm3 in volume, Alb-caged-exatecan was injected intraperitoneally (5mg / kg free exatecan equivalent), with 1 7[_U-PSMA-617 (74.5 MBq) injected 48h later. After an additional 24h, tissues were harvested to assess for biodistribution and drug release of Alb- caged-exatecan as outlined below.

[0282] Preclinical dosimetry.

[0283] 99mTc-FAPI-34 uptake across tissues over 24 h was fitted with a triexponential decay curve using Python (curve_fit package on SciPy) to estimate the time-integrated activity curves. The absorbed dose was estimated using the MIRD-formalism, with literature-derived S-values (Kesner et al., MIRD Pamphlet No. 28, Part 1 : MIRDcalc — a software tool for medical internal radiation dosimetry. J Nucl Med. 2023;64:1117- 1124.; Hindie et al., Dose Deposits from 90Y, 177Lu, 1111n, and 161Tb in Micrometastases of Various SizesJmplications for Radiopharmaceutical Therapy.J Nucl Med. 2016;57:759.).

[0284] Anti-tumor efficacy assay

[0285] 5X1 C)5 TBP3743 cells in PBS were implanted subcutaneously in 5-12 weeks old female B6129SF1 / J mice. When tumors were palpable (4-5 mm diameter), mice were randomized and injected intraperitoneally with vehicle control or caged-MMAE (Alb-caged-MMAE, 5 mg / kg equivalent free MMAE) in 100 pL PBS. 48 h later, 18.5 Mbq 99mTc-FAPI-34 or 17?Lu-FAPI-04 were injected by tail vein. Subsequent doses of caged-MMAE and 99m-rc_FAp|_34were given as indicated in FIG. 6, including 4h after 99mTc-FAPI-34. Body weights were monitored and tumor volumes were calculated using two sets of digital caliper measurements and the equation V = length x width^ / 2. Prespecified euthanasia criteria included ulceration, tumor sizes greater than 20 mm in diameter (or 10 mm if there were multiple tumors per mouse), body condition score < 2, or weight loss greater than 20%.

[0286] Longitudinal tumor growth was plotted as a means for each group until any animals in the group reached the predefined humane experimental endpoint. Toxicity was assayed with complete blood counts, blood chemistry, and histological analysis of the liver and kidneys at the experimental endpoint, performed by the MGH Center for Comparative Medicine Veterinary Pathology Core.

[0287] Tissue extraction of activated prodrug:

[0288] Tissues were harvested at defined time points, weighed, then stored in liquid nitrogen until extraction analysis. Extraction was performed by placing tissue in Lysis buffer II (ThermoFisher, 200 pL; with 1X Halt Protease Inhibitor Cocktail). The tissues were finely minced and incubated for 30 minutes on ice, then diluted 4-fold with acetonitrile and centrifuged (5,000 ref for 5 min). The supernatant was filtered through 3 kDa MWCO spin filters to remove any remaining tissue fragments and proteins. The flowthrough was analyzed by LCMS or HPLC as above to determine the amount of drug released. Measurements were collected in triplicate and compared to a drug calibration curve to determine concentrations normalized to the mass of the collected tissues (extraction normalization).

[0289] In silico modeling of isotope-dependent drug release efficacy

[0290] Simulations of Alb-caged-MMAE interactions with radioisotopes were performed with the Monte Carlo toolkit OpenTOPAS and its extension TOPAS-nBio(14) for nanoscopic and microscopic simulations. Simulations were performed using geometries at two spatial scales, (a) First, the vial in which radioisotopes and Alb-caged-MMAE were incubated was modeled as a liquid water cylinder of 12.74 mm in height and 10 mm in diameter, similar to that used for the in vitro experiments described above. Different concentrations of Alb-caged-MMAE were added by randomly placing the corresponding number of macromolecules, considered to approximate spheres of 10 nm in diameter. In turn, radioisotopes were assumed to be uniformly distributed in the water so that positions for the emission were randomly selected within the volume of the vial. For each case, 10 histories were simulated, scaling by the actual number of decays to provide each history with an appropriate statistical weight. Using the g4-livermore physics model for electromagnetic processes, we calculated the absorbed dose delivered by each radionuclide to the entire vial, considering all the energy-delivering particles and discriminating by each particle. Particles considered were: a-particles, all electrons, electrons producing ionization; electrons undergoing multiple scattering; photons; low-energy electrons (LEEs) defined as those with energy lower than 30 eV; and positrons. Additionally, virtual spheres of 10 pm in diameter surrounding the macromolecules were used to store the phase space reaching their surfaces, i.e., the number, type, energy, position, and momentum of all the particles impinging the spheres. This was stored as phase space files and used as the source for (b) microscopic simulations. In this case, a single nanoparticle of 10 nm diameter was set in the middle of a virtual sphere of 10 pm in diameter made of liquid water. The particles recorded at all the bigger spheres from the macroscopic simulations were simulated using the track-structure geant4-dna (option 2) physics models, which provide more detailed ionization clouds than the g4-livermore physics. We determined the spectrum of electrons reaching macromolecules using the detailed track structure simulations.

[0291] Feasibility assessment of radioisotope-mediated drug release in patients

[0292] Dosimetry of cancer lesions in patients was estimated using MIRDcalc (Kesner et al., MIRD Pamphlet No. 28, Part 1 : MIRDcalc — a software tool for medical internal radiation dosimetry. J Nucl Med. 2023;64:1117-1124.) based on time activity curves inferred from publicly available imaging datasets or published reports of commonly used radiopharmaceutical agents (Velikyan et al., Quantitative and qualitative intrapatient comparison of 68Ga-DOTATOC and 68Ga-DOTATATE: net up-take rate for accurate quantification. J Nucl Med. 2014;55:204.; Jia et al., The role of [99mTc]Tc-HFAPi SPECT / CT in patients with malignancies of digestive system: first clinical experience. EurJ Nucl Med Mol Imaging.2023; 50: 1228- 1239.; Schuchardt et al., Prostate-Specific Membrane Antigen Radioligand Therapy Using (177)Lu-PSMA l&T and (177)Lu-PSMA-617 in Patients with Metastatic Castration- Resistant Prostate Cancer: Comparison of Safety, Biodistribution, and Dosimetry. J Nucl Med.

[0293] 2022;63:1199-1207.). If required, curves were fitted with a triexponential decay curve to enable estimation of the time-integrated activity curve using Python. Dose per unit activity (Gy / MBq) metrics were calculated for major organs and for tumors of varying sizes and soft tissue composition. Alb- caged-MMAE drug release in tumors was then estimated by applying the expected tumor dose delivered with clinically relevant radioisotope activities to drug release efficiency factors measured in FIG. 2B.

[0294] Statistical analysis

[0295] Data were analyzed using GraphPad Prism, MATLAB, and Excel. Normality was tested with the Shapiro-Wilk Test. Specific details of the tests applied are reported in the results. P<0.05 was deemed statistically significant.

[0296] Example 2: Enhancing Bispecific Antibody Efficacy with Radiation-Triggered Payload Release

[0297] Therapeutic antibody drug conjugates (ADCs) have demonstrated efficacy in cancer therapy, but adverse effects due to off-target tissue uptake and payload activity pose challenges. To address these concerns, we engineered Radiation-AMPlified Antibody-Drug Conjugates (RAMP-ADCs) that release active drug payloads only upon spatially localized exposure to ionizing radiation. To further improve tumor targeting, we developed RAMP-ADCs using a bispecific antibody that binds to both EGFR and MET receptors, carrying either the microtubule destabilizing monomethyl auristatin E (MMAE) or the topoisomerase 1 inhibitor exatecan as payloads. Our results showed that the constructs enhance cytotoxic efficacy up to 2,100-fold and enhance the selectivity of tumor payload delivery by up to 760-fold while minimizing systemic toxicity. RAMP-ADC combined with radiation blocked colony formation in cell lines that were resistant to either radiation or amivantamab individually. RAMP-ADCs effectively blocked tumor growth in a mouse xenograft model, whereas the bispecific antibody had little effect alone (P = 0.0045).

[0298] Therapeutic antibody drug conjugates (ADCs) have demonstrated efficacy in cancer therapy, but adverse effects due to off-target tissue uptake and payload activity pose challenges. To address this problem, we have examined whether a radiation-amplified ADC (RAMP-ADC) based on EGFR- and MET-targeted amivantamab could selectively deliver potent drug payloads to irradiated tumors while sparing off-target tissues. In this case, the RAMP-ADC described below binds to both EGFR and MET receptors, carrying either the microtubule destabilizing monomethyl auristatin E (MMAE) or the topoisomerase 1 inhibitor exatecan as payloads. We synthesized amivantamab RAMP-ADC and confirmed its ability to function as designed in vitro and in vivo. Bioinformatic analysis of clinical and cell-line data highlighted potential applicability across multiple cancer types over- expressing EGFR and MET. Experiments validated RAMP-ADC activity in diverse cancer-types such as sarcoma, triple negative breast cancer, and anaplastic thyroid cancer, which often are poorly responsive to traditional EGFR or MET targeted therapies. Our results showed that the constructs enhance cytotoxic efficacy up to 2,100-fold and enhance the selectivity of tumor payload delivery by up to 760-fold while minimizing systemic toxicity. RAMP-ADC combined with radiation blocked colony formation in cell lines that were resistant to either radiation or amivantamab individually. RAMP-ADCs effectively blocked tumor growth in a mouse xenograft model, whereas the bispecific antibody had little effect alone (P = 0.0045).

[0299] This study has demonstrated that bispecific RAMP-ADC as a clinical strategy for safer and more effective chemoradiotherapy, and demonstrates the potential of combining RT with dual receptor targeting to locally co-deliver multiple chemotherapy payloads.

[0300] RESULTS

[0301] RAMP-ADC chemical design and characterization

[0302] \Ne designed and synthesized amivantamab RAMP-ADC guided (see, for example, Quintana et al., ACS Cent Sci. 2024, 10(7) 1371-1382 and Quintana et al., J Nucl Med. 2025, 66(1) 91). Briefly, we used a phenyl azide as the radiation-responsive moiety, which is chemically reduced upon in situ exposure to radiolysis products such as hydrogen radicals and hydrated electrons. Chemical reduction to the primary amine triggers a cascade of electron transfer and self-immolation that releases free and active payload from the antibody (FIG. 20A). We used the microtubule disrupting MMAE (monomethyl auristatin E) as one drug payload, forming RAMP-ADCM- MMAE is radiosensitizing and has clinical activity in traditional ADCs where its activation is promoted by cathepsin-mediated linker proteolysis.

[0303] Caged MMAE payload was conjugated to amivantimab using maleimide chemistry, yielding the conjugate RAMP-ADCM and achieving a molar drug antibody ratio (DAR) of 4.1 ± 0.6 (std. dev., FIG. 35) determined by LCMS of payload (FIG. 29) and supported by mass spectrometry of intact conjugate (FIG. 35). For maximizing cytotoxic dose without negatively effecting conjugate stability, we rationalized that a DAR of 4 would be an ideal preliminary ratio based on prior literature of maleimide-based ADCs. This ratio may need to be further optimized for future work with this and other antibodies. Spontaneous chemical payload release from amivantimab was < 3% over 3 days at 45°C (FIG. 35D) in PBS in the absence of radiation. X-ray exposure triggered linear MMAE release of 147 ± 5 nM Gy1 (FIG. 20B), and serial fractions of X-ray exposure triggered cumulatively increasing payload release (FIG. 20C). RAMP- ADCM thus exhibits stability and responsiveness to repeat RT treatments, which is important since typical ADCs circulate for roughly 1 week and patients often receive conventional 2 Gy daily RT fractions.

[0304] EGFR and MET receptor expression across diverse cancer-types

[0305] Amivantamab binds with high affinity to both EGFR (Kd 1 .4 nM) and MET (Kd 0.04 nM), and we hypothesized that such co-targeting could yield high overall levels of targetable receptor on cancer cells. Using data from the Cancer Genome Atlas (TCGA), pan-cancer analysis of tumor specimens across 10,953 patients showed copy-number amplification, high protein expression, and / or high mRNA expression (conservatively defined here as relative to diploid samples; see Methods) for EGFR or MET in 12% and 7% of specimens, respectively. 18% showed at least one such dysregulation in either EGFR or MET. Therefore, if high target expression were an enrollment criterion for RAMP-ADC treatment, bispecific co-targeting of EGFR and MET would expand the fraction of patients considered for treatment compared to a traditional antibody targeting a single antigen.

[0306] Only 1 .5% of patients met the conservative definition for high mRNA expression in both receptors, but this co-occurrence was statistically significant (odds ratio 3.0, 95% confidence interval 2.5-3.7, P < 0.001 , Fisher’s exact t-test), consistent with the known ability of EGFR and MET to co-promote oncogenic signaling; furthermore, both EGFR and MET are on chromosome 7, and chromosome 7 gain is common in some cancers. RNA expression was on average highest in renal non-clear and clear cell carcinomas, ocular melanoma, glioblastoma, NSCLC, and cancers of the thyroid, head & neck, and upper gastrointestinal tract (FIG. 21 A). We analyzed the protein levels of EGFR and MET in cancer cell lines using mass spectrometry data from the Cancer Cell Line Encyclopedia, and found high levels in cells derived from cancers of the kidney, upper aerodigestive tract (including head and neck carcinomas), and thyroid (FIG. 21 B).

[0307] EGFR has been reported to promote radioresistance, and we calculated a low but statistically significant correlation between in vitro radioresistance and [EGFR+MET] RNA expression (Spearman’s correlation r = 0.10; P = 0.017) across 524 cancer cell lines (FIG. 26A- B). Subset analysis within matched tissues of origin showed no significant correlation between radioresistance and [EGFR+MET] protein, phosphoprotein, or mRNA levels after multiple hypothesis corrections (Figure 26C). This suggests EGFR and / or MET are not the sole drivers of radioresistance across the diverse range of cancer-types represented in the Cancer Cell Line Encyclopedia. Nonetheless, co-targeting EGFR and MET with a bispecific could, in principle, increase the delivery of a radiosensitizing drug payload compared to traditional targeting of individual receptors, and potentially broaden the range of cancers that might be responsive to RAMP-ADC treatment.

[0308] RAMP-ADC cytotoxic activity across diverse cancer types.

[0309] Guided by the above analysis, we examined the impact of RAMP-ADCM on cell lines of lung adenocarcinoma (HCC-827), triple-negative breast cancer (MDA-MB-231), fibrosarcoma (HT-1080), and aggressive anaplastic thyroid cancer (8505c). These cell lines express moderate-to-high levels of EGFR and MET (FIG. 2B) and exhibit moderate radioresistance compared to other cancer cell lines (FIG. 21 A- B). Treatment with amivantamab alone had little impact on proliferation (Fig. 21 D) or colony formation (Fig. 21 C), partly explained by cells lacking EGFR exon 20 insertions, grown without exogenous EGFR or MET ligands, and except for HCC-827, harboring mutations in downstream PI3K and MAPK / ERK pathways (FIG. 21C). Treatment with RAMP-ADCM similarly had little impact on colony formation in the absence of radiation, consistent with stable chemical caging of the MMAE payload that rendered it inactive. In contrast, 8 Gy radiation with RAMP-ADCM restored the ability of MMAE to reduce colony formation (FIG. 21 C) and cell proliferation (FIG. 21 D, FIG. 27). Response did not strongly correlate with receptor expression across these EGFR+ MET+ cell lines (FIG. 27D), suggesting free MMAE payload released by radiation drives effective cytotoxicity across a range of targeted receptor expression in these in vitro experiments.

[0310] Radiation restores MMAE activity and promotes bystander payload effects.

[0311] \Ne investigated cellular mechanisms of RAMP-ADCM efficacy in combining with RT. We focused on the HT1080 cell line, since it expresses only moderate EGFR and MET (FIG. 21 B), it harbors NRAS 1 mutation suggesting constitutive ligand-independent MAPK / ERK signaling and amivantamab resistance, and it is relatively resistant to RT and to therapies targeting EGFR and MET. Microscopy confirmed the ability of both amivantamab and RAMP-ADCM to similarly bind HT1080 cells in culture, indicating that the ADC payload has little impact on receptor target binding (FIG. 22A). As seen with other cell lines, RAMP- ADCM did not substantially affect colony formation or proliferation in the absence of RT, but irradiation (including with the lowest tested dose of 2 Gy, FIG. 37) restored the cytotoxicity of its MMAE payload; further control experiments showed little effect of RT on response to parent amivantamab lacking MMAE (FIG. 22B-D).

[0312] Immunofluorescence staining for a-tubulin indicated that RAMP-ADCM had little impact on cellular microtubule structures in the absence of RT. In contrast, irradiation restored the ability of the RAMP-ADCM MMAE payload to destabilize both cytoskeletal and mitotic spindle microtubule structures, in addition to reducing live cell count (FIG. 22E-H). Thus RAMP-ADCM payload is chemically caged and inactive in the absence of radiation, becomes cytotoxic once irradiated, and efficiently kills cancer cells that are resistant to treatments with RT and amivantamab.

[0313] Membrane-permeable ADC drug payloads including MMAE are known to affect cells that directly internalize ADC, as well as nearby cells that are exposed to the payload as it diffuses into surrounding tissue (FIG. 23A). This “bystander” effect is thought to be a clinically meaningful contributor to overall efficacy since antibodies deposit heterogeneously through tumors. To evaluate RAMP-ADCM bystander effects, we treated HT1080 cells with RAMP-ADCM, washed them to remove extracellular / unbound antibodies, irradiated the cells, and then transferred supernatant to a fresh plate of otherwise untreated HT1080 cells (FIG. 23B). In this experiment, RT and RAMP-ADCM alone showed no significant bystander effect on microtubule structure or cell viability, but their combination phenocopied the bystander effects of free MMAE in disrupting cytoskeletal and mitotic spindle microtubule structures, as well as reducing live cell count (FIG. 23C-F). Mitotic count was increased by RT and RAMP-ADCM individually, indicating potential bystander effects unrelated to MMAE (FIG. 23E). Nonetheless, data show RAMP-ADCM combined with radiation restores MMAE bystander microtubule and cytotoxicity effects.

[0314] Applying the RAMP-ADC design to a topoisomerase inhibitor payload

[0315] \Ne tested a second model RAMP-ADC payload designed to evaluate generalizability of the concept Furthermore, clinical data indicates potential benefits of co-delivering multiple ADC payloads, such as a microtubule-targeted toxin and a topoisomerase 1 inhibitor (TOP1i), and we hypothesized RAMP-ADC could deliver payload combinations specifically to irradiated tissues. As a proof of principle, we combined RAMP-ADC bearing two distinct payloads: MMAE as above (RAMP-ADCM), or the TOP1 i exatecan (RAMP-ADCT; FIG. 24A). We synthesized RAMP-ADCT and confirmed its stability, purity, drug- antibody-ratio (DAR = 3.8 ± 0.3), and radiation release sensitivity were similar to behaviors seen with RAMP-ADCM (FIG. 28F). 8 Gy irradiation increased RAMP-ADCT cytotoxicity on HT1080 cells by 83- fold (FIG. 24B). These results show the RAMP-ADC strategy is compatible with both MMAE and exatecan as chemically distinct payloads.

[0316] To guide the co-dosing of RAMP-ADCM and RAMP-ADCT, we examined how varied molar ratios of MMAE and exatecan affected cytotoxicity. We treated cell lines from three representative cancer types with mixed EGFR / MET expression (anaplastic thyroid cancer, triple-negative breast cancer, lung adenocarcinoma, and sarcoma as above, along with prostate cancer) with a dose-response of MMAE and exatecan mixtures (FIGs. 24C-D, 28). MMAE was generally more potent compared to exatecan. Although MMAE and exatecan showed modestly synergistic effects in some cell lines and dosing ratios according to the combination index (Cl < 1 ) described by Chou and Talalay (FIGs. 24C, 28G), results were variable and in aggregate did not substantially deviate from additivity (Cl~ 1 ). Nonetheless, a ratio of 5:1 MMAE : exatecan maintained statistically similar IC50 values compared to IC50 values of the best performing monotherapy for each cell line (P = 0.48 across n = 6 cell lines, ratio paired t-test; FIGs. 24C, 28H). As an advantage, the combination IC50 was 8- to >500-fold lower than the worse performing monotherapy in all six cell lines (Fig. 5D; P = 0.005, ratio paired t-test; FIGs. 24C, 28H), including showing more potency than MMAE monotherapy in RM1 cells (FIG. 24D). This data suggests the payload combination may more reliably elicit cytotoxicity across diverse cancers compared to monotherapy, motivating future investigation.

[0317] RAMP-ADC co-delivers payload combinations to irradiated tumors and blocks tumor growth

[0318] We next evaluated the ability of RAMP-ADC to accumulate in tumor tissue and deliver local combinations of therapy upon irradiation. We first assessed the biodistribution of RAMP-ADCM by covalently labeling the antibody with the near infrared fluorophore AlexaFluor647 using a non-cleavable stable linker. Fluorescent RAMP-ADCM was injected by tail vein into nu / nu mice bearing subcutaneous HT1080 xenografts. 24 hrs later, mice were perfused with PBS and tissues were scanned for biodistribution. Results showed the highest accumulation in tumors compared to other tissues in the body, at 7.9% injected dose per gram tissue (% I D / g ; FIGs. 25A, 29C). This moderate accumulation is consistent with the moderate expression of EGFR and MET on HT 1080 cells, and likely includes uptake into tumor cells and phagocytes as seen with other antibodies and ADCs. In our model, non-tumor accumulation is likely driven by target-independent uptake pathways, since amivantamab is a weak binder to mouse EGFR, and tumor-to-tissue ratios in uptake were 2.6 ± 0.3 for kidney, 3.3 ± 0.3 for liver, and 5.2 ± 0.2 for spleen.

[0319] We measured the ability of radiation to uncage RAMP-ADC payloads in tissue using liquid chromatography / mass spectrometry (LCMS) of digested tissue lysate. 3 hrs after intravenous injection of RAMP-ADCM and RAMP-ADCT, tumors were exposed to a localized beam of X-rays at 2 Gy, and 24 hrs later mice were dissected for analysis. Guided by in vitro results, RAMP- ADCM and RAMP-ADCT were co-dosed to achieve a molar payload ratio of 5:1 MMAE:exatecan, using a total drug dose of 0.1 mg kg-1(~5 mg kg-1antibody). LCMS showed that irradiation released RAMP-ADC payloads with a total combined yield of 154 ± 14 nmol Gy-1, similar to that observed in vitro and as reported with other phenylazide designs (FIG. 25B-C). Our data also showed that measured molar ratio in payload accumulation (4.7:1 MMAE:exatecan) matched the 5:1 ratio that was initially dosed. The high drug concentrations were detected 24 hrs following irradiation, indicating sustained accumulation as predicted by the tight binding affinity of the pay loads to their targets, along with diffusion and permeability properties. Sustained accumulation in irradiated tumors yielded tumor-to-tissue ratios of active payload that were much greater than those seen with total antibody: 760 ± 210 for kidney, 12 ± 3 for liver, and 63 ± 19 for spleen. Based on this metric, localized radiation improved tumor-to-tissue specificity in payload delivery by 4x in the liver, 12x in the spleen, and 290x in the kidney, compared to total antibody biodistribution.

[0320] We further tested RAMP-ADC’s enhancement of bispecific antibody efficacy in the HT 1080 xenograft model, which is known to be relatively resistant to radiation and EGFR / MET targeted therapy (Huang et al., Exp Hematol Oncol. 2024, 13(1) 97)- Tumor-bearing mice received either isotope control antibody, native amivantamab, RAMP-ADC combination (RAMP-ADCM + RAMP-ADCT), all with or without RT. We used a short course of conventional 2 Gy fractions given for 4 consecutive days (2 Gy x 4), and RAMP- ADC was given 4 hrs prior to RT each day. Monotherapy and control treatments failed to block tumor growth, but the combinations of RAMP-ADC with RT (P = 0.0026) or RAMP-ADCM with RT (P= 0.0092) did (FIGs. 25D-E, 36). These combinations also extended humane survival compared to the combination of native amivantamab with RT (FIGs. 25F, 36). Although RT with the RAMP-ADC combination (RAMP- ADCM + RAMP-ADCT) trended towards a stronger initial response compared to RAMP-ADCM, both were similarly effective in this experiment. Animals tolerated treatments with minimal weight loss compared to control groups (FIG. 25G). A second treatment cohort evaluated shorter-term responses to RAMP-ADC with or without radiation. Consistent with the first experimental cohort, tumor growth was blocked only with the combination of RT and RAMP-ADC, with no noticeable toxicity by body weight (FIG. 30)Histologic analysis of tumor tissue showed that combined RT and RAMP-ADC disrupted microtubule structures and increased apoptosis, as measured by immunofluorescence and the TUNEL (terminal deoxynucleotidyl transferase dUTP nick-end labeling) assay, respectively (FIG. 25H-I). MMAE is a relatively more potent and toxic payload compared to exatecan, and measurement of complete blood count and blood chemistry panels showed no statistically significant effects from RAMP-ADCM in healthy C57BL / 6 background mice, although 1 treated subject showed high alanine aminotransferase (FIG. 31). Taken together, RAMP-ADC payload, released by clinically relevant levels of radiation, can induce cancer-cell killing to block tumor growth.

[0321] SUMMARY

[0322] This study demonstrates how the anti-tumor activity of a bispecific mAb can be enhanced by conjugating radiation-activated drug payloads. The radiation amplified - antibody drug conjugate (RAMP-ADC) strategy demonstrated 3 orders of magnitude improvement in cytotoxic cancer cell killing in vitro, and 1 -2 orders of magnitude improved specificity in payload tumor localization in vivo. The strategy enabled local and simultaneous co-delivery of two payloads at precise stoichiometric ratios in vivo, which provides advantages and broad applicability given the clinical importance of multidrug combinations in chemotherapy treatments. RAMP-ADC synergized with radiation to kill cells derived from diverse cancertypes that are resistant to amivantamab treatment or radiation alone. Thus, amplifying the therapeutic activity of bispecific antibodies using radiation-triggered drug to safely radiosensitize cancers and improve their response to standard-of-care radiation treatments.

[0323] The strategy presented herein has several advantages. As a bispecific, amivantamab targets both EGFR and MET with high affinity, it can bind to either receptor individually, and analyses of clinical and cancer cell line data suggest that such dual targeting increases the total level of targetable receptor expression on cancer cells. A greater fraction of patients express high levels of EGFR and / or MET compared to each receptor individually, and clinical studies with traditional tumor-targeted ADCs have shown a correlation between target receptor expression and response rates. In one recent example, MET-targeted telisotuzumab vedotin showed an overall response rate of 34.6% in patients with non-squamous NSCLC with high MET levels, but responses were lower with squamous histology or intermediate / low MET expression (NCT03539536). This example exemplifies how target expression promotes response to MMAE-containing ADCs, and bispecific targeting leverages this property. Such effects are important in vivo where pharmacokinetics / pharmacodynamics dictate drug delivery, and may be less noticeable in vitro when comparing cell lines that all express moderate-to-high levels of target receptors, as we observed with RAMP-ADC (FIG. 27D).

[0324] Although bispecific targeting may improve drug delivery efficiency, it may also amplify potential toxicities arising from undesired off-site accumulation in non-malignant tissues. EGFR and MET are both expressed in healthy tissues, and therapeutic EGFR-targeted mAbs are limited by on-target toxicities such as skin rash. Target-independent toxicities common to particular ADC payloads include peripheral neuropathy as observed with MMAE and MET-targeted telisotuzumab-vedotin, ocular toxicities observed with monomethyl auristatin F (MMAF) and the EGFR-targeted depatuxizumab mafodotin, and interstitial lung disease as seen with TOP1i payloads. Two patient deaths were attributed to treatment related adverse events (interstitial lung disease and respiratory failure) in a recent trial of MET-targeted telisotuzumab-vedotin. The amivantamab package insert warnings include interstitial lung disease, rash, and ocular toxicity. Thus, both target-dependent and target-indepedent toxicities are of concern for the development of an EGFR- and MET-targeted bispecific ADC, and the RAMP-ADC strategy addresses both types of toxicity by caging payload activity in non-irradiated tissues. Of note, the potency of RAMP- ADC payloads used in this study allow for activity to be observed with lower antibody doses than are used for amivantamab alone, which is dosed in patients at 1050 mg for under 80 kg body weight (14.3 mg kg1for an average 70 kg individual), therefore minimizing antibody-related on-target toxicities. Other bispecifics with co-affinity for immune or microenvironment targets can accumulate in both tumor tissue and leukocyte-rich tissues such as the spleen. For these reasons, confining drug payload activity only to irradiated tumor tissue using the RAMP-ADC design offers an attractive solution to mitigating unwanted off-site activity.

[0325] Our example with MMAE and exatecan show how multiple drug payloads can be released in a stoichiometrically controlled ratio and selectively in irradiated tissue. The combination of traditional ADCs carrying these two payloads has shown promising response rates in a Phase I trial evaluating enfortumab vedotin and sacituzumab govitecan for bladder cancer. Using payload combinations potentially i) creates a synergistic response to multi-pathway targeting in individual cells, ii) overcomes heterogeneity in cancer cell resistance to any individual drug, and iii) offers more even coverage over the tumor microenvironment, since distinct drug payloads exhibit distinct physicochemical and PK / PD properties, including bystander effect behaviors. Future work is needed to dissect these potential factors. Although we did not find that the payload combination substantially improved HT 1080 response compared to the MMAE payload of RAMP-ADCM alone, we demonstrate that multiple drug payloads can be co-delivered in vivo with predictable efficiency, can be locally retained in irradiated tissues hours after activation, and are effective at safely blocking tumor growth in mice. Subsequent studies may investigate alternative payload combinations and potential benefits of using combinations to address patient-to-patient heterogeneity and emergent resistance to certain payload classes.

[0326] Our example largely focuses on evaluating RAMP-ADC in disease models where amivantamab alone showed little cytotoxic effect. By taking this focus, we specifically evaluated the added benefits of radiation-mediated drug delivery. Nonetheless, the role of EGFR and downstream signaling activity on radiation response has been extensively studied, including clinical trials testing the combination of EGFR- targeted agents concurrently with radiation. We hypothesize that any radiosensitizing effects from EGFR / MET blockade are dramatically improved with the codelivery of potent radiation-activated cytotoxic payloads including MMAE and exatecan. We did not investigate specific dependence on EGFR versus MET contributions, nor how RAMP-ADC affected Fc-dependent activity. However, amivantamab has been extensively evaluated in these regards. Amivantamab is Fc-enhanced with a low-fucose lgG1 backbone that increases affinity to FcyRllla on natural killer (NK) cells and macrophages, therefore enhancing antibody-dependent cellular cytotoxicity, trogocytosis, and cytokine responses. Radiation can enrich the tumor microenvironment for phagocytic macrophage infiltration, therefore suggesting the possibility for enhanced Fc-dependent activity in the days and weeks following radiation treatment. Future studies are needed to investigate these hypotheses. The radiation-activated chemistry is compatible with local radiation delivered by radiopharmaceuticals such as fibroblast-activated protein targeted lutetium 177, and there is potential that RAMP-ADC could radiosensitize disseminated metastatic disease treated with radiopharmaceuticals. We hypothesize that higher, clinically relevant doses of radiation — both using external beam and radiopharmaceutical approaches — will yield greater drug payload activation, with corresponding improvements in disease control.

[0327] Overall, this Example demonstrates radiation-activated drug delivery using a tumor-targeted bispecific mAb, and sets a foundation for evaluating alternative bispecifics and next-generation biologies using the radiation-activated approach.

[0328] The above-described results were obtained using the following materials and methods.

[0329] MATERIALS AND METHODS

[0330] Materials and general methods:

[0331] Monomethyl auristatin E (MMAE), exatecan, and amivantamab were purchased from MedChemExpress (NJ, USA). Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) was purchased from Millipore Sigma. PBS(Phosphate Buffered Saline (1X)) was purchased from Corning (NY, USA), FBS(Fetal Bovine Serum), RPMI(RPMI 1640 Medium) was purchased from Gibco (MA, USA), paraformaldehyde (Paraformaldehyde 16% Aqueous Solution EM Grade) was purchased from Electron Microscopy Sciences (PA, USA), Tween-20 was purchased from BostonBioProducts (MA, USA), Triton X-100 and BSA (Bovine Serum Albumin) were purchased from Sigma Aldrich (MO, USA). Mouse cell lines underwent mouse pathogen testing (IDEXX, ME, USA) before use, and all cells were routinely tested for mycoplasma contamination (Mycoplasma PCR test Kit) was purchased from Applied Biological Materials (BC, Canada).

[0332] TBP3743 (provided by Dr. Sareh Parangi, Mass General Hospital), HT1080 (ATCC), MDAMB231 (ATCC), and RM1 (ATCC) cell lines were cultured in DMEM, while HCC827 (ATCC) and 8505c (provided by Dr. Sareh Parangi) were cultured in RPMI, all with 10% FBS and penicillin / streptomycin under an atmosphere of 5% CO2-

[0333] RAMP-ADC synthesis:

[0334] Caged RAMP-ADC payload precursors, pATFB-SIL-Mal-MMAE and pATFB-SIL-Mal-Exatecan, were prepared as described previously(Quintana et al., ACS Cent Sci. 2024, 10(7) 1371 -1382 and Quintana et al., J Nucl Med. 2025, 66(1) 91) and used to synthesize RAMP-ADCM and RAMP- ADCT as amivantamab conjugated with caged MMAE or the topoisomerase 1 inhibitor exatecan, respectively. For payload-mAb conjugation, amivantamab (1 mg / mL, 6.7 pM) in PBS was treated with TCEP hydrochloride (50 pM) at room temperature for 30 min. The TCEP was then removed by spin filtration (50kDa MWCO, 10,000 ref for 5 min; Amicon, Millipore Sigma, MA, USA). Caged payload was then added to a concentration of 50 pM and the mixture was incubated at room temperature for 2 hrs. Unbound payload was removed by repeated spin filtration and washing in PBS (Amicon 50kDa MWCO, 10,000 ref for 5 min x 5, Millipore Sigma). The final antibody concentration was determined by absorbance at 280 nm (Nanodrop 1000, ThermoFisher) using the Beer-Lambert equation ( I = sbC), where the extinction coefficient was 210,000 M"1 cm"1 . The drug-to-antibody ratio (DAR) was determined by incubating an aliquot of the conjugate in 100 pM TCEP for 1 hr and measuring drug concentration via LCMS (Waters instrument equipped with a Waters 2424 ELS Detector, Waters 2998 UV-Vis Diode array Detector, and a Waters 3100 Mass Detector). An Xterra MS C18 Column (Waters; 124A, 5 pm, 4.6 x 50 mm) was used, with a gradient of 5-95% acetonitrile in water with 0.1 % formic acid as the mobile phase. Drug concentration from LCMS samples were quantified by comparing the area under the curve from the isolated mass chromatographs (+ESI 718.8 Da for MMAE, 436.4 Da for exatecan) of each sample to an 8-point standard calibration curve (FIG. 29A-B).

[0335] Cytotoxicity:

[0336] Cells were seeded in 96 well plates (-5,000 cells per well in 100 pL of media) (Corning, Corning, NY) and incubated overnight (37 °C, 5% CO2). When indicated, prodrug was X-ray irradiated using an X-rad 320 (8 Gy, 270 cGy / min; Precision X-ray, Madison, CT). 3-fold serial dilutions were prepared of drug / prodrug in growth media and treated to cells for 48 hrs. The media was then replaced with 100 pL PrestoBlue solution (Thermofisher, Waltham, MA) diluted in growth media; cells were incubated for 1 hr, fluorescence was measured by plate reader (Ex / Em:560 / 590; Spark, Tecan, Mannedorf, Switzerland). Fluorescence values were normalized to wells that did not receive treatment and data were processed in Prism (GraphPad Software, Boston, MA).

[0337] Colony formation assay:

[0338] Concentrations approximating the half-maximum inhibitory concentration (IC50) of free MMAE for each cell line, from the cytotoxicity assay, were used to assess clonogenic effects. Following 48 hrs of treatment, cells were washed and trypsinized (0.25% Trypsin-EDTA, Thermofisher). The plate was then incubated for 5 minutes before cells (-200 each) were added to a 6-welllate with 2 mL media per well. Irradiation used an X-rad 320 (8 Gy, 270 cGy / min) and the 6 well plates were incubated (37 °C, 5% CO2) for 7-10 days, until visible colony formation. Colonies were then fixed in methanol for 20 min., rinsed and stained with 0.1 mg mL"1 crystal violet (Sigma Aldrich) in distilled water for 30 min., rinsed with distilled water and imaged.

[0339] Microscopy:

[0340] Amivantamab immunofluorescence: 5,000 HT1080 cells were plated per well of a 96-well plate (I bidi, Wl, USA) overnight. Unfixed cells were washed and blocked from nonspecific binding by incubating with 10% goat serum in PBS for 10 minutes at 4°C. Primary antibody cocktail was prepared containing either amivantamab or human lgG1 isotype (1 mg mL" "1 ,1 :1000; BioXcell, Lebanon, NH, USA) and Hoechst (10 pg mL"1 > Jnvitrogen, Waltham, MA, USA) in 10% goat serum and 0.05% Tween in PBS. Cells were incubated with cocktail for 2 hrs at 4°C, rinsed, and stained with Alexa Fluor 488-labeled goat anti-human lgG1 for 1 hr at 4°C (1 mg mL"1 , 1 :1000, 3H60L9, Invitrogen, Waltham, MA, USA). Cells were rinsed in PBS and imaged using a laser scanning confocal microscope (Nikon AX R, NY, USA). a-tubulin immunofluorescence: 1 x 1 C)4 HT1080 cells per well were plated overnight in 96-well plates (I bidi) and treated the following day for 16 hrs with 25 nM MMAE or RAMP-ADCM (MMAE concentration) + / - radiation (8 Gy). Cells were fixed with 4% paraformaldehyde for 20 min, washed with PBS, permeabilized with 0.5% Triton X-100 in PBS for 10 min, incubated with 10% bovine serum albumin (BSA) in PBS for 20 min., and incubated overnight with Alexa Fluor 488- conjugated a-tubulin antibody (DM1 A, 1 :100) diluted in PBS containing 10% BSA. Following staining, the cells were washed, mounted with a mounting solution containing DAPI to label nuclei and imaged using a fluorescence microscope (Revolve, Discover Echo, CA, USA). The same procedure was followed with fixed tissue sections obtained from treated mice groups.

[0341] Tumor tissue samples were fixed overnight in 4% PFA at 4°C. They were later placed in 15% sucrose in PBS overnight, then transferred to 30% sucrose in PBS overnight. Tissues were embedded using O.C.T. compound (Tissue-Tek, CA, USA), snap frozen, cryosectioned into 10 pm sections, immunostained and imaged as above.

[0342] Apoptosis analysis: TUNEL (terminal deoxynucleotidyl transferase dUTP nick-end labeling) was used to assess apoptotic response to treatment, following manufacturer guidelines (DeadEndTM, Promega, Wl, USA). Cryosections were fixed by immersion in 4% PFA in PBS for 25 minutes at 4°C, followed by two PBS washes for 5 min. Permeabilization was achieved by immersing the slides in 0.2% Triton X-100 in PBS for 5 minutes, followed by another wash.

[0343] Equilibration was carried out by adding 100 pL of Equilibration Buffer to the slides and incubating them at room temperature for 10 minutes. Next, 50 pL of TdT reaction mix was applied to cells. Slides were covered with plastic coverslips and incubated at 37°C for 60 minutes in a humidified, dark chamber. After incubation, the reaction was stopped by immersing the slides in 2X SSC for 15 minutes, followed by three washes. The slides were then mounted with a cover glass (ThermoFisher) using Vectashield containing DAPI (Vectorlabs, CA, USA) to visualize all nuclei. Apoptotic cells exhibiting localized green fluorescence were analyzed using fluorescence microscopy (Revolve, Discover Echo).

[0344] Bystander effect:

[0345] HT 1080 cells were seeded overnight in a 96-well plate (Ibidi) at 1 x 10^ cells per well and treated the following day with 50 nM MMAE or the equivalent RAMP-ADC or amivantamab. 24 hrs later, cells were rinsed with PBS 3 times to remove extracellular antibody and / or payload and media was replaced. Freshly rinsed cells were exposed to 8 Gy radiation, and 48 hrs. later cells were discarded and their conditioned media was transferred to untreated HT1080 cells.

[0346] Cells were fixed 24 hrs. later for immunofluorescence or further incubated and analyzed for cytotoxicity using the PrestoBlue assay as above after 48 hrs. post-treatment with conditioned media.

[0347] Xenograft study

[0348] Animal research was performed with approval from the Institutional Animal Care and Use Committee (IACUC) at Mass General Hospital (MGH). Experiments were conducted using female nu / nu mice 6-10 weeks old (JAX 002019, Bar Harbor, ME, USA). A total of 1 x 106 cells in 50 pL PBS were implanted subcutaneously into flanks. Approximately 10 days later, once tumors reached an average size of 6 mm in diameter, mice were randomly allotted into treatment groups. Treatments were given (0.1 mg kg-1MMAE + exatecan dose in 100 pL of PBS, injected i.p.) for four consecutive days, and local X-ray radiation was given (2 Gy at 270 ± 10 cGy min-1) 4 hrs later each day of treatment. Digital caliper measured tumor size by two independent researchers and volumes were calculated by the formulate V = Measurement 1x Measurement 2 x (Smaller measurement) / 2. Criteria for humane survival included a body condition score < 2, weight loss > 20%, and tumor size exceeding 1 .5 cm diameter.

[0349] Toxicity

[0350] 8-10 week-old female mice bred on a C57BL6 / J congenic background (JAX 028071 , Bar Harbor, ME, USA) were evenly distributed into treatment groups receiving intraperitoneal saline solution (100 pL) or RAMP-ADCM (100 pL IP at an effective MMAE dose of 0.1 mg / kg) for four consecutive days. Following the treatment period, blood samples were collected and analyzed to evaluate a comprehensive metabolic panel and complete blood count. Blood samples (about 200-500 pL) were collected directly from the heart in anesthetized mice (inhaled isoflurane) into anti-coagulant (EDTA)-coated tubes (SAI Infusion Technologies) during a terminal procedure. Whole blood or serum, prepared from the collected whole blood samples by centrifugation, was analyzed using the services of the Center for Comparative Medicine (CCM) Veterinary Clinical Pathology Laboratory at MGH.

[0351] Biodistribution and tumor immunofluorescence.

[0352] RAMP-ADCM (6.7 pM protein concentration) was conjugated with AF647-NHS (Lumiprobe, 67 pM) in carbonate buffer (pH 8, 500 pL) for 1 hr prior to removal of free dye via spin filtration (Amicon, 10 kDa MWCO, 10,000 ref for 5 min x 5). The purified conjugate was prepared to a concentration of 1 mg mL-1protein (6.7 pM). 6-10 weeks old female nu / nu mice (JAX 002019, Bar Harbor, ME, USA) were injected subcutaneously with 1 x 106HT1080 cells at four flank sites. Once tumors reached approximately 5 mm in diameter, fluorescently labeled RAMP- ADCM was injected intravenously (100 pL, 5 mg kg-1protein). Radiation treatment (RT, 2 Gy) was applied to selected groups 4 h after RAMP-ADC administration to assess the effect of RT on drug localization. After 24 hrs, mice were sacrificed, perfused with 10 mL PBS, and tissues were harvested, weighed, and imaged on a Sapphire FL Imager (Azure Biosystems, CA, USA) . The tissues were finely minced in 200 pL Lysis buffer II (ThermoFisher) and incubated for 30 min. on ice, then diluted 4-fold with acetonitrile (600 pL) and centrifuged (5,000 ref for 5 min). The supernatant was filtered through 3 kDa MWCO spin filters (Amicon, 10,000 ref for 5 min) to remove any remaining tissue fragments and proteins. The flow-through was analyzed by LCMS (n = 3), as described above, to determine the amount of drug released.

[0353] Data analysis

[0354] Data from The Cancer Genome Atlas (TCGA) PanCancer Atlas Studies were downloaded on August 30th, 2024, from cbioportal. Data included EGFR and MET mutation counts, structural variant counts, putative copy number alteration counts from GISTIC (Genomic Identification of Significant Targets in Cancer), counts of high mRNA expression (when there is a z-score of 2 or higher compared to the expression distribution of each gene tumors that are diploid for this gene) relative to diploid samples (RNA Seq V2 RSEM), and counts of high protein expression z- scores (when there is a z-score of 2 or higher measured by reverse-phase protein array) from 32 studies in 10953 patient samples(de Bruijn et al., Cancer Res. 2023, 83(23) 3861-3867 and Cerami et al., Cancer Discov. 2012, 2(5) 401-404). The RSEM mRNA expression of EGFR and MET for each sample was summed to analyze the normalized count of [MET + EGFR] mRNA. In order to plot the alteration frequency percentage of EGFR and MET, we counted as positive the presence of either a copy number alteration (excluding deletions), high expression of mRNA, high levels of protein and / or the presence of a structural variant. CCLE data were downloaded on September 25th 2024 from the DepMap portal (mRNA expression) (Ghandi et al., Nature. 2019, 569(7757) 503-508 and Li et al., Nat Med. 2019, 25(5) 850-860) the Gygi lab website(Nusinow et al., Cell. 2020, 180(2) 387-402. e16) (CCLE proteomics, RPPA protein data and approximate normalized Iog2-transformed ratios for the MS protein data), and Yard et al., Nat Commun. 2016, 7 11428 (CCLE radiosensitivity) where cell lines where categorized by tissue origin. GraphPad Prism v10.3.1 was used to plot the data and perform statistical analysis. Statistical methods included one-way and two-way ANOVA models with Dunnett’s test for multiple comparisons, Spearman rank correlation test, two-tailed Mann- Whitney tests, and with Benjamini-Hochberg correction for false discovery rates all when indicated in figure captions. Data are reported as mean ± standard deviation (s.d.) unless otherwise stated.

[0355] Example 3: Uncaging of caged prodrug ADC with Co-administered Targeted Radioemitter

[0356] This study assessed the viability of utilizing a co-administered radioemitter to uncage the caged prodrug ADC’s of Example 2.

[0357] Three Caged prodrug ADC’s were prepared, as outlined in Example 2, and include amivantamab-MMAE, amivantamab-Exatecan (amivantamab-Ex), and rosopatamab-MMAE. To test the efficacy of uncaging multiple caged prodrug ADCs simultaneously, a ratio of 5:1 of MMAE and Exatecan containing amivantamab ADC were provided to 6 reaction vessels at a final concentration of 10 pM ADC. Similarly, to test the viability of uncaging utilizing a different antibody handle, an amount equal to 10 pM ADC of the rosopatamab-MMAE caged prodrug ADC was provided to 6 separate reaction vessels.

[0358] Next, radioemitters bearing99mTc or177Lu were added, at a dose of 1 mCi / mL, to the reaction vessels containing the mixture of amivantamab ADCs or the rosopatamab ADCs, so that each combination of antibody and radioemitter had a sample size of 3. The mixture were allowed to incubate for 48 hours, after which, the concentration of the uncaged Drug Moiety was determined (FIG. 32).

[0359] Results demonstrate that caged prodrug ADCs may be uncaged utilizing a co-administered radionuclide, as with the peptide based targeted caged prodrugs of Example 1 , and further demonstrates that uncaging of multiple caged prodrug ADCs may be done simultaneously, opening up options for use with uncaging stoichiometric ratios of multi-Drug Moiety compositions and methods of treatment. Also, the successful uncaging of rosopatamab-MMAE caged ADCs demonstrates that the approach may be utilized with various antibodies. It should also be noted, that although the two radioemitters used contained99mTc or177Lu, which have different half-lives, 6 hours versus 6.6 days, the amount of uncaging for both radioemitters were nearly identical.

[0360] Example 4: Preparation of PBD Dimer Caged Prodrug

[0361] An example synthesis pathway for the production of the caged prodrug containing the PBD dimer is described below in detail (Scheme 4).

[0362] Scheme 4.

[0363] Step 1 : PBD (4.5 mg, 6.2 pmol; SGD-1882, MedChemExpress) was added to a solution of pATFB-SIL- PNP (6.0 mg, 10.1 pmol) dissolved in dry DMF (1 mL). The mixture was vortexed until all solid was dissolved, then DIPEA (5 uL, 28.7 pmol) and DMAP (5 mg, 40.9 pmol) were added and the mixture shaken at room temperature for 18 hours. The reaction mixture was then loaded directly onto a reverse phase column (6 g) and purified using a gradient of 5-95% acetonitrile in water (0.1 % formic acid). Fractions containing the product were identified by LCMS, combined, and evaporated to provide a white solid (5.1 mg, 70% yield).

[0364] Step 2: pATFB-SIL-PBD (5.1 mg, 4.3 pmol, formed in step 1) was dissolved in 1 :1 methanoLDMF (2 mL) and treated with 0.5M LiOH (500 pL) for 1 hour. The presence of the hydrolysis intermediate was identified by LCMS before the mixture was quenched with acidic resin (-250 mg), filtered, and dried by rotary evaporation (concentrated in DMF). Mal-PEG4-amine TFA (50 pL of a 100 mg / mL solution in DMF, 15.8 pmol) was added to the previous pATFB- SIL-PBD mixture with DIPEA (5 pL, 28.7 pmol). The mixture was then shaken at room temperature for 20 hours, loaded directly onto a reverse phase column (6 g), and purified using a gradient of 5-95% acetonitrile in water (0.1 % formic acid). Fractions containing the product were identified using LCMS, combined, and evaporated to provide a pale yellow solid (2.1 mg, 33% yield). Example 5: PBD Caged Prodrug in vivo Efficacy

[0365] This study assessed the safety and efficacy of delivering a pyrrolobenzodiazepine dimer (PBD dimer) containing caged prodrug and the release of the PBD dimer utilizing a targeted radioemitter in vivo. The method of delivery and efficacy of in vivo uncaging of a caged prodrug utilizing ionizing radiation using a targeted radioemitter has been previously discussed (e.g., Example 1).

[0366] Two mice types were chosen for experimentation, C57BL / 6 and nude mice. The population of C57BL / 6 mice (6-8 weeks old) were inoculated with a mixture of RM1.WT cells (0.5x105cells) and RM1.PSMA cells (2x105) cell in 100 pL of PBS, subcutaneously, generating “hPSMA+ murine Pea” mice. These mice were allowed to grow for 7 days, after which a sub population of 7 mice were injected with the targeted radioemitter, PSMA-617, containing177Lu, at a dose of 0.5 mCi per mouse. The radioemitter was allowed to localize within the PSMA expressing tumor cells for 24 hours, after which 4 of the 7 mice were further injected with the PBD caged prodrug of Formula X, at a dose of 0.1 mg / kg.

[0367] Formula X

[0368] The free maleimide form of the PBD caged prodrug was chosen, which will react with free albumin within the serum, effectively increasing the circulation stability of the caged prodrug, while providing a targeting moiety which favors tumor cells.

[0369] The C57BL / 6 mice injected with the targeted radioemitter PSMA-617 (“Lu-177 PSMA”) and the PSMA- 617 in combination with the PBD caged prodrug (“Lu-177 PSMA + prodrug”) were monitored for 8 says after the injection, weighing the mice as well as measuring the tumor size (see FIG. 33).

[0370] As is shown in FIG. 33, the subset of mice injected with the PBD caged prodrug maintained a much smaller tumor size as compared to mice injected with PSMA-617 alone. It is thus our belief that the caged prodrug has localized to the tumor cells and was released via the ionizing radiation generated by the177Lu, which ultimately led to the death of the tumor cells, and maintained a relatively stable tumor size.

[0371] Similar to the above outlined experiment on C57BL / 6 mice, the nude mice (6-8 weeks old) were inoculated with 22Rv1 cells (2x106cells) in 100 pL of PBS, subcutaneously, generating “PSMA+ Pea” mice. These mice were allowed to grow for 7 days, after which a sub population of 7 mice were injected with the targeted radioemitter, PSMA-617, containing177Lu, at a dose of 0.5 mCi per mouse. The radioemitter was allowed to localize within the PSMA expressing tumor cells for 24 hours, after which 4 of the 7 mice were further injected with the PBD caged prodrug of Formula X, at a dose of 0.1 mg / kg. The nude mice injected with the targeted radioemitter PSMA-617 (“Lu-177 PSMA”), the PSMA-617 in combination with the PBD caged prodrug (“Lu-177 PSMA + prodrug”), and the control mice not injected with either the radioemitter nor the caged prodrug were monitored for 7 days after the injection, weighing the mice as well as measuring the tumor size (see FIG. 34). As is shown in FIG. 34, the subset of mice injected with the PBD caged prodrug and the targeted radioemitter maintained a much smaller tumor size as compared to control mice or mice injected with PSMA-617 alone. It is thus our belief that the caged prodrug has localized to the tumor cells and was released via the ionizing radiation generated by the177Lu, which ultimately led to the death of the tumor cells, and maintained a relatively stable tumor size. Furthermore, it should be noted that none of the mice given PSMA-617 and the PBD caged prodrug died during the experiment, showing the safety of utilizing a targeted radioemitter and a targeted PBD caged prodrug for delivery of the highly toxic (IC50 = 100 pM) drug.

Claims

CLAIMS1 . A method of killing a cell in a subject, the method comprising: a) administering to the subject a targeted caged prodrug; and b) administering to the subject a targeted radioemitter comprising67Ga,68Ga,99mTc,103Pd, 1111 n ,123l,125l,131l,131Cs,133Xe,153Sm,149Pm,161Tb,169Er,177Lu,201TI, or203Pb that produces ionizing radiation, wherein the ionizing radiation releases drug from the caged prodrug; and each of the targeted caged prodrug and the targeted radioemitter is configured to target the same cell type or tissue.

2. The method of claim 1 , wherein the targeted radioemitter administered to the subject, during step b), comprises67Ga,99mTc,1111 n, or123l.

3. A method of killing a cell in a subject, the method comprising: a) administering to the subject a targeted caged prodrug conjugated to a protein; and b) following from 8 hours to 48 hours after the completion of step a), administering to the subject a targeted radioemitter that produces ionizing radiation, wherein the ionizing radiation releases drug from the caged prodrug; and each of the targeted caged prodrug and the targeted radioemitter is configured to target the same cell type or tissue.

4. The method of claim 3, wherein the targeted radioemitter administered to the subject, during step b), comprises "mTc,1111 n ,177Lu, or225Ac.

5. A method of killing a cell in a subject, the method comprising: a) administering to the subject a targeted radioemitter comprising a radiolabeled peptide pharmaceutical, radiolabeled small molecule, or a radiolabeled protein that produces ionizing radiation; and b) following from 4 hours to 48 hours after the completion of step a), administering to the subject a targeted caged prodrug; wherein the ionizing radiation releases drug from the caged prodrug; and each of the targeted caged prodrug and the targeted radioemitter is configured to target the same cell type or tissue.

6. The method of claim 5, wherein the targeted radioemitter administered to the subject, during step a), comprises64Cu,68Ga, "Y,99mTc,1111 n,123l,131l,177Lu,223Ra, or225Ac.

7. The method of any one of claims 1-4, wherein the targeted radioemitter comprises a radiolabeled small molecule, a radiolabeled peptide pharmaceutical, or a radiolabeled protein.

8. The method of any one of claims 5-7, wherein the radiolabeled small molecules comprises methylene diphosphonate, phytate, or meta-iodobenzylguanidine.

9. The method of any one of claims 5-7, wherein the radiolabeled peptide pharmaceuticals comprise one of PSMA-11 , PSMA-617, PSMA-1007, DOTA-TATE, DOTA-TOC, FAPI-04, FAPI-34, FAPI-mFS, H6F, NNS309, NeoBOMBI , RM2, BAY 86-7548, AMBA, NOTA-PRGD2, pentixafor, CCZ01048, CP04, PP-F11 N, DOTA-MG11 , and DOTA-MSH.

10. The method of any one of claims 5-7, wherein the radiolabeled protein comprises one ofABY- 025, MIRC213, VHH1 , 2Rs15d, ZHER2:2891 affibody, ZEGFR:2377 affibody, R3B23, CEA61 , DARPin 9_29, Adnectin CT-322, AKY 1189, CD105-CD3 BiTE nanobody, PD-L1xCD3 BiTE nanobody, Anti-CD20 x CD3 BiTE nanobody, CD3-FAP BiTE nanobody, and HER2-scFvCD3 x HER2-EGFR BiTE nanobody.

11. The method of any one of claims 1-10, wherein the targeted caged prodrug comprises a peptide, small molecule, a protein, an antibody, a nanoparticle, or a polymer.

12. The method of claim 11 , wherein the targeted caged prodrug is a peptide conjugate comprising a targeting moiety that is a radical of a radiolabeled peptide pharmaceutical selected from PSMA- 11 , PSMA-617, PSMA-1007, DOTA-TATE, DOTA-TOC, FAPI-04, FAPI-34, FAPI-mFS, H6F, NNS309, NeoBOMBI , RM2, BAY 86-7548, AMBA, NOTA-PRGD2, pentixafor, CCZ01048, CP04, PP-F11 N, DOTA-MG11 , DOTA-MSH, and MIBG.

13. The method of claim 11 , wherein the protein is an albumin.

14. The method of claim 11 , wherein the antibody is any one of amivantamab, loncastuximab, camidanlumab, rovalpituzumab, vadastuximab, trastuzumab, gemtuzumab, brentuximab, ado- trastuzumab, inotuzumab, polatuzumab, enfortumab, fam-trastuzumab, scituzumab, tisotumab, mirvetuximab belantamab, moxetumomab, cetuximab, distamab, ibritumomab, metuximab, tositumomab, rosopatamab, girentuximab, 9MW2821 , ABBV-383, AK109, AMG 160, AMG 199, AMG 211 , AMG 596, AMG 910, anbenitamab, anvatabart opadotin, apamistamab, ASKB589, atezolizumab, avelumab, AZD0901 , AZD5863, ifinatamab, GSK5764227, YL201 , BCA-101 , BL- M07D1 , BNT323, DP303c, FDA018, HLX22, HPN424, IAH0968, IBIT343, JSKN003, IMC-F106C, M108, IA0968, MDX1110, MRGO02, RO6958688, SHR-A1904, SHR-A1921 , SHR-A2009, SYSA1801 , SYS6010, TF2, TNB-383B, TORL-1-23, TQB2102, becotatug, bevacizumab, belantamab, bemarituzumab, camidanlumab, catumaxomab, caxmotabart, cetuximab, cosibelimab, datopotamab, dinutuximab, distamab, durvalumab, edrecolomab, elranatamab, enfortumab, ficlatuzumab, gemtuzumab, girentuximab, ianalumab, ibritumomab, ifinatamab, inotuzumab, izalontamab, kintuximab, labetuzumab, ligufalimab, linvoseltamab, livmoniplimab, Im-302, loncastuximab, luveltamab, margetuximab, metuximab, mirvetuximab, monalizumab, moxetumomab, naxitamab, necitumumab, olaratumab, oleclumab, onfekafusp alfa, ozekibart, panitumumab, pasotuxizumab, patritumab, panitumumab, pertuzumab, petosemtamab, polatuzumab, raludotatug, ramucirumab, 76acituzumab, scituzumab, sigvotatug, sugemalimab, tarlatamab, tebentafusp, telisotuzumab, tifcemalimab, tisotumab, tositumomab, solitomab, trastuzumab, uliledlimab, vadastuximab, varlilumab, xaluritamig, zanidatamab, zenocutuzumab, zilovertamab, and zolbetuximab.

15. The method of any one of claims 1-14, wherein the targeted caged prodrug comprises one of a cytotoxic, cytostatic, or immunomodulatory agent.

16. The method of claim 15, wherein the targeted caged prodrug comprises any one of an antitubulin agents, a DNA replication inhibitors, an alkylating agents, an antifolates, an antimetabolites, a chemotherapy sensitizers, a topoisomerase inhibitors, and a vinca alkaloids.

17. The method of claim 15, wherein the targeted caged prodrug comprises any one of pyrrolobenzodiazepine dimer (PBD dimer), exatecan, monomethyl auristatin E (MMAE), doxorubicin, and gardiquimod.

18. The method of any one of claims 1-17, wherein the caged prodrug has the structure of Formula I:[RSM]-Linker-Drug MoietyFormula I wherein RSM is a radiation-sensitive moiety, or a pharmaceutically acceptable salt thereof.

19. The method of claim 18, wherein the linker of Formula I comprises a carbamate group.

20. The method of claims 18 or 19, wherein the caged prodrug has the structure of Formula I -A:Formula l-A wherein: ring A is a 5-6 member heteroaryl or a phenyl; each Ri is independently halogen, azido, or C1-C6 alkoxy, wherein not more than one of Ri is azido; m is 2, 3, 4, or 5; each R2 is independently halogen, C1-C6 alkyl, or C1-C6 haloalkyl; n is 0, 1 , 2, 3, or 4;R3is -NHRaor -NHRb;Rais -H or C1-C6 alkyl;R4 is a small molecule, a peptide, a protein, or an antibody;X is an electrophilic group; p, q, r, s, t, u, and v is each independently an integer from 2-20; and D is a drug moiety.21 . The method of claim 20, wherein ring A is phenyl.

22. The method of claim 20 or 21 , wherein m is 2 or 5.

23. The method of any one of claims 20-22, wherein m is 2 and each R1 is methoxy.

24. The method of any one of claims 20-22, wherein m is 5, one R1 is azido, and the remaining R1 are each fluoro.

25. The method of any one of claims 20-24, wherein n is 0.

26. The method of any one of claims 20-25, wherein R3 is -NHRb.

27. The method of any one of claims 20-26, wherein28. The method of any one of claims 20-26, wherein29. The method of any one of claims 20-26, wherein30. The method of any one of claims 20-26, wherein31 . The method of any one of claims 20-26, wherein32. The method of any one of claims 20-26, wherein33. The method of any one of claims 20-26, whereinThe method of any one of claims 20-26, wherein35. The method of any one of claims 20-26, wherein36. The method of any one of claims 20-26, wherein37. The method of claim 36, wherein v is 4 and caged prodrug has a structure of Formula II:Formula II.

38. The method of any one of claims 20-26, wherein R3 is -NHRa.

39. The method of any one of claims 20-26 or 38, wherein Rais -H or Rais C1-C6 alkyl.

40. The method of claim 18, wherein the linker (L) has the structure:HR6)a- (R7)b“ (R8)c- (R9)d-(R1°)e-(R11)f-(R12)g-FFormula VIII wherein a, b, c, e, f, and g are each, independently, 0 or 1 , d is 0, 1 , 2, or 3, each of R6, R8, R10, and R12, is, independently, optionally substituted Ci-Ce alkylene, optionally substituted Ci-Ce heteroalkylene, optionally substituted C2-C6 alkenylene, optionally substituted C2-Ce alkynylene, or optionally substituted Ce-Cio arylene, O, S, Se, and NR13, R7and R11are each, independently, carbonyl, thiocarbonyl, sulfonyl, or phosphoryl, wherein, if R7is phosphoryl, -(R9)d- is a bond, and e, f, and g are 0, then at least one of R6or R8is not O, and if R11is phosphoryl, -(R9)d- is a bond, and a, b, and c are 0, then at least one of R10or R12is not O, each R9is optionally substituted C1-C10 alkylene, optionally substituted C2-C10 alkenylene, optionally substituted C2-C10 alkynylene, optionally substituted C2-C10 heterocyclylene, optionally substituted C6-C12 arylene, optionally substituted C2-C100 polyethylene glycolene, or optionally substituted C1-C10 heteroalkylene, or a bond linking (R6)a-(R7)b-(R8)c to (R10)e-(R11)f-(R12)g, wherein if -(R9)d- is a bond, then at least one of a, b, c, e, f, or g is 1 , and R13is hydrogen, optionally substituted C1-C4 alkyl, optionally substituted C2-C4 alkenyl, optionally substituted C2-C4 alkynyl, optionally substituted C2-C6 heterocyclyl, optionally substituted C6-C12 aryl, or optionally substituted C1-C7 heteroalkyl.

41. The method of any one of claims 1-40, wherein (i) the targeted caged prodrug is a conjugate comprising a first targeting moiety, and (ii) the targeted radioemitter is a conjugate comprising asecond targeting moiety, wherein the first targeting moiety and the second targeting moiety target the same cell type, tissue, or receptor.

42. The method of 41 , wherein the first targeting moiety and the second targeting moiety are identical.

43. The method of claim 41 or 42, wherein the first targeting moiety and the second targeting moiety are selected from small molecules and peptides and nanobodies.

44. The method of any one of claims 1-43, the method further comprising administering to the subject a first caged prodrug comprising a first cytotoxic agent and administering to the subject a second caged prodrug comprising a second cytotoxic agent, wherein the first cytotoxic agent and the second cytotoxic agent are different.

45. The method of claim 44, where in the first cytotoxic agent is MMAE and the second cytotoxic agent is exatecan.

46. The method of any one of claims 1-45, wherein the cell is a cancer cell.

47. A composition comprising a caged prodrug of Formula III:Formula III, or a pharmaceutically acceptable salt thereof; and wherein R3 is a linker bound to a targeting moiety.

48. A composition comprising a caged prodrug of Formula IV:Formula IV, or a pharmaceutically acceptable salt thereof; and wherein R3 is a linker bound to a targeting moiety.

49. The composition of claim 47 or 48, wherein the targeting moiety is a peptide, small molecule, a protein, an antibody, a nanoparticle, or a polymer.

50. The composition of any one of claims 47-49, wherein the targeted moiety is a is a radical of a radiolabeled peptide pharmaceutical selected from PSMA-11 , PSMA-617, PSMA-1007, DOTA- TATE, DOTA-TOC, FAPI-04, FAPI-34, FAPI-mFS, H6F, NNS309, NeoBOMBI , RM2, BAY 86- 7548, AMBA, NOTA-PRGD2, pentixafor, CCZ01048, CP04, PP-F11 N, DOTA-MG11 , DOTA-MSH, and MIBG.

51. The composition of any one of claims 47-49, wherein the targeting moiety comprises methylene diphosphonate, phytate, or meta-iodobenzylguanidine.

52. The composition of any one of claims 47-49, wherein the protein is an albumin.

53. The composition of any one of claims 47-49, wherein the antibody is any one of amivantamab, loncastuximab, camidanlumab, rovalpituzumab, vadastuximab, trastuzumab, gemtuzumab, brentuximab, ado-trastuzumab, inotuzumab, polatuzumab, enfortumab, fam-trastuzumab, scituzumab, tisotumab, mirvetuximab belantamab, moxetumomab, cetuximab, distamab, ibritumomab, metuximab, tositumomab, rosopatamab, girentuximab, 9MW2821 , ABBV-383, AK109, AMG 160, AMG 199, AMG 211 , AMG 596, AMG 910, anbenitamab, anvatabart opadotin, apamistamab, ASKB589, atezolizumab, avelumab, AZD0901 , AZD5863, ifinatamab, GSK5764227, YL201 , BCA-101 , BL-M07D1 , BNT323, DP303c, FDA018, HLX22, HPN424, IAH0968, IBIT343, JSKN003, IMC-F106C, M108, IA0968, MDX1110, MRGO02, RO6958688, SHR-A1904, SHR-A1921 , SHR-A2009, SYSA1801 , SYS6010, TF2, TNB-383B, TORL-1-23, TQB2102, becotatug, bevacizumab, belantamab, bemarituzumab, camidanlumab, catumaxomab,caxmotabart, cetuximab, cosibelimab, datopotamab, dinutuximab, distamab, , durvalumab, edrecolomab, elranatamab, enfortumab, ficlatuzumab, gemtuzumab, girentuximab, ianalumab, ibritumomab, ifinatamab, inotuzumab, izalontamab, kintuximab, labetuzumab, ligufalimab, linvoseltamab, livmoniplimab, Im-302, loncastuximab, luveltamab, margetuximab, metuximab, mirvetuximab, monalizumab, moxetumomab, naxitamab, necitumumab, olaratumab, oleclumab, onfekafusp alfa, ozekibart, panitumumab, pasotuxizumab, patritumab, panitumumab, pertuzumab, petosemtamab, polatuzumab, raludotatug, ramucirumab, 83acituzumab, scituzumab, sigvotatug, sugemalimab, tarlatamab, tebentafusp, telisotuzumab, tifcemalimab, tisotumab, tositumomab, trastuzumab, uliledlimab, vadastuximab, varlilumab, xaluritamig, zanidatamab, zenocutuzumab, zilovertamab, and zolbetuximab.

54. A method of killing a cell in a subject, the method comprising: a) contacting the cell with a radioemitter comprising67Ga,68Ga,99mTc,103Pd,1111 n ,123l,125l, 131l,131Cs,133Xe,153Sm,149Pm,161Tb,169Er,177Lu,201TI, or203Pb to produce ionizing radiation within the cell; and b) contacting the cell with cytotoxic drug, wherein the cytotoxic drug is generated in situ by exposing a caged prodrug to the ionizing radiation to produce the cytotoxic drug.

55. The method of claim 54, wherein the radioemitter comprises67Ga,99mTc,111In , or123l.

56. A method of killing a cell in a subject, the method comprising: a. contacting the cell with a caged prodrug of a cytotoxic drug; b. following from 4 hours to 48 hours after the completion of step a), contacting the cell with a radioemitter to produce ionizing radiation within the cell; and c. generating the cytotoxic drug in situ by exposing the caged prodrug to the ionizing radiation to produce the cytotoxic drug.

57. The method of claim 56, wherein the radioemitter comprises "mTc,111In ,177Lu, or225Ac.

58. A method of killing a cell in a subject, the method comprising: a. contacting the cell with a radioemitter to produce ionizing radiation within the cell; b. following from 4 hours to 48 hours after the completion of step a), contacting the cell with a caged prodrug of a cytotoxic drug; and c. generating the cytotoxic drug in situ by exposing the caged prodrug to the ionizing radiation to produce the cytotoxic drug.

59. The method of claim 58, wherein the radioemitter comprises64Cu,68Ga, "Y, "mTc,111ln,123l,131l, 177Lu,223Ra, or225Ac.

60. The method of any one of claims 54-59, wherein the cytotoxic drug is selected from antitubulin agents, DNA replication inhibitors, alkylating agents, antifolates, antimetabolites, chemotherapy sensitizers, topoisomerase inhibitors, and vinca alkaloids.61 . The method of any one of claims 54-60, wherein the cytotoxic drug is selected from pyrrolobenzodiazepine dimer (PBD dimer), exatecan, monomethyl auristatin E (MMAE), doxorubicin, and gardiquimod.

62. The method of any one of claims 54-61 , wherein the caged prodrug has the structure of Formula l-B:Formula l-B wherein: ring A is a phenyl; m is 5; one Ri is azido, and each remaining Ri is fluoro; each R2 is independently halogen, C1 -C6 alkyl, or C1-C6 haloalkyl; n is 0, 1 , 2, 3, or 4;Rs is -NRaRb, or a targeting moiety;Rais -H or C1-C6 alkyl;Rb is -H or C1-C6 alkyl; andD is a drug moiety comprising a radical of the cytotoxic drug.

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