Connectin-4 small protein conjugates

Abstract

Provided herein are peptides and conjugates useful in compositions and methods of treating, diagnosing, monitoring, and / or imaging a disease, disorder, or condition associated with the expression of one or more targets, including connin-4, including radionuclide conjugates.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 359,146, filed on July 7, 2022, the entire contents of which are incorporated by reference. Background Art

[0002] Cancer is the leading cause of death worldwide. Classical cancer therapies (e.g., radiotherapy, chemotherapy, and surgery) can be associated with severe side effects, including those resulting from the killing of healthy, non-cancerous cells. Newer therapeutic agents enhance the targeting of cytotoxic drugs to tumor cells relative to earlier therapies, including those using conjugates of biologics. Summary of the invention

[0003] The present disclosure provides technologies, such as compositions and methods of use and manufacture thereof, to meet the needs of the field of cancer. For example, in contrast to classical cancer diagnosis or therapy, targeting molecules can be designed to increase the specificity of, for example, imaging or treatment modalities and reduce toxicity. For example, the use of polypeptides to deliver therapeutic agents such as chelators and / or radionuclides (e.g., alpha emitters) to specifically target the therapeutic agent to the tumor microenvironment provides targeted treatment of tumor cells and avoids or reduces the risk of toxicity to surrounding healthy tissues by targeting therapeutic agents such as tumors.

[0004] Compared with classical cancer therapy, radionuclide therapy is more targeted and less toxic. For example, the specific delivery of radionuclides to the tumor microenvironment allows radiation to be selectively directed to tumor tissue, effectively killing malignant cells while retaining surrounding healthy tissue. For example, radionuclides can adopt targeting molecules, which specifically bind to antigens expressed at increased levels and / or density on the surface of tumor cells relative to non-tumor cells. The combination of radionuclides and antigen-positive tumor cells will target these cells by radiation, without targeting healthy tissue. Full-length antibodies have been evaluated as targeting parts before; however, due to considerations such as their large size, full-length antibodies or even antibody fragments (for example, fragments larger than polypeptides provided herein) will have several challenges, such as poor penetration of tumor tissue, and challenges such as administration, manufacture and storage around, and other. Therefore, there is still a need for a novel method for specific targeted tumors, particularly solid tumors. This disclosure provides a technology that meets this demand and other needs. Among other things, the present disclosure provides compositions comprising polypeptides and conjugates thereof having improved tumor penetration, reduced off-target toxicity and / or accumulation, and improved affinity for Connexin-4 compared to the current prior art (e.g., antibodies, e.g., antibody-drug conjugates).

[0005] In some aspects, the disclosure provides polypeptides comprising certain amino acid sequences. In some embodiments, these polypeptides bind to connexin-4 with a certain affinity. In some embodiments, the disclosure provides polypeptides comprising an amino acid sequence, wherein the amino acid sequence comprises Formula I: CX1YDX2X3FFTALX4X5LRGX6DICX7YIX8X9X10FX11X12X13X14X15X16CIX17EILX18X19LGCX20 (SEQ ID NO: 161), wherein X20 is an optional amino acid or carboxyl terminus comprising -OH and wherein X1 is D or E; X2 is E or G; X3 is Q or E; X4 is A, K or S; X5 is A, R, Q, K, S or Cit; X6 is A, D, G or S; X7 is D, Q, E, L, S or Y; X8 is Q, L or S; X9 is A, Q, E, K; X10 is A , Q, K, S, Y, OH-norleucine or norleucine; X11 is A, N, Q or S; X12 is N, T or Y; X13 is L, Y or V; X14 is P or E; X15 is A, D, Q, G or K; X16 is D, Q, E, I or L; X17 is Q or E; X18 is D, Q or E; X19 is N or Q; and X20, when present as an amino acid, is S.

[0006] In some embodiments, X1 is E; X2 is E; X3 is E; X4 is K; X5 is R; X6 is G; X7 is Y; X8 is Q; X9 is A; X10 is S; X11 is Q; X12 is Y; X13 is L; X14 is P; X15 is G; X16 is L; X17 is E; X18 is D; X19 is N; and X20 is S.

[0007] In some embodiments, the polypeptide further comprises one or more of a linker, a chelator, and a radionuclide. In some embodiments, the linker comprises or consists of a polyethylene glycol (PEG) linker of PEG4, PEG, PEG2, PEG6, PEG8, PEG12, PEG24, an ester linker, an amide linker, a maleimide linker, a succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) linker, a propionic acid linker, a decenoic acid linker, or (Gly)n-(gGlu)n- or (PEG)n (wherein n is 1 to 10), (Gly)1-10, or any fragment or combination thereof via a covalent bond. In some embodiments, the chelator comprises or consists of DOTA, DOPA, a macrocyclic ligand (Macropa) or a crown (Crown). In some embodiments, the radionuclide is selected from Ac-225, Ga-68, In-111, Pb-212, Lu-177, Cu-67, Cu-64, La-132, La-135 or Ce-134. In some embodiments, when present, the linker is attached to the C-terminus of the polypeptide. In some embodiments, when present, the chelator is attached to the polypeptide or the linker. In some embodiments, when present, the radionuclide is attached to the chelator. In some embodiments, the polypeptide comprises one or more additional N-terminal amino acids. In some embodiments, the polypeptide comprises one or more amino acids at the N-terminus and / or C-terminus of the polypeptide. In some embodiments, the polypeptide is a monomer. In some embodiments, the polypeptide is a dimer, trimer or tetramer. In some embodiments, the polypeptide comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 78. In some embodiments, the polypeptide comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence of any one of SEQ ID NO: 1-158 or 177. In some embodiments, the polypeptide comprises or consists of a consensus sequence according to any one of Formulas IIA-IIO (SEQ ID NOs: 161-176) in Table IB, further comprising substitutions according to those shown in Table 1C.

[0008] In some embodiments, the present disclosure provides a composition comprising a small protein (M), an optional linker (L), and one or both of a chelator (C) and a radionuclide (R), which is selected from (M) x -LCR, (M) x -LC, (M) x -CR, (M) x -LR, (M) x -C, (M) x -L and (M) x-R (wherein x is 1, 2, 3 or 4) is represented by the formula of one or more. In some embodiments, M comprises or consists of a sequence selected from any one of SEQ ID NO: 78 or SEQ ID NO: 1-77, 79-158, 177, 161-176 (including amino acid substitutions as shown in Table 1C) or a functional variant or portion thereof. In some embodiments, when L is present, L comprises or consists of a polyethylene glycol (PEG) linker of PEG4, PEG, PEG2, PEG6, PEG8, PEG12, PEG24, an ester linker, an amide linker, a maleimide linker, a valine-citrulline linker, a hydrazone linker, N-succinimidyl-4-(2-pyridyldithio)butyrate (SPDB) linker, succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) linker, a vinyl sulfone-based linker, a propionic acid linker, a decenoic acid linker, or (Gly)n-(gGlu)n- or (PEG)n (wherein n is 1 to 10), (Gly)1-10, or any fragment or combination thereof via a covalent bond. In some embodiments, when C is present, C comprises or consists of: i) DOTA ii)Crown iii) NOPO, or iv) Macrocyclic ligands

[0009] Among other things, the present disclosure provides conjugates comprising polypeptides (e.g., small proteins) targeting tumor microenvironments and / or tumor cells conjugated to one or more additional components, including, for example, linkers, chelators, and / or radionuclides (e.g., alpha emitters). In some embodiments, such conjugates are used to treat cells expressing a target (e.g., connexin-4). In some such embodiments, the cell is a cancer cell.

[0010] In some embodiments, the composition comprises a linker and a chelator.

[0011] In some embodiments, the composition comprises a linker, a chelator, and a radionuclide.

[0012] In some embodiments, the composition comprises a small protein, optionally a linker, a chelator, and / or a radionuclide.

[0013] In some embodiments, the composition is selected from (M) x -LCR, (M)x -LC, (M) x -CR, (M) x -LR, (M) x -C, (M) x -L and (M) x -R, wherein M comprises a small protein (M), L comprises a linker (L), C comprises a chelator (C), R comprises a radionuclide (R), and x is 1, 2, 3 or 4, wherein M comprises a small protein that binds to connexin-4 or a functional variant or portion thereof.

[0014] In some embodiments, the polypeptide according to the present disclosure is manufactured using a solid phase peptide synthesis method. In some embodiments, the polypeptide is recombinant. In some embodiments, the polypeptide comprises or consists of a small protein. In some embodiments, the small protein comprises or consists of: a cysteine-dense polypeptide, a knotting factor, a conjugate, an affibody, an engineered Kunitz domain, a monoclonal antibody, an anticalin, a designed ankyrin repeat domain (DARPin) or an avimer. In some embodiments, the small protein comprises or consists of: about 100 or less amino acids. In some embodiments, the small protein is a cysteine-dense polypeptide. In some embodiments, the small protein comprises at least one cysteine-dense region. In some embodiments, the amino acid sequence of the small protein comprises at least two cysteines (which can form at least one disulfide bridge). In some embodiments, the small protein comprises one or more disulfide bridges. In some embodiments, the amino acid sequence of the small protein comprises at least three cysteines. In some embodiments, the small protein comprises at least two disulfide bridges or consists of at least two disulfide bridges. In some embodiments, the small protein comprises a non-disulfide amino acid sequence or consists of a non-disulfide amino acid sequence. In some embodiments, the small protein comprises three alpha helices with 44-45 amino acids. In some such embodiments, the small protein has a molar mass of about 6kDa or less. In some embodiments, the small protein is stable under high temperature and acidic or alkaline conditions. In some embodiments, the small protein comprises an engineered protein derived from lipocalin. In some embodiments, the small protein comprises an eight-stranded β-barrel. In some embodiments, the small protein exhibits (i) high structural plasticity due to sequence variation and (ii) improved conformational flexibility, thereby allowing induction to adapt to targets with different shapes. In some embodiments, the small protein comprises a class of antibody mimics, which consists of two or more peptide sequences each having 30 to 35 amino acids, which are derived from the A domains of various membrane receptors and are connected by linker peptides. In some embodiments, the small protein comprises a peptide derived from ankyrin. In some embodiments, the small protein comprises ankyrin repeats, a 33-residue motif consisting of two alpha helices and a β turn. In some embodiments, the small protein comprises a peptide derived from a 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 small protein comprises a molecule based on the 10th extracellular domain of human fibronectin III (10Fn3), which adopts a 94-residue Ig-like b sandwich fold with 2 to 3 exposed loops, but lacks a central disulfide bridge.

[0015] In some embodiments, the present disclosure provides compositions having various advantages over compositions comprising full-length proteins or non-small proteins. For example, in some embodiments, the compositions of the present disclosure include one or more improved properties selected from the following: increased protein expression, increased thermal activity, increased thermal stability, 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 chemical selectivity, improved solvent stability, increased tolerance to acidic pH, increased tolerance to proteolytic activity (e.g., reduced sensitivity to proteolysis), reduced aggregation, increased solubility, and / or reduced immunogenicity.

[0016] 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 connexin-4. In some embodiments, the cell is a tumor cell (which may or may not have the characteristics of having cancer, but is 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 taken from a sample of a subject (e.g., from a biopsy, e.g., from a tumor). In some embodiments, the cell is from a cell line.

[0017] In some embodiments, the small protein or its conjugate selectively binds to a target. In some embodiments, the target is a protein or part thereof expressed on the cell surface. 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 located in (i.e., is part of) or near a tumor. In some embodiments, the cancer cell is a circulating cell.

[0018] 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 comprises or consists of: connexin-4 or a portion thereof.

[0019] In some embodiments, small proteins or fragments thereof can be used to produce binding proteins, such as antibodies that specifically bind to at least one epitope on a target (e.g., recombinant antibodies, polyclonal, monoclonal, monospecific, multispecific (e.g., bispecific, trispecific, tetraspecific, etc.), chimeric, synthetic, natural, humanized, transplanted, full-length antibodies, antigen-binding regions of antibodies (e.g., Fab, Fab'F(ab')2, single chain (Fv, sFv, scFv, bispecific scFv), diabodies, single chains (e.g., single heavy chain, single light chain), nanobodies, VHH, etc.)). In some embodiments, the target is connexin-4. In some embodiments, connexin-4 is human connexin-4. In some such embodiments, such antibodies can be used, for example, for diagnostic applications or blocking applications, etc.

[0020] In a related aspect, the present disclosure provides pharmaceutical compositions comprising the conjugate.

[0021] In some embodiments, the conjugate comprises one or more of a small protein, a linker, a chelator, and a radionuclide.

[0022] In some embodiments, one or more components of the conjugate are provided using a vector (eg, a recombinant expression vector comprising a nucleic acid encoding a polypeptide).

[0023] In some embodiments, the present disclosure provides vectors comprising an isolated polynucleotide provided herein.

[0024] In some embodiments, the disclosure provides host cells comprising a vector encoding one or more components of the conjugates provided herein.

[0025] In some embodiments, the disclosure provides compositions capable of binding, modulating and / or inhibiting connexin-4. In some embodiments, connexin-4 is human connexin-4. In some embodiments, the compositions of the disclosure are or comprise the following: monoclonal antibody therapy, peptide therapy, peptide receptor radionuclide therapy, immunotherapy, radioimmunotherapy, gene therapy, or RNA therapy.

[0026] 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 composition is 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, treatment comprises providing a composition (e.g., by administration, e.g., by contacting a cell or cell population, such as a tumor), wherein the composition comprises a component that specifically binds to connexin-4. In some embodiments, connexin-4 is expressed on a cell or cell population. In some embodiments, a composition is provided to treat a disease, disorder, or condition.

[0027] In some embodiments, the present disclosure provides methods for regulating connexin-4. That is, in some embodiments, the method for regulating the biological activity of connexin-4 includes providing (e.g., by administering, e.g., by contacting a cell or cell population) a composition comprising one or more components of an amount that effectively regulates connexin-4 and its activity. For example, in some embodiments, the composition comprises a small protein that specifically binds to connexin-4 and one or more of a linker, a chelator, and / or a radionuclide. In some such embodiments, the composition binds to cells expressing connexin-4 and targets one or more therapies (e.g., a chelator, e.g., a radionuclide) to cells expressing connexin-4, e.g., by internalizing the composition or its components into the cell.

[0028] In some embodiments, the present disclosure provides methods capable of activating or inhibiting immune cell responses and compositions used therein. In some embodiments, the composition provided is administered for the treatment of cancer. In some embodiments, cancer is associated with the expression or overexpression of connexin-4. In some embodiments, cancer is selected from non-small cell lung cancer (NSCLC), squamous cell carcinoma of the skin, pancreatic cancer, primary hepatocellular carcinoma, colorectal cancer, clear cell renal carcinoma, breast cancer and prostate cancer. See, for example, Yang, Shuo et al. "Connexin-4, a checkpoint molecule, as a target for cancer immunotherapy. [Connexin-4, a checkpoint molecule, as a target for cancer immunotherapy]" International journal of biological sciences [International Journal of Biological Sciences] Volume 16, 11 1767-1773. March 25, 2020, doi: 10.7150 / ijbs.41105.

[0029] In some embodiments, the 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 cancer or is at risk of having cancer. In some embodiments, the subject diagnosed with cancer has been treated with one or more treatments such as compositions provided herein. In some embodiments, the method of detecting (e.g., monitoring / determining prognosis, diagnosis, etc.) includes measuring a target or a small protein specifically binding to a target in a sample containing one or more cells from a subject (e.g., using a cell-based assay) or in a subject (e.g., using in vivo scanning or measurement). In some such embodiments, by comparing the level with a control level or with the level at different time points from the same patient, the level of the target or small protein is used to determine the presence and / or extent of cancer in the subject.

[0030] In some embodiments, the disclosure provides kits. In some such embodiments, the kits comprise one or more components, such as small proteins, linkers, chelators, and / or radionuclides, which can be combined in one or more methods for use in binding to a target (e.g., connexin-4).

[0031] In some embodiments, the present disclosure provides a composition comprising a small protein (M), an optional linker (L), and one or both of a chelator (C) and a radionuclide (R), which is selected from (M) x -LCR, (M) x -LC, (M) x -CR, (M) x -LR, (M) x -C, (M) x -L and (M) x-R (wherein x is 1, 2, 3 or 4) is represented by the formula of one or more. In some embodiments, M comprises or consists of: a cysteine ​​dense peptide, a knottin peptide, a conjugate, an affibody, an engineered Kunitz domain, a monoclonal antibody, an anticalin, a designed ankyrin repeat domain (DARPin) or an avimer. In some embodiments, the conjugate comprises or consists of: a linear polypeptide and / or a non-disulfide sequence. In some embodiments, M is characterized in that it comprises (i) no more than 1,000 amino acids, 900 amino acids, 800 amino acids, 700 amino acids, 600 amino acids, 500 amino acids, 400 amino acids, 300 amino acids, 200 amino acids or 100 amino acids. In some preferred embodiments, M is characterized in that it comprises (i) no more than 100 amino acids and / or 12 kDa; (ii) at least one secondary structure element; (iii) an isolated hydrophobic core; and / or exhibits cooperative folding. In some embodiments, M comprises no more than 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, 10 amino acids, or 5 amino acids.

[0032] In some embodiments, the small protein comprises at least one disulfide bridge. In some embodiments, the small protein comprises zero, one, two or more disulfide bonds. In some embodiments, the small protein comprises at least one disulfide bridge. In some embodiments, the amino acid sequence of the small protein comprises at least three cysteine ​​(C) amino acids. In some embodiments, the small protein comprises at least two disulfide bridges.

[0033] In some embodiments, the disclosure provides a composition as represented by LC, wherein L comprises or consists of a linker, C comprises or consists of a chelator, and wherein the linker is designed to be conjugated to a polypeptide.

[0034] In some embodiments, the present disclosure provides a composition as shown in LCR, wherein L comprises or consists of a linker, C comprises or consists of a chelator, and R comprises or consists of a radionuclide, and wherein the composition is capable of being conjugated to a small protein. In some embodiments, L, when present, comprises or consists of a polyethylene glycol (PEG) linker of PEG4, PEG, PEG2, PEG6, PEG8, PEG12, PEG24, an ester linker, an amide linker, a maleimide linker, a succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) linker, a propionic acid linker, a decenoic acid linker, or (Gly)n-(γGlu)n- or (PEG)n (wherein n is 1 to 10), (Gly) 1-10 , or any fragment or combination thereof via a covalent bond. In some embodiments, C, when present, comprises or consists of: i) DOTA ii)Crown iii) NOPO, or iv) Macrocyclic ligands

[0035] In some embodiments, C, when present, comprises or consists of: a derivative of tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), crown, NOPO, or a macrocyclic ligand. In some embodiments, when L is present and C is absent, L is covalently attached to M. In some embodiments, R, when present, comprises or consists of: Ac-225, In-111, Ga-68, Pb-212, Lu-177, Cu-67, Cu-64, La-132, La-135, or Ce-134. 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, a small protein bound to a target modulates a biological function. In some embodiments, when M is present, M selectively binds to connexin-4 or a portion thereof.

[0036] In some embodiments, the disclosure provides compositions comprising small proteins that bind to Connexin-4 with an affinity about as shown in Table 1C.

[0037] In some embodiments, the compositions provided herein comprise a small protein (M) comprising an amino acid sequence selected from any one of SEQ ID NOs: 1-158 or 177. In some embodiments, the compositions comprise a small protein (M) comprising an amino acid sequence selected from any one of SEQ ID NOs: 161-176. In some embodiments, M comprises an amino acid substitution at any one of SEQ ID NOs: 161-176 as shown in Table 1C.

[0038] In some embodiments, the present disclosure provides compositions comprising an isolated compound or a pharmaceutically acceptable salt thereof, comprising an optional linker (L), and one or more of a miniprotein (M), a chelator (C), or a radionuclide (R). In some embodiments, the miniprotein comprises an amino acid sequence selected from any one of SEQ ID NOs: 1-158 or 177 or an amino acid sequence according to any one of SEQ ID NOs: 161-176 and / or Table 1C.

[0039] In some embodiments, the present disclosure provides a pharmaceutical composition comprising a small protein (M), wherein the small protein selectively binds to a target.

[0040] In some embodiments, the present disclosure provides pharmaceutical compositions comprising one or more miniproteins (M) x, wherein the miniproteins selectively bind to connexin-4, and x is 1, 2, 3, or 4.

[0041] In some embodiments, the composition exhibits adhesion in a tumor. In some embodiments, the composition exhibits adhesion through the kidney, liver, bone marrow, or spleen.

[0042] In some embodiments, the present disclosure provides a pharmaceutical composition comprising a small protein (M) x, an optional linker (L), and one or both of a chelator (C) and a radionuclide (R), wherein x is 1, 2, 3, or 4. In some embodiments, when C is present, C is covalently attached to M. In some embodiments, the chelation efficiency is >90%. In some embodiments, the pharmaceutical composition further comprises a radionuclide R. In some embodiments, R is an alpha emitter when present. In some embodiments, R is Ac-225, In-111, Ga-68, Pb-212, Lu-177, Cu-67, Cu-64, La-132, La-135, or Ce-134 when present. In some embodiments, M specifically binds to a target. In some embodiments, the target is connexin-4. In some embodiments, connexin-4 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 small protein comprises or consists of: a cysteine ​​dense peptide, a knotting peptide, a conjugate, an affibody, an engineered Kunitz domain, a monoclonal antibody, an anticalin, a designed ankyrin repeat domain (DARPin) or an avimer. In some embodiments, the small protein comprises one or more disulfide bonds. In some embodiments, the small protein is characterized in that it has nM or sub-nM binding affinity to the target in vivo or in a cell-based assay. In some embodiments, the small protein has a binding affinity of 1 pM to 100 nM for the connexin-4 on a cell line expressing human connexin-4. In some embodiments, the binding affinity of the small protein is about the binding affinity disclosed for the compounds in Table 2. In some embodiments, the miniprotein has an amino acid sequence of no more than 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, 10 amino acids, or 5 amino acids.

[0043] In some embodiments, administration of the pharmaceutical composition to a subject in need thereof does not induce an immune response or the immune response elicited therein is tolerable to the subject. In some embodiments, the composition comprises high tumor tissue penetration. In some embodiments, the composition exhibits adhesion in a tumor. In some embodiments, the composition is not taken up and / or retained by the kidney or liver. In some embodiments, the composition is internalized in a cell expressing human connexin-4. In some embodiments, the small protein comprises an amino acid sequence selected from any one of SEQ ID NOs: 1-158 or 177. In some embodiments, the small protein comprises an amino acid sequence according to any one of SEQ ID NOs: 161-176. In some embodiments, the amino acid sequence of the small protein further comprises one or more substitutions shown in Table 1C.

[0044] In certain embodiments, compositions include small protein-drug conjugates, which include small proteins and at least one drug portion. In certain embodiments, pharmaceutical compositions include small protein-drug conjugates, which include small proteins and at least one drug portion. In certain embodiments, drug portions include but are not limited to V-ATPase inhibitors, pro-apoptotic agents, Bcl2 inhibitors, MCL1 inhibitors, HSP90 inhibitors, IAP inhibitors, mTor inhibitors, microtubule stabilizers, microtubule destabilizers, auristatins, dolastatins, maytansinoids (maytansinoid), MetAP (methionine aminopeptidase), protein CRM1 nuclear export inhibitors, DPPIV inhibitors, proteasome inhibitors, inhibitors of phosphoryl transfer reactions in mitochondria, 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, topoisomerase inhibitors, immunotoxins and / or DHFR inhibitors or their functional derivatives, variants or portions.

[0045] In other embodiments, drug moieties include, but are not limited to, alkylating agents such as thioTEPA and cyclophosphamide (CYTOXAN TM); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa and uredopa; ethyleneimines and methylamelamines including hexamethylmelamine, trothamide, triethylenephosphoramide, triethylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); camptothecins (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its adolesine derivative); ozelesin, carzelesin and bizelesin synthetic analogs) and their derivatives; cryptophycines (specifically cryptophycin 1 and cryptophycin 8); dolastatins, auristatins (including the analogs monomethyl-auristatin E and monomethyl-auristatin F (see, e.g., U.S. Published Application No. 2005-0238649, published on October 27, 2005, incorporated herein in its entirety); duocarmycins (including the analogs monomethyl-auristatin E and monomethyl-auristatin F (see, e.g., U.S. Published Application No. 2005-0238649, published on October 27, 2005, incorporated herein in its entirety); ) (including synthetic analogs, KW-2189 and CBI-TMI); eleutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards, such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, nitrogen mustard, mechlorethamine hydrochloride, oxide hydrochloride), melphalan, novembichin, phenesterine, prednimustine; trofosfamide, uramustine; nitrosureas, such as carmustine, chlorozotocin, fotemustine, cyclohexyl lomustine, nimustine, ranimustine;Antibiotics, such as enediyne antibiotics (e.g., calicheamicins, particularly calicheamicin gamma 1I and calicheamicin phill (see, e.g., Agnew, Chem. Intl. Ed. Engl. [Applied Chemistry English International Edition], 33: 183-186; dynemicins, including dynemicin A; bisphosphonates, such as clodronate; esperamicin; and neocarzinostatin chromophores (see, e.g., Agnew, Chem. Intl. Ed. Engl. [Applied Chemistry English International Edition], 33: 183-186); ... chromophore) and related chromophores (enediyne antibiotic chromophores), aclacinomysin, actinomycin, authramycin, azaserine, bleomycin, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, adriamycin; TM) (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin (e.g., mitomycin C), mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zidovudine, antimetabolites, such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs, such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs, such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs, such as ancitabine, azacitidine, 6-azauridine, carmofur; armofur), cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens, such as calusterone, drostanolone propionate, cyclothiodine, melastane, testolactone; antiadrenal agents, such as aminoglutethimide, mitotane, trilostane; folic acid supplements, such as folinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil luracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; democolcine; diaziquone; elfornithine; elliptinium acetate; epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine;Maytansine alkaloids, such as maytansine and ansamitocins; mitoxantrone, mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; razoxane; rhizoxin; siroxan; spirogermanium; tenuazonic acid; triiminoquinone; -trichlorotriethylamine; trichothecenes (particularly T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitabronitol; dibromodulanol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxanes, for example, paclitaxel (TAXOL TM , Bristol-Myers Squibb Oncology, Princeton, NJ) and docetaxel ( Rhone-Poulenc Rorer, Antony, France); Chlorambucil; Gemcitabine TM ); 6-thioguanine; mercaptopurine; methotrexate; platinum analogs, such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine (Navelbine TM ); novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine; and any pharmaceutically acceptable salts, acids or derivatives thereof.

[0046] In some embodiments, the drug moiety is or comprises an auristatin. In some such embodiments, an auristatin includes analogs, such as monomethyl-auristatin E and monomethyl-auristatin F (see, e.g., U.S. Published Application No. 2005-0238649, published on October 27, 2005, incorporated herein in its entirety.

[0047] In some embodiments, the drug moiety includes, but is not limited to, anti-hormonal agents used to modulate or inhibit the effects of hormones on tumors, such as antiestrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including Nolvadex TM ), raloxifene, droloxifene, 4-hydroxytamoxifen, troloxifene, ketoxifen, LY117018, onapristone, and toremifene (Fareston TM ); aromatase inhibitors that inhibit aromatase (which regulates estrogen production in the adrenal glands), such as 4(5)-imidazole, aminoglutamine, megestrol acetate (Megace TM ), exemestane, formestane, fadroxil, vorazole (Rivisor TM ), letrozole (Femara TM ) and Arimidex TM ); and antiandrogens such as flutamide, nilutamide, bicalutamide, leuprolide and goserelin; and any pharmaceutically acceptable salts, acids or derivatives thereof.

[0048] In some embodiments, the drug moiety is or comprises a topoisomerase inhibitor, including but not limited to irinotecan, topotecan, camptothecin, etoposide, etoposide phosphate, doxorubicin, epirubicin, indolocarbazole and derivatives, phenanthridine and derivatives, indenoisoquinoline and derivatives, amsacrine, teniposide, daunomycin, mitoxantrone, idarubicin, indotecan, innotecan, lamellae D, SN-38, exotecan, lortotecan, diflutecan, gimatecan, lortotecan, belotecan, edotecarin, rosetacin, cositocan, etirinotecan pegol, HOCPT, silatecan, elmotecan, topoisomerase inhibitor RFS2000; or a functional derivative or portion thereof.

[0049] In some embodiments, the drug moiety is or comprises an immunotoxin or a functional derivative, variant or portion thereof. In some embodiments, the immunotoxin is or comprises Erb38, scFv(FRP5)-ETA, SSIP(SSI(dsFv)-PE38, LMB-I, LMB-7, LMB-9, SGN-10, OvB3-PE, TP40, denileukin diftitox, moxetumomab pasudotox, LMB-2, UCHTI, D2219ARL, SSIP, MOC31PE, oportuzumab monatox, HuMI95-polygonatum toxin, RG7787, VB8-845 hSGZ, D2c7-(scdsfv)-PE38KDEI or a functional derivative, variant or portion thereof.

[0050] 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 a further embodiment, 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 in that it exhibits improved resistance to peptidases, proteases, or heat.

[0051] In some embodiments, a method of producing a composition represented by one or more of the formula selected from (M)xLCR, (M)xLC, (M)xCR, (M)xLR, (M)xC, (M)xL, and (M)xR, wherein M comprises a small protein (M), L comprises a linker (L), C comprises a chelator (C), R comprises a radionuclide (R), and x is 1, 2, 3, or 4, the method comprising synthesizing the small protein (M) and / or the linker (L), and optionally reacting the chelator (C) and / or the radionuclide (R), and conjugating the small protein (M) conjugating one or more of the small proteins (M) to a linker (L), conjugating one or more of the small proteins (M) to a chelator (C), conjugating one or more of the small proteins (M) to a radionuclide (R), conjugating one or more of the small proteins (M) to a linker (L) to a chelator (C), conjugating one or more of the small proteins (M) to a linker (L) to a chelator (C), conjugating one or more of the small proteins (M) to a linker (L) to a chelator (C) and a radionuclide (R), conjugating one or more of the small proteins (M) to a chelator (C) to a radionuclide (R), or conjugating one or more of the small proteins (M) to a linker (L) to a radionuclide (R).

[0052] In some embodiments, the method involves reacting the chelator (C) and the radionuclide (R) at a temperature between about 10°C and 65°C during incubation. In some embodiments, the reaction of the chelator (C) and the radionuclide (R) is carried out within an incubation period of about 5 minutes to about 30 minutes. In further embodiments, the reaction of the chelator (C) and the radionuclide (R) is carried out at a pH in the range of about 5.0 to 7.4. In some embodiments, the reaction of the chelator (C) and the radionuclide (R) is carried out in an aqueous solution that is substantially free of alcohol.

[0053] In some embodiments, a method of delivering a radionuclide to a selected location in a patient involves administering a composition represented by one or more of the formula selected from (M)xLCR, (M)xLC, (M)xCR, (M)xLR, (M)xC, (M)xL, and (M)xR, wherein M comprises a small protein (M), L comprises a linker (L), C comprises a chelator (C), R comprises a radionuclide (R), and x is 1, 2, 3, or 4. In some embodiments, the radionuclide is selected from Ac-225, In-111, Ga-68, Pb-212, Lu-177, Cu-67, Cu-64, La-132, La-135, or Ce-134. In some embodiments, the method further comprises performing an imaging procedure to evaluate the localization of the radionuclide in vivo, wherein the imaging procedure optionally comprises positron emission tomography (PET) imaging or single photon emission computed tomography (SPECT) imaging. In further embodiments, the imaging procedure allows selection of a patient. In certain embodiments, the imaging procedure allows monitoring of a patient. In some embodiments, imaging procedures allow for determination of appropriate dosages for treating patients in need of a pharmaceutical composition comprising one or more small proteins.

[0054] In some embodiments, the present disclosure provides a composition comprising one or both of a miniprotein (M), an optional linker (L), and / or a chelator (C) and a radionuclide (R), which is represented by a formula selected from one or more of (M)xLCR, (M)xLC, (M)xCR, (M)xLR, (M)xC, (M)xL and (M)xR (wherein x is 1, 2, 3 or 4). In some embodiments, M comprises or consists of a sequence selected from any one of the following: SEQ ID NO: 1-158 or 177 or a functional variant or portion thereof or SEQ ID NO: 161-176 and / or according to Table 1B and / or 1C or a functional variant or portion thereof. In some embodiments, L, when present, comprises or consists of: a polyethylene glycol (PEG) linker of PEG4, PEG, PEG2, PEG6, PEG8, PEG12, PEG24, an ester linker, an amide linker, a maleimide linker, a valine-citrulline linker, a hydrazone linker, N-succinimidyl-4-(2-pyridyldithio)butyrate (SPDB) linker, succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) linker, a vinyl sulfone-based linker, a propionic acid linker, a decenoic acid linker, or (Gly)n-(γGlu)n- or (PEG)n (wherein n is 1 to 10), (Gly)1-10, or any fragment or combination thereof via a covalent bond. In some embodiments, C, when present, comprises or consists of: i) DOTA ii)Crown iii) NOPO, or iv) Macrocyclic ligands

[0055] In some embodiments, R, when present, comprises or consists of Ac-225, In-111, Ga-68, Pb-212, Lu-177, Cu-67, Cu-64, La-132, La-135, or Ce-134.

[0056] In some embodiments, the disclosure provides methods of treating a subject in need thereof, the methods comprising administering a composition comprising a miniprotein (M), an optional linker (L), and one or both of a chelator (C) and a radionuclide (R).

[0057] 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 propionic acid linker, a decenoic acid linker, or any fragment or combination thereof. In some embodiments, C comprises or consists of: i) DOTA ii)Crown iii) NOPO, or iv) Macrocyclic ligands

[0058] In some embodiments, R comprises or consists of: Ac-225, In-111, Ga-68, Pb-212, Lu-177, Cu-67, Cu-64, La-132, La-135, or Ce-134. In some embodiments, M comprises or consists of: a cysteine ​​dense polypeptide, a knottin, a binder, an affibody, an engineered Kunitz domain, a monoclonal antibody, an anticalin, a designed ankyrin repeat domain (DARPin), or an avimer. In some embodiments, M is characterized in that it comprises (i) no more than 1,000 amino acids, 900 amino acids, 800 amino acids, 700 amino acids, 600 amino acids, 500 amino acids, 400 amino acids, 300 amino acids, 200 amino acids, or 100 amino acids. In some preferred embodiments, M is characterized in that it comprises (i) no more than 100 amino acids and / or 12 kDa; (ii) at least two secondary structural elements; (iii) an isolated hydrophobic core; and / or exhibits cooperative folding. In some embodiments, the composition comprises at least one additional plus component. In some embodiments, the composition can penetrate tumor tissue. In some embodiments, the small protein comprises no more than 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, 10 amino acids or 5 amino acids. In some embodiments, the small protein comprises at least one disulfide bridge. In some embodiments, the small protein specifically binds to the target. In some embodiments, the composition exhibits mm or nM binding affinity to the target in an in vitro assay.

[0059] In some embodiments, the composition binds to the target with an affinity of 100 pM to 100 nM as measured by an in vitro binding assay. In some embodiments, the composition binds to Connexin-4 with an affinity about as shown for any of the compounds in Table 1C.

[0060] In some embodiments, the composition is characterized in that it has high tissue penetration properties relative to a composition comprising a full-sized protein that binds the same target. In some embodiments, the small protein that binds to the target modulates a biological function.

[0061] In some embodiments, administration of the composition to a subject in need thereof does not elicit an immune response or wherein the elicited immune response is tolerable to the subject. In some embodiments, the tolerable immune response comprises a systemic immune response or a local immune response.

[0062] In some embodiments, the subject is diagnosed with cancer. In some embodiments, cancer cells from the subject express connexin-4 or a portion thereof. In some embodiments, expression of the target in cancer cells is higher than expression in non-cancerous cells.

[0063] In some embodiments, the cancer is selected from 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 lymphoma, non-Hodgkin 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 neck cancer, multiple myeloma and other plasma cell neoplasms, mycosis fungoides and Sezary 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.

[0064] In some embodiments, the composition is administered intravenously or subcutaneously. In some embodiments, cancer is treated following administration of the composition.

[0065] In some embodiments, the disclosure provides a method for targeting a cancer cell population expressing connexin-4, the method comprising: (i) determining the expression level of a target in a cancer cell population; (ii) administering to a subject in need thereof a composition comprising a polypeptide provided herein, a composition as provided herein, or a pharmaceutical composition as provided herein, wherein the composition comprises at least one component that specifically binds to connexin-4, wherein the composition targets cells expressing connexin-4 and is internalized into cells expressing connexin-4; (iii) wherein the patient is treated after administration compared to before administration; and the treatment does not damage cells that do not express connexin-4. In some embodiments, the polypeptide comprises or consists of a small protein (M), the small protein comprising an amino acid sequence selected from any one of SEQ ID NOs: 1-158 or 177 or 161-176 and / or Table 1C.

[0066] In some embodiments, the present disclosure provides a small protein conjugate comprising: (i) a small protein (M) that specifically binds to connexin-4; (ii) a chelator (C) conjugated to (M) via an optional linker (L), wherein (C) comprises DOTA and (L) when present comprises PEG; and (iii) a radionuclide (R) chelated to (C), wherein (R) is actinium-225.

[0067] In some embodiments, the present disclosure provides a small protein conjugate comprising: (i) a small protein (M) that specifically binds to connexin-4; (ii) a chelator (C) conjugated to (M) via an optional linker (L), wherein (C) comprises DOTA and (L) when present comprises PEG; and (iii) a radionuclide (R) chelated to (C), wherein (R) is indium-111.

[0068] In some embodiments, the present disclosure provides a small protein conjugate comprising: (i) a small protein (M) that specifically binds to connexin-4; (ii) a chelator (C) conjugated to (M) via an optional linker (L), wherein (C) comprises DOTA and (L) when present comprises PEG; and (iii) a radionuclide (R) chelated to (C), wherein (R) is gallium-68.

[0069] In some embodiments, the present disclosure provides a small protein conjugate comprising: (i) a small protein (M) that specifically binds to connexin-4; (ii) a chelator (C) conjugated to (M) via an optional linker (L), wherein (C) comprises DOTA and (L) when present comprises PEG; and (iii) a radionuclide (R) chelated to (C), wherein (R) is copper-64.

[0070] In some embodiments, a small protein conjugate as provided herein comprises a small protein (M) comprising an amino acid sequence selected from any one of SEQ ID NOs: 1-158 or 177 or 161-176 and / or Table 1C.

[0071] 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 comprises or consists of: connexin-4 or a portion thereof. In some embodiments, the target comprises an amino acid sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO: 159 or SEQ ID NO: 160.

[0072] In some embodiments, the miniprotein specifically binds to a target. In some embodiments, the miniprotein binds to a protein comprising or consisting of: an amino acid sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence as shown in any one of SEQ ID NOs: 1-158 or 177 or 161-176 and / or according to Tables 1B and / or 1C, or any functional variant or portion thereof.

[0073] In some embodiments, the miniprotein comprises an amino acid sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in any one of SEQ ID NOs: 1-158 or 177 or 161-176 and / or according to Tables 1B and / or 1C, or any functional variant or portion thereof.

[0074] In some embodiments, the target is connexin-4. In some embodiments, the small protein selectively binds to connexin-4 or a portion thereof. In some embodiments, the small protein comprises an amino acid sequence selected from any one of SEQ ID NO: 1-158 or 177 or 161-176 and / or according to Table 1B and / or 1C, or any functional variant or portion thereof. In some embodiments, at least 2 amino acids in the amino acid sequence bind to a position on the target. In some embodiments, the small protein exhibits μm or nM binding affinity to the target in an in vitro assay. In some embodiments, the small protein has an amino acid sequence that is at least 98% identical to an amino acid sequence shown in any one of SEQ ID NO: 1-1-158 or 177 or 161-176 and / or according to Table 1B and / or 1C, or any functional variant or portion thereof. In some embodiments, the small protein has an amino acid sequence that is at least 99% identical to an amino acid sequence shown in any one of SEQ ID NO: 1-1-158 or 177 or 161-176 and / or according to Table 1B and / or 1C, or any functional variant or portion thereof. In some embodiments, the composition is composed of any one of SEQ ID NO: 1-158 or 177 or 161-176 and / or according to Table 1B and / or 1C or any functional variant or portion thereof. In some embodiments, the small protein binds to the target with an affinity of about 1 pM to about 500 nM. In some embodiments, the small protein binds to the target with an affinity of about 1 pM to about 500 nM, for example, as measured by an in vitro binding assay. In some embodiments, the affinity is about 100 pM to about 10 nM. In some embodiments, the small protein is characterized in that it has higher tissue penetration properties relative to the full-size protein corresponding to the same target. In some embodiments, the small protein is stable under ambient conditions. In some embodiments, the small protein is stable at 80 degrees Celsius.

[0075] In some embodiments, the small protein selectively binds to connexin-4 or a portion thereof. In some embodiments, the small protein comprises an amino acid sequence selected from any one of SEQ ID NO: 1-158 or 177 or 161-176 and / or according to Table 1B and / or 1C or any functional variant or portion thereof. In some embodiments, at least 2 amino acids in the amino acid sequence bind to a position on the target. In some embodiments, the small protein binds to the target with an affinity of about 1 pM to about 500 nM, for example, as measured by an in vitro binding assay. In some embodiments, the small protein is characterized in that it has a higher tissue penetration property relative to the full-size protein corresponding to the same target.

[0076] In some embodiments, the disclosure provides a polynucleotide encoding a polypeptide comprising or consisting of an amino acid sequence of any one of SEQ ID NOs: 1-158 or 177 or 161-176 or a functional fragment or portion thereof. In some embodiments, the sequence is codon optimized.

[0077] In some embodiments, the present disclosure provides one or more vectors comprising one or more polynucleotides, wherein the one or more polynucleotides encode one or more polypeptides, and the one or more polypeptides comprise or consist of an amino acid sequence, and the amino acid sequence comprises or consists of: SEQ ID NO: 1-158 or 177 or 161-176 and / or according to any one of Tables 1B and / or 1C or any functional variant or portion thereof.

[0078] In some embodiments, the disclosure provides isolated polynucleotides comprising one or more nucleic acid sequences encoding one or more of SEQ ID NOs: 1-158 or 177 or 161-176 and / or according to Tables 1B and / or 1C, or any functional variants or portions thereof. In some such embodiments, such nucleic acids encode polypeptides comprising at least 90%, 95%, 96%, 97%, 98%, 99% or higher identity to an amino acid sequence of SEQ ID NOs: 1-158 or 177 or 161-176 and / or according to Tables 1B and / or 1C, or any functional variants or portions thereof.

[0063] In some embodiments, the present disclosure provides a host cell comprising a vector encoding a polypeptide comprising or consisting of: any one of SEQ ID NO: 1-158 or 177 or 161-176 and / or according to Table 1B and / or 1C or any functional variant or portion thereof.

[0064] In some embodiments, the disclosure provides an isolated polypeptide having an amino acid sequence comprising or consisting of: one or more amino acid sequences selected from SEQ ID NOs: 161-176 according to Tables 1B and 1C; or one or more amino acid sequences having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NOs: 161-176 according to Tables 1B and 1C. In some embodiments, the polypeptide comprises no more than 100 amino acids. In some embodiments, the polypeptide specifically binds to connexin-4 or a portion thereof.

[0065] In some embodiments, the disclosure provides an isolated polynucleotide comprising one or more nucleic acid sequences encoding a polypeptide selected from SEQ ID NOs: 161-176 and / or according to Tables 1B and 1C; or a nucleic acid sequence encoding a polypeptide having at least 90%, 95%, 96%, 97%, 98%, 99% or higher identity with a polypeptide of SEQ ID NOs: 161-176 and / or according to Tables 1B and / or 1C. In some embodiments, the polypeptide does not bind to a non-target (e.g., a non-target protein) with an affinity greater than 10 nM. In some embodiments, the non-target does not include connexin-4 or a functional variant or portion thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0066] Figure 1A and 1B Binding affinities (BIACORE) of exemplary connexin-4 peptides are shown.

[0067] Figure 2A and 2B Binding affinities (BIACORE) of exemplary connexin-4 peptides are shown.

[0068] Figure 3A and 3B Binding affinities (BIACORE) of exemplary conjugates comprising exemplary connexin-4 peptides are shown.

[0069] Figure 4A and 4B Association (4A) and dissociation (4B) kinetics are shown for exemplary compounds comprising miniproteins provided herein.

[0070] Figure 5A and 5B Association (5A) and dissociation (5B) of exemplary compounds comprising the miniproteins provided herein are shown.

[0071] Fig. 6A and 6BInternalization of the exemplary radioconjugate C215 in HT1376 (6A) and MCF7 (6B) cell lines is shown. TB = total binding, NSB = non-specific binding.

[0072] Figure 7 A graph depicting thermal stability is shown, showing the percentage of exemplary compound C215 within 60 minutes when heated to 75 degrees Celsius. DETAILED DESCRIPTION

[0073] Among other things, the present disclosure provides compositions and methods of use thereof. In some embodiments, the composition selectively binds to a target (e.g., connexin-4). In some embodiments, the composition comprises one or more therapeutic agents (e.g., chelators, radionuclides), wherein the therapeutic agent selectively targets cells expressing connexin-4, such that cells expressing connexin-4 are treated while cells expressing connexin-4 are not treated. The present disclosure recognizes that the problem of treating cells expressing a target (e.g., cancer cells) stems from the fact 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 insights into the combination of selective targeting with specific therapeutic agents (e.g., chelators and / or radionuclides (e.g., alpha emitters)) that provide advantages over previously used therapeutic agents (e.g., antibodies, beta emitters, etc.).

[0074] Unless otherwise defined herein, scientific and technical terms used in conjunction with this disclosure will have the meanings commonly understood by those of ordinary skill in the art. Further, unless the context otherwise requires, singular terms shall include the plural and plural terms shall include the singular. Typically, the nomenclature and techniques used in connection with biochemistry, enzymology, molecular and cell biology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization as described herein are those well known and commonly used in the art.

[0075] The methods and techniques of the present disclosure are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989); Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992, and supplement in 2002); Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, [Cold Spring Harbor], New York (1990); Wittrup and Van Antwerp, Fine Affinity Discrimination by Yeast Surface Display and Flow Spectroscopy, 1996; Cytometry [Refined affinity identification by yeast surface display and flow cytometry], Biotechnol. Prog. [Progress in Biotechnology] 2002, (16) 31-37; C. Queen et al., A humanized antibody that binds to the interleukin 2 receptor [Humanized antibody that binds to the interleukin 2 receptor], Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America] 1989, 86 (24) 10029-10033; Scheinberg DA and McDevitt MR. Actinium-225 in targeted alpha-particle therapeutic applications. [Actinium-225 in targeted alpha-particle therapeutic applications] Curr Radiopharm. [Contemporary Radiopharmaceuticals] 2011; 4 (4): 306-320.

[0076] All publications, patents, and other references mentioned herein are hereby incorporated by reference in their entirety. In the event of a conflict, the present specification (including definitions) shall prevail. The materials, methods, and examples disclosed herein are illustrative only and are not intended to be limiting. definition

[0077] Unless otherwise defined, all technical terms and scientific terms used herein have the same meanings as those commonly understood by those of ordinary skill in the art to which the present disclosure belongs. Further, unless the context otherwise requires, singular terms shall include the plural and plural terms shall include the singular. Typically, the nomenclature and techniques used in connection with the 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.

[0078] Throughout the present specification and claims, the word "comprise" or variations 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.

[0079] As used herein, ranges and amounts may be expressed as "about" a particular value or range. About further includes an exact amount. Thus, "about 100 nucleotides" means "about 100 nucleotides" and also means "100 nucleotides". In general, the term "about" as used herein includes amounts that are expected to be within the range of experimental error. If "about" appears before a quantitative value, the disclosure also includes the specific quantitative value itself, unless otherwise expressly stated. In this case, "about" may also refer to a variation of ±10% from the nominal value, unless otherwise stated or inferred.

[0080] Unless otherwise specified, and as an example of all sequences described herein in the general format "SEQ.ID NO:", "a nucleic acid comprising SEQ.ID NO: 1" refers to a nucleic acid, at least a portion of which has (i) the sequence of SEQ.ID NO: 1 or (ii) a sequence complementary to SEQ.ID NO: 1. The choice between the two depends on the context. For example, if the nucleic acid is used as a probe, the choice between the two depends on the requirement that the probe is complementary to the desired target.

[0081] As used herein, the term "administering" refers to providing a composition to a subject or system. Administration to a subject can be by any appropriate route, dose and / or dosage regimen.

[0082] As used herein, the term "affibomer" refers to a subgenus of small proteins. Affibodies are molecules derived from the Z domain of Staphylococcal protein A, which consist of three alpha helices of 58 amino acids and have a molar mass of about 6 kDa. See, for exemplary details of affibody structure and use, Orlova, A; Magnusson, M; Eriksson, TL; Nilsson, M; Larsson, B; -Guthenberg, I; C; Carlsson, J et al. (2006). "Tumor imaging using a picomolar affinity HER2 binding affibody molecule", Cancer Res. 66(8): 4339-48. Affibodies are commercially available from Abcam Corp., Cambridge, Massachusetts. Affibodies are stable at high temperatures and acidic or alkaline conditions. Target specificity is obtained by randomization of 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).

[0083] As used herein, the term "affinity maturation" generally refers to the process of making sequential changes (e.g., sequential mutations) to a sequence and performing polypeptide sequence selection to select one or more sequences with increased affinity relative to a "starting" sequence or another sequence with lower affinity compared to a sequence with higher affinity.

[0084] As used herein, the terms "amino acid sequence" and "polypeptide" refer to amino acid polymers connected by one or more peptide bonds. The polypeptides disclosed herein encompass naturally occurring and non-naturally occurring proteins, and any fragments, parts, peptides, mutants, derivatives and analogs thereof. The polypeptide may be monomeric or polymeric. In addition, the polypeptide may comprise many different domains, each of which has one or more different activities. The polypeptide may be fully or partially synthesized or otherwise modified (i.e., comprising one or more synthetically produced amino acids and / or modifications thereof). The term "peptide" may be used to refer to short polypeptides, such as polypeptides comprising less 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).

[0085] As used herein, the term "Anticalins" refers to a subgenus of small proteins. 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-chain b-barrel that forms a highly conserved core unit in lipocalins and naturally forms a binding site for ligands via four structurally variable loops at the open ends. Although not homologous to the IgG superfamily, anticalins exhibit features that have been hitherto considered typical of antibody binding sites: (i) high structural plasticity due to sequence variation and (ii) increased conformational flexibility, allowing induced adaptation to targets with different shapes.

[0086] As used herein, the term "attenuation" as used herein generally refers to functional loss, including mutations, partial or complete deletions, insertions or other variations of gene sequences or sequences controlling transcription of gene sequences, which reduce or inhibit the production of gene products, or render gene products non-functional. In some cases, functional loss is described as knockout mutations. Attenuation also includes changing the amino acid sequence by changing the nucleic acid sequence, placing the gene under the control of a promoter with lower activity, down-regulating, expressing interfering RNA, ribozymes or antisense sequences targeting the target gene, or by any other technology known in the art. In one example, the sensitivity of a specific enzyme to feedback inhibition or inhibition (non-pathway-specific feedback) caused by a composition of a non-product or reactant is attenuated so that the enzyme activity is not affected by the presence of the compound. In other cases, enzymes that have been changed to be less active can be referred to as attenuated.

[0087] As used herein, the term "avimer" refers to a subgenus of small proteins. Avimer is a class of antibody mimics, which consists of two or more peptide sequences each having preferably 30 to 35 amino acids, which are derived from the A domains of various membrane receptors and connected by linker peptides. The binding of the target molecule occurs through the A domain, and the domain with the desired binding specificity can be selected, for example, by phage display technology. The binding specificity of the different A domains contained in avimer can be but not necessarily the same (Weidle UH, et al., (2013), Cancer Genomics Proteomics [Cancer Genomics and Proteomics]; 10 (4): 155-68). For more detailed information, see Nature Biotechnology [Natural Biotechnology] 23 (12), 1556-1561 (2005) and Expert Opinion on Investigational Drugs [Expert Opinions on Research Drugs] 16 (6), 909-917 (June 2007).

[0088] As used herein, the term "conjugate" refers to a subgenus of small proteins. The conjugate is characterized in that it comprises a polypeptide (e.g., a peptide) or is composed of a polypeptide that is capable of binding or having the ability to engage and associate with a known target or part thereof. The conjugate typically comprises a cysteine-containing peptide containing one or more disulfide bonds, but some conjugates do not contain cysteine ​​residues and / or disulfide bonds. When administered systemically to a mammalian subject, the conjugate is preferably rapidly cleared from the circulation. As will be appreciated, in the case of a given context, reference to the conjugate may be or include its nucleic acid sequence or the amino acid sequence encoding it. A vector, a host cell, etc. and / or any form of combination may be used to provide the conjugate, for example as a polynucleotide, a polypeptide. The conjugate may be derived or manufactured using any method known to those skilled in the art. For example, in some embodiments, the conjugate may be recombinant (i.e., produced using a recombinant nucleic acid encoding a polypeptide). In some embodiments, the conjugate can be synthesized (e.g., for example, using standard solid phase synthesis methods known to those of skill in the art, such as solid phase peptide synthesis (see, e.g., Palomo, J. RSC Adv. [Progress of the Royal Society of Chemistry], 2014, 4, 32658-32672) and described herein).

[0089] As used herein, the term "chelator" refers to any molecule or moiety that is capable of forming a complex (i.e., a "chelate") with a metal ion. Chelators typically have two or more unshared electron pairs that can be used to provide metal ions. Metal ions are typically coordinated to the chelator through two or more pairs of electrons.

[0090] As used herein, the term "conjugated" refers to the joining of two compounds or agents by covalent or non-covalent means.

[0091] As used herein, a "conservative amino acid substitution" is one in which an amino acid residue is substituted with another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, conservative amino acid substitutions do not significantly change the functional properties of the protein. In the case where two or more amino acid sequences differ from each other due to conservative substitutions, the percentage of sequence identity or degree of homology may be adjusted upward to correct for the conservative nature of the substitution. Means for making such adjustments are well known to those skilled in the art. See, for example, Pearson, 1994, Methods Mol. Biol. [Molecular Biology Methods] 24: 307-31 and 25: 365-89 (incorporated herein by reference). The following six groups each contain amino acids that are conservative substitutions for each other: 1) serine (S), threonine (T); 2) aspartic acid (D), glutamic acid I; 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).

[0092] As used herein, the terms "cysteine ​​dense peptide" and "CDP" are used interchangeably and refer to a subgenus of small proteins that generally contain a high density of cysteines (e.g., at least one, two, three, four or more cysteines within a range of about 10 to about 90 amino acids, or about 13 to about 80 amino acids in a polypeptide). In some embodiments, such a CDP may contain at least two independent folded domains. In some embodiments, the CDP contains at least one, two, three, four or more cysteine ​​residues within a range of about 10 to about 90 amino acid residues, preferably 13 to 80 amino acid residues. (pubmed.ncbi.nlm.nih.gov / 29483648 / ) In some embodiments, the CDP contains a restricted cysteine ​​distribution, 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).

[0093] As used herein, the term "deletion" generally refers to the removal of one or more nucleotides from a nucleic acid molecule, or the removal of one or more amino acids from a protein, with the regions on either side being joined together.

[0094] 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 an amino acid sequence identical to the amino acid sequence translated from the reference nucleic acid sequence. The term "degenerate oligonucleotide" or "degenerate primer" is used to refer to an oligonucleotide capable of hybridizing to target nucleic acid sequences that are not necessarily identical in sequence but are homologous to each other in one or more specific segments.

[0095] As used herein, the term "derived from" when referring to a nucleic acid sequence refers to a nucleic acid sequence that has at least 85% sequence identity to the reference naturally occurring nucleic acid sequence from which it is derived. The term "derived from" when referring to an amino acid sequence refers to an amino acid sequence that has at least 85% sequence identity to the reference naturally occurring amino acid sequence from which it is derived. As used herein, the term "derived from" does not imply 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.

[0096] As used herein, the term "designed ankyrin repeat domain (DARPin)" refers to a subgenus of small proteins. DARPins are peptides derived from ankyrin, a family of proteins that mediate the attachment of integral membrane proteins to the cytoskeleton. A single ankyrin repeat is preferably a 33-residue motif consisting of two α helices and a β turn. They can be engineered to bind different target antigens by randomizing the residues in the first α helix and β turn of each repeat. Their binding interface can be increased by increasing the number of modules (a method of affinity maturation). For more details, see J. Mol. Biol. 332, 489-503 (2003), PNAS 100 (4), 1700-1705 (2003) and J. Mol. Biol. 369, 1015-1028 (2007) and US20040132028 A1. DARPins generally provide a rigid interface and lack structural flexibility (Gebauer and Skerra, 2009).

[0097] As used herein, the term "domain" as used herein refers to a biomolecule structure that contributes to a 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.

[0098] As used herein, the term "engineered Kunitz domain" refers to a subgenus of small proteins. The engineered Kunitz domain is preferably a peptide derived from a Kunitz domain of a Kunitz-type protease inhibitor (e.g., bovine pancreatic trypsin inhibitor (BPTI), amyloid precursor protein (APP), or tissue factor pathway inhibitor (TFPI). The Kunitz domain has a molecular weight of approximately 6 kDa, and domains with desired target specificity can be selected by display technology (e.g., phage display) (Weidle et al., (2013), Cancer Genomics Proteomics [Cancer Genomics and Proteomics]; 10 (4): 155-68).

[0099] As used herein, the term "expression control sequence" as used herein refers to a polynucleotide sequence necessary for affecting the expression of a coding sequence operably connected thereto. An expression control sequence is a sequence that controls transcription, post-transcriptional events, and translation of a nucleic acid sequence. An expression control sequence includes appropriate transcription initiation, termination, promoter, and enhancer sequences; effective 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 sequences that enhance protein secretion when necessary. The properties of such control sequences vary according to the host organism; in prokaryotes, such control sequences typically include promoters, ribosome binding sites, and transcription termination sequences. The term "control sequence" is intended to include at least all components whose presence is necessary for expression, and may also include additional components whose presence is advantageous, such as leader sequences and fusion partner sequences.

[0100] As used herein, the term "functional variant" refers to a polypeptide that comprises a portion of the sequence of a polypeptide provided herein or consists of a portion of the sequence of a polypeptide provided herein, and still retains one or more functions of a polypeptide comprising or consisting of the complete amino acid sequence as provided herein (e.g., still binds to a target, such as connexin-4).

[0101] 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. The fusion protein contains at least 10 consecutive amino acids from the target polypeptide, more preferably at least 20 or 30 amino acids, even more preferably at least 40, 50 or 60 amino acids, and still more preferably at least 75, 100 or 125 amino acids. The fusion comprising the entirety of the protein disclosed herein has a specific utility. The length of the heterologous polypeptide included in the fusion protein disclosed herein is at least 6 amino acids, generally at least 8 amino acids in length, and usefully at least 15, 20 and 25 amino acids in length. Fusions including larger polypeptides (e.g., IgG Fc regions) and even whole proteins (e.g., proteins containing green fluorescent protein ("GFP") chromophores) have specific utilities. Fusion proteins can be recombinantly produced by constructing a nucleic acid sequence encoding a polypeptide or a fragment thereof in the same reading frame as 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 a polypeptide or fragment thereof to another protein.

[0102] 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 two proteins have similar amino acid sequences.) Homology between two regions of amino acid sequence (particularly in terms of predicted structural similarity) can be interpreted as implying functional similarity. Homologous proteins or peptides with non-identical residue positions are generally considered to be different due to conservative amino acid substitutions.

[0103] As used herein, the term "identical" refers to a nucleic acid sequence or an amino acid sequence, and refers to at least two nucleic acids or at least two amino acid sequences or subsequences having a specific percentage of identical nucleotides or amino acids, respectively, as measured using a sequence comparison algorithm or by visual inspection, when comparing and aligning for maximum correspondence. For sequence comparison, typically, a sequence is used as a reference sequence, and a test sequence is compared with the reference sequence. The length of sequence identity comparison can be any number of nucleotides or amino acid segments. When using a sequence comparison algorithm, the test sequence and the reference sequence are input into a computer (if necessary, specifying subsequence coordinates), and the sequence algorithm program parameters are specified. Then, the sequence comparison algorithm calculates the sequence identity percentage of one or more test sequences relative to the reference sequence based on the specified program parameters. Many algorithms are known in the art. Non-limiting examples of algorithms suitable for determining percent sequence identity and sequence similarity are BLAST and BLAST 2.0 algorithms, which are described in, for example, Altschul et al. (1990) J. Mol. Biol. [Molecular Biology] 215: 403-410 and Altschul et al. (1977) Nucleic Acids Res. [Nucleic Acids Research] 25: 3389-3402, respectively. Software for performing BLAST analysis is available through the National Center for Biotechnology Information. Additionally or alternatively, FASTA, Gap or Bestfit can be used to compare sequences, which are programs in Wisconsin Package Version 10.0, Genetics Computer Group (GCG), Madison, Wisconsin. FASTA provides an alignment and a percent sequence identity of the best overlapping region between the query sequence and the search sequence. Pearson, Methods Enzymol. 183:63-98 (1990) (incorporated herein by reference in its entirety). For example, percent sequence identity can be determined using FASTA and its default parameters (word length of 6 and NOPAM factor for the scoring matrix) or using Gap and its default parameters as provided in GCG Version 6.1 (incorporated herein by reference).

[0104] As used herein, the term "isolated" polynucleotide or polypeptide is a polynucleotide or polypeptide that is substantially separated from other cellular components that naturally accompany the native polynucleotide in its native host cell (e.g., ribosomes, polymerases, and genomic sequences with which it is naturally associated). For example, an isolated molecule is one that, by virtue of its origin or source of derivation, (1) is not associated with naturally associated components that accompany it in its native state, (2) is present in a purity not found in nature, where purity can be judged by the presence of other cellular material (e.g., it is free of other proteins from the same species), (3) is expressed by a cell from a different species, or (4) does not occur in nature (e.g., it is a fragment of a polynucleotide or polypeptide found in nature, or it includes amino acid analogs or derivatives or linkages other than standard peptide bonds not found in nature). Thus, a polynucleotide or polypeptide that is chemically synthesized or synthesized in a cellular system different from the cell from which it is naturally derived will be "separated" from its naturally associated components. A polynucleotide or polypeptide may also be rendered substantially free of naturally associated components by separation using protein purification techniques well known in the art. As so defined, "isolated" does not necessarily require that any molecule described has been physically removed from its natural environment. In some embodiments, when used in reference to isolated constructs, isolated means in the absence of pharmaceutically acceptable salts.

[0105] As used herein, the term "K D "Kd" or "Kd" refers to the dissociation equilibrium constant for a particular antibody-antigen interaction. Typically, the antibodies of the present disclosure dissociate at a constant of less than about 10 -7 M (e.g., less than about 10 -8 M, 10 -9 M or 10 -10 M or less) dissociation equilibrium constant (K D ) binds to nexin-4, e.g., as determined using surface plasmon resonance (SPR) technology in a BIACORE instrument.

[0106] As used herein, the term "knockout" generally refers to a gene whose expression level or activity has been reduced to zero. In some instances, a gene is knocked out by deleting part or all of its coding sequence. In other instances, a gene is knocked out via the introduction of one or more nucleotides into its open reading frame, which results in the translation of nonsense or other non-functional protein products.

[0107] As used herein, the term "knotin" refers to a structural motif of a small protein containing three disulfide bridges.

[0108] As used herein, the term "knotin peptides" refers to a subgenus of small proteins that contain at least one knotin.

[0109] As used herein, the term "linker" refers to a moiety used to conjugate a small protein to a chelator.

[0110] As used herein, the term "small protein" refers to a short protein of less than or equal to 100 amino acids, with a well-defined fold comprising two or more secondary structural elements, an isolated hydrophobic core and / or a cooperative fold. CDPs, knottins, affibodies, engineered Kunitz domains, monoclonal antibodies (adnectins), anticalins, designed ankyrin repeat domains (DARPins), avimers and conjugates as disclosed herein are examples of small proteins.

[0111] As used herein, the term "modification" refers to a nucleic acid sequence that comprises a substitution, change, inversion, addition or deletion of at least one nucleotide compared to a reference nucleic acid sequence when it is related to a nucleic acid sequence. As used herein, the term "modification" refers to a nucleic acid sequence that comprises a substitution, change, inversion, addition or deletion of at least one amino acid residue compared to a reference amino acid sequence when it is related to an amino acid sequence.

[0112] As used herein, the term "modified derivative" refers to a polypeptide or fragment thereof that is substantially homologous in the primary structural sequence, but these polypeptides or fragments thereof include, for example, in vivo or in vitro chemical and biochemical modifications or the incorporation of amino acids not found in the native polypeptide. Such modifications include, for example, acetylation, carboxylation, phosphorylation, glycosylation, ubiquitination, labeling (e.g., labeling with a radionuclide), and various enzymatic modifications, as will be readily understood by those skilled in the art. Various methods for labeling polypeptides and substituents or labels for such purposes are well known in the art and include radioisotopes such as 125I, 32P, 35S, and 3H, ligands bound to labeled anti-ligands (e.g., antibodies), fluorophores, chemiluminescent agents, enzymes, and anti-ligands that can be used as members of specific binding pairs of labeled ligands. The choice of label depends on the desired sensitivity, the ease of conjugation with primers, stability requirements, and available instruments. 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 supplement 2002) (hereby incorporated by reference).

[0113] 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.

[0114] As used herein, the term "monoantibody" or "fibronectin" is used interchangeably and refers to a subgenus of a small protein. Monoantibodies relate to molecules, preferably based on the 10th extracellular domain of human fibronectin III (10Fn3), which employ an Ig-like b sandwich fold of preferably 94 residues and 2 to 3 exposed loops, but lack a central disulfide bridge (Gebauer and Skerra (2009) Curr Opinion in Chemical Biology [Chemical Biology Current Views] 13: 245-255). By introducing modifications in the specific loops of the protein, it is possible to carry out genetic engineering to fibronectin with a desired target specificity.

[0115] As used herein, the term "mutant protein" or "mutant protein" or "variant" means a protein comprising an amino acid sequence having at least one variation (e.g., 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 the nucleotides in the nucleic acid sequence or the amino acids in the amino acid sequence may be inserted, deleted or changed compared to the reference sequence. A single change (point mutation) may be made at a locus, or multiple nucleotides or amino acids may be inserted, deleted or changed at a single locus. In addition, 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 in which PCR is performed under conditions in which the replication 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 capable of generating 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, resulting in the production of different polypeptides having one or more amino acid changes that may or may not confer greater fitness on the polypeptide); and site-directed mutagenesis (e.g., specific directed changes in a sequence).

[0116] 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 bonds in its folded and active form. In some embodiments, a small protein may comprise or consist of a non-disulfide sequence.

[0117] As used herein, the term "non-peptide analog" refers to a compound that has properties similar to those of a reference polypeptide. Non-peptide compounds may also be referred to as "peptide mimetics" or "mimetic of a peptide." 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., WH Freeman and Co., 1992); Evans et al., J. Med. Chem. 30:1229 (1987); Fauchere, J. Adv. Drug. Res. [Journal of Advanced Pharmaceutical Research] 15:29 (1986); Veber and Freidinger, Trends Neurosci. [Trends in Neuroscience], 8:392-396 (1985); and references located in each of the above, which are incorporated herein by reference. Such compounds are generally developed with the aid of computer molecular modeling. Peptide mimetics that are structurally similar to the useful peptides of the present disclosure can be used to produce equivalent effects and are therefore contemplated as part of the present disclosure.

[0118] As used herein, the terms "nucleic acid sequence" and "polynucleotide" are used interchangeably to refer to nucleotide polymers. 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-natural internucleoside bonds, 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, hairpin, circular or padlocked conformations. Nucleic acid sequences can contain natural, non-natural or altered nucleotides; and contain natural, non-natural or altered internucleotide connections, such as phosphoramidate connections or phosphorothioate connections, rather than phosphodiesters found between nucleotides of 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 a recombinant library or a cell genome using common cloning techniques and polymerase chain reaction, and by synthetic means. The polynucleotides disclosed herein may include RNA, cDNA, the sense strand and antisense strand of genomic DNA and the above-mentioned synthetic forms and mixed polymers. As will be readily understood by those skilled in the art, they may be chemically or biochemically modified or may contain non-natural or derived nucleotide bases. Such modifications include, for example, labeling, methylation, substitution of one or more naturally occurring nucleotides, internucleotide modifications such as uncharged connections (e.g., methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.) with analogs, charged connections (e.g., phosphorothioates, phosphorodithioates, etc.), pendant moieties (e.g., polypeptides), intercalators (e.g., acridine, psoralens, etc.), chelators, alkylating agents, and modified connections (e.g., α-anomeric nucleic acids, etc.). Also included are synthetic molecules that simulate the ability of polynucleotides to bind to a specified sequence through hydrogen bonds and other chemical interactions. Such molecules are known in the art, and include, for example, those in which peptides are connected to replace the phosphate connections in the main chain of the molecule. Other modifications may include, for example, analogs in which the ribose ring includes a bridging moiety or other structures (e.g., modifications found in "locked" nucleic acids).

[0119] As used herein, the term "operatively linked" or "operably linked" expression control sequence refers to a linkage in which the expression control sequence is adjacent to the target gene to control the target gene, as well as an expression control sequence that acts in trans or remotely to control the target gene.

[0120] As used herein, the term "polypeptide mutant" or "mutant" refers to a polypeptide whose sequence contains one or more insertions, duplications, deletions, rearrangements or substitutions of amino acids compared to the amino acid sequence of a natural or wild-type protein. A mutant protein may have one or more amino acid point substitutions, wherein a single amino acid at one position has been changed to another amino acid, one or more insertions and / or deletions (wherein one or more amino acids are inserted or deleted, respectively, in the sequence of a naturally occurring protein), and / or a truncation of the amino acid sequence at one or both of the amino or carboxyl termini. Compared to a naturally occurring protein, a mutant protein may have the same biological activity, but preferably has different biological activities. A mutant protein has at least 85% overall sequence homology with its wild-type counterpart. Even more preferred is a mutant protein with at least 90% overall sequence homology with a wild-type protein. In an even more preferred embodiment, a mutant protein exhibits at least 95% sequence identity, even more preferably 98%, even more preferably 99%, and even more preferably 99.9% overall sequence identity. Sequence homology can be measured by any common sequence analysis algorithm, such as Gap or Bestfit. Amino acid substitutions may include those that: (1) reduce susceptibility to proteolysis; (2) reduce susceptibility to oxidation; (3) alter binding affinity for forming protein complexes; (4) alter binding affinity or enzymatic activity, and (5) confer or alter other physiochemical or functional properties of such analogs.

[0121] As used herein, the term "polypeptide fragment" as used herein refers to a polypeptide having a deletion (e.g., an amino-terminal and / or carboxyl-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 positions in the naturally occurring sequence. A fragment is typically at least 5, 6, 7, 8, 9 or 10 amino acids long, preferably at least 12, 14, 16 or 18 amino acids long, more preferably at least 20 amino acids long, more preferably at least 25, 30, 35, 40 or 45 amino acids long, even more preferably at least 50 or 60 amino acids long, and even more preferably at least 70 amino acids long.

[0122] The term "radionuclide" as used herein refers to an atom capable of undergoing radioactive decay.

[0123] As used herein, the term "recombinant" refers to a biological molecule, such as a gene or protein, that (1) has been removed from its naturally occurring environment, (2) is not associated with all or part of a polynucleotide to which the gene is found in nature, (3) is operably linked to a polynucleotide to which it is not linked in nature, and / or (4) does not occur in nature. The term "recombinant" may be used to refer to cloned DNA isolates, chemically synthesized polynucleotide analogs, or polynucleotide analogs biologically synthesized by a heterologous system, and proteins and / or mRNA encoded by such nucleic acids. As used herein, an endogenous nucleic acid sequence (or the encoded protein product of the sequence) in the genome of an organism is considered "recombinant" herein if the heterologous sequence is placed adjacent to an 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 an endogenous nucleic acid sequence, whether the heterologous sequence itself is endogenous (derived from the same host cell or its progeny) or exogenous (derived from a different host cell or its progeny). For example, a promoter sequence can be replaced by the natural promoter of a gene in the host cell genome (e.g., by homologous recombination) so that the gene has an altered expression pattern. The gene will now become "recombinant" because it is separated from at least some of the sequences of its natural flanks. If a nucleic acid contains any modification that does not occur naturally in the corresponding nucleic acid in the genome, the nucleic acid is also considered to be "recombinant". For example, if an endogenous coding sequence comprises an insertion, a deletion or a point mutation introduced artificially (e.g., by human intervention), it is considered to be "recombinant". "Recombinant nucleic acid" also includes nucleic acids integrated at heterologous sites in host cell chromosomes and nucleic acid constructs present as episomes.

[0124] As used herein, the term "recombinant host cell" (or simply "host cell") as used herein means a cell into which a recombinant vector has been introduced. It should be understood that this term refers not only to the specific subject cell, but also to the progeny of such a cell. Because certain modifications may occur in subsequent generations due to mutations or environmental influences, such progeny may not actually be the same as the parent cell, but are still included in the scope of the term "host cell" as used herein. Recombinant host cells can be isolated cells or cell lines grown in culture, or can be cells present in living tissues or organisms.

[0125] As used herein, the term "region" as used herein refers herein 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 the protein.

[0126] As used herein, the phrase "secondary structural element" refers to a local folded structure formed in a polypeptide due to the interaction between its main chain atoms. Examples of secondary structural elements may include alpha helices, beta sheets, 310 helices, π helices, and random coils. The small protein disclosed herein may contain one or more of any of such secondary structures (e.g., one or more alpha helices, one or more alpha helices, and one or more beta folds). It will be appreciated by those skilled in the art that secondary structural elements may be connected by loop regions, which may or may not be modified to change the interaction of the secondary structural elements of the polypeptide. As will be appreciated by those skilled in the art, in some embodiments, a loop may be a secondary structural element. In some embodiments, a loop may be an inter-structural element, which is not necessarily considered to be a secondary structural element.

[0127] As used herein, "sequence homology" of a polypeptide, also referred to as "percentage of sequence identity", is typically measured using sequence analysis software. See, e.g., the sequence analysis software package of the Genetic Computing Group (GCG), University of Wisconsin Biotechnology Center, 910 University Avenue, Madison, Wisconsin 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 (e.g., homologous polypeptides from different organism species) or between a wild-type protein and its mutant protein. See, e.g., GCG version 6.1. A preferred algorithm when comparing a specific 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); Zhang and Madden, Genome Res. 7:649-656 (1997)). Res. [Nucleic Acids Research] 25: 3389-3402 (1997)). Preferred parameters for BLASTp are: expectation: 10 (default); filter: segment (default); penalty for opening a gap: 11 (default); penalty for extending a gap: 1 (default); maximum alignment: 100 (default); word length: 11 (default); number of descriptions: 100 (default); penalty matrix: BLOSUM62. The length of polypeptide sequences to be compared for homology is usually 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 more than about 35 residues. When searching a database containing sequences from a large number of different organisms, it is preferred to compare amino acid sequences. Database searches using amino acid sequences can be measured by algorithms other than blastp known in the art. For example, the program FASTA in GCG version 6.1 can be used to compare polypeptide sequences. FASTA provides an alignment and percentage of sequence identity of the best overlapping region between the query sequence and the search sequence.Pearson, Methods Enzymol. 183:63-98 (1990) (incorporated herein by reference). For example, the percentage of sequence identity between amino acid sequences can be determined using FASTA as provided in GCG Version 6.1 (incorporated herein by reference) and its default parameters (word length of 2 and PAM250 scoring matrix).

[0128] As used herein, the term "specificity" generally refers to a sequence that selectively or "specifically" binds to a specific target when in a conformation that can bind. As used herein, "specific binding" means that the binding of a polynucleotide, polypeptide or protein is selective for a specific antigen and can be distinguished from unwanted or non-specific interactions. For example, the ability of a protein (e.g., a cysteine-dense peptide) to bind to a specific antigenic determinant can be measured by techniques familiar to those skilled in the art, such as by enzyme-linked immunosorbent assay (ELISA) or surface plasmon resonance. Between two molecules, "specific binding" refers to the ability of two molecules to bind to each other in preference to binding to other molecules in the environment. Typically, "specific binding" differs from accidental binding in a reaction by at least two times, more typically by at least 10 times, and often by at least 100 times. Typically, the affinity or avidity of a specific binding reaction, as quantified by a dissociation constant, is about 10-7M or stronger (e.g., about 10-8M, 10-9M or even stronger). Specific binding requires specific first entities (e.g., polypeptides) to specific second entities (e.g., antigen binding sequences).

[0129] As used herein, the term "stabilizer" in the context of a pharmaceutical composition refers to an agent, molecule or compound that acts to affect one or more active pharmaceutical ingredients to maintain desired properties (e.g., therapeutic properties or properties that allow a therapeutic effect to be achieved) until it is administered to a subject.

[0130] As used herein, "stringent hybridization conditions" and "stringent washing conditions" in the context of nucleic acid hybridization experiments depend on many different physical parameters. Nucleic acid hybridization will be affected by conditions such as salt concentration, temperature, solvent, base composition of the hybridization species, length of the complementary region, and the number of nucleotide base mismatches between the hybridized nucleic acids, as will be readily understood by those skilled in the art. One of ordinary skill in the art knows how to change these parameters to achieve a particular hybridization stringency. In general, "stringent hybridization" is performed under a specific set of conditions at about 25°C below the thermal melting temperature (Tm) of a specific DNA hybrid. "Stringent washing" is performed at a temperature about 5°C lower than the Tm of a specific DNA hybrid under a specific set of conditions. Tm is the temperature at which 50% of the target sequence hybridizes to a fully matched probe. See, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989), p. 9.51, incorporated herein by reference. For the purposes of this document, "stringent conditions" for solution phase hybridization are defined as aqueous hybridization (i.e., without formamide) in 6xSSC (wherein 20xSSC contains 3.0 M NaCl and 0.3 M sodium citrate), 1% SDS at 65° C. for 8-12 hours, followed by two washes in 0.2xSSC, 0.1% SDS at 65° C. for 20 minutes each. It will be appreciated by those skilled in the art that hybridization at 65° C. will occur at varying rates, depending on a variety of factors, including the length and percent identity of the sequences being hybridized.

[0131] As used herein, the term "synthetic" is used to refer to an entity that is made in a laboratory, rather than an entity that is produced in nature or that is 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, biosynthesis (e.g., by a host cell, etc.).

[0132] As used herein, the term "subject" is a mammal. A subject can be a human or a non-human mammal. Subject can be used interchangeably with patient, individual, donor, etc., given the context.

[0133] As used herein, the term "substantial homology" or "substantial similarity" when referring to a polynucleotide or polypeptide means that when optimally aligned with another reference molecule (or, where appropriate, its complementary strand) 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 of the nucleic acid or amino acid residues, as measured by any well-known algorithm for sequence identity (e.g., FASTA, BLAST, Gap, etc.). Alternatively or additionally, substantial homology or similarity exists when, for example, a nucleic acid or fragment thereof hybridizes to another nucleic acid, a strand of another nucleic acid, or its complementary strand, under stringent hybridization conditions.

[0134] As used herein, the term "target" refers to a protein or a functional part or variant thereof. The target may be expressed on the surface of a specific cell ("target cell") or in a cell population (e.g., on its surface). The target may have a certain percentage of identity with a reference protein and still be referred to as a target by a specific name (e.g., connexin-4). The target may also refer to a protein in a pathway associated with another protein. For example, if the target is connexin-4, the target may also be a protein in a pathway essential for connexin-4 activity. The target may be or comprise a binding region (e.g., epitope) bound by a small protein (e.g., CDP, knottin, conjugate, affibody, engineered Kunitz domain, monoclonal antibody, anticalin, designed ankyrin repeat domain (DARPin), avimer) disclosed herein.

[0135] As used herein, "thermostability" refers to the ability of a small protein to remain stable (eg, not unfolded, eg, structurally intact) over a period of time. In some embodiments, the small proteins of the present disclosure retain at least 95% stability for at least one hour.

[0136] As used herein, a treatment that is "tolerated" by a subject refers to a therapeutic administration and / or regimen that is not terminated due to dose-limiting toxicity.

[0137] As used herein, the term "treatment" (as well as "treat" or "treating") refers to the partial or complete alleviation, amelioration, alleviation, prevention, reduction in the risk of onset, alleviation, suppression, delay of onset, reduction in severity, reduction in frequency or incidence of one or more causes, features and / or symptoms of, or associated with, a specific disease, disorder and / or condition.

[0138] As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid connected thereto. One type of vector is a "plasmid", which generally refers to a circular double-stranded DNA loop in which another DNA segment can be connected, but also includes a linear double-stranded molecule, such as a molecule produced by amplifying by polymerase chain reaction (PCR) or treating a circular plasmid with a restriction endonuclease. Other vectors include cosmids, bacterial artificial chromosomes (BACs) and yeast artificial chromosomes (YACs). Another type of vector is a viral vector, in which another DNA segment can be connected to a viral genome (discussed in more detail below). Some vectors can replicate autonomously in the host cell into which they are introduced (for example, a vector with a replication origin that works in a host cell). Other vectors can be integrated into the genome of the host cell after being introduced into the host cell, thereby replicating together with the host genome. In addition, some preferred vectors can guide the expression of a gene operably connected thereto. Such a vector is referred to herein as a "recombinant expression vector" (or simply "expression vector"). Composition

[0139] 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 the following or consists of the following: a polypeptide (i.e., a small protein), an optional linker, and a chelator and / or a radionuclide. In some embodiments, the chelator and / or the radionuclide are conjugated to the small protein through a linker. In some embodiments, the small protein disclosed herein comprises the following or consists of the following: a CDP, a knotting element, a conjugate, an affibody, an engineered Kunitz domain, a monoclonal antibody, an anticalin, a designed ankyrin repeat domain (DARPin) and / or an avimer. In some embodiments, the small protein comprises a CDP or consists of it. In some such embodiments, the small protein comprises a knotting element or consists of it. In some such embodiments, the small protein comprises a conjugate or consists of it. In some such embodiments, the small protein comprises an affibody or consists of it. In some such embodiments, the small protein comprises or consists of an engineered Kunitz domain. In some such embodiments, the small protein comprises or consists of a monoclonal antibody. In some such embodiments, the small protein comprises or consists of an ankyrin repeat domain (DARPin). In some such embodiments, the small protein comprises or consists of an avimer. In some embodiments, the small protein (e.g., CDP, knottin, binder, affibody, engineered Kunitz domain, monoclonal antibody, ankyrin, designed ankyrin repeat domain (DARPin), avimer) is designed to be connected to one or more other components. For example, in some embodiments, the small protein (e.g., CDP, knottin, binder, affibody, engineered Kunitz domain, monoclonal antibody, ankyrin, designed ankyrin repeat domain (DARPin), avimer) can be connected (conjugated) to another component (e.g., chelator and / or 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 small protein via a linker.

[0140] Without wishing to be bound by any particular theory, the present disclosure contemplates that the compositions disclosed herein are more effective than previously described compositions (e.g., compositions comprising antibodies and / or β-emitter radionuclides). Such small proteins or compositions comprising small proteins can be used to treat subjects in need, with improved target specificity, increased clearance rates, and reduced off-target effects (e.g., compared to conjugates with non-α-emitter radionuclides, e.g., compared to compositions comprising antibodies or antibody-drug-conjugates, etc.). For example, although small proteins (e.g., for use in compositions as provided herein) have several key features of antibody-based therapeutic agents (e.g., affinity, efficacy, specificity, and the ability to disrupt protein: protein interactions), they can avoid undesirable limitations, such as large size, expensive manufacturing, and the necessity of chimerization or humanization. For example, in some embodiments, the small proteins disclosed herein (e.g., CDPs, knottins, binders, affibodies, engineered Kunitz domains, monoclonal antibodies, anticalins, designed ankyrin repeat domains (DARPins), avimers) are no more than about 100 amino acids in length. In some embodiments, such small proteins (e.g., CDPs, knottins, binders, affibodies, engineered Kunitz domains, monoclonal antibodies, anticalins, designed ankyrin repeat domains (DARPins), avimers) can be or comprise cysteine ​​dense peptides. In some embodiments, small proteins (e.g., CDPs, knottins, binders, affibodies, engineered Kunitz domains, monoclonal antibodies, anticalins, designed ankyrin repeat domains (DARPins), avimers) comprise one or more disulfide bridges. In some embodiments, small proteins (e.g., CDPs, knottins, binders, affibodies, engineered Kunitz domains, monoclonal antibodies, anticalins, designed ankyrin repeat domains (DARPins), avimers) comprise multiple cysteine ​​residues that are cross-linked to maintain a very stable folded state for peptides of their length (e.g., relative to peptides of the same length without as many cysteine ​​residues). Without being bound by any particular theory, the present disclosure contemplates that in some embodiments, small proteins do not comprise multiple cysteine ​​residues, such as small proteins comprising a single cysteine ​​residue. In some such embodiments, the small protein can form a dimer, such as forming a dimer with another small protein (e.g., self-dimerization). In some embodiments, two small proteins are connected together to form a dimer. In other embodiments, two small proteins are each connected to a joint to form a dimer. In other embodiments, two different small proteins are each connected to a joint to form a dimer. The present disclosure contemplates that the stability conferred by cross-linked cysteine ​​helps to reduce the immunogenicity of small proteins or small proteins comprising such proteins.In some embodiments, such stability can also confer resistance to the harsher conditions provided for effective chelation (eg, high temperature, low pH incubation, etc.) while continuing to retain biological activity (eg, ability to bind target).

[0141] In some embodiments, the small protein provided herein works as a targeting part, such as specifically binding to a target expressed on the surface of a tumor cell. In some such embodiments, a small protein is designed so that it can be connected to one or more other components. For example, without being bound by any particular theory, the small protein of the present disclosure can be formulated so that they are combined with other components, and the other components are such as therapeutic molecules (e.g., chelating agent compositions and / or radionuclides) and / or detectable agents (e.g., visualization agents, such as metabolizable and visualization agents). In some such embodiments, such small proteins conjugated with one or more other components can be used, for example, in one or more diseases, obstacles or illnesses (e.g., one or more diseases, obstacles or illnesses expressing specific targets on a specific cell population), diagnosis, prognosis, monitoring and / or treatment.

[0142] In some embodiments, small proteins (e.g., CDPs, knottins, binders, affibodies, engineered Kunitz domains, monoclonal antibodies, anticalins, designed ankyrin repeat domains (DARPins), avimers) have lower immunogenicity relative to larger proteins. In some such embodiments, lower immunogenicity increases adaptability to harsher environmental conditions (e.g., high temperature and low pH incubation) while retaining biological activity. Thus, in some embodiments, conjugates comprising small proteins have lower immunogenicity than compositions comprising larger proteins or different targeting moieties (i.e., in addition to the small protein).

[0143] In some embodiments, the composition comprising a linker, a chelator, and / or a radionuclide can effectively penetrate a tumor.

[0144] In some embodiments, small proteins (e.g., CDPs, knottins, conjugates, affibodies, engineered Kunitz domains, monoclonal antibodies, anticalins, designed ankyrin repeat domains (DARPins), avimers) have superior penetration efficiency relative to larger proteins. That is, in some embodiments, small proteins or compositions comprising small proteins can penetrate solid tumors better than larger proteins or compositions comprising proteins larger than small proteins. For example, in some embodiments, the conjugate has superior tumor penetration efficiency and has a hydrodynamic radius of about 1 nm-25 nm. In some embodiments, the hydrodynamic radius is between about 1 nm-5 nm. In some embodiments, the hydrodynamic radius is about 1 nm-3 nm. In some embodiments, the hydrodynamic radius is about 1 nm, 2 nm, 3 nm, 4 nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm, 11 nm, 12 nm, 13 nm, 14 nm, 15 nm, 16 nm, 17 nm, 18 nm, 19 nm, 20 nm, 21 nm, 22 nm, 23 nm, 24 or 25 nm.

[0145] As described herein, small proteins (e.g., CDPs, knottins, binders, affibodies, engineered Kunitz domains, monoclonal antibodies, anticalins, designed ankyrin repeat domains (DARPins), avimers) are conjugated to chelators. In some embodiments, the chelator binds a radionuclide (e.g., an alpha-emitting radionuclide, e.g., actinium). In some such embodiments, such radionuclide conjugates combine the specific binding capabilities and properties of small proteins (e.g., CPDs, knottins, binders, affibodies, engineered Kunitz domains, monoclonal antibodies, anticalins, designed ankyrin repeat domains (DARPins), avimers) with a radionuclide. That is, without being bound by any particular theory, the present disclosure provides a conjugate wherein in some embodiments, a small protein (e.g., CDP, knottin, binder, affibody, engineered Kunitz domain, monoclonal antibody, anticalin, designed ankyrin repeat domain (DARPin), avimer) targets a radioisotope that is conjugated to a cell expressing a target. In some embodiments, the target is expressed on the cell surface. In some embodiments, the target is connexin-4. In some embodiments, the cell is a tumor cell. In some embodiments, the conjugate binds to connexin-4 on the surface of a tumor cell. In some such embodiments, the radionuclide targets a tumor cell. In some embodiments, the radionuclide is an alpha emitter radionuclide and, when internalized, is used to specifically target (e.g., without damaging surrounding tissue / cells) a tumor cell. Target

[0146] Any cell expressing a target can be targeted by a small protein (e.g., CDP, knottin, binder, affibody, engineered Kunitz domain, monoclonal antibody, anticalin, designed ankyrin repeat domain (DARPin), avimer) as provided herein.

[0147] 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 from a sample of a subject, such as from a tumor or from a corresponding tissue without a tumor (e.g., from another region of an organ or from a healthy donor). In some embodiments, the cell is an in vitro cell (e.g., a primary cell, a cell line, etc.). In some embodiments, the cell is an in vivo cell (e.g., in a subject, for example, in a human subject, for example, in a tumor of a human subject.) In some embodiments, the cell expresses or has been induced to express (e.g., by recombinant technology) a target. In some embodiments, the target is expressed on the cell surface. In some embodiments, the cell is contacted by a composition that is bound to a target expressed on its surface. In some embodiments, after binding (e.g., after binding of a small protein provided by the present disclosure), the target and any bound protein and / or payload are internalized into the cell. In some embodiments, the cell is killed by a payload (e.g., a radionuclide and / or a chelator, etc.) after internalization.

[0148] In some embodiments, the target is a protein or part thereof that is upregulated or overexpressed on cancer cells compared to non-cancerous cells. That is, in some embodiments, the target is expressed or overexpressed in a tumor or tumor microenvironment relative to the target level in a non-diseased tissue (e.g., a tissue without a tumor or tumor microenvironment). In some such embodiments, the target is not present or undetectable in a non-diseased (e.g., healthy) tissue. In some embodiments, the target is a biomarker of cancer (e.g., for cancer cells, for tumors).

[0149] 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 that the surface cell protein acts upon. 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 be refractory to treatment. In some embodiments, a tumor-associated cell surface molecule or a tumor-specific cell surface molecule may be targeted by a small protein or a composition comprising a small protein as provided herein.

[0150] In some embodiments, a small protein or a composition comprising a small protein specifically binds to a target expressed on a cell surface. In some embodiments, the target is cut from the cell surface. In some such embodiments, if the target is in an organism, the cutting of the target causes the target to circulate in the entire organism system. In some such embodiments, a specific level of target is found in blood, serum, plasma, etc. However, in some embodiments, a large part of the expressed target is located on the cell surface; therefore, in some embodiments, the measurement of the target level may not accurately reflect the number of targets in a cell population (e.g., a tumor). In some embodiments, the target is a secreted protein. In some such embodiments, a specific level of target is found in blood, serum, plasma, etc. In some such embodiments, the small protein binds to an area of ​​the target, such as an epitope. In some embodiments, a small protein or a composition comprising a small protein specifically binds to a target expressed on the surface of a cancer cell. In some embodiments, the cancer cell is located in, on, or near a solid tumor. In some embodiments, the cancer cell is a circulating cancer cell. In some embodiments, a small protein or a composition comprising a small protein specifically binds to a target or expresses at a higher level on a cancer cell than a reference cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell.

[0151] In some embodiments, the small protein or a composition comprising the small protein specifically binds to connexin-4. In some embodiments, the target comprises or consists of connexin-4. In some embodiments, the small protein specifically binds to a target comprising the amino acid sequence shown in Table 1A or a portion thereof. Connexin-4

[0152] Connexin proteins are involved in cell adhesion, migration, and polarization. It was found that connexin-4 in most human organs is uniformly expressed at weak to moderate levels, but is specifically overexpressed in most samples from metastatic urothelial tumors. An ADC, enfortumab vedotin ("EV"; Padcev; Astellas; Tokyo, Japan; and Seattle Genetics; Bothell, Washington, USA) specifically targets connexin-4 overexpressed on the surface of bladder tumor cells. EV is conjugated with a microtubule inhibitor (monomethyl auristatin E), resulting in G2 / M cell cycle arrest and apoptosis. In some embodiments, the small protein disclosed herein targets connexin-4 on tumor cells. (Bednova O. & Leyton JV. Int J Mol Sci. [International Journal of Molecular Sciences] October 1, 2020; 21(19): 7268).

[0153] EV's clinical trials have shown that, in some embodiments, targeting connexin-4 can be all or part of a successful treatment strategy for cancer treatment, depending on the context (e.g., cancer type). For example, EV's approval in the United States followed the results of the PhI and II clinical trials. In the PhI study, patients previously treated with ICI therapy had an overall tumor objective response rate (ORR) of 42%, and patients with particularly high tumor burden (e.g., liver metastases) had an ORR of 36%. In addition, in the EV PhII trial, patients with locally advanced or metastatic bladder cancer who had previously received platinum-containing chemotherapy or ICI therapy received EV treatment, and all tumors were positive for connexin-4 and were characterized as having "strong" expression levels. PD-L1 expression was also evaluated, but the results of EV therapy showed that at 10.2 months (median follow-up time), the ORR was 44% and the complete response rate (CRR) was 12%; PD-L1 status had no effect on ORR or CRR. The studies suggest that connexin-4 is a relevant target for bladder cancer, that PD-L1 status or therapy does not negatively impact the efficacy of connexin-4-based therapy, and that it provides a targeted alternative or supplement to ICI-based therapy.

[0154] EV is currently being explored in a Phase III study and is being developed for a Phase II combination study with ICI therapy in patients who are not eligible for cisplatin (Bednova O. & Leyton JV. Int J Mol Sci. 2020 Oct 1;21(19):7268).

[0155] In some embodiments, connexin-4 is an important target, alone or in combination with one or more therapies, for use with the miniproteins of the present disclosure.

[0156] In some embodiments, the compositions provided by the present disclosure more specifically and effectively target cells that overexpress Connexin-4 while minimizing or eliminating damage to surrounding cells that do not express or overexpress Connexin-4 by providing a targeting composition that, in some embodiments, includes a chelator and / or an alpha-emitter that, when combined with a small protein as provided herein, provides a specific, efficient, and effective approach to targeting cells that overexpress Connexin-4.

[0157] Importantly, the novel compositions provided by the present disclosure are able to specifically, efficiently and effectively target cells that overexpress connexin-4, and have reduced toxicity compared to currently available treatments. That is, in some embodiments, the compositions targeting connexin-4 provided by the present disclosure provide improved treatments compared to currently available treatments.

[0158] In some embodiments, the target of the composition of the present disclosure comprises or consists of connexin-4. In some embodiments, connexin-4 is expressed on the cell surface. In some embodiments, the cell is a cancer cell. In some such embodiments, the cancer cell is a tumor cell and the tumor is a solid tumor. In some embodiments, the level of connexin-4 expressed in a tumor cell or a tumor cell group is higher than the level expressed in a non-tumor cell. In some embodiments, the composition or one or more components thereof (e.g., chelating agents and / or radionuclides) are specifically targeted to cancer cells or tumor microenvironments (e.g., a location containing a cancer cell group or a cell at risk of becoming a cancer cell) by targeting connexin-4 with a small protein provided by the present disclosure or a composition comprising a small protein.

[0159] In some embodiments, a miniprotein according to the present disclosure specifically binds to Connexin-4. Without limitation, exemplary Connexin-4 miniproteins are provided in Table 2A, and exemplary Connexin-4 sequences are shown in Table 1A.

[0160] In some embodiments, the small protein disclosed herein comprises or consists of a polypeptide sequence corresponding to a polypeptide sequence shown in Table 1B, 1C and / or 2A, or a polypeptide sequence that binds to a polypeptide shown in Table 1A. In some embodiments, the small protein comprises or consists of an amino acid sequence having a sequence at least 85% identical to a polypeptide sequence shown in one or more tables. In some embodiments, the small protein disclosed herein has 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 higher identity with a polypeptide sequence according to Table 2A, 1B and / or 1C.

[0161] In some embodiments, a composition according to the present disclosure specifically binds to Connexin-4 (eg, via a small protein that specifically binds to Connexin-4).

[0162] In some embodiments, the composition comprising a small protein comprises or consists of a protein comprising a specific amino acid sequence that binds to connexin-4 or a portion thereof. In some such embodiments, such a connexin-4 small protein comprises or consists of an amino acid sequence selected from any one of SEQ ID NOs: 1-158 or 177 or a functional variant or portion thereof (e.g., a functional fragment, such as a small protein that folds and binds to connexin-4 or a portion thereof). In some embodiments, such a connexin-4 small protein is a binding protein as shown in Table 2A or a part of a conjugate comprising such a binding protein.

[0163] In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence that is 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 identical to any one of SEQ ID NO: 1-158 or 177 or a functional variant or portion thereof.

[0164] In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO: 1. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO: 2. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO: 3. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO: 4. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO: 5. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO: 6. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO: 7. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO: 8. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO: 9. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO: 10. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO: 11. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO: 12. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO: 13. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO: 14. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO: 15. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO: 16. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO: 17. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO: 18. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO: 19. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO: 20. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO: 21. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO: 22. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO: 23.In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO:24. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO:25. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO:26. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO:27. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO:28. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO:29. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO:30. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO:31. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO:32. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO:33. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO:34. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO:35. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO:36. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO:37. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO:38. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO:39. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO:40. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO:41. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO:42. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO:43. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO: 44. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO: 45.In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO:46. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO:47. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO:48. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO:49. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO:50. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO:51. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO:52. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO:53. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO:54. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO:55. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO:56. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO:57. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO:58. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO:59. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO:60. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO:61. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO:62. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO:63. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO:64. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO:65. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO: 66. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO: 67.In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO: 68. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO: 69. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO: 70. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO: 71. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO: 72. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO: 73. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO: 74. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO: 75. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO: 76. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO: 77. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO:78. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO:79. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO:80. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO:81. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO:82. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO:83. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO:84. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO:85. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO:86. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO:87. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO: 88. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO: 89.In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO: 90. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO: 91. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO: 92. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO: 93. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO: 94. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO: 95. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO: 96. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO: 97. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO: 98. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO: 99. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO: 100. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO: 101. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO: 102. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO: 103. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO: 104. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO: 105. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO: 106. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO: 107. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO: 108. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO: 109. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO: 110. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO: 111.In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO: 112. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO: 113. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO: 114. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO: 115. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO: 116. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO: 117. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO: 118. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO: 119. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO: 120. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO: 121. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO: 122. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO: 123. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO: 124. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO: 125. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO: 126. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO: 127. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO: 128. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO: 129. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO: 130. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO: 131. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO: 132. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO: 133.In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO: 134. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO: 135. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO: 136. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO: 137. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO: 138. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO: 139. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO: 140. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO: 141. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO: 142. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO: 143. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO: 144. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO: 145. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO: 146. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO: 147. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO: 148. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO: 149. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO: 150. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO: 151. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO: 152. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO: 153. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO: 154. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO: 155.In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO: 156. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO: 157. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO: 158. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence according to SEQ ID NO: 177.

[0165] In some embodiments, the miniproteins of the present disclosure bind to a connexin-4 protein as shown in SEQ ID NO: 159 and / or SEQ ID NO: 160. In some embodiments, the miniproteins of the present disclosure bind to a portion of connexin-4 as shown in SEQ ID NO: 159 and / or SEQ ID NO: 160.

[0166] In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence that is 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 identical to any one of SEQ ID NOs: 161-176 or a functional variant or portion thereof. In some embodiments, the miniproteins of SEQ ID NOs: 161-176 have one or more substitutions as shown in Table 1C. In some embodiments, the connexin-4 miniprotein comprises or consists of an amino acid sequence that is 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 identical to any one of SEQ ID NOs: 161-176, or a functional variant or portion thereof having one or more substitutions shown in Table 1C.

[0167] In some embodiments, the disclosure provides polynucleotides encoding polypeptides, the polypeptides comprising or consisting of: one or more parts of a composition as provided herein. In some embodiments, the disclosure provides vectors and / or host cells comprising sequences encoding one or more components of a composition as provided herein. In some embodiments, the disclosure provides methods for detecting targets. In some embodiments, the methods provided herein include detecting the presence of, for example, a target for, for example, imaging, for example, diagnosis, prognosis, and / or monitoring purposes, for example, treatment. In some embodiments, the disclosure provides methods for treating and / or manufacturing using a composition as provided herein (e.g., a small protein, such as a linker-chelator, for example, a small protein comprising one or more of a linker, a chelator, and a radionuclide, etc.). In some embodiments, the treatment method includes administering a composition provided herein to a subject in need. Small protein

[0168] Provided herein are novel polypeptides (i.e., small proteins) and methods of use thereof. In some embodiments, the polypeptide comprises or consists of a small protein. In some such embodiments, the small protein comprises or consists of: a CDP, a knotting agent, and / or a conjugate. In some embodiments, the small protein is designed to connect one or more other components. For example, in some embodiments, the small protein can be connected (conjugated) to another component, such as a chelating agent and / or a radionuclide. In some embodiments, conjugation is through lysine or cysteine ​​residues. For example, in some embodiments, the small protein is engineered to remove all lysine residues except one lysine residue, and in some embodiments, the residue is used for conjugation. In some embodiments, conjugation occurs through an optional linker. In some embodiments, conjugation between the small protein and the chelating agent and / or the radionuclide is direct. Without wishing to be bound by any particular theory, the present disclosure contemplates therapeutic agents comprising compositions provided by the present disclosure, characterized by several features relative to other (e.g., antibody-based) therapeutic agents. For example, in some embodiments, small proteins exhibit several key features of antibody-based therapeutic agents (e.g., affinity, potency, specificity, and the ability to disrupt protein: protein interactions), but also have several advantages over antibody-based therapeutic agents, such as smaller size, cheaper manufacture, and elimination of the need for chimerization or humanization of proteins. In addition, the size and specificity of the combination increases tumor penetration and uptake into cells of a target expressing a small protein or a composition (e.g., conjugate) comprising the small protein.

[0169] In some embodiments, the length of the small protein of the present disclosure does not exceed about 100 amino acids. In some embodiments, the length of the small protein is about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or more amino acids. However, in some such embodiments, the length of the small protein of the present disclosure does not exceed about 100 amino acids. In some embodiments, the length of the small protein is about 20 to about 40, about 30 to about 50, about 40 to about 60, about 45 to about 65, about 50 to about 70, about 55 to about 75, about 65 to about 85 or more amino acids, but the length does not exceed about 100 amino acids. In some preferred embodiments, the small protein is about 65 amino acids or less. In some preferred embodiments, the small protein is about 50 amino acids or less.

[0170] In some embodiments, the small protein of the present disclosure is no more than about 12kDa. In some embodiments, the small protein of the present disclosure is about 2kDa, 2.5kDa, 3kDa, 3.5kDa, 4kDa, 4.5kDa, 5kDa, 5.5kDa, 6kDa, 6.5kDa, 7kDa, 7.5kDa, 8kDa, 8.5kDa, 9kDa, 9.5kDa, 10kDa, 10.5kDa, 11kDa, 11.5kDa or more. However, in some such embodiments, the small protein of the present disclosure is no more than about 12kDa.

[0171] In some embodiments, the small proteins of the present disclosure comprise or consist of a cysteine-dense peptide, a knottin, a binder, an affibody, an engineered Kunitz domain, a monoclonal antibody, an ankyrin, a designed ankyrin repeat domain (DARPin), and / or an avimer.

[0172] In some embodiments, the small protein comprises one or more disulfide bridges. In some embodiments, the small protein comprises at least two disulfide bridges. In some embodiments, the small protein comprises at least three cysteine ​​residues. In some embodiments, the small protein comprises multiple (e.g., more than three) cysteine ​​residues. In some such embodiments, the cysteine ​​residues are cross-linked to maintain a very stable folded state of a peptide of its length (e.g., relative to a peptide of the same length without so many cysteine ​​residues). The present disclosure contemplates that such cross-linking imparts improved stability while reducing (i.e., extremely low to no) immunogenicity and / or maintaining or improving the ability to maintain biological activity in harsh but effective chelating conditions (e.g., high temperature and low pH).

[0173] In some embodiments, a small protein or a composition comprising a small protein (eg, a radionuclide conjugate) has low immunogenicity relative to a larger protein or a composition comprising or consisting of a larger protein (eg, an antibody).

[0174] In some embodiments, small proteins (e.g., CDPs, knottins, conjugates, affibodies, engineered Kunitz domains, monoclonal antibodies, anticalins, designed ankyrin repeat domains (DARPins), avimers) have superior penetration efficiency relative to larger proteins. That is, in some embodiments, small proteins or compositions comprising small proteins can penetrate solid tumors better than larger proteins or compositions comprising proteins larger than small proteins. For example, in some such embodiments, small proteins or compositions comprising small proteins have a hydrodynamic radius of about 1 nm to about 25 nm. In some embodiments, the hydrodynamic radius is in the range of about 1-25 nm, 10-20 nm, 5-15 nm, 1-5 nm, 2-4 nm, or 1-3 nm. In some embodiments, the hydrodynamic radius is measured using light scattering methods known to those skilled in the art.

[0175] In some embodiments, the small protein of the present disclosure is characterized in that it has one or more properties relative to a protein greater than 100 amino acids (such as an antibody, antibody fragment, VHH domain, single-chain antibody or other protein or conjugate greater than 12 kDa). In some embodiments, the property is selected from increased protein expression, increased thermal activity, increased thermal stability, 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 chemical selectivity, improved solvent stability, increased tolerance to acidic pH, increased tolerance to proteolytic activity (i.e., reduced sensitivity 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.

[0176] In some embodiments, the present disclosure provides a small protein or a composition comprising a small protein (e.g., a conjugate, e.g., a radionuclide conjugate) that exhibits binding affinity to connexin-4. In some embodiments, connexin-4 is human connexin-4. In some embodiments, human connexin-4 is located on a cell. In some embodiments, the cell is a cell line, a primary cell, or a cell in the human body (e.g., in a tumor).

[0177] In some embodiments, the small protein or a composition comprising the small protein (e.g., a conjugate, e.g., a radionuclide conjugate) exhibits nM or sub-nM binding affinity to connexin-4. 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 a subject (e.g., an in vitro assay using a biological sample (e.g., blood or cell biopsy) from a subject).

[0178] In some embodiments, the small protein or its conjugate exhibits a binding affinity for connexin-4. In some embodiments, the small protein or its conjugate has a binding affinity to human connexin-4 of about 500 nM. In some embodiments, the small protein comprises a picomolar binding affinity. In some embodiments, the miniprotein or conjugate thereof comprises a binding affinity characterized by a dissociation constant ranging from about 900 nM to about 1 nM, e.g., 900 nM, 800 nM, 700 nM, 600 nM, 500 nM, 400 nM, 300 nM, 200 nM, 100 nM, 90 nM, 80 nM, 70 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, .9 nM, .8 nM, .7 nM, .6 nM, .5 nM, .4 nM or less for human connexin-4. In some embodiments, the binding is selective for human connexin-4, but not, e.g., non-human connexin-4.

[0179] In some embodiments, the disclosure provides a small protein or a conjugate thereof having a high affinity for connexin-4. In some such embodiments, connexin-4 is human connexin-4. In some embodiments, the disclosure provides a small protein that is stable, including in the presence of one or more additional molecules (e.g., a cytotoxic molecule, such as radiation).

[0180] In some embodiments, the binding ability of the small protein or its conjugate to the target is improved by one or more modifications. For example, in some embodiments, chemical cross-linking is used to improve the binding ability of the small protein or its conjugate provided herein to connexin-4. In some embodiments, binding can be enhanced by using one or more of lysine residues, fusion proteins, non-natural amino acids or other chemical moieties to enhance binding activity and / or functional activity.

[0181] In some embodiments, to ensure proper folding and ligation, selected cysteine ​​pairs can be replaced with selenocysteine. Consider the following: In some embodiments, diselenide crosslinks are easier to form than disulfide crosslinks because of their lower redox potentials, and such substitutions can cross-couple the remaining cysteine ​​residues.

[0182] In some embodiments, the small protein or its conjugate provided by the present disclosure comprises or consists of the following: monomers constituting dimers, polymers or multimers. In some such embodiments, the monomers all bind to the same target. For example, in some embodiments, wherein there is more than one small protein, the length of each small protein is no more than about 30-40 amino acids or the total size is about 8kDa (two small proteins). In some embodiments, each monomer binds to a different target. In some embodiments, some monomers bind to one target, while other monomers bind to one or more additional targets.

[0183] In some embodiments, the miniproteins disclosed herein comprise or consist of an antigen for use in generating an antibody that specifically binds to at least one epitope on connexin-4. In some embodiments, such antibodies can be used, for example, for diagnostic purposes, blocking (e.g., antagonism), etc.

[0184] In some embodiments, the small protein comprises one or more disulfide bridges. In some embodiments, the small protein comprises at least two disulfide bridges.

[0185] In some embodiments, the small proteins provided herein or their conjugates do not comprise one or more cysteine ​​residues. In some embodiments, the small proteins do not comprise one or more disulfide bridges.

[0186] In some embodiments, the miniproteins provided herein or their conjugates are specific for a target. In some embodiments, the miniproteins are specific for connexin-4 or a fragment thereof.

[0187] In some embodiments, the miniproteins provided herein or their conjugates comprise or consist of a specific amino acid sequence.

[0188] In some embodiments, a small protein or a composition comprising a small protein (e.g., a radionuclide conjugate) is conjugated to a chelator that optionally binds a radionuclide (e.g., actinium). In some embodiments, the conjugation is through a linker. In some embodiments, the conjugation is direct conjugation. In some embodiments, such radionuclide conjugates combine and act synergistically to provide target specificity (e.g., via a small protein) and superior treatment (e.g., via directed radioisotope delivery to cells expressing the target).

[0189] As used herein and known to those skilled in the art, twenty kinds of amino acids and their abbreviations follow conventional usage.See Immunology-A Synthesis [Immunology-A Comprehensive] (Golub and Gren edited, Sinauer Associates, Sunderland, Massachusetts, 2nd edition 1991), which is incorporated herein by reference. In certain embodiments, the amino acid disclosed herein can be a stereoisomer (e.g., D-amino acid) of twenty kinds of conventional amino acids. In certain embodiments, the amino acid in the polypeptide disclosed herein can be a non-natural amino acid. For example, amino acids such as α-, α-disubstituted amino acids, N-alkyl amino acids and other unconventional amino acids can also be suitable components of the polypeptide disclosed herein. Examples of unconventional amino acids include: 4-hydroxyproline, γ-carboxyglutamate, ε-N,N,N-trimethyllysine, ε-N-acetylysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, N-methylarginine, and other similar amino acids and imino acids (e.g., 4-hydroxyproline). According to standard usage and convention, the arrangement of polypeptide sequence symbols used herein has a left-hand end corresponding to the amino terminus and a right-hand end corresponding to the carboxyl terminus.

[0190] In some embodiments, a miniprotein as provided herein is specific for a polypeptide or a portion thereof having an amino acid sequence as shown in Table 1A or a portion thereof or a functional variant thereof. Table 1A: Exemplary target protein amino acid sequences

[0191] In some embodiments, the miniprotein comprises or consists of a specific amino acid sequence. In some embodiments, the miniprotein 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 one of SEQ ID NOs: 1-158 or 177.

[0192] In some embodiments, the miniprotein comprises or consists of a specific amino acid sequence. In some embodiments, the miniprotein 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 one of SEQ ID NOs: 161-176.

[0193] In some embodiments, the miniprotein comprises or consists of a specific amino acid sequence. In some embodiments, the miniprotein 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 shown in any one of Tables 1B, 1C and / or 2A.

[0194] As used herein and known to those skilled in the art, twenty kinds of amino acids and their abbreviations follow conventional usage.See Immunology-A Synthesis [immunology-A comprehensive] (Golub and Gren edited, Sinoer United Company (Sinauer Associates), Sunderland (Sunderland), Massachusetts, the 2nd edition 1991), which is incorporated herein by reference. In certain embodiments, the amino acid disclosed herein can be a stereoisomer (e.g., D-amino acid) of twenty kinds of conventional amino acids. In certain embodiments, the amino acid in the polypeptide disclosed herein can be a non-natural amino acid. For example, amino acids such as α-, α-disubstituted amino acids, N-alkyl amino acids and other unconventional amino acids can also be suitable components of the polypeptide disclosed herein. Examples of unconventional amino acids include: 4-hydroxyproline, γ-carboxyglutamate, ε-N,N,N-trimethyllysine, ε-N-acetylysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, N-methylarginine, and other similar amino acids and imino acids (e.g., 4-hydroxyproline). According to standard usage and convention, the arrangement of polypeptide sequence symbols used herein has a left-hand end corresponding to the amino terminus and a right-hand end corresponding to the carboxyl terminus.

[0195] In some embodiments, the present disclosure provides miniproteins set forth in one or more consensus sequences provided in Table 1 B. In some embodiments, miniproteins having sequences set forth in Table 1 B have amino acid substitutions provided in Table 1C. Table 1B: Consensus sequences of exemplary connexin-4 miniproteins Table 1C: Consensus sequence substitutions

[0196] In some embodiments, the disclosure provides small proteins. In some embodiments, the small protein is or comprises a polypeptide, the polypeptide comprising: an amino acid sequence, wherein the amino acid sequence comprises formula I: CX1YDX2X3FFTALX4X5LRGX6DICX7YIX8X9X10FX11X12X13X14X15X16CIX17EILX18X19LGCX20(SEQ ID NO:161) wherein X20 is an optional -OH-containing amino acid or a carboxyl terminus, and wherein X1 is D or E; X2 is E or G; X3 is Q or E; X4 is A, K or S; X5 is A, R, Q, K, S or Cit; X6 is A, D, G or S; X7 is D, Q, E, L, S or Y; X8 is Q, L or S; X9 is A, Q, E, K; X10 is A, Q, K, S, Y, OH-norleucine or norleucine; X11 is A, N, Q or S; X12 is N, T or Y; X13 is L, Y or V; X14 is P or E; X15 is A, D, Q, G or K; X16 is D, Q, E, I or L; X17 is Q or E; X18 is D, Q or E; X19 is N or Q; and X20, when present as an amino acid, is S.

[0197] In some embodiments, the miniprotein comprises or consists of: a polypeptide according to the amino acid sequence of SEQ ID NO: 161, wherein X1 is E; X2 is E; X3 is E; X4 is K; X5 is R; X6 is G; X7 is Y; X8 is Q; X9 is A; X10 is S; X11 is Q; X12 is Y; X13 is L; X14 is P; X15 is G; X16 is L; X17 is E; X18 is D; X19 is N; and X20 is S.

[0198] In some embodiments, the disclosure provides small proteins. In some embodiments, the small protein is or comprises a polypeptide, the polypeptide comprising: an amino acid sequence, wherein the amino acid sequence comprises Formula IIA: CEYDEX1FFTALX2X3LRGX4DICX5YIQX6X7FX8YLPX9LCIEEILDNLGCS(SEQ ID NO:162) wherein X1 is A, Q or E; X2 is A, K or S; X3 is A, R, Q, K or S; X4 is A, D, G or S; X5 is D, Q, E, L, S or Y; X6 is A, Q, E or K; X7 is A, Q, K, S, Y, OH-norleucine or norleucine; X8 is A, N, Q or S; and X9 is A, D, Q, G or K.

[0199] In some embodiments, the disclosure provides small proteins. In some embodiments, the small protein is or comprises a polypeptide, the polypeptide comprising: an amino acid sequence, wherein the amino acid sequence comprises formula II-B: CX1YDEX2FFTALX3X4LRGX5DICX6YIQX7X8FX9YLPX10LCIEEILDNLGCS(SEQ ID NO:163) wherein X1 is D or E; X2 is A, Q or E; X3 is A, K or S; X4 is A, R, Q, K or S; X5 is A, D, G or S; X6 is D, Q, E, L, S or Y; X7 is A, Q, E or K; X8 is A, Q, K, S, Y, OH-norleucine or norleucine; X9 is A, N, Q or S; and X10 is A, D, Q, G or K.

[0200] In some embodiments, the disclosure provides small proteins. In some embodiments, the small protein is or comprises a polypeptide, the polypeptide comprising: an amino acid sequence, wherein the amino acid sequence comprises formula II-C: CX1YDEEFFTALX2X3LRGX4DICX5YIQX6X7FQYLPGLCIEEILDNLGCS(SEQ ID NO:164) wherein X1 is D or E; X2 is A, K or S; X3 is A, R, Q, K or S; X4 is A, D, G or S; X5 is D, Q, E, L, S or Y; X6 is A, Q, E or K; and X7 is A, Q, K, S, Y, norleucine or OH-norleucine.

[0201] In some embodiments, the disclosure provides small proteins. In some embodiments, the small protein is or comprises a polypeptide, the polypeptide comprising: an amino acid sequence, wherein the amino acid sequence comprises formula II-D: CX1YDEX2FFTALX3RLRGX4DICX5YIQAX6FQYLPX7LCIEEILDNLGCS(SEQ ID NO:165) wherein X1 is D or E; X2 is A, Q or E; X3 is A, K or S; X4 is A, D, G or S; X5 is D, Q, E, L, S or Y; X6 is A, Q, K, S, Y, OH-norleucine or norleucine; and X7 is A, N, Q or S.

[0202] In some embodiments, the disclosure provides small proteins. In some embodiments, the small protein is or comprises a polypeptide, the polypeptide comprising: an amino acid sequence, wherein the amino acid sequence comprises formula II-E: CEYDEX1FFTALX2X3LRGGDICX4YIQX5X6FX7YLPX8LCIEEILDNLGCS(SEQ ID NO:166) wherein X1 is A, Q or E; X2 is A, L or S; X3 is A, R, Q, K or S; X4 is D, Q, E, L, S or Y; X5 is A, Q, E or K; X6 is A, Q, K, S, Y, OH-norleucine or norleucine; X7 is A, N, Q, S; and X8 is A, D, Q, G or K.

[0203] In some embodiments, the disclosure provides a small protein. In some embodiments, the small protein is or comprises a polypeptide, the polypeptide comprising: an amino acid sequence, wherein the amino acid sequence comprises formula II-F: CX1YDEX2FFTALX3X4LRGX5DICX6YIQX7X8FX9YLPX10LCIEEILDNLGCS(SEQ ID NO:167) wherein X1 is D or E; X2 is A, Q or E; X3 is A, K or S; X4 is A, R, Q, K or S; X5 is A, D, G or S; X6 is D, Q, E, L, S or Y; X7 is A, Q, E or K; X8 is A, Q, K, S, Y, OH-norleucine or norleucine; X9 is A, N, Q or S; and X10 is A, D, Q, G or K.

[0204] In some embodiments, the disclosure provides small proteins. In some embodiments, the small protein is or comprises a polypeptide, the polypeptide comprising: an amino acid sequence, wherein the amino acid sequence comprises formula II-G: CX1YDEX2FFTALX3RLRGX4DICX5YIQAX6FQYLPGLCIEEILDNLGCS(SEQ ID NO:168) wherein X1 is D or E; X2 is A, Q or E; X3 is A, K or S; X4 is A, D, G or S; X5 is D, Q, E, L, S or Y; and X6 is A, Q, K, S, Y, OH-norleucine or norleucine.

[0205] In some embodiments, the disclosure provides small proteins. In some embodiments, the small protein is or comprises a polypeptide, the polypeptide comprising: an amino acid sequence, wherein the amino acid sequence comprises formula II-H: CX1YDEX2FFTALX3X4LRGX5DICX6YIQAX7FQYLPX8LCIEEILDNLGCS(SEQ ID NO:169) wherein X1 is D or E; X2 is A, Q or E; X3 is A, K or S; X4 is A, R, Q, K or S; X5 is A, D, G or S; X6 is D, Q, E, L, S or Y; and X7 is A, Q, K, S, Y, OH-norleucine or norleucine; and X8 is A, D, Q, G or K.

[0206] In some embodiments, the disclosure provides small proteins. In some embodiments, the small protein is or comprises a polypeptide, the polypeptide comprising: an amino acid sequence, wherein the amino acid sequence comprises formula II-I: CX1YDEX2FFTALX3X4LRGX5DICX6YIQAX7FQYLPX8LCIEEILDNLGCS(SEQ ID NO:170) wherein X1 is D or E; X2 is A, Q or E; X3 is A, K or S; X4 is A, R, Q, K or S; X5 is A, D, G or S; X6 is D, Q, E, L, S or Y; X7 is A, Q, K, S, Y, OH-norleucine or norleucine; and X8 is A, D, Q, G or K.

[0207] In some embodiments, the disclosure provides small proteins. In some embodiments, the small protein is or comprises a polypeptide, the polypeptide comprising: an amino acid sequence, wherein the amino acid sequence comprises formula II-J: CX1YDEX2FFTALX3X4LRGX5DICX6YIQX7X8FX9YLPX10LCIEEILDNLGCS(SEQ ID NO:171) wherein X1 is D or E; X2 is A, Q or E; X3 is A, K or S; X4 is A, R, Q, K or S; X5 is A, D, G or S; X6 is D, Q, E, L, S or Y; X7 is A, Q, E or K; X8 is A, Q, K, S, Y, OH-norleucine, norleucine; X9 is A, N, Q or S; and X10 is A, D, Q, G or K.

[0208] In some embodiments, the disclosure provides a small protein. In some embodiments, the small protein is or comprises a polypeptide, the polypeptide comprising: an amino acid sequence, wherein the amino acid sequence comprises formula II-K: CX1YDEX2FFTALX3X4LRGX5DICX6YIQX7X8FX9YLPGLCIEEILDNLGCS(SEQ ID NO:172) wherein X1 is D or E; X2 is A, Q or E; X3 is A, K or S; X4 is A, R, Q, K or S; X5 is A, D, G or S; X6 is D, Q, E, L, S or Y; X7 is A, Q, E or K; X8 is A, Q, K, S, Y, OH-norleucine or norleucine; and X9 is A, N, Q or S.

[0209] In some embodiments, the disclosure provides small proteins. In some embodiments, the small protein is or comprises a polypeptide comprising: an amino acid sequence, wherein the amino acid sequence comprises formula II-L: CX1YDEX2FFTALX3RLRGX4DICX5YIQAX6FX7YLPX8LCIEEILDNLGCS(SEQ ID NO:173) wherein X1 is D or E; X2 is A, Q or E; X3 is A, K or S; X4 is A, D, G or S; X5 is D, Q, E, L, S or Y; X6 is A, Q, K, S, Y, OH-norleucine or norleucine; X7 is A, N, Q or S; and X8 is A, D, Q, G or K.

[0210] In some embodiments, the disclosure provides small proteins. In some embodiments, the small protein is or comprises a polypeptide, the polypeptide comprising: an amino acid sequence, wherein the amino acid sequence comprises formula II-M: CX1YDEX2FFTALX3X4LRGX5DICX6YIQX7X8FX9YLPX10LCIEEILDNLGCS(SEQ ID NO:174) wherein X1 is D or E; X2 is A, Q or E; X3 is A, K or S; X4 is A, R, Q, K or S; X5 is A, D, G or S; X6 is D, Q, E, L, S or Y; X7 is A, Q, E or K; X8 is A, Q, K, S, Y, OH-norleucine or norleucine; X9 is A, N, Q or S; and X10 is A, D, Q, G or K.

[0211] In some embodiments, the disclosure provides small proteins. In some embodiments, the small protein is or comprises a polypeptide, the polypeptide comprising: an amino acid sequence, wherein the amino acid sequence comprises formula II-N: CX1YDEX2FFTALX3X4LRGX5DICX6YIQX7X8FX9YLPGLCIEEILDNLGCS(SEQ ID NO:175) wherein X1 is D or E; X2 is A, Q or E; X3 is A, K or S; X4 is A, R, Q, K or S; X5 is A, D, G or S; X6 is D, Q, E, L, S or Y; X7 is A, Q, E or K; X8 is A, Q, K, S, Y, OH-norleucine or norleucine; and X9 is A, N, Q or S.

[0212] In some embodiments, the disclosure provides small proteins. In some embodiments, the small protein is or comprises a polypeptide, the polypeptide comprising: an amino acid sequence, wherein the amino acid sequence comprises formula II-O: CX1YDEX2FFTALX3X4LRGX5DICX6YIQX7X8FX9YLPX10LCIEEILDNLGCS(SEQ ID NO:176) wherein X1 is D or E; X2 is A, Q or E; X3 is A, K or S; X4 is A, R, Q, K or S; X5 is A, D, G or S; X6 is D, Q, E, L, S or Y; X7 is A, Q, E or K; X8 is A, Q, K, S, Y, OH-norleucine or norleucine; X9 is A, N, Q or S; and X10 is A, D, Q, G or K.

[0213] In some embodiments, the polypeptide according to any one of Formula I or Formula IIA-IIO further comprises one or more of a linker, a chelator, and a radionuclide. In some embodiments, the linker may be or comprise a polyethylene glycol (PEG) linker comprising or consisting of PEG4, PEG, PEG2, PEG6, PEG8, PEG12, PEG24, an ester linker, an amide linker, a maleimide linker, a succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) linker, a propionic acid linker, a decenoic acid linker, or (Gly)n-(gGlu)n- or (PEG)n (wherein n is 1 to 10), (Gly)1-10, or any fragment or combination thereof via a covalent bond. In some embodiments, the chelator comprises or consists of DOTA, DOPA, a macrocyclic ligand, and a crown. In some embodiments, the radionuclide is Ac-225, In-111, Ga-68, Pb-212, Lu-177, Cu-67, Cu-64, La-132, La-135, or Ce-134.

[0214] In some embodiments, when present, the linker is attached to the C-terminus of the polypeptide. In some embodiments, when present, the chelator is attached to the polypeptide or the linker. In some embodiments, when present, the radionuclide is attached to the chelator. In some embodiments, the polypeptide comprises one or more additional N-terminal amino acids. In some embodiments, one or more amino acids are located at the N-terminus and / or the C-terminus of the polypeptide.

[0215] In some embodiments, the small protein comprising any one of SEQ ID NOs: 161-176 and / or an amino acid sequence according to any one of Tables 1B, 1C, and / or 2A has at least one disulfide bridge.

[0216] In some embodiments, the small protein has at least two disulfide bridges.

[0217] In some embodiments, disulfide bridges are formed between cysteines 1 and 34 and between cysteines 20 and 44. In some embodiments, a small protein comprising four cysteines has two disulfide bridges, and Cys1 is linked to Cys34, and Cys20 is linked to Cys44.

[0218] In some embodiments, the miniprotein has one or more additional amino acids inserted into the amino acids within the CDP loop region (amino acids in the secondary structure loop between cysteines connected to each other, e.g., amino acids in the structure formed between Cys1 and Cys 34 and Cys 20 and Cys 44). As will be appreciated by those skilled in the art, the retention of conformation is key to binding affinity and behavior; therefore, in some embodiments, any addition within the CDP loop region can be performed in a manner that retains the appropriate conformation and stability, so that binding affinity is not reduced and / or conformation or stability is not impaired or destroyed.

[0219] In some embodiments, the polypeptide is a monomer. In some embodiments, the polypeptide is a dimer, trimer or tetramer. In some embodiments, the polypeptide comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 78. In some embodiments, the polypeptide comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence of any one of SEQ ID NO: 1-158 or 177. In some embodiments, the polypeptide comprises a consensus sequence according to any one of Formulas IIA-IIO (SEQ ID NO: 161-176) of Table 1B, further comprising substitutions according to those shown in Table 1C.

[0220] In some embodiments, the sequences comprising variable positions (e.g., in Table 1B) can also include substitutions with any homologous amino acids. As used herein, a homologous amino acid is an amino acid that has one or more similar characteristics to another amino acid, such as, for example, a similar charge (e.g., a negatively charged amino acid, with reference to the charge at physiological pH), or, in some embodiments, a set of substitutions (e.g., more than one amino acid) that creates a similar charge profile to the polypeptide prior to the substitution or set of substitutions, etc.

[0221] In some embodiments, the small proteins disclosed herein exhibit binding specificity to human connexin-4. For example, in some embodiments, the small proteins provided by the present disclosure, such as those represented by any one of SEQ ID NOs: 1-158 or 177 or 161-176, exhibit binding when expressed on the surface of yeast and are tested for binding to connexin-4 by flow cytometry. In some embodiments, the small proteins provided by the present disclosure, such as those represented by any one of SEQ ID NOs: 1-158 or 177 or 161-176 or according to Tables 1B, 1C and / or 2A, exhibit binding specificity via flow cytometry when, for example, such connexin-4 small proteins (e.g., small proteins such as those represented by any one of SEQ ID NOs: 1-158, 177, 161-176 or according to any one of Tables 1B, 1C and / or 2A) bind only to connexin-4 without binding to other antigens.

[0222] In some embodiments, the small proteins of the present disclosure, e.g., small proteins represented by SEQ ID NOs 1-158 or 177 or 161-176 or any of the small proteins according to Tables 1B, 1C and / or 2A, exhibit a potency greater than 10 nM. In some embodiments, the small proteins exhibit a potency greater than 1 nM, 2 nM, 3 nM, 4 nM, 5 nM, 6 nM, 7 nM, 8 nM, 9 nM or more.

[0223] In some embodiments, the miniprotein is part of a conjugate comprising one or more modifications or components, e.g., as provided herein (see e.g., Table 2A).

[0224] In some embodiments, miniproteins according to the present disclosure exhibit binding specificity to human connexin-4. In some embodiments, the miniprotein comprises a binding affinity characterized by a dissociation constant ranging from about 500 nM to about 1 pM, e.g., 500 nM, 400 nM, 300 nM, 200 nM, 100 nM, 90 nM, 80 nM, 70 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 100 pM, 90 pM, 80 pM, 70 pM, 60 pM, 50 pM, 40 pM, 30 pM, 20 pM, 10 pM, 9 pM, 8 pM, 7 pM, 6 pM, 5 pM, 4 pM, 3 pM, 2 pM, or 1 pM binding affinity for human connexin-4. Without being bound by theory, the present disclosure contemplates that, in some embodiments, the preferred dissociation constant for small proteins 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 (ie, in the picomolar range).

[0225] In some embodiments, the small proteins of the present disclosure bind to Connexin-4 with a binding affinity of about 1 pM to 100 nM. In some embodiments, a small protein according to the disclosure binds to connexin-4 with a binding affinity of about 1 pM, 2 pM, 3 pM, 4 pM, 5 pM, 6 pM, 7 pM, 8 pM, 9 pM, 10 pM, 15 pM, 20 pM, 25 pM, 30 pM, 35 pM, 40 pM, 45 pM, 50 pM, 55 pM, 60 pM, 65 pM, 70 pM, 75 pM, 80 pM, 85 pM, 90 pM, 95 pM, 100 pM, 150 pM, 200 pM, 250 pM, 300 pM, 350 pM, 400 pM, 450 pM, 500 pM, 550 pM, 600 pM, 650 pM, 700 pM, 750 pM, 800 pM, 850 pM. pM, 900pM, 950pM; 1nM, 2nM, 3nM, 4nM, 5nM, 6nM, 7nM, 8nM, 9nM, 10nM, 15nM, 20 nM, 25nM, 30nM, 35nM, 40nM, 45nM, 50nM, 55nM, 60nM, 65nM, 70nM, 75nM, 80nM, 8 100nM, 150nM, 200nM, 250nM, 300nM, 350nM, 400nM, 450nM, 500nM, 550nM, 600nM, 650nM, 700nM, 750nM, 800nM, 850nM, 900nM, 950 or 1000nM. In some embodiments, the small protein according to the present disclosure binds to connexin-4 with a binding affinity of about 1pM to 100pM, 10pM to 1nM, 100pM to 10nM, or 1nM to 100nM. CDP

[0226] In some embodiments, the small protein disclosed herein comprises or is composed of a cysteine-dense peptide (CDP). In some embodiments, the conjugate provided herein comprises a CDP. In some embodiments, the CDP acts as a targeting moiety, such as specifically binding to a protein target or antigen expressed on the surface of a target tumor cell. In some embodiments, the CDP comprises or is composed of: at least two independent folding domains and a high-density cysteine. In some embodiments, the CDP comprises at least one, two, three, four, five, six or more than six cysteine ​​residues within a range 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. [Natural Structure and Molecular Biology] March 2018; 25 (3): 270-278, exemplary CDPs and their features). In some embodiments, the CDP comprises a restricted cysteine ​​distribution, Cys-X[0-15]-Cys-X[0-15]-Cys-X[0-15]-Cys-X[0-15]-Cys-X[0-15]-Cys (where X represents any amino acid). In some embodiments, the CDP comprises one or more cysteine-dense regions, wherein the cysteine-dense regions comprise at least one cysteine ​​residue, preferably at least two, three, four or more cysteine ​​residues, within a range of about 10 to 80 amino acid residues. In some embodiments, the CDP can be further engineered to modify binding, folding and / or related properties.

[0227] In some embodiments, the CDP specifically binds to a target. In some embodiments, the target is located in a cell, on a cell, or near a cell. In some embodiments, the CDP specifically binds to connexin-4 or a fragment thereof. In some embodiments, the CDP is conjugated to a chelator and / or a radionuclide. In some embodiments, the conjugation is through a linker. It will be appreciated by those skilled in the art that in some embodiments, the specific CDP employed in the conjugates of the present disclosure may vary depending on the target protein or antigen of interest.

[0228] In some embodiments, in small proteins with cysteine ​​residues, selected cysteine ​​pairs may be replaced with selenocysteine ​​to ensure proper folding and connectivity. In some embodiments, diselenide crosslinks may be easier to form than disulfide crosslinks because of their lower redox potentials. In some such embodiments, this replacement may result in cross-coupling of the remaining cysteines. Knotting agent

[0229] In some embodiments, the small protein disclosed herein comprises or consists of a knotting peptide. In some embodiments, the conjugate provided herein comprises a knotting peptide. In some embodiments, the knotting peptide acts as a targeting portion, for example, specifically binding to an antigen expressed on the surface of a target tumor cell. In some embodiments, the knotting comprises at least three disulfide bonds, which are connected in an arrangement to produce a so-called "cysteine ​​knot", from which the knotting is named. (See, for example, Kintzing & Cochran et al., Curr Opin Chem Biol. [New Views in Chemical Biology] October 2016; 34: 143-150). In some embodiments, the knotting has high stability (e.g., heat, proteolysis, chemistry, etc.). In some embodiments, the knotting can be further engineered to modify binding, folding and / or related properties.

[0230] In some embodiments, a given knottin is highly specific for a given target. In some embodiments, the knottin specifically binds to a target. In some embodiments, the target is located within a cell, on a cell, or near a cell. In some embodiments, the knottin specifically binds to connexin-4 or a fragment thereof. In some embodiments, the knottin is conjugated to a chelator and / or a radionuclide. In some embodiments, the conjugation is through a linker. It will be appreciated by those skilled in the art that in some embodiments, the specific knottin employed in the conjugates of the present disclosure may vary depending on the target protein or antigen of interest.

[0231] In some embodiments, the folded structure of small proteins (e.g., CDPs, knottins, binders, affibodies, engineered Kunitz domains, monobodies, anticalins, designed ankyrin repeat domains (DARPins), avimers) renders them rigid, providing very tight and efficient binding to a target protein or antigen (relative to poorly structured peptides). In some such embodiments, small proteins (e.g., CDPs, knottins, binders, affibodies, engineered Kunitz domains, monobodies, anticalins, designed ankyrin repeat domains (DARPins), avimers) exhibit extraordinary stability, with resistance to heat, peptidase cleavage, and pH. Conjugate

[0232] In some embodiments, the small protein of the present disclosure comprises or consists of a conjugate. In some embodiments, the conjugate acts as a targeting moiety, for example, specifically binds to a target expressed on the surface of a tumor cell.

[0233] In some embodiments, the conjugate has certain structural features; for example, in some embodiments, the conjugate may be rich in alpha-helices, such as helix-helix-helix structures (see, for example, 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 conjugate comprises sufficient surface to functionalize molecules on different surfaces as binding surfaces. In some embodiments, the conjugate comprises an isolated hydrophobic core. In some embodiments, the conjugate exhibits cooperative folding. In some embodiments, the conjugate has two or more of the following features: (i) represented by a sequence of 100 amino acids or less; (ii) at least two secondary structural elements; (iii) an isolated hydrophobic core; and / or (iv) cooperative folding.

[0234] In some embodiments, a given conjugate is highly specific for a given target. In some embodiments, the conjugate specifically binds to a target. In some embodiments, the target is located in a cell, on a cell, or near a cell. In some embodiments, the conjugate specifically binds to connexin-4 or a fragment thereof. In some embodiments, the conjugate is conjugated to a chelating agent and / or a radionuclide. In some embodiments, the conjugation is through a linker. It will be appreciated by those skilled in the art that in some embodiments, the specific conjugate employed in the conjugate of the present disclosure may vary depending on the target protein or antigen of interest. Affibody

[0235] In some embodiments, the small protein disclosed herein comprises an affibody or is composed of it. In some embodiments, the conjugate provided herein comprises an affibody. In some embodiments, the affibody acts as a targeting moiety, such as specifically binding to a protein target or antigen expressed on the surface of a target tumor cell. In some embodiments, the affibody comprises or is composed of: no more than 100 amino acids, 90 amino acids, 80 amino acids, 70 amino acids, 60 amino acids, 50 amino acids, 40 amino acids, 30 amino acids, 20 amino acids or 10 amino acids. In some embodiments, the affibody comprises or is composed of: at least three alpha helices with 58 amino acids. In some embodiments, the affibody comprises a target specificity obtained by randomization of 13 amino acids located in two alpha helices involving the binding activity of the parent protein domain (Feldwisch J, Tolmachev V.; (2012) Methods Mol Biol. [Molecular Biology Methods] 899: 103-26). In some embodiments, the affibody can be further engineered to modify binding, folding and / or related properties.

[0236] In some embodiments, the affibody specifically binds to a target. In some embodiments, the target is located in a cell, on a cell, or near a cell. In some embodiments, the affibody specifically binds to connexin-4 or a fragment thereof. In some embodiments, the affibody is conjugated to a chelator and / or a radionuclide. In some embodiments, the conjugation is through a linker. It will be appreciated by those skilled in the art that in some embodiments, the specific affibody employed in the conjugates of the present disclosure may vary depending on the target protein or antigen of interest. Engineered Kunitz domain

[0237] In some embodiments, the small protein disclosed herein comprises or consists of an engineered Kunitz domain. In some embodiments, the conjugate provided herein comprises an engineered Kunitz domain. In some embodiments, the engineered Kunitz domain acts as a targeting moiety, such as specifically binding to a protein target or antigen 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 a Kunitz domain of a Kunitz-type protease inhibitor (e.g., 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.

[0238] 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 connexin-4 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 through a linker. It will be appreciated by those skilled in the art that in some embodiments, the specific engineered Kunitz domain employed in the conjugates of the present disclosure may vary depending on the target protein or antigen of interest. Monoclonal antibody

[0239] In some embodiments, the small protein disclosed herein comprises a single antibody or is composed of it. In some embodiments, the conjugate provided herein comprises a single antibody. In some embodiments, the single antibody acts as a targeting portion, such as specifically binding to a protein target or antigen expressed on the surface of a target tumor cell. In some embodiments, the single antibody comprises or is composed of the following: a molecule based on the 10th extracellular domain of human fibronectin III (10Fn3), the molecule adopts an Ig-like b sandwich fold of about 94 residues, has 2 to 3 exposed loops, but lacks a central disulfide bridge. In some embodiments, the single antibody can be further engineered to modify binding, folding and / or related properties.

[0240] In some embodiments, the monoclonal antibody specifically binds to a target. In some embodiments, the target is located in a cell, on a cell, or near a cell. In some embodiments, the monoclonal antibody specifically binds to connexin-4 or a fragment thereof. In some embodiments, the monoclonal antibody is conjugated to a chelator and / or a radionuclide. In some embodiments, the conjugation is through a linker. It will be appreciated by those skilled in the art that in some embodiments, the specific monoclonal antibody employed in the conjugates of the present disclosure may vary depending on the target protein or antigen of interest. Anticalin

[0241] In some embodiments, the small protein disclosed herein comprises or consists of anticalin. In some embodiments, the conjugates provided herein comprise anticalin. In some embodiments, anticalin acts as a targeting moiety, for example, specifically binding to a protein target or antigen expressed on the surface of a target tumor cell. In some embodiments, anticalin comprises or consists of: an eight-chain β barrel, which forms a highly conserved core unit in lipocalin and naturally forms a binding site for a ligand through four structurally variable loops at the open end. In some embodiments, anticalin can be further engineered to modify binding, folding and / or related properties.

[0242] In some embodiments, the anticalin specifically binds to a target. In some embodiments, the target is located within, on, or near a cell. In some embodiments, the anticalin specifically binds to connexin-4 or a fragment thereof. In some embodiments, the anticalin is conjugated to a chelator and / or a radionuclide. In some embodiments, the conjugation is through a linker. It will be appreciated by those skilled in the art that in some embodiments, the specific anticalin employed in the conjugates of the present disclosure may vary depending on the target protein or antigen of interest. Designed ankyrin repeat domain

[0243] In some embodiments, the small protein of the present disclosure comprises or is composed of the ankyrin repeat domain of design. In some embodiments, the conjugate provided herein comprises the ankyrin repeat domain of design. In some embodiments, the ankyrin repeat domain of design acts as a targeting portion, such as specifically binding to a protein target or antigen expressed on the surface of a target tumor cell. In some embodiments, the ankyrin repeat domain of design comprises a peptide derived from ankyrin. In some embodiments, the ankyrin repeat domain of design comprises a single ankyrin repeat, preferably comprising a 33 residue motif containing two α helices and a β turn. In some embodiments, the ankyrin repeat domain of design provides a rigid interface and lacks structural flexibility. In some embodiments, the ankyrin repeat domain of design can be further engineered to modify binding, folding and / or related properties.

[0244] In some embodiments, the ankyrin repeat domain of design specifically binds to a target. In some embodiments, the target is located in a cell, on a cell, or near a cell. In some embodiments, the ankyrin repeat domain of design specifically binds to connexin-4 or a fragment thereof. In some embodiments, the ankyrin repeat domain of design is conjugated to a chelating agent and / or a radionuclide. In some embodiments, conjugation is through a linker. It will be appreciated by those skilled in the art that in some embodiments, the ankyrin repeat domain of design specifically used in the conjugate of the present disclosure may vary according to the target protein or antigen of interest. Avimer

[0245] In some embodiments, the small protein disclosed herein comprises avimer or is composed thereof. In some embodiments, the conjugate provided herein comprises avimer. In some embodiments, avimer acts as a targeting moiety, such as specifically binding to a protein target or antigen expressed on the surface of a target tumor cell. In some embodiments, avimer comprises a peptide of about 10 amino acids, 20 amino acids, 30 amino acids, 40 amino acids, 50 amino acids, 60 amino acids, 70 amino acids, 80 amino acids, 90 amino acids or 100 amino acids. In some embodiments, avimer comprises at least one peptide sequence of about 30 to 35 amino acids. In some embodiments, avimer comprises two or more of the following: two peptide sequences of about 30 to 35 amino acids. In some embodiments, avimer comprises one or more peptide sequences derived from the A domain of various membrane receptors. (Weidle UH, et al., (2013), Cancer Genomics Proteomics [Cancer Genomics and Proteomics]; 10 (4): 155-68). For more details, see Nature Biotechnology 23(-2), 1556-1561 (2005) and Expert Opinion on Investigational Drugs 16(6), 909-917 (June 2007). In some embodiments, avimers can be further engineered to modify binding, folding and / or related properties.

[0246] In some embodiments, the avimer specifically binds to a target. In some embodiments, the target is located in a cell, on a cell, or near a cell. In some embodiments, the avimer specifically binds to connexin-4 or a fragment thereof. In some embodiments, the avimer is conjugated to a chelator and / or a radionuclide. In some embodiments, the conjugation is through a linker. It will be appreciated by those skilled in the art that in some embodiments, the specific avimer employed in the conjugate of the present disclosure may vary depending on the target protein or antigen of interest. Connectors

[0247] In some embodiments, the disclosure provides a linker for use in one or more conjugates. For example, in some embodiments, the linker is connected to a chelator. In some embodiments, the linker is connected to a chelator, and the chelator itself is coupled to a radionuclide. In some embodiments, a small protein is conjugated to a chelator and / or a radionuclide. In some embodiments, a small protein is optionally conjugated to a chelator via a linker. In some embodiments, the compositions provided herein comprise one or more linkers.

[0248] As described herein, in some embodiments, the small protein conjugate comprises a linker. In some embodiments, the role of the linker is to connect the chelator to the small protein. In some embodiments, the linker is not cleavable. In some embodiments, the linker is cleavable. In some embodiments, selecting and placing one or more linkers and chelators on the small protein helps to maintain the desired efficacy and receptor engagement spectrum, enhance the affinity of the conjugate and optimize the physicochemical and pharmacokinetic properties of the small protein or its conjugate. Any suitable linker known in the art can be used. 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-pyridyl dithio) butyrate (SPDB) linkers, succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) linkers, vinyl sulfone-based linkers, propionic acid linkers, decenoic acid linkers or linkers including any combination thereof. One or more additional linkers as known to those skilled in the art can be considered, and selected according to 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 any one of PEG (2-24).

[0249] In some embodiments, the linker is used to evaluate the lead polypeptide sequence bound to the target, the target expressed on the cell, and the target selectivity and / or affinity. For example, in some embodiments, Biacore can be used to evaluate the target binding in vitro and the confirmation of the affinity of the lead polypeptide sequence and the lead polypeptide sequence-linker-fluorophore reagent. In some embodiments, other linkers, such as fast clear linkers or halogen linkers, can also be considered. Chelating agents

[0250] In some embodiments, the compositions (e.g., conjugates) provided herein include a linker. In some embodiments, the composition includes a linker and a chelator. In some embodiments, the composition includes a linker, a chelator, and a radionuclide. In some embodiments, the composition includes a small protein, an optional linker, a chelator, and / or a radionuclide. In some embodiments, a chelator is covalently attached to a small protein. In some embodiments, a chelator binds a radionuclide. In some embodiments, a chelator refers to any molecule or part that "binds" to a metal ion in a solution (effectively collecting / binding metal ions so that they can, for example, no longer participate in one or more cellular activities or processes). In some embodiments, a chelator chelates one or more components (e.g., metal ions, e.g., copper, iron, zinc, etc.) of a metabolic pathway in a cell. In some such embodiments, a chelator destroys the life cycle of a cancer cell, and in some embodiments, can reduce its vigor, function, and / or ability to grow or proliferate. In some embodiments, a chelator chelates one or more toxins produced by targeted radiotherapy (e.g., to reduce the toxicity of the therapy).

[0251] In some embodiments, the chelating agent comprises or consists of, but is not limited to, tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), diethylenetriaminepentaacetic acid (DTPA), ethylenediaminetetraacetic acid (EDTA), 1,4,7-triazacyclo'on"ne-N,N',N"-triacetic acid (NOTA), ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), 1,4,7-triazacyclo'on"ne-N,N',N"-triacetic acid (NOTA) , ({4-[2-(bis-carboxymethyl-amino)-ethyl]-7-carboxymethyl-[1,4,7]triazolidin-1-yl}acetic acid (NETA), macrocyclic ligands and p-bromoacetamidobenzyl-tetraethylaminetetraacetic acid (TETA), porphyrins, polyamines, crown ethers, bis-thiosemicarbazones, polyoximes. In some embodiments, the chelating agent is 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA). In some embodiments, the chelating agent is a macrocyclic ligand. In some embodiments, the chelating agent comprises or consists of: (i) DOTA: (ii)Crown (iii) NOPO ;or (iv) Macrocyclic ligands

[0252] In some embodiments, the chelating agent comprises or consists of, but is not limited to, diethylenetriaminepentaacetic acid (DTPA), tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), ethylenediaminetetraacetic acid (EDTA), 1,4,7-triazacyclo'on"ne-N,N',N"-triacetic acid (NOTA), ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), 1,4,7-triazacyclo'on"ne-N,N',N"-triacetic acid (NOTA), ({ 4-[2-(bis-carboxymethyl-amino)-ethyl]-7-carboxymethyl-[1,4,7]triazolidine-1-yl}acetic acid (NETA), macrocyclic ligands and p-bromoacetamidobenzyl-tetraethylaminetetraacetic acid (TETA), porphyrins, polyamines, crown ethers, bis-thiosemicarbazones, polyoximes. In some embodiments, the chelating agent is 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA). In some embodiments, the chelating agent is a macrocyclic ligand. In some embodiments, the chelating agent comprises or consists of:

[0253] In further embodiments, the chelation conditions are optimized using methods known to those skilled in the art (see, e.g., J Nucl Med. 1998 Dec; 39(12):2105-10). In some embodiments, the 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%.

[0254] In some embodiments, the chelating agent used in the compositions described herein is selected based on whether a radionuclide is present and which radionuclide is present. As provided herein, in some embodiments, the chelating agent is DOTA, NOPO, a crown, or a macrocyclic ligand. In some embodiments, DOTA is a chelating agent and the radionuclide is Ac-225, In-111, Ga-68, Pb-212, Lu-177, Cu-67, Cu-64, La-132, La-135, or Ce-134. In some embodiments, the crown is a chelating agent and the radionuclide is Ac-225, In-111, Ga-68, Pb-212, Lu-177, Cu-67, Cu-64, La-132, La-135, or Ce-134. In some embodiments, the NOPO is a chelator and the radionuclide is Ac-225, In-111, Ga-68, Pb-212, Lu-177, Cu-67, Cu-64, La-132, La-135, or Ce-134. In some embodiments, the macrocyclic ligand is a chelator and the radionuclide is Ac-225, In-111, Ga-68, Pb-212, Lu-177, Cu-67, Cu-64, La-132, La-135, or Ce-134.

[0255] In some embodiments, a particular chelator or chelator type may be selected for certain applications. In some embodiments, DOTA is used for diagnostic applications, theranostic applications, and / or therapeutic applications. For example, in some embodiments, NOPO is used for diagnostic applications or theranostic applications. In some embodiments, corona is used for therapeutic applications. In some embodiments, macrocyclic ligands are used for diagnostic applications, theranostic applications, and / or therapeutic applications.

[0256] It should be appreciated that screening for certain characteristics of chelators is within the scope of the present disclosure, and methods for such screening are known to those skilled in the art. For example, in some embodiments, chelators are screened for their ability to bind radionuclides (e.g., Ac225 and one or more daughter nuclides of Ac225 (Bi213), In-111, Ga68) and to exhibit serum stability.

[0257] In some embodiments, the small protein conjugates described herein include a chelating agent. Any suitable chelating agent known in the art can be used. In some embodiments, the chelating agent is directly conjugated to the small protein. In some embodiments, the chelating agent is indirectly connected to the small protein through a joint. In some embodiments, the chelating agent is indirectly connected to the small protein through a joint (e.g., a joint described below). Radionuclides

[0258] In some embodiments, the present disclosure provides one or more radionuclides for use in a composition (eg, a conjugate).

[0259] In some embodiments, the small protein conjugate comprises a radionuclide combined with a chelating agent. As will be appreciated by those skilled in the art, any suitable radionuclide known in the art may be used. In some embodiments, the radionuclide is selected for imaging tumors in a human body suffering from 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.

[0260] 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 particles, 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, an alpha emitter has a more localized area of ​​influence, such that when internalized into a cell, it will act, for example, to kill cancer cells, but may spare surrounding tissues from extensive damage, such as that which may occur using beta or gamma emitters.

[0261] Studies have evaluated alpha therapy versus beta therapy, with alpha therapy providing stronger clinical outcomes. In some embodiments, a benefit of alpha therapy is the short path length, meaning that patients do not have to maintain physical distance from family and healthcare providers, making treatment more tolerable. Additionally, in some embodiments, alpha therapy exhibits better cell killing efficacy due to its ability to induce double-stranded DNA breaks.

[0262] In some embodiments, the composition comprises a linker, a chelating agent, and a radionuclide. In some embodiments, the composition comprises a small protein, an optional linker, a chelating agent, and a radionuclide. Without being bound by any particular theory, the present disclosure contemplates that a wide variety of radionuclides can be used in pharmaceutical compositions or as diagnostic agents. Exemplary radionuclides include, but are not limited to, actinium-225, indium-111, astatine-211, bismuth-212, bismuth-213, cesium-137, chromium-51, cobalt-60, copper-64, dysprosium-165, erbium-169, fermium-255, fluorine-18, gallium-67, gallium-68, gold-198, holmium-166, iodine-123, iodine-124, iodine-125, iodine-13 1. Iridium-192, iron-59, 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. Therefore, in some embodiments, the radionuclide is selected from: actinium (225Ac), indium (111In), iodine (131I or 125I), yttrium (90Y), lutetium (177Lu), praseodymium, astatine (211At), rhenium (186Re), bismuth (212Bi or 213Bi), technetium (99Tc), phosphorus (32P), rhodium (188Rh), sulfur (35S), carbon (14C), tritium (3H), chromium (51Cr), chlorine (36Cl), cobalt (57Co or 58Co), iron (59Fe), selenium (75Se) or gallium (67Ga) or (68Ga). In some embodiments, the present disclosure contemplates that certain radioisotopes may be used in therapeutic agents or may be used as therapeutic agents, including but not limited to yttrium (90Y), lutetium (177Lu), actinium (225Ac), praseodymium, astatine (211At), rhenium (186Re), bismuth (212Bi or 213Bi) and rhodium (188Rh). In some embodiments, radioisotopes may be used as labels, for example, for use in diagnosis. In some such embodiments, such radioisotopes may include but are not limited to iodine (131I or 125I), technetium (99Tc), phosphorus (32P), carbon (14C), lead (212Pb) or tritium (3H). See, for example, U.S. Patent No. 7514078.

[0263] In some embodiments, radionuclides are conjugated to different complexing agents and chelators. In some embodiments, chelators are identified and attached / bound to small proteins through linkers or through acyclic, cyclic and macrocyclic chelates such as, for example, 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, corona, etc. In some embodiments, certain chelators may be preferred for certain radionuclides, such as Ac-225 with DOTA or corona, Ga-68 with NOPO, etc. In some embodiments, preferred combinations of chelators and radionuclides include one or more of the following: DOTA and Ac-225, In-111, Ga-68, Pb-212, Lu-177, Cu-67, Cu-64, La-132, La-135, or Ce-134; crown and Ac-225, In-111, Ga-68, Pb-212, Lu-177, Cu-67, Cu-64, La-132, La-135, or Ce-134; La-135 or Ce-134; NOPO and Ac-225, In-111, Ga-68, Pb-212, Lu-177, Cu-67, Cu-64, La-132, La-135 or Ce-134; and / or a macrocyclic ligand and Ac-225, In-111, Ga-68, Pb-212, Lu-177, Cu-67, Cu-64, La-132, La-135 or Ce-134.

[0264] Preferably, in some embodiments, the preferred radionuclide complex comprises Ac-225, In-111, Ga-68, Pb-212, Lu-177, Cu-67, Cu-64, La-132, La-135 or Ce-134. In some such embodiments, a complex with desired stability is selected. That is, in some embodiments, a complex comprising Ac-225, In-111, Ga-68, Pb-212, Lu-177, Cu-67, Cu-64, La-132, La-135 or Ce-134 is characterized as having better in vivo stability compared with other complexes. Without being bound by any particular theory, the present disclosure considers that, in some embodiments, a radionuclide complex comprising a small protein is formed with a small protein target (e.g., connexin-4 or a fragment thereof). In some such embodiments, such a complex is internalized into a target cell.

[0265] In some embodiments, radionuclide complexes are formed with chelators (eg, DOTA, NOPO, corona, macrocyclic ligands, etc.) and are more stable in vivo. In some embodiments, small proteins form internalization complexes with targets (eg, connexin-4).

[0266] In some embodiments, the compositions provided by the present disclosure include actinium-225 (Ac-225). 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 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 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 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 cerium (Ce-134). For example, in some embodiments, radioimmunotherapy comprising Ac-225 can provide i) a limited range of a few cell diameters in tissue; ii) high linear energy transfer, resulting in dense radiation damage along each alpha trajectory; iii) a half-life of 10 days; and / or iv) emission of four net alpha particles per decay (see, e.g., as described in: Scheinberg, David A, and Michael R McDevitt. "Actinium-225 in targeted alpha-particle therapeutic applications." Current radiopharmaceuticals, Vol. 4, 4 (2011): 306-20).

[0267] In some embodiments, targeting constructs (e.g., 225-Ac-drug constructs, e.g., 68-Ga-constructs) have the potential to be used in cancer. For example, in some such embodiments, such constructs can be used to treat cancer, such as 225-Ac-drug constructs. In some embodiments, such constructs can be used for imaging, such as for prognosis, diagnosis and / or monitoring, such as constructs based on Ga-68 or Cu-64.

[0268] In some embodiments, Ac-225 is conjugated to a small protein as provided herein. In some embodiments, actinium is conjugated to a chelator and an optional linker may be included to connect it to a small protein that targets the conjugate to cells expressing the target (e.g., connexin-4).

[0269] In some embodiments, Ga-68 is conjugated to a small protein as provided herein. In some embodiments, gallium is conjugated to a chelator and an optional linker may be included to connect it to a small protein that targets the conjugate to cells expressing the target (e.g., connexin-4).

[0270] In some embodiments, Cu-64 is conjugated to a small protein as provided herein. In some embodiments, copper is conjugated to a chelator and an optional linker may be included to connect it to a small protein that targets the conjugate to cells expressing the target (e.g., connexin-4).

[0271] In some embodiments, In-111 is conjugated to a small protein as provided herein. In some embodiments, indium is conjugated to a chelator and may include an optional linker to connect it to a small protein that targets the conjugate to cells expressing a target (e.g., connexin-4).

[0272] In some embodiments, Lu-177 is conjugated to a small protein as provided herein. In some embodiments, lutetium is conjugated to a chelator and an optional linker may be included to connect it to a small protein that targets the conjugate to cells expressing a target (e.g., connexin-4).

[0273] In some embodiments, Pb-212 is conjugated to a small protein as provided herein. In some embodiments, lead is conjugated to a chelator and may include an optional linker to connect it to a small protein that targets the conjugate to cells expressing the target (e.g., connexin-4).

[0274] In some embodiments, Cu-67 is conjugated to a small protein as provided herein. In some embodiments, copper is conjugated to a chelator and an optional linker may be included to connect it to a small protein that targets the conjugate to cells expressing the target (e.g., connexin-4).

[0275] In some embodiments, La-132 is conjugated to a small protein as provided herein. In some embodiments, lanthanum is conjugated to a chelator and an optional linker may be included to connect it to a small protein that targets the conjugate to cells expressing the target (e.g., connexin-4).

[0276] In some embodiments, La-135 is conjugated to a small protein as provided herein. In some embodiments, lanthanum is conjugated to a chelator and an optional linker may be included to connect it to a small protein that targets the conjugate to cells expressing the target (e.g., connexin-4).

[0277] In some embodiments, Ce-134 is conjugated to a small protein as provided herein. In some embodiments, cerium is conjugated to a chelator and may include an optional linker to connect it to a small protein that targets the conjugate to cells expressing a target (e.g., connexin-4).

[0278] In some embodiments, the alpha particles (e.g., alpha particles of actinium-225, etc.) are positively charged. In some such embodiments, the penetration range in tissue varies between 5 and 10 cell diameters (40 to 100 μm), depending on their energy (Radiobiologic principles in radionuclide therapy. Kassis AI, Adelstein SJ J Nucl Med. 2005 Jan;46 Suppl 10:4S-12S). In some such embodiments, such penetration allows local irradiation of target cells with minimal toxicity to surrounding normal cells and allows internalization by cancer cells with as few as 1-3 tracks across the nucleus, leading to cell death (Humm 1987; Macklis et al. 1988; Humm and Chin 1993; Couturier et al. 2005) causing single-strand and double-strand DNA breaks. 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

[0279] In some embodiments, the dose of radiation therapy is calculated. In some such embodiments, the calculation of absorbed dose (D) is necessary to quantitatively relate tumor response to a particular radiation therapy modality and to predict the potential effects of other radiation therapy modalities or strategies of use. That is, in some embodiments, the absorbed dose from a target site is defined as the energy (E) absorbed by a particular tissue mass, 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 decays (δ) within a particular volume of interest, the energy emitted per decay (ε), and the fraction of the emitted energy (f) absorbed by the particular volume of interest (target mass): E = δ × ε × f. For relatively long-range beta emitters, the dose assessment of the target site includes not only the energy emitted by the local radionuclides within the target volume, but also the energy emitted by the radionuclides accumulated in adjacent organs or regions along their path through the target volume of interest (Kolbert et al. 2003). In other words, in some embodiments, the calculated total absorbed dose is the sum of the dose contributions from all regions containing radionuclides 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 the composition comprises an alpha particle emitter, such cross-organ absorbed dose may not be meaningful due to its short recoil distance. In some embodiments, given the appropriate context, microdose assessment is used to assess the dose or "hits" obtained by cancer cells within a micrometastatic cluster, on the order of microns and at distances comparable to a few cells (Palm et al. 2002).

[0280] In some embodiments, the small protein conjugate comprising a radionuclide exhibits binding specificity for human connexin-4. In some embodiments, the miniprotein comprises a binding affinity characterized by a dissociation constant ranging from about 500 nM to about 1 pM, e.g., 500 nM, 400 nM, 300 nM, 200 nM, 100 nM, 90 nM, 80 nM, 70 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 100 pM, 90 pM, 80 pM, 70 pM, 60 pM, 50 pM, 40 pM, 30 pM, 20 pM, 10 pM, 9 pM, 8 pM, 7 pM, 6 pM, 5 pM, 4 pM, 3 pM, 2 pM, or 1 pM binding affinity for human connexin-4. Without being bound by any particular theory, the present disclosure contemplates that, in some embodiments, the preferred dissociation constant for small proteins 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 (ie, in the picomolar range).

[0281] In some embodiments, a small protein comprising a radionuclide according to the disclosure binds to Connexin-4 with a binding affinity of about 1 pM to 100 nM. In some embodiments, a small protein according to the disclosure binds to connexin-4 with a binding affinity of about 1 pM, 2 pM, 3 pM, 4 pM, 5 pM, 6 pM, 7 pM, 8 pM, 9 pM, 10 pM, 15 pM, 20 pM, 25 pM, 30 pM, 35 pM, 40 pM, 45 pM, 50 pM, 55 pM, 60 pM, 65 pM, 70 pM, 75 pM, 80 pM, 85 pM, 90 pM, 95 pM, 100 pM, 150 pM, 200 pM, 250 pM, 300 pM, 350 pM, 400 pM, 450 pM, 500 pM, 550 pM, 600 pM, 650 pM, 700 pM, 750 pM, 800 pM, 850 pM. pM, 900pM, 950pM; 1nM, 2nM, 3nM, 4nM, 5nM, 6nM, 7nM, 8nM, 9nM, 10nM, 15nM, 20 nM, 25nM, 30nM, 35nM, 40nM, 45nM, 50nM, 55nM, 60nM, 65nM, 70nM, 75nM, 80nM, 8 100nM, 150nM, 200nM, 250nM, 300nM, 350nM, 400nM, 450nM, 500nM, 550nM, 600nM, 650nM, 700nM, 750nM, 800nM, 850nM, 900nM, 950 or 1000nM. In some embodiments, the small protein according to the present disclosure binds to connexin-4 with a binding affinity of about 1pM to 100pM, 10pM to 1nM, 100pM to 10nM, or 1nM to 100nM.

[0282] In some embodiments, the compositions as provided herein are characterized by one or more of absorbed dose, dose rate, tumor penetration spectrum of the radionuclide, intracellular localization spectrum 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).

[0283] As known to those skilled in the art, due to the toxicity of radionuclides, doses need to be carefully controlled and considered. Therefore, in some embodiments, the compositions comprising the radionuclides of the present disclosure address the dose-limiting toxicity of the composition, such that the radionuclides do not accumulate significantly in vital organs (e.g., in a toxicity-limiting manner).

[0284] In some embodiments, alpha particle emitting isotopes participate in targeted cell killing while minimizing toxic effects (eg, to surrounding tissues, eg, as compared to, for example, beta emitters, etc.).

[0285] In some embodiments, the compositions provided herein (including radionuclides) are administered in a single step, e.g., using ligands such as small proteins, which result in improved biodistribution (e.g., specific targeting), a pK with partial and acceptable damage or no damage to normal tissues, and enhanced penetration of the pharmaceutical composition into the heterogeneous interstitial space of a tumor.

[0286] In some embodiments, one or more radionuclides are conjugated to small proteins. Relatedly, in some embodiments, the radiolabeling efficiency of small proteins is optimized to radiolabel the desired number of radionuclides. In some embodiments, the ratio of radionuclides conjugated to small proteins is 1: 1, 2: 1, 3: 1, 4: 1, 5: 1, 6: 1, 7: 1, 8: 1, 9: 1, 10: 1. In some embodiments, there is no toxicity in the radionuclides conjugated to small proteins. In some embodiments, the composition comprising small proteins and radionuclides does not accumulate in the liver, spleen and / or pancreas, and is rapidly cleared when administered to a subject. For example, in some embodiments, after administration to a subject, the biodistribution in the kidney at 24 hours is> 10% of the injected dose (ID), and the biodistribution in the tumor at 24 hours is> 3% ID.

[0287] In some embodiments, after administration to a subject, the t1 / 2 is shorter than the t1 / 2 of, e.g., a connexin-4 antibody, e.g., vitin-enfortumab. Radionuclides and chelation

[0288] Radionuclides can be combined with chelators by any method known in the art. In some embodiments, the chelation method may vary depending on the selected radionuclide and chelator. For example, in some embodiments, chelation can be performed in one step by incubating a small protein-chelator conjugate with a radionuclide at a predetermined temperature for a predetermined time to achieve a sufficient amount of chelation. In some embodiments, the small protein-chelator conjugate comprises a chelator or a variant thereof provided herein (e.g., DOTA, for example, NOPO, for example, a crown, for example, a macrocyclic ligand, etc.). In some embodiments, the small protein-chelator conjugate can be chelated to a radionuclide (e.g., actinium-225, indium-111, gallium-68, copper-64, lutetium-177, lead-212, etc.) by incubating with the radionuclide at 70°C for about 1 hour. In some embodiments, the small protein-chelator conjugate can be chelated to a radionuclide (e.g., actinium-225, indium-111, gallium-68, copper-64, lutetium-177, lead-212, etc.) by incubating with the radionuclide at 70°C for about 1 hour.

[0289] In some embodiments, the chelation process produces a preparation in which at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the small protein-chelator is bound to the radionuclide. In some embodiments, the chelation process produces a preparation in which more than 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the small protein-chelator is bound to the radionuclide. Excess radionuclides can be removed from the preparation by purification methods known in the art. Peptides

[0290] Among other things, the present disclosure provides polypeptides. In some embodiments, the polypeptides are assembled using solid phase synthesis methods. In some embodiments, the polypeptides are recombinant. In some embodiments, the polypeptides comprise or consist of miniproteins. In some such embodiments, miniproteins comprise or consist of conjugates. In some embodiments, the polypeptides of the present disclosure (including mutant proteins, allelic variants, fragments, derivatives and analogs) are encoded by polynucleotides described and provided herein.

[0291] In some embodiments, the miniprotein of the present disclosure comprises or consists of a polypeptide capable of binding to a target shown in Table 1A.

[0292] In some embodiments, the disclosure provides a conjugate comprising or consisting of a fragment of a polypeptide provided herein. In some such embodiments, the fragment comprises at least 20 consecutive amino acids, more preferably at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or more consecutive amino acids.

[0293] In some embodiments, the small protein disclosed herein may also include fusions or conjugates with one or more other components (e.g., heterologous polypeptides). For example, in some embodiments, the heterologous sequence may include or consist of the following: sequences designed to facilitate visualization of purified (e.g., histidine tags) and / or recombinantly expressed proteins. Other non-limiting examples of such fusions or conjugates include those that allow the encoded protein to be displayed on a phage or cell surface, including any detectable or visualized components, such as green fluorescent protein (GFP) and fusions with IgG Fc regions.

[0294] In some embodiments, the miniprotein comprises or consists of a specific amino acid sequence. In some embodiments, the miniprotein 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 shown in any one of SEQ ID NOs: 1-158 or 177 or 161-176 and / or according to Tables 1B and / or 1C. In some embodiments, the miniprotein 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 shown in SEQ ID NO: 78. In some embodiments, the miniprotein comprises or consists of the amino acid sequence shown in SEQ ID NO: 78.

[0295] In some embodiments, the miniprotein 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 shown in any of the amino acid sequences shown in Table 2A.

[0296] As used herein and known to those skilled in the art, the twenty amino acids and their abbreviations follow conventional usage. See Immunology-A Synthesis (Golub and Gren, eds., Sinauer Associates, Sunderland, NY, USA). an d), Massachusetts, 2nd edition 1991), which is incorporated herein by reference. In some embodiments, the amino acids disclosed herein may be stereoisomers of twenty conventional amino acids (e.g., D-amino acids). In some embodiments, the amino acids in the polypeptides disclosed herein may be non-natural 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 components of the polypeptides disclosed herein. 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). According to standard usage and convention, the arrangement of the polypeptide sequence symbols used herein has a left end corresponding to the amino terminus and a right end corresponding to the carboxyl terminus.

[0297] In some embodiments, small proteins of the disclosure comprising two or more cysteine ​​residues, such as those set forth in SEQ ID NOs: 1-158 or 177 or 161-176, have the cysteine ​​residues linked via a disulfide bridge (eg, via native folding).

[0298] In some embodiments, the cysteine ​​linkages are located between Cys1 and Cys34 and between Cys20 and Cys44.

[0299] In some embodiments, the present disclosure provides a small protein comprising or consisting of: an amino acid sequence shown in SEQ ID NO: 1-158 or 177 or 161-176 and / or as shown in Tables 1B, 1C and / or 2A, or a portion or functional variant thereof. In some embodiments, the miniprotein comprises or consists of an amino acid sequence having at least 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 more sequence identity to SEQ ID NO: 1-158 or 177 or 161-176 and / or as shown in Tables 1B, 1C and / or 2A, or a portion or functional variant thereof. In some embodiments, the miniprotein comprises or consists of an amino acid sequence having at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20 or more amino acid residue differences from SEQ ID NO: 1-158 or 177 or 161-176 and / or as shown in Tables 1B, 1C and / or 2A, or a portion or functional variant thereof. In some embodiments, the miniprotein comprising or consisting of selectively binds to the target connexin-4: SEQ ID NO: 1-158 or 177 or 161-176 and / or as shown in Tables 1B, 1C and / or 2A, or a portion or functional variant thereof. Nucleic Acids

[0300] Among other things, the disclosure provides herein polynucleotides and methods of use thereof. In some embodiments, all or part of the polynucleotide encodes a polypeptide (e.g., a small protein) that specifically binds to connexin-4. In some embodiments, connexin-4 is mouse or human connexin-4. In some embodiments, the nucleic acid sequence has a specific sequence. In some embodiments, the polynucleotides of the disclosure are codon optimized (i.e., the nucleic acid sequence is codon optimized).

[0301] In some embodiments, the polynucleotides of the present disclosure comprise or consist of a nucleic acid sequence encoding a polypeptide that is or comprises a miniprotein that specifically binds to connexin-4 or any portion, fragment, or variant thereof.

[0302] In some embodiments, a small protein is represented by a nucleic acid molecule encoding amino acids that, when folded, contain one or more disulfide bridges.

[0303] In some embodiments, for example, nucleic acid molecules (i.e., polynucleotides) may be different from reference sequences as provided herein, but still encode binding substances as provided by the present disclosure. In some such embodiments, for example, provided polynucleotides (i.e., encoding miniproteins or their analogs) hybridize under stringent conditions as disclosed herein.

[0304] In some embodiments, the disclosure provides nucleic acid molecules comprising a fragment of any polynucleotide provided herein. In some embodiments, the polynucleotide fragment comprises or consists of: a portion of a continuous nucleic acid residue. 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.

[0305] In some embodiments, the fragments disclosed herein show utility in various 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 DNA or RNA. The target nucleic acid sequence can be graded (for example, by gel electrophoresis) before hybridization, or in situ hybridization can be performed on the sample. It will be appreciated by those skilled in the art that nucleic acid probes of known sequences can be used to determine chromosome structure (for example, by Southern blotting) and to measure gene expression (for example, by Northern blotting). In such experiments, the sequence fragments are preferably detectably labeled so that their specific hybridization with the target sequence can be detected and optionally quantified. In some embodiments, the fragments can be used as probes, for example, such as when fixed on a microarray. The method of creating a microarray by depositing and fixing nucleic acids on a supporting substrate is well known in the art. Reviewed in 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 comprising nucleic acid sequence fragments, such as those disclosed herein, is an established utility of sequence fragments in the fields of cell and molecular biology.Other uses of 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).

[0306] In some embodiments, the polynucleotides of the present disclosure comprise or consist of a nucleic acid sequence encoding up to 100 amino acids of SEQ ID NO: 159 or SEQ ID NO: 160. In some embodiments, the polynucleotides of the present disclosure comprise or consist of a sequence encoding a polypeptide of SEQ ID NO: 1-158 or 177 or 161-176 and / or as shown in Tables 1B, 1C and / or 2A, or a portion or functional variant thereof. In some such embodiments, the polynucleotides encode polypeptides, such as those shown in SEQ ID NO: 1-158 or 177 or 161-176 and / or as shown in Tables 1B, 1C and / or 2A, or a portion or functional variant thereof, which bind to a target represented by SEQ ID NO 159 or 160.

[0307] In some embodiments, the polynucleotides disclosed herein comprise or consist of a nucleic acid sequence encoding a polypeptide that is or comprises a small protein that binds connexin-4 or any portion, fragment, or variant thereof. In some embodiments, the polynucleotides encode a polypeptide that comprises or consists of an amino acid sequence as shown in any one of SEQ ID NOs: 1-158 or 177 or 161-176 and / or as shown in Tables 1B, 1C, and / or 2A, or a portion or functional variant thereof. In some embodiments, the polynucleotide encodes a polypeptide sequence 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 an amino acid sequence provided in a polypeptide according to a polypeptide set forth in Table 1B and / or 1C or a compound of Table 2A.

[0308] In some embodiments, the small protein comprises one or more disulfide bridges. In some embodiments, the small protein is represented by a nucleic acid sequence encoding a polypeptide that, when folded, comprises one or more disulfide bridges.

[0309] In some embodiments, the disclosure provides a nucleic acid molecule comprising or consisting of the sequence shown in SEQ ID NO: 159 or SEQ ID NO: 160, or a variant thereof (eg, a codon-optimized variant).

[0310] In some embodiments, for example, a nucleic acid molecule (i.e., a polynucleotide) may not be identical to a reference sequence as provided herein, but still encodes a miniprotein or close analog as provided by the present disclosure (e.g., a miniprotein or a portion or functional variant thereof as shown in any one of SEQ ID NOs: 1-158 or 177 or 161-176 and / or as shown in Tables 1B, 1C and / or 2A, as provided herein). In some such embodiments, for example, a provided polynucleotide (i.e., encoding a miniprotein or an analog thereof) hybridizes under stringent conditions as disclosed herein. In some embodiments, the 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 continuous nucleic acid residues identical to continuous nucleic acid residues of polynucleotides of any one of SEQ ID NO: 1-158 or 177 or 161-176 and / or as shown in Table 1B, 1C and / or 2A or a portion or functional variant thereof. 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 encoding a polypeptide or fragment thereof as shown in any one of SEQ ID NO: 1-158 or 177 or 161-176 and / or as shown in Table 1B, 1C and / or 2A or a portion or functional variant thereof or some or all of the polypeptide or fragment thereof.

[0248] It will be appreciated by those skilled in the art that the nucleic acid fragments disclosed herein can be used in a variety of capture and / or detection techniques not specifically described herein. Carrier

[0311] Also provided herein are vectors, including expression vectors, comprising, among other things, a nucleic acid containing or consisting of: a sequence encoding a small protein that specifically binds to connexin-4. In some embodiments, the vector is used to produce a polypeptide encoding a binder that binds to connexin-4. In some embodiments, connexin-4 is mouse or human connexin-4. In some embodiments, given the appropriate context, the small protein is represented by an amino acid sequence having a corresponding nucleic acid sequence that has been codon-optimized. In some such embodiments, one skilled in the art is able to design and optimize polynucleotides corresponding to the amino acids of a small protein that binds to a target (e.g., connexin-4), exemplary amino acid sequences of the target being listed in Table 1A. In some embodiments, the vector comprises a nucleic acid sequence comprising or consisting of: a sequence encoding connexin-4.

[0312] In some embodiments, the vector comprises a nucleic acid sequence encoding connexin-4 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. In some embodiments, the vector of the present disclosure comprises or consists of the following: a nucleic acid sequence encoding an amino acid sequence of a small protein. In some embodiments, the vector of the present disclosure further comprises a nucleic acid sequence as provided herein operably linked to one or more expression control sequences.

[0313] Also provided herein are vectors, including expression vectors, comprising, among other things, nucleic acids containing or consisting of: those described herein. In some embodiments, the vector is used to produce a polypeptide encoding a small protein that binds to connexin-4. In some embodiments, connexin-4 is mouse or human connexin-4. In some embodiments, given the appropriate context, the connexin-4 small protein (e.g., as provided in Table 2A) has a corresponding nucleic acid sequence that has been codon-optimized. In some such embodiments, one skilled in the art is able to design and optimize polynucleotides corresponding to the amino acids of a particular connexin-4 small protein, for example, such as a polynucleotide comprising a nucleic acid sequence corresponding to the amino acid sequence of Table 2A. In some embodiments, the vector comprises a nucleic acid sequence encoding the amino acid sequence shown in SEQ ID NO: 159. In some embodiments, the vector comprises a nucleic acid sequence encoding the amino acid sequence shown in SEQ ID NO: 160. In some embodiments, the vector comprises a nucleic acid sequence comprising or consisting of a sequence that is 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 identical to wild-type connexin-4. In some embodiments, the vector comprises a nucleic acid sequence encoding connexin-4 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 SEQ ID NO: 1-158 or 177 or 161-176 and / or any one of those shown in Table 1B, 1C and / or 2A or a portion or functional variant thereof up to 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 amino acids. In some embodiments, the vector of the present disclosure comprises a nucleic acid sequence or consists of a nucleic acid sequence, wherein the nucleic acid sequence encodes an amino acid sequence comprising those amino acid sequences shown in Table 1B, 1C and / or 2A or a portion or functional variant thereof. In some embodiments, the nucleic acid sequence is a codon-optimized nucleic acid sequence. In some embodiments, the vector of the present disclosure further comprises a nucleic acid sequence as provided herein that is operably connected to one or more expression control sequences. Host cell transformants

[0314] In some embodiments, the disclosure provides host cells transformed with polynucleotides, polypeptides and / or vectors of the disclosure, and any combination thereof, and any offspring. In some embodiments, the host cell comprises and carries the nucleic acid sequence of the disclosure on a vector. In some embodiments, the host cell is a cell line. In some embodiments, the host cell is a primary cell, such as an immune cell. In some embodiments, such a primary cell is derived from a subject or is compatible with a subject. In some embodiments, the subject is a mammal. In some embodiments, the mammal is a human. In some embodiments, a human is at risk of cancer or has been diagnosed with cancer.

[0315] In some such embodiments, such a vector may, but need not be, a freely replicating vector.In some embodiments, the nucleic acid sequences or polynucleotides provided by the present disclosure have been integrated into the genome of a host cell.

[0316] In some embodiments, a host cell of the disclosure can be mutated by recombination with a disruption, deletion, or mutation of an isolated nucleic acid of the 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 the mutation.

[0317] Without limitation, and as will be appreciated by those skilled in the art, a variety of host cells are contemplated in various embodiments for expressing the conjugates of the present disclosure (via use of, for example, nucleic acid sequences, amino acid sequences, and / or additional components as provided herein). Pharmaceutical composition

[0318] Among other things, the present disclosure provides pharmaceutical compositions comprising polypeptides, polynucleotides, vectors and / or host cells encoding small proteins (e.g., CDPs, knottins, binders, affibodies, engineered Kunitz domains, monoclonal antibodies, anticalins, designed ankyrin repeat domains (DARPins), avimers) as provided herein. It should be understood that a pharmaceutical composition comprising a small protein (e.g., a CDP, a knottin, a binder, an affibody, an engineered Kunitz domain, a monoclonal antibody, an ankyrin, a designed ankyrin repeat domain (DARPin), an avimer) is to be construed as a pharmaceutical composition comprising the small protein (e.g., a CDP, a knottin, a binder, an affibody, an engineered Kunitz domain, a monoclonal antibody, an ankyrin, a designed ankyrin repeat domain (DARPin), an avimer) itself and / or one or more components (e.g., a vector, e.g., a host cell) encoding the small protein (e.g., a CDP, a knottin, a binder, an affibody, an engineered Kunitz domain, a monoclonal antibody, an ankyrin, a designed ankyrin repeat domain (DARPin), an 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 small protein, an optional linker, and a chelator. In some embodiments, the composition comprises a small protein, an optional linker, a chelator, and a radionuclide. In some embodiments, the pharmaceutical compositions provided by the present disclosure comprise a small protein (e.g., CDP, knottin, conjugate, affibody, engineered Kunitz domain, monoclonal antibody, anticalin, designed ankyrin repeat domain (DARPin), avimer) that selectively binds to connexin-4. In some embodiments, connexin-4 is human connexin-4.

[0319] In certain embodiments, the pharmaceutical composition comprises a small protein (e.g., CDP, knottin, binder, affibody, engineered Kunitz domain, monoclonal antibody, anticalin, designed ankyrin repeat domain (DARPin), avimer) containing one or more cysteine-rich domains. In some embodiments, the pharmaceutical composition comprises a small protein (e.g., CDP, knottin, binder, affibody, engineered Kunitz domain, monoclonal antibody, anticalin, designed ankyrin repeat domain (DARPin), avimer) having one or more disulfide bonds. In some embodiments, the pharmaceutical composition comprises a small protein (e.g., a CDP, a knottin, a binder, an affibody, an engineered Kunitz domain, a monoclonal antibody, an ankyrin, a designed ankyrin repeat domain (DARPin), an avimer) represented or encoded by an amino acid sequence having <100AA, <90AA, <80AA, <85AA, <75AA, <70AA, <65AA, <60AA, <55AA, <50AA, <45AA, <40AA, <35AA, <30AA, <25AA, <20AA, <15AA, <10AA, or <5AA).

[0320] In some embodiments, a pharmaceutical composition comprising a small protein (e.g., a CDP, a knottin, a binder, an affibody, an engineered Kunitz domain, a monoclonal antibody, an anticalin, a designed ankyrin repeat domain (DARPin), an avimer) as provided herein is characterized as having a molecular weight equal to or less than 12 kDa.

[0321] In some embodiments, pharmaceutical compositions comprising small proteins (e.g., CDPs, knottins, binders, affibodies, engineered Kunitz domains, monoclonal antibodies, anticalins, designed ankyrin repeat domains (DARPins), avimers) do not elicit an undesirable immune response or elicit a tolerable immune response. In some embodiments, pharmaceutical compositions of the present disclosure comprise small proteins (e.g., CDPs, knottins, binders, affibodies, engineered Kunitz domains, monoclonal antibodies, anticalins, designed ankyrin repeat domains (DARPins), avimers) with high tissue penetrating properties.

[0322] In some embodiments, the pharmaceutical composition comprising a small protein has an 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 a tumor or tumor microenvironment. By way of non-limiting example, for example, in some embodiments, the small protein disclosed herein has a stability of about 2.5 hours in serum. In some embodiments, the stability of the small protein 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 a tumor or tumor microenvironment is 24, 36, 48, 60, 72, 84, 96 hours or longer.

[0323] In certain embodiments, the pharmaceutical composition is characterized by being stable in vivo.In certain embodiments, the pharmaceutical composition provided herein is not taken up by kidney or liver.In certain embodiments, when taken up in kidney or liver, the pharmaceutical composition provided herein is cleared from kidney and / or liver faster than the pharmaceutical composition not comprising the polypeptide provided herein.In certain embodiments, the biodistribution of the pharmaceutical composition as provided herein is measured four hours after administration, and in the tumor of the subject (e.g., mouse subject, e.g., human subject) between about 1 to about 3 (in %ID / g).In certain embodiments, the concentration in the tumor is greater than the concentration in the kidney or liver of the subject of the pharmaceutical composition comprising the polypeptide provided herein.

[0324] In some embodiments, the 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.

[0325] In some embodiments, the pharmaceutical compositions provided by the present disclosure exhibit >80%, >81%, >82%, >83%, >84%, 85%, >86%, >87%, >88%, >89%, >90%, >91%, >92%, >93%, >94%, 95%, >96%, >97%, >98%, or >99% stability.

[0326] In some embodiments, the pharmaceutical composition of the present disclosure is characterized as comprising a certain purity, expressed as a percentage of the parent molecule that is still intact. In some embodiments, the pharmaceutical composition of the present disclosure comprises a purity of about 85% or more at room temperature for 5 days. In some embodiments, the pharmaceutical composition of the present disclosure is characterized in that it has a purity of about 90% or more at 40°C for 4 hours. In some embodiments, the pharmaceutical composition of the present disclosure comprises a cyclic or non-cyclic sequence.

[0327] In some embodiments, pharmaceutical compositions according to the present disclosure comprise a small protein (e.g., a CDP, a knottin, a conjugate, an affibody, an engineered Kunitz domain, a monoclonal antibody, an anticalin, a designed ankyrin repeat domain (DARPin), an avimer) and one or more additional components. For example, in some embodiments, one or more additional components may be a linker and / or a conjugate, such as a cytotoxic payload or a detectable moiety for use in diagnosis and / or imaging. In some such embodiments, the pharmaceutical composition comprises a linker, a chelator, and / or a radionuclide provided herein.

[0328] In some embodiments, pharmaceutical compositions are provided that modulate, bind or inhibit human connexin-4 (or any associated activity thereof). 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., such as for imaging, e.g., MRI, CT, PET, etc.).

[0329] In a preferred embodiment, one or more characteristics of the pharmaceutical composition are identified to optimize administration parameters, including but not limited to dose, effective dose, dose rate, tumor penetration spectrum, intracellular localization spectrum, 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.

[0330] 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, such as a larger targeting peptide or a different radionuclide (eg, beta emitters, etc.).

[0331] In some embodiments, the pharmaceutical compositions of the present disclosure do not accumulate in the liver, spleen and / or pancreas and are rapidly cleared. For example, in the biodistribution in some embodiments, after administration to a subject, the biodistribution in the kidney at 24 hours is >10% of the injected dose (ID), and the biodistribution in the tumor at 24 hours is >3% ID. In some embodiments, after administration to a subject, the t1 / 2 is shorter than the t1 / 2 of, for example, a connexin-4 antibody, such as Vitin-Enfortuzumab. Theranostic compositions

[0332] In some embodiments, therapeutic diagnostic compositions are provided. In some embodiments, the disclosure provides diagnosis or screening to detect the presence or absence and / or level of connexin-4 in a subject or sample. In some embodiments, the subject is a mammal. In some embodiments, the subject is a rodent (e.g., mouse) subject and connexin-4 is rodent (e.g., mouse) connexin-4. In some embodiments, the subject is a human subject and connexin-4 is human connexin-4. In some embodiments, the presence of connexin-4 in a subject is associated with a risk of developing a disease, disorder, or condition. In some embodiments, the presence of a specific level of connexin-4 indicates an increased risk of developing or diagnosing a disease, disorder, or condition. In some embodiments, a reduction in connexin-4 levels (e.g., compared to a previous measurement) is associated with the treatment of a diagnosed disease.

[0333] In certain aspects, theranostic compositions are provided. In some embodiments, the disclosure provides diagnostics or screens to detect the presence or absence and / or levels of human connexin-4 in a subject or sample.

[0334] In certain aspects, the disclosure provides methods for defining a structure-activity relationship of a pharmaceutical composition comprising: (i) a connexin-4-specific miniprotein; (ii) an optional linker; (iii) a chelator; and (iv) a radioactive molecule, wherein the modified polypeptide sequence modulates human connexin-4 activity. Screening and development methods

[0335] In some embodiments, directed evolution and computational folding algorithms can be combined for the de novo creation of small proteins (e.g., CDPs, knottins, binders, affibodies, engineered Kunitz domains, monomers, anticalins, designed ankyrin repeat domains (DARPins), avimers). For example, in some embodiments, hundreds of small protein backbones with various secondary structure elements, orientations, and loop lengths can be matched to hot spot binding motifs on a protein target or antigen of interest (e.g., connexin-4). In some such embodiments, if the binding motif of the small protein does not conflict with the backbone of the target, the monomer and interaction energies are optimized using programs known to those skilled in the art (e.g., such as AlphaFold, Rosetta combinatorial sequence optimization, etc.).

[0336] In some embodiments, the oligonucleotide pool encoding the designed sequence selected by the computational approach can be synthesized, amplified and co-transformed into yeast. The resulting yeast library displaying the designed sequence can be incubated with a fluorescently labeled target protein or antigen. The cells displaying the design of binding target can be retrieved by fluorescence activated cell sorting (FACS) and deep sequencing is performed. Once a small protein (e.g., CDP, knotting element, binder, affibody, engineered Kunitz domain, monomer, anticalin, designed ankyrin repeat domain (DARPin), avimer) is identified by affinity maturation or original design, they can be chemically synthesized or expressed in, for example, Escherichia coli, and purified and characterized in solution.

[0337] In some embodiments, libraries of stable small proteins (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. Such libraries design the hydrophobic core of the small proteins (e.g., CDPs, knottins, binders, affibodies, engineered Kunitz domains, monobodies, anticalins, designed ankyrin repeat domains (DARPins), avimers) to enable folding in addition to cysteine ​​cross-links, thereby improving the number of folded structures in the library.

[0338] In some embodiments, once small proteins (e.g., CDPs, knottins, binders, affibodies, engineered Kunitz domains, monomers, anticalins, designed ankyrin repeat domains (DARPins), avimers) are identified or engineered, they can be produced via chemical synthesis or recombinant expression. In some embodiments, small proteins (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-fluorenylmethoxycarbonyl (Fmoc)-based solid phase peptide chemistry can be employed. 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 and then purified, for example, by reverse phase high performance liquid chromatography (RP-HPLC). Approaches using recombinant DNA can also be used to produce small proteins as provided herein (e.g., CDPs, knottins, binders, affibodies, engineered Kunitz domains, monoclonal antibodies, anticalins, designed ankyrin repeat domains (DARPins), avimers).

[0339] Iteration between data-driven model improvements and experimental testing of small proteins (e.g., CDPs, knottins, binders, affibodies, engineered Kunitz domains, monomers, anticalins, designed ankyrin repeat domains (DARPins), avimers) may optimize the folding and binding capabilities of small proteins (e.g., CDPs, knottins, binders, affibodies, engineered Kunitz domains, monomers, anticalins, designed ankyrin repeat domains (DARPins), avimers) to develop pharmaceutically superior specific molecules. Characterization, Analysis and Synthesis

[0340] In some embodiments, small proteins of the present disclosure (e.g., CDPs, knottins, binders, affibodies, engineered Kunitz domains, monoclonal antibodies, anticalins, designed ankyrin repeat domains (DARPins), avimers) are characterized. For example, in some embodiments, binding specificity, binding affinity, binding localization, and the like are performed using methods known to those skilled in the art. For example, in some embodiments, binding localization is performed using one or more techniques, such as immunohistochemistry / immunocytochemistry (e.g., using cell lines or tissue biopsy samples). In some embodiments, binding affinity is performed using surface plasmon resonance measurements. In some such embodiments, binding affinity (e.g., dissociation constant expressed as K D) is measured in one or more assays (e.g., a yeast-based assay in which the target is recombinantly expressed in yeast and exposed to a small protein provided by the disclosure (e.g., a CDP, knottin, binder, affibody, engineered Kunitz domain, monoclonal antibody, anticalin, designed ankyrin repeat domain (DARPin), avimer)).

[0341] In some embodiments, synthetic and analytical techniques (including but not limited to HPLC, LCMS, CD, quantitative thin layer chromatography, and other techniques known to those skilled in the art) are used to efficiently synthesize small proteins via solid phase peptide synthesis methods, characterize small proteins (e.g., CDPs, knottins, binders, affibodies, engineered Kunitz domains, monoclonal antibodies, anticalins, designed ankyrin repeat domains (DARPins), avimers), and fully optimized clinical drug composition candidates.

[0342] In some embodiments, internalization assays can be performed. For example, in some embodiments, labeled small proteins (e.g., radiolabeled, e.g., fluorescently labeled, etc.) can be used to determine the internalization of connexin-4 small proteins. In some embodiments, the small proteins can be coupled to a detectable marker (e.g., a pH-sensitive fluorescent dye, e.g., a radiolabeled marker). The cells can then be examined. In some embodiments, after fixation and permeabilization, cells can be labeled with antibodies to the target and / or one or more antibodies to endocytic or lysosomal markers (e.g., clathrin, LAMP-1, etc.), followed by microscopic analysis.

[0343] In some embodiments, internalization assays can be performed using other methods known to those of skill in the art and provided herein (eg, such as in Example 13A). Screening and development methods for small proteins

[0344] Directed evolution and computational folding algorithms can be combined to create small proteins provided herein from scratch. In some embodiments, hundreds of small protein backbones with various secondary structure elements, orientations, and loop lengths can be matched with hotspot binding motifs on the target of interest (e.g., connexin-4). In some such embodiments, if the binding motif of the small protein does not conflict with the main chain of the target, the monomer and interaction energy are optimized using Rosetta combined sequence optimization.

[0345] In some embodiments, the oligonucleotide pool encoding the designed sequence selected by the computational approach can be synthesized, amplified and co-transformed into yeast. In some embodiments, the resulting yeast library displaying the designed sequence can be incubated with a fluorescently labeled target. In some embodiments, cells displaying the design of binding targets can be retrieved by fluorescence activated cell sorting (FACS) and deep sequencing can be performed. In some embodiments, once a small protein is identified by affinity maturation or original design, the small protein can be chemically synthesized or expressed in Escherichia coli, purified, and characterized in solution.

[0346] In some embodiments, libraries of stable CDP or knottin peptides can be developed to allow screening against specifically selected targets. The libraries design hydrophobic cores for small proteins to enable folding in addition to cystine cross-links, thereby improving the number of folded structures in the library.

[0347] In certain embodiments, once identified or engineered small protein, they can be produced by chemical synthesis or recombinant expression. In certain embodiments, small protein peptides can be produced by solid phase peptide synthesis and then folding in vitro. Standard solid phase peptide chemistry based on 9-fluorenylmethoxycarbonyl (Fmoc) can be adopted. In some such embodiments, then linear peptides can be folded under the conditions of promoting cysteine ​​side chain thiol oxidation to form disulfide bonds, and then purified, for example, by reversed phase high performance liquid chromatography (RP-HPLC). In certain embodiments, the approach using recombinant DNA can also be adopted to produce the desired small protein.

[0348] In some embodiments, iterations between data-driven model refinement and experimental testing of small proteins may optimize the folding and binding capabilities of small proteins in order to develop pharmaceutically superior specific molecules. In some embodiments, the miniprotein or portion thereof is engineered at the DNA level (e.g., degenerate codons can be introduced by oligonucleotide assembly using overlap extension PCR; or flanking primers with sufficient overlap with a yeast display vector can be used to amplify the genetic material for homologous recombination). Modification of small proteins

[0349] In some embodiments, the disclosure further provides one or more modifying components. In some embodiments, the modifying component comprises or consists of the following: an inducible or repressible promoter operably connected to the coding sequence of a small protein as provided herein. In some embodiments, the expression profile of a small protein or its basic amino acid sequence can be changed via the promoter of the nucleic acid sequence encoding it. In some aspects, the expression profile of a small protein can be changed or controlled in time by changing or controlling the promoter function in time. In some embodiments, the promoter can be controlled in space and / or environment. In some embodiments, the modifying component comprises or consists of an enhancer. In some such embodiments, the enhancer is used to modify the expression profile of the conjugate, but is not necessarily operably connected to the coding sequence of the conjugate; on the contrary, in some embodiments, the enhancer is located upstream or downstream of the coding sequence of the conjugate disclosed herein. In some embodiments, the enhancer can be controlled in time. In some embodiments, the enhancer can be controlled in space and / or environment.

[0350] In certain embodiments, the binding substances of the present disclosure and / or the expression spectrum of the sequence (for example nucleotide sequence, for example amino acid sequence, for example, such as gene or its part) encoding it can be changed via one or more modifications.In some such embodiments, one or more modifications comprise one or more sudden changes in the sequence (for example nucleotide sequence, for example amino acid sequence) provided by the present disclosure.In some respects, the sequence of the present disclosure comprises the disappearance relative to the parental sequence or its part.

[0351] Modification can also be performed using changes to the amino acid sequence and the bond. For example, chemical crosslinking can be used to improve the binding ability or affinity of small proteins to the target. In certain embodiments, changes such as amino acid residues (such as lysine, such as non-natural amino acids, etc.) fusion proteins or other chemical moieties can be used to generate small proteins with enhanced binding and functional activity, such as compared to those without modification. In certain embodiments, the feature of small proteins is that they have a small disulfide-rich peptide scaffold, and may be difficult to fold. For example, in certain embodiments, small proteins can form various isomers (for example, small proteins with three core disulfide bonds can form at least 15 different isomers in some embodiments). In some such embodiments, the diversity of isomers can affect yield. In certain embodiments, small proteins without cysteine ​​residues (such as two or more cysteines, such as at least one disulfide bridge) can be modified to improve stability without the need for additional chemical crosslinking by increasing the number of disulfide bonds.

[0352] For example, in some embodiments, the SAR approach can be used to improve the binding affinity of small proteins. For example, various amino acid residues within the small protein structure can be replaced with optimal substitutions to improve binding affinity. In some embodiments, these substitutions can include natural and / or non-natural amino acids, conjugated chemical moieties, and / or other small molecule attachments.

[0353] In some embodiments, chemical cross-linking can be used to provide appropriate structural conformation and stability. Appropriate structural conformation is crucial for retaining certain binding affinity (e.g., in improved connexin-4 small proteins, such as compared to small proteins that are not improved using SAR or other approaches). In some embodiments, small proteins have small disulfide-rich peptide scaffolds and are difficult to fold. In some such embodiments, using techniques and approaches known to those of ordinary skill in the art, optimized conditions for folding and purification via reversed-phase HPLC can be used to ensure that the final (e.g., optimized) compound (including small proteins as provided herein) has the correct structure, conformation and purity. constraint

[0354] Modifications, such as constraints, can be introduced (e.g., engineered) into proteins, such as any small proteins provided herein. Constraints can change the function (e.g., binding) and / or structure (e.g., folding) of a protein. For example, constraints can help maintain the secondary structure of a given protein (e.g., a small protein as provided herein, e.g., a conjugate as provided herein). Common protein secondary structures are α-helices. These helices play a key role not only in the structure of the protein, but also in function, affecting how a particular protein interacts with a binding partner. For example, α-helices can mediate protein-protein interactions (PPIs) by acting as recognition motifs. Introducing constraints on α-helices in proteins (e.g., small proteins) can change one or more features of the protein in some embodiments, such as protein affinity for a target (e.g., increasing affinity), cell penetration (e.g., cell penetration increases), and resistance to proteolysis (e.g., resistance to proteolytic degradation increases). Examples of constraints (e.g., α-helical constraints) can include, but are not limited to, disulfide bridges / bonds, staples (e.g., hydrocarbon staples), salt bridges between charged amino acid side chain residues, lactam bridges, disulfide bonds, hydrogen bond surrogates, hydrophobic interactions, metal linkages, triazole staples synthesized from alkenyl and azido side chain residues, photo-controllable macrocycles, and the introduction of amino acids, such as, for example, α,α-disubstituted amino acids. As known to those skilled in the art, a staple can refer to a synthetic constraint (e.g., a brace) between two previously independent entities. For example, a staple can be formed (e.g., to form, for example, a peptide macrocycle) via a covalent linkage between two previously independent entities (e.g., such as amino acid side chains).

[0355] In some embodiments, the small protein provided herein comprises one or more constraints (e.g., disulfide bridges, e.g., spikes). In some embodiments, the small protein disclosed herein comprises one or more disulfide bridges. In some embodiments, the small protein provided by the present disclosure comprises two or more disulfide bridges. For example, the small protein disclosed herein may contain a group of amino acids that together support the formation of a constraint or are part of a constraint (the constraint is, for example, a disulfide bridge, e.g., a spike). In some embodiments, the structural feature of the small protein is that it has at least two cysteine ​​residues that are positioned relative to each other so that a disulfide bridge can be formed (e.g., as provided herein, see, e.g., Table 2A). In some embodiments, the small protein provided by the present disclosure has at least three or at least four cysteine ​​residues that form one or two disulfide bridges. In some such embodiments, such a small protein further includes one or more additional constraints (e.g., spikes).

[0356] In some embodiments, the miniproteins disclosed herein comprise spikes. In some embodiments, the disclosure provides spiked miniproteins that bind to connexin-4, wherein the presence of the spikes changes at least one binding characteristic (e.g., increased binding affinity to connexin-4, e.g., a change in affinity to connexin-4) relative to miniproteins without the spikes. Those skilled in the art who read the disclosure will understand that any desired spike technology can be used to prepare spiked miniproteins. Binding assay

[0357] In some embodiments, binding assays are used to determine binding affinity and / or binding / dissociation constants or composition or one or more components thereof (e.g., of a small protein with or without one or more additional components as provided herein). For example, in some embodiments, the equilibrium dissociation constant (Kd) is determined using fluorescent labels and detection methods. In some embodiments, an engineered cell population (e.g., yeast cells) is generated to express a library of small proteins (e.g., CDPs, knottins, binders, affibodies, engineered Kunitz domains, monomers, 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 a group of cells expresses a target or a small protein (e.g., a CDP, a knottin, a binder, an affibody, an engineered Kunitz domain, a monoclonal antibody, an ankyrin, a designed ankyrin repeat domain (DARPin), an avimer), in some embodiments, a library of cells is incubated with a target (e.g., connexin-4) or a small protein (e.g., a CDP, a knottin, a binder, an affibody, an engineered Kunitz domain, a monoclonal antibody, an ankyrin, a designed ankyrin repeat domain (DARPin), an avimer) as provided herein or a group of small proteins (e.g., a CDP, a knottin, a binder, an affibody, an engineered Kunitz domain, a monoclonal antibody, an ankyrin, a designed ankyrin repeat domain (DARPin), an avimer). In some such embodiments, cells and small proteins (e.g., CDPs, knottins, binders, affibodies, engineered Kunitz domains, monomers, anticalins, designed ankyrin repeat domains (DARPins), avimers) are evaluated using flow cytometry and / or FACS analysis to determine binding affinity using methods known to those skilled in the art. (See, e.g., "Chapter Nine - Engineering CDPs as Novel Binding Agents." Methods in Enzymology, K. Dane Wittrup and Gregory L. Verdine, eds., Vol. 503, Academic Press, 2012, pp. 223-51. ScienceDirect, doi: 10.1016 / B978-0-12-396962-0.00009-4.). Affinity Maturation

[0358] In some embodiments, the small proteins disclosed herein (e.g., CDPs, knottins, binders, affibodies, engineered Kunitz domains, monoclonal antibodies, anticalins, designed ankyrin repeat domains (DARPins), avimers) comprise or consist of sequences that exhibit certain desired affinity ranges for a target. In some embodiments, the sequences as provided by the present disclosure are affinity matured, wherein the affinity matured sequences exhibit the same or better selectivity and / or affinity for connexin-4 compared to the starting sequence or another sequence having a "lower" affinity compared to the affinity matured sequence. In some embodiments, affinity maturation is performed using a connexin-4 antigen, and the sequence that binds to the antigen is a sequence of a small protein (e.g., CDPs, knottins, binders, affibodies, engineered Kunitz domains, monoclonal antibodies, anticalins, designed ankyrin repeat domains (DARPins), avimers) as provided herein.

[0359] In some embodiments, the sequence that selectively binds to Connexin-4 is affinity matured. In some embodiments, Connexin-4 is human Connexin-4.

[0360] In preferred aspects of the disclosure, the modified polypeptide sequence of the pharmaceutical composition comprises nM or sub-nM binding affinity to a target on a cell line expressing human connexin-4, binding potency to a protein target or in a cell-based assay.

[0361] In certain embodiments, the modified polypeptide sequence comprises a binding affinity for human connexin-4 of 900nM, 800nM, 700nM, 600nM, 500nM, 400nM, 300nM, 200nM, 100nM, 90nM, 80nM, 70nM, 60nM, 50nM, 40nM, 30nM, 20nM, 10nM, 9nM, 8nM, 7nM, 6nM, 5nM, 4nM, 3nM, 2nM, 1nM.

[0362] In one embodiment, the modified polypeptide sequence comprises a binding affinity for human connexin-4 of 500 nM binding affinity.

[0363] In more preferred embodiments, the modified polypeptide sequence comprises a picomolar binding affinity.

[0364] In other preferred embodiments, the modified polypeptide sequence exhibits selectivity, wherein the sequence only binds to the connexin-4 protein target. 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 skilled in the art. Small protein production method

[0365] In some embodiments, the synthesis of small proteins includes solid phase synthesis. In some embodiments, small proteins are synthesized using standard solid phase peptide synthesis known to those skilled in the art. (See, for example, Johannes Meienhofer, Hormonal Proteins and Peptides, Volume II, 1973, pages 45-267). In some embodiments, SPS includes synthesis using methods known to those skilled in the art, including, for example, Fmoc or Boc amino protecting groups. In some embodiments, synthesis includes preventing reaction with introduced N-protected amino acids. In some embodiments, the synthesized polypeptide is analyzed using HPLC / LC-MS to determine sequence, structure, and related properties.

[0366] In some embodiments, small proteins are made using recombinant production methods as known to those of skill in the art, including, for example, yeast-based pathways and chemical synthesis. Conjugation method

[0367] In some embodiments, the conjugate of the present disclosure comprises a linker and a chelator. In some embodiments, the chelator has a bound radionuclide. In some embodiments, a linker and a chelator, or a linker, a chelator and a radionuclide are conjugated to a small protein. In some embodiments, a chelator and / or a radionuclide are conjugated to a small protein. In some embodiments, the small protein is conjugated to a chelator directly or through a linker (e.g., a linker described below). Any known conjugation chemistry can be used to conjugate a small protein directly to a chelator or to conjugate a linker to a small protein and to a chelator.

[0368] In some embodiments, the small protein comprises surface exposed functional groups to allow site-specific conjugation. In some embodiments, the small protein comprises surface exposed lysine or cysteine ​​residues that can be used for site-specific conjugation. In some embodiments, the small protein conjugate comprises one or more non-naturally occurring amino acids that can be used for site-specific conjugation.

[0369] Those of ordinary skill in the art will recognize that a variety of chemical conjugation strategies provide ready access to the present technology whereby exposed amino acid residues on the protein undergo well-known reactions with reactive moieties on the chelator.

[0370] Those of ordinary skill in the art will recognize that cysteine ​​coupling reactions can be used to conjugate chelators with thiol-reactive ends to protein surfaces through thiol side chains exposed on cysteine ​​residues on the protein surface. (See generally Tsuchikama and An, supra, pages 36-37; see also, for example, Pierre Adumeau et al., Thiol-Reactive Bifunctional Chelators for the Creation of Site-Selectively Modified Radioimmuno conjugates with Improved Stability [thiol-reactive bifunctional chelators for the creation of site-selectively modified radioimmunoconjugates with improved stability], 29 Bioconjugate Chem. [Biological conjugation chemistry] 1364 (2018)). In some embodiments, since cysteine ​​residues are easily connected to nearby cysteine ​​residues under physiological conditions, rather than existing as free thiols, some cysteine ​​coupling strategies may rely on the selective reduction of disulfides to generate a greater 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, for example, CR Behrens et al., Antibody-Drug Conjugates (ADCs) Derived from Interchain Cysteine ​​Cross-Linking Demonstrates Improved Homogeneity and Other Pharmacological Properties Over Conventional Heterogeneous ADCs [Antibody-Drug Conjugates (ADCs) Derived from Interchain Cysteine ​​Cross-Linking Demonstrates Improved Homogeneity and Other Pharmacological Properties Over Conventional Heterogeneous ADCs [Antibody-Drug Conjugates (ADCs) Derived from Interchain Cysteine ​​Cross-Linking Demonstrates Improved Homogeneity and Other Pharmacological Properties Over Conventional Heterogeneous ADCs], 12 Mol. Pharm. [Molecular Pharmacy] 3986 (2015); Vinogradova et al., Organometallic Palladium Reagents for Cysteine ​​Bioconjugation [Organometallic Palladium Reagents for Cysteine ​​Bioconjugation], 526 Nature [Nature] 687 (2015); See also Tsuchikama, supra, at 37.

[0371] Protein conjugation strategies using non-natural amino acid side chains are also well known in the art. For example, in some embodiments, "click chemistry" provides access to conjugated proteins by rapid and selective chemical transformations under various reaction conditions. In some embodiments, click chemistry is known to produce peptide conjugates under aqueous conditions with limited formation of byproducts despite the presence of unprotected functional groups. For example, in some embodiments, the click reaction to form the conjugated peptide is a copper (I)-catalyzed azide-alkyne 1,3-dipolar cycloaddition reaction (CuAAC). (See Liyuan Liang and Didier Astruc, The Copper(I)-Catalyzed Alkyne-Azide Cycloaddition (CuAAC) "Click" Reaction and Its Applications: An Overview, 255 COORD.CHEM.Rev 2933 (2011); see also, e.g., Herman S. Gill and 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 increase the reaction rate. In some such embodiments, such ligands may include, for example, polydentate nitrogen donors, including amines (e.g., tris(triazolyl)methylamine) and pyridine. (See Liang & Astruc, supra, at 2934 (collected examples); PL Goias et al., 39 Macromolecules [macromolecules] 6451 (2006)). In some embodiments, other widely used click reactions include, but are not limited to, thiol-ene, oxime, Diels-Alder, Michael addition, and pyridine thioether reactions.

[0372] In some embodiments, copper-free (Cu-free) click methods are also known in the art for delivering therapeutic and / or diagnostic agents, such as radionuclides (e.g., 18F), chemotherapeutic agents, dyes, contrast agents, fluorescent markers, chemiluminescent markers or other markers to protein surfaces. In some embodiments, copper-free click methods may allow stable covalent attachment between target molecules and prosthetic groups. In some embodiments, copper-free click chemistry may include reacting an antibody or antigen-binding fragment modified with a non-natural amino acid side chain with a prosthetic group presenting a corresponding or complementary reactive moiety (e.g., azide, nitrone or cyclooctyne (e.g., DBCO)), wherein the non-natural amino acid side chain includes an activated moiety (e.g., cyclooctyne (e.g., dibenzocyclooctyne (DBCO)), nitrone or azide group). (See, e.g., J. Donnelly et al., Synthesis and Biologic Evaluation of a Novel 18F-Labeled Adnectin as a PET Radio ligand for Imaging PD-L1 Expression [Synthesis and biological evaluation of novel 18F-labeled fibronectin as a PET radioligand for PD-L1 expression imaging], 59 J. NUCL. MED. 529 (2018). For example, in some embodiments, when the targeting molecule comprises a cyclooctyne, the auxiliary group may include an azide, a nitrone, or a similar reactive moiety. In some embodiments, when the targeting molecule comprises an azide or a nitrone, the auxiliary group may present a complementary cyclooctyne, an alkyne, or a similar reactive moiety. In some embodiments, the copper-free click reaction may be performed at room temperature, in an aqueous solution, in the presence of phosphate buffered saline (PBS). In some such embodiments, the auxiliary group may be radiolabeled (e.g., with 18F) or may be conjugated to any alternative diagnostic agent and / or therapeutic agent (e.g., a chelating agent). (See supra, at 531.)

[0373] In some embodiments, conjugation chemistries such as the Huisgen cycloaddition ("click" reaction) can be used for the synthesis of chelates and peptides. In some embodiments, an efficient, high-yield three-step synthesis of multifunctional monofluorosubstituted cyclooctynes ​​(MFCO) has been demonstrated to facilitate various bioconjugation processes (M. Martin et al., 2013). In some embodiments, MFCO can be used to prepare DOTA derivatives for copper-free click chemistry additions to internal azide-modified lysine residues of CDP or knottin peptides.

[0374] In some embodiments, the small protein conjugates provided herein have lysines at specific positions (e.g., in cysteine ​​knots or cysteine-dense regions) and can be replaced with azide derivatives for "click" chemistry with DOTA-MFCO.

[0375] In some embodiments, a DOTA-MFCO-CDP conjugate can be prepared by first coupling amine-modified DOTA to MFCO and then conjugating DOTA-MFCO to the azide on the desired lysine of a small protein.

[0376] In some embodiments, the chelator and the small protein are linked together via a cycloaddition reaction in the presence of a transition metal catalyst. In some embodiments, the metal catalyst is based on Cu or Rh.

[0377] In some embodiments, the chelator (DOTA) and the small protein are linked with 1-ethyl-3-[3-(dimethylamino)propyl] (EDC) and N-hydroxysulfosuccinimide (SNHS) in water (pH 5.5) at room temperature for 40 min using solution phase conjugation 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 sulfosuccinimide ester of DOTA (DOTA-OSSu) described above. In some such embodiments, a molar excess of DOTA-OSSu is used to drive conjugation on the N-terminus of the peptide (see, e.g., Kimura, Richard H et al. "Engineeredknottin peptides: a new class of agents for imaging integrin expression in living subjects." Cancer research 69, 6 (2009): 2435-42. doi: 10.1158 / 0008-5472. CAN-08-2495).

[0378] In some embodiments, a new DOTA derivative α-amino-DOTA was prepared with the purpose of attaching DOTA to the C-terminus of a peptide, since in some scenarios, peptide function may be compromised due to conjugation of DOTA to the N-terminus or to a lysine side chain.

[0379] In certain embodiments, small albumen is produced by solid phase peptide synthesis (SPPS).The tri-tert-butyl ester of DOTA is a bifunctional ligand (in salt-free, zwitterionic form), soluble in most organic solvents, and the tert-butyl ester protection is fully compatible with standard SPPS technology.The most convenient conjugation mode is included in the N-terminal as the last amino acid added to the protected peptide chain by DOTA in an automatic peptide synthesizer, and then cuts and removes the acid-unstable protecting group from the resin. It can also be attached to the Lys side chain. The activated NHS ester of preformed DOTA-three (tert-butyl ester) has also been synthesized, and the reagent does not need a coupling agent to couple DOTA with free amino groups. The DOTA unit is connected to the peptide by one in the acetic acid side arm, and the conjugate has four amino groups, three carboxylates and an amide group that can be used for metal bonding. See, e.g., DeLeón-Rodríguez 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.

[0380] In some embodiments, a more general approach involves the use of preformed DOTA-amino acid derivatives that allow the introduction of the DOTA unit at any desired position in the 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 acetic acid sidearm of the DOTA unit) have been synthesized that are fully compatible with standard SPPS conditions. These DOTA amino acids can be used in SPPS to construct peptides incorporating the DOTA moiety at any desired position. See, e.g., De León-Rodríguez 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.

[0381] General methods for coupling DOTA-type macrocycles to targeting groups via a linker (e.g., by activating one of the carboxylates of DOTA to form an active ester, which 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.

[0382] A linker can be incorporated between the chelator and the targeting carrier to affect the pharmacokinetic properties of the conjugate. Hydrocarbon, PEG or polypeptide linkers can alter the pharmacokinetics and biodistribution by changing the overall charge and hydrophilicity of the radiopharmaceutical. See, e.g., De León-Rodríguez LM, Kovacs Z. The synthesis and chelation chemistry of DOTA-peptide conjugates. Bioconjug Chem. 2008 Feb; 19(2): 391-402. doi: 10.1021 / bc700328s. Electronic publication Dec. 12, 2007. PMID: 18072717. Small protein conjugate orientation

[0383] In some embodiments, the conjugation has the following orientation: linker-chelator, linker-chelator-radionuclide, linker-radionuclide, chelator-radionuclide. In some such embodiments, the conjugation has the following orientation: small protein-linker-radionuclide, small protein-chelator, chelator-small protein, small protein-linker-chelator, chelator-linker-small protein, small protein-chelator-radionuclide, radionuclide-chelator-small protein, small protein-linker-chelator-radionuclide, or radionuclide-chelator-linker-small protein.

[0384] In some embodiments, the conjugates provided by the present disclosure include small proteins. In some such embodiments, the small proteins act as targeting moieties, such as specifically binding to a target, such as a protein expressed on the surface of a ...

Claims

1. A polypeptide comprising: an amino acid sequence, wherein the amino acid sequence comprises formula I: CX1YDX2X3FFTALX4X5LRGX6DICX7YIX8X9X10FX11X12X13X14X15X16CIX17EILX18X19LGCX20(SEQ ID NO:161) wherein X20 is an optional -OH-containing amino acid or a carboxyl terminus, and wherein X1 is D or E; X2 is E or G; X3 is Q or E; X4 is A, K or S; X5 is A, R, Q, K, S or Cit; X6 is A, D, G or S; X7 is D, Q, E, L, S or Y; X8 is Q, L or S; X9 is A, Q, E, K; X10 is A, Q, K, S, Y, OH-norleucine or norleucine; X11 is A, N, Q or S; X12 is N, T or Y; X13 is L, Y or V; X14 is P or E; X15 is A, D, Q, G or K; X16 is D, Q, E, I or L; X17 is Q or E; X18 is D, Q or E; X19 is N or Q; and X20, when present as an amino acid, is S.

2. The polypeptide of claim 1, wherein X1 is E; X2 is E; X3 is E; X4 is K; X5 is R; X6 is G; X7 is Y; X8 is Q; X9 is A; X10 is S; X11 is Q; X12 is Y; X13 is L; X14 is P; X15 is G; X16 is L; X17 is E; X18 is D; X19 is N; and X20 is S.

3. The polypeptide of claim 1 or claim 2, further comprising one or more of a linker, a chelator and a radionuclide.

4. The polypeptide of claim 3, wherein the linker comprises or consists of: PEG2, PEG, PEG4, PEG6, PEG8, PEG12, PEG24, a polyethylene glycol (PEG) linker, an ester linker, an amide linker, a maleimide linker, a succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) linker, a propionic acid linker, a decenoic acid linker, or (Gly)n-(gGlu)n- or (PEG)n, (Gly)1-10 wherein n is 1 to 10, or any fragment or combination thereof via a covalent bond.

5. The polypeptide of claim 3, wherein the chelating agent comprises or consists of: DOTA, DOPA, macrocyclic ligands or coronas.

6. The polypeptide of claim 3, wherein the radionuclide is selected from Ac-225, Ga-68, In-111, Pb-212, Lu-177, Cu-67, Cu-64, La-132, La-135 or Ce-134.

7. The polypeptide of any one of claims 3-6, wherein the linker, when present, is attached to the C-terminus of the polypeptide.

8. The polypeptide of claims 3-7, wherein the chelator, when present, is attached to the polypeptide or the linker.

9. The polypeptide of any one of claims 3-8, wherein the radionuclide, when present, is attached to the chelator.

10. A polypeptide as claimed in any preceding claim comprising one or more additional N-terminal amino acids.

11. A polypeptide as claimed in any one of the preceding claims, comprising one or more additional amino acids located at the N-terminus and / or C-terminus of the polypeptide.

12. A composition as claimed in the preceding claim, wherein the polypeptide is a monomer.

13. The composition of any one of claims 1-11, wherein the polypeptide is a dimer, trimer or tetramer.

14. The polypeptide of claim 1, comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:

78.

15. The polypeptide of claim 1, comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 1-158 and 177.

16. A polypeptide comprising or consisting of a consensus sequence according to any one of Formulas IIA-IIO (SEQ ID NOs: 161-176) of Table 1B, further comprising substitutions according to those shown in Table 1C.

17. A composition represented by one or more formulas selected from (M)xLCR, (M)xLC, (M)xCR, (M)xLR, (M)xC, (M)xL and (M)xR, wherein M comprises a small protein (M), L comprises a linker (L), C comprises a chelator (C), R comprises a radionuclide (R), and x is 1, 2, 3 or 4.

18. A composition as described in claim 17, wherein M comprises or consists of a sequence selected from any one of SEQ ID NO: 78, or SEQ ID NO: 1-77, 79-158, 177, 161-176 (including amino acid substitutions as shown in Table 1C), or a functional variant or portion thereof.

19. The composition of claim 18, wherein when L is present, L comprises or consists of: PEG4, PEG, PEG2, PEG6, PEG8, PEG12, PEG24, polyethylene glycol (PEG) linker, ester linker, amide linker, maleimide linker, valine-citrulline linker, hydrazone linker, N-succinimidyl-4-(2-pyridyldithio)butyrate (SPDB) linker, succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) linker, vinyl sulfone-based linker, propionic acid linker, decenoic acid linker, or (Gly)n-(gGlu)n- or (PEG)n, (Gly)1-10, or any fragment or combination thereof via a covalent bond.

20. A composition as described in any one of claims 17-19, wherein when C is present, C comprises or consists of: i) DOTA ii)Crown iii) NOPO, or iv) Macrocyclic ligands 21. A composition as described in any one of claims 17-20, wherein when R is present, R comprises or consists of: Ac-225, In-111, Ga-68, Pb-212, Lu-177, Cu-67, Cu-64, La-132, La-135 or Ce-134.

22. A method selected from (M) x -LCR, (M) x -LC, (M) x -CR, (M) x -LR, (M) x -C, (M) x -L and (M) x -R, wherein M comprises a small protein (M), L comprises a linker (L), C comprises a chelator (C), R comprises a radionuclide (R), and x is 1, 2, 3 or 4, wherein M comprises a small protein that binds to connexin-4 or a functional variant or portion thereof.

23. The composition of claim 22, wherein M comprises or consists of a cysteine ​​dense peptide, a knottin peptide, a conjugate, an affibody, an engineered Kunitz domain, a monoclonal antibody, an anticalin, a designed ankyrin repeat domain (DARPin), or an avimer.

24. The composition of claim 23, wherein the conjugate comprises or consists of a linear polypeptide and / or a non-disulfide sequence.

25. A composition as described in any of claims 22-24, wherein M is characterized in that it comprises (i) no more than 100 amino acids and / or 12 kDa; (ii) at least one secondary structure element; (iii) an isolated hydrophobic core; and / or exhibits cooperative folding.

26. The composition of any one of claims 22-25, wherein M comprises no more than 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, 10 amino acids, or 5 amino acids.

27. The composition of any one of claims 22-26, wherein the small protein comprises at least one disulfide bridge.

28. The composition of any one of claims 22-27, wherein the amino acid sequence of the small protein comprises at least three cysteine ​​(C) amino acids.

29. The composition of any one of claims 22-28, wherein the small protein comprises at least two disulfide bridges.

30. A composition as represented by LC, wherein L comprises or consists of a linker, C comprises or consists of a chelator, and wherein the linker is conjugated to the polypeptide.

31. A composition as shown in LCR, wherein L comprises or consists of a linker, C comprises or consists of a chelator, and R comprises or consists of a radionuclide, and wherein the composition is conjugated to a small protein.

32. A composition as described in any one of claims 22-31, wherein L, when present, comprises or consists of: PEG4, PEG, PEG2, PEG6, PEG8, PEG12, PEG24, polyethylene glycol (PEG) linker, ester linker, amide linker, maleimide linker, valine-citrulline linker, hydrazone linker, N-succinimidyl-4-(2-pyridyldithio)butyrate (SPDB) linker, succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) linker, vinyl sulfone-based linker, propionic acid linker, decenoic acid linker, or (Gly)n-(γGlu)n- or (PEG)n, (Gly) wherein n is 1 to 10. 1-10 , or any fragment or combination thereof via a covalent bond.

33. A composition as described in any one of claims 22-31, wherein there is, when present, comprising or consisting of: i) DOTA ii)Crown iii) NOPO, or iv) Macrocyclic ligands 34. A composition as described in any one of claims 22-33, wherein C, when present, comprises or consists of: Derivatives of tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), crown, NOPO or macrocyclic ligands.

35. The composition of any one of claims 22-29, wherein when L is present and C is absent, L is covalently attached to M.

36. A composition as described in any one of claims 22-35, wherein R, when present, comprises or consists of: Ac-225, In-111, Ga-68, Pb-212, Lu-177, Cu-67, Cu-64, La-132, La-135 or Ce-134.

37. The composition of any one of claims 22-36, wherein the composition binds to Connexin-4 with an affinity of 1 pM to 100 nM.

38. The composition of any one of claims 22-37, wherein when the small protein is present, binding of the small protein to the target modulates a biological function.

39. The composition of any one of claims 22-38, wherein when M is present, M selectively binds to connexin-4 or a portion thereof.

40. The composition of any one of claims 22-39, comprising a small protein (M) comprising an amino acid sequence selected from any one of SEQ ID NOs: 1-158 or 177.

41. The composition of any one of claims 22-40, wherein the miniprotein (M) comprises an amino acid sequence selected from any one of SEQ ID NOs: 161-176.

42. The composition of claim 41, wherein M comprises an amino acid substitution at any one of SEQ ID NOs: 161-176 as shown in Table 1C.

43. A composition comprising a small protein-drug conjugate comprising a small protein and at least one drug moiety.

44. The composition of claim 43, wherein the drug moiety is selected from a V-ATPase inhibitor, a pro-apoptotic agent, a Bcl2 inhibitor, a MCL1 inhibitor, a HSP90 inhibitor, an IAP inhibitor, an mTor inhibitor, a microtubule stabilizer, a microtubule destabilizer, an auristatin, a dolastatin, a maytansinoid, a MetAP (methionine aminopeptidase), an inhibitor of protein CRM1 nuclear export, a DPPIV inhibitor, a proteasome inhibitor, an inhibitor of phosphoryl transfer reactions in mitochondria, a protein synthesis inhibitor, a kinase inhibitor, a CDK2 inhibitor, a CDK9 inhibitor, a kinesin inhibitor, an HDAC inhibitor, a DNA damaging agent, a DNA alkylating agent, a DNA intercalating agent, a DNA minor groove binder, a DHFR inhibitor, a topoisomerase inhibitor, an auristatin (e.g., monomethyl auristatin E), and an immunotoxin.

45. A composition comprising an isolated compound or a pharmaceutically acceptable salt thereof, wherein the isolated compound or a pharmaceutically acceptable salt thereof comprises an optional linker (L), and one or more of a miniprotein (M), a chelator (C), or a radionuclide (R).

46. ​​The composition of claim 45, wherein the small protein comprises an amino acid sequence selected from any one of SEQ ID NOs: 1-158 or 177 or an amino acid sequence according to any one of SEQ ID NOs: 161-176 and / or Table 1C.

47. A pharmaceutical composition comprising one or more miniproteins (M)x, wherein the miniproteins selectively bind to connexin-4, and x is 1, 2, 3 or 4.

48. A pharmaceutical composition comprising one or more of: a miniprotein (M) x, an optional linker (L), and one or both of a chelator (C) and a radionuclide (R), wherein x is 1, 2, 3 or 4.

49. The pharmaceutical composition of claim 48, wherein when C is present, C is covalently attached to M.

50. The pharmaceutical composition of claim 48 or 49, wherein the chelation efficiency is >90%.

51. The pharmaceutical composition of claim 50, further comprising a radionuclide R.

52. A pharmaceutical composition as described in any one of claims 48-51, wherein R, when present, is an alpha emitter.

53. A pharmaceutical composition as described in any one of claims 48-52, wherein R when present is Ac-225, In-111, Ga-68, Pb-212, Lu-177, Cu-67, Cu-64, La-132, La-135 or Ce-134.

54. The pharmaceutical composition of any one of claims 28-53, wherein the small protein specifically binds to connexin-4.

55. The pharmaceutical composition of claim 47 or 54, wherein the connexin-4 is expressed on a cell.

56. The pharmaceutical composition of claim 55, wherein the cell is a human cell.

57. The pharmaceutical composition of claim 56, wherein the human cell is a tumor cell.

58. The pharmaceutical composition of claim 57, wherein the tumor cell is a solid tumor cell.

59. A pharmaceutical composition as described in any of claims 47-58, wherein the small protein comprises or consists of a cysteine-dense peptide, a knottin peptide, a conjugate, an affibody, an engineered Kunitz domain, a monoclonal antibody, an anticalin, a designed ankyrin repeat domain (DARPin) or an avimer.

60. The pharmaceutical composition of any one of claims 47-59, wherein the small protein comprises zero, one, two or more disulfide bonds.

61. The pharmaceutical composition of any one of claims 47-60, wherein the small protein is characterized in that it has nM or sub-nM binding affinity for connexin-4 in vivo or in a cell-based assay.

62. The pharmaceutical composition of any one of claims 47-61, wherein the small protein has a binding affinity of 1 pM to 100 nM for connexin-4 on a cell line expressing human connexin-4.

63. The pharmaceutical composition of any one of claims 47-62, wherein the small protein has a binding affinity that is about the binding affinity disclosed for the compounds in Table 2.

64. A pharmaceutical composition as described in any of claims 47-63, wherein the miniprotein has an amino acid sequence of no more than 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, 10 amino acids, or 5 amino acids.

65. A pharmaceutical composition as described in any one of claims 47-64, wherein administration of the pharmaceutical composition to a subject in need thereof does not elicit an immune response or wherein the immune response elicited is tolerable to the subject.

66. The pharmaceutical composition of any one of claims 47-65, wherein the composition comprises increased tumor tissue penetration compared to the full-size protein.

67. The pharmaceutical composition of any one of claims 47-66, wherein the composition exhibits adhesion in a tumor.

68. The pharmaceutical composition of any one of claims 47-67, wherein the composition exhibits transit through the kidney, liver, bone marrow or spleen.

69. The pharmaceutical composition of any one of claims 47-68, wherein the composition is internalized in a cell expressing human connexin-4.

70. The pharmaceutical composition of any one of claims 47-69, wherein the small protein comprises an amino acid sequence selected from any one of SEQ ID NO: 1-158 or 177.

71. The pharmaceutical composition of any one of claims 47-69, wherein the small protein comprises an amino acid sequence according to any one of SEQ ID NOs: 161-176.

72. The pharmaceutical composition of claim 71, wherein the amino acid sequence of the miniprotein further comprises one or more substitutions as shown in Table 1C.

73. A pharmaceutical composition comprising a small protein-drug conjugate comprising a small protein and at least one drug moiety.

74. A pharmaceutical composition as described in claim 73, wherein the drug moiety is selected from V-ATPase inhibitors, pro-apoptotic agents, Bcl2 inhibitors, MCL1 inhibitors, HSP90 inhibitors, IAP inhibitors, mTor inhibitors, microtubule stabilizers, microtubule destabilizers, auristatins, dolastatins, maytansinoids, MetAP (methionine aminopeptidase), protein CRM1 nuclear export inhibitors, DPPIV inhibitors, proteasome inhibitors, inhibitors of phosphoryl transfer reactions in mitochondria, 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.

75. The pharmaceutical composition of any one of claims 47-74, comprising one or more antioxidant molecules, wherein the antioxidant molecules neutralize free radicals.

76. The pharmaceutical composition of any one of claims 47-74, comprising at least one stabilizer.

77. The pharmaceutical composition of claim 76, wherein 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.

78. The pharmaceutical composition of any one of claims 47-77, wherein the pharmaceutical composition is characterized by exhibiting improved resistance to peptidases, proteases, and / or heat.

79. A pharmaceutical composition as described in any of claims 47-78, wherein the pharmaceutical composition is characterized in that it exhibits improved stability and / or reduced degradation.

80. A method of treating a subject in need thereof, the method comprising administering a composition comprising at least one of: a miniprotein (M), an optional linker (L), and one or both of a chelator (C) and a radionuclide (R).

81. The method of claim 80, wherein L comprises or consists of: PEG4, PEG, PEG2, PEG6, PEG8, PEG12, PEG24, polyethylene glycol (PEG) linker, ester linker, amide linker, maleimide linker, valine-citrulline linker, hydrazone linker, N-succinimidyl-4-(2-pyridyldithio)butyrate (SPDB) linker, succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) linker, vinyl sulfone-based linker, propionic acid linker, decenoic acid linker, or (Gly)n-(γGlu)n- or (PEG)n, (Gly) wherein n is 1 to 10. 1-10 , or any fragment or combination thereof via a covalent bond.

82. The method of claim 80 or 81, wherein C comprises or consists of: i) DOTA ii)Crown iii) NOPO, or iv) Macrocyclic ligands 83. The method of any one of claims 80-82, wherein R comprises or consists of: Ac-225, In-111, Ga-68, Pb-212, Lu-177, Cu-67, Cu-64, La-132, La-135 or Ce-134.

84. The method of any one of claims 80-83, wherein M comprises or consists of a cysteine ​​dense peptide, a knottin peptide, a binder, an affibody, an engineered Kunitz domain, a monoclonal antibody, an ankyrin, a designed ankyrin repeat domain (DARPin), or an avimer.

85. The method of any one of claims 80-84, wherein M is characterized in that it comprises (i) no more than 100 amino acids and / or 12 kDa; (ii) at least two secondary structure elements; (iii) an isolated hydrophobic core; and / or exhibits cooperative folding.

86. The method of any one of claims 80-85, wherein the composition is capable of penetrating tumor tissue.

87. The method of any one of claims 80-86, wherein the small protein comprises no more than 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, 10 amino acids, or 5 amino acids.

88. The method of any one of claims 80-87, wherein the small protein comprises at least one disulfide bridge.

89. The method of any one of claims 80-88, wherein the small protein specifically binds to a target.

90. The method of any one of claims 80-89, wherein the composition exhibits mm or nM binding affinity for Connexin-4 in an in vitro assay.

91. The method of any one of claims 80-90, wherein the composition binds to Connexin-4 with an affinity of 1 pM to 100 nM.

92. The method of any one of claims 80-90, wherein the composition binds Connexin-4 with about an affinity shown for any of the compounds of Table 1C.

93. The method of any one of claims 80-92, wherein the composition is characterized by having high tissue penetration properties relative to a composition comprising a full-size protein that binds connexin-4.

94. The method of any one of claims 80-93, wherein the small protein that binds to the target modulates a biological function.

95. The method of any one of claims 80-94, wherein administration of the composition to a subject in need thereof does not elicit an immune response or wherein the immune response elicited is tolerable to the subject.

96. The method of claim 95, wherein the tolerable immune response comprises a systemic immune response or a local immune response.

97. The method of claim 95 or 96, wherein the subject is diagnosed with cancer.

98. The method of claim 97, wherein the cancer cells from the subject express connexin-4 or a portion thereof.

99. The method of claim 98, wherein expression of the target in the cancer cells is higher than in non-cancerous cells.

100. The method of any one of claims 97-99, wherein the cancer is selected from 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 cancer, liver cancer, male breast cancer, malignant mesothelioma, medulloblastoma, Merkel cell cancer, metastatic squamous cell carcinoma of the neck, multiple myeloma and other plasma cell neoplasms, mycosis fungoides and Sezary 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.

101. The method of any one of claims 80-100, wherein the composition is administered intravenously or subcutaneously.

102. The method of claim 101, wherein the cancer is treated after administering the composition.

103. A method for targeting a cancer cell population expressing connexin-4, the method comprising: (i) determining the expression level of connexin-4 in a cancer cell population; (ii) administering to a subject in need thereof a composition comprising a polypeptide according to any one of claims 1 to 16, a composition according to any one of claims 17 to 45, or a pharmaceutical composition according to any one of claims 46 to 79, wherein the composition comprises at least one component that specifically binds to connexin-4, wherein the composition is targeted to the cell expressing connexin-4 and is internalized into the cell expressing connexin-4; as well as (iii) wherein said patient is treated after said administering compared to before said administering; and said treatment preferentially damages cells expressing connexin-4.

104. The method of claim 103, wherein the polypeptide comprises or consists of: A small protein (M), comprising an amino acid sequence selected from any one of SEQ ID NOs: 1-158 or 177 or 161-176 and / or Table 1C.

105. A small protein conjugate comprising: (i) a small protein that specifically binds to connexin-4 (M); (ii) a chelator (C) conjugated to (M) via an optional linker (L), wherein (C) comprises DOTA and (L) when present comprises PEG; and (iii) a radionuclide (R) chelated to (C), wherein (R) is actinium-225.

106. A small protein conjugate comprising: (i) a small protein that specifically binds to connexin-4 (M); (ii) a chelator (C) conjugated to (M) via an optional linker (L), wherein (C) comprises DOTA and (L) when present comprises PEG; and (iii) a radionuclide (R) chelated to (C), wherein (R) is indium-111.

107. A small protein conjugate comprising: (i) a small protein that specifically binds to connexin-4 (M); (ii) a chelator (C) conjugated to (M) via an optional linker (L), wherein (C) comprises DOTA and (L) when present comprises PEG; and (iii) a radionuclide (R) chelated to (C), wherein (R) is gallium-68.

108. A small protein conjugate comprising: (i) a small protein that specifically binds to connexin-4 (M); (ii) a chelator (C) conjugated to (M) via an optional linker (L), wherein (C) comprises DOTA and (L) when present comprises PEG; and (iii) a radionuclide (R) chelated to (C), wherein (R) is copper-64.

109. The small protein conjugate of any of the preceding claims, comprising a small protein (M) comprising an amino acid sequence selected from any one of SEQ ID NOs: 1-158 or 177 or 161-176 and / or Table 1C.

110. An isolated polypeptide having an amino acid sequence comprising or consisting of: One or more amino acid sequences selected from SEQ ID NO: 1-158 or 177; or One or more amino acid sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 1-158 or 177.

111. An isolated polypeptide having an amino acid sequence comprising or consisting of: One or more amino acid sequences selected from SEQ ID NOs: 161-176 according to Tables 1B and 1C; or one or more amino acid sequences having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity with SEQ ID NOs: 161-176 according to Tables 1B and 1C.

112. The isolated polypeptide of claim 110 or 111, wherein the polypeptide comprises no more than 100 amino acids.

113. The isolated polypeptide of any one of claims 110-112, wherein the polypeptide specifically binds to connexin-4 or a portion thereof.

114. An isolated polynucleotide comprising one or more nucleic acid sequences encoding a polypeptide selected from SEQ ID NO: 1-158 or 177; or a nucleic acid sequence encoding a polypeptide having at least 90%, 95%, 96%, 97%, 98%, 99% or higher identity with SEQ ID NO: 1-158 or 177.

115. An isolated polynucleotide comprising one or more nucleic acid sequences encoding a polypeptide selected from SEQ ID NOs: 161-176 and / or according to Tables 1B and 1C; or a nucleic acid sequence encoding a polypeptide having at least 90%, 95%, 96%, 97%, 98%, 99% or higher identity with a polypeptide of SEQ ID NOs: 161-176 and / or according to Tables 1B and / or 1C.

116. A vector comprising the isolated polynucleotide of claim 114 or 115.

117. A host cell transformed with the isolated polynucleotide of claim 114 or 115 or the vector of claim 116.

118. A method of producing a product selected from (M) x -LCR, (M) x -LC, (M) x -CR, (M) x -LR, (M) x -C, (M) x -L and (M) x -R, wherein M comprises a small protein (M), L comprises a linker (L), C comprises a chelator (C), R comprises a radionuclide (R), and x is 1, 2, 3 or 4, the method comprising a. Synthetic small protein (M) and / or linker (L), and b. optionally reacting a chelating agent (C) and / or a radionuclide (R), and c. Conjugation i. one or more of the miniproteins (M) to the linker (L), ii. one or more of said small proteins (M) to said chelating agent (C), iii. one or more of said small proteins (M) to said radionuclide (R), iv. one or more of said small proteins (M) to said linker (L) to said chelator (C), v. one or more of said miniproteins (M) to said linker (L) to said chelator (C) and said radionuclide (R), vi. one or more of said small proteins (M) to said chelating agent (C) to said radionuclide (R), or vii. One or more of said miniproteins (M) to said linker (L) to said radionuclide (R).

119. The method of claim 118, wherein the reaction of the chelating agent (C) and the radionuclide (R) is performed at a temperature between 10°C and 65°C during incubation.

120. The method of claim 118, wherein the reaction of the chelating agent (C) and the radionuclide (R) is carried out within an incubation period of 5 minutes to 30 minutes.

121. The method of claim 118, wherein the reaction of the chelating agent (C) and the radionuclide (R) is carried out at a pH ranging from 5.0 to 7.

4.

122. The method of claim 118, wherein the reaction of the chelating agent (C) and the radionuclide (R) is carried out in an aqueous solution that is substantially free of alcohol.

123. A method of delivering a radionuclide to a selected location in a patient, the method comprising administering to the patient a composition comprising a radionuclide as provided herein.

124. The method of claim 123, wherein the radionuclide is selected from Ac-225, In-111, Ga-68, Pb-212, Lu-177, Cu-67, Cu-64, La-132, La-135, or Ce-134.

125. The method of claim 123 or 124, further comprising performing an imaging procedure to evaluate the localization of the radionuclide in vivo, wherein the imaging procedure optionally comprises positron emission tomography (PET) imaging or single photon emission computed tomography (SPECT) imaging.

126. The method of claim 125, wherein the imaging procedure allows for selection of a patient.

127. The method of claim 126, wherein the imaging procedure allows monitoring of the patient.

128. The method of claim 125, wherein the imaging procedure allows determination of an appropriate dose for treating a patient in need of a pharmaceutical composition provided herein.

129. A method selected from (M) x -LCR, (M) x -LC, (M) x -CR, (M) x -LR, (M) x -C, (M) x -L and (M) x -R, wherein M comprises a small protein (M), L comprises a linker (L), C comprises a chelator (C), R comprises a radionuclide (R), and x is 1, 2, 3 or 4.

130. The composition of claim 129, wherein M comprises or consists of a sequence selected from any one of the following: SEQ ID NO: 1-158 or 177 or a functional variant or part thereof or SEQ ID NO: 161-176 and / or according to Table 1B and / or 1C or a functional variant or part thereof.

131. The composition of claim 129, wherein L, when present, comprises or consists of: PEG4, PEG, PEG2, PEG6, PEG8, PEG12, PEG24, polyethylene glycol (PEG) linker, ester linker, amide linker, maleimide linker, valine-citrulline linker, hydrazone linker, N-succinimidyl-4-(2-pyridyldithio)butyrate (SPDB) linker, succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) linker, vinyl sulfone-based linker, propionic acid linker, decenoic acid linker, or (Gly)n-(γGlu)n- or (PEG)n, (Gly) wherein n is 1 to 10. 1-10 , or any fragment or combination thereof via a covalent bond.

132. The composition of any one of claims 129-131, wherein C, when present, comprises or consists of: i) DOTA ii)Crown iii) NOPO, or iv) Macrocyclic ligands 133. The composition of any one of claims 129-132, wherein R, when present, comprises or consists of: Ac-225, In-111, Ga-68, Pb-212, Lu-177, Cu-67, Cu-64, La-132, La-135 or Ce-134.

134. The composition of any one of claims 129-133, wherein M does not bind to a non-target with an affinity greater than 10 nM.

135. The composition of claim 134, wherein the non-target does not comprise connexin-4 or a functional variant or portion thereof.

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