TGF-beta theranostics and use thereof

Abstract

The compositions provided herein including embodiments thereof include, inter alia, TGF-beta-binding agents that bind to TGF-beta in its active / mature form (i.e., active TGF-beta, mature TGF-beta). Further provided are methods of treatment including, inter alia, administering to a patient a diagnostic and a therapeutic TGF-beta-binding agents both including a radioisotope. The compositions and methods provided herein are, inter alia, useful as theranostics for the treatment of cancer (e.g., solid tumors such as carcinoma or sarcoma).

Description

PATENT Attorney Docket No.: 048536-803001WO Client Ref. No. SF2024-230 TGF-BETA THERANOSTICS AND USE THEREOF CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This International Application claims the benefit of priority under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 63 / 690,238, filed on September 3, 2024 which is hereby incorporated by reference in its entirety and for all purposes. STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

[0002] This invention was made with government support under R01 NS109911 awarded by the National Institutes of Health. The government has certain rights in the invention. BACKGROUND

[0003] Despite earlier detection of cancer, the rate of metastatic breast cancer at initial diagnosis in the United States has not changed since 1975. Metastatic spread is most frequent in women who have triple-negative breast cancer (TNBC), but also occurs in women with hormone receptor- positive (HR+) with or without human epidermal growth factor receptor 2 (HER2) -positive disease. Metastatic breast cancer can spread to any part of the body, but upon presentation, it has most commonly has spread to bone (51%), followed by lung (17%), brain (16%), and liver (6%). The remaining 10% of patients have multiple metastatic sites. Of appreciable concern, brain metastases that occur in 30% of breast cancer patients are associated with poor prognosis and very limited therapeutic options (1, 2).

[0004] External beam radiotherapy (EBT) is employed in metastatic disease setting when patients present with single or multiple symptomatic brain metastases (2-5). Whole brain irradiation is used when there are numerous lesions or a large total volume of intracranial disease but engenders neurocognitive compromise that negatively impacts the quality of life (6). In patients with limited numbers of small brain metastases, commonly one to three metastases, stereotactic radiosurgery (delivers high doses to small volumes that provide equivalent control but spare normal brain,thereby reducing the morbidity of whole brain irradiation. Use of stereotactic radiotherapy has the advantage of minimizing dose to uninvolved brain tissue with modest to good local control of brain metastases of approximately 70% at one year (7) and is associated with less neurocognitive decline (8). Moreover, in contrast to whole brain radiotherapy EBT can be performed iteratively to address subsequent new intracranial disease frequently observed in patients with metastatic disease (4). However, the repeated treatment comes with a quality-of-life detriment in cognitive function and does not cure metastatic disease. The compositions and methods provided herein address these and other needs in the art. BRIEF SUMMARY OF THE INVENTION

[0005] In an aspect is provided an active TGF-beta binding agent including a radioisotope.

[0006] In an aspect is provided a pharmaceutical composition including a pharmaceutically acceptable excipient and an active TGF-beta binding agent as provided herein including embodiments thereof.

[0007] In an aspect is provided a method of treating cancer in a subject in need thereof. The method includes administering an effective amount of an active TGF-beta binding agent as provided herein including embodiments thereof, or an effective amount of a pharmaceutical composition of a pharmaceutical composition provided herein including embodiments to the subject, thereby treating cancer in the subject.

[0008] In an aspect is provided a method of treating cancer in a subject in need thereof. The method includes (i) administering to the subject an effective amount of a first active TGF-beta binding agent including a first radioisotope; and (ii) administering to the subject an effective amount of a second active TGF-beta binding agent including a second radioisotope, thereby treating cancer in the subject.

[0009] In an aspect is provided a method of treating cancer in a subject in need thereof. The method includes (i) administering to the subject an effective amount of a first active TGF-beta binding agent including a first radioisotope; (ii) administering a TGFbeta-activating amount of anexternal beam therapy to the subject, and (iii) administering to the subject an effective amount of a second active TGF-beta binding agent including a second radioisotope, thereby treating cancer in the subject. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] FIG. 1 External Beam Therapy (EBT) versus radiopharmaceutical therapy (RPT). A. An external beam delivers the same absorbed dose per cell regardless of the number of cells and has both an entry and exit field that may affect normal organs, which is decreased by using conformal delivery. B. In radiopharmaceutical therapy, the absorbed dose delivered per cell by emissions originating from cells is influenced by the range of the isotope emissions, the density of the target in the volume, and the specificity of the tumor vs normal distribution. When these are optimized, normal tissue receives much less dose than tumor.

[0011] FIG. 2 Conjugation, purification and competition binding assay of 89Zr-Fresolimumab. A. Schematic representation of the process of conjugation and radiolabeling of fresolimumab. B. Competition binding assay of 89Zr-DFO-fresolimumab against unlabeled fresolimumab (IC50 = 16nM). C. 89Zr-GC1008 (Fresolimumab) was injected into mice with intra-cranial tumors (left) or PBS injected (right) and imaged with microPET-CT scan 72 hours later.

[0012] FIG. 3 PET imaging of 89Zr-fresolimumab detects active TGFbeta in vivo. A. PET imaging of 89Zr-fresolimumab in mice bearing latent TGFbeta producing B9 tumors and active TGFbeta producing C19 cells. Representative PET / CT transverse sections (lower image) and coronal sections (right image) are shown. B. Quantification of radioactivity measured in flank tumors (p=0.004, paired-test). C. PET imaging of mice bearding subcutaneous LLC beta8 LLC tumors. D. Quantification of radioactivity measured in flank tumors (p=0.004, paired t-test). E. One tumor of bilateral flank tumors was irradiated with a single dose of 15Gy while the contralateral tumor served as a sham control. Mice were injected with 89Zr-fresolimumab and ex-vivo radioactivity was measured after 72 hours. F. Sham and irradiated tumors were harvested and stained with antibodies for active TGFbeta and the downstream signaling pathway readout pSmad2. G. Quantification of TGFbeta mean fluorescence intensity and percentage of pSmad2 positive cellsper high-power field (HPF). Each symbol represents the average of 5 representative images from one tumor. Paired student t test, ** indicates p,0.001

[0013] FIG. 4 Detection of active TGFbeta in brain tumors by 89Zr-fresolimumab PET imaging. Mice bearing intracranial A. SB28 and B. GL261 intracranial tumors imaged with 18F-FDG PET (upper panel) and 89Zr-fresolimumab PET (bottom panel). Coronal (left), sagittal (middle) and BLI (right) are shown. C. Total intracranial radioactivity in mice bearing SB28 tumors or PBS injected controls 120 h after injection (p=0.006, t-test). D. Total intracranial radioactivity in mice bearing SB28 tumors or GL261 tumors or PBS injected controls analyzed 48 h after injection (p=0.01, t- test). E. Ex vivo radioactivity of tissues from mice injected with 89Zr-fresolimumab. Ex vivo radioactivity for lung, liver, and brain of mice bearing orthotopic SB28, GL261 or 4T1 tumors 7 days post injection with 89Zr-fresolimumab. One-way ANOVA, p,0.0001.

[0014] FIG. 5 Pharmacokinetics of 89Zr-fresolimumab and external beam response of 4T1 tumor bearing mice. A. Temporal PET / CT images up to 10 days after the i.v. injection of about 190 uCi of 89Zr-fresolimumab. Images show left flank tumor with ROI in red (axial images). The scale represents the % injected does (ID) per gram (0-36% ID / g). B. Time-activity graph demonstrating the retention of 89Zr-fresolimumab in the tumor.

[0015] FIG.6 Tumor uptake and control by 177Lu-Freso. Mice bearing subcutaneous 4T1-BrA tumors were randomized on day 9 to 2 Gy priming EBT (n=4) or none (n=2). All mice received about 0.8 mCi 177Lu-freso on day 10 and were imaged by SPECT / CT on day 14. A. Representative coronal and axial SPECT / CT projections are shown with tumor uptake (T). B. Tumor uptake (%ID / g) based on tumor region of interest. C. Mice bearing subcutaneous 4T1-BrA tumors were randomized and day 10 (arrow) to sham (circles, n=7). Tumors were measured daily by calipers. D. Mice bearing subcutaneous 4T1-BrA tumors were randomized on day 12 (arrow) to sham (circles n=7) EBT with 10 Gy (squares, n=10). Mean and SEM are shown, one way ANOVA. Doubling time (DT) estimate and 95% confidence intervals based on exponential growth are shown.

[0016] FIG. 7 Intracranial 4T1-BrA metastatic progression. A. Bioluminescence of intracranial 4T1-BrA tumors and leptomeningeal spread. B. Ex vivo bioluminescence of brain and concurrent metastasis to lungs.

[0017] FIG. 8 Digital autoradiography image and relative activity map for 177Lu-PSMA-167. (A) DAR image and relative activity distribution of 177Lu-PSMA-167 in cryosectioned tumor slices harvested from prostate cancer xenograft. (B) Example of dose-rate-area histogram and dose distribution map (78)

[0018] FIG. 9 Isotype uptake and survival in intracranial iGL261 brain tumor model.

[0019] FIG. 10 Survival in intracranial iGL261 brain tumor model.

[0020] FIG. 11 Effect of177Lu-fresolimumab in 4T1 breast cancer model. DETAILED DESCRIPTION I. Definitions

[0021] While various embodiments and aspects of the present invention are shown and described herein, it will be obvious to those skilled in the art that such embodiments and aspects are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention.

[0022] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in the application including, without limitation, patents, patent applications, articles, books, manuals, and treatises are hereby expressly incorporated by reference in their entirety for any purpose.

[0023] The abbreviations used herein have their conventional meaning within the chemical and biological arts. The chemical structures and formulae set forth herein are constructed according to the standard rules of chemical valency known in the chemical arts.

[0024] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art. See, e.g., Singleton et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY 2nd ed., J. Wiley & Sons (New York, NY 1994); Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, Cold Springs Harbor Press (Cold Springs Harbor, NY 1989). Any methods, devices and materials similar or equivalent to those described herein can be used in the practice of this invention. The following definitions are provided to facilitate understanding of certain terms used frequently herein and are not meant to limit the scope of the present disclosure.

[0025] Where substituent groups are specified by their conventional chemical formulae, written from left to right, they equally encompass the chemically identical substituents that would result from writing the structure from right to left, e.g., -CH2O- is equivalent to -OCH2-.

[0026] The term "alkyl," by itself or as part of another substituent, means, unless otherwise stated, a straight (i.e., unbranched) or branched non-cyclic carbon chain (or carbon), or combination thereof, which may be fully saturated, mono- or polyunsaturated and can include di- and multivalent radicals, having the number of carbon atoms designated (i.e., C1-C10 means one to ten carbons). Examples of saturated hydrocarbon radicals include, but are not limited to, groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, (cyclohexyl)methyl, homologs and isomers of, for example, n-pentyl, n-hexyl, n-heptyl, n-octyl and the like. An unsaturated alkyl group is one having one or more double bonds or triple bonds. Examples of unsaturated alkyl groups include, but are not limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4- pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl, and the higher homologs and isomers. An alkoxy is an alkyl attached to the remainder of the molecule via an oxygen linker (-O-). An alkyl moiety may be an alkenyl moiety. An alkyl moiety may be an alkynyl moiety. An alkyl moiety may be fully saturated.

[0027] The term "alkylene," by itself or as part of another substituent, means, unless otherwise stated, a divalent radical derived from an alkyl, as exemplified, but not limited by, -CH2CH2CH2CH2-. Typically, an alkyl (or alkylene) group will have from 1 to 24 carbon atoms, with those groups having 10 or fewer carbon atoms being preferred in the present invention. A "lower alkyl" or "lower alkylene" is a shorter chain alkyl or alkylene group, generally having eight or fewer carbon atoms. The term "alkenylene," by itself or as part of another substituent, means, unless otherwise stated, a divalent radical derived from an alkene.

[0028] The term "heteroalkyl," by itself or in combination with another term, means, unless otherwise stated, a stable non-cyclic straight or branched chain, or combinations thereof, including at least one carbon atom and at least one heteroatom (e.g. O, N, P, Si or S) and wherein the nitrogen and sulfur atoms may optionally be oxidized, and the nitrogen heteroatom may optionally be quaternized. The heteroatom(s) O, N, P, S, and Si may be placed at any interior position of the heteroalkyl group or at the position at which the alkyl group is attached to the remainder of the molecule. Examples include, but are not limited to: -CH2-CH2-O-CH3, -CH2-CH2-NH-CH3, -CH2-CH2-N(CH3)-CH3, -CH2-S-CH2-CH3, -CH2-CH2, -S(O)-CH3, -CH2-CH2-S(O)2-CH3, -CH=CH-O-CH3, -Si(CH3)3, -CH2-CH=N-OCH3, -C H=CH-N(CH3)-CH3, -O-CH3, -O-CH2-CH3, and -CN. Up to two or three heteroatoms may be consecutive, such as, for example, -CH2-NH-OCH3 and –CH2-O-Si(CH3)3. A heteroalkyl moiety may include one heteroatom (e.g., O, N, S, Si, or P). A heteroalkyl moiety may include two optionally different heteroatoms (e.g., O, N, S, Si, or P). A heteroalkyl moiety may include three optionally different heteroatoms (e.g., O, N, S, Si, or P). A heteroalkyl moiety may include four optionally different heteroatoms (e.g., O, N, S, Si, or P). A heteroalkyl moiety may include five optionally different heteroatoms (e.g., O, N, S, Si, or P). A heteroalkyl moiety may include up to 8 optionally different heteroatoms (e.g., O, N, S, Si, or P).

[0029] Similarly, the term "heteroalkylene," by itself or as part of another substituent, means, unless otherwise stated, a divalent radical derived from heteroalkyl, as exemplified, but not limited by, -CH2-CH2-S-CH2-CH2- and -CH2-S-CH2-CH2-NH-CH2-. For heteroalkylene groups, heteroatoms can also occupy either or both of the chain termini (e.g., alkyleneoxy, alkylenedioxy,alkyleneamino, alkylenediamino, and the like). Still further, for alkylene and heteroalkylene linking groups, no orientation of the linking group is implied by the direction in which the formula of the linking group is written. For example, the formula -C(O)2R'- represents both -C(O)2R'- and -R'C(O)2-. As described above, heteroalkyl groups, as used herein, include those groups that are attached to the remainder of the molecule through a heteroatom, such as -C(O)R', -C(O)NR', -NR'R'', -OR', -SR', and / or -SO2R'. Where “heteroalkyl” is recited, followed by recitations of specific heteroalkyl groups, such as -NR'R'' or the like, it will be understood that the terms heteroalkyl and -NR'R'' are not redundant or mutually exclusive. Rather, the specific heteroalkyl groups are recited to add clarity. Thus, the term "heteroalkyl" should not be interpreted herein as excluding specific heteroalkyl groups, such as -NR'R'' or the like.

[0030] The terms "cycloalkyl" and "heterocycloalkyl," by themselves or in combination with other terms, mean, unless otherwise stated, non-aromatic cyclic versions of "alkyl" and "heteroalkyl," respectively, wherein the carbons making up the ring or rings do not necessarily need to be bonded to a hydrogen due to all carbon valencies participating in bonds with non-hydrogen atoms. Additionally, for heterocycloalkyl, a heteroatom can occupy the position at which the heterocycle is attached to the remainder of the molecule. Examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, 3- hydroxy-cyclobut-3-enyl-1,2, dione, 1H-1,2,4-triazolyl-5(4H)-one, 4H-1,2,4-triazolyl, and the like. Examples of heterocycloalkyl include, but are not limited to, 1-(1,2,5,6-tetrahydropyridyl), 1- piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1-piperazinyl, 2-piperazinyl , and the like. A "cycloalkylene" and a "heterocycloalkylene," alone or as part of another substituent, means a divalent radical derived from a cycloalkyl and heterocycloalkyl, respectively. A heterocycloalkyl moiety may include one ring heteroatom (e.g., O, N, S, Si, or P). A heterocycloalkyl moiety may include two optionally different ring heteroatoms (e.g., O, N, S, Si, or P). A heterocycloalkyl moiety may include three optionally different ring heteroatoms (e.g., O, N, S, Si, or P). A heterocycloalkyl moiety may include four optionally different ring heteroatoms (e.g., O, N, S, Si, or P). A heterocycloalkyl moiety may include five optionally different ring heteroatoms(e.g., O, N, S, Si, or P). A heterocycloalkyl moiety may include up to 8 optionally different ring heteroatoms (e.g., O, N, S, Si, or P).

[0031] The terms "halo" or "halogen," by themselves or as part of another substituent, mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom. Additionally, terms such as "haloalkyl" are meant to include monohaloalkyl and polyhaloalkyl. For example, the term "halo(C1- C4)alkyl" includes, but is not limited to, fluoromethyl, difluoromethyl, trifluoromethyl, 2,2,2- trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the like.

[0032] The term "acyl" means, unless otherwise stated, -C(O)R where R is a substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.

[0033] The term "aryl" means, unless otherwise stated, a polyunsaturated, aromatic, hydrocarbon substituent, which can be a single ring or multiple rings (preferably from 1 to 3 rings) that are fused together (i.e., a fused ring aryl) or linked covalently. A fused ring aryl refers to multiple rings fused together wherein at least one of the fused rings is an aryl ring. The term "heteroaryl" refers to aryl groups (or rings) that contain at least one heteroatom such as N, O, or S, wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atom(s) are optionally quaternized. Thus, the term "heteroaryl" includes fused ring heteroaryl groups (i.e., multiple rings fused together wherein at least one of the fused rings is a heteroaromatic ring). A 5,6-fused ring heteroarylene refers to two rings fused together, wherein one ring has 5 members and the other ring has 6 members, and wherein at least one ring is a heteroaryl ring. Likewise, a 6,6-fused ring heteroarylene refers to two rings fused together, wherein one ring has 6 members and the other ring has 6 members, and wherein at least one ring is a heteroaryl ring. And a 6,5-fused ring heteroarylene refers to two rings fused together, wherein one ring has 6 members and the other ring has 5 members, and wherein at least one ring is a heteroaryl ring. A heteroaryl group can be attached to the remainder of the molecule through a carbon or heteroatom. Non-limiting examples of aryl and heteroaryl groups include phenyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl,2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3- isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2- thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, and 6-quinolyl. Substituents for each of the above noted aryl and heteroaryl ring systems are selected from the group of acceptable substituents described below. An "arylene" and a "heteroarylene," alone or as part of another substituent, mean a divalent radical derived from an aryl and heteroaryl, respectively. Non-limiting examples of aryl and heteroaryl groups include pyridinyl, pyrimidinyl, thiophenyl, thienyl, furanyl, indolyl, benzoxadiazolyl, benzodioxolyl, benzodioxanyl, thianaphthanyl, pyrrolopyridinyl, indazolyl, quinolinyl, quinoxalinyl, pyridopyrazinyl, quinazolinonyl, benzoisoxazolyl, imidazopyridinyl, benzofuranyl, benzothienyl, benzothiophenyl, phenyl, naphthyl, biphenyl, pyrrolyl, pyrazolyl, imidazolyl, pyrazinyl, oxazolyl, isoxazolyl, thiazolyl, furylthienyl, pyridyl, pyrimidyl, benzothiazolyl, purinyl, benzimidazolyl, isoquinolyl, thiadiazolyl, oxadiazolyl, pyrrolyl, diazolyl, triazolyl, tetrazolyl, benzothiadiazolyl, isothiazolyl, pyrazolopyrimidinyl, pyrrolopyrimidinyl, benzotriazolyl, benzoxazolyl, or quinolyl. The examples above may be substituted or unsubstituted and divalent radicals of each heteroaryl example above are non-limiting examples of heteroarylene. A heteroaryl moiety may include one ring heteroatom (e.g., O, N, or S). A heteroaryl moiety may include two optionally different ring heteroatoms (e.g., O, N, or S). A heteroaryl moiety may include three optionally different ring heteroatoms (e.g., O, N, or S). A heteroaryl moiety may include four optionally different ring heteroatoms (e.g., O, N, or S). A heteroaryl moiety may include five optionally different ring heteroatoms (e.g., O, N, or S). An aryl moiety may have a single ring. An aryl moiety may have two optionally different rings. An aryl moiety may have three optionally different rings. An aryl moiety may have four optionally different rings. A heteroaryl moiety may have one ring. A heteroaryl moiety may have two optionally different rings. A heteroaryl moiety may have three optionally different rings. A heteroaryl moiety may have four optionally different rings. A heteroaryl moiety may have five optionally different rings.

[0034] A fused ring heterocycloalkyl-aryl is an aryl fused to a heterocycloalkyl. A fused ring heterocycloalkyl-heteroaryl is a heteroaryl fused to a heterocycloalkyl. A fused ring heterocycloalkyl-cycloalkyl is a heterocycloalkyl fused to a cycloalkyl. A fused ring heterocycloalkyl-heterocycloalkyl is a heterocycloalkyl fused to another heterocycloalkyl. Fused ring heterocycloalkyl-aryl, fused ring heterocycloalkyl-heteroaryl, fused ring heterocycloalkyl- cycloalkyl, or fused ring heterocycloalkyl-heterocycloalkyl may each independently be unsubstituted or substituted with one or more of the substituents described herein.

[0035] The term "oxo," as used herein, means an oxygen that is double bonded to a carbon atom.

[0036] The term "alkylsulfonyl," as used herein, means a moiety having the formula -S(O2)-R', where R' is a substituted or unsubstituted alkyl group as defined above. R' may have a specified number of carbons (e.g., "C1-C4alkylsulfonyl").

[0037] Each of the above terms (e.g., "alkyl," "heteroalkyl,", "cycloalkyl", "heterocycloalkyl", "aryl," and "heteroaryl") includes both substituted and unsubstituted forms of the indicated radical. Preferred substituents for each type of radical are provided below.

[0038] Substituents for the alkyl and heteroalkyl radicals (including those groups often referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl) can be one or more of a variety of groups selected from, but not limited to, -OR', =O, =NR', =N-OR', -NR'R'', -SR', -halogen, -SiR'R''R''', -OC(O)R', -C(O)R', -CO2R', -CONR'R'', -OC(O)NR'R'', -NR''C(O)R', -NR'-C(O)NR''R''', -NR''C(O)2R', -NR-C(NR'R'')=NR''', -S(O)R', -S(O)2R', -S(O)2N(R)('R''-NRSO2R'), -CN, and -NO2in a number ranging from zero to (2m'+1), where m' is the total number of carbon atoms in such radical. R', R'', R''', and R'''' each preferably independently refer to hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl (e.g., aryl substituted with 1-3 halogens), substituted or unsubstituted alkyl, alkoxy, or thioalkoxy groups, or arylalkyl groups. When a compound of the invention includes more than one R group, for example, each of the R groups is independently selected as are each R', R'', R''', and R'''' group when more than one ofthese groups is present. When R' and R'' are attached to the same nitrogen atom, they can be combined with the nitrogen atom to form a 4-, 5-, 6-, or 7-membered ring. For example, -NR'R'' includes, but is not limited to, 1-pyrrolidinyl and 4-morpholinyl. From the above discussion of substituents, one of skill in the art will understand that the term “alkyl” is meant to include groups including carbon atoms bound to groups other than hydrogen groups, such as haloalkyl (e.g., -CF3 and -CH2CF3) and acyl (e.g., -C(O)CH3, -C(O)CF3, -C(O)CH2OCH3, and the like).

[0039] Similar to the substituents described for the alkyl radical, substituents for the aryl and heteroaryl groups are varied and are selected from, for example: -OR', -NR'R'', -SR', -halogen, -SiR'R''R''', -OC(O)R', -C(O)R', -CO2R', -CONR'R'', -OC(O)NR'R'', -NR''C(O)R', -NR'-C(O)NR''R''', NR''C(O)2R', NRC(NR'R'')=NR''', S(O)R', -S(O)2R', -S(O)2N(R')(R'', -NRSO2R'), -CN, -NO2, -R', -N3, -CH(Ph)2, fluoro(C1-C4)alkoxy, and fluoro(C1- C4)alkyl, in a number ranging from zero to the total number of open valences on the aromatic ring system; and where R', R'', R''', and R'''' are preferably independently selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl. When a compound of the invention includes more than one R group, for example, each of the R groups is independently selected as are each R', R'', R''', and R'''' groups when more than one of these groups is present.

[0040] Where a moiety is substituted with an R substituent, the group may be referred to as "R- substituted." Where a moiety is R-substituted, the moiety is substituted with at least one R substituent and each R substituent is optionally different. For example, where a moiety herein is R1A-substituted or unsubstituted alkyl, a plurality of R1Asubstituents may be attached to the alkyl moiety wherein each R1Asubstituent is optionally different. Where an R-substituted moiety is substituted with a plurality of R substituents, each of the R-substituents may be differentiated herein using a prime symbol (') such as R', R'', etc. For example, where a moiety is R3A-substituted or unsubstituted alkyl, and the moiety is substituted with a plurality of R3Asubstituents, the plurality ofR3Asubstituents may be differentiated as R3A', R3A'', R3A''', etc. In some embodiments, the plurality of R substituents is 3.

[0041] Two or more substituents may optionally be joined to form aryl, heteroaryl, cycloalkyl, or heterocycloalkyl groups. Such so-called ring-forming substituents are typically, though not necessarily, found attached to a cyclic base structure. In one embodiment, the ring-forming substituents are attached to adjacent members of the base structure. For example, two ring-forming substituents attached to adjacent members of a cyclic base structure create a fused ring structure. In another embodiment, the ring-forming substituents are attached to a single member of the base structure. For example, two ring-forming substituents attached to a single member of a cyclic base structure create a spirocyclic structure. In yet another embodiment, the ring-forming substituents are attached to non-adjacent members of the base structure.

[0042] Two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally form a ring of the formula -T-C(O)-(CRR')q-U-, wherein T and U are independently -NR-, -O-, -CRR'-, or a single bond, and q is an integer of from 0 to 3. Alternatively, two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula -A-(CH2)r-B-, wherein A and B are independently -CRR'-, -O-, -NR-, -S-, -S(O) -, -S(O)2-, -S(O)2NR'-, or a single bond, and r is an integer of from 1 to 4. One of the single bonds of the new ring so formed may optionally be replaced with a double bond. Alternatively, two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula -(CRR')s-X'- (C''R''R''')d-, where variables s and d are independently integers of from 0 to 3, and X' is -O-, -NR'-, -S-, -S(O)-, -S(O)2-, or -S(O)2NR'-. The substituents R, R', R'', and R''' are preferably independently selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl.

[0043] As used herein, the terms "heteroatom" or "ring heteroatom" are meant to include, oxygen (O), nitrogen (N), sulfur (S), phosphorus (P), and silicon (Si).

[0044] A "substituent group," as used herein, means a group selected from the following moieties: (A) oxo, halogen, -CF3, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO2Cl, -SO3H, -SO4H, -SO2NH2, −NHNH2, −ONH2, −NHC=(O)NHNH2, −NHC=(O) NH2, -NHSO2H, -NHC= (O)H, -NHC(O)-OH, -NHOH, -OCF3, -OCHF2, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl, and (B) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, substituted with at least one substituent selected from: (i) oxo, halogen, -CF3, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO2Cl, -SO3H, -SO4H, -SO2NH2, −NHNH2, −ONH2, −NHC=(O)NHNH2, −NHC=(O) NH2, -NHSO2H, -NHC= (O)H, -NHC(O)-OH, -NHOH, -OCF3, -OCHF2, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl, and (ii) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, substituted with at least one substituent selected from: (a) oxo, halogen, -CF3, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO2Cl, -SO3H, -SO4H, -SO2NH2, −NHNH2, −ONH2, −NHC=(O)NHNH2, −NHC=(O) NH2, - NHSO2H, -NHC= (O)H, -NHC(O)-OH, -NHOH, -OCF3, -OCHF2, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl, and (b) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, substituted with at least one substituent selected from: oxo, halogen, -CF3, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO2Cl, -SO3H, -SO4H, -SO2NH2, −NHNH2, −ONH2, −NHC=(O)NHNH2, −NHC=(O) NH2, -NHSO2H, -NHC= (O)H, -NHC(O)-OH, -NHOH, -OCF3, -OCHF2, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl.

[0045] A “chemical linker,” as provided herein, is a covalent linker, a non-covalent linker, a peptide linker (a linker including a peptide moiety), a cleavable peptide linker, a substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene or substituted or unsubstituted heteroarylene or any combination thereof. Thus, a chemical linker as provided herein may include a plurality of chemical moieties, wherein each of the plurality of chemical moieties is chemically different. Alternatively, the chemical linker may be a non-covalent linker. Examples of non-covalent linkers include without limitation, ionic bonds, hydrogen bonds, halogen bonds, van der Waals interactions (e.g. dipole-dipole, dipole-induced dipole, London dispersion), ring stacking (pi effects), and hydrophobic interactions. In embodiments, a chemical linker is formed using conjugate chemistry including, but not limited to nucleophilic substitutions (e.g., reactions of amines and alcohols with acyl halides, active esters), electrophilic substitutions (e.g., enamine reactions) and additions to carbon-carbon and carbon-heteroatom multiple bonds (e.g., Michael reaction, Diels-Alder addition).

[0046] A "size-limited substituent" or "size-limited substituent group," as used herein, means a group selected from all of the substituents described above for a "substituent group," wherein each substituted or unsubstituted alkyl is a substituted or unsubstituted C1-C20alkyl, each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2 to 20 membered heteroalkyl, each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C3-C8 cycloalkyl, each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 3 to 8 membered heterocycloalkyl, each substituted or unsubstituted aryl is a substituted or unsubstituted C6-C10aryl, and each substituted or unsubstituted heteroaryl is a substituted or unsubstituted 5 to 10 membered heteroaryl.

[0047] A "lower substituent" or "lower substituent group," as used herein, means a group selected from all of the substituents described above for a “substituent group,” wherein each substituted or unsubstituted alkyl is a substituted or unsubstituted C1-C8 alkyl, each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2 to 8 membered heteroalkyl, each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C3-C7 cycloalkyl, each substituted orunsubstituted heterocycloalkyl is a substituted or unsubstituted 3 to 7 membered heterocycloalkyl, each substituted or unsubstituted aryl is a substituted or unsubstituted C6-C10 aryl, and each substituted or unsubstituted heteroaryl is a substituted or unsubstituted 5 to 9 membered heteroaryl.

[0048] In some embodiments, each substituted group described in the compounds herein is substituted with at least one substituent group. More specifically, in some embodiments, each substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, substituted alkylene, substituted heteroalkylene, substituted cycloalkylene, substituted heterocycloalkylene, substituted arylene, and / or substituted heteroarylene described in the compounds herein are substituted with at least one substituent group. In other embodiments, at least one or all of these groups are substituted with at least one size-limited substituent group. In other embodiments, at least one or all of these groups are substituted with at least one lower substituent group.

[0049] In other embodiments of the compounds herein, each substituted or unsubstituted alkyl may be a substituted or unsubstituted C1-C20alkyl, each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2 to 20 membered heteroalkyl, each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C3-C8 cycloalkyl, each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 3 to 8 membered heterocycloalkyl, each substituted or unsubstituted aryl is a substituted or unsubstituted C6-C10 aryl, and / or each substituted or unsubstituted heteroaryl is a substituted or unsubstituted 5 to 10 membered heteroaryl. In some embodiments of the compounds herein, each substituted or unsubstituted alkylene is a substituted or unsubstituted C1-C20alkylene, each substituted or unsubstituted heteroalkylene is a substituted or unsubstituted 2 to 20 membered heteroalkylene, each substituted or unsubstituted cycloalkylene is a substituted or unsubstituted C3-C8 cycloalkylene, each substituted or unsubstituted heterocycloalkylene is a substituted or unsubstituted 3 to 8 membered heterocycloalkylene, each substituted or unsubstituted arylene is a substituted or unsubstituted C6-C10 arylene, and / or each substituted or unsubstituted heteroarylene is a substituted or unsubstituted 5 to 10 membered heteroarylene.

[0050] In some embodiments, each substituted or unsubstituted alkyl is a substituted or unsubstituted C1-C8 alkyl, each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2 to 8 membered heteroalkyl, each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C3-C7cycloalkyl, each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 3 to 7 membered heterocycloalkyl, each substituted or unsubstituted aryl is a substituted or unsubstituted C6-C10 aryl, and / or each substituted or unsubstituted heteroaryl is a substituted or unsubstituted 5 to 9 membered heteroaryl. In some embodiments, each substituted or unsubstituted alkylene is a substituted or unsubstituted C1-C8 alkylene, each substituted or unsubstituted heteroalkylene is a substituted or unsubstituted 2 to 8 membered heteroalkylene, each substituted or unsubstituted cycloalkylene is a substituted or unsubstituted C3-C7cycloalkylene, each substituted or unsubstituted heterocycloalkylene is a substituted or unsubstituted 3 to 7 membered heterocycloalkylene, each substituted or unsubstituted arylene is a substituted or unsubstituted C6-C10arylene, and / or each substituted or unsubstituted heteroarylene is a substituted or unsubstituted 5 to 9 membered heteroarylene. In some embodiments, the compound is a chemical species set forth in the Examples section, figures, or tables below.

[0051] As used herein, the term "conjugate" refers to the association between atoms or molecules. The association can be direct or indirect. For example, a conjugate between a nucleic acid and a protein can be direct, e.g., by covalent bond, or indirect, e.g., by non-covalent bond (e.g. electrostatic interactions (e.g. ionic bond, hydrogen bond, halogen bond), van der Waals interactions (e.g. dipole-dipole, dipole-induced dipole, London dispersion), ring stacking (pi effects), hydrophobic interactions and the like). In embodiments, conjugates are formed using conjugate chemistry including, but are not limited to nucleophilic substitutions (e.g., reactions of amines and alcohols with acyl halides, active esters), electrophilic substitutions (e.g., enamine reactions) and additions to carbon-carbon and carbon-heteroatom multiple bonds (e.g., Michael reaction, Diels- Alder addition). These and other useful reactions are discussed in, for example, March, ADVANCED ORGANIC CHEMISTRY, 3rd Ed., John Wiley & Sons, New York, 1985; Hermanson, BIOCONJUGATE TECHNIQUES, Academic Press, San Diego, 1996; and Feeney etal., MODIFICATION OF PROTEINS; Advances in Chemistry Series, Vol. 198, American Chemical Society, Washington, D.C., 1982.

[0052] Useful reactive moieties or functional groups used for conjugate chemistries (including "click chemistries" as known in the art) herein include, for example: (a) carboxyl groups and various derivatives thereof including, but not limited to, N- hydroxysuccinimide esters, N-hydroxybenztriazole esters, acid halides, acyl imidazoles, thioesters, p-nitrophenyl esters, alkyl, alkenyl, alkynyl and aromatic esters; (b) hydroxyl groups which can be converted to esters, ethers, aldehydes, etc. (c) haloalkyl groups wherein the halide can be later displaced with a nucleophilic group such as, for example, an amine, a carboxylate anion, thiol anion, carbanion, or an alkoxide ion, thereby resulting in the covalent attachment of a new group at the site of the halogen atom; (d) dienophile groups which are capable of participating in Diels-Alder reactions such as, for example, maleimido groups; (e) aldehyde or ketone groups such that subsequent derivatization is possible via formation of carbonyl derivatives such as, for example, imines, hydrazones, semicarbazones or oximes, or via such mechanisms as Grignard addition or alkyllithium addition; (f) sulfonyl halide groups for subsequent reaction with amines, for example, to form sulfonamides; (g) thiol groups, which can be converted to disulfides, reacted with acyl halides, or bonded to metals such as gold; (h) amine or sulfhydryl groups, which can be, for example, acylated, alkylated or oxidized; (i) alkenes, which can undergo, for example, cycloadditions, acylation, Michael addition, etc.; (j) epoxides, which can react with, for example, amines and hydroxyl compounds; (k) phosphoramidites and other standard functional groups useful in nucleic acid synthesis; (l) metal silicon oxide bonding; (m) metal bonding to reactive phosphorus groups (e.g. phosphines) to form, for example, phosphate diester bonds; and(n) sulfones, for example, vinyl sulfone.

[0053] Chemical synthesis of compositions by joining small modular units using conjugate (“click”) chemistry is well known in the art and described, for example, in H. C. Kolb, M. G. Finn and K. B. Sharpless ((2001). "Click Chemistry: Diverse Chemical Function from a Few Good Reactions". Angewandte Chemie International Edition 40 (11): 2004–2021); R. A. Evans ((2007). "The Rise of Azide–Alkyne 1,3-Dipolar 'Click' Cycloaddition and its Application to Polymer Science and Surface Modification". Australian Journal of Chemistry 60 (6): 384–395; W.C. Guida et al. Med. Res. Rev. p 31996; Spiteri, Christian and Moses, John E. ((2010). "Copper-Catalyzed Azide–Alkyne Cycloaddition: Regioselective Synthesis of 1,4,5-Trisubstituted 1,2,3-Triazoles". Angewandte Chemie International Edition 49 (1): 31–33); Hoyle, Charles E. and Bowman, Christopher N. ((2010). "Thiol–Ene Click Chemistry". Angewandte Chemie International Edition 49 (9): 1540–1573); Blackman, Melissa L. and Royzen, Maksim and Fox, Joseph M. ((2008). "Tetrazine Ligation: Fast Bioconjugation Based on Inverse-Electron-Demand Diels−Alder Reactivity". Journal of the American Chemical Society 130 (41): 13518–13519); Devaraj, Neal K. and Weissleder, Ralph and Hilderbrand, Scott A. ((2008). "Tetrazine Based Cycloadditions: Application to Pretargeted Live Cell Labeling". Bioconjugate Chemistry 19 (12): 2297–2299); Stöckmann, Henning; Neves, Andre; Stairs, Shaun; Brindle, Kevin; Leeper, Finian ((2011). "Exploring isonitrile-based click chemistry for ligation with biomolecules". Organic & Biomolecular Chemistry), all of which are hereby incorporated by reference in their entirety and for all purposes.

[0054] The reactive functional groups can be chosen such that they do not participate in, or interfere with, the chemical stability of the proteins or nucleic acids described herein. By way of example, the nucleic acids can include a vinyl sulfone or other reactive moiety (e.g., maleimide). Optionally, the nucleic acids can include a reactive moiety having the formula -S-S-R. R can be, for example, a protecting group. Optionally, R is hexanol. As used herein, the term hexanol includes compounds with the formula C6H13OH and includes, 1-hexanol, 2-hexanol, 3-hexanol, 2-methyl-1- pentanol, 3-methyl-1-pentanol, 4-methyl-1-pentanol, 2-methyl-2-pentanol, 3-methyl-2-pentanol, 4- methyl-2-pentanol, 2-methyl-3-pentanol, 3-methyl-3-pentanol, 2,2-dimethyl-1-butanol, 2,3-dimethyl-1-butanol, 3,3-dimethyl-1-butanol, 2,3-dimethyl-2-butanol, 3,3-dimethyl-2-butanol, and 2- ethyl-1-butanol. Optionally, R is 1-hexanol.

[0055] As used herein, the term "about" means a range of values including the specified value, which a person of ordinary skill in the art would consider reasonably similar to the specified value. In embodiments, the term "about" means within a standard deviation using measurements generally acceptable in the art. In embodiments, about means a range extending to + / - 10% of the specified value. In embodiments, about means the specified value.

[0056] The term “chelator,” “chelating agent,” or “complexing agent” is used in accordance with its plain ordinary meaning and refers to any agent capable of donating a pair of electrons to a metal to form a chelate. An example of a commonly used chelating agent is ethylenediaminetetraacetic acid (EDTA) to chelate calcium, magnesium, lead, and iron. Chelating agents are also found in nuclear medicine for the generation of theranostics and include, but are not limited to, macrocyclic and acyclic chelators. A chelating moiety is a monovalent chelating agent. Examples of chelating agents used for theranostics include, but are not limited to, DOTA (1,4,7,10-tetra-azacyclododecane- 1,4,7,10-tetraacetic acid; used for theranostics containing111In,177Lu,86 / 90Y,225Ac,44 / 47Sc, or68Ga), NOTA (1,4,7-triazacyclononane-1,4,7- triacetic acid; used for theranostics containing67 / 68Ga,64Cu,177Lu,86 / 90Y, or212 / 213Bi), TETA (1,4,8,11-tetra-azacyclotetradecane-1,4,8,11-tetraacetic acid; used for theranostics containing64Cu), and DTPA (diethylenetriaminepentaacetic acid; used for theranostics containing111In or177Lu) (Price et al. Chem Soc Rev. 2014 Jan 7;43(1):260-90).

[0057] The terms “theranostic” or “theranostics” as provided herein, refer to an agent or a combination of two agents with diagnostic imaging and therapeutic capabilities. Radiotheranostics represent as an exemplary example of theranostics and typically harbor the following structural design: . The cancer-targeting ligand directs the radiotheranostic to the tumor site by binding to target receptors differentially expressed on the surface of tumor cells, which may facilitate its internalization by the tumor cell. Following its entry, the radioisotope moiety induces radiative damage to the tumor cell. (Herrmann et al. Lancet Oncol. 2020 Mar; 21(3): e146-e156; Perera et al. J Nucl Med. 2022; 63(12):1793-1801). In 2022, the FDAapproved Pluvicto ™, a theranostic that contains a cancer-targeting ligand moiety capable of binding to prostate-specific membrane antigen (PMSA), radioactive payload177Lu, and a linker between the two moieties. Once localized within tumor cells,177Lu emits β particles, which induces cellular injury and DNA damage. The use of177Lu in this theranostic permits the evaluation of its localization and therapeutic efficacy using PET scans, and thus, enables177Lu to serve as a diagnostic imaging and therapeutic modality (Herrmann et al. Lancet Oncol. 2020 Mar; 21(3): e146- e156; Perera et al. J Nucl Med. 2022; 63(12):1793-1801). Additional examples of theranostics are known in the art, for example, Lee et al. Acc Chem Res. 2015 Nov 17; 48(11): 2935-2946).

[0058] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art. See, e.g., Singleton et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY 2nd ed., J. Wiley & Sons (New York, NY 1994); Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, Cold Springs Harbor Press (Cold Springs Harbor, NY 1989). Any methods, devices and materials similar or equivalent to those described herein can be used in the practice of this invention. The following definitions are provided to facilitate understanding of certain terms used frequently herein and are not meant to limit the scope of the present disclosure.

[0059] "Nucleic acid" refers to nucleotides (e.g., deoxyribonucleotides or ribonucleotides) and polymers thereof in either single-, double- or multiple-stranded form, or complements thereof; or nucleosides (e.g., deoxyribonucleosides or ribonucleosides). In embodiments, “nucleic acid” does not include nucleosides. The terms “polynucleotide,” “oligonucleotide,” “oligo” or the like refer, in the usual and customary sense, to a linear sequence of nucleotides. The term “nucleoside” refers, in the usual and customary sense, to a glycosylamine including a nucleobase and a five-carbon sugar (ribose or deoxyribose). Non limiting examples, of nucleosides include cytidine, uridine, adenosine, guanosine, thymidine and inosine. The term “nucleotide” refers, in the usual and customary sense, to a single unit of a polynucleotide, i.e., a monomer. Nucleotides can be ribonucleotides, deoxyribonucleotides, or modified versions thereof. Examples of polynucleotides contemplated herein include single and double stranded DNA, single and double stranded RNA, and hybrid molecules having mixtures of single and double stranded DNA and RNA. Examples of nucleic acid,e.g. polynucleotides contemplated herein include any types of RNA, e.g. mRNA, siRNA, miRNA, and guide RNA and any types of DNA, genomic DNA, plasmid DNA, and minicircle DNA, and any fragments thereof. The term “duplex” in the context of polynucleotides refers, in the usual and customary sense, to double strandedness. Nucleic acids can be linear or branched. For example, nucleic acids can be a linear chain of nucleotides or the nucleic acids can be branched, e.g., such that the nucleic acids comprise one or more arms or branches of nucleotides. Optionally, the branched nucleic acids are repetitively branched to form higher ordered structures such as dendrimers and the like.

[0060] Nucleic acids, including e.g., nucleic acids with a phosphothioate backbone, can include one or more reactive moieties. As used herein, the term reactive moiety includes any group capable of reacting with another molecule, e.g., a nucleic acid or polypeptide through covalent, non-covalent or other interactions. By way of example, the nucleic acid can include an amino acid reactive moiety that reacts with an amino acid on a protein or polypeptide through a covalent, non-covalent or other interaction.

[0061] The terms also encompass nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides. Examples of such analogs include, without limitation, phosphodiester derivatives including, e.g., phosphoramidate, phosphorodiamidate, phosphorothioate (also known as phosphothioate having double bonded sulfur replacing oxygen in the phosphate), phosphorodithioate, phosphonocarboxylic acids, phosphonocarboxylates, phosphonoacetic acid, phosphonoformic acid, methyl phosphonate, boron phosphonate, or O-methylphosphoroamidite linkages (see Eckstein, OLIGONUCLEOTIDES AND ANALOGUES: A PRACTICAL APPROACH, Oxford University Press) as well as modifications to the nucleotide bases such as in 5-methyl cytidine or pseudouridine.; and peptide nucleic acid backbones and linkages. Other analog nucleic acids include those with positive backbones; non-ionic backbones, modified sugars, and non-ribose backbones (e.g. phosphorodiamidate morpholino oligos or locked nucleic acids (LNA) as known in the art), including those described in U.S. Patent Nos. 5,235,033 and 5,034,506, andChapters 6 and 7, ASC Symposium Series 580, CARBOHYDRATE MODIFICATIONS IN ANTISENSE RESEARCH, Sanghui & Cook, eds. Nucleic acids containing one or more carbocyclic sugars are also included within one definition of nucleic acids. Modifications of the ribose- phosphate backbone may be done for a variety of reasons, e.g., to increase the stability and half-life of such molecules in physiological environments or as probes on a biochip. Mixtures of naturally occurring nucleic acids and analogs can be made; alternatively, mixtures of different nucleic acid analogs, and mixtures of naturally occurring nucleic acids and analogs may be made. In embodiments, the internucleotide linkages in DNA are phosphodiester, phosphodiester derivatives, or a combination of both.

[0062] Nucleic acids can include nonspecific sequences. As used herein, the term "nonspecific sequence" refers to a nucleic acid sequence that contains a series of residues that are not designed to be complementary to or are only partially complementary to any other nucleic acid sequence. By way of example, a nonspecific nucleic acid sequence is a sequence of nucleic acid residues that does not function as an inhibitory nucleic acid when contacted with a cell or organism.

[0063] A polynucleotide is typically composed of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); and thymine (T) (uracil (U) for thymine (T) when the polynucleotide is RNA). Thus, the term “polynucleotide sequence” is the alphabetical representation of a polynucleotide molecule; alternatively, the term may be applied to the polynucleotide molecule itself. This alphabetical representation can be input into databases in a computer having a central processing unit and used for bioinformatics applications such as functional genomics and homology searching. Polynucleotides may optionally include one or more non-standard nucleotide(s), nucleotide analog(s) and / or modified nucleotides.

[0064] The term “complement,” as used herein, refers to a nucleotide (e.g., RNA or DNA) or a sequence of nucleotides capable of base pairing with a complementary nucleotide or sequence of nucleotides. As described herein and commonly known in the art the complementary (matching) nucleotide of adenosine is thymidine and the complementary (matching) nucleotide of guanosine is cytosine. Thus, a complement may include a sequence of nucleotides that base pair withcorresponding complementary nucleotides of a second nucleic acid sequence. The nucleotides of a complement may partially or completely match the nucleotides of the second nucleic acid sequence. Where the nucleotides of the complement completely match each nucleotide of the second nucleic acid sequence, the complement forms base pairs with each nucleotide of the second nucleic acid sequence. Where the nucleotides of the complement partially match the nucleotides of the second nucleic acid sequence only some of the nucleotides of the complement form base pairs with nucleotides of the second nucleic acid sequence. Examples of complementary sequences include coding and a non-coding sequences, wherein the non-coding sequence contains complementary nucleotides to the coding sequence and thus forms the complement of the coding sequence. A further example of complementary sequences are sense and antisense sequences, wherein the sense sequence contains complementary nucleotides to the antisense sequence and thus forms the complement of the antisense sequence.

[0065] As described herein the complementarity of sequences may be partial, in which only some of the nucleic acids match according to base pairing, or complete, where all the nucleic acids match according to base pairing. Thus, two sequences that are complementary to each other, may have a specified percentage of nucleotides that are the same (i.e., about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region).

[0066] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, γ-carboxyglutamate, and O- phosphoserine. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an α carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the generalchemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid. The terms “non-naturally occurring amino acid” and “unnatural amino acid” refer to amino acid analogs, synthetic amino acids, and amino acid mimetics which are not found in nature.

[0067] Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.

[0068] The terms "polypeptide," "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues, wherein the polymer may In embodiments be conjugated to a moiety that does not consist of amino acids. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. A "fusion protein" refers to a chimeric protein encoding two or more separate protein sequences that are recombinantly expressed as a single moiety.

[0069] An amino acid or nucleotide base "position" is denoted by a number that sequentially identifies each amino acid (or nucleotide base) in the reference sequence based on its position relative to the N-terminus (or 5'-end). Due to deletions, insertions, truncations, fusions, and the like that must be considered when determining an optimal alignment, in general the amino acid residue number in a test sequence determined by simply counting from the N-terminus will not necessarily be the same as the number of its corresponding position in the reference sequence. For example, in a case where a variant has a deletion relative to an aligned reference sequence, there will be no amino acid in the variant that corresponds to a position in the reference sequence at the site of deletion. Where there is an insertion in an aligned reference sequence, that insertion will not correspond to a numbered amino acid position in the reference sequence. In the case of truncations or fusions there can be stretches of amino acids in either the reference or aligned sequence that do not correspond to any amino acid in the corresponding sequence.

[0070] The terms "numbered with reference to" or "corresponding to," when used in the context of the numbering of a given amino acid or polynucleotide sequence, refers to the numbering of the residues of a specified reference sequence when the given amino acid or polynucleotide sequence is compared to the reference sequence. An amino acid residue in a protein "corresponds" to a given residue when it occupies the same essential structural position within the protein as the given residue. One skilled in the art will immediately recognize the identity and location of residues corresponding to a specific position in a protein (e.g., TGF-beta) in other proteins with different numbering systems. For example, by performing a simple sequence alignment with a protein (e.g., TGF-beta) the identity and location of residues corresponding to specific positions of the protein are identified in other protein sequences aligning to the protein. For example, a selected residue in a selected protein corresponds to glutamic acid at position 138 when the selected residue occupies the same essential spatial or other structural relationship as a glutamic acid at position 138. In some embodiments, where a selected protein is aligned for maximum homology with a protein, the position in the aligned selected protein aligning with glutamic acid 138 is the position to correspond to glutamic acid 138. Instead of a primary sequence alignment, a three dimensional structural alignment can also be used, e.g., where the structure of the selected protein is aligned for maximum correspondence with the glutamic acid at position 138, and the overall structures compared. In this case, an amino acid that occupies the same essential position as glutamic acid 138 in the structural model is said to correspond to the glutamic acid 138 residue.

[0071] "Conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, "conservatively modified variants" refers to those nucleic acids that encode identical or essentially identical amino acid sequences. Because of the degeneracy of the genetic code, a number of nucleic acid sequences will encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are "silent variations," which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silentvariation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence.

[0072] As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the disclosure.

[0073] The following eight groups each contain amino acids that are conservative substitutions for one another: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins (1984)).

[0074] The terms "identical" or percent "identity," in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have aspecified percentage of amino acid residues or nucleotides that are the same (i.e., about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region, when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection (see, e.g., NCBI web site http: / / www.ncbi.nlm.nih.gov / BLAST / or the like). Such sequences are then said to be "substantially identical." This definition also refers to, or may be applied to, the compliment of a test sequence. The definition also includes sequences that have deletions and / or additions, as well as those that have substitutions. As described below, the preferred algorithms can account for gaps and the like. Preferably, identity exists over a region that is at least about 25 amino acids or nucleotides in length, or more preferably over a region that is 50- 100 amino acids or nucleotides in length.

[0075] "Percentage of sequence identity" is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.

[0076] A "comparison window", as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of, e.g., a full length sequence or from 20 to 600, about 50 to about 200, or about 100 to about 150 amino acids or nucleotides in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman (1970) Adv. Appl. Math.2:482c, by the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, by the search for similarity method of Pearson and Lipman (1988) Proc. Nat’l. Acad. Sci. USA 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by manual alignment and visual inspection (see, e.g., Ausubel et al., Current Protocols in Molecular Biology (1995 supplement)).

[0077] An example of an algorithm that is suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1977) Nuc. Acids Res. 25:3389-3402, and Altschul et al. (1990) J. Mol. Biol. 215:403-410, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always < 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, an expectation (E) or 10, M=5, N=-4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word length of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA89:10915) alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and a comparison of both strands.

[0078] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.

[0079] An indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the antibodies raised against the polypeptide encoded by the second nucleic acid, as described below. Thus, a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions, as described below. Yet another indication that two nucleic acid sequences are substantially identical is that the same primers can be used to amplify the sequence.

[0080] The phrase "specifically (or selectively) binds" to an antibody or "specifically (or selectively) immunoreactive with," when referring to a protein or peptide, refers to a binding reaction that is determinative of the presence of the protein, often in a heterogeneous population of proteins and other biologics. Thus, under designated immunoassay conditions, the specified antibodies bind to a particular protein at least two times the background and more typically more than 10 to 100 times background. Specific binding to an antibody under such conditions requires an antibody that is selected for its specificity for a particular protein. For example, polyclonal antibodies can be selected to obtain only a subset of antibodies that are specifically immunoreactivewith the selected antigen and not with other proteins. This selection may be achieved by subtracting out antibodies that cross-react with other molecules. A variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein (see, e.g., Harlow & Lane, Using Antibodies, A Laboratory Manual (1998) for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity).

[0081] A "ligand" refers to an agent, e.g., a polypeptide or other molecule, capable of binding to a receptor or antibody, antibody variant, antibody region or fragment thereof.

[0082] Techniques for conjugating therapeutic agents to antibodies are well known (see, e.g., Arnon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”in Controlled Drug Delivery (2ndEd.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review" in Monoclonal Antibodies ‘84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); and Thorpe et al., "The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates", Immunol. Rev., 62:119-58 (1982)). As used herein, the term “antibody-drug conjugate” or “ADC” refers to a therapeutic agent conjugated or otherwise covalently bound to to an antibody.

[0083] For specific proteins described herein, the named protein includes any of the protein’s naturally occurring forms, variants or homologs that maintain the protein transcription factor activity (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to the native protein). In some embodiments, variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring form. In other embodiments, the protein is the protein as identified by its NCBI sequence reference. In other embodiments, the protein is the protein as identified by its NCBI sequence reference, homolog or functional fragment thereof.

[0084] The term "gene" means the segment of DNA involved in producing a protein; it includes regions preceding and following the coding region (leader and trailer) as well as intervening sequences (introns) between individual coding segments (exons). The leader, the trailer as well as the introns include regulatory elements that are necessary during the transcription and the translation of a gene. Further, a "protein gene product" is a protein expressed from a particular gene.

[0085] The terms "plasmid", "vector" or "expression vector" refer to a nucleic acid molecule that encodes for genes and / or regulatory elements necessary for the expression of genes. Expression of a gene from a plasmid can occur in cis or in trans. If a gene is expressed in cis, the gene and the regulatory elements are encoded by the same plasmid. Expression in trans refers to the instance where the gene and the regulatory elements are encoded by separate plasmids.

[0086] The terms "transfection", "transduction", "transfecting" or "transducing" can be used interchangeably and are defined as a process of introducing a nucleic acid molecule or a protein to a cell. Nucleic acids are introduced to a cell using non-viral or viral-based methods. The nucleic acid molecules may be gene sequences encoding complete proteins or functional portions thereof. Non- viral methods of transfection include any appropriate transfection method that does not use viral DNA or viral particles as a delivery system to introduce the nucleic acid molecule into the cell. Exemplary non-viral transfection methods include calcium phosphate transfection, liposomal transfection, nucleofection, sonoporation, transfection through heat shock, magnetifection and electroporation. In some embodiments, the nucleic acid molecules are introduced into a cell using electroporation following standard procedures well known in the art. For viral-based methods of transfection any useful viral vector may be used in the methods described herein. Examples for viral vectors include, but are not limited to retroviral, adenoviral, lentiviral and adeno-associated viral vectors. In some embodiments, the nucleic acid molecules are introduced into a cell using a retroviral vector following standard procedures well known in the art. The terms ″transfection″ or ″transduction″ also refer to introducing proteins into a cell from the external environment. Typically, transduction or transfection of a protein relies on attachment of a peptide or protein capable of crossing the cell membrane to the protein of interest. See, e.g., Ford et al. (2001) Gene Therapy 8:1- 4 and Prochiantz (2007) Nat. Methods 4:119-20.

[0087] A "label" or a "detectable moiety" is a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means. For example, useful labels include 32P, fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in an ELISA), biotin, digoxigenin, or haptens and proteins or other entities which can be made detectable, e.g., by incorporating a radiolabel into a peptide or antibody specifically reactive with a target peptide. Any appropriate method known in the art for conjugating an antibody to the label may be employed, e.g., using methods described in Hermanson, Bioconjugate Techniques 1996, Academic Press, Inc., San Diego.

[0088] When the label or detectable moiety is a radioactive metal or paramagnetic ion, the agent may be reacted with another long-tailed reagent having a long tail with one or more chelating groups attached to the long tail for binding to these ions. The long tail may be a polymer such as a polylysine, polysaccharide, or other derivatized or derivatizable chain having pendant groups to which the metals or ions may be added for binding. Examples of chelating groups that may be used according to the disclosure include, but are not limited to, ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), DOTA, NOTA, NETA, TETA, porphyrins, polyamines, crown ethers, bis-thiosemicarbazones, polyoximes, and like groups. The chelate is normally linked to the TGF-beta antibody or functional antibody fragment by a group, which enables the formation of a bond to the molecule with minimal loss of immunoreactivity and minimal aggregation and / or internal cross-linking. The same chelates, when complexed with non-radioactive metals, such as manganese, iron and gadolinium are useful for MRI, when used along with the antibodies and carriers described herein. Macrocyclic chelates such as NOTA, DOTA, and TETA are of use with a variety of metals and radiometals including, but not limited to, radionuclides of gallium, yttrium and copper, respectively. Other ring-type chelates such as macrocyclic polyethers, which are of interest for stably binding nuclides, such as223Ra for RAIT may be used. In certain embodiments, chelating moieties may be used to attach a PET imaging agent, such as an Al-18F complex, to a targeting molecule for use in PET analysis.

[0089] Radioactive substances (e.g., radioisotopes) that may be used as imaging, labeling or therapeutic agents in accordance with the embodiments of the disclosure include, but are not limitedto,18F,32P,33P,45Ti,47Sc,52Fe,59Fe,62Cu,64Cu,67Cu,67Ga,68Ga,77As,86Y,90Y,89Sr,89Zr,94Tc,94Tc,99mTc,99Mo,105Pd,105Rh,111Ag,111In,123I,124I,125I,131I,142Pr,143Pr,149Pm,153Sm,154-158Gd, inand lanthanide metals (e.g., metals having atomic numbers of 21-29, 42, 43, 44, or 57-71). These metals include ions of Cr, V, Mn, Fe, Co, Ni, Cu, La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb and Lu.

[0090] Antibodies are large, complex molecules (molecular weight of ~150,000 or about 1320 amino acids) with intricate internal structure. A natural antibody molecule contains two identical pairs of polypeptide chains, each pair having one light chain and one heavy chain. Each light chain and heavy chain in turn consists of two regions: a variable (“V”) region, involved in binding the target antigen, and a constant (“C”) region that interacts with other components of the immune system. The light and heavy chain variable regions (also referred to herein as light chain variable (VL) domain and heavy chain variable (VH) domain, respectively) come together in 3-dimensional space to form a variable region that binds the antigen (for example, a receptor on the surface of a cell). Within each light or heavy chain variable region, there are three short segments (averaging 10 amino acids in length) called the complementarity determining regions (“CDRs”). The six CDRs in an antibody variable domain (three from the light chain and three from the heavy chain) fold up together in 3-dimensional space to form the actual antibody binding site which docks onto the target antigen. The position and length of the CDRs have been precisely defined by Kabat, E. et al., Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, 1983, 1987. The part of a variable region not contained in the CDRs is called the framework ("FR"), which forms the environment for the CDRs.

[0091] An “antibody variant” as provided herein refers to a polypeptide capable of binding to an antigen and including one or more structural domains (e.g., light chain variable domain, heavy chain variable domain) of an antibody or fragment thereof. Non-limiting examples of antibody variants include single-domain antibodies or nanobodies, monospecific Fab2, bispecific Fab2, trispecificFab3, monovalent IgGs, scFv, bispecific antibodies, bispecific diabodies, trispecific triabodies, scFv- Fc, minibodies, IgNAR, V-NAR, hcIgG, VhH, or peptibodies. A “peptibody” as provided herein refers to a peptide moiety attached (through a covalent or non-covalent linker) to the Fc (crystallisable fragment) domain of an antibody. Further non-limiting examples of antibody variants known in the art include antibodies produced by cartilaginous fish or camelids. A general description of antibodies from camelids and the variable regions thereof and methods for their production, isolation, and use may be found in references WO97 / 49805 and WO 97 / 49805 which are incorporated by reference herein in their entirety and for all purposes. Likewise, antibodies from cartilaginous fish and the variable regions thereof and methods for their production, isolation, and use may be found in WO2005 / 118629, which is incorporated by reference herein in its entirety and for all purposes.

[0092] The terms "CDR L1", "CDR L2" and "CDR L3" as provided herein refer to the complementarity determining regions (CDR) 1, 2, and 3 of the variable light (L) chain of an antibody. In embodiments, the variable light chain provided herein includes in N-terminal to C- terminal direction a CDR L1, a CDR L2 and a CDR L3. Likewise, the terms "CDR H1", "CDR H2" and "CDR H3" as provided herein refer to the complementarity determining regions (CDR) 1, 2, and 3 of the variable heavy (H) chain of an antibody. In embodiments, the variable heavy chain provided herein includes in N-terminal to C-terminal direction a CDR H1, a CDR H2 and a CDR H3.

[0093] The terms "FR L1", "FR L2", "FR L3" and "FR L4" as provided herein are used according to their common meaning in the art and refer to the framework regions (FR) 1, 2, 3 and 4 of the variable light (L) chain of an antibody. In embodiments, the variable light chain provided herein includes in N-terminal to C-terminal direction a FR L1, a FR L2, a FR L3 and a FR L4. Likewise, the terms "FR H1", "FR H2", "FR H3" and "FR H4" as provided herein are used according to their common meaning in the art and refer to the framework regions (FR) 1, 2, 3 and 4 of the variable heavy (H) chain of an antibody. In embodiments, the variable heavy chain provided herein includes in N-terminal to C-terminal direction a FR H1, a FR H2, a FR H3 and a FR H4.

[0094] An exemplary immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one “light” (about 25 kD) and one “heavy” chain (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms variable light chain (VL), variable light chain (VL) domain or light chain variable region and variable heavy chain (VH), variable heavy chain (VH) domain or heavy chain variable region refer to these light and heavy chain regions, respectively. The terms variable light chain (VL), variable light chain (VL) domain and light chain variable region as referred to herein may be used interchangeably. The terms variable heavy chain (VH), variable heavy chain (VH) domain and heavy chain variable region as referred to herein may be used interchangeably. The Fc (i.e. fragment crystallizable region) is the "base" or "tail" of an immunoglobulin and is typically composed of two heavy chains that contribute two or three constant domains depending on the class of the antibody. By binding to specific proteins, the Fc region ensures that each antibody generates an appropriate immune response for a given antigen. The Fc region also binds to various cell receptors, such as Fc receptors, and other immune molecules, such as complement proteins.

[0095] The terms “KD”, “Kd”, “KD” or “Kd” are used according to its commonly known meaning in the art. A dissociation constant is a specific type of equilibrium constant that measures the propensity of a larger object to separate (dissociate) reversibly into smaller components, as when a complex falls apart into its component molecules, or when a salt splits up into its component ions. The dissociation constant is the inverse of the association constant. KD is the equilibrium dissociation constant, a ratio of koff / kon, between the antibody and its antigen. KD and affinity are inversely related. The KD value relates to the concentration of antibody (the amount of antibody needed for a particular experiment) and so the lower the KD value (lower concentration) and thus the higher the affinity of the antibody.

[0096] The term "antibody" is used according to its commonly known meaning in the art. Antibodies exist, e.g., as intact immunoglobulins or as a number of well-characterized fragments produced by digestion with various peptidases. Thus, for example, pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)'2, a dimer of Fab which itself is a lightchain joined to VH-CH1by a disulfide bond. The F(ab)'2may be reduced under mild conditions to break the disulfide linkage in the hinge region, thereby converting the F(ab)'2 dimer into a Fab' monomer. The Fab' monomer is essentially Fab with part of the hinge region (see Fundamental Immunology (Paul ed., 3d ed. 1993). While various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such fragments may be synthesized de novo either chemically or by using recombinant DNA methodology. Thus, the term antibody, as used herein, also includes antibody fragments either produced by the modification of whole antibodies, or those synthesized de novo using recombinant DNA methodologies (e.g., single chain Fv) or those identified using phage display libraries (see, e.g., McCafferty et al., Nature 348:552- 554 (1990)). The term “antibody” as referred to herein further includes antibody variants such as single domain antibodies. Thus, in embodiments an antibody includes a single monomeric variable antibody domain. Thus, in embodiments, the antibody, includes a variable light chain (VL) domain or a variable heavy chain (VH) domain. In embodiments, the antibody is a variable light chain (VL) domain or a variable heavy chain (VH) domain. The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as the myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.

[0097] For preparation of monoclonal or polyclonal antibodies, any technique known in the art can be used (see, e.g., Kohler & Milstein, Nature 256:495-497 (1975); Kozbor et al., Immunology Today 4:72 (1983); Cole et al., pp. 77-96 in Monoclonal Antibodies and Cancer Therapy (1985)). "Monoclonal" antibodies (mAb) refer to antibodies derived from a single clone. Techniques for the production of single chain antibodies (U.S. Pat. No. 4,946,778) can be adapted to produce antibodies to polypeptides of this invention. Also, transgenic mice, or other organisms such as other mammals, may be used to express humanized antibodies. Alternatively, phage display technology can be used to identify antibodies and heteromeric Fab fragments that specifically bind to selected antigens (see, e.g., McCafferty et al., Nature 348:552-554 (1990); Marks et al., Biotechnology 10:779-783 (1992)).

[0098] The epitope of a mAb is the region of its antigen to which the mAb binds. Two antibodies bind to the same or overlapping epitope if each competitively inhibits (blocks) binding of the other to the antigen. That is, a 1x, 5x, 10x, 20x or 100x excess of one antibody inhibits binding of the other by at least 30% but preferably 50%, 75%, 90% or even 99% as measured in a competitive binding assay (see, e.g., Junghans et al., Cancer Res. 50:1495, 1990). Alternatively, two antibodies have the same epitope if essentially all amino acid mutations in the antigen that reduce or eliminate binding of one antibody reduce or eliminate binding of the other. Two antibodies have overlapping epitopes if some amino acid mutations that reduce or eliminate binding of one antibody reduce or eliminate binding of the other.

[0099] A single-chain variable fragment (scFv) is typically a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of immunoglobulins, connected with a short linker peptide of 10 to about 25 amino acids. The linker may usually be rich in glycine for flexibility, as well as serine or threonine for solubility. The linker can either connect the N-terminus of the VH with the C- terminus of the VL, or vice versa.

[0100] For preparation of suitable antibodies of the invention and for use according to the invention, e.g., recombinant, monoclonal, or polyclonal antibodies, many techniques known in the art can be used (see, e.g., Kohler & Milstein, Nature 256:495-497 (1975); Kozbor et al., Immunology Today 4: 72 (1983); Cole et al., pp. 77-96 in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. (1985); Coligan, Current Protocols in Immunology (1991); Harlow & Lane, Antibodies, A Laboratory Manual (1988); and Goding, Monoclonal Antibodies: Principles and Practice (2d ed. 1986)). The genes encoding the heavy and light chains of an antibody of interest can be cloned from a cell, e.g., the genes encoding a monoclonal antibody can be cloned from a hybridoma and used to produce a recombinant monoclonal antibody. Gene libraries encoding heavy and light chains of monoclonal antibodies can also be made from hybridoma or plasma cells. Random combinations of the heavy and light chain gene products generate a large pool of antibodies with different antigenic specificity (see, e.g., Kuby, Immunology (3rd ed. 1997)). Techniques for the production of single chain antibodies or recombinant antibodies (U.S. Patent 4,946,778, U.S. Patent No. 4,816,567) can be adapted to produce antibodies to polypeptides of this invention. Also,transgenic mice, or other organisms such as other mammals, may be used to express humanized or human antibodies (see, e.g., U.S. Patent Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, Marks et al., Bio / Technology 10:779-783 (1992); Lonberg et al., Nature 368:856-859 (1994); Morrison, Nature 368:812-13 (1994); Fishwild et al., Nature Biotechnology 14:845-51 (1996); Neuberger, Nature Biotechnology 14:826 (1996); and Lonberg & Huszar, Intern. Rev. Immunol. 13:65-93 (1995)). Alternatively, phage display technology can be used to identify antibodies and heteromeric Fab fragments that specifically bind to selected antigens (see, e.g., McCafferty et al., Nature 348:552-554 (1990); Marks et al., Biotechnology 10:779-783 (1992)). Antibodies can also be made bispecific, i.e., able to recognize two different antigens (see, e.g., WO 93 / 08829, Traunecker et al., EMBO J. 10:3655-3659 (1991); and Suresh et al., Methods in Enzymology 121:210 (1986)). Antibodies can also be heteroconjugates, e.g., two covalently joined antibodies, or immunotoxins (see, e.g., U.S. Patent No. 4,676,980 , WO 91 / 00360; WO 92 / 200373; and EP 03089).

[0101] Methods for humanizing or primatizing non-human antibodies are well known in the art (e.g., U.S. Patent Nos. 4,816,567; 5,530,101; 5,859,205; 5,585,089; 5,693,761; 5,693,762; 5,777,085; 6,180,370; 6,210,671; and 6,329,511; WO 87 / 02671; EP Patent Application 0173494; Jones et al. (1986) Nature 321:522; and Verhoyen et al. (1988) Science 239:1534). Humanized antibodies are further described in, e.g., Winter and Milstein (1991) Nature 349:293. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as import residues, which are typically taken from an import variable domain. Humanization can be essentially performed following the method of Winter and co-workers (see, e.g., Morrison et al., PNAS USA, 81:6851- 6855 (1984), Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); Morrison and Oi, Adv. Immunol., 44:65-92 (1988), Verhoeyen et al., Science 239:1534- 1536 (1988) and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992), Padlan, Molec. Immun., 28:489- 498 (1991); Padlan, Molec. Immun., 31(3):169-217 (1994)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such humanized antibodies are chimeric antibodies (U.S. Patent No. 4,816,567), wherein substantially less than anintact human variable domain has been substituted by the corresponding sequence from a non- human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies. For example, polynucleotides comprising a first sequence coding for humanized immunoglobulin framework regions and a second sequence set coding for the desired immunoglobulin complementarity determining regions can be produced synthetically or by combining appropriate cDNA and genomic DNA segments. Human constant region DNA sequences can be isolated in accordance with well known procedures from a variety of human cells.

[0102] A "chimeric antibody" is an antibody molecule in which (a) the constant region, or a portion thereof, is altered, replaced or exchanged so that the antigen binding site (variable region) is linked to a constant region of a different or altered class, effector function and / or species, or an entirely different molecule which confers new properties to the chimeric antibody, e.g., an enzyme, toxin, hormone, growth factor, drug, etc.; or (b) the variable region, or a portion thereof, is altered, replaced or exchanged with a variable region having a different or altered antigen specificity. The preferred antibodies of, and for use according to the invention include humanized and / or chimeric monoclonal antibodies.

[0103] Antibody-drug conjugates as provided herein are composed of a cytotoxic payload conjugated to tumor targeting monoclonal antibody (mAb) via chemical linker that is susceptible to self-immolation upon internalization by the tumor cell. As of 2023, the FDA approved thirteen antibody-drug conjugates, which include Mylotarg™, Adcentris®, Kadcyla®, Besponsa®, Lumoxiti®, Polivy®, Padcev®, Enhertu®, Trodelvy®, Blenrep, Zynlonta®, Tivdak®, and Elahere™. Gemtuzumab ozogamicin (Mylotarg™), inotuzumab ozogamicin (Besponsa®), and mirvetuximab soravtansine (Elahere™) are examples of disulfide linked antibody-drug conjugates.

[0104] "Contacting" is used in accordance with its plain ordinary meaning and refers to the process of allowing at least two distinct species (e.g. antibodies and antigens) to become sufficiently proximal to react, interact, or physically touch. It should be appreciated, however, that the resultingreaction product can be produced directly from a reaction between the added reagents or from an intermediate from one or more of the added reagents which can be produced in the reaction mixture.

[0105] The term "contacting" may include allowing two species to react, interact, or physically touch, wherein the two species may be, for example, a pharmaceutical composition as provided herein and a cell. In embodiments contacting includes, for example, allowing a pharmaceutical composition as described herein to interact with a cell.

[0106] A "cell" as used herein, refers to a cell carrying out metabolic or other function sufficient to preserve or replicate its genomic DNA. A cell can be identified by well-known methods in the art including, for example, presence of an intact membrane, staining by a particular dye, ability to produce progeny or, in the case of a gamete, ability to combine with a second gamete to produce a viable offspring. Cells may include prokaryotic and eukaryotic cells. Prokaryotic cells include but are not limited to bacteria. Eukaryotic cells include, but are not limited to, yeast cells and cells derived from plants and animals, for example mammalian, insect (e.g., spodoptera) and human cells.

[0107] The term "recombinant" when used with reference, e.g., to a cell, nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all. Transgenic cells and plants are those that express a heterologous gene or coding sequence, typically as a result of recombinant methods.

[0108] The term "isolated", when applied to a nucleic acid or protein, denotes that the nucleic acid or protein is essentially free of other cellular components with which it is associated in the natural state. It can be, for example, in a homogeneous state and may be in either a dry or aqueous solution. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. A protein that is the predominant species present in a preparation is substantially purified.

[0109] The term "heterologous" when used with reference to portions of a nucleic acid indicates that the nucleic acid comprises two or more subsequences that are not found in the same relationship to each other in nature. For instance, the nucleic acid is typically recombinantly produced, having two or more sequences from unrelated genes arranged to make a new functional nucleic acid, e.g., a promoter from one source and a coding region from another source. Similarly, a heterologous protein indicates that the protein comprises two or more subsequences that are not found in the same relationship to each other in nature (e.g., a fusion protein).

[0110] The term "exogenous" refers to a molecule or substance (e.g., a compound, nucleic acid or protein) that originates from outside a given cell or organism. For example, an "exogenous promoter" as referred to herein is a promoter that does not originate from the cell or organism it is expressed by. Conversely, the term "endogenous" or "endogenous promoter" refers to a molecule or substance that is native to, or originates within, a given cell or organism.

[0111] As defined herein, the term "inhibition", "inhibit", "inhibiting" and the like in reference to cell proliferation (e.g., cancer cell proliferation) means negatively affecting (e.g., decreasing proliferation) or killing the cell. In some embodiments, inhibition refers to reduction of a disease or symptoms of disease (e.g., cancer, cancer cell proliferation). Thus, inhibition includes, at least in part, partially or totally blocking stimulation, decreasing, preventing, or delaying activation, or inactivating, desensitizing, or down-regulating signal transduction or enzymatic activity or the amount of a protein (e.g. a cancer-associated protein). Similarly an "inhibitor" is a compound or protein that inhibits a receptor or another protein, e.g., by binding, partially or totally blocking, decreasing, preventing, delaying, inactivating, desensitizing, or down-regulating activity (e.g., a receptor activity or a protein activity).

[0112] As defined herein, the term “inhibition”, “inhibit”, “inhibiting” and the like in reference to a protein-inhibitor interaction means negatively affecting (e.g. decreasing) the activity or function of the protein (e.g., TGF-beta protein) relative to the activity or function of the protein in the absence of the inhibitor. In embodiments inhibition means negatively affecting (e.g. decreasing) the concentration or levels of TGF-beta relative to the concentration or level of the protein in theabsence of the inhibitor. In embodiments inhibition refers to reduction of a disease or symptoms of disease. In embodiments, inhibition refers to a reduction in the activity of TGF-beta. Thus, inhibition includes, at least in part, partially or totally blocking stimulation, decreasing, preventing, or delaying activation, or inactivating, desensitizing, or down-regulating signal transduction or activity or the amount TGF-beta. In embodiments, inhibition refers to a reduction of activity of TGF-beta resulting from a direct interaction (e.g., an inhibitor binds to TGF-beta). In embodiments, inhibition refers to a reduction of activity of TGF-beta from an indirect interaction (e.g., an inhibitor binds to a protein that activates TGF-beta, thereby preventing target protein activation).

[0113] Thus, the terms “inhibitor,” “repressor” or “antagonist” or “downregulator” interchangeably refer to a substance capable of detectably decreasing the expression or activity of a given gene or protein (e.g., TGF-beta protein). The antagonist can decrease TGF-beta expression or activity 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more in comparison to a control in the absence of the antagonist. In certain instances, TGF-beta expression or activity is 1.5-fold, 2- fold, 3-fold, 4-fold, 5-fold, 10-fold or lower than the expression or activity in the absence of the antagonist.

[0114] The term "expression" includes any step involved in the production of the polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post- translational modification, and secretion. Expression can be detected using conventional techniques for detecting protein (e.g., ELISA, Western blotting, flow cytometry, immunofluorescence, immunohistochemistry, etc.).

[0115] “Biological sample” or “sample” refer to materials obtained from or derived from a subject or patient. A biological sample includes sections of tissues such as biopsy and autopsy samples, and frozen sections taken for histological purposes. Such samples include bodily fluids such as blood and blood fractions or products (e.g., serum, plasma, platelets, red blood cells, and the like), sputum, tissue, cultured cells (e.g., primary cultures, explants, and transformed cells) stool, urine, synovial fluid, joint tissue, synovial tissue, synoviocytes, fibroblast-like synoviocytes, macrophage-like synoviocytes, immune cells, hematopoietic cells, fibroblasts, macrophages, T cells, etc. A biologicalsample is typically obtained from a eukaryotic organism, such as a mammal such as a primate e.g., chimpanzee or human; cow; dog; cat; a rodent, e.g., guinea pig, rat, mouse; rabbit; or a bird; reptile; or fish.

[0116] A “control” or “standard control” refers to a sample, measurement, or value that serves as a reference, usually a known reference, for comparison to a test sample, measurement, or value. For example, a test sample can be taken from a patient suspected of having a given disease (e.g. cancer) and compared to a known normal (non-diseased) individual (e.g. a standard control subject). A standard control can also represent an average measurement or value gathered from a population of similar individuals (e.g. standard control subjects) that do not have a given disease (i.e. standard control population), e.g., healthy individuals with a similar medical background, same age, weight, etc. A standard control value can also be obtained from the same individual, e.g. from an earlier- obtained sample from the patient prior to disease onset. For example, a control can be devised to compare therapeutic benefit based on pharmacological data (e.g., half-life) or therapeutic measures (e.g., comparison of side effects). Controls are also valuable for determining the significance of data. For example, if values for a given parameter are widely variant in controls, variation in test samples will not be considered as significant. One of skill will recognize that standard controls can be designed for assessment of any number of parameters (e.g. RNA levels, protein levels, specific cell types, specific bodily fluids, specific tissues, etc).

[0117] “Patient” or “subject in need thereof” refers to a living organism suffering from or prone to a disease or condition that can be treated by administration of a composition or pharmaceutical composition as provided herein. Non-limiting examples include humans, other mammals, bovines, rats, mice, dogs, monkeys, goat, sheep, cows, deer, and other non-mammalian animals. In some embodiments, a patient is human.

[0118] The terms “disease” or “condition” refer to a state of being or health status of a patient or subject capable of being treated with the compounds or methods provided herein. The disease may be a cancer. The cancer may refer to a solid tumor malignancy. Solid tumor malignancies include malignant tumors that may be devoid of fluids or cysts. For example, the solid tumor malignancymay include breast cancer, ovarian cancer, pancreatic cancer, cervical cancer, gastric cancer, renal cancer, head and neck cancer, bone cancer, skin cancer or prostate cancer. In some further instances, “cancer” refers to human cancers and carcinomas, sarcomas, adenocarcinomas, including solid cancers, kidney, breast, lung, bladder, colon, ovarian, prostate, pancreas, stomach, brain, head and neck, skin, uterine, testicular, glioma, esophagus, and liver cancer, including hepatocarcinoma.

[0119] As used herein, the term “cancer” refers to all types of cancer, neoplasm or malignant tumors found in mammals (e.g., humans), including, metastasis, carcinomas and sarcomas. Exemplary cancers that may be treated with a compound or method provided herein include breast cancer, colon cancer, kidney cancer, lung cancer, melanoma, ovarian cancer, prostate cancer, pancreatic cancer, brain cancer, liver cancer, gastric cancer or a sarcoma.

[0120] The term “sarcoma” generally refers to a tumor which is made up of a substance like the embryonic connective tissue and is generally composed of closely packed cells embedded in a fibrillar or homogeneous substance. Sarcomas that may be treated with a compound or method provided herein include a chondrosarcoma, fibrosarcoma, lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma, Abemethy's sarcoma, adipose sarcoma, liposarcoma, alveolar soft part sarcoma, ameloblastic sarcoma, botryoid sarcoma, chloroma sarcoma, chorio carcinoma, embryonal sarcoma, Wilms' tumor sarcoma, endometrial sarcoma, stromal sarcoma, Ewing's sarcoma, fascial sarcoma, fibroblastic sarcoma, giant cell sarcoma, granulocytic sarcoma, Hodgkin's sarcoma, idiopathic multiple pigmented hemorrhagic sarcoma, immunoblastic sarcoma of B cells, lymphoma, immunoblastic sarcoma of T-cells, Jensen's sarcoma, Kaposi's sarcoma, Kupffer cell sarcoma, angiosarcoma, leukosarcoma, malignant mesenchymoma sarcoma, parosteal sarcoma, reticulocytic sarcoma, Rous sarcoma, serocystic sarcoma, synovial sarcoma, or telangiectaltic sarcoma.

[0121] The term “melanoma” is taken to mean a tumor arising from the melanocytic system of the skin and other organs. Melanomas that may be treated with a compound or method provided herein include, for example, acral-lentiginous melanoma, amelanotic melanoma, benign juvenile melanoma, Cloudman's melanoma, S91 melanoma, Harding-Passey melanoma, juvenile melanoma,lentigo maligna melanoma, malignant melanoma, nodular melanoma, subungal melanoma, or superficial spreading melanoma.

[0122] The term “carcinoma” refers to a malignant new growth made up of epithelial cells tending to infiltrate the surrounding tissues and give rise to metastases. Exemplary carcinomas that may be treated with a compound or method provided herein include, for example, medullary thyroid carcinoma, familial medullary thyroid carcinoma, acinar carcinoma, acinous carcinoma, adenocystic carcinoma, adenoid cystic carcinoma, carcinoma adenomatosum, carcinoma of adrenal cortex, alveolar carcinoma, alveolar cell carcinoma, basal cell carcinoma, carcinoma basocellulare, basaloid carcinoma, basosquamous cell carcinoma, bronchioalveolar carcinoma, bronchiolar carcinoma, bronchogenic carcinoma, cerebriform carcinoma, cholangiocellular carcinoma, chorionic carcinoma, colloid carcinoma, comedo carcinoma, corpus carcinoma, cribriform carcinoma, carcinoma en cuirasse, carcinoma cutaneum, cylindrical carcinoma, cylindrical cell carcinoma, duct carcinoma, carcinoma durum, embryonal carcinoma, encephaloid carcinoma, epiermoid carcinoma, carcinoma epitheliale adenoides, exophytic carcinoma, carcinoma ex ulcere, carcinoma fibrosum, gelatiniforni carcinoma, gelatinous carcinoma, giant cell carcinoma, carcinoma gigantocellulare, glandular carcinoma, granulosa cell carcinoma, hair-matrix carcinoma, hematoid carcinoma, hepatocellular carcinoma, Hurthle cell carcinoma, hyaline carcinoma, hypernephroid carcinoma, infantile embryonal carcinoma, carcinoma in situ, intraepidermal carcinoma, intraepithelial carcinoma, Krompecher's carcinoma, Kulchitzky-cell carcinoma, large-cell carcinoma, lenticular carcinoma, carcinoma lenticulare, lipomatous carcinoma, lymphoepithelial carcinoma, carcinoma medullare, medullary carcinoma, melanotic carcinoma, carcinoma molle, mucinous carcinoma, carcinoma muciparum, carcinoma mucocellulare, mucoepidermoid carcinoma, carcinoma mucosum, mucous carcinoma, carcinoma myxomatodes, nasopharyngeal carcinoma, oat cell carcinoma, carcinoma ossificans, osteoid carcinoma, papillary carcinoma, periportal carcinoma, preinvasive carcinoma, prickle cell carcinoma, pultaceous carcinoma, renal cell carcinoma of kidney, reserve cell carcinoma, carcinoma sarcomatodes, schneiderian carcinoma, scirrhous carcinoma, carcinoma scroti, signet-ring cell carcinoma, carcinoma simplex, small-cell carcinoma, solanoid carcinoma, spheroidal cell carcinoma, spindle cell carcinoma, carcinoma spongiosum, squamous carcinoma,squamous cell carcinoma, string carcinoma, carcinoma telangiectaticum, carcinoma telangiectodes, transitional cell carcinoma, carcinoma tuberosum, tuberous carcinoma, verrucous carcinoma, or carcinoma villosum.

[0123] As used herein, the terms "metastasis," "metastatic," and "metastatic cancer" can be used interchangeably and refer to the spread of a proliferative disease or disorder, e.g., cancer, from one organ or another non-adjacent organ or body part. Cancer occurs at an originating site, e.g., breast, which site is referred to as a primary tumor, e.g., primary breast cancer. Some cancer cells in the primary tumor or originating site acquire the ability to penetrate and infiltrate surrounding normal tissue in the local area and / or the ability to penetrate the walls of the lymphatic system or vascular system circulating through the system to other sites and tissues in the body. A second clinically detectable tumor formed from cancer cells of a primary tumor is referred to as a metastatic or secondary tumor. When cancer cells metastasize, the metastatic tumor and its cells are presumed to be similar to those of the original tumor. Thus, if lung cancer metastasizes to the breast, the secondary tumor at the site of the breast consists of abnormal lung cells and not abnormal breast cells. The secondary tumor in the breast is referred to a metastatic lung cancer. Thus, the phrase metastatic cancer refers to a disease in which a subject has or had a primary tumor and has one or more secondary tumors. The phrases non-metastatic cancer or subjects with cancer that is not metastatic refers to diseases in which subjects have a primary tumor but not one or more secondary tumors. For example, metastatic lung cancer refers to a disease in a subject with or with a history of a primary lung tumor and with one or more secondary tumors at a second location or multiple locations, e.g., in the breast.

[0124] The term “associated” or “associated with” in the context of a substance or substance activity or function associated with a disease (e.g., a protein associated disease, a cancer associated with TGF-beta activity, TGF-beta associated cancer, TGF-beta associated disease (e.g., cancer,)) means that the disease (e.g., cancer) is caused by (in whole or in part), or a symptom of the disease is caused by (in whole or in part) the substance or substance activity or function. As used herein, what is described as being associated with a disease, if a causative agent, could be a target for treatment of the disease. For example, a cancer associated with TGF-beta activity or function or aTGF-beta associated disease (e.g., cancer), may be treated with a TGF-beta modulator or TGF-beta inhibitor, in the instance where increased TGF-beta activity or function (e.g. signaling pathway activity) causes the disease (e.g., cancer).

[0125] The term “signaling pathway” as used herein refers to a series of interactions between cellular and optionally extra-cellular components (e.g. proteins, nucleic acids, small molecules, ions, lipids) that conveys a change in one component to one or more other components, which in turn may convey a change to additional components, which is optionally propagated to other signaling pathway components. In embodiments, the compositions provided herein bind and / or directly or indirectly affect activity of the TGF-beta signaling pathway.

[0126] The term "aberrant" as used herein refers to different from normal. When used to describe enzymatic activity, aberrant refers to activity that is greater or less than a normal control or the average of normal non-diseased control samples. Aberrant activity may refer to an amount of activity that results in a disease, wherein returning the aberrant activity to a normal or non-disease- associated amount (e.g. by using a method as described herein), results in reduction of the disease or one or more disease symptoms.

[0127] A "therapeutic agent" as referred to herein, is a composition useful in treating or preventing a disease such as cancer (e.g., brain cancer, breat cancer). In embodiments, the therpaeutic agent is an anti-cancer agent. “Anti-cancer agent” is used in accordance with its plain ordinary meaning and refers to a composition (e.g. compound, drug, antagonist, inhibitor, modulator) having antineoplastic properties or the ability to inhibit the growth or proliferation of cells. In embodiments, an anti- cancer agent is a chemotherapeutic. In embodiments, an anti-cancer agent is an agent identified herein having utility in methods of treating cancer. In embodiments, an anti-cancer agent is an agent approved by the FDA or similar regulatory agency of a country other than the USA, for treating cancer.

[0128] An “anticancer agent” as used herein refers to a molecule (e.g., compound, peptide, protein, nucleic acid) used to treat cancer through destruction or inhibition of cancer cells or tissues. Anticancer agents may be selective for certain cancers or certain tissues. In embodiments,anticancer agents herein may include epigenetic inhibitors and multi-kinase inhibit “Anti-cancer agent” and “anticancer agent” are used in accordance with their plain ordinary meaning and refers to a composition (e.g. compound, drug, antagonist, inhibitor, modulator) having antineoplastic properties or the ability to inhibit the growth or proliferation of cells. In some embodiments, an anti- cancer agent is a chemotherapeutic. In some embodiments, an anti-cancer agent is an agent identified herein having utility in methods of treating cancer. In some embodiments, an anti-cancer agent is an agent approved by the FDA or similar regulatory agency of a country other than the USA, for treating cancer. Examples of anti-cancer agents include, but are not limited to, MEK (e.g. MEK1, MEK2, or MEK1 and MEK2) inhibitors (e.g. XL518, CI-1040, PD035901, selumetinib / AZD6244, GSK1120212 / trametinib, GDC-0973, ARRY-162, ARRY-300, AZD8330, PD0325901, U0126, PD98059, TAK-733, PD318088, AS703026, BAY 869766), alkylating agents (e.g., cyclophosphamide, ifosfamide, chlorambucil, busulfan, melphalan, mechlorethamine, uramustine, thiotepa, nitrosoureas, nitrogen mustards (e.g., mechloroethamine, cyclophosphamide, chlorambucil, meiphalan), ethylenimine and methylmelamines (e.g., hexamethlymelamine, thiotepa), alkyl sulfonates (e.g., busulfan), nitrosoureas (e.g., carmustine, lomusitne, semustine, streptozocin), triazenes (decarbazine)), anti-metabolites (e.g., 5- azathioprine, leucovorin, capecitabine, fludarabine, gemcitabine, pemetrexed, raltitrexed, folic acid analog (e.g., methotrexate), or pyrimidine analogs (e.g., fluorouracil, floxouridine, Cytarabine), purine analogs (e.g., mercaptopurine, thioguanine, pentostatin), etc.), plant alkaloids (e.g., vincristine, vinblastine, vinorelbine, vindesine, podophyllotoxin, paclitaxel, docetaxel, etc.), topoisomerase inhibitors (e.g., irinotecan, topotecan, amsacrine, etoposide (VP16), etoposide phosphate, teniposide, etc.), antitumor antibiotics (e.g., doxorubicin, adriamycin, daunorubicin, epirubicin, actinomycin, bleomycin, mitomycin, mitoxantrone, plicamycin, etc.), platinum-based compounds (e.g. cisplatin, oxaloplatin, carboplatin), anthracenedione (e.g., mitoxantrone), substituted urea (e.g., hydroxyurea), methyl hydrazine derivative (e.g., procarbazine), adrenocortical suppressant (e.g., mitotane, aminoglutethimide), epipodophyllotoxins (e.g., etoposide), antibiotics (e.g., daunorubicin, doxorubicin, bleomycin), enzymes (e.g., L-asparaginase), inhibitors of mitogen-activated protein kinase signaling (e.g. U0126, PD98059, PD184352, PD0325901, ARRY-142886, SB239063,SP600125, BAY 43-9006, wortmannin, or LY294002, Syk inhibitors, mTOR inhibitors, antibodies (e.g., rituxan), gossyphol, genasense, polyphenol E, Chlorofusin, all trans-retinoic acid (ATRA), bryostatin, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), 5-aza-2'- deoxycytidine, all trans retinoic acid, doxorubicin, vincristine, etoposide, gemcitabine, imatinib (Gleevec.RTM.), geldanamycin, 17-N-Allylamino-17-Demethoxygeldanamycin (17-AAG), flavopiridol, LY294002, bortezomib, trastuzumab, BAY 11-7082, PKC412, PD184352, 20-epi-1, 25 dihydroxyvitamin D3; 5-ethynyluracil; abiraterone; aclarubicin; acylfulvene; adecypenol; adozelesin; aldesleukin; ALL-TK antagonists; altretamine; ambamustine; amidox; amifostine; aminolevulinic acid; amrubicin; amsacrine; anagrelide; anastrozole; andrographolide; angiogenesis inhibitors; antagonist D; antagonist G; antarelix; anti-dorsalizing morphogenetic protein-1; antiandrogen, prostatic carcinoma; antiestrogen; antineoplaston; antisense oligonucleotides; aphidicolin glycinate; apoptosis gene modulators; apoptosis regulators; apurinic acid; ara-CDP-DL- PTBA; arginine deaminase; asulacrine; atamestane; atrimustine; axinastatin 1; axinastatin 2; axinastatin 3; azasetron; azatoxin; azatyrosine; baccatin III derivatives; balanol; batimastat; BCR / ABL antagonists; benzochlorins; benzoylstaurosporine; beta lactam derivatives; beta-alethine; betaclamycin B; betulinic acid; bFGF inhibitor; bicalutamide; bisantrene; bisaziridinylspermine; bisnafide; bistratene A; bizelesin; breflate; bropirimine; budotitane; buthionine sulfoximine; calcipotriol; calphostin C; camptothecin derivatives; canarypox IL-2; capecitabine; carboxamide- amino-triazole; carboxyamidotriazole; CaRest M3; CARN 700; cartilage derived inhibitor; carzelesin; casein kinase inhibitors (ICOS); castanospermine; cecropin B; cetrorelix; chlorins; chloroquinoxaline sulfonamide; cicaprost; cis-porphyrin; cladribine; clomifene analogues; clotrimazole; collismycin A; collismycin B; combretastatin A4; combretastatin analogue; conagenin; crambescidin 816; crisnatol; cryptophycin 8; cryptophycin A derivatives; curacin A; cyclopentanthraquinones; cycloplatam; cypemycin; cytarabine ocfosfate; cytolytic factor; cytostatin; dacliximab; decitabine; dehydrodidemnin B; deslorelin; dexamethasone; dexifosfamide; dexrazoxane; dexverapamil; diaziquone; didemnin B; didox; diethylnorspermine; dihydro-5- azacytidine; 9-dioxamycin; diphenyl spiromustine; docosanol; dolasetron; doxifluridine; droloxifene; dronabinol; duocarmycin SA; ebselen; ecomustine; edelfosine; edrecolomab;eflornithine; elemene; emitefur; epirubicin; epristeride; estramustine analogue; estrogen agonists; estrogen antagonists; etanidazole; etoposide phosphate; exemestane; fadrozole; fazarabine; fenretinide; filgrastim; finasteride; flavopiridol; flezelastine; fluasterone; fludarabine; fluorodaunorunicin hydrochloride; forfenimex; formestane; fostriecin; fotemustine; gadolinium texaphyrin; gallium nitrate; galocitabine; ganirelix; gelatinase inhibitors; gemcitabine; glutathione inhibitors; hepsulfam; heregulin; hexamethylene bisacetamide; hypericin; ibandronic acid; idarubicin; idoxifene; idramantone; ilmofosine; ilomastat; imidazoacridones; imiquimod; immunostimulant peptides; insulin-like growth factor-1 receptor inhibitor; interferon agonists; interferons; interleukins; iobenguane; iododoxorubicin; ipomeanol, 4-; iroplact; irsogladine; isobengazole; isohomohalicondrin B; itasetron; jasplakinolide; kahalalide F; lamellarin-N triacetate; lanreotide; leinamycin; lenograstim; lentinan sulfate; leptolstatin; letrozole; leukemia inhibiting factor; leukocyte alpha interferon; leuprolide+estrogen+progesterone; leuprorelin; levamisole; liarozole; linear polyamine analogue; lipophilic disaccharide peptide; lipophilic platinum compounds; lissoclinamide 7; lobaplatin; lombricine; lometrexol; lonidamine; losoxantrone; lovastatin; loxoribine; lurtotecan; lutetium texaphyrin; lysofylline; lytic peptides; maitansine; mannostatin A; marimastat; masoprocol; maspin; matrilysin inhibitors; matrix metalloproteinase inhibitors; menogaril; merbarone; meterelin; methioninase; metoclopramide; MIF inhibitor; mifepristone; miltefosine; mirimostim; mismatched double stranded RNA; mitoguazone; mitolactol; mitomycin analogues; mitonafide; mitotoxin fibroblast growth factor-saporin; mitoxantrone; mofarotene; molgramostim; monoclonal antibody, human chorionic gonadotrophin; monophosphoryl lipid A+myobacterium cell wall sk; mopidamol; multiple drug resistance gene inhibitor; multiple tumor suppressor 1-based therapy; mustard anticancer agent; mycaperoxide B; mycobacterial cell wall extract; myriaporone; N-acetyldinaline; N-substituted benzamides; nafarelin; nagrestip; naloxone+pentazocine; napavin; naphterpin; nartograstim; nedaplatin; nemorubicin; neridronic acid; neutral endopeptidase; nilutamide; nisamycin; nitric oxide modulators; nitroxide antioxidant; nitrullyn; O6-benzylguanine; octreotide; okicenone; oligonucleotides; onapristone; ondansetron; ondansetron; oracin; oral cytokine inducer; ormaplatin; osaterone; oxaliplatin; oxaunomycin; palauamine; palmitoylrhizoxin; pamidronic acid; panaxytriol; panomifene;parabactin; pazelliptine; pegaspargase; peldesine; pentosan polysulfate sodium; pentostatin; pentrozole; perflubron; perfosfamide; perillyl alcohol; phenazinomycin; phenylacetate; phosphatase inhibitors; picibanil; pilocarpine hydrochloride; pirarubicin; piritrexim; placetin A; placetin B; plasminogen activator inhibitor; platinum complex; platinum compounds; platinum-triamine complex; porfimer sodium; porfiromycin; prednisone; propyl bis-acridone; prostaglandin J2; proteasome inhibitors; protein A-based immune modulator; protein kinase C inhibitor; protein kinase C inhibitors, microalgal; protein tyrosine phosphatase inhibitors; purine nucleoside phosphorylase inhibitors; purpurins; pyrazoloacridine; pyridoxylated hemoglobin polyoxyethylerie conjugate; raf antagonists; raltitrexed; ramosetron; ras farnesyl protein transferase inhibitors; ras inhibitors; ras-GAP inhibitor; retelliptine demethylated; rhenium Re 186 etidronate; rhizoxin; ribozymes; RII retinamide; rogletimide; rohitukine; romurtide; roquinimex; rubiginone B1; ruboxyl; safingol; saintopin; SarCNU; sarcophytol A; sargramostim; Sdi 1 mimetics; semustine; senescence derived inhibitor 1; sense oligonucleotides; signal transduction inhibitors; signal transduction modulators; single chain antigen-binding protein; sizofuran; sobuzoxane; sodium borocaptate; sodium phenylacetate; solverol; somatomedin binding protein; sonermin; sparfosic acid; spicamycin D; spiromustine; splenopentin; spongistatin 1; squalamine; stem cell inhibitor; stem-cell division inhibitors; stipiamide; stromelysin inhibitors; sulfinosine; superactive vasoactive intestinal peptide antagonist; suradista; suramin; swainsonine; synthetic glycosaminoglycans; tallimustine; tamoxifen methiodide; tauromustine; tazarotene; tecogalan sodium; tegafur; tellurapyrylium; telomerase inhibitors; temoporfin; temozolomide; teniposide; tetrachlorodecaoxide; tetrazomine; thaliblastine; thiocoraline; thrombopoietin; thrombopoietin mimetic; thymalfasin; thymopoietin receptor agonist; thymotrinan; thyroid stimulating hormone; tin ethyl etiopurpurin; tirapazamine; titanocene bichloride; topsentin; toremifene; totipotent stem cell factor; translation inhibitors; tretinoin; triacetyluridine; triciribine; trimetrexate; triptorelin; tropisetron; turosteride; tyrosine kinase inhibitors; tyrphostins; UBC inhibitors; ubenimex; urogenital sinus-derived growth inhibitory factor; urokinase receptor antagonists; vapreotide; variolin B; vector system, erythrocyte gene therapy; velaresol; veramine; verdins; verteporfin; vinorelbine; vinxaltine; vitaxin; vorozole; zanoterone; zeniplatin; zilascorb; zinostatin stimalamer, Adriamycin, Dactinomycin, Bleomycin, Vinblastine,Cisplatin, acivicin; aclarubicin; acodazole hydrochloride; acronine; adozelesin; aldesleukin; altretamine; ambomycin; ametantrone acetate; aminoglutethimide; amsacrine; anastrozole; anthramycin; asparaginase; asperlin; azacitidine; azetepa; azotomycin; batimastat; benzodepa; bicalutamide; bisantrene hydrochloride; bisnafide dimesylate; bizelesin; bleomycin sulfate; brequinar sodium; bropirimine; busulfan; cactinomycin; calusterone; caracemide; carbetimer; carboplatin; carmustine; carubicin hydrochloride; carzelesin; cedefingol; chlorambucil; cirolemycin; cladribine; crisnatol mesylate; cyclophosphamide; cytarabine; dacarbazine; daunorubicin hydrochloride; decitabine; dexormaplatin; dezaguanine; dezaguanine mesylate; diaziquone; doxorubicin; doxorubicin hydrochloride; droloxifene; droloxifene citrate; dromostanolone propionate; duazomycin; edatrexate; eflornithine hydrochloride; elsamitrucin; enloplatin; enpromate; epipropidine; epirubicin hydrochloride; erbulozole; esorubicin hydrochloride; estramustine; estramustine phosphate sodium; etanidazole; etoposide; etoposide phosphate; etoprine; fadrozole hydrochloride; fazarabine; fenretinide; floxuridine; fludarabine phosphate; fluorouracil; fluorocitabine; fosquidone; fostriecin sodium; gemcitabine; gemcitabine hydrochloride; hydroxyurea; idarubicin hydrochloride; ifosfamide; iimofosine; interleukin I1 (including recombinant interleukin II, or rlL.sub.2), interferon alfa-2a; interferon alfa-2b; interferon alfa-n1; interferon alfa-n3; interferon beta-1a; interferon gamma-1b; iproplatin; irinotecan hydrochloride; lanreotide acetate; letrozole; leuprolide acetate; liarozole hydrochloride; lometrexol sodium; lomustine; losoxantrone hydrochloride; masoprocol; maytansine; mechlorethamine hydrochloride; megestrol acetate; melengestrol acetate; melphalan; menogaril; mercaptopurine; methotrexate; methotrexate sodium; metoprine; meturedepa; mitindomide; mitocarcin; mitocromin; mitogillin; mitomalcin; mitomycin; mitosper; mitotane; mitoxantrone hydrochloride; mycophenolic acid; nocodazoie; nogalamycin; ormaplatin; oxisuran; pegaspargase; peliomycin; pentamustine; peplomycin sulfate; perfosfamide; pipobroman; piposulfan; piroxantrone hydrochloride; plicamycin; plomestane; porfimer sodium; porfiromycin; prednimustine; procarbazine hydrochloride; puromycin; puromycin hydrochloride; pyrazofurin; riboprine; rogletimide; safingol; safingol hydrochloride; semustine; simtrazene; sparfosate sodium; sparsomycin; spirogermanium hydrochloride; spiromustine; spiroplatin; streptonigrin; streptozocin; sulofenur; talisomycin;tecogalan sodium; tegafur; teloxantrone hydrochloride; temoporfin; teniposide; teroxirone; testolactone; thiamiprine; thioguanine; thiotepa; tiazofurin; tirapazamine; toremifene citrate; trestolone acetate; triciribine phosphate; trimetrexate; trimetrexate glucuronate; triptorelin; tubulozole hydrochloride; uracil mustard; uredepa; vapreotide; verteporfin; vinblastine sulfate; vincristine sulfate; vindesine; vindesine sulfate; vinepidine sulfate; vinglycinate sulfate; vinleurosine sulfate; vinorelbine tartrate; vinrosidine sulfate; vinzolidine sulfate; vorozole; zeniplatin; zinostatin; zorubicin hydrochloride, agents that arrest cells in the G2-M phases and / or modulate the formation or stability of microtubules, (e.g. Taxol.TM (i.e. paclitaxel), Taxotere.TM, compounds comprising the taxane skeleton, Erbulozole (i.e. R-55104), Dolastatin 10 (i.e. DLS-10 and NSC-376128), Mivobulin isethionate (i.e. as CI-980), Vincristine, NSC-639829, Discodermolide (i.e. as NVP-XX- A-296), ABT-751 (Abbott, i.e. E-7010), Altorhyrtins (e.g. Altorhyrtin A and Altorhyrtin C), Spongistatins (e.g. Spongistatin 1, Spongistatin 2, Spongistatin 3, Spongistatin 4, Spongistatin 5, Spongistatin 6, Spongistatin 7, Spongistatin 8, and Spongistatin 9), Cemadotin hydrochloride (i.e. LU-103793 and NSC-D-669356), Epothilones (e.g. Epothilone A, Epothilone B, Epothilone C (i.e. desoxyepothilone A or dEpoA), Epothilone D (i.e. KOS-862, dEpoB, and desoxyepothilone B), Epothilone E, Epothilone F, Epothilone B N-oxide, Epothilone A N-oxide, 16-aza-epothilone B, 21- aminoepothilone B (i.e. BMS-310705), 21-hydroxyepothilone D (i.e. Desoxyepothilone F and dEpoF), 26-fluoroepothilone, Auristatin PE (i.e. NSC-654663), Soblidotin (i.e. TZT-1027), LS- 4559-P (Pharmacia, i.e. LS-4577), LS-4578 (Pharmacia, i.e. LS-477-P), LS-4477 (Pharmacia), LS- 4559 (Pharmacia), RPR-112378 (Aventis), Vincristine sulfate, DZ-3358 (Daiichi), FR-182877 (Fujisawa, i.e. WS-9885B), GS-164 (Takeda), GS-198 (Takeda), KAR-2 (Hungarian Academy of Sciences), BSF-223651 (BASF, i.e. ILX-651 and LU-223651), SAH-49960 (Lilly / Novartis), SDZ- 268970 (Lilly / Novartis), AM-97 (Armad / Kyowa Hakko), AM-132 (Armad), AM-138 (Armad / Kyowa Hakko), IDN-5005 (Indena), Cryptophycin 52 (i.e. LY-355703), AC-7739 (Ajinomoto, i.e. AVE-8063A and CS-39.HCl), AC-7700 (Ajinomoto, i.e. AVE-8062, AVE-8062A, CS-39-L-Ser.HCl, and RPR-258062A), Vitilevuamide, Tubulysin A, Canadensol, Centaureidin (i.e. NSC-106969), T-138067 (Tularik, i.e. T-67, TL-138067 and TI-138067), COBRA-1 (Parker Hughes Institute, i.e. DDE-261 and WHI-261), H10 (Kansas State University), H16 (Kansas StateUniversity), Oncocidin A1 (i.e. BTO-956 and DIME), DDE-313 (Parker Hughes Institute), Fijianolide B, Laulimalide, SPA-2 (Parker Hughes Institute), SPA-1 (Parker Hughes Institute, i.e. SPIKET-P), 3-IAABU (Cytoskeleton / Mt. Sinai School of Medicine, i.e. MF-569), Narcosine (also known as NSC-5366), Nascapine, D-24851 (Asta Medica), A-105972 (Abbott), Hemiasterlin, 3- BAABU (Cytoskeleton / Mt. Sinai School of Medicine, i.e. MF-191), TMPN (Arizona State University), Vanadocene acetylacetonate, T-138026 (Tularik), Monsatrol, lnanocine (i.e. NSC- 698666), 3-IAABE (Cytoskeleton / Mt. Sinai School of Medicine), A-204197 (Abbott), T-607 (Tuiarik, i.e. T-900607), RPR-115781 (Aventis), Eleutherobins (such as Desmethyleleutherobin, Desaetyleleutherobin, lsoeleutherobin A, and Z-Eleutherobin), Caribaeoside, Caribaeolin, Halichondrin B, D-64131 (Asta Medica), D-68144 (Asta Medica), Diazonamide A, A-293620 (Abbott), NPI-2350 (Nereus), Taccalonolide A, TUB-245 (Aventis), A-259754 (Abbott), Diozostatin, (-)-Phenylahistin (i.e. NSCL-96F037), D-68838 (Asta Medica), D-68836 (Asta Medica), Myoseverin B, D-43411 (Zentaris, i.e. D-81862), A-289099 (Abbott), A-318315 (Abbott), HTI-286 (i.e. SPA-110, trifluoroacetate salt) (Wyeth), D-82317 (Zentaris), D-82318 (Zentaris), SC- 12983 (NCI), Resverastatin phosphate sodium, BPR-OY-007 (National Health Research Institutes), and SSR-250411 (Sanofi)), steroids (e.g., dexamethasone), finasteride, aromatase inhibitors, gonadotropin-releasing hormone agonists (GnRH) such as goserelin or leuprolide, adrenocorticosteroids (e.g., prednisone), progestins (e.g., hydroxyprogesterone caproate, megestrol acetate, medroxyprogesterone acetate), estrogens (e.g., diethlystilbestrol, ethinyl estradiol), antiestrogen (e.g., tamoxifen), androgens (e.g., testosterone propionate, fluoxymesterone), antiandrogen (e.g., flutamide), immunostimulants (e.g., Bacillus Calmette-Guérin (BCG), levamisole, interleukin-2, alpha-interferon, etc.), monoclonal antibodies (e.g., anti-CD20, anti- HER2, anti-CD52, anti-HLA-DR, and anti-VEGF monoclonal antibodies), immunotoxins (e.g., anti- CD33 monoclonal antibody-calicheamicin conjugate, anti-CD22 monoclonal antibody-pseudomonas exotoxin conjugate, etc.), radioimmunotherapy (e.g., anti-CD20 monoclonal antibody conjugated to111In,90Y, or131I, etc.), triptolide, homoharringtonine, dactinomycin, doxorubicin, epirubicin, topotecan, itraconazole, vindesine, cerivastatin, vincristine, deoxyadenosine, sertraline, pitavastatin, irinotecan, clofazimine, 5-nonyloxytryptamine, vemurafenib, dabrafenib, erlotinib, gefitinib, EGFRinhibitors, epidermal growth factor receptor (EGFR)-targeted therapy or therapeutic (e.g. gefitinib (Iressa ™), erlotinib (Tarceva ™), cetuximab (Erbitux™), lapatinib (Tykerb™), panitumumab (Vectibix™), vandetanib (Caprelsa™), afatinib / BIBW2992, CI-1033 / canertinib, neratinib / HKI-272, CP-724714, TAK-285, AST-1306, ARRY334543, ARRY-380, AG-1478, dacomitinib / PF299804, OSI-420 / desmethyl erlotinib, AZD8931, AEE788, pelitinib / EKB-569, CUDC-101, WZ8040, WZ4002, WZ3146, AG-490, XL647, PD153035, BMS-599626), sorafenib, imatinib, sunitinib, dasatinib, or the like.

[0129] As used herein, “treating” or “treatment of” a condition, disease or disorder or symptoms associated with a condition, disease or disorder refers to an approach for obtaining beneficial or desired results, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of condition, disorder or disease, stabilization of the state of condition, disorder or disease, prevention of development of condition, disorder or disease, prevention of spread of condition, disorder or disease, delay or slowing of condition, disorder or disease progression, delay or slowing of condition, disorder or disease onset, amelioration or palliation of the condition, disorder or disease state, and remission, whether partial or total. “Treating” can also mean prolonging survival of a subject beyond that expected in the absence of treatment. “Treating” can also mean inhibiting the progression of the condition, disorder or disease, slowing the progression of the condition, disorder or disease temporarily, although in some instances, it involves halting the progression of the condition, disorder or disease permanently. As used herein the terms treatment, treat, or treating refers to a method of reducing the effects of one or more symptoms of a disease or condition characterized by expression of the protease or symptom of the disease or condition characterized by expression of the protease. Thus in the disclosed method, treatment can refer to a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in the severity of an established disease, condition, or symptom of the disease or condition. For example, a method for treating a disease is considered to be a treatment if there is a 10% reduction in one or more symptoms of the disease in a subject as compared to a control. Thus the reduction can be a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any percent reduction in between 10% and 100% as compared to nativeor control levels. It is understood that treatment does not necessarily refer to a cure or complete ablation of the disease, condition, or symptoms of the disease or condition. Further, as used herein, references to decreasing, reducing, or inhibiting include a change of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater as compared to a control level and such terms can include but do not necessarily include complete elimination.

[0130] The terms “dose” and “dosage” are used interchangeably herein. A dose refers to the amount of active ingredient given to an individual at each administration. The dose will vary depending on a number of factors, including the range of normal doses for a given therapy, frequency of administration; size and tolerance of the individual; severity of the condition; risk of side effects; and the route of administration. One of skill will recognize that the dose can be modified depending on the above factors or based on therapeutic progress. The term “dosage form” refers to the particular format of the pharmaceutical or pharmaceutical composition, and depends on the route of administration. For example, a dosage form can be in a liquid form for nebulization, e.g., for inhalants, in a tablet or liquid, e.g., for oral delivery, or a saline solution, e.g., for injection.

[0131] By “therapeutically effective dose or amount” as used herein is meant a dose that produces effects for which it is administered (e.g. treating or preventing a disease). The exact dose and formulation will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Remington: The Science and Practice of Pharmacy, 20th Edition, Gennaro, Editor (2003), and Pickar, Dosage Calculations (1999)). For example, for the given parameter, a therapeutically effective amount will show an increase or decrease of at least 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90%, or at least 100%. Therapeutic efficacy can also be expressed as “-fold” increase or decrease. For example, a therapeutically effective amount can have at least a 1.2-fold, 1.5-fold, 2-fold, 5-fold, or more effect over a standard control. A therapeutically effective dose or amount may ameliorate one or more symptoms of a disease. A therapeutically effective dose or amount may prevent or delay the onset of a disease or one or more symptoms of a disease when the effect for which it is being administered is to treat a person who is at risk of developing the disease.As is understood in the clinical context, a therapeutically effective dosage of a drug, compound or pharmaceutical composition may or may not be achieved in conjunction with another drug, compound or pharmaceutical composition.

[0132] An “effective amount” of a drug, compound, or pharmaceutical composition is an amount sufficient to affect any one or more beneficial or desired results. For the diagnostic use of the compounds and methods provided herein, beneficial or desired results include detecting a cell expressing detectable levels of TGF-beta using the compositions and methods provided herein, including embodiments thereof, while at the same time not inhibiting the activity of TGF-beta on said cell relative to a standard control. Thus, an effective amount for diagnostic use is an amount that does not inhibit TGF-beta activity relative to a standard control, whereas a therapeutically effective amount inhibits TGF-beta activity relative to a standard control. In embodiments, the effective amount is less than the therapeutically effective amount. For the purposes of this invention, an effective dosage or amount of a drug, compound or pharmaceutical composition is an amount sufficient to accomplish diagnostic analysis either directly or indirectly.

[0133] As used herein, the term "administering" means oral administration, administration as a suppository, topical contact, intravenous, intraperitoneal, intramuscular, intralesional, intrathecal, intranasal or subcutaneous administration, or the implantation of a slow-release device, e.g., a mini- osmotic pump, to a subject. Administration is by any route, including parenteral and transmucosal (e.g., buccal, sublingual, palatal, gingival, nasal, vaginal, rectal, or transdermal). Parenteral administration includes, e.g., intravenous, intramuscular, intra-arteriole, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial. Other modes of delivery include, but are not limited to, the use of liposomal formulations, intravenous infusion, transdermal patches, etc. By "co-administer" it is meant that a composition described herein is administered at the same time, just prior to, or just after the administration of one or more additional therapies, for example cancer therapies such as chemotherapy, hormonal therapy, radiotherapy, or immunotherapy. The compounds of the invention can be administered alone or can be co-administered to the patient. Coadministration is meant to include simultaneous or sequential administration of the compounds individually or in combination (more than one compound). Thus, the preparations can also becombined, when desired, with other active substances (e.g. to reduce metabolic degradation). The compositions of the present invention can be delivered by transdermally, by a topical route, formulated as applicator sticks, solutions, suspensions, emulsions, gels, creams, ointments, pastes, jellies, paints, powders, and aerosols.

[0134] The compositions of the present invention may additionally include components to provide sustained release and / or comfort. Such components include high molecular weight, anionic mucomimetic polymers, gelling polysaccharides and finely-divided drug carrier substrates. These components are discussed in greater detail in U.S. Pat. Nos. 4,911,920; 5,403,841; 5,212,162; and 4,861,760. The entire contents of these patents are incorporated herein by reference in their entirety for all purposes. The compositions of the present invention can also be delivered as microspheres for slow release in the body. For example, microspheres can be administered via intradermal injection of drug-containing microspheres, which slowly release subcutaneously (see Rao, J. Biomater Sci. Polym. Ed. 7:623-645, 1995; as biodegradable and injectable gel formulations (see, e.g., Gao Pharm. Res. 12:857-863, 1995); or, as microspheres for oral administration (see, e.g., Eyles, J. Pharm. Pharmacol. 49:669-674, 1997). In embodiments, the formulations of the compositions of the present invention can be delivered by the use of liposomes which fuse with the cellular membrane or are endocytosed, i.e., by employing receptor ligands attached to the liposome, that bind to surface membrane protein receptors of the cell resulting in endocytosis. By using liposomes, particularly where the liposome surface carries receptor ligands specific for target cells, or are otherwise preferentially directed to a specific organ, one can focus the delivery of the compositions of the present invention into the target cells in vivo. (See, e.g., Al-Muhammed, J. Microencapsul. 13:293-306, 1996; Chonn, Curr. Opin. Biotechnol. 6:698-708, 1995; Ostro, Am. J. Hosp. Pharm. 46:1576-1587, 1989). The compositions of the present invention can also be delivered as nanoparticles.

[0135] As used herein, the term “pharmaceutically acceptable” is used synonymously with “physiologically acceptable” and “pharmacologically acceptable”. A pharmaceutical composition will generally comprise agents for buffering and preservation in storage, and can include buffers and carriers for appropriate delivery, depending on the route of administration.

[0136] "Pharmaceutically acceptable excipient" and "pharmaceutically acceptable carrier" refer to a substance that aids the administration of an active agent to and absorption by a subject and can be included in the compositions of the present invention without causing a significant adverse toxicological effect on the patient. Non-limiting examples of pharmaceutically acceptable excipients include water, NaCl, normal saline solutions, lactated Ringer’s, normal sucrose, normal glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavors, salt solutions (such as Ringer's solution), alcohols, oils, gelatins, carbohydrates such as lactose, amylose or starch, fatty acid esters, hydroxymethycellulose, polyvinyl pyrrolidine, and colors, and the like. Such preparations can be sterilized and, if desired, mixed with auxiliary agents such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, and / or aromatic substances and the like that do not deleteriously react with the compounds of the invention. One of skill in the art will recognize that other pharmaceutical excipients are useful in the present invention.

[0137] The term "pharmaceutically acceptable salt" refers to salts derived from a variety of organic and inorganic counter ions well known in the art and include, by way of example only, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, and the like; and when the molecule contains a basic functionality, salts of organic or inorganic acids, such as hydrochloride, hydrobromide, tartrate, mesylate, acetate, maleate, oxalate and the like.

[0138] The term "preparation" is intended to include the formulation of the active compound with encapsulating material as a carrier providing a capsule in which the active component with or without other carriers, is surrounded by a carrier, which is thus in association with it. Similarly, cachets and lozenges are included. Tablets, powders, capsules, pills, cachets, and lozenges can be used as solid dosage forms suitable for oral administration.

[0139] The pharmaceutical preparation is optionally in unit dosage form. In such form the preparation is subdivided into unit doses containing appropriate quantities of the active component. The unit dosage form can be a packaged preparation, the package containing discrete quantities of preparation, such as packeted tablets, capsules, and powders in vials or ampoules. Also, the unitdosage form can be a capsule, tablet, cachet, or lozenge itself, or it can be the appropriate number of any of these in packaged form. The unit dosage form can be of a frozen dispersion.

[0140] One of skill in the art will understand which standard controls are most appropriate in a given situation and be able to analyze data based on comparisons to standard control values. Standard controls are also valuable for determining the significance (e.g. statistical significance) of data. For example, if values for a given parameter are widely variant in standard controls, variation in test samples will not be considered as significant.

[0141] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes. II. Compositions

[0142] The compositions provided herein including embodiments thereof are TGF-beta-binding agents that bind to TGF-beta in its active / mature form (i.e., active TGF-beta, mature TGF-beta). The agents may be antibodies (e.g., humanized antibodies, bispecific antibodies, antibody drug conjugates (ADC)), peptides or small molecules having the ability to bind the active form of TGF- beta, while not binding inactive or latent TGF-beta at a detectable level relative to a standard control. The compositions provided herein are, inter alia, useful as theranostics for the treatment of cancer (e.g., solid tumors such as carcinoma or sarcoma).

[0143] In an aspect is provided an active TGF-beta binding agent including a radioisotope. In embodiments, the agent is a protein, a nucleic acid, or a small molecule. In embodiments, the agent is a protein. In embodiments, the agent is a nucleic acid. In embodiments, the agent is a small molecule. In embodiments, the agent is an antibody or a peptide. In embodiments, the agent is an antibody. In embodiments, the agent is a peptide.

[0144] In embodiments, the agent is an active TGF-beta binding antibody. An “active TGF-beta binding antibody” as provided herein refers to an antibody that specifically binds to the active form of TGF-beta also referred to therein as “active TGF-beta” or “mature TGF-β.” An active TGF-beta binding antibody as provided herein does not bind to the inactive form of TGF-beta (i.e., latent TGF-beta) at a detectable level and relative to a standard control.

[0145] The transforming growth factor beta-1 proprotein is a polypeptide that includes a latency- associated peptide (LAP) peptide (LAP portion) and a TGF-beta 1 peptide (TGF-beta 1 portion). In embodiments, the transforming growth factor beta-1 proprotein includes the sequence of the protein identified by UniProt reference P01137. In embodiments, the transforming growth factor beta-1 proprotein is the sequence of the protein identified by UniProt reference P01137. In embodiments, the active TGF-beta is formed by proteolytic cleavage of the transforming growth factor beta-1 proprotein. In embodiments, the active TGF-beta includes the N-terminal region of the TGF-beta proprotein. In embodiments, the active TGF-beta is the N-terminal region of the TGF-beta proprotein. In embodiments, the active TGF-beta is a TGF-beta homodimer. In embodiments, the active TGF-beta does not include the C-terminal region of the TGF-beta proprotein. In embodiments, the active TGF-beta is not the C-terminal region of the TGF-beta proprotein. In embodiments, the active TGF-beta does not include a latency-associated peptide (LAP).

[0146] Proteolytic cleavage separates the LAP portion from the TGF-beta 1 portion and provides an active TGF-beta protein. A “latency-associated peptide (LAP)” as provided herein refers to a peptide chain that forms part of the protein identified by UniProt reference P01137. The protein identified by UniProt reference P01137 includes two peptide chains, the LAP, and a transforming growth factor beta-1 (TGF-beta 1) peptide. Upon activation the LAP portion is separated from the TGF-beta 1 portion and an active TGF-beta is provided.

[0147] The C-terminal pro-region, or latency-associated peptide (LAP), is cleaved from the mature TGF-beta during intracellular modification before secretion. When the mature TGF-beta is associated with the LAP, it is called L-TGF-beta, which is inactive and cannot interact with its receptor. Activation of TGF-beta is the process that releases TGF-beta from the latent complex,freeing the ligand to bind to TGF-beta receptors to initiate signaling. The activation process occurs by different mechanisms acting on the latent complex. These include but are not limited to, exposure to proteases or reactive oxygen, or binding to other proteins (e.g., thrombospondin) or binding by cell surface proteins that include GARP, LCC33 and integrins.

[0148] "TGF-beta" as referred to herein includes any of the recombinant or naturally-occurring isoforms (TGF-beta 1, 2, and 3) of the Transforming growth factor beta proteins or variants or homologs thereof that comprise TGF-beta activity (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity). In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring TGF-beta protein. In embodiments, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence compared to the TGF-beta protein identified by the sequence of UniProt reference number P01137.

[0149] In embodiments, the active TGF-beta includes parts of the protein that is substantially identical to the protein identified by the UniProt reference number P01137 or a variant or homolog having substantial identity thereto. In embodiments, the active TGF-beta includes parts of the protein that is substantially identical to the protein identified by the UniProt reference number P01137 or a variant or homolog having substantial identity thereto and does not include the latency- associated peptide (LAP) protein. In embodiments, the active TGF-beta forms part of the protein that is substantially identical to the protein identified by the UniProt reference number P61812 or a variant or homolog having substantial identity thereto. In embodiments, the active TGF-beta forms part of the protein that is substantially identical to the protein identified by the UniProt reference number P10600 or a variant or homolog having substantial identity thereto. In embodiments, the TGF-beta protein is substantially identical to the protein identified by the UniProt reference number P01137 or a variant or homolog having substantial identity thereto.

[0150] In embodiments, the active TGF-beta binding antibody is a humanized active TGF-beta binding antibody. In embodiments, the active TGF-beta binding antibody binds all three isoforms of TGF-beta. In embodiments, the active TGF-beta binding antibody binds TGF-beta 1. Inembodiments, the active TGF-beta binding antibody binds TGF-beta 2. In embodiments, the active TGF-beta binding antibody binds TGF-beta 3. In embodiments, the active TGF-beta binding antibody is fresolimumab. In the usual and customary sense the term “fresolimumab” is the human monoclonal antibody identified by CAS reg. no. 948564-73-6. In embodiments, the active TGF- beta binding agent neutralizes TGF-beta. Neutralizing when used in the context of an agent or antibody, refers in its customary sense to the ability of an agent or antibody to inhibit the activity of the ligand (e.g., TGF-beta) that agent or antibody binds to, to a level that is not detectable relative to a standard control. In embodiments, the agent neutralizes active TGF-beta. In embodiments, the agent is a bispecific antibody. In embodiments, the bispecific antibody binds to active TGF-beta and a cancer antigen. In a further embodiment, the cancer antigen is EGFR.

[0151] In embodiments, the active TGF-beta binding antibody binds TGF-beta 2. In embodiments, the active TGF-beta binding antibody is lerdelimumab. In the usual and customary sense the term “lerdelimumab” is the human monoclonal antibody identified by CAS reg. no. 285985-06-0.

[0152] In embodiments, the active TGF-beta binding antibody binds TGF-beta 1. In embodiments, the active TGF-beta binding antibody is metelimumab. In the usual and customary sense the term “metelimumab” is the human monoclonal antibody identified by CAS reg. no. 272780-74-2.

[0153] In embodiments, the active TGF-beta binding antibody binds TGF-beta 1. In embodiments, the active TGF-beta binding antibody is LY2382770. In embodiments, the active TGF-beta binding antibody is NIS793.

[0154] In embodiments, the active TGF-beta binding antibody binds the GARP-TGF-beta 1 complex. In embodiments, the active TGF-beta binding antibody is ABBV-151. In embodiments, the active TGF-beta binding antibody is PF-06952229.

[0155] In embodiments, the active TGF-beta binding agent is a TGF-beta trap. A “TGF-beta trap” as provided herein refers in its customary meaning to a protein that binds TGF-beta in its activeform and prevents it from binding to its receptor. A TGF-beta trap may be the extracellular domain of the TGF-βRII receptor which binds to TGF-beta and prevents it from binding to its cognate receptor. In embodiments, the TGF-beta trap binds to TGF-beta 1 and TGF-beta 3. In embodiments, the TGF-beta trap is AVID200. In embodiments, the TGF-beta trap binds to TGF- beta 1, TGF-beta 2 and TGF-beta 3. In embodiments, the TGF-beta trap is BIN-1.

[0156] The active TGF-beta binding agent may be a bispecific antibody or fusion protein including a TGF-beta trap and an EGFR binding domain. In embodiments, the active TGF-beta binding agent is BCA-101. In embodiments, the active TGF-beta binding agent is Bintrafusp alfa (M7824).

[0157] The radioisotopes provided herein including embodiments thereof may be used for diagnostic purposes and / or therapeutic purposes. In embodiments, the radioisotope is a therapeutic radioisotope or a diagnostic radioisotope. In embodiments, the radioisotope is a therapeutic radioisotope. In embodiments, the radioisotope is a diagnostic radioisotope. In embodiments, the radioisotope is225Ac,211At,134Ce,64Cu,67Cu,68Ga,123I,124I,131I,111In,132La,133La,177Lu,44Sc,47Sc,153Sm,149Tb,152Tb,161Tb,203Tb,212Tb,227Th,86Y,90Y, or89Zr. In embodiments, the radioisotope is225Ac. In embodiments, the radioisotope is211At. In embodiments, the radioisotope is134Ce. In embodiments, the radioisotope is64Cu. In embodiments, the radioisotope is67Cu. In embodiments, the radioisotope is68Ga. In embodiments, the radioisotope is123I. In embodiments, the radioisotope is124I. In embodiments, the radioisotope is131I. In embodiments, the radioisotope is111In. In embodiments, the radioisotope is132La. In embodiments, the radioisotope is133La. In embodiments, the radioisotope is177Lu. In embodiments, the radioisotope is44Sc. In embodiments, the radioisotope is47Sc. In embodiments, the radioisotope is153Sm. In embodiments, the radioisotope is149Tb. In embodiments, the radioisotope is152Tb. In embodiments, the radioisotope is161Tb. In embodiments, the radioisotope is203Tb. In embodiments, the radioisotope is212Tb. In embodiments, the radioisotope is227Th. In embodiments, the radioisotope is86Y. In embodiments, the radioisotope is90Y. In embodiments, the radioisotope is89Zr. In embodiments, the radioisotope is any of the radioisotopes described in Sharma S. et al. (Radiometals in Imaging and Therapy:Highlighting two decades of Research. Pharmaceuticals 2023, 16, 1460), which is hereby incorporated by references in its entirety and for all purposes.

[0158] In embodiments, the active TGF-beta binding antibody further includes a therapeutic moiety. In embodiments, the radioisotope or the therapeutic moiety are covalently attached to the active TGF-beta binding antibody. In embodiments, the active TGF-beta binding antibody further includes a detectable moiety.

[0159] Methods and compositions of attaching a radioisotope to a protein through chelation are well known in the chemical art and are without limitation contemplated for forming the active TGF- beta biding agents including a radioisotope. Thus, in embodiments, the radioisotope is bound to the active TGF-beta binding agent through a covalent linker. In embodiments, the radioisotope is bound to the active TGF-beta binding agent through a chemical linker. In embodiments, the radioisotope is bound to the active TGF-beta binding agent through a chelate. Any of the chelating agents or chelates described in Chem. Soc. Rev., 2014, 43, 260; Chem. Med. Chem 2021, 16, 2909-2941; or J. Am. Chem. Soc. 2021, 143, 10429-10440; which are hereby incorporated by reference in their entirety and for all purposes may be used for binding a radioisotope to an active TGF-beta binding agent as provided herein including embodiments thereof.

[0160] A “chemical linker,” as provided herein, is a covalent linker, a non-covalent linker, a peptide or peptidyl linker (a linker including a peptide moiety), a cleavable peptide linker, a substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene or substituted or unsubstituted heteroarylene or any combination thereof.

[0161] The chemical linker as provided herein may be a bond, -O-, -S-, -C(O)-, -C(O)O-, -C(O)NH-, -S(O)2NH-, -NH-, -NHC(O)NH-, substituted (e.g., substituted with a substituent group, a size-limited substituent or a lower substituent group) or unsubstituted alkylene, substituted (e.g., substituted with a substituent group, a size-limited substituent or a lower substituent group) or unsubstituted heteroalkylene, substituted (e.g., substituted with a substituent group, a size-limited substituent or a lower substituent group) or unsubstituted cycloalkylene, substituted (e.g., substitutedwith a substituent group, a size-limited substituent or a lower substituent group) or unsubstituted heterocycloalkylene, substituted (e.g., substituted with a substituent group, a size-limited substituent or a lower substituent group) or unsubstituted arylene or substituted (e.g., substituted with a substituent group, a size-limited substituent or a lower substituent group) or unsubstituted heteroarylene.

[0162] The chemical linker as provided herein may be a bond, -O-, -S-, -C(O)-, -C(O)O-, C(O)NH-, -S(O)2NH-, -NH-, -NHC(O)NH-, substituted or unsubstituted (e.g., C1-C20, C1-C10, C1-C5) alkylene, substituted or unsubstituted (e.g., 2 to 20 membered, 2 to 10 membered, 2 to 5 membered) heteroalkylene, substituted or unsubstituted (e.g., C3-C8, C3-C6, C3-C5) cycloalkylene, substituted or unsubstituted (e.g., 3 to 8 membered, 3 to 6 membered, 3 to 5 membered) heterocycloalkylene, substituted or unsubstituted (e.g., C6-C10, C6-C8, C6-C5) arylene or substituted or unsubstituted (e.g., 5 to 10 membered, 5 to 8 membered, 5 to 6 membered,) heteroarylene. III. Pharmaceutical compositions

[0163] The active TGF-beta binding agents provided herein including embodiments thereof may be used for diagnostic / imaging and therapeutic purposes as theranostics. Thus, in an aspect is provided a pharmaceutical composition including a pharmaceutically acceptable excipient and an active TGF-beta binding agent as provided herein including embodiments thereof. The pharmaceutical compositions provided herein may include an active TGF-beta binding agent bound to a therapeutic or a diagnostic radioisotope. In embodiments, the pharmaceutical composition includes a TGF-beta antibody bound to a therapeutic radioisotope. In embodiments, the pharmaceutical composition includes a TGF-beta antibody bound to a diagnostic radioisotope.

[0164] In embodiments, the active TGF-beta binding agent is bound to a therapeutic radioisotope. In embodiments, the active TGF-beta binding agent includes a therapeutic radioisotope. In embodiments, the therapeutic radioisotope is present at a concentration of about 30-300 mCi. In embodiments, the therapeutic radioisotope is present at a concentration of about 30-250 mCi. In embodiments, the therapeutic radioisotope is present at a concentration of about 30-200 mCi. Inembodiments, the therapeutic radioisotope is present at a concentration of about 30-150 mCi. In embodiments, the therapeutic radioisotope is present at a concentration of about 30-100 mCi. In embodiments, the therapeutic radioisotope is present at a concentration of about 30-50 mCi.

[0165] In embodiments, the therapeutic radioisotope is present at a concentration of about 50-300 mCi. In embodiments, the therapeutic radioisotope is present at a concentration of about 100-300 mCi. In embodiments, the therapeutic radioisotope is present at a concentration of about 150-300 mCi. In embodiments, the therapeutic radioisotope is present at a concentration of about 200-300 mCi. In embodiments, the therapeutic radioisotope is present at a concentration of about 250-300 mCi. In embodiments, the therapeutic radioisotope is present at a concentration of about 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 mCi.

[0166] In embodiments, the therapeutic radioisotope is present at a concentration of 30-300 mCi. In embodiments, the therapeutic radioisotope is present at a concentration of 40-300 mCi. In embodiments, the therapeutic radioisotope is present at a concentration of 50-300 mCi. In embodiments, the therapeutic radioisotope is present at a concentration of 60-300 mCi. In embodiments, the therapeutic radioisotope is present at a concentration of 70-300 mCi. In embodiments, the therapeutic radioisotope is present at a concentration of 80-300 mCi. In embodiments, the therapeutic radioisotope is present at a concentration of 90-300 mCi. In embodiments, the therapeutic radioisotope is present at a concentration of 100-300 mCi. In embodiments, the therapeutic radioisotope is present at a concentration of 110-300 mCi. In embodiments, the therapeutic radioisotope is present at a concentration of 120-300 mCi. In embodiments, the therapeutic radioisotope is present at a concentration of 130-300 mCi. In embodiments, the therapeutic radioisotope is present at a concentration of 140-300 mCi. In embodiments, the therapeutic radioisotope is present at a concentration of 150-300 mCi. In embodiments, the therapeutic radioisotope is present at a concentration of 160-300 mCi. In embodiments, the therapeutic radioisotope is present at a concentration of 170-300 mCi. In embodiments, the therapeutic radioisotope is present at a concentration of 180-300 mCi. In embodiments, the therapeutic radioisotope is present at a concentration of 190-300 mCi. Inembodiments, the therapeutic radioisotope is present at a concentration of 200-300 mCi. In embodiments, the therapeutic radioisotope is present at a concentration of 210-300 mCi. In embodiments, the therapeutic radioisotope is present at a concentration of 220-300 mCi. In embodiments, the therapeutic radioisotope is present at a concentration of 230-300 mCi. In embodiments, the therapeutic radioisotope is present at a concentration of 240-300 mCi. In embodiments, the therapeutic radioisotope is present at a concentration of 250-300 mCi. In embodiments, the therapeutic radioisotope is present at a concentration of 260-300 mCi. In embodiments, the therapeutic radioisotope is present at a concentration of 270-300 mCi. In embodiments, the therapeutic radioisotope is present at a concentration of 280-300 mCi. In embodiments, the therapeutic radioisotope is present at a concentration of 290-300 mCi. In embodiments, the therapeutic radioisotope is present at a concentration of 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 mCi. In embodiments, the therapeutic radioisotope is present at a concentration of about 200 mCi. In embodiments, the therapeutic radioisotope is present at a concentration of 200 mCi. In one embodiment, the therapeutic radioisotope is177Lu.

[0167] In embodiments, the active TGF-beta binding agent includes a diagnostic radioisotope. In embodiments, the active TGF-beta binding agent is bound a diagnostic radioisotope. In embodiments, the diagnostic radioisotope is present at a concentration of about 0.5-5 mCi. In embodiments, the diagnostic radioisotope is present at a concentration of about 0.6-5 mCi. In embodiments, the diagnostic radioisotope is present at a concentration of about 0.7-5 mCi. In embodiments, the diagnostic radioisotope is present at a concentration of about 0.8-5 mCi. In embodiments, the diagnostic radioisotope is present at a concentration of about 0.9-5 mCi. In embodiments, the diagnostic radioisotope is present at a concentration of about 1-5 mCi.

[0168] In embodiments, the diagnostic radioisotope is present at a concentration of about 1.1-5 mCi. In embodiments, the diagnostic radioisotope is present at a concentration of about 1.2-5 mCi. In embodiments, the diagnostic radioisotope is present at a concentration of about 1.3-5 mCi. In embodiments, the diagnostic radioisotope is present at a concentration of about 1.4-5 mCi. In embodiments, the diagnostic radioisotope is present at a concentration of about 1.5-5 mCi. Inembodiments, the diagnostic radioisotope is present at a concentration of about 1.6-5 mCi. In embodiments, the diagnostic radioisotope is present at a concentration of about 1.7-5 mCi. In embodiments, the diagnostic radioisotope is present at a concentration of about 1.8-5 mCi. In embodiments, the diagnostic radioisotope is present at a concentration of about 1.9-5 mCi. In embodiments, the diagnostic radioisotope is present at a concentration of about 2-5 mCi.

[0169] In embodiments, the diagnostic radioisotope is present at a concentration of about 2.1-5 mCi. In embodiments, the diagnostic radioisotope is present at a concentration of about 2.2-5 mCi. In embodiments, the diagnostic radioisotope is present at a concentration of about 2.3-5 mCi. In embodiments, the diagnostic radioisotope is present at a concentration of about 2.4-5 mCi. In embodiments, the diagnostic radioisotope is present at a concentration of about 2.5-5 mCi. In embodiments, the diagnostic radioisotope is present at a concentration of about 2.6-5 mCi. In embodiments, the diagnostic radioisotope is present at a concentration of about 2.7-5 mCi. In embodiments, the diagnostic radioisotope is present at a concentration of about 2.8-5 mCi. In embodiments, the diagnostic radioisotope is present at a concentration of about 2.9-5 mCi. In embodiments, the diagnostic radioisotope is present at a concentration of about 3-5 mCi.

[0170] In embodiments, the diagnostic radioisotope is present at a concentration of about 3.1-5 mCi. In embodiments, the diagnostic radioisotope is present at a concentration of about 3.2-5 mCi. In embodiments, the diagnostic radioisotope is present at a concentration of about 3.3-5 mCi. In embodiments, the diagnostic radioisotope is present at a concentration of about 3.4-5 mCi. In embodiments, the diagnostic radioisotope is present at a concentration of about 3.5-5 mCi. In embodiments, the diagnostic radioisotope is present at a concentration of about 3.6-5 mCi. In embodiments, the diagnostic radioisotope is present at a concentration of about 3.7-5 mCi. In embodiments, the diagnostic radioisotope is present at a concentration of about 3.8-5 mCi. In embodiments, the diagnostic radioisotope is present at a concentration of about 3.9-5 mCi. In embodiments, the diagnostic radioisotope is present at a concentration of about 4-5 mCi.

[0171] In embodiments, the diagnostic radioisotope is present at a concentration of about 4.1-5 mCi. In embodiments, the diagnostic radioisotope is present at a concentration of about 4.2-5 mCi.In embodiments, the diagnostic radioisotope is present at a concentration of about 4.3-5 mCi. In embodiments, the diagnostic radioisotope is present at a concentration of about 4.4-5 mCi. In embodiments, the diagnostic radioisotope is present at a concentration of about 4.5-5 mCi. In embodiments, the diagnostic radioisotope is present at a concentration of about 4.6-5 mCi. In embodiments, the diagnostic radioisotope is present at a concentration of about 4.7-5 mCi. In embodiments, the diagnostic radioisotope is present at a concentration of about 4.8-5 mCi. In embodiments, the diagnostic radioisotope is present at a concentration of about 4.9-5 mCi.

[0172] In embodiments, the diagnostic radioisotope is present at a concentration of 0.5-5 mCi. In embodiments, the diagnostic radioisotope is present at a concentration of 0.6-5 mCi. In embodiments, the diagnostic radioisotope is present at a concentration of 0.7-5 mCi. In embodiments, the diagnostic radioisotope is present at a concentration of 0.8-5 mCi. In embodiments, the diagnostic radioisotope is present at a concentration of 0.9-5 mCi. In embodiments, the diagnostic radioisotope is present at a concentration of 1-5 mCi.

[0173] In embodiments, the diagnostic radioisotope is present at a concentration of 1.1-5 mCi. In embodiments, the diagnostic radioisotope is present at a concentration of 1.2-5 mCi. In embodiments, the diagnostic radioisotope is present at a concentration of 1.3-5 mCi. In embodiments, the diagnostic radioisotope is present at a concentration of 1.4-5 mCi. In embodiments, the diagnostic radioisotope is present at a concentration of 1.5-5 mCi. In embodiments, the diagnostic radioisotope is present at a concentration of 1.6-5 mCi. In embodiments, the diagnostic radioisotope is present at a concentration of 1.7-5 mCi. In embodiments, the diagnostic radioisotope is present at a concentration of 1.8-5 mCi. In embodiments, the diagnostic radioisotope is present at a concentration of 1.9-5 mCi. In embodiments, the diagnostic radioisotope is present at a concentration of 2-5 mCi.

[0174] In embodiments, the diagnostic radioisotope is present at a concentration of 2.1-5 mCi. In embodiments, the diagnostic radioisotope is present at a concentration of 2.2-5 mCi. In embodiments, the diagnostic radioisotope is present at a concentration of 2.3-5 mCi. In embodiments, the diagnostic radioisotope is present at a concentration of 2.4-5 mCi. Inembodiments, the diagnostic radioisotope is present at a concentration of 2.5-5 mCi. In embodiments, the diagnostic radioisotope is present at a concentration of 2.6-5 mCi. In embodiments, the diagnostic radioisotope is present at a concentration of 2.7-5 mCi. In embodiments, the diagnostic radioisotope is present at a concentration of 2.8-5 mCi. In embodiments, the diagnostic radioisotope is present at a concentration of 2.9-5 mCi. In embodiments, the diagnostic radioisotope is present at a concentration of 3-5 mCi.

[0175] In embodiments, the diagnostic radioisotope is present at a concentration of 3.1-5 mCi. In embodiments, the diagnostic radioisotope is present at a concentration of 3.2-5 mCi. In embodiments, the diagnostic radioisotope is present at a concentration of 3.3-5 mCi. In embodiments, the diagnostic radioisotope is present at a concentration of 3.4-5 mCi. In embodiments, the diagnostic radioisotope is present at a concentration of 3.5-5 mCi. In embodiments, the diagnostic radioisotope is present at a concentration of 3.6-5 mCi. In embodiments, the diagnostic radioisotope is present at a concentration of 3.7-5 mCi. In embodiments, the diagnostic radioisotope is present at a concentration of 3.8-5 mCi. In embodiments, the diagnostic radioisotope is present at a concentration of 3.9-5 mCi. In embodiments, the diagnostic radioisotope is present at a concentration of 4-5 mCi.

[0176] In embodiments, the diagnostic radioisotope is present at a concentration of 4.1-5 mCi. In embodiments, the diagnostic radioisotope is present at a concentration of 4.2-5 mCi. In embodiments, the diagnostic radioisotope is present at a concentration of 4.3-5 mCi. In embodiments, the diagnostic radioisotope is present at a concentration of 4.4-5 mCi. In embodiments, the diagnostic radioisotope is present at a concentration of 4.5-5 mCi. In embodiments, the diagnostic radioisotope is present at a concentration of 4.6-5 mCi. In embodiments, the diagnostic radioisotope is present at a concentration of 4.7-5 mCi. In embodiments, the diagnostic radioisotope is present at a concentration of 4.8-5 mCi. In embodiments, the diagnostic radioisotope is present at a concentration of 4.9-5 mCi. In embodiments, the diagnostic radioisotope is present at a concentration of 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4., 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9 or 5 mCi. In embodiments, thediagnostic radioisotope is present at a concentration of 0.8 mCi. In one embodiment, the diagnostic radioisotope is89Zr. IV. Methods of use

[0177] In an aspect is provided a method of treating cancer in a subject in need thereof. The method includes administering an effective amount of an active TGF-beta binding agent as provided herein including embodiments thereof, or an effective amount of a pharmaceutical composition provided herein including embodiments thereof to the subject, thereby treating cancer in the subject. In embodiments, the method further includes prior to administering the active TGF-beta binding agent to the subject administering an external beam therapy to the subject. In embodiments, the method further includes prior to administering the effective amount of a pharmaceutical composition to the subject administering an external beam therapy to the subject.

[0178] In embodiments, the external beam therapy is administered at about 1-10 Gy. In embodiments, the external beam therapy is administered at about 2-10 Gy. In embodiments, the external beam therapy is administered at about 3-10 Gy. In embodiments, the external beam therapy is administered at about 4-10 Gy. In embodiments, the external beam therapy is administered at about 5-10 Gy. In embodiments, the external beam therapy is administered at about 6-10 Gy. In embodiments, the external beam therapy is administered at about 7-10 Gy. In embodiments, the external beam therapy is administered at about 8-10 Gy. In embodiments, the external beam therapy is administered at about 9-10 Gy. In embodiments, the external beam therapy is administered at about 1-9 Gy. In embodiments, the external beam therapy is administered at about 1-8 Gy. In embodiments, the external beam therapy is administered at about 1-7 Gy. In embodiments, the external beam therapy is administered at about 1-6 Gy. In embodiments, the external beam therapy is administered at about 1-5 Gy. In embodiments, the external beam therapy is administered at about 1-4 Gy. In embodiments, the external beam therapy is administered at about 1-3 Gy. In embodiments, the external beam therapy is administered at about 1-2 Gy. In embodiments, the external beam therapy is administered at about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 Gy.

[0179] In embodiments, the external beam therapy is administered at 1-10 Gy. In embodiments, the external beam therapy is administered at 2-10 Gy. In embodiments, the external beam therapy is administered at 3-10 Gy. In embodiments, the external beam therapy is administered at 4-10 Gy. In embodiments, the external beam therapy is administered at 5-10 Gy. In embodiments, the external beam therapy is administered at 6-10 Gy. In embodiments, the external beam therapy is administered at 7-10 Gy. In embodiments, the external beam therapy is administered at 8-10 Gy. In embodiments, the external beam therapy is administered at 9-10 Gy. In embodiments, the external beam therapy is administered at 1-9 Gy. In embodiments, the external beam therapy is administered at 1-8 Gy. In embodiments, the external beam therapy is administered at 1-7 Gy. In embodiments, the external beam therapy is administered at 1-6 Gy. In embodiments, the external beam therapy is administered at 1-5 Gy. In embodiments, the external beam therapy is administered at 1-4 Gy. In embodiments, the external beam therapy is administered at 1-3 Gy. In embodiments, the external beam therapy is administered at 1-2 Gy. In embodiments, the external beam therapy is administered at 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 Gy.

[0180] For any of the embodiments provided herein the external beam administered may include photons, protons or electrons. External beam radiation may include any of the state of the art available procedures, including without limitation, 3-D conformal radiation therapy, intensity- modulated radiation therapy (IMRT), image-guided radiation therapy (IGRT), or tomotherapy.

[0181] The compositions provided herein including embodiments thereof may be used for methods of detecting activated TGF-beta followed by treatment, where a first effective amount of a first active TGF-beta binding agent including a first radioisotope is administered. The first effective amount of that first active TGF-beta binding agent including a first radioisotope may not be a therapeutic amount. The first effective amount of that first active TGF-beta binding agent including a first radioisotope is an amount effective to detect activated and radiation-induced TGF-beta activity after administration of, for example, positron emission tomography (PET) to a patient. The first effective amount may not be a therapeutically effective amount which results in treatment (e.g., neutralization of TGF-beta, inhibition of TGF-beta activity), but may provide for detection of active TGF-beta in the patient. Furthermore, the first effective amount of the first active TGF-beta bindingagent including a first radioisotope is effective to detect active TGF-beta and does not detect latent (inactive) TGF-beta. In embodiments, the first active TGF-beta binding agent includes a diagnostic radioisotope. In embodiments, the diagnostic radioisotope is present at a diagnostic amount. In embodiments, the first effective amount is a diagnostic amount.

[0182] In embodiments, the diagnostic amount is about 0.5-5 mCi. In embodiments, the diagnostic amount is about 0.6-5 mCi. In embodiments, the diagnostic amount is about 0.7-5 mCi. In embodiments, the diagnostic amount is about 0.8-5 mCi. In embodiments, the diagnostic amount is about 0.9-5 mCi. In embodiments, the diagnostic amount is about 1-5 mCi.

[0183] In embodiments, the diagnostic amount is about 1.1-5 mCi. In embodiments, the diagnostic amount is about 1.2-5 mCi. In embodiments, the diagnostic amount is about 1.3-5 mCi. In embodiments, the diagnostic amount is about 1.4-5 mCi. In embodiments, the diagnostic amount is about 1.5-5 mCi. In embodiments, the diagnostic amount is about 1.6-5 mCi. In embodiments, the diagnostic amount is about 1.7-5 mCi. In embodiments the diagnostic amount is about 1.8-5 mCi. In embodiments, the diagnostic amount is about 1.9-5 mCi. In embodiments, the diagnostic amount is about 2-5 mCi.

[0184] In embodiments, the diagnostic amount is about 2.1-5 mCi. In embodiments, the diagnostic amount is about 2.2-5 mCi. In embodiments, the diagnostic amount is about 2.3-5 mCi. In embodiments, the diagnostic amount is about 2.4-5 mCi. In embodiments, the diagnostic amount is about 2.5-5 mCi. In embodiments, the diagnostic amount is about 2.6-5 mCi. In embodiments, the diagnostic amount is about 2.7-5 mCi. In embodiments, the diagnostic amount is about 2.8-5 mCi. In embodiments, the diagnostic amount is about 2.9-5 mCi. In embodiments, the diagnostic amount is about 3-5 mCi.

[0185] In embodiments, the diagnostic amount is about 3.1-5 mCi. In embodiments, the diagnostic amount is about 3.2-5 mCi. In embodiments, the diagnostic amount is about 3.3-5 mCi. In embodiments, the diagnostic amount is about 3.4-5 mCi. In embodiments, the diagnostic amount is about 3.5-5 mCi. In embodiments, the diagnostic amount is about 3.6-5 mCi. In embodiments, the diagnostic amount is about 3.7-5 mCi. In embodiments, the diagnostic amount is about 3.8-5mCi. In embodiments, the diagnostic amount is about 3.9-5 mCi. In embodiments, the diagnostic amount is about 4-5 mCi.

[0186] In embodiments, the diagnostic amount is about 4.1-5 mCi. In embodiments, the diagnostic amount is about 4.2-5 mCi. In embodiments, the diagnostic amount is about 4.3-5 mCi. In embodiments, the diagnostic amount is about 4.4-5 mCi. In embodiments, the diagnostic amount is about 4.5-5 mCi. In embodiments, the diagnostic amount is about 4.6-5 mCi. In embodiments, the diagnostic amount is about 4.7-5 mCi. In embodiments, the diagnostic amount is about 4.8-5 mCi. In embodiments, the diagnostic amount is about 4.9-5 mCi.

[0187] In embodiments, the first effective amount is a diagnostic amount. In embodiments, the diagnostic amount is 0.5-5 mCi. In embodiments, the diagnostic amount is 0.6-5 mCi. In embodiments, the diagnostic amount is abot 0.7-5 mCi. In embodiments, the diagnostic amount is 0.8-5 mCi. In embodiments, the diagnostic amount is 0.9-5 mCi. In embodiments, the diagnostic amount is 1-5 mCi.

[0188] In embodiments, the diagnostic amount is 1.1-5 mCi. In embodiments, the diagnostic amount is 1.2-5 mCi. In embodiments, the diagnostic amount is 1.3-5 mCi. In embodiments, the diagnostic amount is 1.4-5 mCi. In embodiments, the diagnostic amount is 1.5-5 mCi. In embodiments, the diagnostic amount is 1.6-5 mCi. In embodiments, the diagnostic amount is 1.7-5 mCi. In embodiments the diagnostic amount is 1.8-5 mCi. In embodiments, the diagnostic amount is 1.9-5 mCi. In embodiments, the diagnostic amount is 2-5 mCi.

[0189] In embodiments, the diagnostic amount is 2.1-5 mCi. In embodiments, the diagnostic amount is 2.2-5 mCi. In embodiments, the diagnostic amount is 2.3-5 mCi. In embodiments, the diagnostic amount is 2.4-5 mCi. In embodiments, the diagnostic amount is 2.5-5 mCi. In embodiments, the diagnostic amount is 2.6-5 mCi. In embodiments, the diagnostic amount is 2.7-5 mCi. In embodiments, the diagnostic amount is 2.8-5 mCi. In embodiments, the diagnostic amount is 2.9-5 mCi. In embodiments, the diagnostic amount is 3-5 mCi.

[0190] In embodiments, the diagnostic amount is 3.1-5 mCi. In embodiments, the diagnostic amount is 3.2-5 mCi. In embodiments, the diagnostic amount is 3.3-5 mCi. In embodiments, the diagnostic amount is 3.4-5 mCi. In embodiments, the diagnostic amount is 3.5-5 mCi. In embodiments, the diagnostic amount is 3.6-5 mCi. In embodiments, the diagnostic amount is 3.7-5 mCi. In embodiments, the diagnostic amount is 3.8-5 mCi. In embodiments, the diagnostic amount is 3.9-5 mCi. In embodiments, the diagnostic amount is 4-5 mCi.

[0191] In embodiments, the diagnostic amount is 4.1-5 mCi. In embodiments, the diagnostic amount is 4.2-5 mCi. In embodiments, the diagnostic amount is 4.3-5 mCi. In embodiments, the diagnostic amount is 4.4-5 mCi. In embodiments, the diagnostic amount is 4.5-5 mCi. In embodiments, the diagnostic amount is 4.6-5 mCi. In embodiments, the diagnostic amount is 4.7-5 mCi. In embodiments, the diagnostic amount is 4.8-5 mCi. In embodiments, the diagnostic amount is 4.9-5 mCi. In embodiments, the diagnostic amount is 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4., 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9 or 5 mCi. In embodiments, the diagnostic amount is 0.8 mCi. In one embodiment, the diagnostic radioisotope is89Zr.

[0192] The methods provided herein include administering an effective amount of a TGF-beta binding agent including a therapeutic radioisotope. In embodiments, a first active TGF-beta binding agent including a first radioisotope is administered at a first effective amount and a second active TGF-beta binding agent including a second radioisotope is administered at a second effective amount. The second effective amount of the second active TGF-beta binding agent including a second radioisotope may be effective to treat TGF-beta related diseases. In embodiments, the second active TGF-beta binding agent includes a therapeutic radioisotope. In embodiments, the therapeutic radioisotope is present at a therapeutic amount. In embodiments, the second effective amount is a therapeutic amount.

[0193] In embodiments, the therapeutic amount is about 30-300 mCi. In embodiments, the therapeutic amount is about 30-250 mCi. In embodiments, the therapeutic amount is about 30-200 mCi. In embodiments, the therapeutic amount is about 30-150 mCi. In embodiments, thetherapeutic amount is about 30-100 mCi. In embodiments, the therapeutic amount is about 30-50 mCi.

[0194] In embodiments, the therapeutic amount is about 50-300 mCi. In embodiments, the therapeutic amount is about 100-300 mCi. In embodiments, the therapeutic amount is about 150- 300 mCi. In embodiments, the therapeutic amount is about 200-300 mCi. In embodiments, the therapeutic amount is about 250-300 mCi. In embodiments, the therapeutic amount is about 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 mCi.

[0195] In embodiments, the therapeutic amount is 30-300 mCi. In embodiments, the therapeutic amount is 40-300 mCi. In embodiments, the therapeutic amount is 50-300 mCi. In embodiments, the therapeutic amount is 60-300 mCi. In embodiments, the therapeutic amount is 70-300 mCi. In embodiments, the therapeutic amount is 80-300 mCi. In embodiments, the therapeutic amount is 90-300 mCi. In embodiments, the therapeutic amount is 100-300 mCi. In embodiments, the therapeutic amount is 110-300 mCi. In embodiments, the therapeutic amount is 120-300 mCi. In embodiments, the therapeutic amount is 130-300 mCi. In embodiments, the therapeutic amount is 140-300 mCi. In embodiments, the therapeutic amount is 150-300 mCi. In embodiments, the therapeutic amount is 160-300 mCi. In embodiments, the therapeutic amount is 170-300 mCi. In embodiments, the therapeutic amount is 180-300 mCi. In embodiments, the therapeutic amount is 190-300 mCi. In embodiments, the therapeutic amount is 200-300 mCi. In embodiments, the therapeutic amount is 210-300 mCi. In embodiments, the therapeutic amount is 220-300 mCi. In embodiments, the therapeutic amount is 230-300 mCi. In embodiments, the therapeutic amount is 260-300 mCi. In embodiments, the therapeutic amount is 270-300 mCi. In embodiments, the therapeutic amount is 280-300 mCi. In embodiments, the therapeutic amount is 290-300 mCi. In embodiments, the therapeutic amount is 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 mCi. In embodiments, the therapeutic amount is about 200 mCi. In embodiments, the therapeutic amount is 200 mCi. In one embodiment, the therapeutic radioisotope is177Lu.

[0196] In another aspect is provided a method of treating cancer in a subject in need thereof. The method includes (i) administering to the subject an effective amount of a first active TGF-beta binding agent including a first radioisotope; (ii) administering a TGF-beta-activating amount of an external beam therapy to the subject, thereby detecting active TGF-beta in the subject and (iii) administering to the subject an effective amount of a second active TGF-beta binding agent including a second radioisotope, thereby treating cancer in the subject. In embodiments, the TGF- beta-activating amount of an external beam therapy is 1-10 Gy.

[0197] In another aspect is provided a method of treating cancer in a subject in need thereof. The method includes (i) administering to the subject an effective amount of a first active TGF-beta binding agent including a first radioisotope; and (ii) administering to the subject an effective amount of a second active TGF-beta binding agent including a second radioisotope, thereby treating cancer in the subject. In embodiments, the first active TGF-beta binding agent is administered at a first time point and the second active TGF-beta binding agent is administered at a second time point. In embodiments, the first time point precedes the second time point. In embodiments, the method includes after the step (i), administering an external beam therapy. The external beam therapy is administered at a diagnostic amount to detect the first active TGF-beta binding agent. In embodiments, the external beam therapy is not administered at an amount to affect treatment when administered after the step (i).

[0198] In embodiments, the external beam therapy is administered at about 1-10 Gy. In embodiments, the external beam therapy is administered at about 2-10 Gy. In embodiments, the external beam therapy is administered at about 3-10 Gy. In embodiments, the external beam therapy is administered at about 4-10 Gy. In embodiments, the external beam therapy is administered at about 5-10 Gy. In embodiments, the external beam therapy is administered at about 6-10 Gy. In embodiments, the external beam therapy is administered at about 7-10 Gy. In embodiments, the external beam therapy is administered at about 8-10 Gy. In embodiments, the external beam therapy is administered at about 9-10 Gy. In embodiments, the external beam therapy is administered at about 1-9 Gy. In embodiments, the external beam therapy is administered at about 1-8 Gy. In embodiments, the external beam therapy is administered at about 1-7 Gy. In embodiments, theexternal beam therapy is administered at about 1-6 Gy. In embodiments, the external beam therapy is administered at about 1-5 Gy. In embodiments, the external beam therapy is administered at about 1-4 Gy. In embodiments, the external beam therapy is administered at about 1-3 Gy. In embodiments, the external beam therapy is administered at about 1-2 Gy. In embodiments, the external beam therapy is administered at about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 Gy.

[0199] In embodiments, the external beam therapy is administered at 1-10 Gy. In embodiments, the external beam therapy is administered at 2-10 Gy. In embodiments, the external beam therapy is administered at 3-10 Gy. In embodiments, the external beam therapy is administered at 4-10 Gy. In embodiments, the external beam therapy is administered at 5-10 Gy. In embodiments, the external beam therapy is administered at 6-10 Gy. In embodiments, the external beam therapy is administered at 7-10 Gy. In embodiments, the external beam therapy is administered at 8-10 Gy. In embodiments, the external beam therapy is administered at 9-10 Gy. In embodiments, the external beam therapy is administered at 1-9 Gy. In embodiments, the external beam therapy is administered at 1-8 Gy. In embodiments, the external beam therapy is administered at 1-7 Gy. In embodiments, the external beam therapy is administered at 1-6 Gy. In embodiments, the external beam therapy is administered at 1-5 Gy. In embodiments, the external beam therapy is administered at 1-4 Gy. In embodiments, the external beam therapy is administered at 1-3 Gy. In embodiments, the external beam therapy is administered at 1-2 Gy. In embodiments, the external beam therapy is administered at 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 Gy.

[0200] In embodiments, the method includes detecting the first radioisotope after administering the external beam therapy.

[0201] In embodiments, the first active TGF-beta binding agent and the second active TGF-beta binding agent are administered intravenously. In embodiments, the first active TGF-beta binding agent and the second active TGF-beta binding agent are administered intraarterially. In embodiments, the first active TGF-beta binding agent and the second active TGF-beta binding agent are administered intraperitoneal. In embodiments, the first active TGF-beta binding agent and the second active TGF-beta binding agent are administered intraosseously. In embodiments, the firstactive TGF-beta binding agent and the second active TGF-beta binding agent are administered intratumor.

[0202] In embodiments, the first radioisotope is a diagnostic radioisotope and the second radioisotope is a therapeutic radioisotope.

[0203] In embodiments, the first radioisotope and the second radioisotope are independently225Ac,211At,134Ce,64Cu,67Cu,68Ga,123I,124I,131I,111In,132La,133La,177Lu,44Sc,47Sc,153Sm,149Tb,152Tb,161Tb,203Tb,212Tb,227Th,86Y,90Y, or89Zr. In embodiments, the first radioisotope is225Ac,211At,134Ce,64Cu,67Cu,68Ga,123I,124I,131I,111In,132La,133La,177Lu,44Sc,47Sc,153Sm,149Tb,152Tb,161Tb,203Tb,212Tb,227Th,86Y,90Y, or89Zr. In embodiments, the first radioisotope is225Ac. In embodiments, the first radioisotope is211At. In embodiments, the first radioisotope is134Ce. In embodiments, the first radioisotope is64Cu. In embodiments, the first radioisotope is67Cu. In embodiments, the first radioisotope is68Ga. In embodiments, the first radioisotope is123I. In embodiments, the first radioisotope is124I. In embodiments, the first radioisotope is131I. In embodiments, the first radioisotope is111In. In embodiments, the first radioisotope is132La. In embodiments, the first radioisotope is133La. In embodiments, the first radioisotope is177Lu. In embodiments, the first radioisotope is44Sc. In embodiments, the first radioisotope is47Sc. In embodiments, the first radioisotope is153Sm. In embodiments, the first radioisotope is149Tb. In embodiments, the first radioisotope is152Tb. In embodiments, the first radioisotope is161Tb. In embodiments, the first radioisotope is203Tb. In embodiments, the first radioisotope is212Tb. In embodiments, the first radioisotope is227Th. In embodiments, the first radioisotope is86Y. In embodiments, the first radioisotope is90Y. In embodiments, the first radioisotope is89Zr.

[0204] In embodiments, the second radioisotope is225Ac,211At,134Ce,64Cu,67Cu,68Ga,123I,124I,131I,111In,132La,133La,177Lu,44Sc,47Sc,153Sm,149Tb,152Tb,161Tb,203Tb,212Tb,227Th,86Y,90Y, or89Zr. In embodiments, the second radioisotope is225Ac. In embodiments, the second radioisotope is211At. In embodiments, the second radioisotope is134Ce. In embodiments, the second radioisotope is64Cu. In embodiments, the second radioisotope is67Cu. In embodiments, the second radioisotope is68Ga. In embodiments, the second radioisotope is123I. In embodiments, the second radioisotopeis124I. In embodiments, the second radioisotope is131I. In embodiments, the second radioisotope is111In. In embodiments, the second radioisotope is132La. In embodiments, the second radioisotope is133La. In embodiments, the second radioisotope is177Lu. In embodiments, the second radioisotope is44Sc. In embodiments, the second radioisotope is47Sc. In embodiments, the second radioisotope is153Sm. In embodiments, the second radioisotope is149Tb. In embodiments, the second radioisotope is152Tb. In embodiments, the second radioisotope is161Tb. In embodiments, the second radioisotope is203Tb. In embodiments, the second radioisotope is212Tb. In embodiments, the second radioisotope is227Th. In embodiments, the second radioisotope is86Y. In embodiments, the second radioisotope is90Y. In embodiments, the second radioisotope is89Zr.

[0205] In embodiments, the first radioisotope and the second radioisotope are independently131I,177Lu,47Sc,161Tb,67Cu,153Sm,64Cu, or149Tb.

[0206] In embodiments, the first radioisotope and the second radioisotope are of the same element. In embodiments, the first radioisotope is64Cu and the second radioisotope is67Cu, the first radioisotope is44Sc and the second radioisotope is47Sc, the first radioisotope is86Y and the second radioisotope is90Y, the first radioisotope is152Tb or161Tb and the second radioisotope is149Tb, the first radioisotope is203Pb and the second radioisotope is212Pb, or the first radioisotope is123I or124I and the second radioisotope is131I.

[0207] In embodiments, the first radioisotope is64Cu and the second radioisotope is67Cu. In embodiments, the first radioisotope is44Sc and the second radioisotope is47Sc. In embodiments, the first radioisotope is86Y and the second radioisotope is90Y. In embodiments, the first radioisotope is152Tb or161Tb and the second radioisotope is149Tb. In embodiments, the first radioisotope is152Tb and the second radioisotope is149Tb. In embodiments, the first radioisotope is161Tb and the second radioisotope is149Tb. In embodiments, the first radioisotope is203Pb and the second radioisotope is212Pb. In embodiments, the first radioisotope is123I or124I and the second radioisotope is131I. In embodiments, the first radioisotope is123I and the second radioisotope is131I. In embodiments, the first radioisotope is124I and the second radioisotope is131I.

[0208] In embodiments, the first radioisotope is68Ga,89Zr,123I,124I,111In,132La,133La, or134Ce and the second radioisotope is177Lu,227Th,211At, or225Ac. In embodiments, the first radioisotope is68Ga, and the second radioisotope is177Lu,227Th,211At, or225Ac. In embodiments, the first radioisotope is89Zr and the second radioisotope is177Lu,227Th,211At, or225Ac. In embodiments, the first radioisotope is123I and the second radioisotope is177Lu,227Th,211At, or225Ac. In embodiments, the first radioisotope is124I and the second radioisotope is177Lu,227Th,211At, or225Ac. In embodiments, the first radioisotope is111In and the second radioisotope is177Lu,227Th,211At, or225Ac. In embodiments, the first radioisotope is132La and the second radioisotope is177Lu,227Th,211At, or225Ac. In embodiments, the first radioisotope is133La and the second radioisotope is177Lu,227Th,211At, or225Ac. In embodiments, the first radioisotope is134Ce and the second radioisotope is177Lu,227Th,211At, or225Ac.

[0209] In embodiments, the first radioisotope is68Ga,89Zr,123I,124I,111In,132La,133La, or134Ce and the second radioisotope is177Lu. In embodiments, the first radioisotope is68Ga,89Zr,123I,124I,111In,132La,133La, or134Ce and the second radioisotope is227Th. In embodiments, the first radioisotope is68Ga,89Zr,123I,124I,111In,132La,133La, or134Ce and the second radioisotope is211At. In embodiments, the first radioisotope is68Ga,89Zr,123I,124I,111In,132La,133La, or134Ce and the second radioisotope is225Ac.

[0210] In embodiments, the first radioisotope is68Ga, and the second radioisotope is177Lu. In embodiments, the first radioisotope is and the second radioisotope is227Th. In embodiments, the first radioisotope is123I and the second radioisotope is211At. In embodiments, the first radioisotope is124I and the second radioisotope is211At. In embodiments, the first radioisotope is68Ga, and the second radioisotope is225Ac. In embodiments, the first radioisotope is89Zr, and the second radioisotope is225Ac. In embodiments, the first radioisotope is111In, and the second radioisotope is225Ac. In embodiments, the first radioisotope is132La, and the second radioisotope is225Ac. In embodiments, the first radioisotope is133La, and the second radioisotope is225Ac. In embodiments, the first radioisotope is134Ce, and the second radioisotope is225Ac.

[0211] In embodiments, the first radioisotope is89Zr and wherein the second radioisotope is177Lu.

[0212] In embodiments, the method includes detecting the second radioisotope. In embodiments, the detecting includes administering Positron Emission Tomography (PET) or Single Photon Emission Computed Tomography (SPECT) to the subject. In embodiments, the first radioisotope is administered at 1-20 mCi and the second radioisotope is administered at 0.5-250 mCi.

[0213] In embodiments, the first radioisotope is administered at 1-20 mCi. In embodiments, the first radioisotope is administered at 1-19 mCi. In embodiments, the first radioisotope is administered at 1-18 mCi. In embodiments, the first radioisotope is administered at 1-17 mCi. In embodiments, the first radioisotope is administered at 1-16 mCi. In embodiments, the first radioisotope is administered at 1-15 mCi. In embodiments, the first radioisotope is administered at 1-14 mCi. In embodiments, the first radioisotope is administered at 1-12 mCi. In embodiments, the first radioisotope is administered at 1-11 mCi. In embodiments, the first radioisotope is administered at 1-10 mCi. In embodiments, the first radioisotope is administered at 1-9 mCi. In embodiments, the first radioisotope is administered at 1-8 mCi. In embodiments, the first radioisotope is administered at 1-7 mCi. In embodiments, the first radioisotope is administered at 1- 6 mCi. In embodiments, the first radioisotope is administered at 1-5 mCi. In embodiments, the first radioisotope is administered at 1-4 mCi. In embodiments, the first radioisotope is administered at 1- 3 mCi. In embodiments, the first radioisotope is administered at 1-2 mCi.

[0214] In embodiments, the first radioisotope is administered at 2-20 mCi. In embodiments, the first radioisotope is administered at 3-20 mCi. In embodiments, the first radioisotope is administered at 4-20 mCi. In embodiments, the first radioisotope is administered at 5-20 mCi. In embodiments, the first radioisotope is administered at 6-20 mCi. In embodiments, the first radioisotope is administered at 7-20 mCi. In embodiments, the first radioisotope is administered at 8-20 mCi. In embodiments, the first radioisotope is administered at 9-20 mCi. In embodiments, the first radioisotope is administered at 10-20 mCi. In embodiments, the first radioisotope is administered at 11-20 mCi. In embodiments, the first radioisotope is administered at 12-20 mCi. In embodiments, the first radioisotope is administered at 13-20 mCi. In embodiments, the first radioisotope is administered at 14-20 mCi. In embodiments, the first radioisotope is administered at 15-20 mCi. In embodiments, the first radioisotope is administered at 16-20 mCi. In embodiments,the first radioisotope is administered at 17-20 mCi. In embodiments, the first radioisotope is administered at 18-20 mCi. In embodiments, the first radioisotope is administered at 19-20 mCi. In embodiments, the first radioisotope is administered at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 mCi.

[0215] In embodiments, the first radioisotope is administered at about 1-20 mCi. In embodiments, the first radioisotope is administered at about 1-19 mCi. In embodiments, the first radioisotope is administered at about 1-18 mCi. In embodiments, the first radioisotope is administered at about 1- 17 mCi. In embodiments, the first radioisotope is administered at about 1-16 mCi. In embodiments, the first radioisotope is administered at about 1-15 mCi. In embodiments, the first radioisotope is administered at about 1-14 mCi. In embodiments, the first radioisotope is administered at about 1- 12 mCi. In embodiments, the first radioisotope is administered at about 1-11 mCi. In embodiments, the first radioisotope is administered at about 1-10 mCi. In embodiments, the first radioisotope is administered at about 1-9 mCi. In embodiments, the first radioisotope is administered at about 1-8 mCi. In embodiments, the first radioisotope is administered at about 1-7 mCi. In embodiments, the first radioisotope is administered at about 1-6 mCi. In embodiments, the first radioisotope is administered at about 1-5 mCi. In embodiments, the first radioisotope is administered at about 1-4 mCi. In embodiments, the first radioisotope is administered at about 1-3 mCi. In embodiments, the first radioisotope is administered at about 1-2 mCi.

[0216] In embodiments, the first radioisotope is administered at about 2-20 mCi. In embodiments, the first radioisotope is administered at about 3-20 mCi. In embodiments, the first radioisotope is administered at about 4-20 mCi. In embodiments, the first radioisotope is administered at about 5- 20 mCi. In embodiments, the first radioisotope is administered at about 6-20 mCi. In embodiments, the first radioisotope is administered at about 7-20 mCi. In embodiments, the first radioisotope is administered at about 8-20 mCi. In embodiments, the first radioisotope is administered at about 9- 20 mCi. In embodiments, the first radioisotope is administered at about 10-20 mCi. In embodiments, the first radioisotope is administered at about 11-20 mCi. In embodiments, the first radioisotope is administered at about 12-20 mCi. In embodiments, the first radioisotope is administered at about 13-20 mCi. In embodiments, the first radioisotope is administered at about14-20 mCi. In embodiments, the first radioisotope is administered at about 15-20 mCi. In embodiments, the first radioisotope is administered at about 16-20 mCi. In embodiments, the first radioisotope is administered at about 17-20 mCi. In embodiments, the first radioisotope is administered at about 18-20 mCi. In embodiments, the first radioisotope is administered at about 19-20 mCi. In embodiments, the first radioisotope is administered at about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 mCi.

[0217] In embodiments, the second radioisotope is administered at 1-250 mCi. In embodiments, the second radioisotope is administered at 5-250 mCi. In embodiments, the second radioisotope is administered at 10-250 mCi. In embodiments, the second radioisotope is administered at 20-250 mCi. In embodiments, the second radioisotope is administered at 30-250 mCi. In embodiments, the second radioisotope is administered at 40-250 mCi. In embodiments, the second radioisotope is administered at 50-250 mCi. In embodiments, the second radioisotope is administered at 60-250 mCi. In embodiments, the second radioisotope is administered at 70-250 mCi. In embodiments, the second radioisotope is administered at 80-250 mCi. In embodiments, the second radioisotope is administered at 90-250 mCi. In embodiments, the second radioisotope is administered at 100-250 mCi. In embodiments, the second radioisotope is administered at 110-250 mCi. In embodiments, the second radioisotope is administered at 120-250 mCi. In embodiments, the second radioisotope is administered at 130-250 mCi. In embodiments, the second radioisotope is administered at 140- 250 mCi. In embodiments, the second radioisotope is administered at 150-250 mCi. In embodiments, the second radioisotope is administered at 160-250 mCi. In embodiments, the second radioisotope is administered at 170-250 mCi. In embodiments, the second radioisotope is administered at 180-250 mCi. In embodiments, the second radioisotope is administered at 190-250 mCi. In embodiments, the second radioisotope is administered at 200-250 mCi. In embodiments, the second radioisotope is administered at 210-250 mCi. In embodiments, the second radioisotope is administered at 220-250 mCi. In embodiments, the second radioisotope is administered at 230- 250 mCi. In embodiments, the second radioisotope is administered at 240-250 mCi. In embodiments, the second radioisotope is administered at 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240 or 250 mCi.

[0218] In embodiments, the second radioisotope is administered at about 1-250 mCi. In embodiments, the second radioisotope is administered at about 5-250 mCi. In embodiments, the second radioisotope is administered at about 10-250 mCi. In embodiments, the second radioisotope is administered at about 20-250 mCi. In embodiments, the second radioisotope is administered at about 30-250 mCi. In embodiments, the second radioisotope is administered at about 40-250 mCi. In embodiments, the second radioisotope is administered at about 50-250 mCi. In embodiments, the second radioisotope is administered at about 60-250 mCi. In embodiments, the second radioisotope is administered at about 70-250 mCi. In embodiments, the second radioisotope is administered at about 80-250 mCi. In embodiments, the second radioisotope is administered at about 90-250 mCi. In embodiments, the second radioisotope is administered at about 100-250 mCi. In embodiments, the second radioisotope is administered at about 110-250 mCi. In embodiments, the second radioisotope is administered at about 120-250 mCi. In embodiments, the second radioisotope is administered at about 130-250 mCi. In embodiments, the second radioisotope is administered at about 140-250 mCi. In embodiments, the second radioisotope is administered at about 150-250 mCi. In embodiments, the second radioisotope is administered at about 160-250 mCi. In embodiments, the second radioisotope is administered at about 170-250 mCi. In embodiments, the second radioisotope is administered at about 180-250 mCi. In embodiments, the second radioisotope is administered at about 190-250 mCi. In embodiments, the second radioisotope is administered at about 200-250 mCi. In embodiments, the second radioisotope is administered at about 210-250 mCi. In embodiments, the second radioisotope is administered at about 220-250 mCi. In embodiments, the second radioisotope is administered at about 230-250 mCi. In embodiments, the second radioisotope is administered at about 240-250 mCi. In embodiments, the second radioisotope is administered at about 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240 or 250 mCi.

[0219] In embodiments, the first radioisotope is administered at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 mCi and the second radioisotope is administered at 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240 or 250 mCi. In embodiments, the first radioisotope is administered at about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 mCi and the second radioisotope is administered at about 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240 or 250 mCi.

[0220] In embodiments, the first radioisotope is administered at 0.8 mCi and the second radioisotope is administered at 190 uCi. In embodiments, the first radioisotope is administered at about 0.8 mCi and the second radioisotope is administered at about 190 uCi.

[0221] In embodiments, the first radioisotope is administered at 1-5 mCi and the second radioisotope is administered at 200 mCi. In embodiments, the first radioisotope is administered at 1 mCi and the second radioisotope is administered at 200 mCi. In embodiments, the first radioisotope is administered at 2 mCi and the second radioisotope is administered at 200 mCi. In embodiments, the first radioisotope is administered at 3 mCi and the second radioisotope is administered at 200 mCi. In embodiments, the first radioisotope is administered at 4 mCi and the second radioisotope is administered at 200 mCi. In embodiments, the first radioisotope is administered at 5 mCi and the second radioisotope is administered at 200 mCi.

[0222] In embodiments, the second radioisotope is administered every 6-8 weeks for 1-8 cycles. In embodiments, the second radioisotope is administered every 6-8 weeks for 1 cycle. In embodiments, the second radioisotope is administered every 6-8 weeks for 2 cycles. In embodiments, the second radioisotope is administered every 6-8 weeks for 3 cycles. In embodiments, the second radioisotope is administered every 6-8 weeks for 4 cycles. In embodiments, the second radioisotope is administered every 6-8 weeks for 5 cycles. In embodiments, the second radioisotope is administered every 6-8 weeks for 6 cycles. In embodiments, the second radioisotope is administered every 6-8 weeks for 7 cycles. In embodiments, the second radioisotope is administered every 6-8 weeks for 8 cycles.

[0223] In embodiments, the second radioisotope is administered every 6 weeks for 1-8 cycles. In embodiments, the second radioisotope is administered every 6 weeks for 1 cycle. In embodiments, the second radioisotope is administered every 6 weeks for 2 cycles. In embodiments, the second radioisotope is administered every 6 weeks for 3 cycles. In embodiments, the second radioisotope isadministered every 6 weeks for 4 cycles. In embodiments, the second radioisotope is administered every 6 weeks for 5 cycles. In embodiments, the second radioisotope is administered every 6 weeks for 6 cycles. In embodiments, the second radioisotope is administered every 6 weeks for 7 cycles. In embodiments, the second radioisotope is administered every 6 weeks for 8 cycles.

[0224] In embodiments, the second radioisotope is administered every 7 weeks for 1-8 cycles. In embodiments, the second radioisotope is administered every 7 weeks for 1 cycle. In embodiments, the second radioisotope is administered every 7 weeks for 2 cycles. In embodiments, the second radioisotope is administered every 7 weeks for 3 cycles. In embodiments, the second radioisotope is administered every 7 weeks for 4 cycles. In embodiments, the second radioisotope is administered every 7 weeks for 5 cycles. In embodiments, the second radioisotope is administered every 7 weeks for 6 cycles. In embodiments, the second radioisotope is administered every 7 weeks for 7 cycles. In embodiments, the second radioisotope is administered every 7 weeks for 8 cycles.

[0225] In embodiments, the second radioisotope is administered every 8 weeks for 1-8 cycles. In embodiments, the second radioisotope is administered every 8 weeks for 1 cycle. In embodiments, the second radioisotope is administered every 8 weeks for 2 cycles. In embodiments, the second radioisotope is administered every 8 weeks for 3 cycles. In embodiments, the second radioisotope is administered every 8 weeks for 4 cycles. In embodiments, the second radioisotope is administered every 8 weeks for 5 cycles. In embodiments, the second radioisotope is administered every 8 weeks for 6 cycles. In embodiments, the second radioisotope is administered every 8 weeks for 7 cycles. In embodiments, the second radioisotope is administered every 8 weeks for 8 cycles.

[0226] In embodiments, the cancer is a carcinoma, a sarcoma or a metastatic cancer. In embodiments, the cancer is a carcinoma. In embodiments, the cancer is a sarcoma. In embodiments, the cancer is a metastatic cancer.

[0227] In embodiments, the cancer is breast cancer, lung cancer, prostate cancer, glioblastoma, or metastatic cancer. In embodiments, the cancer is breast cancer. . In embodiments, the cancer is lung cancer. In embodiments, the cancer is prostate cancer. In embodiments, the cancer is glioblastoma. In embodiments, the cancer is metastatic cancer. In embodiments, the cancer is brain metastasis.

[0228] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes. EXAMPLES Example 1: Introduction

[0229] Radiation therapy increases survival in primary breast cancer patients and is used to control brain metastases, which are on the rise as more successful treatment leads to longer-lived breast cancer survivors. Radiopharmaceutical therapy (RPT), which delivers the benefit of radiation as a therapeutic by labeling antibodies or peptides with radioactive isotopes, is emerging as a safe and effective therapeutic approach in several cancers, notably metastatic prostate cancer, yet no RPT is available for breast cancer. A radioligand targeted to breast cancer could control metastatic disease and spare normal tissue, particularly the brain, an outcome that would revolutionize treatment and provide patients with options in this life-threatening scenario.

[0230] TGF-beta is a widely distributed protein whose biological activity is controlled by extracellular processing from a latent to an active form. Whereas activation is exquisitely controlled in normal tissues, TGF-beta activity is highly dysregulated in the tumor microenvironment. We radiolabeled a clinically validated TGF-beta neutralizing antibody, fresolimumab, with89Zr for positron emission tomography (PET) imaging to demonstrate that TGF-beta activity provides a favorable tumor to tissue differential in breast cancer models. Our PET imaging study of breast cancer primary and metastatic disease models demonstrated that89Zr-fresolimumab specifically detects engineered, physiologically activated and radiation-induced TGF-beta activation. When the targeting moiety is labeled with an imaging isotope, the combination of non-invasive patient specific visualization of the target and treatment is called a theranostic.

[0231] Targeted radiopharmaceutical therapy (RPT) extends the benefit of radiation with several improvements: first, radiation is delivered to sites of disease by attaching a radioactive isotope to anantibody or peptide expressed in the cancer, thus limiting dose to normal tissue. Second, it is suitable for metastatic breast cancer, particularly brain metastases, because multiple sites are treated simultaneously. Third, the manner and type of radiation delivery can have better biological effects. Thus, an RPT could revolutionize outcomes for women with this life-threatening diagnosis and provide a substantial benefit over EBT (Fig. 1).

[0232] The potential of RPT is demonstrated in a recent phase III study of metastatic castration- resistant prostate cancer patients treated with177Lu-labeled antibody prostate-specific membrane antigen (PSMA) RPT that resulted in 35% improvement in overall survival (9). Unlike an RPT that targets isotope to cells overexpressing a specific protein in a particular cancer (as described above), we target the product of a process, activation, that is dysregulated in cancer. We develop an RPT targeted to TGFβ based on the idea that TGFβ activation is tightly regulated in normal tissues but highly dysregulated in cancer. We radiolabeled a TGFβ neutralizing antibody, fresolimumab, with89Zr to non-invasively assess TGFβ activity by positron emission tomography (PET) imaging. We showed functional PET imaging of TGFβ across breast, lung, and glioblastoma tumor models and that89Zr-labeled antibody specifically detected TGFβ activity, whether engineered, physiological, or radiation-induced (See also, 10). Based on these data we set out to develop a breast cancer theranostic, in which non-invasive PET imaging with a89Zr-labeled TGFβ antibody will identify patients who will likely benefit from treatment with177Lu-labeled TGFβ antibody. Example 2: Radiopharmaceutical therapy (RPT)

[0233] RPT is emerging as a safe and effective treatment approach, providing the benefit of therapeutic radiation through delivery of radioactive isotopes directly to the tumor and metastases (12). RPT delivers radioactive isotopes to the tumor bed resulting in a cumulative dose that kills cancer cells, over single or multiple cycles. Imageable emissions from the therapeutic isotope or exchanging an imaging isotope for the therapeutic isotope provides a non-invasive visualization of the therapeutic agent pharmacokinetics with PET or SPECT imaging, a so-called theranostic. Targeting with isotope-labeled antibodies or peptides also minimizes unwanted dose deposition to organs at risk (OAR), such as the kidneys, salivary glands, bladder and bone marrow. RPT using a tumor-targeted molecule linked to a therapeutic beta- or alpha-emitting radioactive nuclide is well-established in specific clinical settings (13, 14). RPT is currently approved for treatment of thyroid (131I), neuroendocrine (177Lu-Dotatate), neuroblastoma (131I-MIBG), lymphomas (90Y-anti-CD20,131I-anti-CD20), and prostate cancers (223Ra,177Lu-PSMA) (12). Notable examples of systemically administered177Lu-labeled antibodies include targeting somatostatin receptors for neuroendocrine tumors of the digestive tract and prostate-specific membrane antigen (PSMA) in metastatic prostate cancer. A recent phase III study of patients with metastatic castration-resistant prostate cancer treated with177Lu-PSMA RPT resulted in ~35% improvement in overall survival (9).

[0234] Provided herewith is a novel RPT agent for breast cancer treatment by targeting transforming growth factor β (TGFβ), whose activity is ubiquitously elevated in breast cancer. The key to our approach lies in the fundamental regulation of TGFβ activity. Although TGFβ is expressed by all cells and its receptors are present on all cells, it is secreted in a latent form that is sequestered within the extracellular matrix. Importantly, the control of TGFβ biology is via a highly regulated process, in which the TGFβ latent complex is modified extracellularly to release TGFβ, which binds to ubiquitous receptors. Hence, the key step to TGFβ activity is the release of TGFβ from this latent complex, the process referred to as activation (reviewed in (11, 15). Activation of latent TGFβ occurs by several different mechanisms that include proteolytic cleavage of latency associated peptide, conformational distortion of the latent complex by integrin binding, interaction with the matricellular protein thrombospondin-1, and oxidation of a methionine residue in latency associated peptide by reactive oxygen species.

[0235] In normal tissue, TGFβ activity is exquisitely controlled, and one of its main effects is to potently suppress epithelial proliferation. However, cancer cells evade this control by various molecular overrides that rarely abrogate ligand completely, but rather maintain its production and often signaling capacity (16). This paradoxical activity - in which TGFβ switches from a tumor suppressor to a tumor promoter - drives malignant phenotypes and metastasis (15, 17, 18). Indeed, TGFβ is activated by cancer-associated fibroblasts, tumor-associated macrophages, and cancer cells, contributing to high levels of active TGFβ in many cancers. This exuberant TGFβ activation affects the composition of the tumor microenvironment (TME), promotes angiogenesis, suppresses tumor immunity, and specifically promotes resistance to DNA damage (11, 19, 20). Hence, pronouncedTGFβ activity, inferred from TGFβ protein levels or gene signatures among human cancers (21, 22), is associated with poor prognosis (23-25).

[0236] TGFβ gene signatures are widely used means of assessing TGFβ activity in cancer because of transcriptomic data are readily available. High expression of TGFβ targets indicates obligate active TGFβ and signaling competency. We have identified 50 genes induced by chronic TGFβ exposure and 36 genes suppressed by TGFβ that together report TGFβ activity (22, 25, 26). Notably, we found patients whose cancers exhibit robust TGFβ activity have significantly worse outcomes in response to standard chemoradiation therapy (22, 25). Moreover, TGFβ activity is further induced by radiation, chemotherapy, and immunotherapies where it opposes therapeutic benefit by regulating the DNA damage response, invasiveness, anti-tumor immunity, and angiogenesis (reviewed in (20, 27)). Indeed, inhibiting TGFβ in preclinical models of breast and other cancers increases radiation sensitivity in vitro and tumor control in vivo (26, 28-31). TGFβ inhibition also promotes response to immunotherapy (28, 32). The high value of TGFβ as a therapeutic target is evidenced by more than 20 biologics or small molecules to inhibit TGFβ that are being tested in more than 30 clinical trials (18, 33).

[0237] The goal of personalized medicine is to understand which therapy benefits which specific patient and the key to choosing the most effective therapy for a given patient is understanding the intrinsic sensitivity to cancer therapies and the mechanistic basis for differential outcomes. Example 3:89Zr-freso discriminates between active and latent TGFβ

[0238] Fresolimumab is a fully human monoclonal antibody that effectively neutralizes the three isoforms of both human and mouse TGFβ (there is only 1 amino acid difference between species). The safety of fresolimumab was tested in a Phase I / II trial in patients with advanced malignant melanoma and renal cell carcinoma in a multi-center trial (34) and in combination with radiation for metastatic breast cancer (35). In addition, PET imaging of89Zr-fresolimumab in glioblastoma patients has shown to detect TGFβ activity (36-38).

[0239] To validate that TGFβ activity was specifically detected, we labeled lyophilized, GMP grade fresolimumab with89Zr using DFO conjugation (Fig. 2A).89Zr-fresolimumab (freso,hereafter) yield, radiochemical purity and biological activity were determined after conjugation and radiolabeling (Fig. 2B). PET imaging of mouse bearing a brain tumor demonstrates89Zr-freso specificity of compared to PBS injection (Fig. 2C) (10). To determine if PET imaging of89Zr-freso can effectively discriminate between active and latent TGFβ, we established tumors from human tumor cell line, B9, which is stably transfected with a construct to express wild-type latent TGFβ1, and isogenic C19 cell line, which expressed constitutively active TGFβ1 (39, 40). Mice bearing contralateral flank tumors of each cell line were imaged by PET and computerized tomography (CT). PET imaging of89Zr-freso showed increased signal in the C19 tumors expressing the constitutively active form of TGFβ1, compared to the contralateral B9 tumors, which had minimal PET signal (Fig. 3A). Analysis of the percentage of injected dose (%ID / g) present in tumors 96 h after injection confirmed a significantly greater uptake of89Zr-freso in C19 tumors (Fig. 3B). Activation of TGFβ results in signaling via receptor-mediated phosphorylation of SMAD2, which ultimately mediates expression of TGFβ gene targets (41). To validate differential TGFβ activation, we used tumor sections for immunofluorescence (IF) staining of active TGFβ and phosphorylated SMAD2. Consistent with constitutive TGFβ activation, immunodetection of TGFβ was 5-fold greater in C19 compared to B9 and the percentage of phosphorylated SMAD2p positive cells increased from 2% to 15% (p<0.05). Next, we assessed the discrimination between physiological activation of TGFβ in Lewis lung cell carcinoma (LLC) flank tumors. A physiological mechanism of TGFβ activation at the cell surface involves integrins that bind RGD sequences in TGFβ’s latency associated protein (42). Parental LLC tumors do not express the αvβ8 integrin were compared to stably transfected β8 LLC tumors that express αvβ8 (43). Mice bearing contralateral LLC tumors and β8 LLC tumors were imaged using PET 89Zr-freso (Fig. 3C). Significantly more radioactivity was measured in β8 LLC flank tumors (p=0.004, paired t-test) relative to the minimal uptake of the contralateral parental LLC tumor (Fig. 3D). Tumor immunostaining confirmed significantly greater active TGFβ (p<0.05) as well as an increased frequency of phosphorylated SMAD2 cells from 2% to 14% (p<0.05).Example 4:89Zr-freso detects radiation-induced TGFβ activity

[0240] Radiation elicits rapid TGFβ activation via a redox mechanism (44), which promotes sustained TGFβ activity that is a significant modulator radiation response (reviewed in (11)). We next sought to determine if89Zr-freso PET imaging could detect increased TGFβ activity in irradiated tumors. Mice were injected subcutaneously with 4T1-BrA cells in both flanks to establish bilateral tumors. The right tumor was irradiated with 15 Gy using a small animal radiation research platform and mice were injected with89Zr-freso immediately thereafter. Mice were imaged with PET / CT 96 h later. Irradiated tumors displayed significantly increased signal relative to non- irradiated contralateral tumors (Fig. 3E). Radioactivity was subsequently quantified ex vivo by autoradiography or gamma counting. Autoradiograms of tumor sections confirmed that the irradiated tumors were more radioactive than the contralateral non-irradiated tumors and irradiated tumors contained significantly more radioactive isotope compared to contralateral tumors. The specificity of89Zr-freso was confirmed by injecting 4T1-BrA tumor bearing mice with cold fresolimumab prior to radioactive-fresolimumab. The activity per gram of tissue was lower in mice that were pre-treated with cold fresolimumab (blocked) compared to non-blocked. To confirm biological activity, tumor sections were immunostained for TGFβ and phosphorylated SMAD2 positive cells in irradiated compared to non-irradiated tumors (Fig. 3F). The percentage of phosphorylated SMAD2 positive cells was significantly increased (Fig. 3G). These in situ markers thus confirm the expected biological consequences of radiation-induced TGFβ activation, further validating functional imaging of active TGFβ by89Zr-freso PET in multiple tumor models (10). Example 5: Imaging TGFβ activity in murine intracranial tumors

[0241] Brain tumors are highly aggressive and retrospective analysis of TGFβ activity suggests that it might be important in patient response but is difficult to ascertain prospectively. Due to the intracranial location and the rapid course of the disease, finding non-invasive means to detect TGFβ activity could be of great utility. Here, we used two orthotopic glioblastoma models (SB28 and GL261) and an intracranial breast cancer brain metastasis model (4T1-BrA). Mice were imaged with18F-FDG PET / CT and bioluminescence imaging (BLI) of a luciferase reporter was used to confirm intracranial SB28 (Fig. 4A), GL261 (Fig. 4B) and 4T1-BrA (not shown) tumors. Micereceiving sham surgery were injected intracranially with PBS and were used as control. All mice were subsequently imaged with89Zr-freso PET, which detected TGFβ activity in the tumor-bearing brains but not in control mice 120 h after injection (p=0.006, t-test; Fig. 4C). Signal quantification in SB28 tumor 96 h after injection demonstrated significant increase in tumor-bearing brains compared to sham surgery (Fig. 4D) (10). In contrast, there is minimal accumulation in major organs (Fig. 4E).

[0242] Imaging invasive brain metastases can be challenging due to the simultaneous presence of multiple extra-cranial metastasis. To model this clinical presentation, we injected mice with intracranial and subcutaneous 4T1BrA tumors and imaged with89Zr-freso PET / CT. PET signal was detected in both brain and flank tumors indicating that functional monitoring of multiple sites, such as metastatic disease is possible. Together these data show that functional imaging of active TGFβ detects tumors with different activation levels, in different organs, which suggest that TGFβ targeted RTP is a viable strategy to increase the utility of RTP for cancer therapy. Additionally,89Zr-freso demonstrated radiation-induced TGFβ activation, which is a potential means of increasing its efficacy. Hence, PET imaging of freso provides proof-of-principle that patients can undergo non- invasive imaging to enrich or select for those whose tumor would likely be responsive to TGFβ RPT. Example 6

[0243] We reported PET imaging evidence of different levels of TGFβ activity between human renal sarcoma xenografts B9 vs C19, parent LLC vs β8 LLC, and sham vs irradiated SB28 and GL261 glioblastoma and 4T1 breast cancer (10). Notably, extended PET imaging of orthotopic 4T1 breast tumors shows that the labeled antibody is specifically retained (Fig. 5). This data is consistent with the primary premise that dysregulated TGFβ activation in tumors provides the targeting differential from normal tissue and that the process of activation is continuous.

[0244] We conducted a pilot experiment in which177Lu-freso was administered to mice bearing 4T1 tumors randomized at day 9 post-implantation to receive a 2 Gy EBT priming dose (n =4) or none (n = 3). All mice were injected ~0.8 mCi of177Lu-freso. SPECT / CT imaging conducted onday 14 showed tumor uptake, marked T in the representative images (Fig. 6A). As is common for labeled antibodies, the liver also shows uptake due to clearance at this early (4 days) timepoint. The estimated tumor uptake (% injected dose (ID) / gram) was calculated from SPECT / CT. There is a trend for increased tumor uptake in the EBT primed tumors (n=4) compared to non-primed tumors (n=3) (Fig. 6B).

[0245] Notably, regardless of priming177Lu-freso treated mice showed significant (p<0.0001) tumor control compared to controls (black) (Fig. 6C). For comparison, we show an independent experiment using 10 Gy EBT treated 4T1 tumors (Fig. 6D). The calculated doubling time for177Lu- freso treated tumors was 25% longer, 18.5 days, for RPT compared to 14 days for EBT. The tumor uptake and similar degree of tumor growth control indicate the feasibility of177Lu-freso delivery of isotope to treat metastatic breast cancer. Example 7: Radiolabel fresolimumab with89Zr for PET imaging and177Lu for RPT to ascertain TGFβ activity differentials and therapeutic potential respectively in breast cancer models

[0246] We will use89Zr-freso PET to determine relative levels of endogenous TGFβ activity in models of metastatic disease, orthotopic 4T1 and TS / A that frequently develop lung metastases, and brain-adapted TS / A-BrA and 4T1-BrA intracranial tumors, with and without a EBT priming dose of 2 Gy. Tumor control will be correlated with actual dose reconstructed from quantitative PET / CT and SPECT / CT imaging and a novel ex vivo method of quantifying uptake heterogeneity. The efficacy and toxicities of EBT is based on radiation quality, dose rate and total dose to the volume (45), whose effects are the result of cellular and tissue mechanisms of response, classically described as the 5 R’s of radiobiology (46). In contrast, RPT uses a fixed activity or activity per unit body weight approach in which the dose delivered is integrated over time in the tumor volume or OAR to inform mechanistic comparisons between patients or isotopes (47). We will use the current technology, quantitative PET combined with x-ray computerized tomography (PET / CT) or single photon emission computed tomography (SPECT / CT) and multiple time point imaging of RPT distribution, to calculate total dose and dose rate for comparison to EBT. A technical goal will be toalso determine tissue and tumor dosimetry from the89Zr-freso PET studies for comparison to the177Lu-freso dosimetry and therapy studies.

[0247] Tumor response, following the administration of increasing doses of177Lu-freso, will be monitored by tumor volume and BLI imaging for up to 4 weeks, and early 177Lu-freso SPECT imaging. These studies will determine the optimal dose of177Lu-freso RPT, with and without a TGF-beta priming external beam radiotherapy dose of 2 Gy.177Lu-freso tumor dosimetry will be calculated using in vivo quantitative SPECT imaging and compared to values generated in the89Zr- freso PET studies. Example 8: Radiolabel fresolimumab with89Zr and177Lu

[0248] Labeling freso with89Zr will be carried out as previously described (10) and shown in FIG. 1. Briefly, DFO-SCN (Macrocyclics, Plano TX) will be conjugated to freso. PD10 purified DFO- freso will be characterized by MALDI mass spectrometry and HPLC. Neutralized89Zr-oxalate (3D Imaging, Little Rock, AR) will be added to the DFO-freso and reacted for 30-60 minutes. PD10 purification will provide89Zr-freso in saline, ready to inject into the mouse models.89Zr-freso will be characterized by HPLC and iTLC.

[0249] Labeling freso with177Lu will be carried out using established procedures. pSCN-Bn- DOTA (Macrocyclics, Plano, TX) will be conjugated to freso in pH 9.0 Na2CO3 / NaHCO3 buffer. PD10 purified DOTA-Bn-freso will be characterized by HPLC and MALDI mass Spectrometry.177LuCl3 (DOE, Oakridge, TN) will be reacted with the DOTA-Bn-freso. PD10 purification will give the177Lu-freso ready to inject into the mouse models.177Lu-freso will be characterized by HPLC and iTLC. The molar activity of177Lu-freso will be determined by HPLC so that the mass injected into the mice may be adjusted for uniformity across the study population.

[0250] Alternatively, freso may be conjugated with DOTAGA for labeling with89Zr and177Lu. Recently, DOTAGA has been shown to stably chelate89Zr (48) and has been evaluated as a177Lu chelate for PSMA and FAPI (in humans) targeted radiotherapeutics (49, 50). pNCS-Bz-DOTAGA (ChemMatech, Dijon France) will be conjugated to freso. Radiolabeling and purification will be conducted per the aforementioned literature procedures or as noted above.

[0251] Preclinical Models: Cell lines are authenticated and routinely assayed to confirm that they are free of mycoplasma. 4T1 is considered a model of triple-negative disease whereas TS / A is estrogen-receptor positive (51). Both are metastatic to the lung. To mimic brain metastases, we adapted 4T1 and TS / A parental cancer cell lines to brain 4T1-BrA and TS / A-BrA, as described (10). All are cultured in DMEM containing 10% FBS. Cell lines are stably transfected with integrated Renilla luciferase gene under the control of the constitutively active CMV promoter, allowing for the detection of tumor burden in vivo by BLI and a firefly luciferase gene under the control of a TGFβ–responsive promoter (multiple Smad-binding elements) that reports TGFβ pathway activity by BLI (52). Each cell line will be expanded in vitro for 2 passages to create large stocks that are viably frozen in aliquots for subsequent an-mal inoculation. Notably, 4T1-BrA exhibits leptomeningeal spread (Fig. 7).

[0252] 4T1 or TS / A parental cells (105) will be injected orthotopically in the inguinal mammary fat pad of syngeneic BALB / cJ female mice. Palpable tumor growth will be measured by calipers and BLI imaging using the Ami Imaging System. For intracranial tumors from 4T1-BrA and TS / A- BrA, a stereotaxic device will be used to inject cells 1 mm anterior, 1.8 mm lateral and 3.5 mm beneath the skull surface of the bregma in the right brain hemisphere into the corpus striatum of 6– 7-week-old syngeneic mice anesthetized with ketamine / xylazine (90 mg / kg and 10mg / kg) and buprenorphine (0.5 mg / kg) whilst maintaining body temperature. 4T1-BrA or TS / A-BrA cells (3x103) will be injected into BALB / cJ female mice. Intracranial tumor growth will be evaluated by BLI imaged using the Ami Imaging System every 5 days following intraperitoneal (i.p.) injection of 200 µl (15 mg / mL) luciferin under anesthesia using 2% isoflurane. Tumor burden will be estimated based on bioluminescence flux (photons / sec) and used to randomize mice to treatment groups. Mice bearing intracranial tumors will be monitored for neurological symptoms or weight loss (≥ 15% body weight) and sacrificed in accordance with the Institutional Animal Care and Use Committee guidelines at the institution.

[0253] EBT: A subset of tumors will be primed with 2 Gy EBT delivered to the tumor using a small animal radiation research system that combines high-resolution computerized tomography (CT) imaging and accurate conformal beam therapy. All mice will be irradiated under anesthesiausing 2% isoflurane. EBT dose will be delivered using dual-focus 0.15 mm Cu filtration, constant voltage X-ray source operating up to 225 kVp (typically 60 kV for imaging, and 220 kV for treatment), which is mounted on a rotating gantry with a nominal source-to-isocenter distance of 35 cm and a positioning accuracy of 0.5 mm. The absolute dosimetry will be verified by a certified medical physicist in the department and by K&S Associates Inc. based on AAPM Task Group reports 51 and 61, using a NIST traceable calibrated ion chamber. Mouse tumor BLI will be used to formulate individualized plans (Muriplan, XStrahl) based on arc beam using the XStrahl SARRP.

[0254] Tumor Imaging: We will monitor TGFβ activity in tumors by two means:89Zr-freso PET and BLI based on total cells and a TGFβ reporter construct. We will use the microPET / CT (nanoScan, Mediso) to evaluate the distribution of the 89Zr-freso over time (4h, 24h, 72h, 120h). Four tumor-bearing animals per tumor model, with and without 2Gy EBT TGF-beta priming dose, will be imaged simultaneously in the microPET / CT after i.v. administration of 150 µCi89Zr-freso (32 mice total over 18 months). In embodiments, 72 h was peak time for imaging89Zr-freso accumulation in tumors (Fig. 5A). The longitudinal PET imaging data will be used to estimate organ and tumor dosimetry (method described below). Following the last microPET / CT scanning session the animals will be euthanized, and the absolute radioactivity quantitation will be determined by collecting blood and excised tissues that will be weighed and counted in the HIDEX gamma counter (Turku, Finland). The percent injected dose per gram of tissue will be determined and correlated with the image data.

[0255] For dual BLI imaging, D-luciferin substrate solution for firefly luciferase BLI (for TGFβ activity) or coelenterazine substrate solution (Caliper) for Renilla luciferase BLI (for tumor burden) will be administered i.p. (100 µL / mouse). AMI HT optical imaging system from Spectral Instruments Imaging will be used to measure bioluminescence and fluorescence in vivo imaging of light generated within living animals (53-56). The method is capable of detecting 100-1000 cancer cells and provides a linear measure of tumor burden (57). It will be used to monitor tumor size and metastasis during the course of the experiment. Lung metastases will be quantified by counting nodules at necropsy and by isotope counting as we previously reported (18).

[0256] We will compare the pharmacokinetics of89Zr-freso by PET / CT with that of177Lu-freso by SPECT / CT (below) in the same tumor models at the same timepoints. Although we will use different animals (i.e., no co-injection) to avoid cross-talk of photons at different energies (down scatter to compromise quantitative accuracies), a group average from multiple animal imaging studies will be used to make the comparison of how these two radiopharmaceuticals behave in vivo over time. The outcome from this study will support the future use of89Zr-freso to guide / plan177Lu- freso treatment given the image quality and more robust quantitative imaging of89Zr-freso PET imaging.

[0257] 177Lu-freso treatment. We will define an RPT treatment dose using the tumor dosimetry method described below to deliver a therapeutic effect equivalent to at least 10 Gy EBT (Fig. 6B). In other RPT models, an estimated dose of 10 Gy required ~1 mCi of177Lu-labeled molecules per mouse (58-60). In our preliminary study with subcutaneous 4T1-BRA tumors we found that ~1 mCi controlled tumor growth over 3 weeks (Fig. 6A). The objective will be to define tumor control at 4 graded doses, 0.25 mCi, 0.5 mCi, 0.75 mCi and 1 mCi of177Lu-freso, with and without the 2 Gy EBT priming dose. Tumor-bearing mice will be randomized for treatment based on tumor measurements with calipers for flank and mammary tumors or BLI for intracranial tumors. Ten (10) mice per tumor model (5 irradiated and 5 non-irradiated) will be injected with a given 177Lu-freso dose and monitored by early SPECT / CT imaging (~day 4), as described below, and tumor volume measured by calipers over a total of 4 weeks. There will be 2 control arms of 4 mice each per tumor model – i) non-radioactive freso equivalent to the dose of non-radioactive freso in the177Lu dose and ii) non-radioactive freso plus the 2 Gy priming EBT dose. Optimal dosing will be defined as that in which tumors are controlled / eliminated and in which OAR toxicity is least. The optimal dose for irradiated and non-irradiated tumors determined will be used to compare response in all models as a function of TGFβ activity and biological response. A total of 48 animals per tumor model (192 total) will be evaluated.

[0258] Evaluate acute toxicity. Necropsy will be performed and organs at risk (OAR, i.e., the organs with the highest absorbed dose other than tumors; lung, kidney, bone marrow) harvested for histological analysis using picrosirius to assess collagen content and fibrosis or immune cellanalysis. RPT toxicities are associated with non-tumor dwell time. Fresolimumab safety and efficacy in improving metastatic melanoma, renal cell carcinoma and metastatic breast cancer showed evidence of acute skin toxicity from formation of acanthomas (35, 61), although such toxicities are not expected from the low dose of fresolimumab used to deliver radioisotope. Here, we will evaluate the risk of177Lu-freso induced hematological toxicity using multispectral flow cytometry to quantify immune cell distribution in bone marrow, spleen and blood at termination. Lung and kidney will be prepared for histology and assessed for radiation induced fibrotic changes such as collagen remodeling (fibrosis per se is unlikely at early time points).

[0259] Tumor and OAR dosimetry by SPECT / CT SPECT / CT scans at multiple time points will be used to calculate the total absorbed dose in tumors and OARs delivered by177Lu-freso. We will use a dedicated small animal SPECT / CT (VECTor4CT, MILabs). Up to 30 minutes of SPECT data will be acquired, followed by low-dose CT. The high-energy general-purpose collimator (HE-GP- RM) built for higher energy photons in SPECT applications, and high sensitivity will be used for all animal scans. This collimator has been routinely validated for its quantitative accuracy in our previous177Lu scans on this scanner (62, 63). SPECT reconstructions will include the vendor- provided similarity-regulated OSEM (SROSEM) algorithm, scatter correction, and CT-based attenuation correction to ensure measurement accuracy. CT data will be reconstructed using the vendor-provided conebeam Feldkamp algorithm. All voxels of SPECT data will be calibrated to be in the physical unit of Bq / mL, the standard quantification method for PET and SPECT.

[0260] For tumor and OAR dosimetry, at least 4 time points of SPECT / CT will be acquired. The time points will be selected based on the expected biokinetics of177Lu-freso. The time points will be designed with knowledge of the previous imaging studies of89Zr-freso. Most likely, the time points will be at 4, 24, 72, and 120 hours after injection. From these multi-time SPECT / CT scans, we will delineate volumes of interest (VOIs) of tumors and OAR and derive the percent of injected activity (%IA) – time (in h) curves. Using bi- (i.e., two decay half-lives) or tri- (i.e., one rise and two decay half-lives) exponential curve-fitting methods, we will calculate the area under these curves, which is known as time-integrated activity coefficients (TIACs, also known as residence times). From TIACs, and Monte Carlo methods we have developed (64, 65), or using simple spheremodels for tumors and standard organs already simulated in OLINDA (version 1.1 and 2.0), we will calculate the total absorbed doses in the unit of Gy / MBq.

[0261] Ex vivo digital autoradiography. After the last SPECT / CT scan, the tumors and OARs will be harvested from the animals, and quantitative digital autoradiography will be performed to produce a dose-rate volume histogram to quantify the degree of uptake heterogeneity. Dose rates and total absorbed doses in the whole tumors do not provide suborgan scale (i.e., small scale or microscale) uptake / dose heterogeneity that could be a very important factor in understanding how physical dose (J / kg = Gy) is distributed over the entire volume, and how specific the dose is delivered in the tumors. We have developed methods of calculating dose rates and quantifying dose heterogeneity using digital autoradiography (DAR) with single particle sensitivity (iQID, QScint) (60, 66). The same dose rate calculation and dose distribution quantification will be used for177Lu- freso. An example of DAR image and relative activity map for177Lu-PSMA-617 shows the capability of177Lu sensitivity on iQID (Fig. 8A). Only the relative activity is displayed in this figure. For this project, we will perform the quantification calibration (i.e., correlating known activity and detected counts) for177Lu for quantifying dose-rate – volume / area histogram (Fig. 8B) for which the previous study for an experimental radiopharmaceutical with an alpha emitter, 211At’s dose distribution in a canine lymph node (66). Example 9: Evaluate biological correlates of metastatic breast cancer responses to177Lu-freso.

[0262] The knowledge of the radiobiology of RPT is sparse, and TGFβ is a novel target. Hence, we will analyze the composition of the tumor and immune system of mice from Examples 7 and 8 that are sacrificed at 5 days post RPT. The goal of early response experiments is to evaluate factors contributing to survival by quantifying known cellular mechanisms that mediate response or resistance across breast cancer models and to correlate these with overall survival. These data will inform clinical translation of this approach since a fundamental challenge in breast cancer therapy is the diversity of intrinsic features, such as metabolism and DNA damage deficits, and the extrinsic components that constitute the TME, which includes the vasculature, immune cells and stroma. All of these constitute the growing list that constitutes the “R’s” of radiobiology: repair, radiosensitivity, reassortment, reoxygenation, remodeling and rejection (67-69).

[0263] 177Lu-freso treatment. The optimal177Lu - RPT dose for each model: orthotopic 4T1 and TS / A parental cell lines, and intracranial 4T1-BrA and TS / A-BrA tumors, determined in Example 7 and 8, will be used with and without the 2 Gy TGF-beta priming EBT dose. 4T1 and TS / A are considered representative of TNBC and ER+ breast cancer respectively. Here the optimal177Lu- freso dose ± 2 Gy EBT will be used to validate tumor response (i.e., survival) in all models as a function of TGFβ activity and biological response. Tumor-bearing mice will be randomized for treatment based on tumor measurements with calipers for flank and mammary tumors or BLI for intracranial tumors. Experiments will consist of two control arms in which mice receive unlabeled fresolimumab equivalent to the same dose of labeled177Lu-freso in the treatment arm with and without 2 Gy EBT. Four tumors in each arm will be randomly harvested 5 days after177Lu-freso injection. Mice will be imaged with89Zr-freso, as described above, on the same day as sacrifice to ascertain the levels of TGFβ activity. The tumors will be harvested and portioned as follows: 1 / 4 will be formalin fixed and paraffin embedded (FFPE) for immunostaining of vessels, 1 / 4 frozen as viable explants for DNA repair foci analysis, and 2 / 4 will be dissociated for flow cytometry of immune composition. Blood and spleen will be stored for flow cytometry.

[0264] The tumors of mice remaining in the study will be measured and monitored for 30 days post-treatment initiation or until morbidity. Mice in which tumors regress or stabilize will be monitored for an additional 30 days. In the absence of tumor regrowth, mice will be tested for immune memory (i.e., rejection) by rechallenge with the same tumor and comparison of tumor growth to a second cohort of 3 naïve mice. Mice in which the primary tumor does not recur, and the second tumor is rejected will be considered to have been cured by immunological response. Mice in which the primary tumor does not recur but the second tumor does grow will be considered to have been cured by ablation, i.e. cell kill.

[0265] DNA Damage. To measure unrepaired DNA damage, we will use radiation-induced P53- binding protein 1 (53BP1) foci, whose persistence (>4 hr) and size indicate unrepaired DNA damage. DNA repair foci manifest differently as a function of cell cycle and radiation quality (71- 73). Cells in S / G2 will be measured by geminin co-staining. Phospho-DNA-PK and phospho- XRCC4 co-localization with gamma-H2AX in foci in geminin-negative cells will be consideredindicative of NHEJ usage. RAD51 foci in geminin positive cells will be considered evidence of HR. By this manner, we may assess pathway competency in cancer cells with different contexts. Interpretation of each DNA damage foci pattern will be validated by using cell lines with known DNA repair phenotypes grown as tumors for optimization of immunostaining. To determine the frequency of DDR foci positive cells, we will use multiplex detection and quantitative image analysis of markers using the Akoya system of automated multispectral whole tissue imaging and trainable pattern recognition. Most immunostaining of DNA repair markers is limited to single pathways, but we propose to simultaneously detect multiple proteins at DNA damage sites using advanced multiplex OpalTM immunostaining. We have optimized several panels using Opal immunostaining. Moreover, multi-spectral microscopy and image analysis generates excellent co- localization data.

[0266] To further evaluate unrepaired DNA damage, alkaline comet assay to detect residual DNA breaks after irradiation will be employed. Neutral comet assay data from our publication of primary HNSCC viable explants demonstrate feasibility using tumor fragments in raft cultures (26). The tumor samples will be diced into small pieces and maintained in DMEM medium on raft cultures for 1 day prior to treatments. Tumor explants will be randomly distributed for treatment with radiation (2 Gy, as a positive control), or sham-irradiated. At selected times post treatment, samples are embedded in optimum cutting temperature, OCT (Sigma Aldrich) and are frozen on dry ice. Cryosections cut in our laboratory will be fixed with 4% PFA and permeabilized for immunofluorescence and Opal staining for analysis of DDRD and p-SMAD2 as described above.

[0267] Vascularity. To quantify the vascularization and hypoxia of tumors as a function of treatment, we will use the detection of pimonidazole (PIMO) and carbonic anhydrase IX (CaIX), a long-lived hypoxia inducible protein, and CD31 to quantify microvessel density (MVD). Mice will be injected with PIMO 1 hr prior to sac, and Hypoxyprobe™-1 Omni Kit (Cat# HP3-200 Kit) used as directed by the manufacturer for its detection. In some mice, vascular integrity will be measured by injecting mice with Evans blue 1 hr before sacrificing. Automated image analysis of whole slide images will be used to determine mean size, vessel density and hypoxia per tumor.

[0268] Immune Cell Characterization. Single cells from blood, spleen, tumor collected at termination will be analyzed using high dimensional multispectral flow cytometry to investigate tumor and systemic immune composition. Samples from mice will be processed according to our standardized mass cytometry analysis pipeline that enables samples to be directly compared with minimal technical variability for different cell surface and intracellular markers of immune cell types using panels of fluorescently labeled antibodies on our in-lab Northern Lights Cytek cytometer that is capable of full spectrum unmixing with 3 lasers, 38 fluorescence detectors, and vacuum fluidics capable of detecting more than 26 markers at a high flow rate. The cytometry data generated from these samples will be modeled using T-distributed stochastic neighbor embedding (t- SNE) to visualize high-dimensional data or scaffold maps to that transforms raw data into a graphical map of the immune system into an intuitive graphical network of cell types that have statistically significant changes in frequency or behavior across experimental conditions (88).

[0269] Cytometry provides deep information about the frequency and activation of immune cells, but tissue dissociation prevents acquisition of important spatial association information. We will use Phenoptics™ (Akoya) quantitative pathology to create a spatial co-localization map to detect, measure, visualize, and compare multiple immune-cell phenotypes simultaneously in existing FFPE tumor specimens using the Vectra System in the Barcellos-Hoff lab. We have optimized simultaneous co-localization of 7 antibodies for murine immune infiltrate using multispectral imaging of Opal labeling (Akoya) quantitatively mapped using InForm software analysis. Project specific panels will consist of TGFβ, phospho-SMAD, Ki-67, FoxP3 and CD25 (Treg cells), IFNγ (Th1 cells and CTLs), and IL-17 (Th17 cells), or natural killer cells identified using DX5 (CD49b). We will assess the anti-tumor immune activation by measuring the frequency of proliferating cytotoxic T-cells (CD8 / Ki67 double positive). The expression of PD-L1 on tumor cells and myeloid cells will be assessed. Macrophage polarization will be assessed using differential expression of CD68, F4 / 80, Arg-1 and mannose-6 phosphate receptor. BATF3 antibody will be used as marker for cross-presenting, type 1 cDC (CD103+ in tissue, CD8+ in the spleen), which are the primary antigen-presenting cells that prime CD8 T cell responses (89, 90).

[0270] We will first test if systemic RPT decreases 4T1-BrA leptomeningeal disease, or, we will test RPT injected directly to cerebral spinal fluid. A low EBT priming dose will allow us to inject less RPT to achieve tumor control and less toxicity. The limited range of breast cancer mouse cell lines compared to the diverse biology of human breast cancer is a challenge for preclinical studies translation. Here we will use metastatic TNBC and ER+ cell lines in two settings, orthotopic with metastases to lung and intracranial brain-adapted tumors. Alternatively, we will use our recently established model, mammary tumor-derived transplants (mTDT), which recapitulate a broad range of breast cancer phenotypes, including immune infiltrate patterns of infiltrated, excluded and desert (87). We have published characterization of 12 independent models, 3 of which metastasize to lung. The number has increased to 24 that include 4 ER+ mTDT. We will use mTDT to assess the efficacy of177Lu-freso mTDT to expand the relevance of our studies. Example 10: Intracranial iGL261 brain tumor model

[0271] Mice bearing brain tumors tumors confirmed by bioluminescence were injected with 1 mCi 177Lu-177Lu-fresolimumab. Isotope uptake (counts per minute / gram of tissue is greater in the tumor bearing hemisphere vs control hemisphere (FIG. 9). Survival increased significantly (P= 0.015, Log-Rank test) compared to untreated mice (FIG. 10) Example 11: 4T1 breast cancer model

[0272] Mice bearing 50-75 mm3 subcutaneous flank tumors were injected with 0.1, 0.25 or 0.5 mCi 177Lu-fresolimumab. Growth compared to untreated mice shows significant tumor control (P<0.0012-way ANOVA) across three doses (FIG. 11). Doubling time increased from 7 days for untreated compared to 37 days for those treated with 0.5 mCi (Table below). Repeat experiment of 0.5 mCi (ongoing) shows a similar response. Subcutaneous 4T1Br tumors treated with 0.5 mCi in a replicate experiment shows a similar response. The tumor doubling time in untreated mice is 7.5 days. The doubling time in mice treated with 0.5 mCi is extended significantly (p < 0.00001). Mice bearing intracranial 4T1Br tumors, which is a model of breast-brain metastases, treated with 0.5 mCi 177Lu-fresolimumab exhibit significantly longer survival (P= 0.04) compared to untreated mice.TABLES

[0273] Table 1. Theranostic radioisotopes. Isotope Half-life Use 131 I8.03 dβ− -Therapy, SPECT 177 Lu6.65 dβ− -Therapy, SPECT 47 Sc3.35 d-Therapy, SPECT 161 Tb*6.89 dβ− -Therapy, SPECT 67 Cu*61.83 hβ− -Therapy, SPECT 153 Sm46.28 hβ− -Therapy, SPECT 64 Cu12.7 hβ− -Therapy, PET 149 Tb4.12 h α-Therapy, PET / SPECT

[0274] Table 2. Theranostic pairs. Therapeutic Isotope Half-life Imaging / Therapy 67 64 − / Cu2.7 d / 12.7 hβ-therapy & SPECT / PET 47 / 44 − Sc3.4 d / 4.0 hβ-therapy & SPECT / PET 90 / 86 − Y64.1 h / 14.7 hβ-therapy / PET 149&161 / 152 − Tb*4.1 h & 6.9 d / 17.5 hα&β-Therapy / SPECT / PET 212 / 203 Pb10.6 h / 51.9 h α-Therapy / SPECT 131 / 123,124 - / + I 8.03 d / 13 h, 4.1 dβ- / SPECT, PET

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[0276] P Embodiment 1. An active TGF-beta binding agent comprising a radioisotope.

[0277] P Embodiment 2. The active TGF-beta binding agent of P embodiment 1, wherein said agent is a protein, a nucleic acid, or a small molecule.

[0278] P Embodiment 3. The active TGF-beta binding agent of P embodiment 1 or 2, wherein said agent is an antibody or a peptide.

[0279] P Embodiment 4. The active TGF-beta binding agent of any one of P embodiments 1-3, wherein said agent is an active TGF-beta binding antibody.

[0280] P Embodiment 5. The active TGF-beta binding agent of P embodiment 4, wherein said active TGF-beta binding antibody is a humanized active TGF-beta binding antibody.

[0281] P Embodiment 6. The active TGF-beta binding agent of any one of P embodiments 4-5, wherein said active TGF-beta binding antibody is fresolimumab.

[0282] P Embodiment 7. The active TGF-beta binding agent of any one of P embodiments 1-6, wherein said radioisotope is a therapeutic radioisotope or a diagnostic radioisotope.

[0283] P Embodiment 8. The active TGF-beta binding agent of any one of P embodiments 1-7, wherein said radioisotope is a therapeutic radioisotope.

[0284] P Embodiment 9. The active TGF-beta binding agent of any one of P embodiments 1-8, wherein said radioisotope is 225Ac, 211At, 134Ce, 64Cu, 67Cu, 68Ga, 123I, 124I, 131I, 111In, 132La, 133La, 177Lu, 44Sc, 47Sc, 153Sm, 149Tb, 152Tb, 161Tb, 203Tb, 212Tb, 227Th, 86Y, 90Y, or 89Zr.

[0285] P Embodiment 10. The active TGF-beta binding agent of any one of P embodiments 1-9, wherein said radioisotope is 177Lu.

[0286] P Embodiment 11. The active TGF-beta binding agent of any one of P embodiments 4- 10, wherein said active TGF-beta binding antibody further comprises a therapeutic moiety.

[0287] P Embodiment 12. The active TGF-beta binding agent of any one of P embodiments 4- 11, wherein said radioisotope or said therapeutic moiety are covalently attached to said active TGF- beta binding antibody.

[0288] P Embodiment 13. The active TGF-beta binding agent of any one of P embodiments 1- 12, further comprising a detectable moiety.

[0289] P Embodiment 14. The active TGF-beta binding agent of any one of P embodiments 1- 13, wherein said radioisotope is bound to said active TGF-beta binding agent through a covalent linker.

[0290] P Embodiment 15. A pharmaceutical composition comprising a pharmaceutically acceptable excipient and an active TGF-beta binding agent of any one of P embodiments 1-14.

[0291] P Embodiment 16. A method of treating cancer in a subject in need thereof, said method comprising administering an effective amount of an active TGF-beta binding agent of any one of P embodiments 1-14, or an effective amount of a pharmaceutical composition of P embodiment 15 to said subject, thereby treating cancer in said subject.

[0292] P Embodiment 17. The method of P embodiment 16, said method further comprising prior to administering said active TGF-beta binding agent to said subject administering an external beam therapy to said subject.

[0293] P Embodiment 18. The method of P embodiment 17, wherein said external beam therapy is administered at 1-10 Gy.

[0294] P Embodiment 19. A method of treating cancer in a subject in need thereof, said method comprising: (i) administering to said subject an effective amount of a first active TGF-beta binding agent comprising a first radioisotope; and (ii) administering to said subject an effective amount of a second active TGF-beta binding agent comprising a second radioisotope, thereby treating cancer in said subject.

[0295] P Embodiment 20. The method of P embodiment 19, wherein said first active TGF-beta binding agent is administered at a first time point and said second active TGF-beta binding agent is administered at a second time point.

[0296] P Embodiment 21. The method of P embodiment 20, wherein said first time point precedes said second time point.

[0297] P Embodiment 22. The method of any one of P embodiments 19-21, further comprising after said step (i), administering an external beam therapy.

[0298] P Embodiment 23. The method of P embodiment 22, wherein said external beam therapy is administered at 1-10 Gy.

[0299] P Embodiment 24. The method of P embodiment 22 or 23, further comprising detecting said first radioisotope after said administering said external beam therapy.

[0300] P Embodiment 25. The method of any one of P embodiments 19-24, wherein said first active TGF-beta binding agent and said second active TGF-beta binding agent are administered intravenously.

[0301] P Embodiment 26. The method of any one of P embodiments 19-25, wherein said first radioisotope is a diagnostic radioisotope and said second radioisotope is a therapeutic radioisotope.

[0302] P Embodiment 27. The method of any one of P embodiments 19-26, wherein said first radioisotope and said second radioisotope are independently 225Ac, 211At, 134Ce, 64Cu, 67Cu, 68Ga, 123I, 124I, 131I, 111In, 132La, 133La, 177Lu, 44Sc, 47Sc, 153Sm, 149Tb, 152Tb, 161Tb, 203Tb, 212Tb, 227Th, 86Y, 90Y, or 89Zr.

[0303] P Embodiment 28. The method of any one of P embodiments 19-27, wherein said first radioisotope and said second radioisotope are independently 131I, 177Lu, 47Sc, 161Tb, 67Cu, 153Sm, 64Cu, or 149Tb.

[0304] P Embodiment 29. The method of P embodiment 19, wherein said first radioisotope and said second radioisotope are of the same element.

[0305] P Embodiment 30. The method of any one of P embodiments 19-28, wherein said first radioisotope is 64Cu and said second radioisotope is 67Cu, said first radioisotope is 44Sc and said second radioisotope is 47Sc, said first radioisotope is 86Y and said second radioisotope is 90Y, said first radioisotope is 152Tb or 161Tb and said second radioisotope is 149Tb, said first radioisotope is 203Pb and said second radioisotope is 212Pb, or said first radioisotope is 123I or 124I and said second radioisotope is 131I.

[0306] P Embodiment 31. The method of any one of P embodiments 19-28, wherein said first radioisotope is 68Ga, 89Zr, 123I, 124I, 111In, 132La, 133La, or 134Ce and wherein said second radioisotope is 177Lu, 227Th, 211At, or 225Ac.

[0307] P Embodiment 32. The method of any one of P embodiments 19-28 or 31, wherein said first radioisotope is 89Zr and wherein said second radioisotope is 177Lu.

[0308] P Embodiment 33. The method of any one of P embodiments 19-32, further comprising detecting said second radioisotope.

[0309] P Embodiment 34. The method of P embodiment 33, wherein said detecting comprises administering Positron Emission Tomography (PET) or Single Photon Emission Computed Tomography (SPECT) to said subject.

[0310] P Embodiment 35. The method of any one of P embodiments 19-34, wherein said first radioisotope is administered at 1-20 mCi and said second radioisotope is administered at 0.5-250 mCi.

[0311] P Embodiment 36. The method of any one of P embodiments 19-35, wherein said first radioisotope is administered at 0.8 mCi and said second radioisotope is administered at 190 uCi.

[0312] P Embodiment 37. The method of any one of P embodiments 16-36, wherein said cancer is a carcinoma, a sarcoma or a metastatic cancer.

[0313] P Embodiment 38. The method of any one of P embodiments 16-37, wherein said cancer is breast cancer, lung cancer, prostate cancer, glioblastoma, or metastatic cancer.

[0314] P Embodiment 39. The method of any one of P embodiments 16-37, wherein said cancer is brain metastasis.

Claims

WHAT IS CLAIMED IS:

1. An active TGF-beta binding agent comprising a radioisotope.

2. The active TGF-beta binding agent of claim 1, wherein said agent is a protein, a nucleic acid, or a small molecule.

3. The active TGF-beta binding agent of claim 1, wherein said agent is an antibody or a peptide.

4. The active TGF-beta binding agent of claim 1, wherein said agent is an active TGF-beta binding antibody.

5. The active TGF-beta binding agent of claim 4, wherein said active TGF- beta binding antibody is a humanized active TGF-beta binding antibody.

6. The active TGF-beta binding agent of claim 5, wherein said active TGF- beta binding antibody is fresolimumab.

7. The active TGF-beta binding agent of claim 6, wherein said radioisotope is a therapeutic radioisotope or a diagnostic radioisotope.

8. The active TGF-beta binding agent of claim 7, wherein said radioisotope is a therapeutic radioisotope.

9. The active TGF-beta binding agent of claim 8, wherein said radioisotope is225Ac,211At,134Ce,64Cu,67Cu, 68Ga,123I,124I,131I,111In,132La,133La,177Lu,44Sc,47Sc,153Sm, 149Tb,152Tb,161Tb,203Tb,212Tb,227Th,86Y,90Y, or89Zr.

10. The active TGF-beta binding agent of claim 9, wherein said radioisotope is177Lu.

11. The active TGF-beta binding agent of claim 10, wherein said active TGF- beta binding antibody further comprises a therapeutic moiety.

12. The active TGF-beta binding agent of claim 11, wherein said radioisotope or said therapeutic moiety are covalently attached to said active TGF-beta binding antibody.

13. The active TGF-beta binding agent of claim 12, further comprising a detectable moiety.

14. The active TGF-beta binding agent of claim 13, wherein said radioisotope is bound to said active TGF-beta binding agent through a covalent linker.

15. A pharmaceutical composition comprising a pharmaceutically acceptable excipient and an active TGF-beta binding agent of claim 1.

16. A method of treating cancer in a subject in need thereof, said method comprising administering an effective amount of an active TGF-beta binding agent 1, or an effective amount of a pharmaceutical composition of claim 15 to said subject, thereby treating cancer in said subject.

17. The method of claim 16, said method further comprising prior to administering said active TGF-beta binding agent to said subject administering an external beam therapy to said subject.

18. The method of claim 17, wherein said external beam therapy is administered at 1-10 Gy.

19. A method of treating cancer in a subject in need thereof, said method comprising: (i) administering to said subject an effective amount of a first active TGF-beta binding agent comprising a first radioisotope; and (ii) administering to said subject an effective amount of a second active TGF-beta binding agent comprising a second radioisotope, thereby treating cancer in said subject.

20. The method of claim 19, wherein said first active TGF-beta binding agent is administered at a first time point and said second active TGF-beta binding agent is administered at a second time point.

21. The method of claim 20, wherein said first time point precedes said second time point.

22. The method of claim 21, further comprising after said step (i), administering an external beam therapy.

23. The method of claim 22, wherein said external beam therapy is administered at 1-10 Gy.

24. The method of claim 22 or 23, further comprising detecting said first radioisotope after said administering said external beam therapy.

25. The method of claim 19, wherein said first active TGF-beta binding agent and said second active TGF-beta binding agent are administered intravenously.

26. The method of claim 19, wherein said first radioisotope is a diagnostic radioisotope and said second radioisotope is a therapeutic radioisotope.

27. The method of claim 19, wherein said first radioisotope and said second radioisotope are independently225Ac,211At,134Ce,64Cu,67Cu, 68Ga,123I,124I,131I,111In,132La,133La,177Lu,44Sc,47Sc,153Sm,149Tb,152Tb,161Tb,203Tb,212Tb,227Th,86Y,90Y, or89Zr.

28. The method of claim 19, wherein said first radioisotope and said second radioisotope are independently131I,177Lu,47Sc,161Tb,67Cu,153Sm,64Cu, or149Tb.

29. The method of claim 19, wherein said first radioisotope and said second radioisotope are of the same element.

30. The method of claim 19, wherein said first radioisotope is64Cu and said second radioisotope is67Cu, said first radioisotope is44Sc and said second radioisotope is47Sc, said first radioisotope is86Y and said second radioisotope is90Y, said first radioisotope is152Tb or161Tb and said second radioisotope is149Tb, said first radioisotope is203Pb and said second radioisotope is212Pb, or said first radioisotope is123I or124I and said second radioisotope is131I.

31. The method of claim 19, wherein said first radioisotope is68Ga,89Zr,123I,124I,111In,132La,133La, or134Ce and wherein said second radioisotope is177Lu,227Th,211At, or225Ac.

32. The method of claim 19, wherein said first radioisotope is89Zr and wherein said second radioisotope is177Lu.

33. The method of claim 19, further comprising detecting said second radioisotope.

34. The method of claim 33, wherein said detecting comprises administering Positron Emission Tomography (PET) or Single Photon Emission Computed Tomography (SPECT) to said subject.

35. The method of claim 19, wherein said first radioisotope is administered at 1-20 mCi and said second radioisotope is administered at 0.5-250 mCi.

36. The method of claim 19, wherein said first radioisotope is administered at 0.8 mCi and said second radioisotope is administered at 190 uCi.

37. The method of claim 16, wherein said cancer is a carcinoma, a sarcoma or a metastatic cancer.

38. The method of claim 16, wherein said cancer is breast cancer, lung cancer, prostate cancer, glioblastoma, or metastatic cancer.

39. The method of claim 16, wherein said cancer is brain metastasis.

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