Decreased retention of miniproteins in the kidney

Abstract

Provided herein are compositions and methods for treating, diagnosing, monitoring, and / or imaging diseases, disorders, or conditions using radiopharmaceuticals selective for various targets. More specifically, the compositions and methods disclosed herein reduce renal retention or potential reuptake in the kidney when the target is expressed in the kidney. Various approaches for reducing renal retention of radiopharmaceuticals are disclosed. In various embodiments, compositions are provided that are represented by a formula selected from MLCR, MLC, MCR, MLR, MC, ML, and MR, wherein M comprises a miniprotein (M), L comprises a linear, branched, or enzymatically cleavable linker (L), C comprises a chelator (C), and R comprises a radionuclide (R), and the composition is characterized by its reduced renal uptake.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 425,263, filed November 14, 2022, the disclosure of which is hereby incorporated by reference herein in its entirety for all purposes. [Background technology]

[0002] background Cancer is a leading cause of death worldwide. Classical cancer treatments, such as radiation therapy, chemotherapy, and surgical procedures, can have severe side effects due to the death of healthy, non-cancerous cells. Newer therapeutic agents, including those using biologic conjugates, enhance the targeting of cytotoxic drugs to tumor cells compared to previous treatments. However, after administration, such therapeutic agents often accumulate and / or are excreted rapidly in the kidney. Summary of the Invention [Means for solving the problem]

[0003] overview In various aspects, compositions are provided that are represented by a formula selected from MLCR, MLC, MCR, MLR, MC, ML, and MR, wherein M comprises a miniprotein (M), L comprises a linear, branched, or enzymatically cleavable linker (L), C comprises a chelator (C), and R comprises a radionuclide (R), wherein the composition is characterized by its reduced renal uptake. In some embodiments, the composition comprises a linear polypeptide, a folded polypeptide (e.g., a covalently linked polypeptide, a non-covalently linked polypeptide, or a polypeptide comprising a disulfide linkage), a cysteine-dense peptide, a knottin peptide, a binder, an affibody, an engineered Kunitz domain, a monobody, an anticalin, a designed ankyrin repeat domain (DARPin), or an avimer. In various embodiments, M comprises an amino acid sequence that shares at least 90% identity with any one of SEQ ID NOs: 1-68 in Tables 4A and 4B. In some embodiments, M comprises an amino acid sequence that shares 100% identity with any one of SEQ ID NOs: 1-68. In some embodiments, M comprises an amino acid sequence that shares 100% identity with any one of SEQ ID NOs: 1-3, 5-15, and 47-67. In some embodiments, M comprises an amino acid sequence that shares 100% identity with SEQ ID NO: 16.

[0004] In some aspects, compositions are provided that comprise or consist essentially of decharged molecules. In some embodiments, the compositions comprise an amino acid sequence comprising between 1 and 5% of the total amino acid sequence as a percentage of charged amino acids, wherein the charged amino acids are selected from Lys, Arg, or His. In certain embodiments, the compositions comprise an amino acid sequence comprising between 5 and 10% of the total amino acid sequence as a percentage of charged amino acids, wherein the charged amino acids are selected from Lys, Arg, or His. In additional embodiments, the compositions comprise an amino acid sequence comprising between 10 and 15% of the total amino acid sequence as a percentage of charged amino acids, wherein the charged amino acids are selected from Lys, Arg, His, or non-canonical amino acids, such as trimethyllysine. In further embodiments, the compositions comprise an amino acid sequence comprising between 1 and 5% of the total amino acid sequence as a percentage of charged amino acids, wherein the charged amino acids are selected from Asp or Glu. In additional embodiments, the composition comprises an amino acid sequence comprising between 5-10% of the total amino acid sequence of charged amino acids, wherein the charged amino acids are selected from Asp or Glu. In yet other embodiments, the composition comprises an amino acid sequence comprising between 10-15% of the total amino acid sequence of charged amino acids, wherein the charged amino acids are selected from Asp or Glu. Preferably, the composition is characterized as exhibiting reduced renal uptake in a subject. Thus, in various embodiments, a composition comprising an amino acid sequence in which M comprises a desired percentage of charged amino acids is characterized as exhibiting reduced renal uptake. In some embodiments, M targets any one of the target proteins in Table 10.

[0005] In some embodiments, the composition is characterized as exhibiting greater than 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or greater than 10% ID / g in the tumor 24 hours after administration. In preferred embodiments, the composition is characterized as exhibiting greater than 5% ID / g in the tumor 24 hours after administration. In other embodiments, the composition is characterized as exhibiting less than 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or less than 20% ID / g in the kidney 4 hours after administration. In preferred embodiments, the composition is characterized as exhibiting less than 5% ID / g in the kidney 4 hours after administration. In certain embodiments, the composition is characterized as exhibiting an adhesion ratio of greater than 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1 or 10:1 in tumor and normal non-ablative tissue after 24 hours.

[0006] In an alternative embodiment, there is provided a composition of a miniprotein (M) and a linker (L), wherein L is linked to the C-terminus or N-terminus of a cysteine ​​of M, and L comprises an enzymatically cleavable linker of the formula L:R1-X1-X2-Lys, wherein: R1 is H, PEG(4-36), 4-aminomethyl-phenylacetic acid (AmPA), aminomethylbenzoyl (AmBz) or succinic acid-PEG(4-36); Compositions are provided in which X1 or X2 is Met, Leu, norleu (Nle), Ile, Glu, Methoxinine, Phe, Tyr, beta-Ala, MWK or MVK, dTyr-Gly-Phe (yGF), dArg-Gly-Phe (rGF), Gly(1-10), citrulline, or sarcosine.

[0007] In various related embodiments, there is provided a composition of a miniprotein (M) and a linker (L), wherein L is linked to the Nε- of a lysine of M via a succinic acid derivative, and L comprises an enzymatically cleavable linker of the formula L:R1-X1-X2-Lys, wherein: R1 is H, PEG(4-36), 4-aminomethyl-phenylacetic acid (AmPA), aminomethylbenzoyl (AmBz) or succinic acid-PEG(4-36); Compositions are provided wherein X1 or X2 is Met, Leu, norleu (Nle), Ile, Glu, methoxynine, Phe, Tyr, beta Ala, MWK or MVK, yGF, rGF, Gly(1-10), citrulline, or sarcosine.

[0008] In various related aspects, compositions are provided that are cleaved by one or more proteases in the kidney, hi some embodiments, the compositions are cleaved by cathepsin B or by neutral endopeptidases, metalloproteases, or dipeptidyl peptidases in the kidney brush border membrane.

[0009] Additionally disclosed herein are methods of treating cancer in a patient, comprising administering to the patient a composition disclosed herein. Additionally disclosed herein are methods of co-administering any of the compositions disclosed herein with a peptide comprising an amino acid sequence selected from SEQ ID NO:4, SEQ ID NO:7, SEQ ID NOs:16-52, or SEQ ID NO:68.

[0010] In various embodiments, the method reduces renal uptake of M, L, C and R, and combinations thereof.

[0011] In various embodiments, co-administration of any one of the compositions disclosed herein with a peptide comprising an amino acid sequence selected from SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:16-52, or SEQ ID NO:68 results in competitive inhibition of the peptide.

[0012] In certain embodiments, the compositions are administered intravenously or subcutaneously.

[0013] In various embodiments, any one of the compositions disclosed herein and a peptide comprising an amino acid sequence selected from SEQ ID NO:4, SEQ ID NO:7, SEQ ID NOs:16-52, or SEQ ID NO:68 are co-formulated with a pharmaceutically acceptable buffer.

[0014] In various embodiments, the method results in a treatment characterized as exhibiting reduced uptake of M in the kidney.

[0015] Additionally disclosed herein are compositions and peptides represented by a formula selected from one or more of MLCR, MLC, MCR, MLR, MC and ML, wherein M comprises a miniprotein (M), L comprises a linear, branched or enzymatically cleavable linker (L), C comprises a chelator (C), and R comprises a radionuclide (R), wherein the peptide is characterized as competitively inhibiting a target for uptake in the kidney.

[0016] In various embodiments, the peptide comprises an amino acid sequence selected from SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NOs: 16-52, or SEQ ID NO: 68, and the peptide is present at a concentration of 10-1000x compared to the concentration of the MLCR or MLC.

[0017] In various embodiments, the peptide comprises an amino acid sequence selected from SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NOs: 16-52, or SEQ ID NO: 68, and the peptide is present at a concentration of 100x or greater than 100x relative to the concentration of M.

[0018] In various embodiments, the peptide comprises an amino acid sequence selected from SEQ ID NO:4, SEQ ID NO:7, SEQ ID NOs:16-52, or SEQ ID NO:68, and the peptide is present at a concentration of 10x to 100x, 100x to 300x, and 300x to 1000x relative to the concentration of M.

[0019] In various embodiments, the peptide comprises an amino acid sequence selected from SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NOs: 16-52, or SEQ ID NO: 68, and the peptide is present at a concentration of 100x or greater than 100x relative to the concentration of M.

[0020] Additionally disclosed herein are compositions and peptides comprising an amino acid sequence selected from one or more of MLC, MC, ML, and M, wherein M comprises a miniprotein (M) comprising an amino acid selected from SEQ ID NO:4 or SEQ ID NO:7, L comprises a linear, branched, or enzymatically cleavable linker (L), and C comprises a chelator (C), wherein the peptide is characterized as competitively inhibiting a target for uptake in the kidney.

[0021] In various embodiments, the peptide is present at a concentration of 10-1000x compared to the concentration of M.

[0022] In various embodiments, the peptide is present at a concentration of 100× or greater than 100× compared to the concentration of M.

[0023] In various embodiments, the peptide reduces uptake of MLCR, MLC, or R in the kidney.

[0024] In certain embodiments, the composition comprises one or more chelators selected from DOTA, NOPO, Crown, or Macropa.

[0025] In other embodiments, the composition comprises one or more radionuclides selected from Ac-225, Ga-68, Pb-212, Lu-177, Cu-67, Cu-64, La-132, La-135, In-111, Ce-134, F-18, or At-211.

[0026] Provided herein are pharmaceutical compositions comprising (M)-L-(C)-R and at least one drug moiety.

[0027] In various embodiments, the composition binds to a target expressed on tumor cells with an affinity of between 1 pM and 100 nM as measured by an in vitro binding assay.

[0028] In other embodiments, the composition is characterized by a peptide solubility of 1-100 mg / mL in the assay formulation and a peptide stability of 80-95% at 75° C. for at least 1 hour.

[0029] In some preferred embodiments, the composition is excreted through the kidney at or near the subject's glomerular filtration rate. In other preferred embodiments, the composition is characterized by being transported or passing through one or more kidneys (e.g., kidney, liver, bone marrow or spleen) at a rate slower than glomerular filtration, resulting in minimized uptake or reuptake, increased systemic exposure, and similar or superior tumor targeting.

[0030] Preferably, the composition is directed to cells where expression of the target is higher in cancer cells than in non-cancer cells.

[0031] In various embodiments, the present invention is directed to the treatment of breast cancer, ovarian cancer, melanoma, pancreatic cancer, peripheral neuroma, glioblastoma, adrenocortical carcinoma, AIDS-related lymphoma, anal cancer, bladder cancer, meningioma, glioma, astrocytoma, cervical cancer, chronic myeloproliferative disorders, colon cancer, endometrial cancer, ependymoma, esophageal cancer, Ewing's sarcoma, extracranial germ cell tumor, extrahepatic bile duct cancer, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gestational trophoblastic tumor, hairy cell leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, hypopharyngeal cancer, islet cell carcinoma, Kaposi's sarcoma, laryngeal cancer, leukemia, lip cancer, oral cancer, liver cancer, male breast cancer, malignant mesothelioma, medulloblastoma, Merkel cell carcinoma, metastatic squamous cell carcinoma of the cervix, multiple myeloma and other plasma cell neoplasms, mycosis fungoides fimgoides) and Sézary syndrome, myelodysplastic syndrome, nasopharyngeal carcinoma, neuroblastoma, non-small cell lung cancer, small cell lung cancer, head and neck cancer, skin cancer, oropharyngeal cancer, bone cancer including osteosarcoma and malignant fibrous histiocytoma of bone, sinus cancer, parathyroid cancer, penile cancer, pheochromocytoma, pituitary tumor, prostate cancer, rectal cancer, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, small intestine cancer, soft tissue sarcoma, supratentorial primitive neuroectodermal tumor, pineoblastoma, testicular cancer, thymoma, thymic carcinoma, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter, urethral cancer, uterine sarcoma, vaginal cancer, vulvar cancer, and Wilms' tumor and other pediatric kidney tumors are provided.

[0032] Additionally disclosed herein is a use of any of the compositions disclosed herein for treating cancer in a subject.

[0033] The present disclosure provides technologies, such as compositions and methods of use and manufacture, to address needs in the field of cancer. For example, in contrast to classical cancer diagnostic methods or treatments, targeted molecules can be designed to increase the specificity and reduce toxicity of imaging or treatment modalities, for example. For example, delivery of therapeutic agents, such as chelators and / or radionuclides (e.g., alpha emitters), using polypeptides to specifically target therapeutic agents to the tumor microenvironment provides treatment focused on tumor cells, avoiding or reducing the risk of toxicity to surrounding healthy tissues from, for example, tumor-targeted therapeutic agents.

[0034] In contrast to traditional cancer treatments, radionuclide therapy is more targeted and less toxic. For example, specific delivery of radionuclides to the tumor microenvironment allows for selective radiation of tumor tissue, effectively killing malignant cells while sparing surrounding healthy tissue. For example, radionuclides may use targeting molecules that specifically bind to target proteins expressed at increased levels and / or density on the surface of tumor cells compared to non-tumor cells. Binding of radionuclides to target-positive tumor cells targets radiation to those cells without targeting healthy tissue. Full-length antibodies have previously been evaluated as targeting moieties; however, due to considerations such as their large size, full-length antibodies may have several challenges, such as poor tumor tissue penetration and a long circulating half-life, which results in irradiation of normal tissues. A significant drawback of radionuclide therapy is the potential for systemic exposure or nephrotoxicity in radionuclide therapy. Therefore, new approaches remain needed to specifically target tumors, particularly solid tumors, while maintaining therapeutic efficacy and reducing the undesirable effects of radionuclide therapy. The present disclosure provides techniques that meet this and other needs.

[0035] Among other things, the present disclosure provides conjugates comprising a polypeptide (e.g., a miniprotein) that targets the tumor microenvironment and / or tumor cells, conjugated to one or more additional components including, for example, a linker, a chelator, and / or a radionuclide (e.g., an alpha emitter). In some embodiments, such conjugates are used in the treatment of cells that express a target (e.g., any one of the target proteins in Table 10). In some such embodiments, the cells are cancer cells.

[0036] In some embodiments, the composition comprises a linker and a chelator.

[0037] In some embodiments, the composition comprises a linker, a chelator, and a radionuclide.

[0038] In some embodiments, the composition comprises a miniprotein, an optional linker, a chelator and / or a radionuclide.

[0039] In some embodiments, the composition is represented by a formula selected from one or more of MLCR, MLC, MCR, MLR, MC, ML and MR, wherein M comprises a miniprotein (M), L comprises a linker (L), C comprises a chelator (C), and R comprises a radionuclide (R).

[0040] In some embodiments, polypeptides in accordance with the present disclosure are produced using solid-phase peptide synthesis. In some embodiments, the polypeptide is recombinant. In some embodiments, the polypeptide is a folded polypeptide linked by disulfide bonds, covalent or non-covalent interactions. In some embodiments, the polypeptide comprises or consists of a miniprotein. In some embodiments, the miniprotein comprises or consists of a linear polypeptide, a folded polypeptide (e.g., a covalently linked polypeptide, a non-covalently linked polypeptide, or a polypeptide containing disulfide bonds), a cysteine-rich peptide, a knottin peptide, a binder, an affibody, an engineered Kunitz domain, a monobody, an anticalin, a designed ankyrin repeat domain (DARPin), or an avimer. In some embodiments, the miniprotein comprises or consists of approximately 100 amino acids or less. In some embodiments, the miniprotein is a cysteine-rich protein. In some embodiments, the miniprotein comprises at least one cysteine-rich region. In some embodiments, the miniprotein comprises one or more disulfide bridges. In some embodiments, the miniprotein comprises or consists of a non-disulfide-containing amino acid sequence. In some embodiments, the miniprotein comprises three alpha helices with 58 amino acids and has a molar mass of approximately 6 kDa. In some embodiments, the miniprotein is stable at high temperatures and under acidic or alkaline conditions. In some embodiments, the miniprotein comprises an engineered protein derived from lipocalin. In some embodiments, the miniprotein comprises an eight-stranded β-barrel. In some embodiments, the miniprotein exhibits (i) high structural plasticity as a result of sequence variation and (ii) increased conformational flexibility, allowing for directed fit to targets with different shapes. In some embodiments, the miniprotein comprises a class of antibody mimetics consisting of two or more peptide sequences of 30-70 amino acids each connected by a linker peptide.In some embodiments, the miniprotein comprises a peptide derived from ankyrin. In some embodiments, the miniprotein comprises an ankyrin repeat, a 33-residue motif consisting of two alpha-helices and one beta-turn. In some embodiments, the miniprotein comprises a peptide derived from the Kunitz domain of a Kunitz-type protease inhibitor, such as bovine pancreatic trypsin inhibitor (BPTI), amyloid precursor protein (APP), or tissue factor pathway inhibitor (TFPI).

[0041] In some embodiments, the present disclosure provides compositions that have various advantages over compositions comprising full-length proteins. For example, in some embodiments, the compositions of the present disclosure comprise one or more improved properties selected from increased miniprotein expression, increased thermostability, increased receptor binding specificity and / or affinity, increased chemical stability, increased resistance to acidic pH, increased resistance to proteolytic activity (e.g., reduced susceptibility to proteolysis), reduced aggregation, increased solubility, and / or reduced immunogenicity.

[0042] In some embodiments, the compositions of the present disclosure specifically bind to at least one epitope of a target in, on, or near a cell. In some embodiments, the target is any one of the target proteins in Table 10. In some embodiments, the cell is a tumor cell (which may or may not have the characteristics of having cancer, but may be derived from a tumor sample). In some embodiments, the cell is a cancer cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a primary cell. In some embodiments, the primary cell is obtained from a sample from a subject (e.g., from a biopsy, e.g., from a tumor). In some embodiments, the cell is from a cell line.

[0043] In some embodiments, the miniprotein or conjugate thereof selectively binds to a target. In some embodiments, the target is a protein or portion thereof expressed on the surface of a cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is a cancer cell. In some such embodiments, the cancer cell is in a tumor (i.e., is part of a tumor) or near a tumor. In some embodiments, the cancer cell is a circulating cell.

[0044] In some embodiments, the target is a cell adhesion receptor, a cytokine receptor, a chemokine receptor, a growth factor receptor, an immune cell receptor, or a tumor-associated extracellular matrix polypeptide. In some such embodiments, the target is 5T4, ADAM9, AG-7, AGS-16 / ENPP3, ALPV, ASCT2, AXL, B7H3 / CD276, B7H4, BCMA, C4.4a, CA6, CA9, CAIX, CCR2, CCR7, CD123, CD138, CD142, CD166, CD19, CD20, CD205, CD22, CD228, CD25, CD30, CD33, CD352, CD37, CD38, CD44v6, CD45, CD46, CD47, CD48, CD5, CD51, CD56, CD7 , CD70, CD71, CD74, CD79B, CDH6, CEACAM5, cholecystokinin 2 receptor, cKIT, CLDN18, CLDN18.2, CLDN6, CLDN6+CLDN9, CLL-1, cMET, cMET+EGFR, Cripto, CXCR4, DLL3, DPEP3, EFNA4, EGFR, EGFR+HER3, EGFR+MUC1, EGFRvIII, EPHA2, ETBR, FAP, FAPI, FCRH5, FGFR2, FGFR3, FLT3, FRα, GCC, GD2, GD3, Globo H, GPC3, GPCR5D, gpNMB, GPR20, HER2, HER2+HER3, IGF1R, IL13Ra, IL-4R, integrin β-6, KAAG1, L1CAM, LAMP1, Lewis Y Ag, LHRH receptor, LIV1, LIV1A, LRRC15, LY6E, Ly75 / CD205, MC1R, MELTF, mesothelin, MSLN, MT1-MMP, MUC1, MUC16 / CA-125, NaPi-2b, nectin-4, neurokinin 1 receptor, NKG2D, norepinephrine transporter, NOTCH3, NTSR1, P-cadherin, PDL1, PRLR, PSMA, PTK7, RNF43, ROR1, ROR2, SEZ6, SLAMF7, SLC44A4, SLITRK6, SS2R, STEAP1, TF, TIM1, TNFSF9, Trop-2, or a portion thereof.

[0045] In a related aspect, the present disclosure provides a pharmaceutical composition comprising the conjugate. In some embodiments, the conjugate comprises one or more of a miniprotein, a linker, a chelator, and a radionuclide.

[0046] In some embodiments, one or more components of the conjugate are provided using a vector, eg, a recombinant expression vector, that includes a nucleic acid encoding the polypeptide.

[0047] In some embodiments, the present disclosure provides host cells comprising vectors encoding one or more components of the conjugates provided herein.

[0048] In some embodiments, the present disclosure provides compositions capable of binding to, modulating, and / or inhibiting any one of the target proteins in Table 10. In some embodiments, the target protein selected from Table 10 is a human isoform. In some embodiments, the compositions of the disclosure are or comprise peptide therapy, peptide receptor radionuclide therapy.

[0049] In some embodiments, the compositions of the present disclosure are formulated for administration to a subject in need thereof (e.g., a pharmaceutical composition). In some embodiments, the compositions are administered for the treatment of one or more diseases, disorders, or conditions. In some embodiments, the disease, disorder, or condition is cancer. In some embodiments, the treatment comprises providing (e.g., by administering, e.g., by contacting, a cell or population of cells, e.g., a tumor) a composition, wherein the composition comprises a component that specifically binds to any one of the target proteins in Table 10. In some embodiments, the target protein selected from Table 10 is expressed on a cell or population of cells. In some embodiments, providing the composition treats the disease, disorder, or condition.

[0050] In some embodiments, the present disclosure provides methods for modulating any one of the target proteins in Table 10. That is, in some embodiments, the method of modulating the biological activity of any one of the target proteins in Table 10 comprises providing (e.g., by administering, e.g., by contacting a cell or population of cells) a composition comprising one or more components in an amount effective to modulate any one of the target proteins in Table 10 and its activity. For example, in some embodiments, the composition comprises a miniprotein that specifically binds to any one of the target proteins in Table 10 and one or more of a linker, a chelator, and / or a radionuclide. In some such embodiments, the composition binds to cells expressing any one of the target proteins in Table 10 and targets one or more therapies (e.g., a chelator, e.g., a radionuclide) to the cells expressing the target protein selected from Table 10, such as by internalization of the composition or components thereof into the cells.

[0051] In some embodiments, the present disclosure provides methods and compositions for use therein that can activate or inhibit immune cell responses. In some embodiments, the provided compositions are administered for the treatment of cancer. In some embodiments, the cancer is associated with the expression or overexpression of any one of the target proteins in Table 10. In some embodiments, the cancer is selected from non-small cell lung cancer (NSCLC), cutaneous squamous cell carcinoma, pancreatic cancer, primary hepatocellular carcinoma, colorectal cancer, renal clear cell carcinoma, breast cancer, and prostate cancer.

[0052] In some embodiments, the present disclosure provides methods and compositions for use in detecting the presence or extent of a disease, disorder, or condition in a subject. In some embodiments, the disease, disorder, or condition is cancer. In some embodiments, the subject has been diagnosed with cancer. In some embodiments, the subject is suspected of having or is at risk of having cancer. In some embodiments, a subject diagnosed with cancer has been treated with one or more treatments, e.g., a composition provided herein. In some embodiments, a method of detecting (e.g., monitoring / determining prognosis, diagnosing, etc.) comprises measuring the level of a target, or a miniprotein that specifically binds to a target, in a sample comprising one or more cells from the subject (e.g., using a cell-based assay) or in the subject (e.g., using in vivo scanning or measurement). In some such embodiments, the level of the target or miniprotein is used to determine the presence and / or extent of cancer in a subject by comparing the level with a control level, or with levels from the same patient at different time points.

[0053] In some embodiments, the present disclosure provides kits. In some such embodiments, the kits include one or more components, such as a miniprotein, a linker, a chelator, and / or a radionuclide, that can be combined in one or more ways for use in binding to a target (e.g., any one of the target proteins in Table 10). In some embodiments, the present disclosure provides compositions including a miniprotein (M), an optional linker (L), and one or both of a chelator (C) and a radionuclide (R), represented by a formula selected from one or more of MLCR, MLC, MCR, MLR, MC, ML, and MR. In some embodiments, M comprises or consists of a linear polypeptide, a folded polypeptide (e.g., a covalently linked polypeptide, a non-covalently linked polypeptide, or a polypeptide containing a disulfide linkage), a cysteine-rich peptide, a knottin peptide, a binder, an affibody, an engineered Kunitz domain, a monobody, an anticalin, a designed ankyrin repeat domain (DARPin), or an avimer. In some embodiments, the binder comprises or consists of a linear polypeptide, a folded polypeptide, and / or a non-disulfide sequence. In some embodiments, M is characterized in that it comprises 10-100 amino acids, and 100 amino acids or less. In some preferred embodiments, M is characterized in that it (i) is 100 amino acids and / or 12 kDa or less; (ii) contains at least one secondary structure element; (iii) contains a sequestered hydrophobic core; and / or exhibits cooperative folding. In some embodiments, M comprises about 100 amino acids or less, 90 amino acids, 85 amino acids, 80 amino acids, 75 amino acids, 70 amino acids, 65 amino acids, 60 amino acids, 55 amino acids, 50 amino acids, 45 amino acids, 40 amino acids, 35 amino acids, 30 amino acids, 25 amino acids, 20 amino acids, 15 amino acids, or 10 amino acids or less. In some embodiments, the miniprotein comprises at least one disulfide bridge.In some embodiments, the miniprotein comprises zero, one, two or more disulfide bonds.

[0054] In some embodiments, the disclosure provides a composition denoted as LC, where L comprises or consists of a linker and C comprises or consists of a chelator, and the linker is designed to be conjugated to a polypeptide.

[0055] In some embodiments, the present disclosure provides a composition, designated LCR, wherein L comprises or consists of a linker, C comprises or consists of a chelator, and R comprises or consists of a radionuclide, wherein the composition is capable of being conjugated to a miniprotein. In some embodiments, when L is present, L is a polyethylene glycol (PEG) linker of PEG2, PEG4, PEG6, PEG8, PEG12, PEG24, PEG36, an ester linker, an amide linker, a maleimide linker, a succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) linker, a propanoic acid linker, a caproleic acid linker, or (Gly)n-(γGlu)n- (SEQ ID NO: 79) or (PEG)n, where n is 1 to 10, (Gly)n 1~10 (SEQ ID NO: 80), or any fragment or combination thereof via a covalent bond. In some embodiments, when C is present, C is i) NOPO [ka] ii) Crown [ka] iii) DOTA, or [ka] iv) Macropa [ka] Comprises or consists of

[0056] In some embodiments, when C is present, C comprises or consists of a derivative of NOPO, Crown, Macropa, or tetrazacyclododecane-1,4,7,10-tetraacetic acid (DOTA). In some embodiments, when L is present and C is absent, L is covalently bonded to M. In some embodiments, when R is present, R comprises or consists of Ac-225, Ga-68, Pb-212, Lu-177, Cu-67, Cu-64, La-132, La-135, In-111, Ce-134, F-18, or At-211. In some embodiments, the composition binds to a target with an affinity of 1 pM to 100 nM as measured by an in vitro binding assay. In some embodiments, when M is present, miniprotein binding to a target modulates a biological function. In some embodiments, when M is present, M selectively binds to any one of the target proteins in Table 10, or a portion thereof.

[0057] In some embodiments, the present disclosure provides an isolated construct, or a pharmaceutically acceptable salt thereof, comprising a miniprotein (M), an optional linker (L), and at least one of a chelator (C) or a radionuclide (R).

[0058] In some embodiments, the present disclosure provides a pharmaceutical composition comprising a miniprotein (M), wherein the miniprotein selectively binds to a target.

[0059] In some embodiments, the composition exhibits adhesion in tumors, hi some embodiments, the composition exhibits passage through the kidney, liver, bone marrow, or spleen.

[0060] In some embodiments, the pharmaceutical composition comprises a miniprotein (M), an optional linker (L), and one or both of a chelator (C) and a radionuclide (R). In some embodiments, when C is present, C is covalently bound to M. In some embodiments, the chelation efficiency is >90%. In some embodiments, the pharmaceutical composition further comprises a radionuclide R. In some embodiments, when R is present, it is an alpha-emitter. In some embodiments, when R is present, it is Ac-225, Ga-68, Pb-212, Lu-177, Cu-67, Cu-64, La-132, La-135, In-111, Ce-134, F-18, or At-211. In various embodiments, the radionuclide R is directly conjugated to M via a prosthetic group. In some embodiments, M specifically binds to a target. In some embodiments, the target is any one of the target proteins in Table 10. In some embodiments, the target protein selected from Table 10 is expressed on a cell. In some embodiments, the cell is a human cell. In some embodiments, the human cell is a tumor cell. In some embodiments, the tumor cell is a solid tumor cell. In some embodiments, the miniprotein comprises or consists of a linear polypeptide, a folded polypeptide (e.g., a covalently linked polypeptide, a non-covalently linked polypeptide, or a polypeptide containing disulfide linkages), a cysteine-rich peptide, a knottin peptide, a binder, an affibody, an engineered Kunitz domain, a monobody, an anticalin, a designed ankyrin repeat domain (DARPin), or an avimer. In some embodiments, the miniprotein comprises one or more disulfide bonds. In some embodiments, the miniprotein is characterized by having nM or sub-nM binding affinity for the target in vivo or in cell-based assays. In some embodiments, the miniprotein has a binding affinity of 1 pM to 100 nM for any one of the target proteins in Table 10 on a cell line expressing a human isoform of the target protein selected from Table 10.In some embodiments, the miniprotein has an amino acid sequence of about 100 amino acids or less, 90 amino acids, 85 amino acids, 80 amino acids, 75 amino acids, 70 amino acids, 65 amino acids, 60 amino acids, 55 amino acids, 50 amino acids, 45 amino acids, 40 amino acids, 35 amino acids, 30 amino acids, 25 amino acids, 20 amino acids, 15 amino acids, or 10 amino acids or less.

[0061] In some embodiments, administration of the pharmaceutical composition to a subject in need thereof does not elicit an immune response, or any immune response that is elicited is tolerable to the subject. In some embodiments, the composition has high tumor tissue penetration. In some embodiments, the composition is not taken up and / or retained in the kidney or liver. In some embodiments, the composition is internalized in cells that express a human isoform of a target protein selected from Table 10.

[0062] In some embodiments, the composition comprises a miniprotein-drug conjugate comprising a miniprotein and at least one drug moiety. In some embodiments, the pharmaceutical composition comprises a miniprotein-drug conjugate comprising a miniprotein and at least one drug moiety. In certain embodiments, the drug moiety includes, but is not limited to, V-ATPase inhibitors, proapoptotic agents, Bcl2 inhibitors, MCL1 inhibitors, HSP90 inhibitors, IAP inhibitors, mTor inhibitors, microtubule stabilizers, microtubule destabilizers, auristatins, dolastatins, maytansinoids, MetAP (methionine aminopeptidase), inhibitors of nuclear export of protein CRM1, DPP-IV inhibitors, proteasome inhibitors, inhibitors of mitochondrial phosphoryl transfer reactions, protein synthesis inhibitors, kinase inhibitors, CDK2 inhibitors, CDK9 inhibitors, kinesin inhibitors, HDAC inhibitors, DNA damaging agents, DNA alkylating agents, DNA intercalators, DNA minor groove binders, and DHFR inhibitors.

[0063] In other embodiments, the drug moiety includes alkylating agents such as thiotepa and cyclosphosphamide (CYTOXAN™); alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramine, ethylenimines and methylamelamines, including imido- and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); camptothecins (including the synthetic analog topotecan); bryostatin; callystatin; CC-1065 (including its synthetic analogs adozelesin, carzelesin, and bizelesin); and derivatives thereof; cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatins, auristatins (including the analogs monomethyl-auristatin E and monomethyl-auristatin F (see, e.g., U.S. Patent Application Publication No. 2005-0238649, published October 27, 2005, which is hereby incorporated by reference in its entirety)); duocarmycins (including the synthetic analogs KW-2189 and CBI-TMI); eleutherobin; pancratistatin n); sarcodictyin; spongistatin; nitrogen mustards, such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine; trofosfamide, uracil mustard;nitrosureas, e.g., carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics, e.g., enediyne antibiotics (e.g., calichemicins, especially calichemicin gamma 11 and calichemicin fill, see e.g., Agnew, Chem. Intl. Ed. Engl. 33:183-186); dynemicin A A), including dynemycins; bisphosphonates, e.g., clodronate; esperamicin; and neocarzinostatin chromophores and related chromoprotein enediyne antibiotic chromophores), aclacinomycin, actinomycin, authramycin, azaserine, bleomycin, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (Adriamycin™) (morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxo rubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate;Purine analogues, such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogues, such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens, such as calsterone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone; anti-adrenals, such as aminoglutethimide, mitotane, trilostane; folic acid supplements, such as folinic acid acid; aceglatone; aldophosphamide glycosides; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatrexate; defofamine; demecolcine; diaziquone; eflornithine; elliptinium acetate; epothilones; etoglucide; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoids, e.g., maytansine and ansamitocin; mitoguazone, mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid acid); 2-ethylhydrazide; procarbazine; PSK®; razoxane; rhizoxin; schizofiran; spirogermanium; tenuazonic acid; triazicon; 2,2',2®-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A, and anguidine); urethane; vindesine; dacarbazine; mannomustine; mitabronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa;Taxoids, such as paclitaxel (TAXOL™, Bristol-Myers Squibb Oncology, Princeton, NJ) and docetaxel (TAXOTERE®, Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine (Gemzar™); 6-thioguanine; mercaptopurine; methotrexate; platinum analogs, such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine (Navelbine™); novantrone; teniposide; edatrexate; daunomycin; aminopterin; Xeloda; ibandronate; CPT-11; topoisomerase inhibitors RFS 2000; difluoromethylornithine (DMFO); retinoids, e.g., retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids, or derivatives of any of the above. Included in this definition are antihormonal agents that act to regulate or inhibit the action of hormones on tumors, such as antiestrogens and selective estrogen receptor modulators (SERMs), including tamoxifen (including Nolvadex™), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston™); and Also included are aromatase inhibitors, which inhibit the enzyme aromatase, which regulates estrogen production, such as 4(5)-imidazole, aminoglutethimide, megestrol acetate (Megace™), exemestane, formestane, fadrozole, vorozole (Rivisor™), letrozole (Femara™), and anastrozole (Arimidex™); and antiandrogens, such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids, or derivatives of any of the above.

[0064] In some embodiments, the pharmaceutical composition comprises one or more antioxidant molecules, wherein the antioxidant molecules neutralize free radicals. In some embodiments, the pharmaceutical composition comprises a stabilizer. In further embodiments, the stabilizer comprises gentisic acid or a salt thereof, ascorbic acid or a salt thereof, methionine, N-acetylcysteine, histidine, melatonin, ethanol, Se-methionine, or a combination thereof. In some embodiments, the pharmaceutical composition is characterized by exhibiting improved resistance to peptidases, proteases, or heat.

[0065] In some embodiments, a method of producing a composition represented by a formula selected from one or more of MLCR, MLC, MCR, MLR, MC, ML and MR, wherein M comprises a miniprotein (M), L comprises a linker (L), C comprises a chelator (C), and R comprises a radionuclide (R), the method comprising the steps of synthesizing the miniprotein (M) and / or the linker (L), and optionally reacting the chelator (C) and / or the radionuclide (R), and reacting one or more miniproteins (M) with the linker ( conjugating one or more miniproteins (M) to a chelator (C), one or more miniproteins (M) to a radionuclide (R), one or more miniproteins (M) to a chelator (C) via a linker (L), one or more miniproteins (M) to a chelator (C) and a radionuclide (R) via a linker (L), one or more miniproteins (M) to a radionuclide (R) via a chelator (C), or one or more miniproteins (M) to a radionuclide (R) via a linker (L).

[0066] In some embodiments, the method comprises reacting the chelator (C) with the radionuclide (R) at a temperature between about 25° C. and 75° C. for an incubation period. In some embodiments, the reacting of the chelator (C) with the radionuclide (R) is carried out for an incubation period of about 5 minutes to about 30 minutes. In further embodiments, the reacting of the chelator (C) with the radionuclide (R) is carried out at a pH in the range of about 5.0 to 7.4. In some embodiments, the reacting of the chelator (C) with the radionuclide (R) is carried out in an aqueous solution that is substantially free of alcohol.

[0067] In some embodiments, a method for delivering a radionuclide to a selected location within a patient comprises administering a composition represented by a formula selected from one or more of MLCR, MLC, MCR, MLR, MC, ML, and MR, wherein M comprises a miniprotein (M), L comprises a linker (L), C comprises a chelator (C), and R comprises a radionuclide (R). In some embodiments, the radionuclide is selected from Ac-225, Ga-68, Pb-212, Lu-177, Cu-67, Cu-64, La-132, La-135, In-111, Ce-134, F-18, or At-211. In some embodiments, the method further comprises performing an imaging procedure to assess localization of the radionuclide within the body, the imaging procedure optionally comprising positron emission tomography (PET) imaging or single photon emission computed tomography (SPECT) imaging. In further embodiments, the imaging procedure allows for patient selection. In certain embodiments, the imaging procedure allows for patient monitoring, hi some embodiments, the imaging procedure allows for the determination of an appropriate dose for treating a patient in need of a pharmaceutical composition comprising one or more miniproteins.

[0068] In some embodiments, the present disclosure provides a method of treating a subject in need thereof, comprising administering a composition comprising a miniprotein (M), an optional linker (L), and one or both of a chelator (C) and a radionuclide (R).

[0069] In some embodiments, L comprises or consists of a polyethylene glycol (PEG) linker, an ester linker, an amide linker, a maleimide linker, a valine-citrulline linker, a hydrazone linker, an N-succinimidyl-4-(2-pyridyldithio)butyrate (SPDB) linker, a succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) linker, a vinyl sulfone-based linker, a propanoic acid linker, a caproleic acid linker, or any fragment or combination thereof. i) NOPO [ka] ii) Crown [ka] iii) DOTA, or [ka] iv) Macropa [ka] In some embodiments, R comprises or consists of Ac-225, Ga-68, Pb-212, Lu-177, Cu-67, Cu-64, La-132, La-135, In-111, Ce-134, F-18, or At-211. In some embodiments, M comprises or consists of a linear polypeptide, a folded polypeptide (e.g., a covalently linked polypeptide, a non-covalently linked polypeptide, or a polypeptide containing disulfide linkages), a cysteine-rich peptide, a knottin peptide, a binder, an affibody, an engineered Kunitz domain, a monobody, an anticalin, a designed ankyrin repeat domain (DARPin), or an avimer. In some embodiments, M is characterized in that it contains between 10 and 100 amino acids, and 100 amino acids or less. In some preferred embodiments, M is characterized by (i) 100 amino acids and / or 12 kDa or less; (ii) at least two secondary structure elements; (iii) a sequestered hydrophobic core; and / or exhibiting cooperative folding. In some embodiments, the composition comprises at least one additional component. In some embodiments, the composition is capable of penetrating tumor tissue. In some embodiments, the miniprotein comprises about 100 amino acids or less, 90 amino acids, 85 amino acids, 80 amino acids, 75 amino acids, 70 amino acids, 65 amino acids, 60 amino acids, 55 amino acids, 50 amino acids, 45 amino acids, 40 amino acids, 35 amino acids, 30 amino acids, 25 amino acids, 20 amino acids, 15 amino acids, or 10 amino acids or less. In some embodiments, the miniprotein comprises at least one disulfide bridge. In some embodiments, the miniprotein specifically binds to a target. In some embodiments, the composition exhibits μm or nM binding affinity for the target in in vitro assays. In some embodiments, the composition binds to a target with an affinity of 1 pM to 100 nM as measured by an in vitro binding assay, hi some embodiments, the composition is characterized by having enhanced tissue penetration properties compared to a composition comprising a full-sized protein that binds to the same target.In some embodiments, mini-protein binding to the target modulates biological function. In some embodiments, administration of the composition to a subject in need thereof does not elicit an immune response, or the elicited immune response is tolerable to the subject. In some embodiments, the tolerable immune response includes a systemic immune response or a local immune response. In some embodiments, the subject is diagnosed with cancer. In some embodiments, cancer cells from the subject express any one of the target proteins in Table 10 or a portion thereof. In some embodiments, target expression is higher in cancer cells than in non-cancerous cells. In some embodiments, the cancer is selected from the group consisting of breast cancer, ovarian cancer, melanoma, pancreatic cancer, peripheral neuroma, glioblastoma, adrenocortical carcinoma, AIDS-related lymphoma, anal cancer, bladder cancer, meningioma, glioma, astrocytoma, cervical cancer, chronic myeloproliferative disorder, colon cancer, endometrial cancer, ependymoma, esophageal cancer, Ewing's sarcoma, extracranial germ cell tumor, extrahepatic bile duct cancer, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gestational trophoblastic tumor, hairy cell leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, hypopharyngeal cancer, islet cell carcinoma, Kaposi's sarcoma, laryngeal cancer, leukemia, lip cancer, oral cavity cancer, liver cancer, male breast cancer, malignant mesothelioma, medulloblastoma, Merkel cell carcinoma, metastatic squamous cell carcinoma of the cervix, multiple myeloma, and The cancer is selected from other plasma cell neoplasms, mycosis fungoides and Sézary syndrome, myelodysplastic syndrome, nasopharyngeal carcinoma, neuroblastoma, non-small cell lung cancer, small cell lung cancer, head and neck cancer, skin cancer, oropharyngeal cancer, bone cancer including osteosarcoma and malignant fibrous histiocytoma of bone, sinonasal cancer, parathyroid cancer, penile cancer, pheochromocytoma, pituitary tumor, prostate cancer, rectal cancer, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, small intestine cancer, soft tissue sarcoma, supratentorial primitive neuroectodermal tumor, pineoblastoma, testicular cancer, thymoma, thymic carcinoma, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter, urethral cancer, uterine sarcoma, vaginal cancer, vulvar cancer, and Wilms' tumor and other pediatric kidney tumors. In some embodiments, the composition is administered intravenously or subcutaneously. In some embodiments, the cancer is treated after administration of the composition.

[0070] Additionally disclosed herein is the use of the compositions disclosed herein for treating cancer in a subject. Additionally disclosed herein is an isolated polynucleotide comprising one or more nucleic acid sequences encoding a polypeptide selected from SEQ ID NOs: 1-68; or a nucleic acid sequence encoding a polypeptide comprising at least 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NOs: 1-68. Additionally disclosed herein is a vector comprising the isolated polynucleotide disclosed herein. Additionally disclosed herein is a host cell transformed with the isolated polynucleotide disclosed herein or the vector disclosed herein.

[0071] Additionally disclosed herein is a method for characterizing renal uptake of a composition, the method comprising: providing a plurality of kidney cells; contacting the plurality of kidney cells with a composition represented by a formula selected from one or more of MLCR, MLC, MCR, MLR, MC, ML, and MR, wherein M comprises a miniprotein (M), L comprises a linear, branched, or enzymatically cleavable linker (L), C comprises a chelator (C), and R comprises a radionuclide (R), and the composition further comprises a biotin group conjugated to fluorescently labeled streptavidin; and measuring renal uptake of the composition by lysing the plurality of kidney cells and detecting fluorescence of the fluorescently labeled streptavidin. In various embodiments, the plurality of kidney cells is provided in a well. In various embodiments, M comprises an amino acid sequence that shares at least 90% identity with any one of SEQ ID NOs: 1-68. In various embodiments, M comprises an amino acid sequence that shares 100% identity with any one of SEQ ID NOs: 1-68.

[0072] BRIEF DESCRIPTION OF THE DRAWINGS [Brief explanation of the drawings]

[0073] [Figure 1]Figure 1 shows analyses generated from SPECT / CT scans to quantify the injected dose per gram (%ID / g) of kidney tissue in mice treated with exemplary Nectin-4 charge variant conjugates (Compound ID Numbers: C14, C45, C48, and C52).

[0074] [Figure 2] Figure 2 shows an analysis generated from SPECT / CT scans to quantify the injected dose per gram (%ID / g) of kidney tissue in mice. Co-administration of an exemplary decoy peptide (Compound ID No.: C15) reduces kidney uptake of an exemplary Nectin-4-targeted mini-protein conjugate (Compound ID No.: C14) scaffold.

[0075] [Figure 3] Figure 3 shows analyses generated from SPECT / CT scans to quantify the injected dose per gram (%ID / g) of kidney tissue in mice. Exemplary B7H3 charge variant conjugates (Compound ID Numbers: C1, C3, C5, C9, and C43) demonstrate reduced levels of kidney retention in mouse biodistribution.

[0076] [Figure 4] Figure 4 shows an analysis generated from a SPECT / CT scan to quantify the injected dose per gram (%ID / g) of kidney tissue in mice. Co-administration of an exemplary decoy peptide (Compound ID No.: C7) reduces kidney uptake of an exemplary B7H3-targeting affibody conjugate (Compound ID No.: C9).

[0077] [Figure 5] Figure 5 shows an analysis generated from SPECT / CT scans to quantify the injected dose per gram (%ID / g) of kidney tissue in mice. Comparison of an exemplary conjugate with an exemplary version 1 cleavable linker (Compound ID No.: C64) with an exemplary Nectin-4 conjugate (Compound ID No.: C14) showed minimal alterations in kidney uptake and retention.

[0078] [Figure 6] FIG. 6 shows the DELFIA saturation binding curve fitting of an exemplary conjugate (Compound ID No.: C34) to estimate KD.

[0079] [Figure 7] FIG. 7 shows the DELFIA competitive binding curve of an exemplary conjugate (Compound ID No.: C40) for estimating Ki.

[0080] [Figure 8] Figure 8 shows the binding kinetics of an exemplary conjugate (Compound ID No.: C11) to an immobilized ligand. The change in signal over time is proportional to peptide binding to the ligand, resulting in a sensorgram. A 1:1 binding model is fitted to the black line to calculate the KD (M). Concentrations correspond to the labels on the figure as follows: a: 25 nM; b: 12.5 nM; c: 6.25 nM; d: 3.13 nM; e: 3.13 nM; f: 1.56 nM; and g: 0.78 nM.

[0081] [Figure 9-1] FIG. 9A shows the quantitative uptake of an exemplary AF647-labeled conjugate (Compound ID No.: C9) in an opossum kidney proximal tubule cell (OK-PTC) uptake assay.

[0082] [Figure 9-2] Figure 9B shows quantitative uptake in an opossum kidney proximal tubule cell (OK-PTC) uptake assay. Co-treatment with an exemplary decoy peptide (Compound ID No.: C15) at 100x and 10x molar excess reduces uptake of an exemplary AF647-labeled conjugate (Compound ID No.: C14).

[0083] [Figure 9-3]Figure 9C shows quantitative uptake in an opossum kidney proximal tubule cell (OK-PTC) uptake assay. Uptake of an exemplary AF647-labeled control conjugate (Compound ID No. C14) is reduced in a dose-dependent manner by pretreatment with a mixture of lysine and arginine amino acids.

[0084] [Figure 10] Figure 10 shows quantitative uptake in an opossum kidney proximal tubule cell (OK-PTC) uptake assay. Co-treatment with a 20-fold excess of exemplary decoys C79, C96, and C78 reduces uptake of test agents C132-C139.

[0085] [Figure 11] Figure 11 shows quantitative uptake in an opossum kidney proximal tubule cell (OK-PTC) uptake assay. Co-treatment with a 10x molar excess of exemplary decoy peptides (Compound ID Nos. C15, C80, and C83) reduces the uptake of an exemplary biotinylated test agent (Compound ID No. C14).

[0086] [Figure 12] Figure 12 shows an analysis generated from SPECT / CT scans to quantify the injected dose per gram (%ID / g) of kidney tissue in mice. Co-administration of an exemplary decoy peptide (Compound ID No.: C79) reduces kidney uptake and retention of an exemplary Nectin-4-targeted mini-protein conjugate (Compound ID No.: C67) scaffold.

[0087] [Figure 13] Figure 13 shows an analysis generated from SPECT / CT scans to quantify the injected dose per gram (%ID / g) of kidney tissue in mice. Comparison of an exemplary conjugate with an exemplary version 2 cleavable linker (Compound ID No.: C68) with an exemplary Nectin-4 conjugate (Compound ID No.: C67) showed no change in early kidney uptake and a modest reduction in kidney retention at later time points.

[0088] [Figure 14] Figure 14 shows an analysis generated from SPECT / CT scans to quantify the injected dose per gram (%ID / g) of kidney tissue in mice. Comparison of an exemplary conjugate with an albumin-binding motif added (Compound ID No.: C69) with an exemplary Nectin-4 conjugate (Compound ID No.: C67) showed a reduction in kidney uptake.

[0089] [Figure 15] Figure 15 shows tumor volume measurements after single-dose treatment of test article in a mouse efficacy study. HT-1376 cells exogenously expressing target were treated with either vehicle or X or 2X nCi of 225Ac-labeled test article (Compound ID No.: C116) and showed a reduction in tumor volume.

[0090] [Figure 16] Figure 16 shows body weight measurements after single dose treatment of test article in a mouse efficacy study. HT-1376 cells exogenously expressing target were treated with either vehicle or X or 2X nCi of 225Ac-labeled test article (Compound ID No.: C116) and showed no change in body weight. DETAILED DESCRIPTION OF THE INVENTION

[0091] Detailed Description Among other things, the present disclosure provides compositions and methods of use thereof. In some embodiments, the compositions selectively bind to a target. In some embodiments, the compositions include one or more therapeutic agents (e.g., chelators, radionuclides), which are selectively targeted to cells expressing, for example, any one of the target proteins in Table 10, such that target-expressing cells are treated and cells that do not express the target are not treated. In various embodiments, the target protein is any one of the target proteins in Table 10. In certain embodiments, the target protein is Nectin-4. The present disclosure recognizes that a source of difficulty in treating target-expressing cells (e.g., cancer cells) is that conventional therapies are not selective enough to specifically target cells and deliver therapeutic agents in a manner that minimizes damage to surrounding cells. The present disclosure provides insight that the combination of selective targeting with a particular therapeutic agent, such as a chelator and / or a radionuclide (e.g., an alpha-emitter), offers advantages over previously used therapeutic agents (e.g., antibodies, beta-emitters, etc.).

[0092] Unless otherwise defined herein, scientific and technical terms used in connection with this disclosure shall have the meanings commonly understood by those skilled in the art. Furthermore, unless the context requires otherwise, singular terms shall include the plural, and plural terms shall include the singular. Generally, the nomenclatures used in connection with, and techniques of, biochemistry, enzymology, molecular and cell biology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein are those well known and commonly used in the art.

[0093] The methods and techniques of the present disclosure are generally, unless otherwise indicated, performed according to conventional methods well known in the art and as described in various general and more specific references cited and discussed throughout the specification. For example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989); Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992, and Supplements to 2002); Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1990);Wittrup and VanAntwerp, Fine Affinity Discrimination by Yeast Surface Display and Flow Cytometry, Biotechnol. Prog. 2002, (16) 31-37;C. Queen et al., A humanized antibody that binds to the interleukin 2 receptor, Proc. Natl. Acad. Sci. USA 1989, 86 (24) 10029-10033;Scheinberg DA and See McDevitt MR. Actinium-225 in targeted alpha-particle therapeutic applications. Curr Radiopharm. 2011;4(4):306-320.

[0094] All publications, patents, and other references mentioned herein are hereby incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. The materials, methods, and examples disclosed herein are illustrative only and are not intended to be limiting. definition

[0095] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Furthermore, unless the context requires otherwise, singular terms shall include the plural, and plural terms shall include the singular. Generally, the nomenclatures used in connection with, and techniques of, biochemistry, enzymology, molecular and cell biology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein are those well known and commonly used in the art.

[0096] Throughout this specification and the claims, the word "comprise" or variations thereof, such as "comprises" or "comprising," will be understood to imply the inclusion of a stated integer or group of integers, but not the exclusion of any other integer or group of integers.

[0097] As used herein, ranges and amounts can be expressed as "about" a particular value or range. About also includes the exact amount. Thus, "about 100 nucleotides" can mean "about 100 nucleotides" and also "100 nucleotides." Generally, the term "about" as used herein includes amounts that are expected to be within experimental error.

[0098] Unless otherwise indicated, as an example for all sequences described herein under the general format "SEQ ID NO: 1," a "nucleic acid comprising SEQ ID NO: 1" refers to a nucleic acid, at least a portion of which has either (i) the sequence of SEQ ID NO: 1, or (ii) a sequence complementary to SEQ ID NO: 1. The choice between the two is dictated by the context. For example, if the nucleic acid is used as a probe, the choice between the two is dictated by the requirement that the probe be complementary to the desired target.

[0099] As used herein, the term "administration" refers to providing a composition to a subject or system. Administration to a subject can be by any appropriate route, dose, and / or dose schedule.

[0100] As used herein, the term "affibody" refers to a subgenus of miniproteins. Affibodies are molecules derived from the Z-domain of Staphylococcus aureus protein A, consisting of three alpha helices with 58 amino acids and a molar mass of approximately 6 kDa. For exemplary details of the structure and use of affibodies, see Orlova, A; Magnusson, M; Eriksson, TL; Nilsson, M; Larsson, B; Hoiden-Guthenberg, I; Widstrom, C; Carlsson, J et al. (2006). "Tumor imaging using a picomolar affinity HER2 binding affibody molecule", Cancer Res. 66 (8): 4339-48. Exemplary Affibody® Molecules are commercially available from Abcam Corp. Cambridge Massachusetts. Affibodies are stable at high temperatures and under acidic or alkaline conditions. Target specificity is achieved by randomizing 13 amino acids located in two alpha-helices involved in the binding activity of the parent protein domain (Feldwisch J, Tolmachev V.; (2012) Methods Mol Biol. 899:103-26).

[0101] As used herein, the term "affinity maturation" generally refers to a process whereby successive changes (e.g., successive mutations) to a sequence are made and selection of polypeptide sequences is performed to select one or more sequences that have increased affinity relative to the "starting" sequence or alternatively, a sequence that has a lower affinity relative to one that has a higher affinity.

[0102] As used herein, the terms "amino acid sequence" and "polypeptide" refer to a polymer of amino acids connected by one or more peptide bonds. Polypeptides of the present disclosure include both naturally occurring and non-naturally occurring proteins, as well as any fragments, portions, peptides, variants, derivatives, and analogs thereof. Polypeptides can be monomeric or polymeric. Furthermore, polypeptides can contain several different domains, each of which has one or more distinct activities. Polypeptides can be fully or partially synthetic or otherwise modified (i.e., contain one or more synthetically produced amino acids and / or modifications thereof). The term "peptide" can be used to refer to short polypeptides, e.g., those containing fewer than about 70 amino acids (e.g., between about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or 70 amino acids).

[0103] As used herein, the term "anticalin" refers to a subgenus of miniproteins.Anticalins are engineered proteins derived from lipocalins (Beste G, Schmidt FS, Stibora T, Skerra A. (1999) Proc Natl Acad Sci US A. 96(5): 1898-903; Gebauer and Skerra (2009) Curr Opinion in Chemical Biology 13:245-255).Anticalins possess an eight-stranded b-barrel, which forms a highly conserved core unit among lipocalins and naturally forms a binding site for ligands through four structurally variable loops at the open end.Although anticalins are not homologous to the IgG superfamily, they exhibit the following features that have been considered typical for antibody binding sites: (i) high structural plasticity as a result of sequence variation, and (ii) increased conformational flexibility, which allows them to be induced to fit targets with different shapes.

[0104] As used herein, a "compound" refers to a miniprotein having at least an amino acid sequence. A compound may include a miniprotein with different modifications, e.g., N- or C-terminal modifications. In various embodiments, a "compound" may include a miniprotein and one or more additional elements, examples of which include a linker, a chelator, and / or a radionuclide. For example, a compound may include a miniprotein conjugated to a chelator and / or a radionuclide, e.g., via a linker. As referred to herein, compounds are identified by a specific compound number, e.g., "C1," "C2," "C3," etc. Different compounds may have different sequences. In various embodiments, different compounds may have the same sequence (e.g., assigned the same SEQ ID NO), but may have one or more of different modifications (e.g., different N- or C-terminal modifications), different linkers, different chelators, and / or different radionuclides.

[0105] As used herein, the term "attenuate" generally refers to a functional deletion, including a mutation, partial or complete deletion, insertion, or other variation made to a gene sequence or a sequence controlling the transcription of a gene sequence, that reduces or inhibits the production of the gene product or makes the gene product non-functional. In some examples, functional deletions are described as knockout mutations. Attenuation also includes amino acid sequence changes by modifying nucleic acid sequences, placing a gene under the control of a less active promoter, down-regulation, expressing interfering RNA, ribozymes or antisense sequences targeting the gene of interest, or any other technique known in the art. In one example, the sensitivity of a particular enzyme to feedback inhibition or inhibition caused by a composition that is neither a product nor a reactant (non-pathway-specific feedback) is reduced so that the enzyme activity is not affected by the presence of a compound. In another example, an enzyme that has been modified to be less active can be referred to as attenuated.

[0106] As used herein, the term "avimer" refers to a subgenus of miniproteins. Avimers are a class of antibody mimics consisting of two or more peptide sequences, preferably 30-35 amino acids each, derived from the A-domains of various membrane receptors and connected by a linker peptide. Binding of target molecules occurs via the A-domains, and domains with desired binding specificities can be selected, for example, by phage display techniques. The binding specificities of different A-domains contained in avimers can be, but do not have to be, identical (Weidle UH, et al., (2013), Cancer Genomics Proteomics; 10(4): 155-68). For further details, see Nature Biotechnology 23(12), 1556-1561 (2005) and Expert Opinion on Investigational Drugs 16(6), 909-917 (June 2007).

[0107] As used herein, the term "binder" refers to a subgenus of miniproteins. Binders are characterized in that they comprise or consist of polypeptides (e.g., peptides) capable of binding to, or having a known ability to engage and associate with, a target or a portion thereof. Binders generally comprise cysteine-containing peptides containing one or more disulfide bonds, although some binders do not contain cysteine ​​residues and / or disulfide bonds. Binders preferably are rapidly cleared from the circulation when systemically administered to a mammalian subject. As will be understood, given the context, reference to a binder can be or include its nucleic acid sequence or the amino acid sequence encoding it. Binders can be provided, for example, as polynucleotides, polypeptides, using vectors, host cells, etc., and / or any combination of modalities. Binders can be derived or produced using any method known to those of skill in the art. For example, in some embodiments, binders can be recombinant (i.e., produced using a recombinant nucleic acid encoding the polypeptide). In some embodiments, the binder may be synthetic (e.g., synthesized using standard solid phase synthesis methods, such as solid phase peptide synthesis, known to those of skill in the art (see, e.g., Palomo, J. RSC Adv., 2014,4, 32658-32672) and described herein).

[0108] The term "chelator," as used herein, refers to any molecule or moiety capable of forming a complex (i.e., a "chelate") with a metal ion. Chelators generally have two or more unshared electron pairs that can be used to donate to a metal ion. The metal ion is usually coordinated to the chelator by two or more pairs of electrons.

[0109] As used herein, the term "conjugated" refers to the joining of two compounds or agents by covalent or non-covalent means. In some embodiments, a "conjugate" may refer to, for example, a peptide coupled to one or more of a linker, a chelator, and / or a radionuclide.

[0110] As used herein, a "conservative amino acid substitution" refers to the replacement of an amino acid residue with another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). Generally, conservative amino acid substitutions do not substantially change the functional properties of a protein. When two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity or degree of homology can be subsequently adjusted to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those skilled in the art. See, e.g., Pearson, 1994, Methods Mol. Biol. 24:307-31 and 25:365-89 (hereby incorporated by reference). The following six groups each contain amino acids that are conservative substitutions for one another: 1) serine (S), threonine (T); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), alanine (A), valine (V), and 6) phenylalanine (F), tyrosine (Y), tryptophan (W).

[0111] As used herein, the terms "cysteine-rich peptide" and "CDP" are used interchangeably and refer to a subgenus of miniproteins that generally contain at least two independently folding domains and a high density of cysteines. In some embodiments, a CDP contains at least one, two, three, four, or more cysteine ​​residues over a span of about 10 to about 90 amino acid residues, preferably 13 to 80 amino acid residues (https: / / pubmed.ncbi.nlm.nih.gov / 29483648 / ). In some embodiments, a CDP contains a constrained distribution of cysteines, Cys-X. [0~15] -Cys-X [0~15] -Cys-X [0~15] -Cys-X [0~15] -Cys-X [0~15] -Cys (wherein X represents any amino acid) (SEQ ID NO: 81).

[0112] As used herein, the term "deletion" generally refers to the removal of one or more nucleotides from a nucleic acid molecule, or one or more amino acids from a protein, with the flanking regions joined together.

[0113] As used herein, the phrase "degenerate variant" of a reference nucleic acid sequence encompasses nucleic acid sequences that can be translated according to the standard genetic code to provide the same amino acid sequence as that translated from the reference nucleic acid sequence. The terms "degenerate oligonucleotide" or "degenerate primer" are used to refer to oligonucleotides that can hybridize to target nucleic acid sequences that are not necessarily identical in sequence to each other within one or more specific segments, but are homologous.

[0114] As used herein, the term "derived from," with respect to a nucleic acid sequence, refers to a nucleic acid sequence that has at least 85% sequence identity to a naturally occurring reference nucleic acid sequence from which it is derived. The term "derived from," with respect to an amino acid sequence, refers to an amino acid sequence that has at least 85% sequence identity to a naturally occurring reference amino acid sequence from which it is derived. The term "derived from," as used herein, does not refer to any particular process or method for obtaining a nucleic acid or amino acid sequence. For example, a nucleic acid or amino acid sequence can be chemically synthesized.

[0115] As used herein, the term "designed ankyrin repeat domain (DARPin)" refers to a subgenus of miniproteins. DARPins are peptides derived from ankyrin, a family of proteins that mediate the binding of integral membrane proteins to the cytoskeleton. A single ankyrin repeat is preferably a 33-residue motif consisting of two alpha-helices and one beta-turn. These can be engineered to bind to different targets by randomizing the residues in the first alpha-helix and beta-turn of each repeat. Their binding interface can be increased by increasing the number of modules (a method of affinity maturation). For further details, see J. Mol. Biol. 332, 489-503 (2003), PNAS 100(4), 1700-1705 (2003) and J. Mol. Biol. 369, 1015-1028 (2007) and US20040132028A1. DARPins typically provide rigid interfaces and lack conformational flexibility (Gebauer and Skerra, 2009).

[0116] As used herein, the term "domain" refers to a structure of a biomolecule that contributes to the known or suspected function of the biomolecule. A domain can be coextensive with a region or portion thereof; a domain can also include distinct, non-contiguous regions of a biomolecule. Examples of protein domains include, but are not limited to, Ig domains, extracellular domains, transmembrane domains, and cytoplasmic domains.

[0117] As used herein, the term "engineered Kunitz domain" refers to a subgenus of mini-proteins.Engineered Kunitz domains are preferably peptides derived from the Kunitz domains of Kunitz-type protease inhibitors, such as bovine pancreatic trypsin inhibitor (BPTI), amyloid precursor protein (APP) or tissue factor pathway inhibitor (TFPI).Kunitz domains have a molecular weight of approximately 6 kDA, and domains with the required target specificity can be selected by display techniques such as phage display (Weidle et al., (2013), Cancer Genomics Proteomics; 10(4): 155-68).

[0118] As used herein, the term "expression control sequence" refers to polynucleotide sequences necessary to affect the expression of coding sequences to which they are operably linked. Expression control sequences are sequences that control the transcription, post-transcriptional events, and translation of nucleic acid sequences. Expression control sequences include appropriate transcription initiation, termination, promoter, and enhancer sequences; efficient RNA processing signals, such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (e.g., ribosome binding sites); sequences that enhance protein stability; and, if desired, sequences that enhance protein secretion. The nature of such control sequences varies depending on the host organism; in prokaryotes, such control sequences generally include promoters, ribosome binding sites, and transcription termination sequences. The term "control sequence" is intended to include, at a minimum, all components whose presence is essential for expression, and may also include additional components whose presence is advantageous, such as leader sequences and fusion partner sequences.

[0119] As used herein, the term "fusion protein" refers to a polypeptide comprising a polypeptide or fragment coupled to a heterologous amino acid sequence. Fusion proteins are useful because they can be constructed to contain two or more desired functional elements from two or more different proteins. A fusion protein comprises at least 10 contiguous amino acids from the polypeptide of interest, more preferably at least 20 or 30 amino acids, even more preferably at least 40, 50, or 60 amino acids, and even more preferably at least 75, 100, or 125 amino acids. Fusions comprising the entire protein of the present disclosure have particular utility. The heterologous polypeptide contained within the fusion protein of the present disclosure is at least 6 amino acids in length, often at least 8 amino acids in length, and usefully at least 15, 20, and 25 amino acids in length. Fusions comprising larger polypeptides, such as IgG Fc regions, and even entire proteins, such as green fluorescent protein ("GFP") chromophore-containing proteins, have particular utility. Fusion proteins can be produced recombinantly by constructing a nucleic acid sequence encoding a polypeptide or fragment thereof in frame with a nucleic acid sequence encoding a different protein or peptide, and then expressing the fusion protein. Alternatively, fusion proteins can be produced chemically by cross-linking the polypeptide or fragment thereof to another protein.

[0120] As used herein, when referring to a protein, "homology" to a second protein may exist if the nucleic acid sequence encoding the protein has a similar sequence to the nucleic acid sequence encoding the second protein. Alternatively, a protein has homology to a second protein if the two proteins have "similar" amino acid sequences (thus the term "homologous proteins" is defined to mean that the two proteins have similar amino acid sequences). Homology between two regions of amino acid sequence (especially with respect to predicted structural similarities) may be interpreted as implying similarity in function. Homologous proteins or peptides with non-identical residue positions are often recognized as differing by conservative amino acid substitutions.

[0121] As used herein, the term "identical" refers to at least two nucleic acid or at least two amino acid sequences or subsequences that have a specified percentage of nucleotides or amino acids, respectively, that are the same when compared and aligned for maximum correspondence, as determined by using a sequence comparison algorithm or by visual inspection. For sequence comparison, typically one sequence serves as a reference sequence to which a test sequence is compared. The length of sequence identity comparison can span any number of nucleotides or amino acids. When using a sequence comparison algorithm, the test sequence and the reference sequence are input into a computer, and subsequence coordinates are designated as necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) compared to the reference sequence based on the designated program parameters. Several algorithms are known in the art. Non-limiting examples of algorithms suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described, for example, in Altschul et al. (1990) J. Mol. Biol. 215: 403-410 and Altschul et al. (1977) Nucleic Acids Res. 25: 3389-3402, respectively. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information. Additionally or alternatively, sequences can be compared using FASTA, Gap, or Bestfit, which are programs in the Wisconsin Package Version 10.0, Genetics Computer Group (GCG), Madison, Wis. FASTA provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences.Pearson, Methods Enzymol. 183:63-98 (1990), which is hereby incorporated by reference in its entirety. For example, percent sequence identity can be determined using FASTA with its default parameters (word size of 6 and NOPAM factor as the scoring matrix), or using Gap with its default parameters, as provided in GCG Version 6.1, which is hereby incorporated by reference.

[0122] As used herein, the term "isolated" polynucleotide or polypeptide is one that is substantially separated from other cellular components that naturally accompany the native polynucleotide in its natural host cell, such as ribosomes, polymerases, and genomic sequences with which it is naturally associated. For example, an isolated molecule, depending on its origin or source, can be: (1) unassociated with the naturally associated components that accompany it in its native state; (2) present in a purity not found in nature, where purity can be declared with respect to the presence of other cellular material (e.g., free from other proteins from the same species); (3) expressed by cells from a different species; or (4) non-naturally occurring (e.g., it is a fragment of a naturally occurring polynucleotide or polypeptide, or it contains amino acid analogs or derivatives not found in nature, or linkages other than standard peptide bonds). Thus, a polynucleotide or polypeptide that is chemically synthesized or synthesized in a cellular system different from the cell in which it naturally originates is "isolated" from its naturally associated components. A polynucleotide or polypeptide can also be rendered substantially free of naturally associated components by isolation using protein purification techniques well known in the art. As defined herein, "isolated" does not necessarily require that any molecule so described be physically removed from its native environment. In some embodiments, when used in reference to an isolated construct, "isolated" refers to the absence of pharmaceutically acceptable salts.

[0123] As used herein, the term "K D " or "Kd" refers to the dissociation equilibrium constant for a particular targeting molecule-targeting protein interaction. Typically, antibodies of the present disclosure have a dissociation equilibrium constant of about 10, as determined, for example, using surface plasmon resonance (SPR) techniques in a BIACORE instrument. -7 Less than m, e.g., about 10 -8 M, 10 -9 M or 10 -10 The dissociation equilibrium constant (KD ) and binds to any one of the target proteins in Table 10.

[0124] As used herein, the term " knockout " generally refers to a gene whose expression or activity level is reduced to zero.In some cases, a gene is knocked out by deleting part or all of its coding sequence.In other cases, a gene is knocked out by introducing one or more nucleotides into its open reading frame, which causes the translation of nonsense or other nonfunctional protein product.

[0125] The term "knottin" as used herein refers to a miniprotein structural motif that contains three disulfide bridges.

[0126] The term "knottin peptides," as used herein, refers to a subgenus of miniproteins that contain at least one knottin.

[0127] The term "linker," as used herein, refers to the moiety used to conjugate the miniprotein to the chelator.

[0128] As used herein, the term "miniprotein" refers to a short protein of 10-100 amino acids with a well-defined fold that includes two or more secondary structure elements, a constrained hydrophobic core, and / or cooperative folding. CDPs, knottins, affibodies, engineered Kunitz domains, monobodies (adnectins), anticalins, designed ankyrin repeat domains (DARPins), avimers, and binders disclosed herein are all examples of miniproteins.

[0129] As used herein, the term "modification," with respect to a nucleic acid sequence, refers to a nucleic acid sequence that contains at least one substitution, alteration, inversion, addition, or deletion of a nucleotide compared to a reference nucleic acid sequence. As used herein, the term "modification," with respect to an amino acid sequence, refers to an amino acid sequence that contains at least one substitution, alteration, inversion, addition, or deletion of an amino acid residue compared to a reference nucleic acid sequence.

[0130] As used herein, the term "modified derivative" refers to a polypeptide or fragment thereof that is substantially homologous in primary structural sequence but includes, for example, in vivo or in vitro chemical and biochemical modifications or incorporates amino acids not found in the native polypeptide. Such modifications include, for example, acetylation, carboxylation, phosphorylation, glycosylation, ubiquitination, labeling with, for example, radionuclides, and various enzymatic modifications, as will be readily understood by those skilled in the art. Various methods for labeling polypeptides and various substituents or labels useful for such purposes are well known in the art, including radioisotopes such as 125I, 32P, 35S, and 3H, ligands that bind to labeled antiligands (e.g., antibodies), fluorophores, chemiluminescent agents, enzymes, and antiligands that can function as specific binding pair members for labeled ligands. The choice of label depends on the required sensitivity, ease of conjugation with primers, stability requirements, and available instrumentation. Methods for labeling polypeptides are well known in the art. See, e.g., Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992, and Supplements to 2002), hereby incorporated by reference.

[0131] As used herein, the term "molecule" means any compound, including but not limited to small molecules, peptides, proteins, sugars, nucleotides, nucleic acids, lipids, etc., and such compounds may be natural or synthetic.

[0132] As used herein, the terms "monobody" and "Adnectin" are used interchangeably and refer to a subgenus of miniproteins. Monobodies refer to molecules that are preferably based on the tenth extracellular domain of human fibronectin III (10Fn3) and adopt a preferably 94-residue Ig-like b-sandwich fold with two to three exposed loops, but lack a central disulfide bridge (Gebauer and Skerra (2009) Curr Opinion in Chemical Biology 13:245-255). Adnectins with desired target specificity can be engineered by introducing modifications into specific loops of the protein.

[0133] As used herein, the term "mutein" or "mutant protein" or "variant" refers to a protein comprising an amino acid sequence having at least one variation (e.g., an insertion, deletion, or substitution, which may be a conservative or non-conservative substitution) compared to a reference sequence. When applied to a sequence (e.g., a nucleic acid sequence, an amino acid sequence), "mutated" means that a nucleotide in a nucleic acid sequence or an amino acid in an amino acid sequence may be inserted, deleted, or changed compared to the reference sequence. A single change may be made at a locus (point mutation), or multiple nucleotides or amino acids may be inserted, deleted, or changed at a single locus. Furthermore, one or more changes may be made at any number of loci within a nucleic acid or amino acid sequence. Nucleic acid or amino acid sequences can be mutated by any method known in the art, including, but not limited to, mutagenesis techniques, such as "error-prone PCR" (a process for performing PCR under conditions in which the copying fidelity of the DNA polymerase is low, resulting in a high rate of point mutations along the entire length of the PCR product; see, e.g., Leung et al., Technique, 1:11-15 (1989) and Caldwell and Joyce, PCR Methods Applic. 2:28-33 (1992)); "oligonucleotide-directed mutagenesis" (a process that allows for the generation of site-specific mutations in any cloned DNA segment of interest; see, e.g., Reidhaar-Olson and Sauer, Science 241:53-57 (1988)); directed evolution (e.g., exposing a polypeptide to different sets of conditions that result in the production of different polypeptides with one or more amino acid changes that may or may not confer greater suitability to the polypeptide); and site-directed mutagenesis (e.g., specifically directed changes in a sequence).

[0134] As used herein, the term "non-disulfide sequence" refers to an amino acid sequence that encodes a polypeptide that does not contain more than one cysteine ​​residue and / or disulfide bond in its folded, active form. In some embodiments, a miniprotein may comprise or consist of a non-disulfide sequence.

[0135] As used herein, the term "non-peptide analog" refers to a compound that has properties similar to those of a reference polypeptide. A non-peptide compound may also be called a "peptide mimetic" or "peptidomimetic." See, e.g., Jones, Amino Acid and Peptide Synthesis, Oxford University Press (1992); Jung, Combinatorial Peptide and Nonpeptide Libraries: A Handbook, John Wiley (1997); Bodanszky et al., Peptide Chemistry—A Practical Textbook, Springer Verlag (1993); Synthetic Peptides: A Users Guide, (Grant, ed., W.H. Freeman and Co., 1992); Evans et al., J. Med. Chem. 30:1229 (1987); Fauchere, J. Adv. Drug Res. 15:29 (1986); Veber and Freidinger, Trends Neurosci., 8:392-396 (1985); and the references cited in each of the above, which are hereby incorporated by reference. Such compounds are often developed with the aid of computerized molecular modeling. Peptide mimetics that are structurally similar to the useful peptides of the present disclosure can be used to produce an equivalent effect and are therefore contemplated as part of the present disclosure.

[0136] As used herein, the terms "nucleic acid sequence" and "polynucleotide" are used interchangeably to refer to a polymer of nucleotides. The term includes DNA molecules (e.g., cDNA or genomic or synthetic DNA) and RNA molecules (e.g., mRNA or synthetic RNA), as well as DNA or RNA analogs containing non-natural nucleotide analogs, non-native internucleoside linkages, or both. Nucleic acids can be in any topological conformation. For example, nucleic acids can be single-stranded, double-stranded, triple-stranded, quadruplexed, partially double-stranded, branched, hairpinned, circular, or padlocked. Nucleic acid sequences can contain natural, non-natural, or modified nucleotides; they can contain natural, non-natural, or modified internucleotide linkages, such as phosphoramidate or phosphorothioate linkages, instead of the phosphodiesters found between nucleotides in unmodified nucleic acid sequences. Nucleic acid sequences include, but are not limited to, all nucleic acid sequences obtained by any means available in the art, including, but not limited to, recombinant means, such as cloning nucleic acid sequences from recombinant libraries or cell genomes using conventional cloning techniques and polymerase chain reaction, as well as synthetic means.Polynucleotides of the present disclosure can include both the sense and antisense strands of RNA, cDNA, genomic DNA, and synthetic forms and mixed polymers of the above.They can be chemically or biochemically modified or contain non-natural or derivatized nucleotide bases, as will be readily understood by those skilled in the art.Such modifications include, for example, labels, methylation, substitution of one or more naturally occurring nucleotides with analogs, internucleotide modifications, such as uncharged linkages (e.g., methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), pendant moieties (e.g., polypeptides), intercalators (e.g., acridines, psoralens, etc.), chelators, alkylating agents, and modified linkages (e.g., alpha-anomeric nucleic acids, etc.). Synthetic molecules that mimic polynucleotides in their ability to bind to designated sequences through hydrogen bonding and other chemical interactions are also included. Such molecules are known in the art and include, for example, those in which peptide linkages replace phosphate linkages in the backbone of the molecule. Other modifications can include, for example, analogs in which the ribose ring contains a bridging moiety or other structure, such as those found in "locked" nucleic acids.

[0137] As used herein, the term "operably linked" or "operably linked" expression control sequences refers to a linkage in which the expression control sequences are contiguous with a gene of interest so as to regulate the gene of interest, as well as expression control sequences that act in trans or at a distance to regulate the gene of interest.

[0138] As used herein, the term "polypeptide variant" or "mutein" refers to a polypeptide whose sequence contains one or more amino acid insertions, duplications, deletions, rearrangements, or substitutions compared to the amino acid sequence of a native or wild-type protein. A mutein may have one or more amino acid point substitutions (a single amino acid at one position is changed to another amino acid), one or more insertions and / or deletions (one or more amino acids are inserted or deleted, respectively) in the sequence of a naturally occurring protein, and / or a shortening of the amino acid sequence at either or both the amino and carboxy termini. A mutein may have the same biological activity as a naturally occurring protein, but preferably has a different biological activity. A mutein has at least 85% overall sequence homology to its wild-type counterpart. Even more preferred is a mutein having at least 90% overall sequence homology to a wild-type protein. In an even more preferred embodiment, the mutein exhibits at least 95% sequence identity, even more preferably 98%, even more preferably 99%, and even more preferably 99.9% overall sequence identity. Sequence identity can be measured by any common sequence analysis algorithm, such as Gap or Bestfit. Amino acid substitutions can include (1) those that reduce susceptibility to proteolysis, (2) those that reduce susceptibility to oxidation, (3) those that alter binding affinity for forming protein complexes, (4) those that alter binding affinity or enzymatic activity, and (5) those that confer or modify other physicochemical or functional properties of such analogs.

[0139] As used herein, the term "polypeptide fragment" refers to a polypeptide that has a deletion, such as an amino-terminal and / or carboxy-terminal deletion, compared to a full-length polypeptide. In a preferred embodiment, a polypeptide fragment is a continuous sequence in which the amino acid sequence of the fragment is identical to the corresponding position in a naturally occurring sequence. The fragment is typically at least 5, 6, 7, 8, 9, or 10 amino acids in length, preferably at least 12, 14, 16, or 18 amino acids in length, more preferably at least 20 amino acids in length, more preferably at least 25, 30, 35, 40, or 45 amino acids in length, even more preferably at least 50 or 60 amino acids in length, and even more preferably at least 70 amino acids in length.

[0140] As used herein, the term "radionuclide" as used herein refers to an atom capable of undergoing radioactive decay.

[0141] As used herein, the term "recombinant" refers to a biological molecule, e.g., a gene or protein, that (1) has been removed from its naturally occurring environment, (2) is not associated with all or a portion of the polynucleotide in which it is found in nature, (3) is operably linked to a polynucleotide to which it is not naturally linked, and / or (4) is not naturally occurring. The term "recombinant" can be used in reference to cloned DNA isolates, chemically synthesized polynucleotide analogs, or polynucleotide analogs biologically synthesized by heterologous systems, as well as proteins and / or mRNAs encoded by such nucleic acids. As used herein, an endogenous nucleic acid sequence in the genome of an organism (or its encoded protein product) is considered "recombinant" herein when a heterologous sequence is positioned adjacent to the endogenous nucleic acid sequence such that expression of the endogenous nucleic acid sequence is altered. In this context, a heterologous sequence is a sequence that is not naturally adjacent to the endogenous nucleic acid sequence, whether the heterologous sequence is itself endogenous (originating from the same host cell or its progeny) or exogenous (originating from a different host cell or its progeny). As an example, a promoter sequence can replace the native promoter of a gene in the genome of a host cell (e.g., by homologous recombination), resulting in the gene having an altered expression pattern. The gene then becomes "recombinant" because it is separated from at least a portion of the sequences that naturally flank it. A nucleic acid is also considered "recombinant" if it contains any alterations that do not naturally occur in the corresponding nucleic acid in the genome. For example, an endogenous coding sequence is considered "recombinant" if it contains an insertion, deletion, or point mutation that has been artificially introduced, e.g., by human intervention. "Recombinant nucleic acid" also includes nucleic acids integrated into a host cell chromosome at a heterologous site and nucleic acid constructs that exist as episomes.

[0142] As used herein, the term "recombinant host cell" (or simply "host cell"), as used herein, is intended to refer to a cell into which a recombinant vector has been introduced. It should be understood that such term is intended to refer not only to the particular subject cell but also to the progeny of such a cell. Because certain modifications may occur in subsequent generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein. A recombinant host cell can be an isolated cell or a cell line grown in culture, or can be a cell present in a living tissue or organism.

[0143] As used herein, the term "region" refers to a physically contiguous portion of the primary structure of a biomolecule. In the case of a protein, a region is defined by a contiguous portion of the amino acid sequence of that protein.

[0144] As used herein, "sequence homology" for polypeptides, also referred to as "percent sequence identity," is typically measured using sequence analysis software. See, for example, the Sequence Analysis Software Package of the Genetics Computer Group (GCG), University of Wisconsin Biotechnology Center, 910 University Avenue, Madison, Wis. 53705. Protein analysis software matches similar sequences using homology measures assigned to various substitutions, deletions, and other modifications, including conservative amino acid substitutions. For example, GCG contains programs such as "Gap" and "Bestfit," which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, for example, between homologous polypeptides from organisms of different species, or between a wild-type protein and its mutein. See, for example, GCG Version 6.1. A preferred algorithm for comparing a particular polypeptide sequence to a database containing a large number of sequences from different organisms is the computer program BLAST (Altschul et al., J. Mol. Biol. 215:403-410 (1990); Gish and States, Nature Genet. 3:266-272 (1993); Madden et al., Meth. Enzymol. 266:131-141 (1996); Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997); Zhang and Madden, Genome Res. 7:649-656 (1997)), in particular blastp or tblastn (Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997)).Preferred parameters for BLASTp are: expectation: 10 (default); filter: seg (default); cost to open a gap: 11 (default); cost to extend a gap: 1 (default); maximum alignment: 100 (default); word size: 11 (default); number of descriptions: 100 (default); penalty matrix: BLOWSUM62. The length of polypeptide sequences compared for homology is generally at least about 16 amino acid residues, usually at least about 20 residues, more usually at least about 24 residues, typically at least about 28 residues, and preferably greater than about 35 residues. When searching a database containing sequences from multiple different organisms, it is preferable to compare amino acid sequences. Database searches using amino acid sequences can be performed by algorithms other than blastp, which are known in the art. For example, polypeptide sequences can be compared using the FASTA program in GCG Version 6.1. FASTA provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences. Pearson, Methods Enzymol. 183:63-98 (1990), hereby incorporated by reference. For example, percent sequence identity between amino acid sequences can be determined using FASTA with its default parameters (word size of 2 and PAM250 scoring matrix), as provided in GCG Version 6.1, hereby incorporated by reference.

[0145] As used herein, the term "specificity" generally refers to a sequence that, when in a conformation capable of binding, selectively or "specifically" binds to a particular target. As used herein, "specifically binds" means that the binding of a polynucleotide, polypeptide, or protein is selective for a specified target and can be distinguished from unwanted or nonspecific interactions. For example, the ability of a protein (e.g., a cysteine-rich peptide) to bind to a specific antigenic determinant can be measured by techniques known to those skilled in the art, such as enzyme-linked immunosorbent assay (ELISA) or surface plasmon resonance. Between two molecules, "specific binding" refers to the ability of the two molecules to bind to each other preferentially over binding to other molecules in the environment. Typically, "specific binding" is at least 2-fold, more typically at least 10-fold, and often at least 100-fold greater than chance binding in the reaction. Typically, the affinity or avidity of a specific binding reaction, as quantified by a dissociation constant, is about 10. -7 M or stronger (e.g., about 10 -8 M, 10 -9 M or even stronger). Specific binding requires the specificity of a particular first entity (e.g., a polypeptide) for a particular second entity (e.g., an antigen-binding sequence).

[0146] As used herein, "stringent hybridization conditions" and "stringent wash conditions" in the context of nucleic acid hybridization experiments depend on several different physical parameters. As will be readily understood by those skilled in the art, nucleic acid hybridization is influenced by conditions such as salt concentration, temperature, solvent, base composition of the hybridizing species, length of the complementary region, and the number of nucleotide base mismatches between hybridizing nucleic acids. Those skilled in the art know how to vary these parameters to achieve a particular stringency of hybridization. Generally, "stringent hybridization" is performed at about 25°C below the thermal melting point (Tm) for a specific DNA hybrid under a particular set of conditions. "Stringent wash" is performed at a temperature about 5°C lower than the Tm for a specific DNA hybrid under a particular set of conditions. Tm is the temperature at which 50% of the target sequence hybridizes to a perfectly matched probe. See Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989), page 9.51, hereby incorporated by reference. For purposes of this specification, "stringent conditions" are defined as solution-phase hybridization as aqueous hybridization (i.e., without formamide) in 6x SSC (20x SSC contains 3.0 M NaCl and 0.3 M sodium citrate), 1% SDS at 65°C for 8-12 hours, followed by two 20-minute washes in 0.2x SSC, 0.1% SDS at 65°C. Those of skill in the art will understand that hybridization at 65°C will occur at different rates depending on several factors, including the length and percent identity of the hybridizing sequences.

[0147] As used herein, the term "synthetic" is used to refer to an entity that is made, created in a laboratory, and is not naturally produced or isolated without modification from a naturally occurring source. A recombinant polymer, such as a recombinant polynucleotide or polypeptide, can be synthetic. A synthetic polymer, such as a polynucleotide or polypeptide, can be produced by any method known to those skilled in the art, including, but not limited to, solid-phase synthesis, solution-phase synthesis, biological synthesis, e.g., by host cells, etc.

[0148] As used herein, the term "subject" refers to a mammal. A subject may be a human or non-human mammal. Given the context, subject may be used interchangeably with patient, individual, donor, etc.

[0149] As used herein, the term "substantial homology" or "substantial similarity," when referring to a polynucleotide or polypeptide, indicates that when optimally aligned with another reference molecule (or its complementary strand, as appropriate), with appropriate nucleotide or amino acid insertions or deletions, there is sequence identity in at least about 70%, 75%, 80%, 85%, preferably at least about 90%, and more preferably at least about 95%, 96%, 97%, 98%, or 99% or more nucleic acid or amino acid residues, as measured by any well-known algorithm of sequence identity, e.g., FASTA, BLAST, Gap, etc. Alternatively, or additionally, substantial homology or similarity exists, for example, when a nucleic acid or a fragment thereof hybridizes to another nucleic acid, to a strand of another nucleic acid, or to its complementary strand, under stringent hybridization conditions.

[0150] As used herein, the term "target" refers to a protein or a functional part or variant thereof. A target can be or include a binding region, e.g., an epitope, to which a miniprotein of the present disclosure (e.g., CDP, knottin, binder, affibody, engineered Kunitz domain, monobody, anticalin, designed ankyrin repeat domain (DARPin), avimer) binds. Furthermore, the term "antigen" refers to a protein or a functional part or variant thereof to which an antibody or variant thereof binds. A target or antigen can be expressed on the surface of a specific cell (a "target cell") or can be expressed intracellularly (e.g., on the surface of a cell) in a population of cells. A target or antigen can have a certain percent identity to a reference protein and can also be referred to by a specific name (e.g., any one of the target proteins in Table 10). A target or antigen can also refer to a protein in a pathway related to another protein. For example, if the target or antigen is any one of the target proteins in Table 10, the target or antigen can also be a protein in a pathway required for the activity of the target protein selected from Table 10.

[0151] As used herein, the term "treatment" (and "treat" or "treating") refers to the partial or complete alleviation, remission, reduction, prevention, reduction in the risk of onset, relief, inhibition, delay in the onset, reduction in the severity, reduction in frequency or incidence of one or more causes, features and / or symptoms of or associated with a particular disease, disorder and / or condition.

[0152] As used herein, the term "vector" is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid," which generally refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated, but also includes linear double-stranded molecules, such as those obtained by amplification via polymerase chain reaction (PCR) or by treating a circular plasmid with a restriction enzyme. Other vectors include cosmids, bacterial artificial chromosomes (BACs), and yeast artificial chromosomes (YACs). Another type of vector is a viral vector, into which additional DNA segments can be ligated into the viral genome (discussed in more detail below). Certain vectors are capable of autonomous replication in the host cell into which they are introduced (e.g., vectors having an origin of replication that functions in the host cell). Other vectors can be integrated into the genome of the host cell upon introduction into the host cell, thereby replicating along with the host genome. Furthermore, certain preferred vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply, "expression vectors").

[0153] As used herein, the term "decharged molecule" refers to a molecule that has been modified to contain a less positively charged or polar feature, a more negatively charged feature, and / or both.

[0154] As used herein, the term "surface charge" refers to an electrical charge present on the surface of a protein (such as a miniprotein). In various embodiments, the surface charge of a miniprotein can affect renal uptake of the miniprotein (e.g., can increase renal uptake of the miniprotein or can decrease renal uptake of the miniprotein).

[0155] As used herein, the term "surface patch" refers to a region on the surface of a protein (e.g., a miniprotein) that has surface characteristics. For example, a surface patch can be defined according to surface charge and / or surface hydrophobicity, which can affect renal uptake of a protein (e.g., a miniprotein).

[0156] As used herein, the term "cleavable linker" refers to a linker that can be cleaved. A cleavable linker contains a cleavable bond that is cleaved in vivo, for example, by acidic pH (a pH less than 7, typically about 4-6), by glutathione, or in the presence of upregulation of enzymes from the proximal tubule, such as matrix proteases or peptidases. Examples of cleavable linkers are linkers containing hydrazine or disulfide bonds or enzymatically cleavable peptide sequences.

[0157] As used herein, the term "decoy peptide" refers to a molecule specifically designed to mimic the role of a particular receptor protein and interact with a particular target entity. composition

[0158] Provided herein are novel compositions comprising one or more of a polypeptide, a linker, a chelator, and / or a radionuclide. In some embodiments, the composition comprises a linker and a chelator. In some embodiments, the composition comprises a linker, a chelator, and a radionuclide. In some embodiments, the composition comprises or consists of a polypeptide (i.e., a miniprotein), an optional linker, and a chelator and / or a radionuclide. In some embodiments, the chelator and / or radionuclide is conjugated to the miniprotein via a linker. In various embodiments, the compositions disclosed herein comprise a compound. In various embodiments, the compound may comprise a miniprotein as described herein. In various embodiments, the compound may comprise one or more additional elements, examples of which include a linker, a chelator, and / or a radionuclide.

[0159] In various embodiments, the compound comprises a miniprotein comprising a sequence disclosed in Table 3 (in the column titled "Sequence"). In various embodiments, the compound comprises a miniprotein comprising a sequence disclosed in Table 3 (in the column titled "Sequence") and further comprises a linker disclosed in Table 3 (in the column titled "Sequence"). In various embodiments, the compound comprises a miniprotein comprising a sequence disclosed in Table 3 (in the column titled "Sequence") and further comprises a radionuclide disclosed herein. In various embodiments, the compound comprises a miniprotein comprising a sequence disclosed in Table 3 (in the column titled "Sequence") and further comprises a chelator disclosed herein. In various embodiments, the compound comprises a miniprotein comprising a sequence disclosed in Table 3 (in the column titled "Sequence") and further comprises a linker disclosed in Table 3 (in the column titled "Sequence") and a chelator disclosed herein. In various embodiments, the compound comprises a miniprotein comprising a sequence disclosed in Table 3 (in the column entitled "Sequence"), and further comprises a linker disclosed in Table 3 (in the column entitled "Sequence") and a radionuclide disclosed herein. In various embodiments, the compound comprises a miniprotein comprising a sequence disclosed in Table 3 (in the column entitled "Sequence"), and further comprises a linker disclosed in Table 3 (in the column entitled "Sequence"), a chelator disclosed herein, and a radionuclide disclosed herein.

[0160] In some embodiments, miniproteins of the present disclosure comprise or consist of CDPs, knottins, binders, affibodies, engineered Kunitz domains, monobodies, anticalins, designed ankyrin repeat domains (DARPins), and / or avimers. In some embodiments, miniproteins comprise or consist of CDPs. In some such embodiments, miniproteins comprise or consist of knottins. In some such embodiments, miniproteins comprise or consist of binders. In some such embodiments, miniproteins comprise or consist of affibodies. In some such embodiments, miniproteins comprise or consist of engineered Kunitz domains. In some such embodiments, miniproteins comprise or consist of monobodies. In some such embodiments, miniproteins comprise or consist of anticalins. In some such embodiments, miniproteins comprise or consist of designed ankyrin repeat domains (DARPins). In some such embodiments, miniproteins comprise or consist of avimers. In some embodiments, miniproteins (e.g., CDPs, knottins, binders, affibodies, engineered Kunitz domains, monobodies, anticalins, designed ankyrin repeat domains (DARPins), avimers) are designed to be linked to one or more other components. For example, in some embodiments, miniproteins (e.g., CDPs, knottins, binders, affibodies, engineered Kunitz domains, monobodies, anticalins, designed ankyrin repeat domains (DARPins), avimers) can be linked (conjugated) to another component, such as a chelator and / or a radionuclide. In some embodiments, the radionuclide of the present disclosure is an alpha emitter. In some such embodiments, the chelator and / or radionuclide is conjugated to the miniprotein via a linker.

[0161] While not wishing to be bound by any particular theory, the present disclosure contemplates that the compositions of the present disclosure are more effective than previously described compositions (e.g., those comprising antibodies and / or beta-emitter radionuclides). For example, miniproteins (e.g., as used in the compositions provided herein) possess some important features of antibody-based therapeutics (e.g., affinity, potency, specificity, and the ability to disrupt protein:protein interactions), but can avoid undesirable limitations, such as large size, costly production, and the need for chimerization or humanization. For example, in some embodiments, miniproteins of the present disclosure (e.g., CDPs, knottins, binders, affibodies, engineered Kunitz domains, monobodies, anticalins, designed ankyrin repeat domains (DARPins), avimers) are about 100 amino acids or less in length. In some embodiments, such miniproteins (e.g., CDPs, knottins, binders, affibodies, engineered Kunitz domains, monobodies, anticalins, designed ankyrin repeat domains (DARPins), avimers) can be or comprise cysteine-rich peptides. In some embodiments, miniproteins (e.g., CDPs, knottins, binders, affibodies, engineered Kunitz domains, monobodies, anticalins, designed ankyrin repeat domains (DARPins), avimers) comprise one or more disulfide bridges. In some embodiments, miniproteins (e.g., CDPs, knottins, binders, affibodies, engineered Kunitz domains, monobodies, anticalins, designed ankyrin repeat domains (DARPins), avimers) comprise multiple cysteine ​​residues that crosslink to maintain a highly stable folded state for a peptide of that length (e.g., compared to a peptide of the same length that does not have as many cysteine ​​residues). Without being bound to any particular theory, the present disclosure contemplates that in some embodiments, the miniprotein does not include multiple cysteine ​​residues, such as a miniprotein that includes a single cysteine ​​residue.The present disclosure contemplates that the stability imparted by cross-linked cysteines contributes to reduced immunogenicity of miniproteins or those comprising such miniproteins. In some embodiments, such stability may also confer resistance to harsher conditions provided for efficient chelation (e.g., high temperature, low pH incubation, etc.) while still retaining biological activity (e.g., the ability to bind to a target).

[0162] In some embodiments, the miniproteins provided herein function as targeting moieties that specifically bind to targets expressed on the surface of tumor cells, for example. In some such embodiments, the miniproteins are designed to be conjugated to one or more additional components. For example, without being bound by any particular theory, the miniproteins of the present disclosure can be formulated to be combined with other components, such as therapeutic molecules (e.g., chelator compositions and / or radionuclides) and / or detectable agents (e.g., visualizeable agents, such as metabolizable and visualizeable agents). In some such embodiments, such miniproteins conjugated to one or more additional components can be used in the diagnosis, prognosis, monitoring, and / or treatment of one or more diseases, disorders, or conditions, such as those involving the expression of specific targets on specific populations of cells.

[0163] In some embodiments, miniproteins (e.g., CDPs, knottins, binders, affibodies, engineered Kunitz domains, monobodies, anticalins, designed ankyrin repeat domains (DARPins), avimers) have reduced immunogenicity compared to larger proteins. In some such embodiments, reduced immunogenicity increases amenability to harsher environmental conditions (e.g., incubation at high temperature and low pH) while retaining biological activity. Thus, in some embodiments, conjugates comprising miniproteins have reduced immunogenicity compared to larger proteins or compositions comprising different targeting moieties (i.e., other than the miniprotein).

[0164] In some embodiments, the composition comprising the linker, chelator and / or radionuclide is capable of efficiently penetrating tumors.

[0165] In some embodiments, miniproteins (e.g., CDPs, knottins, binders, affibodies, engineered Kunitz domains, monobodies, anticalins, designed ankyrin repeat domains (DARPins), avimers) have superior penetration efficiency compared to larger proteins. That is, in some embodiments, miniproteins or compositions comprising miniproteins can penetrate solid tumors better than larger proteins or compositions comprising proteins larger than miniproteins. For example, in some embodiments, binders have superior tumor penetration efficiency with a hydrodynamic radius on the order of about 1 nm to 25 nm. In some embodiments, the hydrodynamic radius is between about 1 nm and 5 nm. In some embodiments, the hydrodynamic radius is between about 1 nm and 3 nm. In some embodiments, the hydrodynamic radius is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nm.

[0166] As described herein, miniproteins (e.g., CDPs, knottins, binders, affibodies, engineered Kunitz domains, monobodies, anticalins, designed ankyrin repeat domains (DARPins), avimers) are conjugated to a chelator. In some embodiments, the chelator binds to a radionuclide (e.g., an alpha-emitter radionuclide, e.g., actinium). In some such embodiments, such radionuclide conjugates combine the specific binding capacity and properties of the miniprotein (e.g., CDPs, knottins, binders, affibodies, engineered Kunitz domains, monobodies, anticalins, designed ankyrin repeat domains (DARPins), avimers) with the radionuclide. That is, without being bound by any particular theory, the present disclosure provides conjugates, wherein in some embodiments, a miniprotein (e.g., CDP, knottin, binder, affibody, engineered Kunitz domain, monobody, anticalin, designed ankyrin repeat domain (DARPin), avimer) targets a radioisotope to which it is conjugated to a cell expressing the target. In some embodiments, the target is expressed on the surface of a cell. In some embodiments, the target is any one of the target proteins in Table 10. In some embodiments, the cell is a tumor cell. In some embodiments, the conjugate binds to any one of the target proteins in Table 10 on the surface of a tumor cell. In some such embodiments, the radionuclide is targeted to the tumor cell. In some embodiments, the radionuclide is an alpha-emitter radionuclide, which, upon internalization, functions to specifically target the tumor cell (e.g., without damaging surrounding tissues / cells). target

[0167] Any cell expressing a target can be targeted by the miniproteins provided herein (e.g., CDPs, knottins, binders, affibodies, engineered Kunitz domains, monobodies, anticalins, designed ankyrin repeat domains (DARPins), avimers).

[0168] In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is from a cell line. In some embodiments, the cell is a primary cell. In some embodiments, the primary cell is derived from a sample from a subject, e.g., from a tumor or from a corresponding tissue that does not have a tumor (e.g., from another region of an organ or from a healthy donor). In some embodiments, the cell is in vitro (e.g., a primary cell, a cell line, etc.). In some embodiments, the cell is in vivo (e.g., in a subject, e.g., in a human subject, e.g., in a tumor in a human subject). In some embodiments, the cell expresses a target or has been induced to express a target (e.g., via recombinant technology). In some embodiments, the target is expressed on the surface of the cell. In some embodiments, the cell is contacted with a composition that binds to a target expressed on its surface. In some embodiments, upon binding (e.g., upon binding of a miniprotein provided by the present disclosure), the target and any bound protein and / or payload are internalized into the cell. In some embodiments, the cells are killed by the payload (eg, a radionuclide and / or a chelator, etc.) after internalization.

[0169] In some embodiments, the target is a protein or a portion thereof that is upregulated or overexpressed in cancer cells compared to non-cancer cells. That is, in some embodiments, the target is expressed in a tumor or in the tumor microenvironment, or is overexpressed in a tumor or in the tumor microenvironment compared to the level of the target in a non-diseased tissue (e.g., a tissue that does not have a tumor or a tumor microenvironment). In some such embodiments, the target is absent or undetectable in a non-diseased (e.g., healthy) tissue. In some embodiments, the target is a biomarker for cancer (e.g., for cancer cells, for tumors). In certain embodiments, the target is any one of the targets identified in Table 10.

[0170] In some embodiments, the target may be associated with a protein, such as a protein in a pathway that is activated or acted upon by another protein. For example, in some embodiments, the protein may be expressed on the surface of a cancer cell, and the target may be a pathway on which the surface-cell protein acts. In some embodiments, the protein may be expressed on a cancer cell, and the target may be a protein on a different cell that causes the cancer cell to proliferate or otherwise become resistant to treatment. In some embodiments, tumor-associated or tumor-specific cell surface molecules may be targeted by the miniproteins or compositions comprising the miniproteins provided herein.

[0171] In some embodiments, the miniprotein or a composition comprising the miniprotein specifically binds to a target expressed on the surface of a cell. In some embodiments, the target is cleaved from the cell surface. In some such embodiments, when the target is in an organism, cleavage of the target results in the circulation of the target throughout the system of the organism. In some such embodiments, the target is found at a particular level, for example, in blood, serum, plasma, etc. However, in some embodiments, a substantial portion of the expressed target is localized to the cell surface; therefore, in some embodiments, measuring the level of the target may not accurately reflect the amount of the target in a population of cells (e.g., a tumor). In some embodiments, the target is a secreted protein. In some such embodiments, the target is found at a particular level, for example, in blood, serum, plasma, etc. In some such embodiments, the miniprotein binds to a region of the target, such as an epitope. In some embodiments, the miniprotein or a composition comprising the miniprotein specifically binds to a target expressed on the surface of a cancer cell. In some embodiments, the cancer cell is in, on, or near a solid tumor. In some embodiments, the cancer cell is a circulating cancer cell. In some embodiments, the miniprotein or a composition comprising the miniprotein specifically binds to a target that is expressed at a higher level on cancer cells than on reference cells. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell.

[0172] In some embodiments, the miniprotein or a composition comprising the miniprotein specifically binds to any one of the target proteins in Table 10. In some embodiments, the target comprises or consists of any one of the target proteins in Table 10. In some embodiments, the miniprotein specifically binds to a target comprising an amino acid sequence shown in Table 6, or a portion thereof. Mini Protein

[0173] Novel polypeptides (i.e., miniproteins) and methods of use thereof are provided herein. In some embodiments, the polypeptide comprises or consists of a miniprotein. In some such embodiments, the miniprotein comprises or consists of a CDP, a knottin, and / or a binder. In some embodiments, the miniprotein is designed to be linked to one or more other components. For example, in some embodiments, the miniprotein may be linked (conjugated) to another component, such as a chelator and / or a radionuclide. In some embodiments, conjugation is via a lysine or cysteine ​​residue. For example, in some embodiments, the miniprotein is engineered to remove all lysine residues except for those used for conjugation in some embodiments. In some embodiments, conjugation occurs via an optional linker. In some embodiments, conjugation between the miniprotein and the chelator and / or radionuclide is direct.

[0174] While not wishing to be bound by any particular theory, the present disclosure contemplates that therapeutics comprising the compositions provided by the present disclosure are characterized by several features compared to other (e.g., antibody-based) therapeutics. For example, in some embodiments, miniproteins exhibit some important features of antibody-based therapeutics (e.g., affinity, potency, specificity, and ability to disrupt protein:protein interactions), but also have several advantages compared to antibody-based therapeutics, such as smaller size, cheaper production, and elimination of the need to chimerize or humanize the protein. Furthermore, the size and specificity of the linkages increase tumor penetration and uptake of the miniprotein or compositions (e.g., conjugates) comprising the miniprotein into cells expressing the target.

[0175] In some embodiments, miniproteins of the present disclosure are about 100 amino acids or less in length. In some embodiments, miniproteins are about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or more amino acids in length. However, in some such embodiments, miniproteins of the present disclosure are no more than about 100 amino acids in length. In some embodiments, miniproteins are between about 20 and about 40, about 30 and about 50, about 40 and about 60, about 45 and about 65, about 50 and about 70, about 55 and about 75, about 65 and about 85, or more amino acids in length, but no more than about 100 amino acids in length. In some preferred embodiments, miniproteins are about 65 amino acids or less. In some preferred embodiments, miniproteins are about 50 amino acids or less.

[0176] In some embodiments, the miniproteins of the present disclosure are no larger than about 12 kDa. In some embodiments, the miniproteins of the present disclosure are about 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5 or more kDa. However, in some such embodiments, the miniproteins of the present disclosure do not exceed about 12 kDa.

[0177] In some embodiments, miniproteins of the present disclosure comprise or consist of a linear polypeptide, a folded polypeptide (e.g., a covalently linked polypeptide, a non-covalently linked polypeptide, or a polypeptide comprising disulfide linkages), a cysteine-rich peptide, a knottin peptide, a binder, an affibody, an engineered Kunitz domain, a monobody, an anticalin, a designed ankyrin repeat domain (DARPin), or an avimer.

[0178] In some embodiments, the miniprotein comprises one or more disulfide bridges. In some embodiments, the miniprotein comprises multiple cysteine ​​residues. In some such embodiments, the cysteine ​​residues are cross-linked in a way that maintains a highly stable folded state for a peptide of that length (e.g., compared to a peptide of the same length that does not have as many cysteine ​​residues). The present disclosure contemplates that such cross-linking confers improved stability with reduced (i.e., very low to no) immunogenicity and / or maintains or improves the ability to maintain biological activity under harsh but efficient chelating conditions (e.g., high temperature and low pH).

[0179] In some embodiments, miniproteins or compositions comprising miniproteins (e.g., radionuclide conjugates) have reduced immunogenicity compared to larger proteins or compositions comprising or consisting of larger proteins (e.g., antibodies).

[0180] In some embodiments, miniproteins (e.g., CDPs, knottins, binders, affibodies, engineered Kunitz domains, monobodies, anticalins, designed ankyrin repeat domains (DARPins), avimers) have superior penetration efficiency compared to larger proteins. That is, in some embodiments, miniproteins or compositions comprising miniproteins can penetrate solid tumors better than larger proteins or compositions comprising proteins larger than miniproteins. For example, in some such embodiments, miniproteins or compositions comprising miniproteins have a hydrodynamic radius of about 1 to about 25 nm. In some embodiments, the hydrodynamic radius is within the range of about 1 to 25 nm, 10 to 20 nm, 5 to 15 nm, 1 to 5 nm, 2 to 4 nm, or 1 to 3 nm. In some embodiments, the hydrodynamic radius is measured using light scattering methods known to those of skill in the art.

[0181] In some embodiments, miniproteins of the present disclosure are characterized in that they have one or more properties compared to proteins larger than 100 amino acids, such as antibodies, antibody fragments, VHH domains, single-chain antibodies, or other proteins or binders larger than 12 kDA. In some embodiments, the properties are selected from increased protein expression, increased thermoactivity, increased thermostability, increased pH activity, increased stability, increased activity, increased receptor binding specificity and / or affinity, increased specific activity, increased resistance to substrate and / or end-product inhibition, increased chemical stability, improved chemoselectivity, improved solvent stability, increased tolerance to acidic pH, increased resistance to proteolytic activity (i.e., reduced susceptibility to proteolysis), reduced aggregation, increased solubility, reduced immunogenicity, and altered temperature profile, increased resistance to liver uptake, kidney uptake, or healthy tissue binding, increased tumor penetration, and / or increased volume of distribution.

[0182] In some embodiments, a miniprotein or a composition comprising a miniprotein (e.g., a conjugate, e.g., a radionuclide conjugate) provided by the present disclosure exhibits binding affinity for any one of the target proteins in Table 10. In some embodiments, the target protein selected from Table 10 is a human isoform. In some embodiments, the human isoform of any one of the target proteins in Table 10 is on a cell. In some embodiments, the cell is a cell line, a primary cell, or a cell in a human (e.g., in a tumor).

[0183] In some embodiments, the miniprotein or a composition comprising the miniprotein (e.g., a conjugate, e.g., a radionuclide conjugate) exhibits nM or sub-nM binding affinity to any one of the target proteins in Table 10. In some embodiments, the affinity is measured in an in vitro assay. In some embodiments, the in vitro assay is a cell-based assay. In some embodiments, the affinity is measured in an in vivo assay (e.g., a PET scan) or using a sample from the subject (e.g., an in vitro assay using a biological specimen, e.g., a blood or cell biopsy, from the subject).

[0184] In some embodiments, the miniprotein or conjugate thereof exhibits binding affinity to any one of the target proteins in Table 10. In some embodiments, the binding affinity of the miniprotein or conjugate thereof to the human isoform of any one of the target proteins in Table 10 is about 500 nM. In some embodiments, the miniprotein comprises picomolar binding affinity. In some embodiments, the miniprotein or conjugate thereof comprises a binding affinity to the human isoform of any one of the target proteins in Table 10 characterized by a dissociation constant in the range of about 900 nM to about 1 nM, for example, 900, 800, 700, 600, 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4 nM or less. In some embodiments, the binding is selective for, for example, a human isoform of any one of the target proteins in Table 10 rather than a non-human target protein selected from Table 10.

[0185] In some embodiments, the miniproteins provided by the present disclosure or conjugates thereof have high affinity for any one of the target proteins in Table 10. In some such embodiments, the target protein selected from Table 10 is a human isoform of a protein selected from Table 10. In some embodiments, the miniproteins of the present disclosure are stable, including in the presence of one or more additional molecules (e.g., cytotoxic molecules, e.g., radiation).

[0186] In some embodiments, the binding ability of the miniprotein or its conjugate to the target is improved by one or more modifications. For example, in some embodiments, the binding ability of the miniprotein or its conjugate provided herein to any one of the target proteins in Table 10 is improved using chemical cross-linking. In some embodiments, binding can be enhanced by using one or more of lysine residues, fusion proteins, unnatural amino acids, or other chemical moieties that enhance binding and / or functional activity.

[0187] In some embodiments, to ensure proper folding and connectivity, selected cysteine ​​pairs may be replaced with selenocysteines. In some embodiments, diselenide bridges form more readily than disulfide bridges due to their lower redox potential, and it is contemplated that such replacements may cross-couple the remaining cysteine ​​residues.

[0188] In some embodiments, miniproteins of the present disclosure comprise or consist of an antigen for use in generating antibodies that specifically bind to at least one epitope on any one of the target proteins in Table 10. In some embodiments, such antibodies can be used, for example, for diagnostic purposes, blocking (e.g., antagonism), etc.

[0189] In some embodiments, the miniprotein comprises one or more disulfide bridges.

[0190] In some embodiments, the miniproteins provided herein or conjugates thereof do not contain one or more cysteine ​​residues. In some embodiments, the miniproteins do not contain one or more disulfide bridges.

[0191] In some embodiments, the miniproteins provided herein or conjugates thereof are specific for a target. In some embodiments, the miniproteins are specific for any one of the target proteins in Table 10 or fragments thereof.

[0192] In some embodiments, the target is represented by a polypeptide set forth in any one of SEQ ID NOs: 69-72 shown in Table 6, or a portion thereof.

[0193] In some embodiments, the miniproteins or conjugates thereof provided herein comprise or consist of a particular amino acid sequence.

[0194] In some embodiments, the miniprotein or a composition comprising the miniprotein (e.g., a radionuclide conjugate) is conjugated to a chelator that optionally binds to a radionuclide (e.g., actinium). In some embodiments, the conjugation is via a linker. In some embodiments, the conjugation is direct conjugation. In some embodiments, such radionuclide conjugates combine or synergize to provide target specificity (e.g., via the miniprotein) and superior treatment (e.g., via directed radioisotope delivery to cells expressing the target).

[0195] As used herein and as known to those skilled in the art, the 20 conventional amino acids and their abbreviations follow conventional usage. See Immunology-A Synthesis (Golub and Gren eds., Sinauer Associates, Sunderland, Mass., 2nd ed. 1991), hereby incorporated by reference. In some embodiments, the amino acids of the present disclosure may be stereoisomers of the 20 conventional amino acids (e.g., D-amino acids). In some embodiments, the amino acids in the polypeptides of the present disclosure may be unnatural amino acids. For example, amino acids such as α-,α-disubstituted amino acids, N-alkyl amino acids, and other unconventional amino acids may also be suitable building blocks for the polypeptides of the present disclosure. Examples of unconventional amino acids include: 4-hydroxyproline, γ-carboxyglutamic acid, ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, N-methylarginine, and other similar amino acids and imino acids (e.g., 4-hydroxyproline). The arrangement of polypeptide sequence notation used herein has the left-hand end corresponding to the amino-terminus and the right-hand end corresponding to the carboxy-terminus, in accordance with standard usage and convention.

[0196] In some embodiments, the miniproteins provided herein are specific for a polypeptide or portion thereof having an amino acid sequence or portion thereof as set forth in Tables 3, 4A and 4B.

[0197] In some embodiments, the mini-protein comprises or consists of a particular amino acid sequence. In some embodiments, the mini-protein has an amino acid sequence that is 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to the amino acid sequence set forth in any of SEQ ID NOs: 1-68.

[0198] As used herein and as known to those skilled in the art, the 20 conventional amino acids and their abbreviations follow conventional usage. See Immunology-A Synthesis (Golub and Gren eds., Sinauer Associates, Sunderland, Mass., 2nd ed. 1991), hereby incorporated by reference. In some embodiments, the amino acids of the present disclosure may be stereoisomers of the 20 conventional amino acids (e.g., D-amino acids). In some embodiments, the amino acids in the polypeptides of the present disclosure may be unnatural amino acids. For example, amino acids such as α-,α-disubstituted amino acids, N-alkyl amino acids, and other unconventional amino acids may also be suitable building blocks for the polypeptides of the present disclosure. Examples of unconventional amino acids include: 4-hydroxyproline, γ-carboxyglutamic acid, ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, N-methylarginine, and other similar amino acids and imino acids (e.g., 4-hydroxyproline). The arrangement of polypeptide sequence notation used herein has the left-hand end corresponding to the amino-terminus and the right-hand end corresponding to the carboxy-terminus, in accordance with standard usage and convention. DOTA-PEG4: alpha-(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid)-4(ethylene glycol) Biotin-PEG4: [ka] FITCI_PEG4: Fluorescein isothiocyanate-4 (ethylene glycol) Norleucine: (S)-(+)-2-aminohexanoic acid, (S)-2-aminocaproic acid [ka] Homoleucin: (S)-3-amino-5-methylhexanoic acid hydrochloride [ka]

[0199] In some embodiments, miniproteins of the present disclosure have glutamine, tryptophan, leucine, phenylalanine, alanine, 3-pyridylalanine, substituted tryptophan, or substituted phenylalanine. In some embodiments, miniproteins of the present disclosure have asparagine, aspartic acid, serine, lysine, glutamine, glutamic acid, leucine, alanine, norleucine, homoleucine, homoserine, or substituted phenylalanine. In some embodiments, miniproteins of the present disclosure have glycine, alanine, lysine, glutamic acid, leucine, serine, proline, phenylalanine, norleucine, homoleucine, homoserine, or substituted phenylalanine. In some embodiments, miniproteins of the present disclosure have glutamic acid, leucine, aspartic acid, methionine, glutamine, tyrosine, phenylalanine, norleucine, homoleucine, homoserine, or substituted phenylalanine.

[0200] In some embodiments, miniproteins of the present disclosure exhibit binding specificity for the human isoform of any one of the target proteins in Table 10. For example, in some embodiments, miniproteins provided by the present disclosure, such as those set forth in any one of SEQ ID NOS: 1-68, demonstrate binding when expressed on the surface of yeast and to any one of the target proteins in Table 10 tested by flow cytometry. In some embodiments, miniproteins provided by the present disclosure, such as those set forth in any one of SEQ ID NOS: 1-68, demonstrate binding specificity via flow cytometry, for example, where a miniprotein for any one of the target proteins in Table 10 (e.g., set forth in any of SEQ ID NOS: 1-68) binds only to a target protein selected from Table 10 and not to other target proteins.

[0201] In some embodiments, miniproteins of the present disclosure, such as any of those set forth by SEQ ID NOs: 1-68, exhibit potencies greater than 10 nM. In some embodiments, miniproteins exhibit potencies greater than 1, 2, 3, 4, 5, 6, 7, 8, 9 nM, or greater. CDP

[0202] In some embodiments, the miniproteins of the present disclosure comprise or consist of a cysteine-rich peptide (CDP). In some embodiments, the conjugates provided herein comprise a CDP. In some embodiments, the CDP functions as a targeting moiety, for example, specifically binding to a protein target expressed on the surface of a target tumor cell. In some embodiments, the CDP comprises or consists of at least two independent folding domains and a high density of cysteines. In some embodiments, the CDP comprises at least one, two, three, four, five, six, or more than six cysteine ​​residues over a span of about 10 to about 90 amino acid residues, preferably 13 to 80 amino acid residues (see, e.g., Correnti et al., Nat Struct Mol Biol. 2018 Mar;25(3):270-278 for exemplary CDPs and their characteristics). Knotchin

[0203] In some embodiments, miniproteins of the present disclosure comprise or consist of knottin peptides. In some embodiments, conjugates provided herein comprise knottin peptides. In some embodiments, knottin peptides function as targeting moieties, specifically binding to target proteins expressed on the surface of target tumor cells, for example. In some embodiments, knottins contain at least three disulfide bonds connected in an arrangement that creates the so-called "cysteine-knot" for which knottins are named (see, e.g., Kintzing & Cochran et al., Curr Opin Chem Biol. 2016 Oct;34:143-150). In some embodiments, knottins have high stability (e.g., thermal, proteolytic, chemical, etc.). In some embodiments, knottins can be further engineered to modify binding, folding, and / or related properties.

[0204] In some embodiments, a given knottin is highly specific for a given target. In some embodiments, the knottin specifically binds to the target. In some embodiments, the target is located in, on, or near a cell. In some embodiments, the knottin specifically binds to any one of the target proteins in Table 10, or a fragment thereof. In some embodiments, the knottin is conjugated to a chelator and / or a radionuclide. In some embodiments, the conjugation is via a linker. It will be understood by those skilled in the art that in some embodiments, the particular knottin used in the conjugates of the present disclosure may vary depending on the target protein of interest.

[0205] In some embodiments, the folded structure of miniproteins (e.g., CDPs, knottins, binders, affibodies, engineered Kunitz domains, monobodies, anticalins, designed ankyrin repeat domains (DARPins), avimers) rigidifies them, providing very tight and strong binding (compared to less structured peptides) to target proteins or antigens. In some such embodiments, miniproteins (e.g., CDPs, knottins, binders, affibodies, engineered Kunitz domains, monobodies, anticalins, designed ankyrin repeat domains (DARPins), avimers) exhibit exceptional stability, with resistance to heat, peptidase cleavage, and pH. binder

[0206] In some embodiments, the miniproteins of the present disclosure comprise or consist of a binder, which in some embodiments functions as a targeting moiety that specifically binds to a target expressed, for example, on the surface of a tumor cell.

[0207] In some embodiments, the binder has certain structural features; for example, in some embodiments, the binder may be rich in alpha-helices, e.g., helix-helix-helix structures (see, e.g., Crook et al., Nat Commun. (2017) 8, 2244; Berger et al., Elife (2016) 5, e20352; and Procko et al., Cell (2014), 157, 1644-1656). In some embodiments, the binder comprises a surface sufficient to functionalize molecules on completely different surfaces into binding surfaces. In some embodiments, the binder comprises a sequestered hydrophobic core. In some embodiments, the binder exhibits cooperative folding. In some embodiments, the binder has two or more of the following features: (i) represented by an amino acid sequence of 100 amino acids or less; (ii) at least two secondary structure elements; (iii) a sequestered hydrophobic core; and / or (iv) cooperative folding.

[0208] In some embodiments, a given binder is highly specific for a given target. In some embodiments, the binder specifically binds to a target. In some embodiments, the target is located in, on, or near a cell. In some embodiments, the binder specifically binds to any one of the target proteins in Table 10, or a fragment thereof. In some embodiments, the binder is conjugated to a chelator and / or a radionuclide. In some embodiments, the conjugation is via a linker. It will be understood by those skilled in the art that in some embodiments, the particular binder used in the conjugates of the present disclosure may vary depending on the target protein or antigen of interest. Affibody

[0209] In some embodiments, the miniproteins of the present disclosure comprise or consist of an affibody. In some embodiments, the conjugates provided herein comprise an affibody. In some embodiments, the affibody functions as a targeting moiety, specifically binding to a protein target or antigen expressed on the surface of a target tumor cell, for example. In some embodiments, the affibody comprises or consists of no more than 100, 90, 80, 70, 60, 50, 40, 30, 20, or 10 amino acids. In some embodiments, the affibody comprises or consists of at least three alpha helices with 58 amino acids. In some embodiments, the affibody comprises target specificity obtained by randomizing 13 amino acids located in two alpha-helices involved in the binding activity of the parent protein domain (Feldwisch J, Tolmachev V.; (2012) Methods Mol Biol. 899:103-26). In some embodiments, the affibody can be further engineered to modify binding, folding, and / or related properties.

[0210] In some embodiments, the affibody specifically binds to a target. In some embodiments, the target is located in, on, or near a cell. In some embodiments, the affibody specifically binds to any one of the target proteins in Table 10, or a fragment thereof. In some embodiments, the affibody is conjugated to a chelator and / or a radionuclide. In some embodiments, the conjugation is via a linker. It will be understood by those skilled in the art that in some embodiments, the particular affibody used in the conjugates of the present disclosure may vary depending on the target protein or antigen of interest. Manipulated Kunitz domains

[0211] In some embodiments, the miniproteins of the present disclosure comprise or consist of an engineered Kunitz domain. In some embodiments, the conjugates provided herein comprise an engineered Kunitz domain. In some embodiments, the engineered Kunitz domain functions as a targeting moiety, for example, specifically binding to a protein target expressed on the surface of a target tumor cell. In some embodiments, the engineered Kunitz domain comprises or consists of at least one peptide derived from the Kunitz domain of a Kunitz-type protease inhibitor, such as bovine pancreatic trypsin inhibitor (BPTI), amyloid precursor protein (APP), or tissue factor pathway inhibitor (TFPI). In some embodiments, the engineered Kunitz domain can be further engineered to modify binding, folding, and / or related properties.

[0212] In some embodiments, the engineered Kunitz domain specifically binds to a target. In some embodiments, the target is located in, on, or near a cell. In some embodiments, the engineered Kunitz domain specifically binds to any one of the target proteins in Table 10 or a fragment thereof. In some embodiments, the engineered Kunitz domain is conjugated to a chelator and / or a radionuclide. In some embodiments, the conjugation is via a linker. In some embodiments, it will be understood by those skilled in the art that the specific engineered Kunitz domain used in the conjugates of the present disclosure may vary depending on the target protein of interest. Monobody

[0213] In some embodiments, the miniproteins of the present disclosure comprise or consist of a monobody. In some embodiments, the conjugates provided herein comprise a monobody. In some embodiments, the monobody functions as a targeting moiety, specifically binding to a protein target expressed, for example, on the surface of a target tumor cell. In some embodiments, the monobody comprises or consists of a molecule based on the tenth extracellular domain of human fibronectin III (10Fn3), adopting an approximately 94-residue Ig-like b-sandwich fold with two to three exposed loops but lacking a central disulfide bridge. In some embodiments, the monobody can be further engineered to modify binding, folding, and / or related properties.

[0214] In some embodiments, the monobody specifically binds to a target. In some embodiments, the target is located in, on, or near a cell. In some embodiments, the monobody specifically binds to any one of the target proteins in Table 10, or a fragment thereof. In some embodiments, the monobody is conjugated to a chelator and / or a radionuclide. In some embodiments, the conjugation is via a linker. It will be understood by those skilled in the art that in some embodiments, the particular monobody used in the conjugates of the present disclosure may vary depending on the target protein of interest. Anticalin

[0215] In some embodiments, the miniprotein of the present disclosure comprises or consists of an anticalin. In some embodiments, the conjugate provided herein comprises an anticalin. In some embodiments, the anticalin functions as a targeting moiety that specifically binds to a protein target expressed on the surface of a target tumor cell, for example. In some embodiments, the anticalin comprises or consists of an eight-stranded β-barrel that forms a highly conserved core unit among lipocalins and naturally forms a binding site for a ligand with four structurally variable loops at the open end. In some embodiments, the anticalin can be further engineered to modify its binding, folding, and / or related properties.

[0216] In some embodiments, the anticalin specifically binds to a target. In some embodiments, the target is located in, on, or near a cell. In some embodiments, the anticalin specifically binds to any one of the target proteins in Table 10, or a fragment thereof. In some embodiments, the anticalin is conjugated to a chelator and / or a radionuclide. In some embodiments, the conjugation is via a linker. It will be understood by those skilled in the art that in some embodiments, the particular anticalin used in the conjugates of the present disclosure may vary depending on the target protein of interest. Engineered ankyrin repeat domains

[0217] In some embodiments, the miniprotein of the present disclosure comprises or consists of a designed ankyrin repeat domain. In some embodiments, the conjugate provided herein comprises a designed ankyrin repeat domain. In some embodiments, the designed ankyrin repeat domain functions as a targeting moiety that specifically binds to a protein target expressed on the surface of target tumor cells, for example. In some embodiments, the designed ankyrin repeat domain comprises a peptide derived from ankyrin. In some embodiments, the designed ankyrin repeat domain preferably comprises a single ankyrin repeat, which comprises a 33-residue motif containing two alpha-helices and one beta-turn. In some embodiments, the designed ankyrin repeat domain provides a rigid interface and lacks structural flexibility. In some embodiments, the designed ankyrin repeat domain can be further engineered to modify binding, folding, and / or related properties.

[0218] In some embodiments, the designed ankyrin repeat domain specifically binds to a target. In some embodiments, the target is located in, on, or near a cell. In some embodiments, the designed ankyrin repeat domain specifically binds to any one of the target proteins in Table 10, or a fragment thereof. In some embodiments, the designed ankyrin repeat domain is conjugated to a chelator and / or a radionuclide. In some embodiments, the conjugation is via a linker. It will be understood by those skilled in the art that in some embodiments, the specific designed ankyrin repeat domain used in the conjugates of the present disclosure may vary depending on the target protein of interest. Abima

[0219] In some embodiments, the miniproteins of the present disclosure comprise or consist of avimers. In some embodiments, the conjugates provided herein comprise avimers. In some embodiments, avimers function as targeting moieties, specifically binding to protein targets expressed on the surface of target tumor cells, for example. In some embodiments, avimers comprise peptides of approximately 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acids. In some embodiments, avimers comprise at least one peptide sequence of approximately 30-35 amino acids. In some embodiments, avimers comprise two or more of two peptide sequences of approximately 30-35 amino acids. In some embodiments, avimers comprise one or more peptide sequences derived from the A-domains of various membrane receptors (Weidle UH, et al., (2013), Cancer Genomics Proteomics; 10(4): 155-68). For further details, see Nature Biotechnology 23(12), 1556-1561 (2005) and Expert Opinion on Investigational Drugs 16(6), 909-917 (June 2007). In some embodiments, avimers can be further engineered to alter binding, folding and / or related properties.

[0220] In some embodiments, the avimer specifically binds to a target. In some embodiments, the target is located in, on, or near a cell. In some embodiments, the avimer specifically binds to any one of the target proteins in Table 10, or a fragment thereof. In some embodiments, the avimer is conjugated to a chelator and / or a radionuclide. In some embodiments, the conjugation is via a linker. It will be understood by those skilled in the art that in some embodiments, the particular avimer used in the conjugates of the present disclosure may vary depending on the target protein of interest. Linker

[0221] In some embodiments, the present disclosure provides a linker for use in one or more conjugates. For example, in some embodiments, the linker is linked to a chelator. In some embodiments, the linker is linked to a chelator that is itself coupled to a radionuclide. In some embodiments, the miniprotein is conjugated to a chelator and / or a radionuclide. In some embodiments, the miniprotein is conjugated to a chelator, optionally via a linker. In some embodiments, the compositions provided herein comprise one or more linkers.

[0222] As described herein, in some embodiments, the miniprotein conjugate comprises a linker. In some embodiments, the linker functions to connect the chelator to the miniprotein. In some embodiments, the linker is non-cleavable. In some embodiments, the linker is cleavable. In some embodiments, the selection of one or more linkers and chelators and their placement on the miniprotein helps maintain the desired potency and receptor engagement profile of the miniprotein or its conjugate, enhance binder affinity, and optimize physicochemical and pharmacokinetic properties. Any suitable linker known in the art may be utilized. Exemplary linkers include, but are not limited to, polyethylene glycol (PEG) linkers, ester linkers, amide linkers, maleimide linkers, valine-citrulline linkers, hydrazone linkers, N-succinimidyl-4-(2-pyridyldithio)butyrate (SPDB) linkers, succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) linkers, vinyl sulfone-based linkers, propanoic acid linkers, caproleic acid linkers, or linkers comprising any combination thereof. One or more additional linkers may be contemplated as known to those skilled in the art and may be selected taking into account the context and components of a given composition. In some embodiments, the linker is a PEG linker. In some embodiments, the linker is a non-cleavable PEG linker. In some embodiments, the PEG linker is PEG(4-24).

[0223] In some embodiments, linkers are used to evaluate the target binding lead polypeptide sequence, the target expressed on cells, and target selectivity and / or affinity.For example, in some embodiments, the in vitro on-target binding and affinity confirmation for the lead polypeptide sequence and the lead polypeptide sequence-linker-fluorophore reagent can be evaluated using Biacore.In some embodiments, other linkers, such as fast clear linkers or halogen linkers, are also contemplated. Chelator

[0224] In some embodiments, the compositions (e.g., conjugates) provided herein comprise a linker. In some embodiments, the compositions comprise a linker and a chelator. In some embodiments, the compositions comprise a linker, a chelator, and a radionuclide. In some embodiments, the compositions comprise a miniprotein, an optional linker, a chelator, and / or a radionuclide. In some embodiments, the chelator is covalently attached to the miniprotein. In some embodiments, the chelator binds to the radionuclide. In some embodiments, a chelator refers to any molecule or moiety that "binds" (e.g., effectively collects / restricts) metal ions in solution so that they no longer participate in one or more cellular activities or processes. In some embodiments, the chelator chelates one or more components of a metabolic pathway in a cell (e.g., metal ions, e.g., copper, iron, zinc, etc.). In some such embodiments, the chelator may disrupt the life cycle of cancer cells, and in some embodiments, reduce their viability, function, and / or ability to grow or proliferate. In some embodiments, the chelator chelates one or more toxins produced as a result of targeted radiation therapy (eg, reduces the toxicity of the treatment).

[0225] In some embodiments, the chelator is diethylenetriaminepentaacetic acid (DTPA), tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), ethylenediaminetetraacetic acid (EDTA), 1,4,7-triazacyclononane-N,N',N"-triacetic acid (NOTA), ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), 1,4,7-triazacyclononane-N,N',N"-triacetic acid (NOTA), ({4-[2-(bis-carboxymethyl-amino)-ethyl]-7-carboxymethyl-[1,4,7 In some embodiments, the chelator includes or consists of, but is not limited to, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA). In some embodiments, the chelator is Macropa. In some embodiments, the chelator is (i) NOPO: [ka] (ii) CROWN [ka] (iii) DOTA [ka] ;or (iv) Macropa [ka] Comprises or consists of

[0226] In additional embodiments, chelation conditions are optimized using methods known to those of skill in the art (see, e.g., J Nucl Med. 1998 Dec;39(12):2105-10). In some embodiments, chelation efficiency is about >99%, >98%, >97%, >96%, >95%, >94%, >93%, >92%, >91%, >90%, >89%, >88%, >87%, >86%, >85%, >84%, >83%, >82%, >81%, or >80%.

[0227] In some embodiments, the chelator for use in the compositions described herein is selected based on whether and which radionuclide is present.As provided herein, in some embodiments, the chelator is DOTA, NOPO, Crown, or Macropa.In some embodiments, DOTA is the chelator, and the radionuclide is Ac-225, Ga-68, Pb-212, Lu-177, Cu-67, Cu-64, La-132, La-135, In-111, Ce-134, F-18, or At-211.In some embodiments, Crown is the chelator, and the radionuclide is Ac-225, Ga-68, Pb-212, Lu-177, Cu-67, Cu-64, La-132, La-135, In-111, Ce-134, F-18, or At-211. In some embodiments, NOPO is the chelator and the radionuclide is Ac-225, Ga-68, Pb-212, Lu-177, Cu-67, Cu-64, La-132, La-135, In-111, Ce-134, F-18, or At-211. In some embodiments, Macropa is the chelator and the radionuclide is Ac-225, Ga-68, Pb-212, Lu-177, Cu-67, Cu-64, La-132, La-135, In-111, Ce-134, F-18, or At-211.

[0228] In some embodiments, a specific chelator or type of chelator can be selected for a specific application. For example, in some embodiments, NOPO is used in diagnostic or theranostic applications. In some embodiments, Crown is used in therapeutic applications. In some embodiments, DOTA is used in diagnostic, theranostic, and / or therapeutic applications. In some embodiments, Macropa is used in diagnostic, theranostic, and / or therapeutic applications.

[0229] It is within the scope of this disclosure to screen chelators for certain characteristics, and it is recognized that methods for such screening are known to those of skill in the art. For example, in some embodiments, chelators are screened for their ability to bind radionuclides (e.g., Ga68, Ac225, and daughter(s) of Ac225 (Bi213)) and exhibit serum stability.

[0230] In some embodiments, the miniprotein conjugates described herein include a chelator. Any suitable chelator known in the art may be utilized. In some embodiments, the chelator is directly conjugated to the miniprotein. In some embodiments, the chelator is indirectly connected to the miniprotein via a linker. In some embodiments, the chelator is indirectly connected to the miniprotein via a linker (e.g., a linker described herein). radionuclides

[0231] In some embodiments, the present disclosure provides one or more radionuclides for use in a composition (eg, a conjugate).

[0232] In some embodiments, the mini-protein conjugate comprises a radionuclide bound to a chelator. As will be understood by those skilled in the art, any suitable radionuclide known in the art can be used. In some embodiments, the radionuclide is selected for imaging tumors in humans with cancer. In some embodiments, the radionuclide is selected for its inability to kill cells in vivo. In some embodiments, the radionuclide is selected for its ability to kill cells in vivo.

[0233] In some embodiments, the compositions of the present disclosure comprise one or more cytotoxic payloads, including particle-emitting isotopes in radiotherapy applications, such as alpha-, beta-particles, and Auger electrons. In some embodiments, the radionuclides of the present disclosure are alpha emitters. As known to those skilled in the art, in some embodiments, alpha emitters, when internalized into cells, have a more localized area of ​​effect, for example, to kill cancer cells, but spare surrounding tissues from widespread damage that may occur with the use of beta or gamma emitters.

[0234] Studies have evaluated alpha nuclide therapy versus beta nuclide therapy, and stronger clinical results have led to a focus on alpha nuclides.In some embodiments, the advantage of alpha therapy is that the short course length means that patients do not need to physically distance themselves from their families and medical providers, making treatment more tolerable.Furthermore, in some embodiments, alpha therapy exhibits better cell killing efficacy due to its ability to induce double-stranded DNA breaks.

[0235] In some embodiments, the composition comprises a linker, a chelator, and a radionuclide. In some embodiments, the composition comprises a miniprotein, an optional linker, a chelator, and a radionuclide. Without being bound to any particular theory, the present disclosure contemplates that a wide variety of radionuclides may be used in pharmaceutical compositions or as diagnostic agents. Exemplary radionuclides include actinium-225, astatine-211, bismuth-212, bismuth-213, cesium-137, chromium-51, cobalt-60, copper-64, dysprosium-165, erbium-169, fermium-255, fluorine (Fluor)-18, gallium-67, gallium-68, gold-198, holmium-166, indium-111, iodine-123, iodine-124, iodine-125, iodine-131, iridium-192, iron-59, These include, but are not limited to, lead-212, lutetium-177, molybdenum-99, palladium-103, phosphorus-32, potassium-42, rhenium-186, rhenium-188, samarium-153, technetium-99m, radium-223, ruthenium-106, sodium-24, strontium-89, terbium-149, thorium-227, xenon-133, ytterbium-169, ytterbium-177, yttrium-90, and zirconium-89. Thus, in some embodiments, the radionuclide is selected from: iodine (I or I), yttrium (Y), lutetium (Lu), actinium (Ac), praseodymium, astatine (At), rhenium (Re), bismuth (Bi or Bi), indium (In), technetium (Tc), phosphorus (P), rhodium (Rh), sulfur (S), carbon (C), tritium (H), chromium (Cr), chlorine (Cl), cobalt (Co or Co), iron (Fe), selenium (Se), or gallium (Ga) or (Ga).In some embodiments, the present disclosure contemplates that certain radioisotopes may be useful in or as therapeutic agents, including, but not limited to, yttrium (Y), lutetium (Lu), actinium (Ac), praseodymium, astatine (At), rhenium (Re), bismuth (Bi or Bi), and rhodium (Rh). In some embodiments, radioisotopes are useful as labels, for example, for use in diagnostic methods. In some such embodiments, such radioisotopes may include, but are not limited to, iodine (I or I), indium (In), technetium (Tc), phosphorus (P), carbon (C), lead (Pb), or tritium (H). See, e.g., U.S. Patent No. 7,514,078.

[0236] In some embodiments, the radionuclide is conjugated to different complexing agents and chelators. In some embodiments, the chelators are identified and attached / bound to the miniprotein via a linker or by acyclic, cyclic, and macrocyclic chelates, such as 1,4,7,10,13,16-hexaazacyclohexadecane-N,N',N",N"',N"",N""'-hexaacetic acid (HEHA), 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid (DOTA), NOPO, Crown, etc. In some embodiments, certain chelators are preferred for certain radionuclides, such as Ac-225 with DOTA or Crown, Ga-68 with NOPO, etc. In some embodiments, preferred combinations of chelators and radionuclides include one or more of the following: DOTA with Ac-225, Ga-68, Pb-212, Lu-177, Cu-67, Cu-64, La-132, La-135, In-111, Ce-134, F-18, or At-211; Crown with Ac-225, Ga-68, Pb-212, Lu-177, Cu-67, Cu-64, La-132, La-135, In-111, Ce NOPO with Ac-225, Ga-68, Pb-212, Lu-177, Cu-67, Cu-64, La-132, La-135, In-111, Ce-134, F-18 or At-211; and / or Macropa with Ac-225, Ga-68, Pb-212, Lu-177, Cu-67, Cu-64, La-132, La-135, In-111, Ce-134, F-18 or At-211.

[0237] Preferably, in some embodiments, preferred radionuclide complexes include Ac-225, Ga-68, Pb-212, Lu-177, Cu-67, Cu-64, La-132, La-135, In-111, Ce-134, F-18, or At-211. In some such embodiments, such complexes are selected to have a desired stability. That is, in some embodiments, complexes including Ac-225, Ga-68, Pb-212, Lu-177, Cu-67, Cu-64, La-132, La-135, In-111, Ce-134, F-18, or At-211 are characterized as having better stability in vivo compared to other complexes. Without being bound by any particular theory, the present disclosure contemplates that, in some embodiments, a radionuclide complex comprising a miniprotein forms with a miniprotein target (e.g., any one of the target proteins in Table 10 or a fragment thereof). In some such embodiments, such a complex is internalized into the target cell.

[0238] In some embodiments, radionuclide complexes are formed with chelators (e.g., DOTA, NOPO, Crown, Macropa, etc.) and are much more stable in vivo. In some embodiments, the miniprotein forms an internalization complex with a target (e.g., any one of the target proteins in Table 10).

[0239] In some embodiments, the compositions provided by the present disclosure include actinium-225. In some embodiments, the compositions provided by the present disclosure include gallium (Ga-68). In some embodiments, the compositions provided by the present disclosure include copper (Cu-64). In some embodiments, the compositions provided by the present disclosure include indium (In-111). In some embodiments, the compositions provided by the present disclosure include lutetium (Lu-177). In some embodiments, the compositions provided by the present disclosure include lead (Pb-212). In some embodiments, the compositions provided by the present disclosure include copper (Cu-67). In some embodiments, the compositions provided by the present disclosure include lutetium (Lu-177). In some embodiments, the compositions provided by the present disclosure include lanthanum (La-132). In some embodiments, the compositions provided by the present disclosure include lanthanum (La-135). In some embodiments, the compositions provided by the present disclosure include indium (In-111). In some embodiments, the compositions provided by the present disclosure include cerium (Ce-134). For example, in some embodiments, radioimmunotherapy including Actinium-225 may provide: i) a tissue-limited range of a few cell diameters; ii) a high linear energy transfer resulting in high density radiation damage along each alpha trajectory; iii) a 10-day half-life; and / or iv) four net alpha particles emitted per decay (see, e.g., Scheinberg, David A, and Michael R McDevitt. "Actinium-225 in targeted alpha-particle therapeutic applications." Current radiopharmaceuticals vol. 4,4 (2011): 306-20).

[0240] In some embodiments, targeting constructs (e.g., 225-Ac-drug constructs, e.g., 68-Ga-constructs) have potential for use in cancer. For example, in some such embodiments, such constructs, such as 225-Ac-drug constructs, may be used in the treatment of cancer. In some embodiments, such constructs, e.g., Ga-68 or Cu-64 based constructs, may be used in imaging, e.g., for prognosis, diagnostics, and / or monitoring.

[0241] In some embodiments, Ac-225 is conjugated to a miniprotein provided herein. In some embodiments, actinium is conjugated onto a chelator, which may include an optional linker connecting it to a miniprotein, which targets the conjugate to cells expressing a target (e.g., any one of the target proteins in Table 10).

[0242] In some embodiments, Ga-68 is conjugated to the miniproteins provided herein. In some embodiments, the gallium is conjugated onto a chelator, which may include an optional linker connecting it to the miniprotein, which targets the conjugate to cells expressing a target (e.g., any one of the target proteins in Table 10).

[0243] In some embodiments, Cu-64 is conjugated to a miniprotein provided herein. In some embodiments, the copper is conjugated onto a chelator, which may include an optional linker connecting it to a miniprotein, which targets the conjugate to cells expressing a target (e.g., any one of the target proteins in Table 10).

[0244] In some embodiments, In-111 is conjugated to a miniprotein provided herein. In some embodiments, indium is conjugated onto a chelator, which may include an optional linker connecting it to a miniprotein, which targets the conjugate to cells expressing a target (e.g., any one of the target proteins in Table 10).

[0245] In some embodiments, Lu-177 is conjugated to a miniprotein provided herein. In some embodiments, lutetium is conjugated onto a chelator, which may include an optional linker connecting it to a miniprotein, which targets the conjugate to cells expressing a target (e.g., any one of the target proteins in Table 10).

[0246] In some embodiments, Pb-212 is conjugated to a miniprotein provided herein. In some embodiments, lead is conjugated onto a chelator, which may include an optional linker connecting it to a miniprotein, which targets the conjugate to cells expressing a target (e.g., any one of the target proteins in Table 10).

[0247] In some embodiments, Cu-67 is conjugated to a miniprotein provided herein. In some embodiments, lead is conjugated onto a chelator, which may include an optional linker connecting it to a miniprotein, which targets the conjugate to cells expressing a target (e.g., any one of the target proteins in Table 10).

[0248] In some embodiments, La-132 is conjugated to a miniprotein provided herein. In some embodiments, lead is conjugated onto a chelator, which may include an optional linker connecting it to a miniprotein, which targets the conjugate to cells expressing a target (e.g., any one of the target proteins in Table 10).

[0249] In some embodiments, La-135 is conjugated to a miniprotein provided herein. In some embodiments, lead is conjugated onto a chelator, which may include an optional linker connecting it to a miniprotein, which targets the conjugate to cells expressing a target (e.g., any one of the target proteins in Table 10).

[0250] In some embodiments, In-111 is conjugated to a miniprotein provided herein. In some embodiments, lead is conjugated onto a chelator, which may include an optional linker connecting it to a miniprotein, which targets the conjugate to cells expressing a target (e.g., any one of the target proteins in Table 10).

[0251] In some embodiments, Ce-134 is conjugated to a miniprotein provided herein. In some embodiments, lead is conjugated onto a chelator, which may include an optional linker connecting it to a miniprotein, which targets the conjugate to cells expressing a target (e.g., any one of the target proteins in Table 10).

[0252] In some embodiments, the alpha particles (e.g., actinium-225) are positively charged. In some such embodiments, the range of penetration in tissues varies between 5 and 10 cell diameters (40-100 μm), depending on their energy (Radiobiologic principles in radionuclide therapy. Kassis AI, Adelstein SJ J Nucl Med. 2005 Jan; 46 Suppl 1():4S-12S). In some such embodiments, such penetration allows for localized irradiation of target cells with minimal toxicity to surrounding normal cells, as well as internalization by cancer cells, where as few as 1-3 tracks traversing the cell nucleus cause single- and double-stranded DNA breaks, resulting in cell death (Humm 1987; Macklis et al. 1988; Humm and Chin 1993; Couturier et al. 2005). See, e.g., Sofou S. Radionuclide carriers for targeting of cancer. Int J Nanomedicine. 2008;3(2):181-199. doi:10.2147 / ijn.s2736. Dose calculation

[0253] In some embodiments, the radiation therapy dose is calculated. In some such embodiments, calculation of the absorbed dose (D) is necessary to quantitatively correlate tumor response to a particular radiation therapy modality and to plan for the potential impact of other radiation therapy modalities or administration strategies. That is, in some embodiments, the absorbed dose from a target site is defined as the energy (E) absorbed by a specific mass of tissue normalized by the tissue mass (M): D = E / M (Sgouros 2005). The absorbed energy is defined as a function of three parameters: the number of disintegrations within a specific volume of interest (δ), the energy emitted per disintegration (ε), and the fraction of emitted energy absorbed by the specific volume of interest (target mass) (f): E = δ × ε × f. For relatively long-range beta emitters, dose assessment at the target site includes not only the energy emitted by radionuclides localized within the target volume, but also the energy emitted by radionuclides accumulated in nearby organs or regions whose radiation traverses the intended target volume along its course (Kolbert et al., 2003). In other words, in some embodiments, the calculated total absorbed dose is the sum of dose contributions from all regions containing radionuclides acting as secondary sources. In some embodiments, the absorbed dose due to photon emission is typically calculated separately and added to the dose due to alpha or beta particles. In some embodiments, when a composition includes an alpha particle emitter, such cross-organ absorbed dose may be insignificant due to their short recoil distance. In some embodiments, given the appropriate context, at the micron scale and at distances comparable to a few cells, microdosimetric assessment is used to assess the dose or "hits" acquired by cancer cells within micrometastatic clusters (Palm et al., 2002).

[0254] In some embodiments, the radionuclide-containing mini-protein conjugate exhibits binding specificity for the human isoform of any one of the target proteins in Table 10. In some embodiments, the mini-protein comprises a binding affinity characterized by a dissociation constant in the range of about 500 nM to about 1 pM for the human isoform of any one of the target proteins in Table 10, for example, a binding affinity of 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 nM, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 pM. Without being bound by any particular theory, the present disclosure contemplates that in some embodiments, the preferred dissociation constant of the miniprotein is about 10 nM or less, about 7.5 nM, about 5 nM or less, about 2.5 nM or less, about 1 nM or less (i.e., in the picomolar range).

[0255] In some embodiments, the compositions provided herein are characterized for one or more of absorbed dose, dose rate, tumor penetration profile of the radionuclide, subcellular localization profile of shorter range radionuclides, and tumor radiosensitivity (see, e.g., Sofou S. Radionuclide carriers for targeting of cancer. Int J Nanomedicine. 2008;3(2):181-199).

[0256] As known to those skilled in the art, due to the toxicity of radionuclides, doses must be carefully controlled and considered. Thus, in some embodiments, compositions comprising the radionuclides of the present disclosure address the dose-limiting toxicity of the composition so that the radionuclides do not accumulate significantly (e.g., in a toxicity-limiting manner) in vital organs.

[0257] In some embodiments, alpha particle-emitting isotopes provide on-target cell killing while minimizing toxic effects (e.g., to surrounding tissues, e.g., compared to, e.g., beta emitters, etc.).

[0258] In some embodiments, the compositions provided herein (including radionuclides) are administered in a single step using a ligand, e.g., a miniprotein, that results in, for example, improved biodistribution (e.g., specific targeting), a pK with partial and acceptable or no damage to normal tissue, or enhanced penetration of the pharmaceutical composition into the heterogeneous interstitial space of a tumor.

[0259] In some embodiments, one or more radionuclides are conjugated to the miniprotein. Relatedly, in some embodiments, the radiolabeling efficiency of the miniprotein is optimized to radiolabel a desired number of radionuclides. In some embodiments, the ratio of radionuclides conjugated to the miniprotein is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1. In some embodiments, the radionuclide conjugated to the miniprotein does not exhibit toxicity. In some embodiments, a composition comprising a miniprotein and a radionuclide does not accumulate in the liver, spleen, and pancreas, and is rapidly excreted upon administration to a subject. For example, in some embodiments, after administration to a subject, the biodistribution and t in the kidney are >10% of the injected dose (ID) in the tumor at 24 hours, and the tumor is >3% ID at 24 hours. Radionuclides and Chelation

[0260] Radionuclides can be bound to chelators via any method known in the art. In some embodiments, chelation methods can vary based on the selected radionuclide and chelator. For example, in some embodiments, chelation can be carried out in one step by incubating the miniprotein-chelator conjugate with the radionuclide at a predetermined temperature for a predetermined period of time to achieve a sufficient amount of chelation. In some embodiments, the miniprotein-chelator conjugate comprises a chelator provided herein or its variant (e.g., DOTA, NOPO, Crown, Macropa, etc.). In some embodiments, the miniprotein-chelator conjugate can be chelated to a radionuclide (e.g., actinium-225, gallium-68, copper-64, lutetium-177, indium-111, lead-212, etc.) by incubation with the radionuclide at 70°C for about 1 hour. In some embodiments, the miniprotein-chelator conjugate can be chelated to a radionuclide (e.g., actinium-225, gallium-68, copper-64, lutetium-177, indium-111, lead-212, etc.) by incubation with the radionuclide at 70°C for about 1 hour.

[0261] In some embodiments, the chelation process results in a preparation in which at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the miniprotein-chelator is bound to a radionuclide. In some embodiments, the chelation process results in a preparation in which more than 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the miniprotein-chelator is bound to a radionuclide. Excess radionuclide can be removed from the preparation by purification methods known in the art. Polypeptides

[0262] Among other things, the present disclosure provides polypeptides. In some embodiments, the polypeptides are assembled using solid phase synthesis. In some embodiments, the polypeptides are recombinant. In some embodiments, the polypeptides comprise or consist of miniproteins. In some such embodiments, the miniproteins comprise or consist of binders. In some embodiments, the polypeptides of the present disclosure (including muteins, allelic variants, fragments, derivatives and analogs) are encoded by polynucleotides described and provided herein.

[0263] In some embodiments, miniproteins of the present disclosure comprise or consist of a polypeptide capable of binding to a target as shown in Tables 3, 4A and 4B.

[0264] In some embodiments, the present disclosure provides binders comprising or consisting of fragments of the polypeptides provided herein. In some such embodiments, the fragments comprise at least 20 contiguous amino acids, more preferably at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or more contiguous amino acids.

[0265] In some embodiments, the miniproteins of the present disclosure may also include one or more other components, such as a fusion or conjugate with a heterologous polypeptide. For example, in some embodiments, the heterologous sequence may comprise or consist of a sequence designed to facilitate purification, such as a histidine tag, and / or a sequence designed to facilitate visualization of the recombinantly expressed protein. Other non-limiting examples of such fusions or conjugates include any detectable or visualizeable component, such as green fluorescent protein (GFP), that allows for display of the encoded protein on the surface of a phage or cell, and fusions to an IgG Fc region.

[0266] In some embodiments, the mini-protein comprises or consists of a particular amino acid sequence. In some embodiments, the mini-protein has an amino acid sequence that is 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to the amino acid sequence set forth in any of SEQ ID NOs: 1-68.

[0267] As used herein and as known to those skilled in the art, the 20 conventional amino acids and their abbreviations follow conventional usage. See Immunology-A Synthesis (Golub and Gren eds., Sinauer Associates, Sunderland, Mass., 2nd ed. 1991), hereby incorporated by reference. In some embodiments, the amino acids of the present disclosure may be stereoisomers of the 20 conventional amino acids (e.g., D-amino acids). In some embodiments, the amino acids in the polypeptides of the present disclosure may be unnatural amino acids. For example, amino acids such as α-,α-disubstituted amino acids, N-alkyl amino acids, and other unconventional amino acids may also be suitable building blocks for the polypeptides of the present disclosure. Examples of unconventional amino acids include: 4-hydroxyproline, γ-carboxyglutamic acid, ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, N-methylarginine, and other similar amino acids and imino acids (e.g., 4-hydroxyproline). The arrangement of polypeptide sequence notation used herein has the left-hand end corresponding to the amino-terminus and the right-hand end corresponding to the carboxy-terminus, in accordance with standard usage and convention.

[0268] In some embodiments, miniproteins of the present disclosure that contain two or more cysteine ​​residues, such as those set forth in SEQ ID NOs: 1-84, have the cysteine ​​residues connected via disulfide bridges (e.g., via native folding). nucleic acid

[0269] Among other things, the present disclosure provides herein polynucleotides and methods of use thereof. In some embodiments, all or a portion of the polynucleotide encodes a polypeptide (e.g., a miniprotein) that specifically binds to any one of the target proteins in Table 10. In some embodiments, the target protein selected from Table 10 is a mouse or human isoform of the target protein selected from Table 10. In some embodiments, the nucleic acid sequence has a specific sequence. In some embodiments, the polynucleotides of the present disclosure are codon-optimized (i.e., the nucleic acid sequence is codon-optimized).

[0270] In some embodiments, a polynucleotide of the present disclosure comprises or consists of a nucleic acid sequence encoding a polypeptide that is or comprises a miniprotein that specifically binds to any one of the target proteins in Table 10, or any part, fragment, or variant thereof.

[0271] In some embodiments, the miniprotein is represented by a nucleic acid molecule that encodes amino acids that, when folded, comprise one or more disulfide bridges.

[0272] In some embodiments, for example, a nucleic acid molecule (i.e., polynucleotide) may be non-identical to a reference sequence provided herein, but still encode a binder provided by the present disclosure. In some such embodiments, for example, a provided polynucleotide (i.e., encoding a miniprotein or analog thereof) hybridizes under stringent conditions disclosed herein.

[0273] In some embodiments, the present disclosure provides nucleic acid molecules comprising fragments of any polynucleotide provided herein.In some embodiments, the polynucleotide fragment comprises or consists of a portion of consecutive nucleic acid residues.For example, in some embodiments, the polynucleotide fragment comprises or consists of 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 100 or more nucleic acid residues.

[0274] In some embodiments, the fragments of the present disclosure are useful in a variety of systems and methods. For example, the fragments can be used as probes in various assays. For example, in some embodiments, the fragments can be used in hybridization techniques. Depending on the method, the target nucleic acid sequence can be either DNA or RNA. The target nucleic acid sequence can be fractionated (e.g., by gel electrophoresis) before hybridization, or hybridization can be performed in situ on the sample. Those skilled in the art will understand that nucleic acid probes of known sequences will find utility in determining chromosome structure (e.g., by Southern blotting) and measuring gene expression (e.g., by Northern blotting). In such experiments, the sequence fragments are preferably detectably labeled so that their specific hybridization to the target sequence can be detected and, if necessary, quantified. In some embodiments, the fragments can be used as probes, such as when immobilized on a microarray. Methods for creating microarrays by depositing and immobilizing nucleic acids on a support substrate are well known in the art. DNA Microarrays: A Practical Approach (Practical Approach Series), Schena (ed.), Oxford University Press (1999) (ISBN: 0199637768); Nature Genet. 21(1)(suppl):1-60 (1999); Microarray Biochip: Tools and Technology, Schena (ed.), Eaton Publishing Company / BioTechniques Books Division (2000) (ISBN: 1881299376), the disclosures of which are hereby incorporated by reference in their entireties. For example, analysis of gene expression using microarrays containing nucleic acid sequence fragments, e.g., the nucleic acid sequence fragments disclosed herein, is a well-established utility of sequence fragments in the fields of cell and molecular biology.Other uses for sequence fragments immobilized on microarrays are described in Gerhold et al., Trends Biochem. Sci. 24:168-173 (1999) and Zweiger, Trends Biotechnol. 17:429-436 (1999); DNA Microarrays: A Practical Approach (Practical Approach Series), Schena (ed.), Oxford University Press (1999) (ISBN: 0199637768); Nature Genet. 21(1)(suppl):1-60 (1999); Microarray Biochip: Tools and Technology, Schena (ed.), Eaton Publishing Company / BioTechniques Books Division (2000) (ISBN: 1881299376).

[0275] In some embodiments, a polynucleotide of the present disclosure comprises or consists of a nucleic acid sequence encoding a polypeptide, or any part, fragment, or variant thereof, that is or comprises a miniprotein that binds to any one of the target proteins in Table 10. In some embodiments, the polynucleotide encodes a polypeptide that comprises or consists of an amino acid sequence set forth in any one of SEQ ID NOs: 1-68. In some embodiments, the polynucleotides encode polypeptide sequences having at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more identity to the amino acid sequences provided in Tables 3, 4A and 4B.

[0276] In some embodiments, the miniprotein comprises one or more disulfide bridges. In some embodiments, the miniprotein is represented by a nucleic acid sequence that encodes a polypeptide that, when folded, comprises one or more disulfide bridges.

[0277] In some embodiments, for example, a nucleic acid molecule (i.e., polynucleotide) may be non-identical to a reference sequence provided herein, but still encode a miniprotein provided by the present disclosure (e.g., a miniprotein according to any one of SEQ ID NOS: 1-68, or a closely related analog provided herein). In some such embodiments, for example, a provided polynucleotide (i.e., encoding a miniprotein or an analog thereto) hybridizes under stringent conditions disclosed herein. In some embodiments, the present disclosure provides nucleic acid molecules comprising a fragment of any polynucleotide provided herein. In some embodiments, a polynucleotide fragment comprises or consists of a portion of contiguous nucleic acid residues identical to those of any polynucleotide of SEQ ID NOS: 1-68. For example, in some embodiments, a polynucleotide fragment comprises or consists of 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 100, or more nucleic acid residues encoding part or all of a polypeptide set forth in any one of SEQ ID NOS: 1-68, or a fragment thereof.

[0278] Those skilled in the art will appreciate that the nucleic acid fragments of the present disclosure can be used in a wide variety of capture and / or detection techniques not specifically described herein. vector

[0279] Also provided herein are vectors, including, inter alia, expression vectors comprising a nucleic acid comprising or consisting of a sequence encoding a miniprotein that specifically binds to any one of the target proteins in Table 10. In some embodiments, the vector is used to produce a polypeptide encoding a binder that binds to any one of the target proteins in Table 10. In some embodiments, the target protein selected from Table 10 is a mouse or human isoform of any one of the target proteins in Table 10. In some embodiments, given the appropriate context, the miniprotein is represented by an amino acid sequence along with a codon-optimized corresponding nucleic acid sequence. In some such embodiments, one skilled in the art can design and optimize a polynucleotide corresponding to the amino acids of a miniprotein that binds to a target (e.g., any one of the target proteins in Table 10), whose exemplary amino acid sequences are shown in Tables 3, 4A, and 4B. In some embodiments, the vector comprises a nucleic acid sequence comprising or consisting of a sequence encoding any one of the target proteins in Table 10.

[0280] In some embodiments, a vector comprises a nucleic acid sequence encoding any one of the target proteins in Table 10, or a fragment or variant thereof, wherein the polynucleotide is codon-optimized (i.e., the nucleic acid sequence is codon-optimized). In some embodiments, a vector comprises a nucleic acid sequence encoding up to 100 amino acids. In some embodiments, a vector of the present disclosure comprises or consists of a nucleic acid sequence encoding the amino acid sequence of a miniprotein. In some embodiments, a vector of the present disclosure further comprises a nucleic acid sequence provided herein operably linked to one or more expression control sequences.

[0281] Also provided herein are vectors, including, inter alia, expression vectors comprising nucleic acids comprising or consisting of those described herein. In some embodiments, the vectors are used to produce polypeptides encoding miniproteins that bind to any one of the target proteins in Table 10. In some embodiments, the target protein selected from Table 10 is a mouse or human isoform of any one of the target proteins in Table 10. In some embodiments, given the appropriate context, a miniprotein for any one of the target proteins in Table 10 (e.g., provided in Tables 3, 4A, and 4B) has a corresponding nucleic acid sequence that is codon-optimized. In some such embodiments, one skilled in the art can design and optimize polynucleotides corresponding to the amino acids of a particular miniprotein for any one of the target proteins in Table 10, such as, for example, polynucleotides comprising nucleic acid sequences corresponding to the amino acid sequences of Tables 3, 4A, and 4B. In some embodiments, the vector comprises a nucleic acid sequence that comprises or consists of a sequence having 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or higher identity to the wild-type isform of any one of the target proteins in Table 10. In some embodiments, the vector comprises a nucleic acid sequence encoding any one of the target proteins in Table 10 or a fragment or variant thereof, wherein the polynucleotide is codon optimized (i.e., the nucleic acid sequence is codon optimized). In some embodiments, the vector comprises a nucleic acid sequence encoding up to 100 amino acids of any one of SEQ ID NOs: 1-68. In some embodiments, the vector of the present disclosure comprises or consists of a nucleic acid sequence, wherein the nucleic acid sequence encodes an amino acid sequence set forth in Tables 3, 4A, and 4B. In some embodiments, the nucleic acid sequence is a codon-optimized nucleic acid sequence.In some embodiments, the vectors of the present disclosure further comprise a nucleic acid sequence provided herein operably linked to one or more expression control sequences. Kidney retention

[0282] In various embodiments, novel methods and compositions are provided for addressing the undesirable effects of radionuclide therapy during renal transport. Accordingly, compositions characterized as exhibiting reduced renal retention are disclosed herein. Renal retention of peptide-based radiopharmaceuticals can be attributed to target expression in the kidney (e.g., PSMA) or protein retention from ultrafiltration and reuptake into proximal tubule cells. Reuptake in proximal tubule cells occurs through the reuptake of proteins or peptides cleavable by renal brush border peptidases, and short peptides, as well as receptor-mediated reuptake. In receptor-mediated reuptake, peptide-based radiopharmaceuticals bind to the megalin / cubulin receptor complex, undergo receptor-mediated endocytosis, and are degraded and retained in lysosomes, resulting in prolonged retention of radioactivity in the proximal tubules of the kidney and dose-limiting toxicity of the radiopharmaceutical. Geenen et al., Nucl. Med. Biol, 2021, 102-103: 1-11. Decharged molecules

[0283] In exemplary aspects for reducing renal retention, in some embodiments, the composition comprises a decharged molecule or a molecule with a lower charge that allows for rapid clearance via the kidney. In some embodiments, a decrease in positively charged or polar molecules, an increase in negatively charged molecules, and / or both results in reduced renal retention.

[0284] In some embodiments, the composition comprising the decharged molecule(s) comprises an amino acid sequence comprising any one of SEQ ID NOs: 1-3, 8-11, or 65-67. In some embodiments, the composition comprising the decharged molecule(s) comprises a compound selected from any of C1-C6, C43-54, or C155-C157.

[0285] In some embodiments, compositions wherein M comprises an amino acid sequence comprising between 1-5% of the total amino acid sequence a percentage of charged amino acids, wherein the charged amino acids are selected from Lys, Arg, or His, are characterized as exhibiting reduced renal uptake. In some embodiments, compositions wherein M comprises an amino acid sequence comprising between 5-10% of the total amino acid sequence a percentage of charged amino acids, wherein the charged amino acids are selected from Lys, Arg, or His, are characterized as exhibiting reduced renal uptake. In some embodiments, compositions wherein M comprises an amino acid sequence comprising between 10-15% of the total amino acid sequence a percentage of charged amino acids, wherein the charged amino acids are selected from Lys, Arg, or His, are characterized as exhibiting reduced renal uptake. In some embodiments, compositions wherein M comprises an amino acid sequence comprising between 15-20% of the total amino acid sequence a percentage of charged amino acids, wherein the charged amino acids are selected from Lys, Arg, or His, are characterized as exhibiting reduced renal uptake. In some embodiments, compositions wherein M comprises an amino acid sequence comprising between 20-25% of the total amino acid sequence of charged amino acids, wherein the charged amino acids are selected from Lys, Arg, or His, are characterized as exhibiting reduced renal uptake. In some embodiments, compositions wherein M comprises an amino acid sequence comprising between 25-30% of the total amino acid sequence of charged amino acids, wherein the charged amino acids are selected from Lys, Arg, or His, are characterized as exhibiting reduced renal uptake. In some embodiments, the reduced percentage of positively charged amino acids in M ​​results in decreased reabsorption of M across the negatively charged membrane of renal proximal tubule cells.

[0286] In some embodiments, compositions comprising an amino acid sequence in which M comprises between 1-5% of the total amino acid sequence a percentage of charged amino acids, wherein the charged amino acids are selected from Asp or Glu, are characterized as exhibiting reduced renal uptake. In some embodiments, compositions comprising an amino acid sequence in which M comprises between 5-10% of the total amino acid sequence a percentage of charged amino acids, wherein the charged amino acids are selected from Asp or Glu, are characterized as exhibiting reduced renal uptake. In some embodiments, compositions comprising an amino acid sequence in which M comprises between 10-15% of the total amino acid sequence a percentage of charged amino acids, wherein the charged amino acids are selected from Asp or Glu, are characterized as exhibiting reduced renal uptake. In some embodiments, compositions comprising an amino acid sequence in which M comprises between 15-20% of the total amino acid sequence a percentage of charged amino acids, wherein the charged amino acids are selected from Asp or Glu, are characterized as exhibiting reduced renal uptake. In some embodiments, compositions wherein M comprises an amino acid sequence comprising between 20-25% of the total amino acid sequence a percentage of charged amino acids, wherein the charged amino acids are selected from Asp or Glu, are characterized as exhibiting reduced renal uptake. In some embodiments, compositions wherein M comprises an amino acid sequence comprising between 25-30% of the total amino acid sequence a percentage of charged amino acids, wherein the charged amino acids are selected from Asp or Glu, are characterized as exhibiting reduced renal uptake. In some embodiments, the reduced percentage of polar amino acids in M ​​results in decreased reabsorption of M across the negatively charged membrane of renal proximal tubule cells.

[0287] Examples 10 and 11 and Figures 1 and 3 provide exemplary data on the level of kidney retention measured in mice treated with radiolabeled peptides and imaged via SPECT / CT. In some embodiments, the percentage of ID / g in the kidney at 4 hours is reduced by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% when the composition includes a certain percentage of charged amino acids, compared to a composition in which the percentage of charged amino acids is not reduced. In some embodiments, the percentage of ID / g in the kidney at 24 hours is reduced by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% when the composition comprises a certain percentage of charged amino acids compared to a composition without an increased percentage of charged amino acids. Albumin-binding domain

[0288] In an exemplary aspect for reducing renal retention, in some embodiments, a composition comprises an amino acid sequence comprising an albumin-binding domain. Thus, such a composition can bind to albumin, which can extend the circulating half-life of the amino acid sequence in the circulation and reduce renal retention. In some embodiments, the composition comprising an amino acid sequence comprising an albumin-binding domain comprises the amino acid sequence of any one of SEQ ID NOs: 7 or 13. In some embodiments, the composition comprising an amino acid sequence comprising an albumin-binding domain comprises a compound selected from any of C12, C23-C25, C29, C30, C32, C33, C66, C69, or C71. Cleavable Linker

[0289] In another aspect for reducing kidney retention, in some embodiments, the composition comprises MLCR, and L is a cleavable linker. In some embodiments, the cleavable linker is distal to the miniprotein so as not to interfere with tumor binding. In some embodiments, the cleavable linker is engineered to optimize physicochemical characteristics. In some embodiments, the cleavable linker has a short length. In some embodiments, the cleavable linker is selectively cleaved in the proximal tubules of the kidney. In some embodiments, the selective cleavage of the cleavable linker in the proximal tubules of the kidney is due to the presence of peptidases in the proximal tubules. In some embodiments, cleavage of the cleavable linker in the proximal tubules releases the chelator-radionuclide-miniprotein, which is filtered into the bladder and avoids renal reuptake. See Arano, Y. Nuclear Medicine and Biology 2021, 92, 149-55; Zhang, M. et. al. Bioconjugate Chemistry 2019, 30, 1745-53.

[0290] In some embodiments, the composition comprising a cleavable linker comprises an amino acid sequence comprising any one of SEQ ID NOs: 7-11, 17-23, 26, or 27. In some embodiments, the composition comprising a cleavable linker comprises a compound selected from any of C15, C17-C22, C26-C28, C34-C42, C55-66, C68, C70, or C72-C77.

[0291] In some embodiments, the cleavable linker is connected to M at the Na-carboxyl of lysine. In some embodiments, the cleavable linker comprises hydrogen, (PEG4)1-4, 4-aminomethyl-phenylacetic acid (AmPA), aminomethylbenzoyl (AmBz), succinic acid-(PEG4)1-4, norleucine, isoleucine (ileucine), glutamine, methoxynine, phenylalanine, tyrosine, beta-alanine, MWK or MVK, glycine, citrulline (Cit), or sarcosine (Sar). In some embodiments, the linker is cleaved by cathepsin B in lysosomes or by neutral endopeptidases, metalloproteases, or dipeptidyl peptidases in renal brush border membranes.

[0292] The level of kidney retention measured in mice treated with radiolabeled peptides and imaged via SPECT / CT is described in Examples 10 and 11. In some embodiments, the cleavable linker reduces kidney uptake of M, as shown in Figures 5 and 13.

[0293] In some embodiments, the cleavable linker is a disulfide bond or protease-sensitive. In further embodiments, groups adjacent to the disulfide bond are modified to control the hindrance of the disulfide bond and, thereby, the rate of cleavage. Published research has established the possibility of modifying the susceptibility of disulfide bonds to reduction by introducing steric hindrance on either side of the disulfide bond (Kellogg et al. (2011) Bioconjugate Chemistry, 22, 717). A higher degree of steric hindrance reduces the rate of reduction by intracellular glutathione and also extracellular (systemic) reducing agents, thereby reducing the ease with which the remainder of the conjugate is released both inside and outside the cell. Therefore, optimal selection of disulfide stability in circulation (minimizing undesirable side effects of the radionuclide) versus efficient release in the intracellular environment (maximizing therapeutic efficacy) can be achieved by careful selection of the degree of hindrance on either side of the disulfide bond. The disorder on either side of the disulfide bond is modulated through the introduction of one or more methyl groups on either the miniprotein or radionuclide side of the molecular construct. Coadministration

[0294] In an alternative embodiment for reducing renal clearance, in some embodiments, the composition comprises MLCR and one or more additional proteins, such as decoy peptides. In some embodiments, co-administration of the composition with decoy peptides results in reduced renal uptake due to competitive inhibition of proximal tubule cell receptors. In some embodiments, co-administration with functional inhibitors results in reduced renal uptake. See Xiong, C. et al. Mol. Pharmaceutics 2019, 16, 808-15; Melis, M. et al. Eur J Nucl Med Imaging, 2009, 36, 1968-76.

[0295] In some embodiments, the decoy peptide has an amino acid sequence comprising any one of SEQ ID NO:4, SEQ ID NO:7, SEQ ID NOs:16-52, or SEQ ID NO:68. In some embodiments, the decoy peptide is a compound selected from any of C7, C31, C100-C108, or C131-C139. In some embodiments, the decoy peptide is present at a concentration that is 10x, 20x, 30x, 40x, 50x, 60x, 70x, 80x, 90x, or 100x the concentration of M. Examples 10 and 11 describe the level of renal retention measured in mice treated with radiolabeled peptides and imaged via SPECT / CT using the approaches provided herein. In some embodiments, the decoy peptide reduces renal uptake of M, as shown, for example, in Figures 2, 4, and 12.

[0296] In some embodiments, the decoy peptide reduces the uptake of M in the kidney by competitive inhibition. In some embodiments, the decoy peptide can be of a desired length. In some aspects, the amino acid residues added to the N-terminus or C-terminus of the decoy peptide disclosed herein can prevent ubiquitination, improve stability, help maintain the three-dimensional structure of the peptide, or a combination thereof.

[0297] In some embodiments, the decoy peptides disclosed herein may further comprise peptides or polypeptides having one or more amino acid residues with modified side chains.In some embodiments, any one or more amino acids of the decoy peptides disclosed herein may have modified side chains.Examples of side chain modifications include but are not limited to the modification of amino acid groups, such as reductive alkylation; amidination with methylacetimidate; acylation with acetic anhydride; carbamoylation of amino groups with cyanate; trinitrobenzylation of amino acids with 2,4,6-trinitrobenzenesulfonic acid (TNBS); alkylation of amino groups with succinic anhydride; and pyridoxylation with pyridoxal-5-phosphate followed by reduction with NaBH4.

[0298] In some embodiments, the decoy peptides described herein can be further modified to improve stability.In some embodiments, any of the amino acid residues of the decoy peptides described herein can be modified to improve stability.In some embodiments, the decoy peptides can have at least one amino acid residue with an acetyl group, a fluorenylmethoxycarbonyl group, a formyl group, a palmitoyl group, a myristyl group, a stearyl group or polyethylene glycol.In some embodiments, an acetyl protecting group can be attached to the decoy peptides described herein.

[0299] As used herein, the term "decoy peptide" may also be used to include functional equivalents of the decoy peptides described herein. As used herein, the term "functional equivalent" may refer to an amino acid sequence variant having an amino acid substitution, addition, or deletion in a portion of the amino acid sequence of the decoy peptide while simultaneously having similar or improved biological activity compared to the decoy peptides described herein. In some embodiments, the amino acid substitution may be a conservative substitution. Examples of naturally occurring conservative amino acid substitutions include, for example, aliphatic amino acids (Gly, Ala, and Pro), hydrophobic amino acids (Ile, Leu, and Val), aromatic amino acids (Phe, Tyr, and Trp), acidic amino acids (Asp and Glu), basic amino acids (His, Lys, Arg, Gln, and Asn), and sulfur-containing amino acids (Cys and Met). In some embodiments, the amino acid deletion may be located in a region not directly involved in the activity of the decoy peptides disclosed herein.

[0300] In some embodiments, the amino acid sequence of the decoy peptide described herein can comprise a peptide sequence that has substantial identity with any of the sequences of the decoy peptide disclosed herein.As used herein, the term "substantial identity" means that when two amino acid sequences are optimally aligned and then analyzed by algorithms commonly used in the art, such as BLAST, GAP or BESTFIT, or by visual inspection, they share at least about 60%, 70%, 80%, 85%, 90% or 95% sequence identity.The alignment method for sequence comparison is known in the art.

[0301] In some embodiments, the amino acid sequence of the decoy peptide described herein may comprise a peptide sequence that has a degree of identity or homology with any of the sequences of the decoy peptides disclosed herein. The degree of identity may vary and can be determined by methods known to those skilled in the art. The terms "homology" and "identity" each refer to the sequence similarity between two polypeptide sequences. Homology and identity can each be determined by comparing positions in each sequence that can be aligned for comparison purposes. If a position in the compared sequences is occupied by the same amino acid residue, the polypeptides can be said to be identical at that position; if an equivalent position is occupied by the same amino acid (e.g., identical) or a similar amino acid (e.g., similar in steric and / or electronic properties), the molecules can be said to be homologous at that position. The percentage of homology or identity between sequences is a function of the number of matching or homologous positions shared by the sequences. The decoy peptides described herein may have at least or about 25%, 50%, 65%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity or homology to the decoy peptide. Host cell transformants

[0302] In some embodiments, the present disclosure provides host cells transformed with the polynucleotides, polypeptides, and / or vectors of the present disclosure, and any combination thereof, and any progeny thereof. In some embodiments, the host cells comprise and carry the nucleic acid sequences of the present disclosure on a vector. In some embodiments, the host cells are cell lines. In some embodiments, the host cells are primary cells, such as immune cells. In some embodiments, such primary cells are derived from or adapted to a subject. In some embodiments, the subject is a mammal. In some embodiments, the mammal is a human. In some embodiments, the human is at risk of having cancer or has been diagnosed with cancer.

[0303] In some such embodiments, such vectors may be, but need not be, freely replicating vectors, hi some embodiments, the nucleic acid sequences or polynucleotides provided by the present disclosure are integrated into the genome of a host cell.

[0304] In some embodiments, a host cell of the present disclosure may be recombinantly altered by disruption, deletion, or mutation of an isolated nucleic acid of the present disclosure such that the activity of one or more functional activities in the host cell is reduced or eliminated compared to a host cell lacking that mutation.

[0305] Without limitation, as will be appreciated by those of skill in the art, a wide variety of host cells are contemplated in various embodiments for expressing the binders of the present disclosure (e.g., through the use of the nucleic acid sequences, amino acid sequences and / or additional components provided herein). Pharmaceutical Composition

[0306] The present disclosure provides, inter alia, pharmaceutical compositions comprising polypeptides, polynucleotides, vectors and / or host cells encoding the miniproteins provided herein (e.g., CDPs, knottins, binders, affibodies, engineered Kunitz domains, monobodies, anticalins, designed ankyrin repeat domains (DARPins), avimers). It should be understood that a pharmaceutical composition comprising a miniprotein (e.g., CDP, knottin, binder, affibody, engineered Kunitz domain, monobody, anticalin, designed ankyrin repeat domain (DARPin), avimer) is to be construed as a pharmaceutical composition comprising the miniprotein (e.g., CDP, knottin, binder, affibody, engineered Kunitz domain, monobody, anticalin, designed ankyrin repeat domain (DARPin), avimer) itself and / or one or more components (e.g., vector, e.g., host cell) encoding the miniprotein (e.g., CDP, knottin, binder, affibody, engineered Kunitz domain, monobody, anticalin, designed ankyrin repeat domain (DARPin), avimer). In some embodiments, the pharmaceutical composition comprises a linker and a chelator. In some embodiments, the pharmaceutical composition comprises a linker, a chelator, and a radionuclide. In some embodiments, the composition comprises a miniprotein, an optional linker, and a chelator. In some embodiments, the composition comprises a miniprotein, an optional linker, a chelator, and a radionuclide. In some embodiments, the pharmaceutical composition provided by the present disclosure comprises a miniprotein (e.g., a CDP, a knottin, a binder, an affibody, an engineered Kunitz domain, a monobody, an anticalin, a designed ankyrin repeat domain (DARPin), an avimer) that selectively binds to any one of the target proteins in Table 10. In some embodiments, the target protein selected from Table 10 is a human target protein selected from Table 10.

[0307] In certain embodiments, pharmaceutical compositions comprise miniproteins comprising one or more cysteine-rich domains (e.g., CDPs, knottins, binders, affibodies, engineered Kunitz domains, monobodies, anticalins, designed ankyrin repeat domains (DARPins), avimers). In some embodiments, pharmaceutical compositions comprise miniproteins having one or more disulfide bonds (e.g., CDPs, knottins, binders, affibodies, engineered Kunitz domains, monobodies, anticalins, designed ankyrin repeat domains (DARPins), avimers). In some embodiments, the pharmaceutical composition comprises a miniprotein (e.g., CDP, knottin, binder, affibody, engineered Kunitz domain, monobody, anticalin, designed ankyrin repeat domain (DARPin), avimer) represented by or encoded by an amino acid sequence having <100 AA, <90 AA, <80 AA, <85 AA, <75 AA, <70 AA, <65 AA, <60 AA, <55 AA, <50 AA, <45 AA, <40 AA, <35 AA, <30 AA, <25 AA, <20 AA, <15 AA, <10 AA or <5 AA).

[0308] In some embodiments, pharmaceutical compositions comprising a miniprotein provided herein (e.g., CDPs, knottins, binders, affibodies, engineered Kunitz domains, monobodies, anticalins, designed ankyrin repeat domains (DARPins), avimers) are characterized as having a molecular weight less than or equal to 12 kDa.

[0309] In some embodiments, pharmaceutical compositions comprising miniproteins (e.g., CDPs, knottins, binders, affibodies, engineered Kunitz domains, monobodies, anticalins, designed ankyrin repeat domains (DARPins), avimers) do not elicit an unwanted immune response or elicit a tolerable immune response. In some embodiments, pharmaceutical compositions of the present disclosure comprise miniproteins (e.g., CDPs, knottins, binders, affibodies, engineered Kunitz domains, monobodies, anticalins, designed ankyrin repeat domains (DARPins), avimers) with high tissue penetration properties.

[0310] In some embodiments, pharmaceutical compositions comprising miniproteins have acceptable half-life and / or stability. In some such embodiments, acceptable stability is between about 30 minutes and 48 hours in serum and 1-4 days or longer in tumors or tumor microenvironments. By way of non-limiting example, in some embodiments, miniproteins of the present disclosure have a stability of about 2.5 hours in serum. In some embodiments, the stability of the miniprotein is about 30 minutes, 60 minutes, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 30, 36, 40, or 48 hours in serum. In some embodiments, the stability in tumors or tumor microenvironments is 24, 36, 48, 60, 72, 84, 96 hours or longer.

[0311] In some embodiments, the pharmaceutical compositions are characterized as being stable in vivo. In some embodiments, the pharmaceutical compositions provided herein are not taken up by the kidney or liver.

[0312] In some embodiments, pharmaceutical compositions of the present disclosure exhibit a solubility of >0.05 mg / mL, >0.1 mg / mL, >0.2 mg / mL, >0.3 mg / mL, >0.4 mg / mL, >0.5 mg / mL, >0.6 mg / mL, >0.7 mg / mL, >0.8 mg / mL, >0.9 mg / mL, >1 mg / mL, >2 mg / mL, >3 mg / mL, >4 mg / mL, >5 mg / mL, >6 mg / mL, >7 mg / mL, >8 mg / mL, >9 mg / mL or >10 mg / mL.

[0313] In some embodiments, pharmaceutical compositions provided by the present disclosure exhibit a stability of >80%, >81%, >82%, >83%, >84%, 85%, >86%, >87%, >88%, >89%, >90%, >91%, >92%, >93%, >94%, 95%, >96%, >97%, >98% or >99%.

[0314] In some embodiments, the pharmaceutical compositions of the present disclosure are characterized as comprising a certain purity expressed as a percentage of the parent molecule that is still intact. In some embodiments, the pharmaceutical compositions of the present disclosure comprise a purity of about 85% or greater at 5 days at room temperature. In some embodiments, the pharmaceutical compositions of the present disclosure are characterized as having a purity of about 90% or greater at 4 hours at 40°C. In some embodiments, the pharmaceutical compositions of the present disclosure comprise a cyclic or acyclic sequence.

[0315] In some embodiments, pharmaceutical compositions according to the present disclosure comprise a miniprotein (e.g., a CDP, a knottin, a binder, an affibody, an engineered Kunitz domain, a monobody, an anticalin, a designed ankyrin repeat domain (DARPin), an avimer) and one or more additional components. For example, in some embodiments, the one or more additional components can be a linker and / or a conjugate, e.g., a cytotoxic payload or a detectable moiety for use in diagnostics and / or imaging. In some such embodiments, the pharmaceutical composition comprises a linker, a chelator and / or a radionuclide as provided herein.

[0316] In some embodiments, pharmaceutical compositions are provided that modulate, bind, or inhibit the human isoform of any one of the target proteins in Table 10 (or any activity associated therewith). In some embodiments, the pharmaceutical composition is or comprises a therapeutic agent. In some embodiments, the pharmaceutical composition is or comprises a detectable moiety (e.g., used for imaging, e.g., MRI, CT, PET, etc.).

[0317] In preferred embodiments, one or more characteristics of the pharmaceutical composition are identified for optimized administration parameters, including but not limited to, dose, effective dose, dose rate, tumor penetration profile, intracellular localization profile, binding specificity, etc. (See Sofou S. Radionuclide carriers for targeting of cancer. Int J Nanomedicine. 2008;3(2):181-199. doi:10.2147 / ijn.s2736).

[0318] In some embodiments, the pharmaceutical compositions of the present disclosure exhibit no toxicity or exhibit less toxicity than compositions comprising one or more different components, e.g., a larger targeting peptide or a different radionuclide (e.g., a beta emitter, etc.).

[0319] In some embodiments, the pharmaceutical compositions of the present disclosure do not accumulate in the liver, spleen, and pancreas, and are rapidly excreted. For example, the biodistribution and t in the kidney are >10% of the injected dose (ID; the initial dose injected) in the tumor at 24 hours, and the tumor is >3% ID at 24 hours. Theranostic compositions

[0320] In some embodiments, theranostic compositions are provided. In some embodiments, the present disclosure provides diagnostic methods or screens for detecting the presence or absence and / or level of any one of the target proteins in Table 10 in a subject or sample. In some embodiments, the subject is a mammalian human subject, and the target protein selected from Table 10 is a human isoform. In some embodiments, the presence of any one of the target proteins in Table 10 in a subject is associated with a risk of developing a disease, disorder, or condition. In some embodiments, the presence of any one of the target proteins in Table 10 at a particular level indicates an increased risk of developing a disease, disorder, or condition, or a diagnosis of the disease, disorder, or condition. In some embodiments, a reduction in the level of any one of the target proteins in Table 10 (e.g., compared to a previous measurement) is associated with treatment of the diagnosed disease.

[0321] In certain aspects, theranostic compositions are provided. In some embodiments, the present disclosure provides diagnostic methods or screens for detecting the presence or absence and / or level of any one of the human target proteins in Table 10 in a subject or sample.

[0322] In certain aspects, the present disclosure provides a method for producing a pharmaceutical composition comprising: (i) a miniprotein specific for any one of the target proteins in Table 10; (ii) an optional linker; (iii) a chelator; and (iv) Radioactive molecules wherein the modified polypeptide sequence modulates the activity of a human isoform of any one of the target proteins in Table 10. Screening and Development Methods

[0323] In some embodiments, directed evolution and computational folding algorithms can be combined for the de novo creation of miniproteins (e.g., CDPs, knottins, binders, affibodies, engineered Kunitz domains, monobodies, anticalins, designed ankyrin repeat domains (DARPins), avimers). For example, in some embodiments, hundreds of miniprotein scaffolds with various secondary structure elements, orientations, and loop lengths can be matched to hotspot binding motifs on a protein target or antigen of interest (e.g., any one of the target proteins in Table 10). In some such embodiments, if the binding motifs of the miniprotein do not clash with the scaffold of the target, the monomers and interaction energies are optimized using Rosetta combinatorial sequence optimization.

[0324] In some embodiments, oligonucleotide pools encoding design sequences selected via computational approaches can be synthesized, amplified, and co-transformed into yeast. The resulting yeast library displaying the design sequences can be incubated with a fluorescently labeled target protein or antigen. Cells displaying designs that bind to the target can be recovered by fluorescence-activated cell sorting (FACS) and deep-sequenced. Once miniproteins (e.g., CDPs, knottins, binders, affibodies, engineered Kunitz domains, monobodies, anticalins, designed ankyrin repeat domains (DARPins), avimers) are identified either through affinity maturation or original design, they can be chemically synthesized or expressed, for example, in Escherichia coli, purified, and characterized in solution.

[0325] In some embodiments, libraries of stable miniproteins (e.g., CDPs, knottins, binders, affibodies, engineered Kunitz domains, monobodies, anticalins, designed ankyrin repeat domains (DARPins), avimers) can be developed to allow screening against specific selected targets. In addition to cysteine ​​cross-linking, such libraries may engineer a hydrophobic core into the miniproteins (e.g., CDPs, knottins, binders, affibodies, engineered Kunitz domains, monobodies, anticalins, designed ankyrin repeat domains (DARPins), avimers) to allow folding, improving the number of folded structures in the library.

[0326] In some embodiments, once miniproteins (e.g., CDPs, knottins, binders, affibodies, engineered Kunitz domains, monobodies, anticalins, designed ankyrin repeat domains (DARPins), avimers) are identified or engineered, they can be produced via chemical synthesis or recombinant expression. In some embodiments, miniproteins (e.g., CDPs, knottins, binders, affibodies, engineered Kunitz domains, monobodies, anticalins, designed ankyrin repeat domains (DARPins), avimers) can be produced by solid-phase peptide synthesis followed by in vitro folding. Standard 9-fluorenylmethyloxycarbonyl (Fmoc)-based solid-phase peptide chemistry can be used. In some such embodiments, linear peptides can then be folded under conditions that promote oxidation of cysteine ​​side chain thiols to form disulfide bonds, and subsequently purified by, for example, reverse-phase high-performance liquid chromatography (RP-HPLC). Recombinant DNA approaches can also be used to produce the miniproteins provided herein (e.g., CDPs, knottins, binders, affibodies, engineered Kunitz domains, monobodies, anticalins, designed ankyrin repeat domains (DARPins), avimers).

[0327] Iterative data-driven model improvements and experimental testing with miniproteins (e.g., CDPs, knottins, binders, affibodies, engineered Kunitz domains, monobodies, anticalins, designed ankyrin repeat domains (DARPins), avimers) are likely to optimize the folding and binding capacity of miniproteins (e.g., CDPs, knottins, binders, affibodies, engineered Kunitz domains, monobodies, anticalins, designed ankyrin repeat domains (DARPins), avimers) to develop pharmaceutically superior specific molecules. Characterization, analysis, and synthesis

[0328] In some embodiments, miniproteins of the present disclosure (e.g., CDPs, knottins, binders, affibodies, engineered Kunitz domains, monobodies, anticalins, designed ankyrin repeat domains (DARPins), avimers) are characterized. For example, in some embodiments, binding specificity, binding affinity, binding localization, etc. are performed using methods known to those of skill in the art. For example, in some embodiments, binding localization is performed using one or more techniques, e.g., immunohistochemistry / immunocytochemistry (e.g., using cell lines or tissue biopsy samples). In some embodiments, binding affinity is performed using surface plasmon resonance measurements.

[0329] In some such embodiments, the binding affinity (e.g., K D The dissociation constant, expressed as , is measured in one or more assays (e.g., yeast-based assays in which the target is recombinantly expressed in yeast and exposed to a miniprotein provided by the present disclosure (e.g., CDP, knottin, binder, affibody, engineered Kunitz domain, monobody, anticalin, designed ankyrin repeat domain (DARPin), avimer).

[0330] In some embodiments, synthetic and analytical techniques, including, without limitation, HPLC, LCMS, CD, quantitative thin layer chromatography, and others known to those skilled in the art, are used to efficiently synthesize and characterize miniproteins (e.g., CDPs, knottins, binders, affibodies, engineered Kunitz domains, monobodies, anticalins, designed ankyrin repeat domains (DARPins), avimers) and fully optimized clinical pharmaceutical composition candidates via solid phase peptide synthesis. Methods for screening and development of miniproteins

[0331] Directed evolution and computational folding algorithms can be combined for the de novo creation of miniproteins provided herein. In some embodiments, hundreds of miniprotein scaffolds with various secondary structure elements, orientations, and loop lengths can be matched with hotspot binding motifs on a target of interest (e.g., any one of the target proteins in Table 10). In some such embodiments, if the binding motifs of the miniprotein do not clash with the scaffold of the target, the monomers and interaction energies are optimized using Rosetta combinatorial sequence optimization.

[0332] In some embodiments, oligo pools encoding the design sequences selected via computational approaches can be synthesized, amplified, and co-transformed into yeast. In some embodiments, the resulting yeast library displaying the design sequences can be incubated with a fluorescently labeled target. In some embodiments, cells displaying designs that bind to the target can be recovered by fluorescence-activated cell sorting (FACS) and deep sequencing. In some embodiments, once a miniprotein is identified either through affinity maturation or original design, the miniprotein can be chemically synthesized or expressed in Escherichia coli, purified, and characterized in solution.

[0333] In some embodiments, libraries of stable CDP or knottin peptides can be developed to allow screening against specific selected targets, where, in addition to cysteine ​​cross-linking, a hydrophobic core is engineered into the miniprotein to allow folding, improving the number of folded structures in the library.

[0334] In some embodiments, once miniproteins are identified or engineered, they can be produced via chemical synthesis or recombinant expression. In some embodiments, miniprotein peptides can be produced by solid-phase peptide synthesis followed by in vitro folding. Standard 9-fluorenylmethyloxycarbonyl (Fmoc)-based solid-phase peptide chemistry can be used. In some such embodiments, the linear peptide can then be folded under conditions that promote oxidation of cysteine ​​side chain thiols to form disulfide bonds, followed by purification by, for example, reverse-phase high-performance liquid chromatography (RP-HPLC). In some embodiments, recombinant DNA approaches can also be used to produce the desired miniproteins.

[0335] In some embodiments, iteration between data-driven model improvement and experimental testing with miniproteins is likely to optimize the folding and binding capabilities of miniproteins to develop specific, pharmaceutically superior molecules.

[0336] In some embodiments, the mini-protein or portions thereof are engineered at the DNA level (e.g., degenerate codons can be introduced by oligonucleotide assembly using overlap extension PCR; or genetic material can be amplified using flanking primers with sufficient overlap with the yeast display vector for homologous recombination). Modifications to the miniprotein

[0337] In some embodiments, the present disclosure further provides one or more modifying components. In some embodiments, the modifying component comprises or consists of an inducible or repressible promoter operably linked to the coding sequence of a miniprotein provided herein. In some embodiments, the expression profile of the miniprotein or its underlying amino acid sequence can be altered via the promoter. In some aspects, the expression profile of the miniprotein can be temporally altered or controlled by temporally altering or controlling promoter function. In some embodiments, the promoter can be spatially and / or environmentally controlled. In some embodiments, the modifying component comprises or consists of an enhancer. In some such embodiments, an enhancer is used to alter the expression profile of the binder, but is not necessarily operably linked to the coding sequence of the binder; rather, in some embodiments, the enhancer is located upstream or downstream of the coding sequence of a binder of the present disclosure. In some embodiments, the enhancer can be temporally controlled. In some embodiments, the enhancer can be spatially and / or environmentally controlled.

[0338] In some embodiments, the expression profile of a binder of the present disclosure and / or the sequence encoding it (e.g., a nucleic acid sequence, e.g., an amino acid sequence, e.g., a gene or portion thereof, etc.) may be altered through one or more modifications. In some such embodiments, the one or more modifications comprise one or more mutations in a sequence (e.g., a nucleic acid sequence, e.g., an amino acid sequence) provided by the present disclosure. In some aspects, a sequence of the present disclosure comprises a deletion compared to a parent sequence or portion thereof. Binding assay

[0339] In some embodiments, binding assays are used to determine the binding affinity and / or association / dissociation constant or composition or one or more components thereof (e.g., of a miniprotein with or without one or more additional components provided herein). For example, in some embodiments, the equilibrium dissociation constant (Kd) is determined using fluorescent labeling and detection methods. In some embodiments, a population of cells (e.g., yeast cells) is produced that are engineered to express a library of miniproteins (e.g., CDPs, knottins, binders, affibodies, engineered Kunitz domains, monobodies, anticalins, designed ankyrin repeat domains (DARPins), avimers) (e.g., binders that bind to a target). In some embodiments, the cells express the target. Depending on whether the set of cells expresses a target or a miniprotein (e.g., CDP, knottin, binder, affibody, engineered Kunitz domain, monobody, anticalin, designed ankyrin repeat domain (DARPin), avimer), in some embodiments, the cell library is incubated with a target (e.g., any one of the target proteins in Table 10) or with a set of miniproteins (e.g., CDP, knottin, binder, affibody, engineered Kunitz domain, monobody, anticalin, designed ankyrin repeat domain (DARPin), avimer) or miniproteins (e.g., CDP, knottin, binder, affibody, engineered Kunitz domain, monobody, anticalin, designed ankyrin repeat domain (DARPin), avimer) provided herein.In some such embodiments, cells and mini-proteins (e.g., CDPs, knottins, binders, affibodies, engineered Kunitz domains, monobodies, anticalins, designed ankyrin repeat domains (DARPins), avimers) are assessed using flow cytometry and / or FACS analysis to determine binding affinity using methods known to those of skill in the art (see, e.g., "Chapter Nine - Engineering CDPs as Novel Binding Agents." Methods in Enzymology, edited by K. Dane Wittrup and Gregory L. Verdine, vol. 503, Academic Press, 2012, pp. 223-51. ScienceDirect, doi:10.1016 / B978-0-12-396962-0.00009-4.).

[0340] In some embodiments, affinity measurements for live cells can be measured using methods known to those of skill in the art, for example, DELFIA® time-resolved fluorescence (TRF) intensity technology. As known to those of skill in the art, DELFIA assays can be performed using europium-labeled ligands (e.g., miniproteins and / or conjugates capable of binding to a target, e.g., any one of the target proteins in Table 10). In some such embodiments, at equilibrium, the binding constant (K D) can be estimated after the addition of increasing amounts of a europium-labeled composition (e.g., a europium-DOTA chelated peptide) capable of binding to mammalian cells expressing any one of the target proteins in Table 10. To measure affinity using this approach, after addition to a saturating concentration, unbound fluorescent peptide can be separated from the bound reagent through a series of washing and aspiration steps. Detection of the remaining fluorescence, also considered a reflection of the bound peptide, is performed after dissociation of europium (e.g., from the DOTA-peptide) at low pH, and fluorescence can be measured on a fluorescent plate reader after addition of a signal enhancement reagent. The concentration-dependent increase in fluorescence can be plotted as a function of peptide concentration, and a model curve fit can be used to estimate the equilibrium binding constant using GraphPad Prism software.

[0341] Another approach to estimating binding affinity uses the peptide inhibition constant (K) to estimate peptide binding affinity after co-addition of a single, subsaturating amount of labeled binding agent (e.g., a europium-DOTA chelated peptide that binds to a target protein selected from Table 10) with increasing concentrations of unlabeled peptide. i ) is used. In some embodiments, it is contemplated that an unlabeled peptide competes for the same binding site on the target (e.g., any one of the target proteins in Table 10) to displace a labeled binding agent (e.g., a europium-DOTA-chelated binding peptide of any one of the target proteins in Table 10). In some such embodiments, using a fluorescence enhancement and detection step similar to a DELFIA europium dissociation measurement, the binding affinity for the unlabeled peptide is estimated according to a model curve fit of the data upon loss of fluorescent signal. affinity maturation

[0342] In some embodiments, miniproteins of the present disclosure (e.g., CDPs, knottins, binders, affibodies, engineered Kunitz domains, monobodies, anticalins, designed ankyrin repeat domains (DARPins), avimers) comprise or consist of sequences that exhibit a particular desired affinity range for a target. In some embodiments, affinity maturation is performed on sequences provided by the present disclosure, and the affinity-matured sequence exhibits the same or better selectivity and / or affinity for any one of the target proteins in Table 10 compared to the starting sequence or another sequence that has a "lower" affinity compared to the affinity-matured sequence. In some embodiments, affinity maturation is performed using an antigen of any one of the target proteins in Table 10 and a sequence that binds to the target protein of a miniprotein provided herein (e.g., CDPs, knottins, binders, affibodies, engineered Kunitz domains, monobodies, anticalins, designed ankyrin repeat domains (DARPins), avimers).

[0343] In some embodiments, affinity maturation is performed on sequences that selectively bind to any one of the target proteins in Table 10. In some embodiments, the target protein selected from Table 10 is a human isoform.

[0344] In preferred embodiments of the present disclosure, the modified polypeptide sequence of the pharmaceutical composition comprises nM or sub-nM binding affinity, binding potency, against a protein target or in a cell-based assay against a target on a cell line expressing a human isoform of any one of the target proteins in Table 10.

[0345] In certain embodiments, the modified polypeptide sequence comprises a binding affinity for a human isoform of any one of the target proteins in Table 10 of 900, 800, 700, 600, 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 nM binding affinity.

[0346] In one embodiment, the modified polypeptide sequence comprises a binding affinity for the human isoform of any one of the target proteins in Table 10 of 500 nM binding affinity.

[0347] In a more preferred embodiment, the modified polypeptide sequence comprises a picomolar binding affinity.

[0348] In other preferred embodiments, the modified polypeptide sequence exhibits selectivity for binding to only target proteins selected from Table 10. In some embodiments, affinity maturation is performed using a magnetic-based assay. In some embodiments, affinity maturation is performed using a flow cytometry / FACS-based assay according to procedures known to those of skill in the art. Methods for producing mini-proteins

[0349] In some embodiments, miniprotein synthesis involves solid-phase synthesis. In some embodiments, miniproteins are synthesized using standard solid-phase peptide synthesis as known to those skilled in the art (see, e.g., Johannes Meienhofer, Hormonal Proteins and Peptides, Volume II, 1973, Pages 45-267). In some embodiments, SPS involves synthesis using methods known to those skilled in the art, including, for example, Fmoc or Boc amino protecting groups. In some embodiments, synthesis includes protection from subsequent reaction with N-protected amino acids. In some embodiments, synthesized polypeptides are analyzed to determine sequence, structure, and related properties using HPLC / LC-MS.

[0350] In some embodiments, mini-proteins are produced using recombinant production methods known to those skilled in the art, including, for example, yeast-based approaches and chemical synthesis. Conjugation Methods

[0351] In some embodiments, the conjugates of the present disclosure comprise a linker and a chelator. In some embodiments, the chelator has an attached radionuclide. In some embodiments, the linker and chelator, or the linker, chelator, and radionuclide, are conjugated to the miniprotein. In some embodiments, the chelator and / or the radionuclide are conjugated to the miniprotein. In some embodiments, the miniprotein is conjugated to the chelator either directly or via a linker (e.g., a linker described herein). Any known conjugation chemistry can be utilized to directly conjugate the miniprotein to the chelator or to conjugate a linker to the miniprotein and chelator.

[0352] In some embodiments, the mini-protein comprises a surface-exposed functional group to enable site-specific conjugation. In some embodiments, the mini-protein comprises a surface-exposed lysine or cysteine ​​residue that can be useful for site-specific conjugation. In some embodiments, the mini-protein conjugate comprises one or more non-naturally occurring amino acids that can be useful for site-specific conjugation.

[0353] Those skilled in the art will recognize that numerous chemical conjugation strategies provide ready access to the techniques of the present invention whereby exposed amino acid residues on the protein undergo well-known reactions with reactive moieties on the chelator.

[0354] Those skilled in the art will recognize that cysteine ​​coupling reactions can be used to conjugate chelators with thiol-reactive termini to protein surfaces via exposed thiol side chains on cysteine ​​residues on the protein surface (see generally Tsuchikama & An, supra, at 36-37; see also, e.g., Pierre Adumeau et al., Thiol-Reactive Bifunctional Chelators for the Creation of Site-Selectively Modified Radioimmunoconjugates with Improved Stability, 29 Bioconjugate Chem. 1364 (2018)). In some embodiments, because cysteine ​​residues readily form disulfide linkages with neighboring cysteine ​​residues under physiological conditions rather than existing as free thiols, some cysteine ​​coupling strategies may rely on selective reduction of disulfides to generate a higher number of reactive free thiols. Cysteine ​​coupling techniques known in the art include, but are not limited to, cys alkylation reactions, cysteine ​​re-bridging reactions, and cys-aryl coupling using organometallic palladium reagents (see, e.g., C.R. Behrens et al., Antibody-Drug Conjugates (ADCs) Derived from Interchain Cysteine ​​Cross-Linking Demonstrates Improved Homogeneity and Other Pharmacological Properties Over Conventional Heterogeneous ADCs, 12 Mol. Pharm. 3986 (2015); Vinogradova et al., Organometallic Palladium Reagents for Cysteine ​​Bioconjugation, 526 Nature 687 (2015); see also Tsuchikama, supra, at 37).

[0355] Protein conjugation strategies using unnatural amino acid side chains are also well known in the art. For example, in some embodiments, "click chemistry" provides access to conjugated proteins through rapid and selective chemical transformations under a diverse range of reaction conditions. In some embodiments, click chemistry is known to produce peptide conjugates with limited by-product formation in aqueous conditions, despite the presence of unprotected functional groups. For example, in some embodiments, the click reaction in forming the conjugated peptide is copper(I)-catalyzed azide-alkyne 1,3-dipolar cycloaddition (CuAAC) (see Liyuan Liang & Didier Astruc, The Copper(I)-CatalyzedAlkyne-Azide Cycloaddition (CuAAC) "Click" Reaction and Its Applications: An Overview, 255 COORD. CHEM. Rev 2933 (2011); see also, e.g., Herman S. Gill & Jan Marik, Preparation of 18F-labeled Peptides using the Copper(I)-Catalyzed Azide-Alkyne 1,3-Dipolar Cycloaddition, 6 Nature Protocols 1718 (2011)). In some embodiments, the CuAAC click reaction can be carried out in the presence of a ligand to enhance the reaction rate. In some such embodiments, such ligands can include multidentate nitrogen donors, including, for example, amines (e.g., tris(triazolyl)methylamine) and pyridines (see Liang & Astruc, supra, at 2934 (collecting examples); P.L. Goias et al., 39 Macromolecules 6451 (2006)). In some embodiments, other widely utilized click reactions include, but are not limited to, thiol-ene, oxime, Diels-Alder, Michael addition, and pyridyl sulfide reactions.

[0356] In some embodiments, copper-free (Cu-free) click methods are also known in the art for delivery of therapeutic and / or diagnostic agents, such as radionuclides (e.g., 18F), chemotherapeutic agents, dyes, imaging agents, fluorescent labels, chemiluminescent labels, or other labels, to protein surfaces. In some embodiments, Cu-free click methods may enable stable covalent linkages between target molecules and prosthetic groups. In some embodiments, Cu-free click chemistry can involve reacting an antibody, antigen-binding fragment, or target protein-binding fragment modified with an activating moiety, e.g., a cyclooctyne (e.g., dibenzocyclooctyne (DBCO)), a non-natural amino acid side chain containing a nitrone, or azide group, with a prosthetic group displaying a corresponding or complementary reactive moiety, e.g., an azide, a nitrone, or a cyclooctyne (e.g., DBCO) (see, e.g., David. J. Donnelly et al., Synthesis and Biologic Evaluation of a Novel 18F-Labeled Adnectin as a PET Radioligand for Imaging PD-L1 Expression, 59 J. NUCL. MED. 529 (2018)). For example, in some embodiments, when the targeting molecule contains a cyclooctyne, the prosthetic group can contain an azide, a nitrone, or a similar reactive moiety. In some embodiments, when the targeting molecule contains an azide or nitrone, the prosthetic group may present a complementary cyclooctyne, alkyne, or similar reactive moiety. In some embodiments, the Cu-free click reaction may be carried out at room temperature in aqueous solution in the presence of phosphate-buffered saline (PBS). In some such embodiments, the prosthetic group may be radiolabeled (e.g., with 18F) or conjugated to any alternative diagnostic and / or therapeutic agent (e.g., a chelator) (see ibid., p. 531).

[0357] In some embodiments, conjugation chemistries, such as Huisgen cycloaddition ("click" reaction), are available for the synthesis of chelates and peptides. In some embodiments, an efficient, high-yielding, three-step synthesis of the versatile monofluoro-substituted cyclooctyne (MFCO) has been shown to facilitate various bioconjugation processes (M. Martin et al., 2013). In some embodiments, MFCO can be utilized to prepare DOTA derivatives for copper-free click chemistry addition at internal azide-modified lysine residues of CDP or knottin peptides.

[0358] In some embodiments, the mini-protein conjugates provided herein have lysines at specific positions (e.g., in cysteine ​​knots or cysteine-rich regions) that can be replaced with azide derivatives for "click" chemistry with DOTA-MFCO.

[0359] In some embodiments, DOTA-MFCO-CDP conjugates can be prepared by first coupling amine-modified DOTA to MFCO, and then conjugating DOTA-MFCO to an azide on a desired lysine of the miniprotein.

[0360] In some embodiments, the chelator and mini-protein are joined together by a cycloaddition reaction in the presence of a transition metal catalyst. In some embodiments, the metal catalyst is Cu or Rh based.

[0361] In some embodiments, using solution-phase conjugation, the chelator (DOTA) and miniprotein are conjugated with 1-ethyl-3-[3-(dimethylamino)propyl] (EDC) and N-hydroxysulfonosuccinimide (SNHS) in water (pH 5.5) at room temperature for 40 minutes using a 1:1:1 molar ratio of DOTA:EDC:SNHS. In some such embodiments, the peptide is dissolved in sodium phosphate buffer and added to the sulfosuccinimidyl ester of DOTA (DOTA-OSSu). In some such embodiments, a molar excess of DOTA-OSSu is used to drive conjugation at the N-terminus of the peptide (see, e.g., Kimura, Richard H et al. "Engineered knottin peptides: a new class of agents for imaging integrin expression in living subjects." Cancer research vol. 69,6 (2009): 2435-42. doi:10.1158 / 0008-5472.CAN-08-2495).

[0362] Since in some scenarios peptide function may be impaired due to DOTA conjugation to the N-terminus or to lysine side chains, in some embodiments a new DOTA derivative, α-amino-DOTA, is prepared with the aim of attaching DOTA to the C-terminus of the peptide.

[0363] In some embodiments, miniproteins are produced by solid-phase peptide synthesis (SPPS). The tris-tert-butyl ester bifunctional ligand of DOTA (salt-free zwitterionic form) is readily soluble in most organic solvents, and the tert-butyl ester protection is fully compatible with standard SPPS techniques. The most convenient method of conjugation involves adding DOTA to the N-terminus of a protected peptide chain as the last amino acid in an automated peptide synthesizer, followed by cleavage from the resin and removal of the acid-labile protecting group. It can also be attached to the Lys side chain. A preformed activated NHS ester of DOTA-tris(tert-butyl ester) has also been synthesized; this reagent does not require a coupling agent to couple DOTA to free amino groups. The DOTA unit is linked to the peptide via one of the acetate side arms, leaving the conjugate with four amino groups, three carboxylic acid groups, and one amide group available for metal binding (see, e.g., De Leon-Rodriguez LM, Kovacs Z. The synthesis and chelation chemistry of DOTA-peptide conjugates. Bioconjug Chem. 2008 Feb;19(2):391-402. doi: 10.1021 / bc700328s. Epub 2007 Dec 12. PMID: 18072717).

[0364] In some embodiments, a more general method involves the use of preformed DOTA-amino acid derivatives, which allow the introduction of a DOTA unit into any desired position in a peptide sequence without the need for orthogonal protection. Protected DOTA-Lys and DOTA-Phe derivatives (NR-Fmoc protection, free carboxyl for coupling, and acid-labile tert-butyl protection of the remaining acetate side arm of the DOTA unit) that are fully compatible with standard SPPS conditions have been synthesized. These DOTA-amino acids can be used in SPPS to construct peptides incorporating DOTA moieties at any desired position (see, for example, De Leon-Rodriguez LM, Kovacs Z. The synthesis and chelation chemistry of DOTA-peptide conjugates. Bioconjug Chem. 2008 Feb;19(2):391-402. doi: 10.1021 / bc700328s. Epub 2007 Dec 12. PMID: 18072717).

[0365] General methods for coupling DOTA-type macrocycles to targeting groups via linkers (e.g., by activation of one of the carboxylic acids of DOTA to form an active ester that then reacts with an amino group on the linker to form a stable amide bond) are known to those skilled in the art (see, e.g., Tweedle et al., U.S. Pat. No. 4,885,363).

[0366] Linkers can be incorporated between the chelator and the targeting vector to affect the pharmacokinetic properties of the conjugate. Carbohydrate, PEG, or polypeptide linkers can alter the overall charge and hydrophilicity of the radiopharmaceutical, thereby modifying its pharmacokinetics and biodistribution (see, e.g., De Leon-Rodriguez LM, Kovacs Z. The synthesis and chelation chemistry of DOTA-peptide conjugates. Bioconjug Chem. 2008 Feb;19(2):391-402. doi: 10.1021 / bc700328s. Epub 2007 Dec 12. PMID: 18072717). Orientation of mini-protein conjugates

[0367] In some embodiments, the conjugate has the following orientation: linker-chelator, linker-chelator-radionuclide, linker-radionuclide, chelator-radionuclide. In some such embodiments, the conjugate has the following orientation: miniprotein-linker-radionuclide, miniprotein-chelator, chelator-miniprotein, miniprotein-linker-chelator, chelator-linker-miniprotein, miniprotein-chelator-radionuclide, radionuclide-chelator-miniprotein, miniprotein-linker-chelator-radionuclide, or radionuclide-chelator-linker-miniprotein.

[0368] In some embodiments, the conjugates provided by the present disclosure comprise a miniprotein. In some such embodiments, the miniprotein functions as a targeting moiety, e.g., specifically binding to a target, e.g., a protein expressed on the surface of a target tumor cell. Thus, in some embodiments, the miniprotein in the conjugates of the present disclosure can vary depending on the target of interest. Exemplary Mini-Protein Conjugates

[0369] The following provide exemplary embodiments of the miniprotein conjugates provided herein. In some such embodiments, the miniprotein specifically binds to any one of the target proteins in Table 10 that are expressed on the surface of cancer cells (e.g., solid tumor cells). In some embodiments, the conjugate comprises a linker, a chelator, and / or a radionuclide.

[0370] In some embodiments, the conjugate comprises a miniprotein, an optional linker, a chelator, and a radionuclide. In some embodiments, the miniprotein comprises or consists of a binder, CDP, knottin, affibody, engineered Kunitz domain, monobody, anticalin, designed ankyrin repeat domain (DARPin), or avimer. In some embodiments, the chelator comprises or consists of DOTA, Crown, NOPO, or Macropa. In some embodiments, the radionuclide comprises or consists of an alpha emitter. In some embodiments, the radionuclide comprises or consists of a beta emitter. In some embodiments, the radionuclide comprises or consists of Ac-225, Ga-68, Pb-212, Lu-177, Cu-67, Cu-64, La-132, La-135, In-111, Ce-134, F-18, or At-211.

[0371] In some embodiments, the conjugate comprises or consists of a miniprotein that specifically binds to any one of the target proteins in Table 10, an optional PEG linker, a DOTA chelator, and Ac-225.

[0372] In some embodiments, the conjugate comprises or consists of a miniprotein that specifically binds to any one of the target proteins in Table 10, an optional PEG linker, a DOTA chelator, and Lu-177.

[0373] In some embodiments, the conjugate comprises or consists of a miniprotein that specifically binds to any one of the target proteins in Table 10, an optional PEG linker, a DOTA chelator, and Ga-68.

[0374] In some embodiments, the conjugate comprises or consists of a miniprotein that specifically binds to any one of the target proteins in Table 10, an optional PEG linker, a DOTA chelator, and La-132.

[0375] In some embodiments, the conjugate comprises or consists of a miniprotein that specifically binds to any one of the target proteins in Table 10, an optional PEG linker, a DOTA chelator, and La-135.

[0376] In some embodiments, the conjugate comprises or consists of a miniprotein that specifically binds to any one of the target proteins in Table 10, an optional PEG linker, a DOTA chelator, and Cu-64.

[0377] In some embodiments, the conjugate comprises or consists of a miniprotein that specifically binds to any one of the target proteins in Table 10, an optional PEG linker, a DOTA chelator, and In-111.

[0378] In some embodiments, the conjugate comprises or consists of a miniprotein that specifically binds to any one of the target proteins in Table 10, an optional PEG linker, a DOTA chelator, and Cu-67.

[0379] In some embodiments, the conjugate comprises or consists of a miniprotein that specifically binds to any one of the target proteins in Table 10, an optional PEG linker, a DOTA chelator, and Ce-134.

[0380] In some embodiments, the conjugate comprises or consists of a miniprotein that specifically binds to any one of the target proteins in Table 10, an optional PEG linker, a Crown chelator, and Ac-225.

[0381] In some embodiments, the conjugate comprises or consists of a miniprotein that specifically binds to any one of the target proteins in Table 10, an optional PEG linker, a Crown chelator, and Lu-177.

[0382] In some embodiments, the conjugate comprises or consists of a miniprotein that specifically binds to any one of the target proteins in Table 10, an optional PEG linker, a Crown chelator, and Ga-68.

[0383] In some embodiments, the conjugate comprises or consists of a miniprotein that specifically binds to any one of the target proteins in Table 10, an optional PEG linker, a Crown chelator, and La-132.

[0384] In some embodiments, the conjugate comprises or consists of a miniprotein that specifically binds to any one of the target proteins in Table 10, an optional PEG linker, a Crown chelator, and La-135.

[0385] In some embodiments, the conjugate comprises or consists of a miniprotein that specifically binds to any one of the target proteins in Table 10, an optional PEG linker, a Crown chelator, and Cu-64.

[0386] In some embodiments, the conjugate comprises or consists of a miniprotein that specifically binds to any one of the target proteins in Table 10, an optional PEG linker, a Crown chelator, and In-111.

[0387] In some embodiments, the conjugate comprises or consists of a miniprotein that specifically binds to any one of the target proteins in Table 10, an optional PEG linker, a Crown chelator, and Cu-67.

[0388] In some embodiments, the conjugate comprises or consists of a miniprotein that specifically binds to any one of the target proteins in Table 10, an optional PEG linker, a Crown chelator, and Ce-134.

[0389] In some embodiments, the conjugate comprises or consists of a miniprotein that specifically binds to any one of the target proteins in Table 10, an optional PEG linker, a NOPO chelator, and Ga-68.

[0390] In some embodiments, the conjugate comprises or consists of a miniprotein that specifically binds to any one of the target proteins in Table 10, an optional PEG linker, a NOPO chelator, and La-132.

[0391] In some embodiments, the conjugate comprises or consists of a miniprotein that specifically binds to any one of the target proteins in Table 10, an optional PEG linker, a NOPO chelator, and La-135.

[0392] In some embodiments, the conjugate comprises or consists of a miniprotein that specifically binds to any one of the target proteins in Table 10, an optional PEG linker, a NOPO chelator, and Cu-64.

[0393] In some embodiments, the conjugate comprises or consists of a miniprotein that specifically binds to any one of the target proteins in Table 10, an optional PEG linker, a NOPO chelator, and In-111.

[0394] In some embodiments, the conjugate comprises or consists of a miniprotein that specifically binds to any one of the target proteins in Table 10, an optional PEG linker, a NOPO chelator, and Cu-67.

[0395] In some embodiments, the conjugate comprises or consists of a miniprotein that specifically binds to any one of the target proteins in Table 10, an optional PEG linker, a NOPO chelator, and Ce-134.

[0396] In some embodiments, the conjugate comprises or consists of a miniprotein that specifically binds to any one of the target proteins in Table 10, an optional PEG linker, a Macropa chelator, and Ac-225.

[0397] In some embodiments, the conjugate comprises or consists of a miniprotein that specifically binds to any one of the target proteins in Table 10, an optional PEG linker, a Macropa chelator, and Lu-177.

[0398] In some embodiments, the conjugate comprises or consists of a miniprotein that specifically binds to any one of the target proteins in Table 10, an optional PEG linker, a Macropa chelator, and Ga-68.

[0399] In some embodiments, the conjugate comprises or consists of a miniprotein that specifically binds to any one of the target proteins in Table 10, an optional PEG linker, a Macropa chelator, and La-132.

[0400] In some embodiments, the conjugate comprises or consists of a miniprotein that specifically binds to any one of the target proteins in Table 10, an optional PEG linker, a Macropa chelator, and La-135.

[0401] In some embodiments, the conjugate comprises or consists of a miniprotein that specifically binds to any one of the target proteins in Table 10, an optional PEG linker, a Macropa chelator, and Cu-64.

[0402] In some embodiments, the conjugate comprises or consists of a miniprotein that specifically binds to any one of the target proteins in Table 10, an optional PEG linker, a Macropa chelator, and In-111.

[0403] In some embodiments, the conjugate comprises or consists of a miniprotein that specifically binds to any one of the target proteins in Table 10, an optional PEG linker, a Macropa chelator, and Cu-67.

[0404] In some embodiments, the conjugate comprises or consists of a miniprotein that specifically binds to any one of the target proteins in Table 10, an optional PEG linker, a Macropa chelator, and Ce-134.

[0405] In some embodiments, the exemplary imaging / diagnostic mini-protein conjugates described herein comprise a mini-protein that specifically binds to any one of the target proteins in Table 10 or a fragment or portion thereof (e.g., expressed on the surface of a solid tumor cell), a PEG linker, a DOTA chelator, and gallium-68.

[0406] In some embodiments, the exemplary imaging / diagnostic mini-protein conjugates described herein comprise a mini-protein that specifically binds to any one of the target proteins in Table 10 or a fragment or portion thereof (e.g., expressed on the surface of a solid tumor cell), a PEG linker, a DOTA chelator, and copper-64.

[0407] In some embodiments, the exemplary imaging / diagnostic mini-protein conjugates described herein comprise a mini-protein that specifically binds to any one of the target proteins in Table 10 or a fragment or portion thereof (e.g., expressed on the surface of a solid tumor cell), a PEG linker, a DOTA chelator, and indium-111.

[0408] In some embodiments, the exemplary imaging / diagnostic mini-protein conjugates described herein comprise a mini-protein that specifically binds to any one of the target proteins in Table 10 or a fragment or portion thereof (e.g., expressed on the surface of a solid tumor cell), a PEG linker, a DOTA chelator, and lutetium-177.

[0409] In some embodiments, an exemplary imaging / diagnostic CDP conjugate described herein comprises a miniprotein that specifically binds to any one of the target proteins in Table 10 or a fragment or portion thereof (e.g., expressed on the surface of a solid tumor cell), a PEG linker, a DOTA chelator, and lead-212.

[0410] In some embodiments, an exemplary imaging / diagnostic CDP conjugate described herein comprises a miniprotein that specifically binds to any one of the target proteins in Table 10 or a fragment or portion thereof (e.g., expressed on the surface of a solid tumor cell), a PEG linker, a DOTA chelator, and cerium-134.

[0411] In some embodiments, pharmaceutical compositions comprising radionuclides are used in imaging scans to detect or diagnose one or more diseases. Further embodiments include use as a companion diagnostic.

[0412] In some embodiments, one or more different linkers and different chelators for both Ga-68 (eg, NOPO) and Ac-225 (eg, Crown or DOTA) are operably linked to the same miniprotein. How to use

[0413] In some embodiments, the present disclosure provides methods of treating or preventing a disease or disorder in a subject, the method comprising administering to the subject an amount of a pharmaceutical composition effective to modulate, bind to, or inhibit a human isoform of any one of the target proteins in Table 10, thereby treating or preventing the disease or disorder in the subject. In preferred embodiments, a disease or disorder associated with any one of the target proteins in Table 10 is treated (e.g., prevented, progression slowed, symptoms relieved, tumor size reduced so as to improve overall survival, etc.).

[0414] In some embodiments, subjects, e.g., patients or patient inclusion criteria include, without limitation, candidates who are positive for any one of the target proteins in Table 10 as shown via imaging (e.g., DOTA PET / CT), candidates with progressive, advanced, or metastatic disease, candidates who are not candidates for surgery, and candidates who are resistant or relapsed.

[0415] In some embodiments, certain side effects, which may include nausea and suppressed blood counts, are managed through one or more drug therapies. In some embodiments, side effects may include nephrotoxicity and myelodysplastic syndrome, although it is contemplated that the pharmaceutical compositions are manageable and treatment is generally well tolerated. The present disclosure relates to addressing, ameliorating, or proactively avoiding such nephrotoxicity before, during, or after radionuclide therapy while maintaining and / or improving therapeutic efficacy through administration of the compositions.

[0416] In some embodiments, the present disclosure includes methods for treatment comprising administering a pharmaceutical composition provided herein in the absence of administration of a targeted conditioning or preconditioning regimen where conditioning is required prior to administration of therapies to ablate certain cells, e.g., adoptive cell therapy and gene therapy.

[0417] In some embodiments, the methods and compositions of the present disclosure include a multi-step or pre-targeting approach. For example, in some embodiments, a radionuclide can be detached from the provided composition and administered subsequently after the initial step of administering a miniprotein or antibody (e.g., a first ligand-binding moiety). In such embodiments, the first ligand-binding moiety is not conjugated to a radionuclide and has the desired affinity and specificity for tumor cells. The first ligand-binding moiety is then targeted by a second moiety carrying a radionuclide. For example, the first ligand-binding moiety can include an antibody against a target protein selected from Table 10, and the second moiety can be a pharmaceutical composition comprising a miniprotein, a linker, a chelator, and a radionuclide, and the miniprotein can exhibit the desired avidity for the first ligand-binding moiety.

[0418] In some embodiments, the present disclosure provides methods of use (e.g., treatment, manufacturing, etc.) of the miniproteins provided herein (e.g., CDPs, knottins, binders, affibodies, engineered Kunitz domains, monobodies, anticalins, designed ankyrin repeat domains (DARPins), avimers).

[0419] In some embodiments, compositions and pharmaceutical compositions directed to a target protein selected from Table 10 are produced using, for example, miniproteins provided herein (e.g., CDPs, knottins, binders, affibodies, engineered Kunitz domains, monobodies, anticalins, designed ankyrin repeat domains (DARPins), avimers).

[0420] In some embodiments, miniproteins (e.g., CDPs, knottins, binders, affibodies, engineered Kunitz domains, monobodies, anticalins, designed ankyrin repeat domains (DARPins), avimers) for any one of the target proteins in Table 10 provided by the present disclosure are formatted to generate peptides, antibodies, antibody and antibody fragments, ADCs, BiTEs, CAR-Ts, and TRuCs, ​​Fc-domain components, portions, or modifications, bispecific antibodies, and the like. In some such embodiments, such compositions and pharmaceutical compositions are used in the treatment of diseases, disorders, or conditions in which expression of any one of the target proteins in Table 10 is suspected or detected. In some such embodiments, the disease, disorder, or condition is associated with overexpression and / or aberrant expression of any one of the target proteins in Table 10. In some embodiments, the disease, disorder, and / or condition is cancer. Accordingly, the present disclosure provides various compositions and pharmaceutical compositions against a target protein selected from Table 10 for the treatment of a disease associated with any one of the target proteins in Table 10.

[0421] In particular, the present disclosure provides methods of treating a subject in need thereof by administering a composition provided herein. In some such embodiments, the composition is or comprises a miniprotein (e.g., a CDP, a knottin, a binder, an affibody, an engineered Kunitz domain, a monobody, an anticalin, a designed ankyrin repeat domain (DARPin), an avimer). In some embodiments, the composition is a miniprotein conjugate comprising a miniprotein and one or more of a chelator and a radionuclide, and optionally a linker (e.g., linking the chelator to the miniprotein).

[0422] In some embodiments, the subject treated herein is at risk of having cancer or has been diagnosed with cancer. In some embodiments, the subject is a mammal. In some embodiments, the mammal is a human. In some embodiments, the human is a fetus, infant, child, adolescent, adult, or elderly. In some embodiments, a human subject with cancer is treated by administering a miniprotein described herein (e.g., CDP, knottin, binder, affibody, engineered Kunitz domain, monobody, anticalin, designed ankyrin repeat domain (DARPin), avimer).

[0423] In some embodiments, the cancer expresses a target protein (e.g., any one of the target proteins in Table 10) that is specifically bound by a miniprotein of the present disclosure (e.g., a CDP, a knottin, a binder, an affibody, an engineered Kunitz domain, a monobody, anticalin, a designed ankyrin repeat domain (DARPin), an avimer). In some embodiments, a target protein selected from Table 10, or a portion thereof, is expressed on the surface of cancer cells of the subject. In some such embodiments, a target protein selected from Table 10 is expressed on cancer cells and has lower or undetectable expression on cells of normal tissue and / or is expressed at a significantly higher density on cancer cells relative to normal cells.

[0424] In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is metastatic. In some embodiments, the cancer is recurrent. In some embodiments, the cancer is in remission. In some embodiments, the cancer is selected from the group consisting of bladder, breast, pancreas, ovary, stomach, gastrointestinal tract, liver, lung, prostate, skin, colon, rectum, colon and rectum, and skin.

[0425] In some embodiments, the miniproteins provided herein (e.g., CDPs, knottins, binders, affibodies, engineered Kunitz domains, monobodies, anticalins, designed ankyrin repeat domains (DARPins), avimers) can be used in conjunction with one or more additional components. For example, in some embodiments, the miniproteins (e.g., CDPs, knottins, binders, affibodies, engineered Kunitz domains, monobodies, anticalins, designed ankyrin repeat domains (DARPins), avimers) can be combined with one or more other components for use in imaging, diagnosis, prognosis / monitoring, and / or treatment of a disease, disorder, or condition. In some embodiments, the disease is cancer.In some embodiments, the miniproteins of the present disclosure (e.g., CDPs, knottins, binders, affibodies, engineered Kunitz domains, monobodies, anticalins, designed ankyrin repeat domains (DARPins), avimers) are used to treat breast cancer, ovarian cancer, melanoma, pancreatic cancer, peripheral neuroma, glioblastoma, adrenocortical carcinoma, AIDS-related lymphoma, anal cancer, bladder cancer, meningioma, glioma, astrocytoma, cervical cancer, chronic myeloproliferative disorder, colon cancer, endometrial cancer, ependymoma, esophageal cancer, Ewing's sarcoma, extracranial germ cell tumor, extrahepatic bile duct cancer, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gestational trophoblastic tumor, hairy cell leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, hypopharyngeal cancer, islet cell carcinoma, Kaposi's sarcoma, laryngeal cancer, leukemia, lip cancer, oral cavity cancer, liver cancer, male breast cancer, malignant tumors, The therapeutic agent may be used in a wide variety of cancers, including, but not limited to, leiomyoma, medulloblastoma, Merkel cell carcinoma, metastatic squamous cell carcinoma of the cervix, multiple myeloma and other plasma cell neoplasms, mycosis fungoides and Sézary syndrome, myelodysplastic syndromes, nasopharyngeal cancer, neuroblastoma, non-small cell lung cancer, small cell lung cancer, head and neck cancer, skin cancer, oropharyngeal cancer, bone cancer including osteosarcoma and malignant fibrous histiocytoma of bone, sinus cancer, parathyroid cancer, penile cancer, pheochromocytoma, pituitary tumors, prostate cancer, rectal cancer, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, small intestine cancer, soft tissue sarcoma, supratentorial primitive neuroectodermal tumor, pineoblastoma, testicular cancer, thymoma, thymic carcinoma, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter, urethral cancer, uterine sarcoma, vaginal cancer, vulvar cancer, and Wilms' tumor and other childhood kidney tumors.

[0426] In some embodiments, treatment (including, for example, with a miniprotein of the present disclosure (e.g., CDP, knottin, binder, affibody, engineered Kunitz domain, monobody, anticalin, designed ankyrin repeat domain (DARPin), avimer)), diagnosis, prognosis / monitoring, or imaging is in a subject who does not exhibit signs or symptoms of the disease, disorder, and / or condition. In some embodiments, treatment is in a subject who exhibits one or more signs or symptoms of the disease, disorder, or condition, even if such signs or symptoms are not objectively observable without further testing, such as laboratory diagnostic methods. In some embodiments, the subject is susceptible to having or is at risk of developing a disease, disorder, or condition (e.g., cancer) based on one or more factors associated with an increased risk of developing the disease, disorder, or condition (e.g., the level of any one of the target proteins in Table 10). In some embodiments, the subject has been diagnosed with a disease, disorder, or condition (e.g., cancer).

[0427] In some embodiments, the present disclosure provides a method of treating or preventing a disease or disorder in a subject, the method comprising administering to the subject a pharmaceutical composition comprising a miniprotein of the present disclosure (e.g., a CDP, a knottin, a binder, an affibody, an engineered Kunitz domain, a monobody, anticalin, a designed ankyrin repeat domain (DARPin), an avimer) in an amount effective to modulate, bind to, or inhibit the human isoform of any one of the target proteins in Table 10, thereby treating or preventing the disease or disorder in the subject. In some embodiments, the disease or disorder is cancer. In some embodiments, the cancer is treated (e.g., eliminated, prevented, progression slowed, symptoms relieved, tumor size reduced to improve overall survival, etc.). Formulation and Administration

[0428] In various embodiments, formulations of the pharmaceutical compositions of the present disclosure include parenteral, e.g., subcutaneous, intravenous, intraarterial, intramuscular, intradermal, intraperitoneal, intraperitoneal, and intrathecal administration. See, e.g., Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins; 22nd ed. (2013), which is described in more detail.

[0429] In some embodiments, a pharmaceutical composition comprising a miniprotein of the present disclosure (e.g., a CDP, knottin, binder, affibody, engineered Kunitz domain, monobody, anticalin, designed ankyrin repeat domain (DARPin), avimer) is administered to a subject in need thereof. In some embodiments, the subject has or is at risk of having cancer. By way of non-limiting example, in some embodiments, the cancer is selected from the group consisting of breast cancer, ovarian cancer, melanoma, pancreatic cancer, peripheral neuroma, glioblastoma, adrenocortical carcinoma, AIDS-related lymphoma, anal cancer, bladder cancer, meningioma, glioma, astrocytoma, cervical cancer, chronic myeloproliferative disorder, colon cancer, endometrial cancer, ependymoma, esophageal cancer, Ewing's sarcoma, extracranial germ cell tumor, extrahepatic bile duct cancer, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gestational trophoblastic tumor, hairy cell leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, hypopharyngeal cancer, islet cell carcinoma, Kaposi's sarcoma, laryngeal cancer, leukemia, lip cancer, oral cavity cancer, liver cancer, male breast cancer, malignant mesothelioma, medulloblastoma, Merkel cell carcinoma, metastatic squamous cell carcinoma of the neck, multiple bone marrow tumors, and the like. Selected from myeloma and other plasma cell neoplasms, mycosis fungoides and Sézary syndrome, myelodysplastic syndromes, nasopharyngeal cancer, neuroblastoma, non-small cell lung cancer, small cell lung cancer, head and neck cancer, skin cancer, oropharyngeal cancer, bone cancer including osteosarcoma and malignant fibrous histiocytoma of bone, cancer of the paranasal sinuses, parathyroid cancer, penile cancer, pheochromocytoma, pituitary tumors, prostate cancer, rectal cancer, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, small intestine cancer, soft tissue sarcoma, supratentorial primitive neuroectodermal tumor, pineoblastoma, testicular cancer, thymoma, thymic carcinoma, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter, urethral cancer, uterine sarcoma, vaginal cancer, vulvar cancer, and Wilms' tumor and other childhood kidney tumors.

[0430] In some embodiments, determination of appropriate doses and regimens of miniproteins of the present disclosure (e.g., CDPs, knottins, binders, affibodies, engineered Kunitz domains, monobodies, anticalins, designed ankyrin repeat domains (DARPins), avimers) can be made by a clinician, using, for example, parameters or factors known or suspected in the art to affect or be predicted to affect treatment. The actual dosage level of an active ingredient (e.g., a miniprotein (e.g., CDPs, knottins, binders, affibodies, engineered Kunitz domains, monobodies, anticalins, designed ankyrin repeat domains (DARPins), avimers)) provided by a composition of the present disclosure can be varied to obtain an amount of active ingredient effective to achieve a desired therapeutic response for a particular patient, composition, and mode of administration without being toxic to the patient. In some embodiments, the selected dosage level will depend on various pharmacokinetic factors, including factors known in the medical art, such as the activity of the particular composition of the present disclosure or its ester, salt, or amide being used, the route of administration, the time of administration, the rate of excretion of the particular compound being used, the duration of treatment, other drugs, compounds, and / or materials used in combination with the particular composition being used, the age, sex, weight, condition, overall health, and prior medical history of the patient being treated, etc.

[0431] In some embodiments, administration is by one or more routes including, but not limited to, bronchial, buccal, enteral, interdermal, intra-arterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, intraspecific organs and / or tissues, mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (including by intratracheal instillation), transdermal, vaginal, and intravitreal.

[0432] In some embodiments, administration may comprise or consist of continuous dosing (eg, intravenous administration) over a period of time.

[0433] In some embodiments, administration may comprise or consist of intermittent dosing.

[0434] In some embodiments, administration may include or consist of dosing with one or more doses separated by a selected period of time based on clinical response and / or activity after one or more doses.

[0435] In some embodiments, the administration is to a subject suffering from the relevant disease, disorder, or condition. In some embodiments, the administration is to a subject who is susceptible to or at risk of developing a disease, disorder, or condition. In some such embodiments, the subject exhibits one or more symptoms or characteristics of a disease, disorder, or condition. In some embodiments, the subject does not exhibit any symptoms or characteristics of a disease, disorder, or condition. In some such embodiments, the subject has one or more features characteristic of susceptibility to a disease, disorder, or condition, or is at risk of developing a disease, disorder, or condition. In some embodiments, the subject has been diagnosed with a disease, disorder, or condition.

[0436] In some embodiments, the present disclosure provides a method for modulating in a subject the biological activity of any one of the target proteins in Table 10. In some such embodiments, the method comprises administering to the subject an amount of a pharmaceutical composition provided by the present disclosure effective to modulate the biological activity of any one of the target proteins in Table 10 in the subject.

[0437] In some embodiments, the present disclosure provides a method for treating or preventing cancer in a subject. In some embodiments, the method includes administering to a subject a pharmaceutical composition provided by the present disclosure, wherein a miniprotein (e.g., a CDP, a knottin, a binder, an affibody, an engineered Kunitz domain, a monobody, anticalin, a designed ankyrin repeat domain (DARPin), an avimer) of the pharmaceutical composition selectively binds to any one of the target proteins in Table 10 in an amount effective to treat or prevent cancer in the subject.

[0438] In some embodiments, the present disclosure provides methods and compositions capable of binding to a target (e.g., any one of the target proteins in Table 10) and activating or inhibiting an immune cell response. In some embodiments, the compositions are administered for the treatment of non-small cell lung cancer (NSCLC), cutaneous squamous cell carcinoma, pancreatic cancer, primary hepatocellular carcinoma, colorectal cancer, renal clear cell carcinoma, breast cancer, and prostate cancer (Yang, S et al., Int J of Bio Sci 2020 Mar 25 (16): 11; 1767-1773).

[0439] In some embodiments, the present disclosure provides a method for detecting the presence or extent of cancer in a subject. In some such embodiments, the method comprises measuring the level of any one of the target proteins in Table 10 in a sample comprising one or more cells from the subject; detecting the level of the target protein selected from Table 10 in the subject compared to the level of the target protein selected from Table 10 in one or more control subjects indicates the presence or extent of cancer.

[0440] In some embodiments, the compositions provided by the present disclosure are used to downregulate inhibitory immune responses in a subject. For example, in some embodiments, the miniproteins of the present disclosure (e.g., CDPs, knottins, binders, affibodies, engineered Kunitz domains, monobodies, anticalins, designed ankyrin repeat domains (DARPins), avimers) specifically bind to deliver cytotoxic payloads and can be used to promote T cell cytotoxicity against specific cells (e.g., tumors) that express any one of the target proteins in Table 10 (see Goodman A, Patel SP, Kurzrock R Nat Rev Clin Oncol. 2017 Apr; 14(4):203-220). In some such embodiments, miniproteins (e.g., CDPs, knottins, binders, affibodies, engineered Kunitz domains, monobodies, anticalins, designed ankyrin repeat domains (DARPins), avimers) modulate IFN-γ, IL-2, IL-10, and IL-13 production during T cell activation. Useful treatments, imaging and diagnostic / prognostic methods

[0441] In some embodiments, the present disclosure provides a method of treating cancer in a human subject by administering a miniprotein conjugate described herein. In some embodiments, the cancer expresses a target (e.g., any one of the target proteins in Table 10) that is specifically bound by the miniprotein of the conjugate. In some embodiments, the target protein is expressed on the surface of malignant cells, with limited expression on cells of normal tissues, and / or is expressed at a significantly higher density on malignant cells relative to normal cells.

[0442] In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is metastatic. In some embodiments, the cancer is recurrent. In some embodiments, the cancer is in remission. In some embodiments, the cancer is selected from the group consisting of bladder, breast, pancreas, ovary, stomach, gastrointestinal tract, liver, lung, prostate, skin, colon, rectum, colon and rectum, and skin.

[0443] In some embodiments, the mini-protein conjugates provided herein are useful for the treatment of breast cancer, ovarian cancer, melanoma, pancreatic cancer, peripheral neuroma, glioblastoma, adrenocortical carcinoma, AIDS-related lymphoma, anal cancer, bladder cancer, meningioma, glioma, astrocytoma, cervical cancer, chronic myeloproliferative disorders, colon cancer, endometrial cancer, ependymoma, esophageal cancer, Ewing's sarcoma, extracranial germ cell tumor, extrahepatic bile duct cancer, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gestational trophoblastic tumor, hairy cell leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, hypopharyngeal cancer, islet cell carcinoma, Kaposi's sarcoma, laryngeal cancer, leukemia, lip cancer, oral cavity cancer, liver cancer, male breast cancer, malignant mesothelioma, medulloblastoma, Merkel cell carcinoma, metastatic squamous cell carcinoma of the cervix, multiple myeloma, and other plasma cell neoplasms. The imaging and treatment methods may be used to image and treat a wide variety of cancers, including, but not limited to, mycosis fungoides and Sézary syndrome, myelodysplastic syndrome, nasopharyngeal cancer, neuroblastoma, non-small cell lung cancer, small cell lung cancer, head and neck cancer, skin cancer, oropharyngeal cancer, bone cancer including osteosarcoma and malignant fibrous histiocytoma of bone, sinus cancer, parathyroid cancer, penile cancer, pheochromocytoma, pituitary tumors, prostate cancer, rectal cancer, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, small intestine cancer, soft tissue sarcoma, supratentorial primitive neuroectodermal tumor, pineoblastoma, testicular cancer, thymoma, thymic carcinoma, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter, urethral cancer, uterine sarcoma, vaginal cancer, vulvar cancer, and Wilms' tumor and other childhood kidney tumors.

[0444] As known to those skilled in the art, the determination of the appropriate dose and regimen of the compositions provided by the present disclosure can be made by the clinician, for example, using parameters or factors known or suspected in the art to affect or be predicted to affect treatment. In some embodiments, the actual dosage level of the active ingredient in the compositions provided by the present disclosure can be varied to obtain an effective amount of the active ingredient to achieve the desired therapeutic response for a particular patient, composition, and mode of administration without being toxic to the patient. In some embodiments, the selected dosage level depends on various pharmacokinetic factors, including factors known in the medical art, such as the activity of the particular composition of the present disclosure or its ester, salt, or amide used, the route of administration, the time of administration, the rate of excretion of the particular compound used, the duration of treatment, other drugs, compounds, and / or materials used in combination with the particular composition used, the age, sex, weight, condition, overall health, and previous medical history of the patient being treated.

[0445] In some embodiments, the present disclosure provides methods of imaging, diagnosing, and / or monitoring (including determining prognosis) the presence of a target in a subject. In some embodiments, the conjugates of the present disclosure (e.g., mini-protein conjugates comprising a chelator and / or a radionuclide, etc.) are useful for PET, SPECT, or MRI imaging.

[0446] In some embodiments, the conjugates of the present disclosure (e.g., mini-protein conjugates comprising chelators and / or radionuclides) can be used in image-guided surgery. For example, in some embodiments, a target tissue suspected of containing cancerous cells or tumors can be contacted with a mini-protein (e.g., CDP, knottin, binder, affibody, engineered Kunitz domain, monobody, anticalin, designed ankyrin repeat domain (DARPin), avimer) targeted to any one of the target proteins in Table 10, such that the mini-protein (e.g., CDP, knottin, binder, affibody, engineered Kunitz domain, monobody, anticalin, designed ankyrin repeat domain (DARPin), avimer) or its component(s) (chelator, radionuclide) accumulate in metastatic cancerous cells. Imaging of tissue labeled with a mini-protein conjugate for any one of the target proteins in Table 10, where the conjugate comprises an additional detectable component (e.g., a chelator, e.g., a radionuclide, e.g., other detectable imaging moiety), can be used, for example, to detect metastatic cells, delineate tumor margins, assess the completeness of resection, and evaluate the effectiveness of treatment.

[0447] In some embodiments, the present disclosure provides a method for imaging cancer in a subject. In some embodiments, the mini-protein conjugate of the present disclosure is useful for PET, SPECT, or MRI imaging. In some embodiments, a detectably effective amount of the mini-protein conjugate is administered to a subject; that is, an amount sufficient to obtain an acceptable image using an imaging device available for clinical use. In some embodiments, the detectably effective amount of the mini-protein conjugate can be administered in more than one injection, if necessary. In some such embodiments, the detectably effective amount of the mini-protein conjugate required for an individual can vary according to factors such as the degree of uptake of the mini-protein conjugate into cancerous tissue, the age, sex, and weight of the individual, and the specific medical imaging method used. Optimizing such factors is within the skill of one of ordinary skill in the art.

[0448] In some embodiments, imaging with mini-protein conjugates can be used to evaluate the effectiveness of therapeutic drugs in treating cancer. For example, images can be acquired after treatment with anti-cancer therapy to determine whether an individual is responding to treatment. In some embodiments, in subjects with cancer, imaging with mini-protein conjugates can be used to evaluate whether tumors are shrinking or growing. Furthermore, the extent of cancerous disease (how far and where the cancer has spread) can be determined to help determine prognosis and evaluate the optimal strategy for treatment (e.g., surgery, radiation, or chemotherapy).

[0449] In some embodiments, the mini-protein conjugate can be used in image-guided surgery.Tissues of interest suspected of containing cancerous cells or tumors can be contacted with the mini-protein conjugate so that the mini-protein or its components (e.g., chelators, e.g., radionuclides) accumulate in metastatic cancerous cells.In some embodiments, imaging of tissues labeled with the mini-protein conjugate in this manner can be used, for example, to detect metastatic cells, delineate tumor margins, assess the completeness of resection, and evaluate the effectiveness of treatment. kit

[0450] In one aspect, provided herein is a kit comprising the pharmaceutical composition described herein for therapeutic use, imaging use, or diagnostic use. In some embodiments, the kit typically includes a label indicating the intended use of the contents of the kit and instructions for use. The term label includes any written or recorded material provided on or with the kit, or otherwise accompanying the kit. Thus, the present disclosure provides a kit for treating a subject suffering from cancer, comprising (a) a dosage of the pharmaceutical composition described herein, and (b) instructions for use in the method of treatment disclosed herein. In certain embodiments for treating human patients, the kit comprises the pharmaceutical composition described herein, comprising the mini-protein conjugate described herein.

[0451] In some embodiments, the kit comprises a cold mini-protein conjugate provided herein (i.e., a mini-protein conjugate that does not contain a radionuclide) and instructions for chelating the mini-protein conjugate to a radionuclide. In some embodiments, the kit comprises a hot mini-protein conjugate provided herein (i.e., a mini-protein conjugate described herein that contains a radionuclide) along with instructions for administration to a subject.

[0452] In some embodiments, the present disclosure provides kits comprising a miniprotein provided herein (e.g., CDP, knottin, binder, affibody, engineered Kunitz domain, monobody, anticalin, designed ankyrin repeat domain (DARPin), avimer). In some embodiments, the kit comprises a composition (e.g., conjugation to one or more detectable moieties) for detecting a miniprotein (e.g., CDP, knottin, binder, affibody, engineered Kunitz domain, monobody, anticalin, designed ankyrin repeat domain (DARPin), avimer) for any one of the target proteins in Table 10.

[0453] In some embodiments, the kit includes a label indicating the intended use of the contents of the kit and instructions for use, The term label includes any written or recorded material supplied on or with the kit, or which otherwise accompanies the kit.

[0454] In some embodiments, the present disclosure provides kits for treating, monitoring, or diagnosing a subject having or suspected of having cells that overexpress any one of the target proteins in Table 10, the kit comprising: (a) a unit of a pharmaceutical composition described herein; and (b) instructions for use in the methods of administration disclosed herein. In certain embodiments for treating a human patient, the kit comprises a pharmaceutical comp...

Claims

1. 1. A composition represented by a formula selected from one or more of M-L-C-R, M-L-C, M-C-R, M-L-R, M-C, M-L and M-R, wherein M comprises a miniprotein (M), L comprises a linear, branched or enzymatically cleavable linker (L), C comprises a chelator (C), and R comprises a radionuclide (R), wherein said composition is characterized as exhibiting reduced renal uptake.

2. 2. The composition of claim 1, wherein M comprises a linear polypeptide, a folded polypeptide (e.g., a covalently linked polypeptide, a non-covalently linked polypeptide, or a polypeptide containing disulfide linkages), a cysteine-rich peptide, a knottin peptide, a binder, an affibody, an engineered Kunitz domain, a monobody, an anticalin, a designed ankyrin repeat domain (DARPin), or an avimer.

3. 2. The composition of claim 1, wherein M comprises an amino acid sequence that shares at least 90% identity with any one of SEQ ID NOs: 1-68.

4. 4. The composition of claim 3, wherein M comprises an amino acid sequence that shares 100% identity with any one of SEQ ID NOs: 1-68.

5. The composition of claim 4, comprising any one of C1 to C139.

6. 5. The composition of claim 4, wherein M comprises an amino acid sequence that shares 100% identity with any one of SEQ ID NOs: 1-3, 5-15, and 47-67.

7. 7. The composition of claim 6, comprising any one of C1-C6, C8-C77, or C118.

8. 5. The composition of claim 4, wherein M comprises an amino acid sequence that shares 100% identity with SEQ ID NO:

16.

9. The composition of claim 8 comprising C78.

10. 10. The composition of any one of claims 1 to 9, wherein M comprises an amino acid sequence comprising a percentage of charged amino acids between 1-5% of the total amino acid sequence, said charged amino acids being selected from Lys, Arg, His, or a non-canonical amino acid, such as trimethyllysine.

11. 10. The composition of any one of claims 1 to 9, wherein M comprises an amino acid sequence comprising a percentage of charged amino acids of between 5-10% of the total amino acid sequence, said charged amino acids being selected from Lys, Arg, His, or a non-canonical amino acid, such as trimethyllysine.

12. 10. The composition of any one of claims 1 to 9, wherein M comprises an amino acid sequence comprising a percentage of charged amino acids of between 10-15% of the total amino acid sequence, said charged amino acids being selected from Lys, Arg, His, or a non-canonical amino acid, such as trimethyllysine.

13. 10. The composition of any one of claims 1 to 9, wherein M comprises an amino acid sequence comprising a percentage of charged amino acids between 1 and 5% of the total amino acid sequence, said charged amino acids being selected from Asp or Glu.

14. 10. The composition of any one of claims 1 to 9, wherein M comprises an amino acid sequence comprising a percentage of charged amino acids between 5-10% of the total amino acid sequence, said charged amino acids being selected from Asp or Glu.

15. 10. The composition of any one of claims 1 to 9, wherein M comprises an amino acid sequence comprising a percentage of charged amino acids of between 10-15% of the total amino acid sequence, said charged amino acids being selected from Asp or Glu.

16. 10. The composition of any one of the preceding claims, characterized as exhibiting an increased circulatory half-life.

17. 13. The composition of any one of claims 10 to 12, characterized as exhibiting an ID / g of greater than 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or 10% in tumors 24 hours after administration.

18. 13. The composition of any one of claims 10 to 12, characterized as exhibiting less than 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or less than 20% ID / g in the kidney 4 hours after administration.

19. 13. The composition of any one of claims 10 to 12, characterized as exhibiting an adhesion ratio of greater than 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1 or 10:1 in tumor and normal non-ablative tissue 24 hours after administration.

20. 14. The composition of claim 13, characterized as exhibiting an ID / g greater than 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or 10% in tumors 24 hours after administration.

21. 14. The composition of claim 13, characterized as exhibiting less than 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% ID / g in the kidney 4 hours after administration.

22. 14. The composition of claim 13, characterized as exhibiting an adhesion ratio of greater than 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1 or 10:1 in tumor and normal non-ablative tissue 24 hours after administration.

23. When the chelator (C) is present, C is i) DOTA 【Chemistry 28】 ii) Crown 【Chemistry 29】 iii) NOPO, or 【Transformation 30】 iv) Macropa 【Chemistry 31】 23. The composition of any one of claims 1 to 22, comprising or consisting of:

24. 24. The composition of any one of claims 1 to 23, wherein R, if present, comprises or consists of Ac-225, Ga-68, Pb-212, Lu-177, Cu-67, Cu-64, La-132, La-135, In-111, Ce-134, F-18, or At-211.

25. 25. The composition of any one of claims 1 to 24, wherein M comprises no more than 100 amino acids, 90 amino acids, 85 amino acids, 80 amino acids, 75 amino acids, 70 amino acids, 65 amino acids, 60 amino acids, 55 amino acids, 50 amino acids, 45 amino acids, 40 amino acids, 35 amino acids, 30 amino acids, 25 amino acids, 20 amino acids, 15 amino acids, 10 amino acids, or 5 amino acids.

26. 26. The composition of any one of claims 1 to 25, wherein the miniprotein comprises at least one disulfide bridge.

27. 27. The composition of any one of claims 1 to 26, characterized as exhibiting reduced renal uptake using a biotin-conjugated test agent-based assay.

28. said biotin-conjugated test agent-based assay comprising: providing a plurality of kidney cells; contacting said plurality of kidney cells with a composition represented by a formula selected from one or more of M-L-C-R, M-L-C, M-C-R, M-L-R, M-C, M-L and M-R, wherein M comprises a miniprotein (M), L comprises a linear, branched or enzymatically cleavable linker (L), C comprises a chelator (C), and R comprises a radionuclide (R), and said composition further comprises a biotin group conjugated to fluorescently labeled streptavidin; and measuring renal uptake of the composition by lysing the plurality of kidney cells and detecting fluorescence of the fluorescently labeled streptavidin.

28. The composition of claim 27, wherein the composition is performed by:

29. 1. A composition represented by a formula selected from one or more of M-L-C-R, M-L-C, M-C-R, M-L-R, M-C, M-L and M-R, wherein M comprises a miniprotein (M), L comprises a linear, branched or enzymatically cleavable linker (L), C comprises a chelator (C), and R comprises a radionuclide (R), wherein said composition is characterized as having a charge-reduced miniprotein or a modification of said miniprotein associated with increased renal reabsorption, and wherein said composition exhibits reduced renal uptake.

30. A composition of a miniprotein (M) and a linker (L), wherein L is linked to the C-terminus or N-terminus of M at the Nα-carboxyl of a cysteine, and L comprises an enzymatically cleavable linker of formula L: R1-X1-X2-Lys, wherein: R1 is H, PEG(4-36), 4-aminomethyl-phenylacetic acid (AmPA), aminomethylbenzoyl (AmBz) or succinic acid-PEG(4-36); The composition, wherein X1 or X2 is Met, Leu, norleu (Nle), Ile, Glu, methoxynine, Phe, Tyr, beta Ala, MWK or MVK, yGF, rGF, Gly(1-10), citrulline, or Sar.

31. A composition of a miniprotein (M) and a linker (L), wherein L is linked to the Nε- of a lysine of M via a succinic acid derivative, and L comprises an enzymatically cleavable linker of formula L: R1-X1-X2-Lys, wherein: R1 is H, PEG(4-36), 4-aminomethyl-phenylacetic acid (AmPA), aminomethylbenzoyl (AmBz) or succinic acid-PEG(4-36); The composition, wherein X1 or X2 is Met, Leu, norleu (Nle), Ile, Glu, methoxynine, Phe, Tyr, beta Ala, MWK or MVK, yGF, rGF, Gly(1-10), citrulline, or Sar.

32. 10. The composition of any one of the preceding claims, wherein L is cleaved in the kidney.

33. 10. The composition of any one of the preceding claims, wherein L is cleaved by cathepsin B in lysosomes or by neutral endopeptidases, metalloproteases or dipeptidyl peptidases in the renal brush border membrane.

34. 34. A method of treating cancer in a patient, comprising administering to the patient a composition of any one of claims 1 to 33.

35. The cancer is breast cancer, ovarian cancer, melanoma, pancreatic cancer, peripheral neuroma, glioblastoma, adrenocortical carcinoma, AIDS-related lymphoma, anal cancer, bladder cancer, meningioma, glioma, astrocytoma, cervical cancer, chronic myeloproliferative disorder, colon cancer, endometrial cancer, ependymoma, esophageal cancer, Ewing's sarcoma, extracranial germ cell tumor, extrahepatic bile duct cancer, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gestational trophoblastic tumor, hairy cell leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, hypopharyngeal cancer, islet cell carcinoma, Kaposi's sarcoma, laryngeal cancer, leukemia, lip cancer, oral cavity cancer, liver cancer, male breast cancer, malignant mesothelioma, medulloblastoma, Merkel cell carcinoma, metastatic squamous cell carcinoma of the cervix, multiple myeloma and other plasma cell neoplasms, 35. The method of claim 34, wherein the cancer is selected from mycosis fungoides and Sézary syndrome, myelodysplastic syndrome, nasopharyngeal carcinoma, neuroblastoma, non-small cell lung cancer, small cell lung cancer, head and neck cancer, skin cancer, oropharyngeal cancer, bone cancer including osteosarcoma and malignant fibrous histiocytoma of bone, sinus cancer, parathyroid cancer, penile cancer, pheochromocytoma, pituitary tumor, prostate cancer, rectal cancer, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, small intestine cancer, soft tissue sarcoma, supratentorial primitive neuroectodermal tumor, pineoblastoma, testicular cancer, thymoma, thymic carcinoma, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter, urethral cancer, uterine sarcoma, vaginal cancer, vulvar cancer, and Wilms' tumor and other childhood kidney tumors.

36. 36. A method of co-administering the composition of any one of claims 1 to 35 and a peptide comprising an amino acid sequence selected from SEQ ID NO:4, SEQ ID NO:7, SEQ ID NOs:16-52 or SEQ ID NO:

68.

37. 37. The method of claim 36, wherein the composition comprises any one of C7, C12-C35, C55, C56, C58, C59, C61-C65, or C131-133.

38. 38. The method of claim 36 or 37, which reduces kidney uptake of M, L, C and R, and combinations thereof.

39. 39. The method of claim 38, wherein co-administering the composition of any one of claims 1 to 27 with a peptide comprising an amino acid sequence selected from SEQ ID NO:4, SEQ ID NO:7, SEQ ID NOs:16-52, or SEQ ID NO:68 results in competitive inhibition of M.

40. 40. The method of claim 39, wherein the composition comprises any one of C7, C12-C35, C55, C56, C58, C59, C61-C65, or C131-133.

41. 41. The method of any one of claims 36 to 40, wherein the composition is administered intravenously or subcutaneously.

42. 42. The method of any one of claims 36 to 41, wherein the composition of any one of claims 1 to 27 and the peptide comprising an amino acid sequence selected from SEQ ID NO:4, SEQ ID NO:7, SEQ ID NOs:16-52, or SEQ ID NO:68 are co-formulated with a pharmaceutically acceptable buffer.

43. 43. The method of claim 42, wherein the composition comprises any one of C7, C12-C35, C55, C56, C58, C59, C61-C65, or C131-133.

44. 44. The method of any one of claims 36 to 43, resulting in a treatment characterized as exhibiting reduced uptake of M in the kidney and / or exhibiting an increased circulating half-life.

45. 1. A composition comprising a compound represented by a formula selected from one or more of M-L-C-R, M-L-C, M-C-R, M-L-R, M-C and M-L, and a peptide, wherein M comprises a miniprotein (M), L comprises a linear, branched or enzymatically cleavable linker (L), C comprises a chelator (C), and R comprises a radionuclide (R), wherein the peptide is characterized as competitively inhibiting a target for uptake in the kidney.

46. 46. ​​The composition of claim 45, wherein the peptide comprises an amino acid sequence selected from SEQ ID NO:4, SEQ ID NO:7, SEQ ID NOs:16-52, or SEQ ID NO:68, and wherein the peptide is present at a concentration of 10-1000x compared to the concentration of M.

47. 47. The composition of claim 46, comprising any one of C7, C12-C35, C55, C56, C58, C59, C61-C65 or C131-133.

48. 48. The composition of claim 47, wherein the peptide comprises an amino acid sequence selected from SEQ ID NO:4, SEQ ID NO:7, SEQ ID NOs:16-52, or SEQ ID NO:68, and wherein the peptide is present at a concentration of 100x or greater than 100x compared to the concentration of M.

49. 49. The composition of claim 48, comprising any one of C7, C12-C35, C55, C56, C58, C59, C61-C65 or C131-133.

50. 1. A composition comprising a compound represented by a formula selected from one or more of M-L-C, M-C, and M-L, and a peptide comprising an amino acid sequence selected from SEQ ID NO:4, SEQ ID NO:7, SEQ ID NOs:16-52, or SEQ ID NO:68, wherein M comprises a miniprotein (M) comprising an amino acid selected from SEQ ID NO:4 or SEQ ID NO:7, L comprises a linear, branched, or enzymatically cleavable linker (L), and C comprises a chelator (C), wherein the peptide is characterized as competitively inhibiting a target for uptake in the kidney.

51. 51. The composition of claim 50, comprising any one of C7, C12-C35, C55, C56, C58, C59, C61-C65 or C131-133.

52. 51. The composition of claim 50, wherein the peptide is present at a concentration of 10-1000x compared to the concentration of M.

53. 51. The peptide of claim 50, wherein the peptide is present at a concentration of 100x or greater than 100x compared to the concentration of M.

54. 54. The composition of any one of claims 45 to 53, wherein the peptide reduces M-L-C-R, M-L-C or R uptake in the kidney.

55. 55. The composition of any one of claims 45 to 54, wherein C, when present, comprises or consists of DOTA, NOPO, Crown, or Macropa.

56. 56. The composition of any one of claims 45 to 55, wherein R, if present, comprises or consists of Ac-225, Ga-68, Pb-212, Lu-177, Cu-67, Cu-64, La-132, La-135, In-111, Ce-134, F-18, or At-211.

57. A pharmaceutical composition comprising (M)-L-(C)-R and at least one drug moiety.

58. 58. The composition of any one of claims 45 to 57, which binds to a target expressed on a tumor cell with an affinity of 1 pM to 100 nM as measured by an in vitro binding assay.

59. 57. The composition of any one of claims 43 to 56, characterized by a peptide solubility of 1-100 mg / mL in the assay formulation and a peptide stability of 80-95% at 75°C for at least 1 hour.

60. 60. The composition of any one of claims 45 to 59, which is excreted by the kidney at or near the subject's glomerular filtration rate.

61. 61. The composition of any one of claims 45 to 60, which exhibits passage via the kidney, liver, bone marrow, or spleen.

62. 62. The composition of any one of claims 45 to 61, wherein expression of the target is higher in cancer cells than in non-cancer cells.

63. The target is 5T4, ADAM9, AG-7, AGS-16 / ENPP3, ALPV, ASCT2, AXL, B7H3 / CD276, B7H4, BCMA, C4.4a, CA6, CA9, CAIX, CCR2, CCR7, CD123, CD138, CD14 2, CD166, CD19, CD20, CD205, CD22, CD228, CD25, CD30, CD33, CD352, CD37, CD38, CD44v6, CD45, CD46, CD47, CD48, CD5, CD51, CD56, CD7, CD70 , CD71, CD74, CD79B, CDH6, CEACAM5, cholecystokinin 2 receptor, cKIT, CLDN18, CLDN18.2, CLDN6, CLDN6 + CLDN9, CLL-1, cMET, cMET + EGFR, Cripto, CXCR4, DLL3, DPEP3, EFNA4, EGFR, EGFR + HER3, EGFR + MUC1, EGFRvIII, EPHA2, ETBR, FAP, FAPI, FCRH5, FGFR2, FGFR3, FLT3, FRα, GCC, GD2, GD3, Globo H, GPC3, GPCR5D, gpNMB, GPR20, HER2, HER2+HER3, IGF1R, IL13Ra, IL-4R, integrin β-6, KAAG1, L1CAM, LAMP1, Lewis Y Ag, LHRH receptor, LIV1, LIV1A, LRRC15, LY6E, Ly75 / CD205, MC1R, MELTF, mesothelin, MSLN, MT1-MMP, MUC1, MUC16 / CA-125, NaPi-2b, nectin-4, neurokinin 1 receptor, NKG2D, norepinephrine transporter, NOTCH3, NTSR1, P-cadherin, PDL1, PRLR, PSMA, PTK7, RNF43, ROR1, ROR2, DLL3, SLAMF7, SLC44A4, SLITRK6, SS2R, STEAP1, TF, TIM1, TNFSF9, or Trop-2.

64. When the chelator (C) is present, C is i) DOTA 【Chemistry 32】 ii) Crown 【Transformation 33】 iii) NOPO, or 【Transformation 34】 iv) Macropa 【Chemistry 35】 64. The composition of any one of claims 45 to 63, comprising or consisting of:

65. 65. The composition of any one of claims 45 to 64, wherein R, if present, comprises or consists of Ac-225, Ga-68, Pb-212, Lu-177, Cu-67, Cu-64, La-132, La-135, In-111, Ce-134, F-18, or At-211.

66. 66. The composition of any one of claims 45 to 65, wherein M comprises no more than 100 amino acids, 90 amino acids, 85 amino acids, 80 amino acids, 75 amino acids, 70 amino acids, 65 amino acids, 60 amino acids, 55 amino acids, 50 amino acids, 45 amino acids, 40 amino acids, 35 amino acids, 30 amino acids, 25 amino acids, 20 amino acids, 15 amino acids, 10 amino acids, or 5 amino acids.

67. 67. The composition of any one of claims 45 to 66, wherein the miniprotein comprises at least one disulfide bridge.

68. 68. A method of treating cancer in a patient, comprising administering to the patient a composition of any one of claims 45 to 67.

69. The cancer is breast cancer, ovarian cancer, melanoma, pancreatic cancer, peripheral neuroma, glioblastoma, adrenocortical carcinoma, AIDS-related lymphoma, anal cancer, bladder cancer, meningioma, glioma, astrocytoma, cervical cancer, chronic myeloproliferative disorder, colon cancer, endometrial cancer, ependymoma, esophageal cancer, Ewing's sarcoma, extracranial germ cell tumor, extrahepatic bile duct cancer, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gestational trophoblastic tumor, hairy cell leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, hypopharyngeal cancer, islet cell carcinoma, Kaposi's sarcoma, laryngeal cancer, leukemia, lip cancer, oral cavity cancer, liver cancer, male breast cancer, malignant mesothelioma, medulloblastoma, Merkel cell carcinoma, metastatic squamous cell carcinoma of the cervix, multiple myeloma and other plasma cell neoplasms, 69. The method of claim 68, wherein the cancer is selected from mycosis fungoides and Sézary syndrome, myelodysplastic syndrome, nasopharyngeal carcinoma, neuroblastoma, non-small cell lung cancer, small cell lung cancer, head and neck cancer, skin cancer, oropharyngeal cancer, bone cancer including osteosarcoma and malignant fibrous histiocytoma of bone, sinus cancer, parathyroid cancer, penile cancer, pheochromocytoma, pituitary tumor, prostate cancer, rectal cancer, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, small intestine cancer, soft tissue sarcoma, supratentorial primitive neuroectodermal tumor, pineoblastoma, testicular cancer, thymoma, thymic carcinoma, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter, urethral cancer, uterine sarcoma, vaginal cancer, vulvar cancer, and Wilms' tumor and other childhood kidney tumors.

70. 10. Use of a composition according to any one of claims 1 to 33 or 45 to 47 for treating cancer in a subject.

71. An isolated polynucleotide comprising one or more nucleic acid sequences encoding a polypeptide selected from SEQ ID NOs: 1-68; or a nucleic acid sequence encoding a polypeptide comprising at least 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NOs: 1-68.

72. 72. A vector comprising the isolated polynucleotide of claim 71.

73. 73. A host cell transformed with the isolated polynucleotide of claim 71 or the vector of claim 72.

74. 1. A method for characterizing renal uptake of a composition, comprising: providing a plurality of kidney cells; contacting said plurality of kidney cells with a composition represented by a formula selected from one or more of M-L-C-R, M-L-C, M-C-R, M-L-R, M-C, M-L and M-R, wherein M comprises a miniprotein (M), L comprises a linear, branched or enzymatically cleavable linker (L), C comprises a chelator (C), and R comprises a radionuclide (R), and said composition further comprises a biotin group conjugated to fluorescently labeled streptavidin; and measuring renal uptake of the composition by lysing the plurality of kidney cells and detecting fluorescence of the fluorescently labeled streptavidin. A method comprising:

75. 75. The method of claim 74, wherein the plurality of kidney cells is provided in a well.

76. 76. The method of claim 74 or 75, wherein M comprises an amino acid sequence that shares at least 90% identity with any one of SEQ ID NOs: 1-68.

77. 77. The method of claim 76, wherein M comprises an amino acid sequence that shares 100% identity with any one of SEQ ID NOs: 1-68.

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