Methods of using surface-expressible activatable epitopes to locate and / or treat diseased cells - Patents.com

Abstract

This application relates to engineered polypeptides, which include an extracellular targeting domain that contains an epitope (e.g., derived from DLL3, PSMA, or SSTR2), an extracellular targeting polypeptide spacer domain, and a transmembrane / membrane affinity domain.Methods of using such engineered polypeptides for cancer detection, imaging, and treatment are also described.Imaging modalities such as immunoPET allow the specificity of antibodies and protein ligands to be combined with the sensitivity of PET imaging.

Description

[Technical field]

[0001] cross reference This application claims the benefit of U.S. Provisional Application No. 63 / 338,435, filed May 4, 2022, entitled "METHODS OF USING SURFACE-EXPRESSABLE ACTIVATABLE EPITOPES TO IMAGE AND / OR TREAT DISEASED CELLS," and U.S. Provisional Application No. 63 / 429,783, filed December 2, 2022, entitled "METHODS OF USING SURFACE-EXPRESSABLE ACTIVATABLE EPITOPES TO DETERMINE THE LOCALIZATION OF DISEASED CELLS AND / OR TREAT DISEASED CELLS," each of which is incorporated by reference in its entirety herein. [Background technology]

[0002] background Imaging modalities such as immunoPET make it possible to combine the specificity of antibodies and protein ligands with the sensitivity of PET imaging.

[0003] In addition, affinity-based reagents, antibodies and protein ligands conjugated with radionuclide moieties, toxins and peptide fragments can be used to induce cell damage and cause cell death in the same locations as PET imaging using antibodies and protein ligands. Summary of the Invention [Means for solving the problem]

[0004] summary The combination of immunoPET (using affinity-based reagents) and affinity-based reagents conjugated with detectable or therapeutic moieties (radionuclide moieties, toxins and peptide fragments used to induce cell damage) and inducing cancer-specific expression of markers targeted by the affinity-based reagents may enable advantageous new modalities for detecting, imaging or treating cancer in situ. Described herein are methods, systems and compositions for enhancing affinity-based targeting of cancer for detection or treatment.

[0005] In some aspects, the disclosure provides a nucleic acid encoding an engineered polypeptide, the polypeptide comprising: (a) an extracellular-targeting domain comprising an epitope capable of binding to a polypeptide (i) an antibody or (ii) a peptide hormone or growth factor, the extracellular-targeting domain not comprising an scFv; (b) an extracellular-targeting polypeptide spacer domain having a length of about 15 to about 40 angstroms when folded; and (c) a transmembrane domain or membrane affinity domain capable of associating with the outer membrane of a cell, wherein at least two of (a), (b), and (c) are heterologous to each other. In some embodiments, the polypeptide does not comprise a light chain variable (VL) domain. In some embodiments, the engineered polypeptide does not comprise an intracellular signaling domain. In some embodiments, the engineered polypeptide does not comprise an intracellular portion of a CD3 zeta, CD137, or CD28 polypeptide. In some embodiments, the intracellular-targeting portion of the polypeptide comprises less than or equal to 100, 75, 50, 25, 10, or 5 residues. In some embodiments, the extracellular-directed polypeptide spacer domain comprises a hinge domain. In some embodiments, (a), (b), and (c) are in the order of N-terminus to C-terminus of the engineered polypeptide. In some embodiments, the engineered polypeptide is capable of being presented at the cell surface. In some embodiments, the epitope further comprises an activatable epitope selectively available for binding to an extracellular ligand in the tumor microenvironment. In some embodiments, the activatable epitope is flanked by at least two copies of a pH-sensitive helix. In some embodiments, the epitope capable of binding (i) an antibody or (ii) a peptide hormone or growth factor is derived from DLL3, PSMA, SSTR2, or any combination thereof. In some embodiments, the epitope is derived from DLL3, and the epitope comprises about 15 to about 260 contiguous residues of an extracellular domain of DLL3 having at least 80% identity to SEQ ID NO: 5 or an extracellular domain of any of the proteins described herein, or a variant thereof.In some embodiments, the epitope comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 4, 5, 19, 20, 64, 65, or a variant thereof. In some embodiments, the epitope can bind to (i) an antibody or (ii) a peptide hormone or growth factor with a Kd of less than or equal to 20 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM. In some embodiments, the extracellular-directing polypeptide spacer domain comprises a hinge sequence from CD8, CD8a, CD8b, IgG4, IgG1, IgG2, IgG3, IgK, CD4, or CD28, or any combination thereof. In some embodiments, the extracellular-directed polypeptide spacer domain comprises a hinge sequence having at least 80% sequence identity to any one of the hinge sequences of SEQ ID NOs: 56, 57, 58, 59, 60, 61, 62, 66, 67 or any of the proteins described herein, or a variant thereof. In some embodiments, the transmembrane domain or membrane affinity domain comprises a transmembrane domain. In some embodiments, the transmembrane domain comprises a single-pass transmembrane domain. In some embodiments, the transmembrane domain comprises a transmembrane domain from DLL3, PSMA, SSTR2, PD-L1, EGFR, CD28, CD4, CD8, CD8a, CD8b, ICOS, or CD73. In some embodiments, the transmembrane domain comprises a sequence having at least 80% sequence identity to any one of the transmembrane domains of SEQ ID NOs: 68-76 or any of the proteins described herein, or a variant thereof. In some embodiments, the transmembrane domain or membrane affinity domain comprises a membrane affinity domain. In some embodiments, the membrane affinity domain comprises a C1, C2, PH, FYVE, PX, or ENTH domain, or a transmembrane helix from bacteriorhodopsin. In some embodiments, the membrane affinity domain comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 77-82, or any membrane affinity domain of the proteins described herein, or a variant thereof.In some embodiments, the engineered polypeptide further comprises a scaffold domain (i) N-terminal to the hinge domain and C-terminal to the epitope; or (ii) N-terminal to the hinge domain and containing the epitope. In some embodiments, the engineered polypeptide further comprises a scaffold domain N-terminal to the hinge domain and C-terminal to the epitope. In some embodiments, the scaffold comprises a sequence having at least 80% sequence identity to SEQ ID NO: 63, or a variant thereof. In some embodiments, the engineered polypeptide further comprises a scaffold domain N-terminal to the hinge domain and containing the epitope. In some embodiments, the scaffold comprises a heavy chain variable (VH) domain and does not comprise a light chain variable (VL) domain. In some embodiments, the VH domain comprises an inactivating mutation in the CDR1, CDR2, or CDR3 region of the VH domain. In some embodiments, the scaffold comprises an epitope inserted into the CDR1, CDR2, or CDR3 region of the VH domain. In some embodiments, the nucleic acid further comprises an ORF encoding the engineered polypeptide. In some embodiments, the nucleic acid further comprises a promoter operably linked to the ORF. In some embodiments, the promoter is not a T cell specific promoter or a TCRA, TCRB, CMV, EF-1, hPGK, CD3, or RPBSA promoter. In some embodiments, the promoter is a cancer specific promoter. In some embodiments, the promoter is a promoter of a gene that is overexpressed in cancer cells compared to normal cells, or a functional fragment thereof. In some embodiments, the promoter is a survivin promoter (BIRC5), a CXCR4 promoter, an ATP-binding cassette subfamily C member 4 (ABCC4) promoter, anterior gradient 2, a protein disulfide isomerase family member (AGR2) promoter, an activation-induced cytidine deaminase (AICDA) promoter, a UDP-GlcNAc:beta Gal beta-1,3-N-acetylglucosaminyltransferase 3 (B3GNT3) promoter, a cadherin 3 (CDH3) promoter, a CEA cell adhesion molecule 5 (CEACAM5) promoter, a centromere protein F (CENPF) promoter, a centrosomal protein 55 (CEP55) promoter, a claudin 3 (CLDN3) promoter, a claudin 4 (CLDN4) promoter, a collagen type XI alpha 1 chain (COL11A1) promoter, a collagen type I alpha 1 chain (COL1A1) promoter, a cystatin SN (CST1) promoter, a dentate-less E3 ubiquitin Deoxyribonuclease protein ligase homolog (DTL) promoter, family 111 member B with sequence similarity (FAM111B) promoter, forkhead box A1 (FOXA1) promoter, kinesin family member 20A (KIF20A), laminin subunit gamma 2 (LAMC2) promoter, mitotic spindle positioning (MISP) promoter, matrix metallopeptidase 1 (MMP1) promoter, matrix metallopeptidase 12 (MMP12) promoter, and matrix metallopeptidase 13 (MMP14) promoter. metallopeptidase 13 (MMP13) promoter, mesothelin (MSLN) promoter, cell surface-associated mucin 1 (MUC1) promoter, phospholipase A2 group IID (PLA2G2D) promoter, regulator of G protein signaling 13 (RGS13) promoter, secretoglobin family 2A member 1 (SCGB2A1) promoter, topoisomerase II alpha (TOP2A) promoter, ubiquitin D (UBD) promoter, ubiquitin-conjugating enzyme E2 (U2) promoter,C (UBE2C), USH1 protein network component harmonin (USH1C), V-set domain-containing T cell activation inhibitor 1 (VTCN1) promoter, hexokinase type II promoter, TRPM4 promoter, stromelysin 3 promoter, surfactant protein A promoter, secretory leukocyte protease inhibitor promoter, tyrosinase promoter, stress-inducible grp78 / BiP promoter, interleukin-10 promoter, alpha-B-crystallin / heat shock protein 27 promoter, epidermal growth factor receptor promoter, mucin-like glycoprotein promoter, mts1 promoter, NSE promoter, somatostatin tin receptor promoter, c-erbB-3 promoter, c-erbB-2 promoter, c-erbB4 promoter, thyroglobulin promoter, α-fetoprotein promoter, villin promoter, albumin promoter, glycoprotein A33 promoter, B cell specific Moloney leukemia virus insertion site 1 promoter, cyclooxygenase-2 promoter, fibroblast growth factor promoter; human epidermal growth factor receptor 2, human telomerase reverse transcriptase promoter; kinase domain insertion-containing receptor promoter; rad51 recombinase promoter; TTF-1, urokinase-type plasminogen activator receptor promoter, ubiquitin-conjugating enzyme E2 T (UBE2T) promoter, checkpoint kinase 1 (CHEK1) promoter, epithelial cell transforming 2 promoter (ECT2), BCL2-like 12 (BCL2L12) promoter, centromere protein I (CENPI) promoter, E2F transcription factor 1 (E2F1) promoter, flavin adenine dinucleotide synthetase 1 (FLAD1) promoter, protein phosphatase, Mg2+ / Mn2+-dependent 1G (PPM1G) promoter, ubiquitin-conjugating enzyme E2S (UBE2S) promoter, aurora kinase A and ninein interacting protein (AUNIP) promoter, cell division cycle 6 (CDC6) promoter, centromere protein L (CENPL) promoter, DNA replicative helicase / nuclease 2 (DNA2) promoter, DSN1 homolog, MIS12 kinetochore complex component (DSN1) promoter, deoxythymidylate kinase (DTYMK) promoter, G protein-regulated inducer of neurite outgrowth 1 (GPRIN1) promoter, mitochondrial fission regulator 2 (MTFR2) promoter, RAD51-associated protein 1 (RAD51AP1) promoter, small nuclear ribonucleoprotein polypeptide A' (SNRPA1) promoter, ATPase family, AAA domain containing 2 (ATAD2) promoter, BUB1 mitotic checkpoint serine / threonine kinase (BUB1) promoter, calcyclin-binding protein (CACYBP) promoter, cell division cycle associated 3 (CDCA3) promoter, centromere protein O (CENPO) promoter promoter, flap structure-specific endonuclease 1 (FEN1) promoter, forkhead box M1 (FOXM1) promoter, cell proliferation-regulated inhibitor of protein phosphatase 2A (KIAA1524) promoter, kinesin family member 2C (KIF2C) promoter, karyopherin subunit alpha 2 (KPNA2) promoter, MYB proto-oncogene-like 2 (MYBL2) promoter, NIMA-related kinase 2 (NEK2) promoter, RAN-binding protein 1 (RANBP1) promoter, small nuclear ribonucleoprotein polypeptides B and B1 (SNRPB) promoter, SPC24 / NDC80 kinetochore complex component (SPC24) promoter, transforming acidic coiled-coil-containing protein 3 (TACC3) promoter, TBC1 domain family member 31 (TBC1D31) promoter, thymidine kinase 1 (TK1) promoter, zinc finger protein 695 (ZNF695) promoter, aurora kinase A (AURKA) promoter, BLMRecQ-like helicase (BLM) promoter, chromosome 17 open reading frame 53 (C17orf53) promoter, chromobox 3 (CBX30) promoter, cyclin B1 (CCNB1) promoter, cyclin E1 (CCNE1) promoter, cyclin F (CCNF), cell division cycle 20 (CDC20) promoter, cell division cycle 45 (CDC45) promoter, cell division cycle associated 5 (CDCA5) promoter, cyclin-dependent kinase inhibitor 3 (CDKN3) promoter, cadherin EGF LAG 7-transmembrane G-type receptor 3 (CELSR3) promoter, centromere protein A (CENPA) promoter, centrosomal protein 72 (CEP72) promoter, CDC28 protein kinase regulatory subunit 2 (CKS2) promoter, type X collagen alpha 1 chain (COL10A1) promoter, chromosome segregation 1-like (CSE1L) promoter, DBF4 zinc finger promoter, GINS complex subunit 1 (GINS1) promoter, G protein-coupled receptor 19 (GPR19) promoter, kinesin family member 18A (KIF18A) promoter, kinesin family member 4A (KIF4A) promoter, kinesin family member C1 ( KIFC1) promoter, minichromosome maintenance 10 replication initiator (MCM10) promoter, minichromosome maintenance complex component 2 (MCM2) promoter, minichromosome maintenance complex component 7 (MCM7) promoter, MRG domain binding protein (MRGBP) promoter, methylenetetrahydrofolate dehydrogenase (NADP+-dependent) 2, methenyltetrahydrofolate cyclohydrolase (MTHFD2) promoter, non-SMC condensin I complex subunit H (NCAPH) promoter, kinetochore complex component (NDC80) promoter, nudix hydrolase 1 (NUDT1) promoter, ribonuclease H2 subunit A (RNASEH2A) promoter, RuvB-like AAA (RcA) promoter,ATPase1 (RUVBL1) promoter, serologically defined breast cancer antigen NY-BR-85 (SGOL1) promoter, SHC-binding and spindle-associated 1 (SHCBP1) promoter, small nuclear ribonucleoprotein polypeptide G (SNRPG) promoter, timeless circadian regulator promoter, thyroid hormone receptor interacting factor 13 (TRIP13) promoter, trophinin-related protein (TROAP) promoter, ubiquitin-conjugating enzyme E2 C (UBE2C) promoter, WD-repeat and HMG box DNA-binding protein 1 (WDHD1) promoter, alpha-fetoprotein (AFP) promoter, functional fragments thereof, any combination thereof, chimeric promoters compiled from multiple elements from the foregoing, or completely synthetic promoters composed of a tiled transcription-factor binding site derived from any of the foregoing.

[0006] In some aspects, the present disclosure provides a vector comprising any of the nucleic acids described herein. In some embodiments, the vector is a recombinant viral vector. In some embodiments, the vector is a non-viral vector.

[0007] In some aspects, the present disclosure provides a method of detecting, imaging, or treating cancer cells, comprising: (a) administering to a subject a composition comprising any of the nucleic acids or vectors described herein; and (b) administering to a subject an antibody or antigen-binding fragment thereof, a protein ligand or functional fragment thereof, or a small molecule configured to bind to an epitope. In some embodiments, the steps of administering to a subject a composition comprising a nucleic acid in (a) and administering to a subject an antibody or antigen-binding fragment thereof in (b) are at least about 8, 12, 16, 24, 36, 48, 60, 72, 84, or 96 hours apart. In some embodiments, the composition, or the antibody or antigen-binding fragment thereof, the protein ligand or functional fragment thereof, or the small molecule configured to bind to an epitope, is administered to a subject by parenteral, intramuscular, subcutaneous, intratumoral, rectal, vaginal, transdermal, or intravenous administration, or by cannula. In some embodiments, the composition comprising a nucleic acid or vector is configured for intravenous administration. In some embodiments, the method further comprises detecting binding of the antibody or antigen-binding fragment, protein ligand or functional fragment thereof, or small molecule to the epitope. In some embodiments, the antibody or antigen-binding fragment thereof further comprises or is chelated to a radioisotope or imaging agent (e.g., an MRI or X-ray imaging agent). In some embodiments, the antibody or antigen-binding fragment thereof further comprises or is chelated to a radioisotope. In some embodiments, the radioisotope comprises a positron-emitting radioisotope, an alpha-emitting radioisotope, a beta-emitting radioisotope, or a gamma-emitting radioisotope. In some embodiments, the radioisotope is a positron-emitting radioisotope, 124 I, 68 Ga, 11 C. 13 N, 15 O. 18 F, 68 Ga, 64 Cu, 52 Mn, 55 Co,89 Zr, 82 In some embodiments, the radioisotope comprises an alpha-emitting radioisotope, 225 Ac, 211 At, 227 Th, 224 In some embodiments, the radioisotope comprises a beta emitting radioisotope, 177 Lu, 67 Cu, 131 I, 90 Y, 89 Sr, 186 Re, 165 Dy, 32 P, 166 Ho, 188 In some embodiments, the radioisotope is a gamma ray emitting radioisotope, 99m Tc, 123 I, or 131In some embodiments, the antibody or antigen-binding fragment thereof further comprises an imaging agent or is chelated to an imaging agent. In some embodiments, the imaging agent comprises iron oxide nanoparticles (IONP), superparamagnetic iron platinum nanoparticles, manganese (II), or gadolinium (III). In some embodiments, the antibody or antigen-binding fragment thereof further comprises an antibody drug conjugate (ADC). In some embodiments, the antibody drug conjugate is conjugated to a pyrrolobenozdiazepine (PBD), a protein toxin, a diphtheria toxin, a glucagon-like peptide (GLP-1), a cytotoxic immunomodulatory protein, a Fas ligand, an auristatin or an analog thereof, a maytansinoid, a calicheamicin, a duocarmycin or an analog thereof, or a doxorubicin or an analog thereof. In some embodiments, the method further comprises detecting the binding of the antibody or antigen-binding fragment, protein ligand or its functional fragment, or small molecule to the epitope by MRI imaging, PET imaging, SPECT imaging, photoacoustic imaging, or luminescence imaging performed on the subject.In some embodiments, the epitope is presented in tumor cells of the subject.In some embodiments, the tumor cells are liver, ovarian, pancreatic, breast, lung, smooth muscle, bladder, kidney, skin, prostate, or bone tumor cells.

[0008] In some aspects, the disclosure provides a cell comprising any of the polypeptides, nucleic acids, or vectors described herein.

[0009] In some aspects, the disclosure provides a composition comprising any of the polypeptides, nucleic acids, or vectors described herein and a pharma- ceutically acceptable carrier. In some embodiments, the composition further comprises a transfection agent. Incorporation by Reference

[0010] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0011] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings. [Brief description of the drawings]

[0012] [Figure 1A] 1A shows an example of a cell surface expressible biomarker design according to some embodiments described herein, from N-terminus to C-terminus. In some cases, the cell surface biomarker comprises at least an extracellular domain and a transmembrane domain. In other cases, the cell surface biomarker comprises a signal peptide that guides membrane insertion, a hinge region, and an intracellular domain.

[0013] [Figure 1B] Figure IB depicts examples of N- to C-terminal linear construct designs for engineered SSTR2 (top and middle) and DLL3 (bottom) constructs. pH Helix: pH sensitive helix.

[0014] [Figure 1C] Figure 1C depicts examples of N- to C-terminal linear construct designs for engineered DLL3 (top) and GFP (bottom) constructs. mIgK: mouse IgK. pHLIP: pH-sensitive membrane affinity protein motif.

[0015] [Diagram 2]FIG. 2 depicts the function of a cell surface expressible biomarker containing an activatable epitope according to some embodiments described herein. In this embodiment, the activatable epitope is inserted in the middle of a pH sensitive helix (e.g., any of the helices described herein) such that at high pH (e.g., pH>6.0), the epitope is prevented by co-association of the pH sensitive helices. Upon transition of the cell surface biomarker in the disease microenvironment to low pH (e.g., <6.0, e.g., in an acidic tumor microenvironment), co-association of the pH sensitive helices is abolished and the epitope becomes accessible for binding, for example, by an antibody or a binding fragment of an antibody configured to bind to the epitope. Administration of an antibody or ligand that binds to the epitope can be used to detect, image, or bind the newly accessible epitope, thus assessing the presence of an accessible epitope in the disease environment.

[0016] [Diagram 3]Figure 3 depicts the function of a second cell surface expressible biomarker containing an activatable epitope according to some embodiments described herein. In this embodiment, a native cell surface receptor is engineered with a cancer activatable epitope, where the epitope sequence is inserted between a distal pH-sensitive helix (e.g., any of the helices described herein) and a proximal sequence that is post-translationally modified only if the protein reaches the cell surface in a cancer microenvironment-specific manner. In its basic conformation, the cancer activating epitope is such that at high pH (e.g., pH>6.0), the epitope is prevented from binding to affinity-based reagents. Upon transition of the cell surface biomarker in the disease microenvironment to low pH (e.g., <6.0, e.g., in an acidic tumor microenvironment), the sequence proximal to the surface-bound protein becomes glycosylated, stabilizing the framework expressing the epitope and simultaneously disrupting the association between the distal pH-sensitive helix and the cell surface receptor, allowing the epitope to become accessible for binding, e.g., by an antibody or antibody binding fragment configured to bind the epitope. Administration of an antibody or ligand that binds the epitope can be used to detect, image, or bind the newly accessible epitope, thus assessing the presence of accessible epitopes in the disease environment.

[0017] [Figure 4] 4 depicts the function of a third cell surface biomarker containing an activatable epitope according to some embodiments described herein. In this embodiment, a native cell surface receptor (e.g., SSTR2) is engineered with multiple pH-sensitive helices on the ends of the protein that co-associate at high pH and disrupt the ligand-bound state ("epitope") of the receptor. Upon transition of the receptor to low pH (e.g., in a tumor microenvironment), the pH-sensitive helices dissociate, allowing binding of a detectable synthetic analog of the native ligand (e.g., 68Ga-DOTATATE-labeled somatostatin "DOTATATE", which can be detected by PET / CT).

[0018] [Diagram 5] FIG. 5 depicts an example of a design for a secretable membrane-bound reporter molecule as described in Example 3.

[0019] [Figure 6] FIG. 6 depicts a proposed mechanism of function for the secretable membrane-bound reporter molecule described in Example 3.

[0020] [Figure 7] FIG. 7 represents an experiment described in Example 3 demonstrating that secretable membrane-bound reporter molecules such as those in FIGS. 5 and 6 propagate through the medium to untransfected cells.

[0021] [Figure 8A] FIG. 8A depicts a schematic for engineered delta-like ligand 3 (DLL3) surface display constructs (top) and the N- to C-terminal linear construct designs (bottom) of NP244, NP245, NP246, NP247, NP248, and NP249. DLL3 constructs were engineered by replacing the domain of the DLL3 protein that serves as the antigen target in a molecular scaffold where the transmembrane tethering domain serves to tether DLL3 on the cell surface. The SP represents the presence of a signaling peptide used to drive expression of the engineered DLL3 protein to the cell surface. The structure of DLL3 wild type (WT) with the SP is depicted on the far left. Two strategies were evaluated for enhanced surface trafficking, utilizing surface trafficking instructions (signal peptide or transmembrane tethering domain) from proteins known to be well expressed on the surface of lung cancer cells (B1, C1, and D1) or surface trafficking instructions commonly used for surface display of chimeric antigen receptors on immune cells (E1, F1, and G1). E1 contains the hIgG4 Fc domain, and F1 and G1 contain the CD8a hinge as an extracellular scaffold that serves to present the DLL3 epitope.

[0022] [Figure 8B]FIG. 8B represents the amount of DLL3 protein that can be measured by FACS analysis at the cell surface after transient transfection with a DNA expression plasmid expressing an engineered DLL3 variant. A fluorescently labeled antibody against DLL3 was used as the detection agent in this experiment. In A2, which corresponds to A1 in FIG. 8A, the H1299 cell line derived from a human cancer was transfected with a formulated DNA nanoplasmid that expressed wild-type DLL3 protein. Panels B2-G2 show the corresponding levels of DLL3 produced at the cell surface when a similar plasmid was introduced into H1299 cells. The data in FIG. 8B matches the scaffold in which DLL3 was modeled in FIG. 8A. B2 did not increase the expression level of DLL3. C2-G2 resulted in an increase in the expression level of DLL3 compared to the wild-type variant in B2.

[0023] [Figure 8C] FIG. 8C shows the detection of engineered DLL3 on the surface of H1299 cells. All engineered constructs except NP244 (B1 in FIG. 8A) increased DLL3 positive cells above wild-type DLL3 levels (red line representing NP116). The top two constructs, NP249 and NP247 (D1 and G1, respectively, in FIG. 8A), increased the positive percentage to about 80% and about 94%, respectively. NP247 (G1 in FIG. 8A) also showed significantly higher mean fluorescence intensity compared to DLL3 wild-type or DLL3 stable cell lines, suggesting that increased amounts of DLL3 protein may be transported to the cell surface. H1299-DLL3: stable DLL3 expressing cell lines. NP116: SURV-DLL3 WT. NP127: CAG-DLL3 WT. GMFI: geometric mean fluorescence intensity.

[0024] [Figure 8D]Figure 8D shows an in vitro cell killing assay. H1299 cells were transfected with NP247 or NP249 to determine the ability of anti-DLL3 antibodies conjugated with cytotoxic payloads (PBDs) to induce cytotoxicity. NP247 and NP249 expressing cells experienced a substantial decrease in viability when treated with anti-DLL3 antibodies conjugated with PBDs (anti-DLL3-PBDs). This suggests that cells expressing NP247 or NP249 DLL3 constructs may be more susceptible to cell death when treated with anti-DLL3-PBDs compared to cells transfected with wild-type DLL3 (e.g., NP116 or NP127).

[0025] [Figure 9A-1] FIG. 9A depicts additional protein changes bioengineered into NP247 (G1 in FIG. 8A) or CD8 short, which served as the parent sequence for the second design set (top), as well as the N- to C-terminal linear construct designs of NP296, NP298, NP299, NP302, NP303, and NP304 (bottom). NP296, NP297, and NP298 were engineered to identify the "minimal domain" of DLL3 sufficient for interaction with the antibody tracer. Importantly, in an attempt to double the antibody binding capacity, NP298 was engineered to include a second epitope in the structure. NP299 was engineered to include two minimal epitopes of DLL3 inserted into the extracellular domain of DLL3. NP302, NP303, NP304 were engineered by exchanging the C2 domain of DLL3 with a more soluble humanized nanobody structure. NP303 and NP304 additionally contain two minimal epitopes of DLL3 in a soluble humanized nanobody structure, with NP304 not containing the extracellular DLL3 domain. [Figure 9A-2] Same as above.

[0026] [Figure 9B]Figure 9B represents the level of DLL3 expression on the cell surface after transfection into H1299 cells. As in Figure 8B, these data demonstrate that DLL3 modeled on a molecular scaffold results in higher expression levels of surface DLL3 than H1299 transfected with a plasmid carrying wild-type DLL3 protein. A fluorescently labeled antibody against DLL3 was used as a detection agent in this experiment. Structural refinement to include the presence of a humanized soluble nanobody domain in NP302 further enhances expression over NP247. Importantly, the addition of two DLL3 binding domains in NP298 indicates that antibody binding capacity can be increased by a multivalent approach, boosting the overall level of antibody binding activity.

[0027] [Figure 9C] Figure 9C shows the level of DLL3 expression from NP247, NP302, NP304, NP296 and NP298 constructs on the cell surface after transfection into H1299 cells. The left graph shows the percentage of DLL3 positive cells, and the right graph shows the geometric mean fluorescence intensity (GMFI) of DLL3 positive cells. Fluorescently labeled antibody against DLL3 was used as a detection agent in this experiment.

[0028] [Figure 10A-B] Figures 10A and 10B show the amount of DLL3 that can be measured by FACS analysis on the cell surface after transfection of H1299 cells with the wild-type DLL3 epitope, NP247 expressing a single epitope of the CD8 scaffold, or NP298 expressing two epitopes of DLL3 on the CD8 scaffold. Fluorescently labeled antibodies against DLL3, phycoerythrin (PE, Figure 10A) or fluorescein isothiocyanate (FITC, Figure 10B) were used as detection agents in this experiment. Approximate antibody binding per cell was assessed using a standard curve (Figure 10B).

[0029] [Figure 10C] Figure 10C shows the physical number of antibody copies bound to DLL3 expressed on the cell surface after transfection of H1299 cells with wild-type DLL3 epitope, NP247 expressing a single epitope of CD8 scaffold, or NP298 expressing two epitopes of DLL3 on CD8 scaffold. A standard curve was used to estimate antibody binding. NP298 showed a 32-fold increase over the level of wild-type DLL3 epitope expression. Fluorescently labeled antibody against DLL3 was used as a detection agent in this experiment.

[0030] [Figure 10D] Figure 10D depicts the geometric mean intensity of internalized DLL3 following transfection of H1299 cells with NP116 (wild type DLL3) or NP247. Fluorescently labeled antibody against DLL3 was used as the detection agent in this experiment.

[0031] [Figure 11] Figure 11 shows images obtained from a positron emission tomography (PET) scan. In this model system, a DNA plasmid expressing a copy of the somatostatin receptor 2 (SSTR2) gene was created and then stably introduced into H1299 cells such that each cell line contained two genomic copies of the stably integrated SSTR2 construct. With the goal of ascertaining the minimum number of cells that would produce a measurable signal, mice were subcutaneously implanted with different numbers of engineered cells with stably integrated SSTR2 at separate locations and then treated with 68Ga-dotate positron-emitting tracer that binds to the SSTR2 protein at the cell surface. These data illustrate that even as few as 31,000 cancer cells can be detected in this mouse model when the cells are induced to express a surrogate biomarker at the cell surface.

[0032] [Figure 12]Figure 12 shows images obtained from a single photon emission computed tomography (SPECT) scan. In this model system, a DNA plasmid expressing a copy of the SSTR2 gene was created and then stably introduced into H1299 cells such that each cell line contained two genomic copies of the stably integrated SSTR2 construct. Mice were subcutaneously implanted with engineered cells with stably integrated SSTR2 and then treated with 177LU-dotate beta-emitting tracer that binds to the SSTR2 protein at the cell surface. These data illustrate that high-energy tracers can be used to detect biomarkers in cells that have been engineered to express that biomarker. Center: Coronal images of kidney and tumor cross sections. Right: Maximum intensity projection (MIP). 4H, 24H, and 120H: 4 hours, 24 hours, and 120 hours after tracer dosing, respectively.

[0033] [Figure 13A] Figure 13A shows the physical number of antibody copies bound to DLL3 expressed at the cell surface (left) and the geometric mean intensity of internalized DLL3 after transfection of H1299 cells with NP116 (wild type DLL3), NP247, or NP298. Antibody binding was estimated using a standard curve. NP247 showed a 21-fold increase over the level of wild type DLL3 epitope expression. NP298 showed a 37-fold increase over the level of wild type DLL3 epitope expression. Fluorescently labeled antibodies against DLL3 were used as detection agents in this experiment.

[0034] [Figure 13B]Figure 13B shows an in vitro cell killing assay. H1299 cells were transfected with NP116, NP247, or NP298 to determine the ability of anti-DLL3 antibodies conjugated with cytotoxic payloads (PBDs) to induce cytotoxicity. NP247 and NP298 expressing H1299 cells experienced a substantial decrease in viability when treated with anti-DLL3 antibodies conjugated with PBDs (anti-DLL3-PBDs). This suggests that cells expressing NP247 or NP298 DLL3 constructs may be more susceptible to cell death when treated with anti-DLL3-PBDs compared to cells transfected with wild-type DLL3 (NP116).

[0035] [Figure 14] Figure 14 shows images obtained from a single photon emission computed tomography (SPECT) scan. In this model system, a DNA plasmid expressing a copy of the SSTR2 gene was created and then stably introduced into H1299 cells using a lentivirus system. Mice were subcutaneously implanted with engineered cells carrying stably integrated SSTR2 constructs, treated with 200μCi of [68GA]-DOTATATE tracer, which binds to SSTR2 protein on the cell surface, and PET / CT imaging was performed 2 hours after tracer administration.

[0036] [Figure 15]Figure 15 represents a schematic of an exemplary workflow of the methods described herein. A DNA (1) construct using activated expression of a cell surface protein can be introduced into a target cell population by transient transfection (2). After transport to the nucleus and unpackaging, the DNA acts as a nucleic acid template for transcription (3-5) to produce an engineered reporter gene. Further protein modifications can occur in the cytoplasm (7) or cell surface (9) in response to the molecular disease status of the cell. Alternatively, the protein can be secreted into the extracellular environment for propagation of a signal to neighboring cells (8). On the cell surface, the target antigen can be easily detected using affinity-based reagents that allow detection by imaging techniques including radioactive tracers or other modalities. The use of high-energy emitters also offers the possibility of therapeutic applications. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0037] Detailed Description overview In some aspects, the disclosure relates to codable (e.g., under the control of a tumor or cancer-specific promoter) cell surface synthetic biomarkers that contribute to the identification, localization, or treatment of tumor cells in a subject.

[0038] In some cases, such codable cell surface synthetic biomarkers include (a) an extracellular domain configured to bind to an affinity reagent, and (b) a membrane-binding or transmembrane domain. When provided encoded in a nucleic acid under the control of a cancer-specific promoter (e.g., a promoter of a gene that is overexpressed in cancer cells compared to normal cells), administration of the nucleic acid to a subject suspected of having cancer drives the production of the selective cell surface biomarker in cancerous cells. The extracellular domain can include a terminal activatable epitope or ligand binding domain. Administration of a suitable affinity reagent (e.g., a ligand that binds to the ligand binding domain, or an affinity reagent / antibody that binds to the epitope) to the subject can then be used to treat neoplastic cells that selectively express the encoded cell surface synthetic biomarker (if the affinity reagent is a suitable therapeutic antibody) or to determine the location or identify those cells (if the affinity reagent is, for example, a labeled antibody).

[0039] An example of a configuration for such a cell surface synthetic biomarker is provided in FIG. 2, where binding of the affinity reagent depends on the cancer-associated transcription and extracellular conditions. In this configuration, the cell surface synthetic biomarker comprises an extracellular domain (201, e.g., derived from any of the receptors described herein) attached to the membrane by a transmembrane domain; an extracellular domain with a terminal epitope (210) flanked by two compatible portions of a pH-sensitive multimerization domain (220 and 230), which may be a pH-sensitive helix as described herein. Proper external localization of the extracellular domain can be driven by providing a signal peptide at the end of the extracellular domain. At high pH, ​​self-association of the pH-sensitive multimerization domain (240) prevents the epitope (250) from binding to an affinity reagent (e.g., an affinity reagent administered to a subject). At low pH, dissociation of the two compatible portions of the pH-sensitive multimerization domain (220 and 230) allows the epitope (210) to reach the extracellular affinity reagent. In this manner, cell surface synthetic biomarkers may depend on both the transcriptional status of cancer cells (e.g., by expression from cancer-specific promoters or promoters of genes that are overexpressed in cancer cells compared to normal cells) and the acidic extracellular environment of a typical tumor. The combination of transcriptional and extracellular conditions for binding to affinity reagents may result in improved specificity for targeting cancer in a subject.

[0040] Another example of a configuration for such a cell surface synthetic biomarker is provided in Figure 3. In this case, the configuration is similar to that of Figure 2, except that the extracellular domain (301) is configured to bind to a pH-sensitive helix (330) at low pH, and the activatable epitope (320) is flanked by the pH-sensitive helix (330) and a cancer-associated post-translational modification site (310), which, when post-translationally modified, contributes to the stabilization of the activatable epitope. In this manner, the accessibility of the activatable epitope can be controlled in a manner that is dependent on both extracellular pH by the pH-sensitive helix and cancer-associated extracellular enzyme expression, if the post-translational modification site is selected as one that is selectively targeted by an extracellular enzyme expressed in cancer. In this design, in the absence of post-translational modification and low pH (e.g., in a non-cancer microenvironment), the activatable epitope (320) is eliminated. In contrast, in the cancer-associated extracellular environment, the activatable epitope (350) is accessible due to dissociation of the pH-sensitive helix (360) and dissociation of the post-translational modification of the flanking post-translational modification sites (340).

[0041] Another example of a configuration for such a cell surface synthetic biomarker is provided in FIG. 4, where the biomarker configuration incorporates a ligand binding domain to allow for the use of modified natural ligands for the ligand binding domain to be used as an affinity reagent. In this configuration, a membrane-bound ligand binding domain (301) is provided linked to a terminal pH-sensitive helix (320) that prevents binding of the ligand (330) to the ligand binding site (310) at low pH. At high pH (e.g., a non-cancer microenvironment), the pH-sensitive helix (350) dissociates, allowing binding of the ligand to the ligand binding site (340) on the surface of the cell. The combination of transcriptional (if the biomarker is encoded under the control of a cancer-specific promoter or a promoter of a gene that is overexpressed in cancer cells compared to normal cells), extracellular pH, and extracellular post-translational conditions required to activate the epitope for binding in this example can result in improved specificity for targeting the cancer in a subject.

[0042] Another example of a configuration for such a cell surface synthetic biomarker is provided in Figure 5, where a receptor (501, 515, 510) of the type described in Figure 2 is modified to replace the signal peptide (501) with a secretion signal (520) and the transmembrane domain (510) with a membrane binding domain (530), e.g., a pH-sensitive membrane binding helix, to provide a second generation cell surface synthetic biomarker (520, 515, 530) that is capable of propagation to neighboring cells when expressed by a cancer cell (e.g., under a cancer cell specific promoter). Such a cell surface synthetic biomarker is thus configured to function as shown in Figure 6, where the secreted biomarker is secreted in the extracellular fluid (601) and does not attach to cells when secreted in a non-acidic extracellular environment (e.g., a non-tumor environment), but attaches to both the secreting cell (610) and neighboring cells (620) when the correct pH allows for assembly of the membrane binding helices. The combination of the reliance on extracellular conditions to label cells and the ability to spread to neighboring cells may provide improved specificity and sensitivity for targeting cancers for this construct. definition

[0043] While various embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be used.

[0044] Before describing the present disclosure in more detail, it should be understood that the present disclosure is not limited to the particular embodiments described, which, for example, can, of course, be modified. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure is limited only by the appended claims.

[0045] When a range of values ​​is provided, it is understood that each intervening value between the upper and lower limits of that range and any other stated range, to the tenth of the unit of the lower limit, or any intervening value in that stated range, is encompassed within the disclosure, unless the context clearly dictates otherwise. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, subject to any specifically excluded limit in the stated range, and are also encompassed within the disclosure. Where a stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0046] It must be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a support" includes a plurality of supports. In this specification and the following claims, reference will be made to certain terms that are intended to be defined to have the following meanings unless a contrary intention is apparent.

[0047] As used herein, the following terms have the meanings ascribed to them unless otherwise stated. In this disclosure, "comprises," "comprising," "containing," "having," and the like can have the meanings ascribed to them in U.S. Patent Law, and can mean "includes," "including," and the like, and "consisting essentially of" or "consists essentially of," and the like, when applied to methods and compositions encompassed by this disclosure, refer to compositions such as those disclosed herein, but which may contain additional structural groups, compositional components, or method steps (or analogs or derivatives thereof as discussed above). Such additional structural groups, compositional components, method steps, and the like, however, do not substantially affect the basic and novel properties of the composition or method as compared to the properties of the corresponding composition or method disclosed herein.

[0048] The term "or" as used herein is generally intended to be inclusive, encompassing both alternatives and either / or.

[0049] The present disclosure includes any variant of the polypeptide described herein with one or more conservative amino acid substitutions.Such conservative substitutions can be made in the amino acid sequence of a polypeptide without disrupting the three-dimensional structure or function of the polypeptide, or with only minimal disruption to the function of the polypeptide.Conservative substitutions can be achieved by replacing amino acids with similar hydrophobicity, polarity, and R-chain length for each other.Additionally or alternatively, by comparing the aligned sequences of homologous proteins from different species, conservative substitutions can be identified by finding amino acid residues (e.g., non-conserved residues) that are mutated between species without changing the basic function of the encoded protein. Such conservatively substituted variants may include variants having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identity with any one of the polypeptide protein sequences described herein. In some embodiments, such conservatively substituted variants are functional variants. Such functional variants may include sequences having substitutions that do not disrupt the activity of one or more important active site residues or residues that support the structure of the polypeptide.

[0050] Conservative substitution tables providing functionally similar amino acids are available in a variety of references (see, for example, Creighton, Proteins: Structures and Molecular Properties (WH Freeman & Co.; 2nd edition (December 1993)). The following eight groups each contain amino acids that are conservative substitutions for one another:

[0051] 1) Alanine (A), Glycine (G);

[0052] 2) Aspartic acid (D), glutamic acid (E);

[0053] 3) Asparagine (N), Glutamine (Q);

[0054] 4) arginine (R), lysine (K);

[0055] 5) isoleucine (I), leucine (L), methionine (M), valine (V);

[0056] 6) phenylalanine (F), tyrosine (Y), tryptophan (W);

[0057] 7) serine (S), threonine (T); and

[0058] 8) Cysteine ​​(C), Methionine (M)

[0059] The term "sequence identity" or "percent identity" in the context of two or more nucleic acid or polypeptide sequences generally refers to two (e.g., in a pairwise alignment) or more (e.g., in a multiple sequence alignment) sequences that are the same or have a certain percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence over a local or global comparison window as measured using a sequence comparison algorithm. Suitable sequence comparison algorithms for polypeptide sequences include, for example, BLASTP using parameters of a word length (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix setting gap costs at 11, an extension of 1, and using a conditional composition scoring matrix adjustment for polypeptide sequences longer than 30 residues; BLASTP using parameters of a word length (W) of 2, an expectation (E) of 1,000,000, and the PAM30 scoring matrix setting gap costs at 9 for open gaps and 1 for extended gaps for sequences shorter than 30 residues (these are the default parameters for BLASTP in the BLAST package available at https: / / blast.ncbi.nlm.nih.gov); CLUSTALW using Smith-Waterman homology search algorithm parameters with a match of 2, a mismatch of -1, and a gap of -1; MUSCLE using default parameters; MAFFT using parameters of retries of 2 and a maximum repeat of 1000; Novafold using default parameters; HMMER hmmalign using default parameters.

[0060] The term "about" or "approximately" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within 1 or more than 1 standard deviation, in accordance with the practice in the art. Alternatively, "about" can mean within a range of up to 20%, up to 15%, up to 10%, up to 5%, or up to 1% of a given value.

[0061] The term "subject" may generally include humans or non-human animals. Thus, the methods and compositions described herein are applicable to both human and veterinary diseases, as well as animal models. A preferred subject is a "patient", i.e., a living human undergoing medical care for a disease or condition. This includes those who do not have a defined disease, who are being investigated for signs of pathology. It also includes those who have or are suspected to be at risk for a defined disease. In some embodiments, the subject has at least one risk factor for cancer, such as Li-Fraumeni syndrome, Lynch syndrome, familial adenomatous polyposis, pulmonary nodules, von Hippel-Lindau disease, aplastic anemia, myelodysplastic syndrome, Cowden syndrome, hereditary breast and ovarian cancer syndrome (HBOC), or a BRCA mutation; is a current smoker, ex-smoker, or has been exposed to heavy passive smoking; has been exposed to carcinogens, excessive sunlight, immunosuppressants, or hepatitis B, hepatitis C, or human papillomavirus; or has obesity.

[0062] "Vector", as used herein, generally refers to a nucleic acid sequence capable of transferring other operably linked heterologous or recombinant nucleic acid sequences to a target cell. In some examples, the vector is a minicircle, a plasmid, a nanoplasmid, a yeast artificial chromosome (YAC), a bacterial artificial chromosome (BAC), a cosmid, a phagemid, a bacteriophage genome, or a baculovirus genome. Suitable vectors include vectors derived from bacteriophages or plants, invertebrates, or animals (including humans) viruses, such as CELiD vectors, dog-bone DNA (dbDNA) vectors, closed-end linear double-stranded DNA vectors (e.g., each end is covalently closed by chemical modification), adeno-associated virus vectors (e.g., AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or pseudotyped combinations thereof, such as AAV2 / AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or pseudotyped combinations thereof, such as AAV2 / A ... In some embodiments, the vector may be a replication-competent virus-derived vector. In some embodiments, the vector may be a replication-incompetent virus-derived vector. In some cases, the vector may include an episomal maintenance element, such as a scaffold / matrix attachment region (S / MAR), to facilitate replication in one or more target cell types. S / MAR elements are particularly useful for facilitating replication in the context of "naked" nucleic acid vectors, such as minicircles.Exemplary suitable S / MAR elements include, but are not limited to, EμMAR from the immunoglobulin heavy chain locus, apoB MAR from the human apolipoprotein B locus, Ch-LysMAR from the chicken lysozyme locus, and huIFNβ MAR from the human IFNβ locus. A vector may contain a coding sequence that can be expressed in a target cell. Thus, as used herein, the terms "vector construct", "expression vector" and "gene transfer vector" may refer to any nucleic acid construct that can direct the expression of a gene of interest and is useful for transferring the gene of interest into a target cell. The vectors described herein may additionally contain one or more cis-acting elements that stabilize or improve the expression of mRNA therefrom. Such cis-acting elements include, but are not limited to, any of the elements described in Johansen et al. The Journal of Gene Medicine. (5)12:1080-1089 (doi: 10.1002 / jgm.444) or Vlasova-St. Louis and Sagarsky. Mammalian Cis-Acting RNA Sequence Elements (doi: 10.5772 / intechopen.72124).

[0063] As a form of vector, the term "minicircle" as used herein generally refers to a small double-stranded circular DNA molecule that provides persistent high-level expression of a sequence of interest present on the vector, where the sequence of interest may code for a polypeptide, shRNA, antisense RNA, siRNA, etc. The sequence of interest is operably linked to a regulatory sequence present on the minicircle vector, which controls its expression. Such minicircle vectors are described, for example, in published US patent application US20040214329, which is specifically incorporated herein by reference. As a different form of vector, "nanoplasmid" refers to a vector that may contain a minimal bacterial ColE1 or R6K replication origin (providing such nanoplasmid capable of replicating in a bacterial host strain), a bacterial RNA selection marker, and a eukaryotic gene region. Further examples of such elements (nanoplasmid origin and RNA-out selection marker) are described, for example, in US9737620B2, which is incorporated herein by reference for purposes of describing nanoplasmid sequence elements.

[0064] The overall length of the minicircle vector is sufficient to include the desired elements described below, so long as it does not unacceptably prevent or substantially inhibit the vector's ability to enter target cells upon contact with the cells, e.g., by systemic administration to a host containing the cells. Thus, minicircle vectors will generally be at least about 0.3 kb in length, and often at least about 1.0 kb in length, while parent vectors may be 6 kb, 10 kb, or longer.

[0065] Minicircle vectors differ from bacterial plasmid vectors in that they lack a replication origin or lack a natural replication origin (e.g., may contain a minimal synthetic bacterial replication origin) and lack the selection markers normally found in bacterial plasmids, such as p-lactamase, tetracycline resistance (tet), kanamycin resistance (kan), or other antibiotic resistance selection markers. Minicircles are therefore smaller in size, allowing for more efficient delivery. Minicircles lack the transgene expression silencing effect associated with the vector backbone nucleic acid sequences of the parental plasmid from which the minicircle vector is excised. Minicircles may be substantially free of vector sequences other than the recombinase hybrid product sequence, as well as the sequences of interest, i.e., the transcriptional and regulatory sequences required for expression.

[0066] The term "Nanoplasmid", as used herein, generally refers to a vector that may include a minimal bacterial ColE1 or R6K origin of replication (which renders such Nanoplasmid replicable in a bacterial host strain), a bacterial RNA selection marker, and a eukaryotic gene region. Some embodiments of Nanoplasmids are described, for example, in US20150275221A1. In some embodiments, the Nanoplasmid may include a fusion bacterial-RNA selection marker / minimal origin of replication. In some embodiments, the fusion bacterial-RNA selection marker / minimal origin of replication may be located within a synthetic intron located within the eukaryotic gene region of the Nanoplasmid.

[0067] RNA selection marker is generally an expressed non-translated RNA with a vector that controls a target gene expressed in chromosome, so as to provide the selection of the vector.It can also be a nonsense-suppressing tRNA with a plasmid that controls a nonsense-suppressible selectable chromosomal target, as described by Crouzet J and Soubrier F, US Patent 6,977,174, 2005, which is incorporated herein by reference. It may also be an antisense repressor RNA carrying a plasmid, an RNA-OUT gene that represses an RNA-IN regulatory target, a pMB1 plasmid origin encoding an RNAI that represses an RNAII regulatory target, an IncB plasmid pMU720 origin encoding an RNAI that represses an RNAII regulatory target, the ParB locus Sok of the plasmid RI that represses a Hok regulatory target, the Flm locus FlmB of the F plasmid that represses a flmA regulatory target, another natural antisense repressor RNA, such as those described in Wagner EGH, Altuvia S, Romby P. 2002. Adv Genet 46:361 and Franch T, and Gerdes K. 2000. Current Opin Microbiol 3: 159, or an engineered repressor RNA, such as small synthetic small RNAs such as the SgrS, MicC or MicF scaffolds described in Park et al. Nature Biotechnology volume 31, pages 170-174 (2013).

[0068] The term "transfection agent" generally encompasses any compound that mediates the incorporation of DNA or RNA into a host cell, such as liposomes. Suitable methods for transforming or transfecting a host cell can be found in Sambrook, et al. (MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989), Ausubel, et al., Short Protocols in Molecular Biology, 3rd ed., Wiley & Sons, 1995, and other laboratory manuals, which are hereby incorporated by reference. Examples of suitable transfection agents include, but are not limited to, linear or branched polyethyleneimine, nanoparticles, liposomes, lipid-soluble particles, solid nanoparticles, amphiphilic peptides, micelles, dendrimers, polymer compositions, hydrogels, synthetic or naturally derived exosomes, virus-like particles, or any combination thereof.

[0069] The terms "nucleic acid molecule" and "polynucleotide" as used herein generally refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three-dimensional structure and may perform any function, known or unknown. Non-limiting examples of polynucleotides include genes, gene fragments, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, shRNA, single-stranded short or long RNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, control regions, isolated RNA of any sequence, nucleic acid probes, and primers. Nucleic acid molecules may be linear or circular.

[0070] The term "promoter" generally refers to a DNA sequence that directs the transcription of a polynucleotide. Typically, a promoter may be located in the 5' region of a polynucleotide to be transcribed, proximal to the transcription start site of such polynucleotide. More typically, a promoter is defined as a region upstream of the first exon, and more typically, as a region upstream of the first of multiple transcription start sites. Frequently, a promoter can direct the transcription of genes located on each of the complementary DNA strands 3' of the promoter. In other words, many promoters are bidirectional and can direct the transcription of downstream genes when present in either orientation (i.e., 5' to 3' or 3' to 5' relative to the coding region of the gene). In addition, a promoter may also contain at least one control element, e.g., an upstream element. Such elements include an upstream activator region (UAR), and optionally other DNA sequences that affect the transcription of the polynucleotide, e.g., synthetic upstream elements. Some promoters may be assembled from fragments of endogenous promoters (e.g., derived from the human genome).

[0071] The terms "coding sequence" and "encode" as used herein in reference to a polypeptide generally refer to a nucleic acid molecule that is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide, for example, when the nucleic acid is present in a living cell (in vivo) and placed under the control of appropriate regulatory sequences (or "control elements"). The boundaries of a coding sequence are typically determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus. Coding sequences may include, but are not limited to, cDNA from viruses, prokaryotic or eukaryotic mRNA, genomic DNA sequences from viruses, eukaryotic or prokaryotic DNA, and synthetic DNA sequences. A transcription termination sequence may be located 3' to the coding sequence, optionally together with additional control sequences such as enhancers, introns, polyadenylation sites, and the like, and a promoter may be located 5' to the coding sequence. A DNA sequence encoding a polypeptide may be optimized for expression in a selected cell by using codons preferred by the selected cell to present a DNA copy of the desired polypeptide coding sequence.

[0072] The term "operably linked" as used herein generally refers to an arrangement of elements, where the components so described are configured to perform their normal functions. Thus, a given promoter (e.g., a reporter expression cassette) operably linked to a coding sequence can cause expression of the coding sequence if the proper enzymes are present. Promoters or other control elements need not be contiguous with the coding sequence, so long as they function to direct its expression. For example, intervening untranslated but transcribed sequences can be present between the promoter sequence and the coding sequence, and the promoter sequence would still be considered "operably linked" to the coding sequence.

[0073] The term "pharmaceutical acceptable carrier" as used herein generally refers to a diluent, adjuvant, excipient, or vehicle administered with the probe of the present disclosure that is approved by a federal or state regulatory agency or listed in the United States Pharmacopoeia or other generally recognized pharmacopoeias for use in animals, and more particularly, in humans. Such pharmaceutical carriers can be liquids, such as water and oils, such as those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Pharmaceutical carriers can be saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea, and the like. When administered to a patient, the probe and pharmaceutical acceptable carrier can be sterile. Water is a useful carrier when the probe is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical carriers also include excipients such as glucose, lactose, sucrose, glycerol monostearate, sodium chloride, glycerol, propylene, glycol, water, ethanol, etc. The present composition can also contain a small amount of wetting or emulsifying agent, or pH buffering agent, if desired. The present composition can advantageously take the form of solution, emulsion, sustained release formulation, or any other form suitable for use.

[0074] The term "antibody" or "immunoglobulin" generally includes five distinct classes of antibodies that can be distinguished biochemically. With regard to the IgG class of immunoglobulin molecules, which can be considered the prototype, immunoglobulins contain two identical light polypeptide chains of approximately 23,000 daltons molecular weight, and two identical heavy chains of 53,000-70,000 molecular weight. The four chains are joined by disulfide bonds in a "Y" configuration, where the light chains surround the heavy chains starting at the mouth of the "Y" and continuing through to the variable domain.

[0075] The light chains of immunoglobulins can be classified as either kappa or lambda (κ, λ). Each heavy chain class can be associated with either a kappa or lambda light chain. Generally, when immunoglobulins are made by either hybridomas, B cells or genetically engineered host cells, the light and heavy chains can be covalently linked to each other, and the "tail" portions of the two heavy chains can be linked to each other by covalent disulfide bonds or non-covalent bonds. In the heavy chains, the amino acid sequence runs from the N-terminus at the forked end of the Y configuration to the C-terminus at the bottom of each chain. Heavy chains can be classified as gamma, mu, alpha, delta, or epsilon (γ, μ, α, δ, ε), with some subclasses of them (e.g., γ1-γ4). It is the nature of this chain that determines the "class" of the antibody as IgG, IgM, IgA, IgG, or IgE, respectively. Immunoglobulin subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, etc., can confer functional specialization.

[0076] Both light and heavy chains can be divided into regions of structural and functional homology. The term "region", when used to describe immunoglobulins or related molecules, generally refers to a single immunoglobulin portion or part (as in the term "Fc region") or a single antibody chain, and includes the constant or variable region and more distinct portions or parts of said domains. For example, the light chain variable domain includes "complementarity determining regions" or "CDRs" interspersed between "framework regions" or "FRs" as defined herein.

[0077] Certain regions of immunoglobulins can be defined as "constant" (C) regions or "variable" (V) regions based on the relative lack of sequence variation within the regions of various class members in the case of "constant regions" or the significant variation within the regions of various class members in the case of "variable regions". The terms "constant region" and "variable region" may also be used functionally. In this regard, it will be recognized that the variable region of an immunoglobulin or antibody determines antigen recognition and specificity. Conversely, the constant region of an immunoglobulin or antibody confers important effector functions such as secretion, transplacental mobility, Fc receptor binding, complement fixation, and the like.

[0078] The constant and variable regions of immunoglobulin heavy and light chains may be folded into domains. The term "domain" generally refers to an independently folded globular region of a heavy or light chain polypeptide, including, for example, a peptide loop stabilized by a β-pleated sheet and / or an intrachain disulfide bond (e.g., including 3-4 peptide loops). The constant region domain on the light chain of an immunoglobulin may be interchangeably referred to as a "light chain constant region domain", "CL region" or "CL domain". The constant domain on the heavy chain (e.g., hinge, CH1, CH2, or CH3 domain) may be interchangeably referred to as a "heavy chain constant region domain", "CH" region domain, or "CH domain". The variable domain on the light chain may be interchangeably referred to as a "light chain variable region domain", "VL region domain" or "VL domain". The variable domain on the heavy chain may be interchangeably referred to as a "heavy chain variable region domain", "VH region domain", or "VH domain".

[0079] By convention, the numbering of the variable and constant region domains increases as they become more distal from the antigen-binding portion or amino terminus of the immunoglobulin or antibody. The N-terminus of each heavy and light immunoglobulin chain is the variable region, the C-terminus is the constant region, and the CH3 and CL domains comprise the carboxy termini of the heavy and light chains, respectively. Thus, the light chain immunoglobulin domains are arranged in a VL-CL orientation, while the heavy chain domains are arranged in a VH-CH1-hinge-CH2-CH3 orientation.

[0080] As used herein, the term "CDR" or "complementarity determining region" refers to the non-contiguous antigen binding sites found in the variable regions of both heavy and light chain polypeptides. These particular regions are described by Kabat et al., J. Biol. Chem. 252, 6609-6616 (1977) and Kabat et al., Sequences of proteins of immunological interest. (1991), as well as Chothia et al., J. Mol. Biol. 196:901-917 (1987), and MacCallum et al., J. Mol. Biol. 262:732-745 (1996), where the definitions include overlapping or subsets of amino acid residues when compared with each other. The amino acid residues encompassing the CDRs defined by each of the above cited references are shown for comparison. Preferably, the term "CDR" refers to the CDRs defined by Kabat based on sequence comparison.

[0081] As used herein, the terms "CH2 domain sequence," "CH3 domain sequence," "CH1 domain sequence," and "CL domain sequence" generally refer to polypeptide sequences derived from the CH2 domain, CH3 domain, CH1 domain, and CL domain, respectively, of a native immunoglobulin molecule.

[0082] Amino acid positions in the heavy chain constant region, including amino acid positions in the CL, CH1, hinge, CH2, and CH3 domains, may be numbered herein according to the EU index numbering system (see, e.g., Kabat et al., in "Sequences of Proteins of Immunological Interest", US Dept. Health and Human Services, 5th edition, 1991, incorporated herein by reference).

[0083] As used herein, the term "VH domain" generally comprises the amino-terminal variable domain of an immunoglobulin heavy chain, and the term "VL domain" comprises the amino-terminal variable domain of an immunoglobulin light chain.

[0084] As used herein, the term "CH1 domain" generally includes the first (most amino terminal) constant region domain of an immunoglobulin heavy chain, e.g., extending from about EU positions 118-215. The CH1 domain is adjacent to the VH domain and amino terminal to the hinge region of the immunoglobulin heavy chain molecule and is not considered part of the Fc region of the immunoglobulin heavy chain.

[0085] As used herein, the term "hinge region" or "hinge domain" generally includes the portion of a heavy chain molecule that joins the CH1 domain to the CH2 domain, or the domain of a non-heavy chain molecule that has sequence similarity to the portion of a heavy chain molecule that joins the CH1 domain to the CH2 domain. This hinge region is flexible, thus allowing the two N-terminal antigen-binding regions to move independently. The hinge region can be further divided into three separate domains: the upper, middle, and lower hinge domains (see, for example, Roux et al. J. Immunol. 1998, 161:4083, which is incorporated herein by reference). The upper hinge domain generally refers to the portion of a heavy chain molecule that extends from the C-terminus of the CH1 domain to the cysteine ​​of the first hinge. The middle hinge domain generally refers to the portion of a heavy chain molecule that extends from the first cysteine ​​to the last cysteine ​​in the hinge. The lower hinge domain generally refers to that portion of the heavy chain molecule spanning from the last cysteine ​​in the hinge to Gly-237, according to EU numbering, in the CH2 domain.

[0086] As used herein, the term "hinge region" or "hinge domain" generally includes the portion of a CD8 molecule (e.g., a CD8a or CD8b molecule) following an immunoglobulin V domain-like region of a beta pleated sheet that is rich in proline, threonine and serine residues and has significant sequence identity to an immunoglobulin heavy chain hinge region or domain.

[0087] As used herein, the term "CH2 domain" generally includes the portion of a heavy chain immunoglobulin molecule extending from, for example, about EU positions 231-340. The CH2 domain is not closely paired with another domain. Rather, two N-linked branched carbohydrate chains are inserted between the two CH2 domains of an intact native IgG molecule.

[0088] As used herein, the term "CH3 domain" generally includes the portion of a heavy chain immunoglobulin molecule extending approximately 110 residues from the C-terminus of the CH2 domain, e.g., from about positions 341 to 447 (EU numbering system). The CH3 domain typically forms the C-terminal portion of the antibody. In some immunoglobulins, however, additional domains may extend from the CH3 domain to form the C-terminal portion of the molecule (e.g., the CH4 domain in the μ chain of IgM and the c chain of IgE).

[0089] As used herein, the term "CL domain" generally includes the first (most amino terminal) constant region domain of an immunoglobulin light chain, e.g., extending from about EU positions 108 to 214. The CL domain is adjacent to the VL domain.

[0090] As used herein, the term "Fc domain" generally refers to that portion of a single immunoglobulin heavy chain starting at the hinge region just upstream of the papain cleavage site (i.e., residue 216 in IgG, the first residue of the heavy chain constant region, which is 118) and ending at the C-terminus of the antibody. Thus, a complete Fc domain includes at least the hinge, CH2, and CH3 domains.

[0091] As used herein, the term "antibody or antigen-binding fragment thereof" is generally used in the broadest sense and includes intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, and antibody fragments. Antibody fragments include portions of intact antibodies that retain antigen-binding activity, and examples include Fab, Fab', F(ab)2, F(abc)2, and Fv fragments, as well as diabodies, linear antibodies, scFv, and multispecific antibodies formed from antibody fragments.

[0092] Single chain Fv ("sFv" or "scFv") polypeptide generally refers to a covalently linked VH-VL heterodimer expressed from a gene fusion comprising VH and VL coding genes linked by a peptide-encoding linker (see, for example, Huston et al., Proc. Nat. Acad. Sci. USA (1988) 85:5879-5883). Several methods have been described for determining and developing chemical structures (linkers) to convert the naturally aggregated but chemically separate light and heavy polypeptide chains derived from antibody V regions into sFv molecules that fold into a three-dimensional structure substantially similar to the structure of an antigen-binding site. See, for example, U.S. Patent Nos. 5,091,513, 5,132,405 and 4,946,778.

[0093] As used herein, the term "epitope" generally refers to a localized region of an antigen to which an antibody can specifically bind. An epitope can be, for example, consecutive amino acids of a polypeptide (linear or continuous epitope), or an epitope can be, for example, two or more non-contiguous regions of one or more polypeptides taken together (conformational, non-linear, discontinuous, or non-contiguous epitope). In certain embodiments, the epitope to which an antibody binds can be determined, for example, by NMR spectroscopy, X-ray diffraction crystallography studies, ELISA assays, hydrogen / deuterium exchange coupled with mass spectrometry (e.g., liquid chromatography electrospray mass spectrometry), array-based oligopeptide scanning assays, and / or mutagenesis mapping (e.g., site-directed mutagenesis mapping). For X-ray crystallography, crystallization can be accomplished using any of a number of documented methods (e.g., Giege R. et al., (1994) Acta Crystallogr D Biol Crystallogr 50(Pt 4): 339-350; McPherson A (1990) Eur J Biochem 189: 1-23; Chayen NE (1997) Structure 5: 1269-1274; McPherson A (1976) J Biol Chem 251: 6300-6303).Antibody:antigen crystals may be studied using well-known X-ray diffraction techniques and refined using computer software such as X-PLOR (Yale University, 1992, distributed by Molecular Simulations, Inc.; see, e.g., Meth Enzymol (1985) volumes 114 & 115, eds Wyckoff HW et al.; US2004 / 0014194), and BUSTER (Bricogne G (1993) Acta Crystallogr D Biol Crystallogr 49(Pt 1): 37-60; Bricogne G (1997) Meth Enzymol 276A: 361-423, ed Carter CW; Roversi P et al., (2000) Acta Crystallogr D Biol Crystallogr 56(Pt 10): 1316-1323). Mutagenesis mapping studies may be accomplished using any of a number of documented methods, such as those described in Champe M et al., (1995) J Biol Chem 270: 1388-1394 and Cunningham BC & Wells JA (1989) Science 244: 1081-1085.

[0094] As used herein, the term "linker domain" generally refers to a sequence that connects two or more domains in a linear sequence. In some embodiments, any of the domains or regions described herein may be connected to each other by a linker domain, which may include, for example, (GGS)n or (GGGS)n.

[0095] The term "transmembrane domain" or "transmembrane region" generally includes an amino acid sequence of about 15 amino acid residues in length that spans the cell membrane. A transmembrane domain or region may also include at least about 20, 25, 30, 35, 40, or 45 amino acid residues and may span the cell membrane. A transmembrane domain may be rich in hydrophobic residues and has an alpha-helical structure. In one embodiment, at least 50%, 60%, 70%, 80%, 90%, 95% or more of the amino acids in a transmembrane domain are hydrophobic, e.g., leucine, isoleucine, tyrosine, or tryptophan. Transmembrane domains are described, for example, in Zagotta WN et al. (1996) Annual Rev. Neurosci. 19: 235-263, the contents of which are incorporated herein by reference. Epitope

[0096] In some aspects, provided herein is an engineered polypeptide that includes an extracellular tropism domain (e.g., an extracellular domain) that includes an epitope. The extracellular domain can optionally include a signal peptide. In some embodiments, the engineered polypeptide sequence can include a sequence for an extracellular domain sequence that contains an epitope and a sequence for an optional signal peptide sequence encoded by a single nucleic acid sequence. In some embodiments, the signal peptide sequence can be cleaved after expression of the engineered polypeptide. In some embodiments, the engineered polypeptide sequence can be designed to include a signal peptide that is heterologous to the extracellular domain of the engineered polypeptide.

[0097] In some embodiments, the epitope can be bound by an antibody. In some embodiments, the epitope can be bound by a peptide hormone or growth factor. In some embodiments, the epitope can be derived from a biomarker. In some embodiments, the biomarker can be a biomarker for a disease or condition, such as cancer. In some embodiments, the epitope can be derived from a biomarker for acute myeloid leukemia, adrenocortical carcinoma, bladder urothelial carcinoma, breast ductal carcinoma, breast lobular carcinoma, cervical carcinoma, cholangiocarcinoma, colorectal adenocarcinoma, esophageal carcinoma, gastric adenocarcinoma, glioblastoma multiforme, head and neck squamous cell carcinoma, hepatocellular carcinoma, renal chromophobe carcinoma, renal clear cell carcinoma, renal papillary cell carcinoma, low grade glioma, lung adenocarcinoma, lung squamous cell carcinoma, mesothelioma, ovarian serous adenocarcinoma, pancreatic ductal adenocarcinoma, paraganglioma and pheochromocytoma, prostate cancer, ovarian cancer, pancreatic ... The epitope may be derived from a biomarker for cancer, including adenocarcinoma, sarcoma, cutaneous melanoma, testicular germ cell carcinoma, thymoma, papillary thyroid carcinoma, uterine carcinosarcoma, uterine endometrioid carcinoma, uveal melanoma, lip melanoma, spindle cell carcinoma, liposarcoma, nasal sarcoma, breast adenocarcinoma, insulinoma, osteosarcoma, mast cell tumor, angiosarcoma, non-small cell lung cancer (NSCLC), marginal zone lymphoma, malignant melanoma, or chronic lymphocytic leukemia. In some embodiments, the epitope may be derived from DLL3, PSMA, SSTR2, CD8a, CD4, TGFR1, IGF1R, PD-L1, EGFR, CD73, IgK, IL-6, or any combination thereof. In some embodiments, the epitope may be derived from DLL3, PSMA, SSTR2, or any combination thereof.

[0098] Other examples of proteins from which epitopes can be derived include, but are not limited to, exotoxin A, Epstein-Barr nuclear antigen 4, melanoma antigen 1 recognized by T cells, phosphoprotein of 65 kDa, immediate early protein IE1, transactivator protein BZLF1, prostate specific antigen, cellular tumor antigen p53 (UniProt: A0A0U1RQC9), envelope glycoprotein B, melanoma associated antigen 1, tyrosinase, Epstein-Barr nuclear antigen 1, protein K8.1, spike glycoprotein, latent infection membrane protein 1, tandem repeat ... Protein Tax-1, Epstein-Barr nuclear antigen 3, apoptosis regulator BHRF1, histone H4, melanoma-associated antigen 4, receptor tyrosine-protein kinase erbB-2 (UniProt: P04626), melanocyte protein PMEL, prostatic acid phosphatase, Gag-Pro-Pol polyprotein, keratin, type I cytoskeleton 18, L-dopachrome tautomerase, envelope glycoprotein gp62, thyroglobulin, melanoma-associated antigen 3, protein E6, POTE ankyrin domain family member F, envelope glycoprotein Protein B, protein LANA1, GTP-binding protein GEM, envelope glycoprotein gp63, Epstein-Barr nuclear antigen 6, latent infection membrane protein 2, small capsomere interacting protein, thymosin beta-10, carcinoembryonic antigen-related cell adhesion molecule 5 (UniProt:P06731), myelin basic protein (UniProt:J3QL64), melanoma-associated antigen 2, cytoplasmic dynein 1 heavy chain 1 (UniProt:Q14204), tyrosine-protein kinase ABL1, capsid protein, Wilms tumor protein (UniProt: ot:A0A0A0MT54), complement factor H (UniProt:P08603), G2 / mitosis-specific cyclin-B1 (fragment) (UniProt:E9PC90), amyloid-beta precursor protein, mRNA export factor ICP27 homolog, B lymphocyte antigen CD20 (UniProt:P11836), protein virilizer homolog, protein E7, immunoglobulin heavy constant gamma 1 (fragment) (UniProt:A0A0A0MS08), immunoglobulin heavy constant gamma 1, immunoglobulin heavy constant gamma 4, endoplasmic reticulum chaperone BiP,Transferrin receptor protein 1 (UniProt: P02786), cellular tumor antigen p53 (UniProt: P04637), neurotrophin receptor interacting factor homolog, U1 small nuclear ribonucleoprotein 70 kDa, melanoma-associated antigen 6, apolipoprotein B-100, mammaglobin-A (UniProt: Q13296), DNA polymerase processivity factor, Gag polyprotein, TCR gamma alternative reading frame protein, 60S ribosomal protein L28 (UniProt: P46779), glutamic acid carboxypeptidase 2, cellular tumor antigen p53 (UniProt: E7EQX7), DNA (cytosine-5)-methyltransferase 1, catenin beta-1 (UniProt: P35222), small nuclear ribonucleoprotein-associated proteins B and B', 60S ribosomal protein L8 (UniProt: P62917), melanoma-associated antigen B2, protein SSX2, small nuclear ribonucleoprotein Sm D1, major capsid protein L1, tenascin (UniProt:P24821), elongation factor 2, matrix protein, ADP / ATP translocase 2, stearoyl-CoA desaturase, glutamine-fructose-6-phosphate transaminase (isomerization), protein Rex, spectrin alpha chain, non-erythroid 1 (UniProt:Q13813), HLA class II histocompatibility antigen DR beta chain, Hom s 1, receptor tyrosine-protein kinase erbB-2 (UniProt:B4DTR1), CCR4-NOT transcription complex subunit 9, protein E6, dihydrolipoyllysine residue acetyltransferase component of pyruvate dehydrogenase complex, mitochondria, heat shock 70 kDa protein 1A (UniProt:P0DMV8), annexin A1, apoptosis regulator Bcl-2 (UniProt:P10415), melanoma-associated antigen 9, melanoma-associated antigen 6 (fragment), 60S ribosomal protein L7a (UniProt:P62424), ELAV-like protein 4, titin (UniProt:Q8WZ42), cancer / testis antigen 1, cellular tumor antigen p53 (UniProt:J3KP33), mucin-1 (UniProt:A0A0C4DGW3), keratin, type I cytoskeleton 10,Ribonucleoside diphosphate reductase large subunit, G protein-coupled receptor 143, bifunctional purine biosynthesis protein ATIC, myosin-14, fibronectin, transcription factor HIVEP2, histone H3 (fragment), histone H3.3 (UniProt:P84243), solute transporter family 45 member 3, E3 ubiquitin protein ligase, polypyrimidine tract-binding protein 1 (UniProt:P26599), replication protein E1, DNA topoisomerase 2-alpha, proteasome ubiquitin receptor ADRM1 (UniProt:Q16186), epidermal growth factor receptor, Golgi apparatus protein 1 (UniProt:Q92896), elongation factor 1-alpha1, elongation factor 1-alpha2, tyrosine-protein kinase JAK1, DEK (UniProt:P35659) and and nuclear pore complex protein Nup214 (UniProt:P35658), DNA-dependent RNA polymerase II subunit RPB1, myeloblastin (UniProt:P24158), phosphatidylinositol 5-phosphate 4-kinase type 2 alpha (UniProt:P48426), RNA helicase (UniProt:A0A7I2V2S0), cytochrome c oxidase subunit 2, RNA binding protein NOB1 (UniProt:Q9ULX3), dermatan sulfate epimerase (UniProt:Q9UL01), dermatan sulfate epimerase (UniProt:A0A2R8YE23), squamous cell carcinoma antigen 3 recognized by T cells (UniProt:A0A499FI31), squamous cell carcinoma antigen 3 recognized by T cells (UniProt:Q15020), Peptidyl-prolyl cis-trans isomerase B, melanoma-associated antigen 10, secreted protein BARF1, triosephosphate isomerase, 3 beta-hydroxysteroid dehydrogenase type 7, HLA class II histocompatibility antigen, DP alpha 1 chain, calreticulin (UniProt:P27797), minor capsid protein L2, filamin-A (UniProt:P21333), alpha-2-macroglobulin, telomerase reverse transcriptase, cytochrome b, HLA class II histocompatibility antigen, DR beta 5 chain, chondroitin sulfate protease proteoglycan 4, follistatin-related protein 1, desmoglein-3, T cell receptor beta chain MC.7.G5, regulatory protein E2, major capsid protein L1, cullin-related NEDD8 dissociation protein 2, major capsid protein L1, transcription activator protein Pur-alpha, importin-8, high-potency G protein-coupled receptor 139, KICSTOR complex protein SZT2, ATP-citrate synthase, heterogeneous nuclear ribonucleoprotein U (UniProt:Q5RI18), Hsp90 co-chaperone Cdc3 7-like 1 (UniProt:Q7L3B6), protein E7, protein E4, regulatory protein E2, high-potential protein E5, regulatory protein E2, minor capsid protein L2, protein E6, protein E6, 14-3-3 protein gamma, polyadenylation-binding protein 1, serine / threonine-protein kinase SMG1, protein transport protein Sec24D (UniProt:O94855), interferon-induced double-stranded RNA-dependent protein kinase activator A, signal transduction factor of transcription 1-alpha / beta Inducer and activator (UniProt:P42224), E3 ubiquitin-protein ligase TRIP12 (UniProt:Q14669), chromodomain-helicase-DNA binding protein 3, bromodomain adjacent to zinc finger domain protein 2A (UniProt:Q9UIF9), 26S proteasome non-ATPase regulatory subunit 2, baculovirus IAP repeat-containing protein 7, E3 ubiquitin-protein ligase TRIM68, putative HTLV-1-associated intrinsic sequence, caspase-8,SAM and SH3 domain-containing protein 1, IgGFc-binding protein, phosphatidic acid cytidylyltransferase 1, WD40 repeat-containing protein SMU1, NAD-dependent protein deacetylase sirtuin-2 (UniProt:Q8IXJ6), tubulin alpha-1B chain, histone H2B, histone H2B type 1-D, THO complex subunit 4 (UniProt:Q86V81), neuroblast differentiation-associated protein AHNAK, ATP-binding cassette subfamily A member 2 (UniProt:Q9BZC7), plexin domain-containing protein 2, membrane-associated phosphatidylinositol transfer protein 1, baculovirus IAP repeat-containing protein 5 (UniProt:O15392), cAMP-dependent protein kinase type II-alpha regulatory subunit, transformation / transcription domain-associated protein (UniProt:H0Y4W2), sodium / potassium transport ATPase subunit alpha-3 (UniProt:P13637), heterogeneous nuclear ribonucleoprotein L-like (fragment), neuronal membrane glycoprotein M6-b (UniProt:Q13491), von Willebrand factor, alpha-synuclein (UniProt:A0A669KB41), trans Gelin-2, periplakin (UniProt:O60437), O-phosphoseryl-tRNA(Sec) selenotransferase (UniProt:Q9HD40), protein disulfide isomerase A3 (UniProt:P30101), nuclear pore membrane glycoprotein 210, cleavage and polyadenylation specificity factor subunit 1, protein PRRC1, histone acetyltransferase p300, sterol O-acyltransferase 1, peroxiredoxin-5, mitochondria, histone H1.5, pericentriolar material 1 protein (UniProt:Q15154), plectin (UniProt:Q15149), lysine-tRNA ligase, trinucleotide repeat-containing gene 6A protein, plasminogen binding protein PgbA, nucleolar RNA helicase 2, enoyl-CoA delta isomerase 2 (UniProt:O75521), cytochrome P450 2E1, collagen alpha-1(VII) chain, alanine-tRNA ligase, cytoplasm,Extracellular calcium-sensing receptor (UniProt:P41180), ELAV-like protein 3, protein-arginine deiminase type 4, lysine-specific demethylase 5C, tubulin beta chain (UniProt:P07437), heparan sulfate 2-O-sulfotransferase 1, glycophorin-C, ATP-dependent RNA helicase DDX3X (UniProt:O00571), unconventional myosin-Ig (UniProt:B0I1T2), tubulin beta-8 chain, high-probability ATP-dependent RNA helicase DDX47, Microtubule-actin cross-linking factor 1, isoforms 1 / 2 / 3 / 5 (UniProt:H3BPE1), homeodomain-interacting protein kinase 1 (UniProt:Q86Z02), zinc finger homeobox protein 3, 40S ribosomal protein S15 (UniProt:P62841), RAS protein activator-like 3, melanoma antigen preferentially expressed in tumors, high probability ubiquitin carboxyl-terminal hydrolase FAF-X, ATP-dependent RNA helicase DDX1 (UniProt:Q92499), alpha-actinin-1 (UniProt:P12814), Tax1-binding protein 1 (UniProt:Q86VP1), myeloperoxidase, protein ORF28, cyclin-dependent kinase inhibitor 2A (UniProt:K7ES20), high-affinity cationic amino acid transporter 1, mucin-16, histone-lysine N-methyltransferase 2A, circadian clock protein PASD1, bromodomain and WD repeat-containing protein 1, signal transducer and activator of transcription 5 B, Rho-associated GTP-binding protein RhoC, transforming protein RhoA (UniProt:C9JX21), WD repeat-containing protein 75 (UniProt:Q8IWA0), multiple C2 and transmembrane domain-containing protein 1, kinesin-like protein KIF16B (UniProt:A0A1B0GTU3), targeting protein for Xklp2, titin (UniProt:A0A0A0MTS7), NBAS subunit of the NRZ tethering complex, ORM1-like protein 1, protein TASOR 2 (UniProt:Q5VWN6),Interleukin-4 receptor subunit alpha (UniProt:P24394), legumain, phosphoglycerate kinase 1, [pyruvate dehydrogenase [acetyltransferase]]-phosphatase 1, mitochondrial, myristoylated alanine-rich C kinase substrate, heterogeneous nuclear ribonucleoprotein L (UniProt:P14866), transmembrane glycoprotein NMB (UniProt:Q14956), type IV collagen of 72 kDa, calreticulin (UniProt:K7EL50), macrosialin, fibromodulin (UniProt:Q06828), lumican, malate dehydrogenase, cytoplasm (UniProt:P40925), collagen alpha-2(V) chain, clusterin (UniProt:P10909) , transport protein particle complex subunit 1, ETS translocation variant 5, E3 ubiquitin-protein ligase Mdm2 (UniProt:Q00987), nicotinic acid-nucleotide pyrophosphorylase [carboxylation] (UniProt:Q15274), procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 (UniProt:O00469), Golgi-specific brefeldin A-resistant guanine nucleotide exchange factor 1 (UniProt:Q92538), E3 ubiquitin-protein ligase RNF126, Abl interactor 2 (UniProt:Q9NYB9), protein NLRC5, ATP-dependent DNA helicase DDX11 (UniProt:Q96FC9), E3 ubiquitin-protein ligase TRIM9, Tripartite motif-containing protein 67 (UniProt:Q6ZTA4), CC motif chemokine 3, receptor tyrosine-protein kinase FLT3, mediator of RNA polymerase II transcription subunit 24, ubiquitin carboxyl-terminal hydrolase (UniProt:G5E9A6), vacuolar protein sorting-associated protein 13B (UniProt:Q7Z7G8), protein SON (UniProt:P18583), nuclear transcription factor Y subunit gamma (fragment) (UniProt:Q5T6K5), E3 ubiquitin-protein ligase UBR1, nuclear pore complex protein Nup107 (UniProt:P57740), neurocan core protein, replication termination factor 2 (UniProt:Q9BY4 2), terminal uridylyltransferase 4 (UniProt:Q5TAX3), hyaluronan-mediated motility receptor, CDK5 regulatory subunit-associated protein 2 (UniProt:Q96SN8), T complex protein 1 subunit zeta, ATP-dependent DNA helicase Q5, RNA polymerase II elongation factor ELL (UniProt:U3KQ90), abnormal spindle-like microcephaly-associated protein (UniProt:Q8IZT6), SWI / SNF-related matrix-associated actin-dependent regulator of chromatin subfamily B member 1 (UniProt:Q12824), putative tRNA(cytidine(32) / guanosine(34)-2'-O)-methyltransferase (UniProt:Q9UET6), acyl-CoA 6-desaturase, U5 small nuclear ribonucleoprotein component of 116 kDa, nuclear protein 56 (UniProt: O00567), nuclear pore complex proteins Nup98-Nup96 (UniProt: P52948), breast cancer anti-estrogen resistance protein 3, unconventional myosin-Ie (UniProt: Q12965), U3 small nucleolar RNA-associated protein 6 homolog, negative elongation factor B (UniProt: Q8WX92), protein mono-ADP-ribosyltransferase PARP4, DNA repair and recombination protein RAD54B (UniProt: Q9Y620), condensin-2 complex subunit G2, golgin subfamily B member 1, bombesin receptor activating protein C6orf89,Chromosomal protein structural integrity (UniProt:G8JLG1), Erbin (UniProt:Q96RT1), transmembrane protein 161B (UniProt:B7Z6T3), E3 ubiquitin-protein ligase RNF213 (UniProt:Q63HN8), neutral alpha-glucosidase AB (UniProt:Q14697), neutral alpha-glucosidase AB (UniProt:E9PKU7), equilibrative nucleobase transporter 1, Lengsin, protein disulfide isomerase A6, nuclear pore complex protein Nup93 (UniProt:Q8N1F7), homocysteine-responsive endoplasmic reticulum-resistant ubiquitin-like domain member 1 protein, short transient receptor potential channel 4-associated protein, vacuolar protein sorting-associated protein 51 homolog, maternal homolog against decapentaplegic 5 (SMAD5), chromodomain-helicase-DNA binding protein 4, integrin beta-8, nuclear pore complex protein Nup85 (UniProt:Q9BW27), pre-mRNA-processing factor 6, zinc finger protein 281, pre-mRNA-processing-splicing factor 8, nuclear pore complex protein Nup214 (UniProt:P35658), A-kinase anchor protein 13 (UniProt:Q12802), Midasin, Ras GTPase-activating protein-binding protein 2 (UniProt:Q9UN86), cell division cycle protein 27 homolog (UniProt:P30260), clathrin heavy chain 1, solute transporter family 2, facilitative glucose transporter member 1, U3 small nucleolar RNA-associated protein 4 homolog, cytokinesis protein desiccator 7, large subunit GTPase 1 homolog (UniProt:Q9H089), E3 ubiquitin-protein ligase HERC2, talin-1, transcription elongation factor SPT6, threonine-tRNA ligase 1, cytoplasmic, transmembrane emp24 domain-containing protein 4 (UniProt:Q7Z7H5), eukaryotic initiation factor 4A-III, structural maintenance of chromosomal proteins 4, splicing factor 3B subunit 4, transcription regulator QRICH1, tyrosine-protein kinase JAK2,Serine / threonine-protein kinase Nek8 (UniProt:Q86SG6), guanine nucleotide-binding protein G(s) subunit alpha isoform short (UniProt:P63092), protein NipSnap homolog 2, structural maintenance of chromosomes flexible hinge domain-containing protein 1, E3 ubiquitin-protein ligase MYLIP, protein transport protein Sec31A (UniProt:O94979), protein transport protein Sec31A (UniProt:D6RHZ5), dysferlin, Pecan ex-like protein 2, transducin-like enhancer protein 2 (UniProt:Q04725), transducin-like enhancer protein 4 (UniProt:Q04727), gamma-interferon-inducible protein 16 (UniProt:Q16666), mitochondrial fission regulator 2, complement decay-accelerating factor (UniProt:H7BY55), sterile alpha motif domain-containing protein 9-like, nicotinamide phosphoribosyltransferase (UniProt:A0A7P0T8L3), ankyrin repeat ubiquitin domain-containing protein 12, signal-induced proliferation-associated 1-like protein 3, spataxin (UniProt:Q96JI7), sacsin (UniProt:Q9NZJ4), iron-responsive element-binding protein 2, E3 ubiquitin-protein ligase UBR4 (UniProt:Q5T4S7), heterogeneous nuclear ribonucleoprotein Q (UniProt:A0A7I2V5Q6), gamma-tubulin complex component (UniProt:F2Z2B9), gamma-tubulin complex component 2 (UniProt:Q9BSJ2), E3 ubiquitin-protein ligase UBR4 (UniProt:Q5T4S7), Protein ligase MYCBP2, insulin-induced gene 1 protein, insulin-induced gene 1 protein (fragment), nuclear protein TPR, bundling and elongation protein zeta-1, phosphatidylinositol-4,5-bisphosphate 3-kinase (UniProt: A0A2R8Y2F6), phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha isoform, Pogo transposable element with KRAB domain (fragment), brefeldin A-inhibitory guanine nucleotide exchange protein 2 (UniProt: Q9Y6D5),Proteins Mpv17 (UniProt:P39210), septin-9, ubiquitin-conjugating enzyme E2 variant 3 (UniProt:Q8IX04), neurofibromin (UniProt:P21359), dynein axonemal intermediate chain 3 (UniProt:Q8IWG1), exportin-7 (UniProt:Q9UIA9), cytoplasmic carboxypeptidase 1, T complex protein 1 subunit delta, mediator of RNA polymerase II transcription subunit 13 (UniProt:Q9UHV7), tubulin folding cofactor B (UniProt:Q99426), Ras GTPase-activating protein-binding protein 1 (UniProt:A0A7I2YQN9), Ras GTPase-activating protein-binding protein 1 (UniProt:Q13283), protein THEM6, cytoskeleton-associated protein 2, protein arginine N-methyltransferase 3, histone-binding protein RBBP4 (UniProt:Q09028), cyclin-G-associated kinase, ubiquitin carboxyl-terminal hydrolase 7, PALM2 and AKAP2 fusion (fragment) (UniProt:C9JVY5), inosine-5'-monophosphate dehydrogenase 2, valine-tRNA ligase, mitochondrial, growth arrest specific protein 7, M-phase inducing protein 7, phosphatase 3, 60S ribosomal export protein NMD3 (UniProt:C9JA08), glycogen [starch] synthase, muscle, WD repeat and FYVE domain-containing protein 4, DNA damage binding protein 1 (UniProt:Q16531), protein PRRC2B, cytoplasmic FMR1-interacting protein 2, cytoplasmic FMR1-interacting protein 1, prohibitin (fragment) (UniProt:E7ESE2), Muskelin (UniProt:Q9UL63), lysine-specific demethylase 2B (UniProt:Q8NHM5), Mediator of RNA polymerase II transcription subunit 14, BTB / POZ domain-containing protein 2, double-stranded RNA-specific editase 1, transcription factor Dp-2, histone-lysine N-methyltransferase 2C, cell division cycle-associated 7-like protein, F-box / WD repeat-containing protein 11 (UniProt:Q9UKB1), store-operated calcium entry-associated regulator, histone-lysine N-methyltransferase EZH2, myeloid-associated differentiation marker, double-stranded RNA-specific adenosine deaminase (UniProt:A0A3B3ISU1), ATPase family AAA domain-containing protein 2 (UniProt:Q6PL18), programmed cell death protein 7, HTLV-1 basic zipper factor, GTPase KRas, guanine nucleotide-binding protein-like 3 (nuclear)-like, isoform CRA_b, melanoma-associated antigen F1, serine / threonine-protein kinase ATR, dynein axonemal heavy chain 17, inositol 1,4,5-trisphosphate receptor type 1, high-potency E3 ubiquitin-protein ligase HERC1, double-strand break repair protein rad21 homolog, intercellular adhesion molecule 2, neck-turning-like protein 1 (UniProt:Q5JY65), inactive rhomboid protein 2, transcription factor ETV6, vacuolar protein sorting-associated protein 16 homolog, TATA box-binding protein-associated factor RNA polymerase I subunit B, A-kinase anchor protein 9 (UniProt:Q99996), ubiquitin catabolite carboxyl-terminal hydrolase 47, polyadenylation-binding protein 3, zinc finger protein 280C, COP9 signalosome complex subunit 2 (UniProt:P61201), endonuclease G, mitochondria, ribosomal L1 domain-containing protein 1, MAX gene-associated protein, mitochondrial tRNA methylthiotransferase CDK5RAP1, high-potency helicase senataxin, carbonic anhydrase, chromodomain-helicase-DNA binding protein 2 (UniProt:O14647), G-patch domain-containing protein 8, Utp12 domain-containing protein, cytochrome c oxidase subunit 7A2, mitochondria (UniProt:P14406),Heparan-alpha-glucosaminide N-acetyltransferase (UniProt:Q68CP4), high-probability ATP-dependent RNA helicase DHX37, coronin-7 (UniProt:P57737), CREB-binding protein, polycomb protein EED (UniProt:O75530), integrator complex subunit 13, paired amphipathic helix protein Sin3a (UniProt:Q96ST3), clathrin interactor 1, ubiquitin-like modifier-activating enzyme 1 (UniProt:P2231 4), protein tyrosine phosphatase receptor type C-related protein, cyclin-dependent kinase 6, Ran-binding protein 9, integrin alpha-1, E3 ubiquitin-protein ligase BRE1B, proliferation marker protein Ki-67, chromosome-associated kinesin KIF4B, breast cancer type 2 susceptibility protein (UniProt:P51587), transforming acidic coiled-coil-containing protein 3 (UniProt:Q9Y6A5), eIF-2-alpha kinase activator GCN1, transcriptional adaptor 1, reticulon-4 receptor (fragment) (UniProt:H7C0V4), P2X purinergic receptor (UniProt:K7EQ78), Gag protein, aggrecan core protein (UniProt:A0A5K1VW97), ATP-binding cassette subfamily C member 12, B lymphocyte antigen CD19, retinoic acid receptor alpha (UniProt:P10276), G1 / S-specific cyclin-D3 (UniProt:P30281), decapentaplegic opposing maternal homolog 4, mitogen-activated protein kinase (UniProt:K7EN18), E3 ubiquitin-protein ligase TRIM23, glycerol-3-phosphate dehydrogenase, mitochondria, casein kinase I isoform epsilon (UniProt:P49674), kinesin-like protein KIF2C, speckle-type POZ protein (UniProt:O43791), U4 / U6.U5 tri-snRNP-associated protein 2 (UniProt:Q53GS9), SWI / SNF-related matrix-associated actin-dependent regulator of chromatin subfamily A member 5, Kelch-like ECH-associated protein 1,Serine palmitoyltransferase 2 (fragment), protein mono-ADP-ribosyltransferase PARP3, enhancer of filament formation 1 (UniProt:Q14511), sister chromatid cohesion protein PDS5 homolog A, beta-1,3-galactosyl-O-glycosyl-glycoprotein beta-1,6-N-acetylglucosaminyltransferase, general transcription factor 3C polypeptide 2 (UniProt:Q8WUA4), myotubularin, citronellol Rho interacting kinase (UniProt:Q8WUA5). rot:O14578), poly[ADP-ribose] polymerase (UniProt:E9PNI7), transport and Golgi organization protein 6 homolog, protein transport protein Sec24A, MMS19 nucleotide excision repair protein homolog, proteasome activator complex subunit 4 (UniProt:Q14997), myocyte-specific enhancer factor 2B (UniProt:Q02080), transient receptor potential cation channel subfamily M member 8 (UniProt:Q7Z2W7), Rho GTPase-activating protein 35, dihydropyrimidinase-related protein 4 (UniProt:Q5T0Q6), guanine nucleotide-binding protein subunit beta-5, aurora kinase A, serine / threonine-protein kinase B-raf, adenomatous polyposis coli protein, Nck-related protein 1, mediator of RNA polymerase II transcription subunit 15 (UniProt:G3V1P5), DNA excision repair protein ERCC-6-like (UniProt:Q2NKX8), HIV Tat-specific factor 1, negative elongation factor A (fragment) (UniProt:H0Y3X6), negative elongation factor A (UniProt:Q9H3P2), cytochrome P450 7B1, intraflagellar transport protein 80 homolog, CIZ1 protein, FAST kinase domain-containing protein 1, mitochondrion, cytochrome c oxidase subunit 4 isoform 1, mitochondrion, ATP synthase subunit g, mitochondrion (UniProt:O75964), dynein axoneme assembly factor 5 (UniProt:Q86Y56), eukaryotic translation initiation factor 2 subunit 1 (fragment), LisH domain-containing protein ARMC9 (UniProt:A0A804HK42),Exostocin-2 (UniProt:Q93063), dynamin-binding protein (UniProt:Q6XZF7), tripartite motif-containing protein 26, protein kintone, perilipin-2 (UniProt:Q99541), quinone oxidoreductase, protein PAT1 homolog 1, neurobeatin-like protein 2 (UniProt:A0A494C1V1), Ufm1-specific protease 2 (UniProt:Q9NUQ7), Werner syndrome ATP-dependent helicase, WD repeat-containing protein 46, DBF4-type zinc Finger-containing protein 2 (UniProt:Q9HCK1), cytochrome b-c1 complex subunit 7, heparan sulfate 6-O-sulfotransferase 1, E3 ubiquitin-protein ligase HUWE1, NADP-dependent malic enzyme, RNA polymerase II subunit AC-terminal domain phosphatase (fragment) (UniProt:K7EJD2), neuronal Wiskott-Aldrich syndrome protein, cocaine esterase, casein kinase I isoform alpha (UniProt:P48729), BCL-6 corepressor - (UniProt:Q6W2J9), putative polycomb group protein ASXL3 (UniProt:Q9C0F0), retinoblastoma-like protein 2, eyeless homolog 3, protein available homolog (UniProt:Q8N8S7), integrin beta-4, F-actin-capping protein subunit beta (UniProt:P47756), endoplasmic reticulum resident protein 29, 4F2 cell surface antigen heavy chain (UniProt:P08195), transketolase (UniProt:P29401), nuclear receptor corepressor 1 (U niProt:A0A088AWL3), RanBP-type and C3HC4-type zinc finger-containing protein 1 (UniProt:Q9BYM8), cell cycle and apoptosis regulator protein 2 (UniProt:Q8N163), B cell receptor-associated protein (fragment) (UniProt:C9JQ75), E3 ubiquitin-protein ligase Praja-2, guanylate-binding protein 1, chromatin target of PRMT1 protein (UniProt:Q9Y3Y2), phagocytosis and cell motility protein 2 (UniProt:Q96JJ3),F-box-only protein 21 (UniProt:Q4G104), cofilin-2 (UniProt:Q9Y281), kinetochore protein Nuf2 (UniProt:Q9BZD4), trans-3-hydroxy-L-proline dehydratase (UniProt:Q96EM0), zinc finger domain-containing helicase 2, DNA (cytosine-5)-methyltransferase 3A, mitogen-activated protein kinase kinase kinase kinase 4 (MAPK4) (UniProt:G5E948), phospholipid transport ATPase IF (UniProt:Q9Y2G3), coatomer subunit gamma-2, MAP kinase interacting serine / threonine-protein kinase 1 (UniProt:A0A499FIS5), Nucleophosmin (UniProt:P06748), BTB / POZ domain-containing protein 7 (UniProt:Q9P203), melanoma inhibitory activity protein 2 (UniProt:G3V599), phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform, E3 ubiquitin-protein ligase DTX3L, FK506-binding protein 15, PH domain leucine-rich repeat-containing protein phosphatase 1, RAD50-interacting protein 1, leucine-rich repeat-containing tandem repeat kinase 1 (PKI1), and phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (PKI1). protein 42, ATP-dependent DNA / RNA helicase DHX36, TRAF-type zinc finger domain-containing protein 1, ATP-dependent RNA helicase DDX19B, AT-rich interacting domain-containing protein 5B (UniProt:Q14865), 164 kDa centrosomal protein, cell division control protein 42 homolog (UniProt:A0A590UJK8), constitutive coactivator of peroxisome proliferator-activated receptor gamma (UniProt:Q96EK7), transmembrane protein 214 (UniProt:Q 6NUQ4), E3 ubiquitin-protein ligase RNF38, protein farnesyltransferase subunit beta (UniProt:B4DL54), chromosome transmission fidelity protein 18 homolog (UniProt:Q8WVB6), glutathione S-transferase kappa, Arf-GAP with Rho-GAP domain, ANK repeat and PH domain-containing protein 1 (UniProt:Q96P48), minichromosome maintenance complex-binding protein, E3 ubiquitin-protein ligase TRIP 12 (UniProt:A0A6Q8PGG9), FAST kinase domain-containing protein 4, serine / threonine-protein kinase WNK2 (UniProt:Q9Y3S1), nonspecific serine / threonine protein kinase (UniProt:F8W9F9), structural maintenance of chromosomal proteins 1B, peroxiredoxin-6, SWI / SNF complex subunit SMARCC2 (UniProt:Q8TAQ2), UDP-glucose:glycoprotein glucosyltransferase 2 (UniProt:Q9NYU1),Phosphatidylinositol 4-phosphate 5-kinase type 1 gamma (UniProt:O60331), myosin light chain kinase, smooth muscle, endoplasmin (UniProt:A0A7P0TAY2), DmX-like protein 1 (UniProt:Q9Y485), tyrosine-protein kinase Lck, cyclin-dependent kinase 4 (UniProt:P11802), cyclin-dependent kinase 13 (UniProt:Q14004), protein FAM136A (UniProt:Q96C01), germ cell-specific gene 1-like protein 2, calpain-7, zonadhesin, Fanconi anemia group M protein, tan Protein FAM3C, Alström syndrome protein 1 (UniProt:Q8TCU4), cyclic AMP-dependent transcription factor ATF-6 beta (UniProt:Q99941), WD repeat and FYVE domain-containing protein 1, tensin-1 (UniProt:Q9HBL0), astrotactin-2 (UniProt:O75129), methionine synthase reductase, PR domain zinc finger protein 10 (UniProt:Q9NQV6), suppressor of G protein pathway 2, matrilysin, dual specificity protein phosphatase 3, serine / threonine-protein kinase PAK 3, damage-regulated phosphatase ARMT1, HEAT repeat-containing protein 1 (UniProt:Q9H583), ATP synthase F(0) complex subunit C2, mitochondrial, bone marrow stromal antigen 2, cell division cycle 5-like protein, regulator of nonsense transcripts 3A, apoptosis inhibitor 5 (UniProt:G3V1C3), apoptosis inhibitor 5 (UniProt:Q9BZZ5), apoptosis inhibitor 5 (fragment), PHD and RING finger domain-containing protein 1 (UniProt:A0A0J9YWD5), 1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase gamma-1, Fc receptor-like A (fragment), eukaryotic translation initiation factor 4E (UniProt:D6RBW1), DNA / RNA binding protein KIN17, fibronectin type III domain-containing protein 3B, WD repeat-containing protein 35 (UniProt:Q9P2L0),Guanine nucleotide-binding protein G(I) / G(S) / G(O) subunit gamma-5, serine / threonine-protein kinase PLK3, ectodermal-neurocortex protein 1, bromodomain 1A adjacent to zinc finger domain protein, neuroepithelial cell transforming gene 1 protein, calcium load-activated calcium channel (UniProt:Q9UM00), peroxisomal multifunctional enzyme type 2 (UniProt:P51659), pleckstrin homology domain-containing family F member 2, TBC1 domain family member 14 (UniProt:Q9P2M4), tyrosine-protein phosphatase non-receptor type 12, lysine-specific demethylase 6B, histone-lysine N-methyltransferase 2D, zinc finger protein 292 (UniProt:J3KNV1), endoplasmic reticulum transmembrane helix translocase, alpha-methylacyl-CoA racemase (UniProt:Q9UHK6), receptor tyrosine-protein phosphatase zeta, high-potency cation-transporting ATPase 13A4, RNA-binding motif protein, X-linked-like-3, ankyrin repeat domain-containing protein 26 (UniProt:Q9UPS8), fructose-bisphosphate aldolase C, nuclear protein MDM1 (UniProt:Q8TC05), ER luminal protein maintenance receptor 3, AN1-type zinc finger protein 5, mRNA export factor RAE1, zinc phosphodiesterase ELAC protein 2 (fragment) (UniProt:E7ES68), insulin-like growth factor 2 mRNA-binding protein 2 (UniProt:Q9Y6M1), leucine-rich acidic nuclear phosphoprotein 32 family member A (UniProt:P39687), centrosomal protein of 55 kDa, solute transporter family 12 member 4 (UniProt:Q9UP95), transforming acidic coiled-coil-containing protein 2 (UniProt:E9PBC6), transforming acidic coiled-coil-containing protein 2 (UniProt:O95359), basal cell adhesion molecule (UniProt:P50895), alkylglycerone-phosphate synthase (UniProt:A0A2R8YEL0), high probability phospholipid transport ATPase IIA,KIF-binding protein (UniProt:Q96EK5), nuclear valosin-containing protein-like (UniProt:O15381), inactive histone-lysine N-methyltransferase 2E (UniProt:Q8IZD2), C-terminal binding protein 1, bromodomain adjacent to zinc finger domain protein 2B (UniProt:Q9UIF8), tumor protein 63, helicase SKI2W, serine-protein kinase ATM (UniProt:Q13315), STAGA complex 65 subunit gamma, endoplasmic reticulum aminopeptidase 1, NPC intracellular cholesterol transporter 1, synaptojanin-2, nonspecific serine / threonine protein kinase (UniProt:A0A804HLI0), Kalirin (UniProt:O60229), Ras suppressor protein 1 , neural cell adhesion molecule (UniProt:C9JYY6), neutrophil cytosolic factor 2, protein DENND6B, ATP-dependent RNA helicase DHX29 (UniProt:Q7Z478), NIF3-like protein 1, E3 ubiquitin-protein ligase ARIH1 (UniProt:Q9Y4X5), ankyrin repeat domain-containing protein 36C (UniProt:Q5JPF3), RecQ-mediated genomic instability protein 1, ATP-dependent Clp protease proteolytic subunit, mitochondria, cyclin-dependent kinase inhibitor 2A (UniProt:P42771), RUN and FYVE domain-containing protein 1, signal peptidase complex catalytic subunit SEC11 (UniProt:H0YNG3), regulatory related protein of mTOR, phospholipid transport ATPase VD, glutamine-fructose-6-phosphate aminotransferase [isomerase] 2, interferon regulatory factor 5, DmX-like protein 2 (UniProt: Q8TDJ6); Pecanex-like protein 3, high-potency ATP-dependent RNA helicase DDX31, general transcription factor IIE subunit 1, a disintegrin and metalloproteinase domain-containing protein 17, bone morphogenetic protein receptor type 1B, multiple epidermal growth factor-like domain protein 8, zinc finger BED domain-containing protein 4, multiple inositol polyphosphate phosphatase 1, solute transporter family 2, facilitative glucose transporter member 5, tau-tubulin kinase 2 (UniProt:Q6IQ55), Kelch-like protein 29, transcriptional enhancer factor TEF-3 (UniProt:A0A 0A0MRF3), PRELI domain-containing protein 1, mitochondria (UniProt:Q9Y255), DNA polymerase theta, G1 / S-specific cyclin-D1 (UniProt:P24385), glutathione peroxidase 1 (UniProt:P07203), kallikrein-2, brevican core protein, plexin-B3, EH domain-containing protein 2, V-type proton ATPase subunit B, brain isoform, cholinesterase, Ras-specific guanine nucleotide-releasing factor 1, TBC1 domain family member 22A (UniProt:Q8WUA7), cadherin EGF LAG 7-transmembrane G-type receptor 1 (UniProt: A0A6I8PRU0), cadherin EGF LAG 7-transmembrane G-type receptor 3, cadherin EGF LAG 7-transmembrane G-type receptor 1 (UniProt: Q9NYQ6), tumor protein p53-induced protein 11 (UniProt: U3KQ32), vesicle-associated membrane protein 3 (UniProt: K7EKX0), ATP-binding cassette subfamily A member 6, peroxiredoxin-4 (UniProt: Q13162), DmX-like protein 1 (UniProt: F5H269), RasGTPase-activating protein nGAP, ryanodine receptor 2 (UniProt:Q92736), Pro-cathepsin H (UniProt:A0A7I2V3T9), baculovirus IAP repeat-containing protein 5 (UniProt:H3BLT4), baculovirus IAP repeat-containing protein 5 (UniProt:A0A0B4J1S3), interleukin-1 beta, eukaryotic translation initiation factor 4E-binding protein 1, melanoma-associated antigen C2, thioredoxin-dependent peroxisome proliferator-activated receptor 1 (UniProt:A0A0B4J1S3), and ribosomal protein 1 (RIP1). α-acetylglucosamine reductase, mitochondria, endothelin receptor nonselective type, actin-related protein 10 (UniProt:Q9NZ32), protocadherin-8, sterile alpha motif domain-containing protein 9, ubiquitin carboxyl-terminal hydrolase 37, cytochrome c oxidase assembly factor 3 homolog, mitochondria, interleukin-27 receptor subunit alpha, 3-oxoacyl-[acyl-carrier-protein] synthase, mitochondria, phosphomannomutase 2, uncharacterized protein KIAA1958, collagen alpha-1(XVI) chain, zinc finger protein ZXDC, ATPase family protein 2 homolog, chondroitin sulfate synthase 2, epsin-3, 192 kDa centrosomal protein (UniProt:Q8TEP8), plasmenylethanolamine desaturase, homeobox protein TGIF2, zinc finger MYM protein 4, sex Protein of unknown quality C6orf132, DNA helicase B (UniProt:Q8NG08), serine / threonine-protein kinase STK11, Runt-related transcription factor 1, nucleoporin NDC1, unconventional myosin-IXa (UniProt:B2RTY4), arginase-1, sterol-4-alpha-carboxylate 3-dehydrogenase, decarboxylation, vacuolar protein sorting-related protein 4B (UniProt:O75351), syncytin-1, ubiquitin-conjugating enzyme E2D2, all-trans retinol 13,14-reductase, 28S ribosomal protein S5, mitochondria, 39S ribosomal protein L39, mitochondria, protein-cysteine ​​N-palmitoyltransferase HHAT, transforming growth factor-beta-induced protein ig-h3, multidrug resistance-associated protein 1, hepatocyte growth factor receptor, potassium voltage-gated channel subfamily C member 3 (UniProt:E7ETH1), sodium channel protein type 4 subunit alpha, isocitrate dehydrogenase [NADP] cytoplasm, sister chromatid cohesion protein PDS5 homolog B, protein kinase C theta type, isocitrate dehydrogenase [NADP], mitochondria, extracellular sulfatase Sulf-2, nuclear protein Plasma-localized protein 4 homolog (UniProt:Q8TAT6), CUB and sushi domain-containing protein 1 (UniProt:E5RIG2), GPI inositol deacylase (UniProt:Q75T13), Teneurin-3, Mitogen-activated protein kinase kinase kinase 2, Transmembrane channel-like protein 8, Serine / Threonine-protein kinase MRCK alpha, Protein-serine O-palmitoleoyltransferase porcupine, Xaa-Pro aminopeptidase 1, Structure-specific endonuclease subunit SLX4, Kinesin-like protein KIF26B (fragment), Neuroligin-4, Y-linked (UniProt:Q8NFZ3), Neuroligin-4, X-linked, P2Y purinergic receptor 4, SLIT-ROBO Rho GTPase-activating protein 1, Breast cancer anti-estrogen resistance protein 1 (UniProt:A0A2R8Y5E4), Protein magonashi homolog 2 (UniProt:Q96A72), carbonic anhydrase 2, inactive hydroxysteroid dehydrogenase-like protein 1, protoporphyrinogen oxidase, dephospho-CoA kinase domain-containing protein, calcium uniporter protein, mitochondrial, armadillo repeat-containing protein 7, transmembrane protein 168, protein MMS22-like, mannosyl-oligosaccharide 1,2-alpha-mannosidase IB, neuron navigator 3 (UniProt:Q8IVL1), neuron navigator 2 (UniProt:A0A0A0MTL4), desiccator of cytokinesis protein 3 (UniProt:Q8IVL0). , neuroblastoma breakpoint family member 11, serine / threonine-protein phosphatase 4 regulatory subunit 1, gamma-tubulin complex component 6 (UniProt:Q96RT7), vacuolar protein sorting-associated protein 8 homolog (UniProt:Q8N3P4), protein asterate homolog 1, TRIO and F-actin binding protein, guanylate cyclase soluble subunit alpha-1, cryptochrome-1, adhesion G protein-coupled receptor A3, apoptosis regulator BAX (UniProt:Q07812), transmembrane protein 231, transmembrane protein 14B (UniProt:Q9NUH8), L-fucose kinase, phospholipid transfer ATPaseABCA1 (UniProt:O95477), mitochondrial inner membrane protein OXA1L (UniProt:Q15070), NACHT, LRR and PYD domain-containing protein 14, serine-rich coiled-coil domain-containing protein 1, glucosyltransferase 1, rapamycin-insensitive companion of mTOR, integrin alpha-9 (UniProt:Q13797), adenylate kinase 7, anoctamin-3, nuclear exosome regulator NRDE2, calcitonin gene-related peptide type 1 receptor, golgin subfamily A member 2, receptor-interacting serine / threonine-protein kinase 3, Krev-interacting capture protein 1, protein argonaute-2, RWD domain-containing protein 3, 60S ribosomal protein L5 (UniProt:A0A2R8Y4A2), protocadherin-11X-linked, cysteine ​​and glycine-rich protein 2, sleep family member 12-like, nebulin, alpha-1,3-mannosyl-glycoprotein 2-beta-N-acetylglucosaminyltransferase, aldehyde dehydrogenase family 16 member A1 (UniProt:Q8IZ83), a disintegrin and metalloproteinase with thrombospondin motifs 7, unconventional myosin-Vb, MYCBP-related protein (UniProt:A0A140VK87), sodium / hydrogen exchanger (UniProt:A0A7I2V2B0), mucin-4 (UniProt:A0A0G2JNL3), zinc finger FYVE domain-containing protein 9, high probability ubiquitin carboxyl-terminal hydrolase MINDY-4, cytoplasmic protein NCK1, [pyruvate dehydrogenase (acetyltransferase)] kinase isoenzyme 3, mitochondria, cyclin-A1, alpha-2,8-sialyltransferase 8E, 28S ribosomal protein S10, mitochondria, CC chemokine receptor type 6, ankyrin repeat and SAM domain-containing protein 1A (UniProt:Q92625), kinesin-like protein KIF16B (UniProt:Q96L93), dual specificity mitogen-activated protein kinase kinase 4, adipocyte plasma membrane-associated protein, CD9 antigen (UniProt:P21926), adhesion G protein-coupled receptor D1, cytosolic iron-sulfur assembly component 3, receptor tyrosine-protein phosphatase beta, RING finger protein 10, myomegalin (UniProt:A0A075B749), NADH dehydrogenase [ubiquinone] flavoprotein 1, mitochondria (UniProt:G3V0I5), Phosphatidylinositol 3,4,5-trisphosphate 3-phosphatase and dual specificity protein phosphatase PTEN, solute transporter family 27 member 3, amiloride-sensitive sodium channel subunit beta, E3 ubiquitin-protein ligase HACE1, proteinase-activated receptor 3, metalloproteinase inhibitor 3, short-chain-specific acyl-CoA dehydrogenase, mitochondria, POC1 centriole protein homolog B (UniProt:Q8TC44), exocyst complex component 8, ATP-binding cassette subfamily B member 6, PAX-interacting protein 1, phospholipid scramblase 4, protein FAM228B (UniProt:P0C875), Ras-related protein Rab-39B , alpha / beta hydrolase domain-containing protein 17B, FAST kinase domain-containing protein 2, mitochondria, protein FAM83G, SH3 and PX domain-containing protein 2A, mannose-6-phosphate isomerase (UniProt:P34949), triokinase / FMN cyclase, tectonin beta-propeller repeat-containing protein 2, immunoglobulin superfamily member 2, signal regulatory protein gamma, segment polarity protein dishveld homolog DVL-2, Arf-GAP with SH3 domain, ANK repeat and PH domain-containing protein 1 (UniProt:Q9ULH1), EF-hand calcium-binding domain-containing protein 13, phospholipid transport ATPase IK (UniProt:O60423), splicing factor 3A subunit 2, glutaminyl-tRNA synthetase (fragment), poly(ADP-ribose) glycohydrolase, ephrin type B receptor 1, protrudin, proline-rich protein 7, c-Jun-amino-terminal kinase interacting protein 2, unconventional myosin-Ib (UniProt:E9PDF6), transcriptional enhancer factor TEF-1 (UniProt:P28347), protocadherin gamma-C5, zinc finger protein 407, zinc finger protein 3, pentraxin-4, Rho GTPase-activating protein 29 (UniProt:Q52LW3), plasma membrane calcium transport ATPase4, neurosecretory protein VGF,Transcription factor MafF (fragment), cancer / testis antigen family 45 member A10, coiled-coil and C2 domain-containing protein 1A, cyclin-dependent kinase inhibitor 1, mitochondrial chaperone BCS1, serpin B3, receptor protein-tyrosine kinase (UniProt:Q504U8), receptor protein-tyrosine kinase (UniProt:E9PFD7), neutral and basic amino acid transporter protein rBAT (UniProt:Q07837), protein FAM161A (fragment), leucine-rich repeat transmembrane protein C CDC168, serpin B4, epithelial discoidin domain-containing receptor 1, serine / threonine-protein kinase 36, collagen alpha-1(XVIII) chain, DCN1-like protein 4 (UniProt:Q92564), high-probability ATP-dependent RNA helicase DDX60-like (UniProt:A0A804HKC9), glutamate receptor ionotropic, kainate 3, Myb-related protein B, NADH dehydrogenase [ubiquinone] iron-sulfur protein 2, mitochondrial, threonine aspartase 1, testin, transcription factor SOX- 10, doublecortin and CaM kinase-like 2, isoform CRA_c, elongation of very long chain fatty acid protein 2, papalysin-2, forkhead box protein O1, inactive tyrosine-protein kinase PRAG1, splicing regulatory glutamine / lysine-rich protein 1 (UniProt:Q8WXA9), beta-1,4-glucuronyltransferase 1, xylosyl and glucuronyltransferase LARGE1, SH3 domain binding protein 2, liprin-beta-1 (UniProt:Q86W92), 5-oxopeptide lorinase, cancer / testis antigen 2, solute transporter family 52, riboflavin transporter, member 2, ATP-binding cassette subfamily C member 3, dipeptidyl peptidase 2, ETS translocation variant 1 (UniProt:P50549), putative glycosyltransferase ALG1-like, erythrocyte membrane protein of 55 kDa, protein dopey-2, N-terminal kinase-like protein (UniProt:Q96KG9), proton-myo-inositol cotransporter (UniProt:Q96QE2), Arf-GAP with GTPase,ANK repeat and PH domain-containing protein 3 (UniProt:Q96P47), zinc finger protein 423, phospholipid transfer ATPase (UniProt:A0A5K1VW70), roundabout homolog 1 (UniProt:Q9Y6N7), protein spire homolog 1 (UniProt:Q08AE8), transcription factor ETV7, small G protein signaling modulator 3, hyaluronidase-2, autism susceptibility gene 2 protein (UniProt:Q8WXX7), hormone-sensitive lipase, protein FAM50 B, signal regulatory protein delta (UniProt:Q9H106), zinc finger protein 385C, tumor necrosis factor receptor superfamily member 11A, spiny-like protein 2 (UniProt:Q7Z3G6), erythroid transformation-specific transcription factor ERG variant 10, synaptotagmin-like protein 4, galactokinase (UniProt:P51570), cytochrome c oxidase assembly protein COX18, mitochondrial, tyrosine-protein kinase ABL2, motility sperm domain-containing protein 1, transmembrane protein KIAA1109 (fragment) (UniProt:H7C0G8), mitogen-activated protein kinase 13, caspase recruitment domain-containing protein 16, glutamate receptor 4, receptor tyrosine-protein phosphatase U, ankyrin repeat domain-containing protein 40, transcription factor MafF, SEZ6L protein, KH domain-containing, RNA-binding, signal transduction-related protein 2 (UniProt:Q5VWX1), DALR anticodon-binding domain-containing protein 3, elongin-C (UniProt:Q15369), isobutyryl CoA dehydrogenase, mitochondria, centromere protein L, transmembrane protease serine 6, WD repeat-containing protein 87 (UniProt:Q6ZQQ6), Supervillin (UniProt:O95425), Sushi, von Willebrand factor type A, EGF and pentraxin domain-containing protein 1 (UniProt:Q4LDE5), protein BANP (UniProt:Q8N9N5), Delrin-2 (UniProt:Q9GZP9), zinc finger protein 7 (UniProt:P17097),HLA class II histocompatibility complex, DR alpha chain (UniProt:Q30118), polycomb group RING finger protein 5, Toll-like receptor 3, protocadherin-16, mitochondrial 2-oxodicarboxylate carrier, STE20-like serine / threonine-protein kinase, TBC1 domain family member 2A, spectrin alpha chain, erythroid 1, cadherin-7 (UniProt:Q9ULB5), contactin-3, terminal nucleotidyltransferase 5D, nuclear receptor subfamily 4 group A melanoma member 1, laminin subunit alpha-5, cystic fibrosis transmembrane conductance regulator, hydrocephalus-inducible protein homolog, alpha-parvin, tubulin delta chain, tenascin-R, ankyrin and armadillo repeat-containing protein (UniProt:Q7Z5J8), MORC family CW-type zinc finger protein 1, KAT8 regulatory NSL complex subunit 1 (UniProt:A0A0G2JNB1), PWWP domain-containing protein 2A, UPF0687 protein C20orf27, SET-binding protein (UniProt: rot:Q9Y6X0), DNA repair protein RAD51 homolog 2 (UniProt:O15315), sodium / myo-inositol cotransporter 2, pleckstrin homology domain-containing family A member 4, Neurofascin (UniProt:O94856), dystrobrevin beta, aromatic-L-amino acid decarboxylase, protein VAC14 homolog, activin receptor type 2A, MAM and LDL receptor class A domain-containing protein 1, sodium- and chloride-dependent neutral and basic amino acid transporter transmembrane 4-domain subfamily A member 18 (UniProt:A0A499FJ34), protein phosphatase slingshot homolog 1, dehydrogenase / reductase SDR family member 1, eyeless homolog 2, importin-13 (UniProt:O94829), Golgi reassembly stacking protein 2, DNA-dependent metalloprotease SPRTN, enoyl-CoA hydratase, protein WWC2,High Potency Asparagine-tRNA Ligase, Mitochondria, Sodium / Glucose Cotransporter 5, Valyl-tRNA Synthetase, Hamartin (UniProt:Q92574), Serine / Threonine-Protein Kinase ULK1, Cyclin-Dependent Kinase-Like 3 (UniProt:Q8IVW4), Protocadherin Fat 2, histone-lysine N-methyltransferase 2B, protein-tyrosine sulfotransferase 1, suppressor of cytokine signaling 6, Slit homolog 3 protein (UniProt:O75094), 40S ribosomal protein S12, cathepsin F (UniProt:Q9UBX1), high-potency 2-oxoglutarate dehydrogenase E1 component DHKTD1, mitochondria, phosphorylase b kinase regulatory subunit alpha, skeletal muscle isoform, DENN domain-containing protein 2B (UniProt:P78524), ATP-binding cassette subfamily A member 2 (fragment) (UniProt:H0Y8D6), protein MTSS 2 (UniProt:Q765P7), aldehyde dehydrogenase X, mitochondria, protein Niban 2, protein FAM200A, breast cancer anti-estrogen resistance protein 1 (UniProt:P56945), amino acid transporter (UniProt:A0A087X0U3), protein maestro (UniProt:Q9BYG7), sodium and chloride-dependent creatine transporter 1, 1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase delta-3, constitutive coactivator of PPAR-gamma-like protein 2 (UniProt:F8W881), caspase-5, Protein wntless homolog (UniProt:Q5T9L3), androgen receptor (UniProt:P10275), enoyl-CoA delta isomerase 2 (UniProt:A0A0C4DGA2), acetyl-CoA carboxylase 2, AP-4 complex accessory subunit RUSC2, protein odr-4 homolog, nuclear receptor coactivator 6 (UniProt:Q14686), neuroblastoma breakpoint family member 1 (UniProt:Q3BBV0), Rap1 GTPase-activating protein 2 (UniProt:A0A1B0GV05), receptor expression enhancer protein 5, acid sphingomyelinase-like phosphodiesterase 3b, junctophilin-2, tyrosine-protein phosphatase nonreceptor type 3, V-type proton ATPase 21 kDa proteolipid subunit c'', neurogenic locus notch homolog protein 3, trophoblast glycoprotein,Rap1 GTPase-activating protein 1 (UniProt:F2Z357), Ras-related protein Rab-1B (UniProt:E9PLD0), inositol polyphosphate 5-phosphatase K, TGF-beta receptor type 2, formin-2, cytochrome c oxidase assembly factor 6 homolog, G protein-coupled receptor-associated sorting protein 2, Bcl-2-like protein 13 (UniProt:Q9BXK5), myocyte-specific enhancer factor 2B (UniProt:B3KQ23), protein FAM135B, inactive tyrosine-linked kinase protein transmembrane receptor ROR1 (UniProt:Q01973), methionine-R-sulfoxide reductase B2, mitochondria, ENTPD4 protein, endothelin-converting enzyme 2, EEF1AKMT4-ECE2 read-through transcription protein, homeobox protein Meis1 (UniProt:H0YNY8), BRD4-interacting chromatin-remodeling complex-associated protein-like, ubiquinone biosynthesis O-methyltransferase, mitochondria (UniProt:Q9NZJ6), integrin beta-5, Frizzled-6 (UniProt:O60353), certain androgen-regulated gene protein, CTD nuclear envelope phosphatase 1, growth arrest specific protein 6, DNA-dependent RNA polymerase III subunit RPC7-like, exophilin-5, fibroblast growth factor receptor 2, coiled-coil domain-containing protein 120, protein chibby homolog 2, kallikrein-6, villin-like protein (UniProt:O15195), laminin subunit alpha-4 (UniProt:Q16363), SH3 and multiple ankyrin repeats domain protein 2 (UniProt:Q9UPX8), diphosphoinositol polyphosphate phosphohydrolase 3-alpha, centrosomal protein of 131 kDa (UniProt:Q9UPN4), leucine zipper putative tumor suppressor 1, olfactory receptor 2T3, HCG1811249, isoform CRA_e, solute transporter family 25 member 48, phosphodiesterase (UniProt:A1E5M1), integrin alpha-7 (UniProt:Q13683), Gem-related protein 2 (UniProt:O14893),Glutamate receptor ionotropic, NMDA 2B, glypican-3, GPI mannosyltransferase 4, mucin-4 (UniProt:Q99102), mitochondrial peptide methionine sulfoxide reductase, acyl-CoA synthetase short chain family member 3, mitochondria, UPF0692 protein C19orf54, Sushi repeat-containing protein SRPX, galactocerebrosidase (UniProt:P54803), transmembrane protein 260, protein disulfide isomerase CRELD2, arginine / serine-rich protein PNISR (UniProt:Q8TF01), protein adenylyltransferase FICD, zinc finger protein 785, metalloreductase STEAP1, mucolipin-2, kynurenine formamidase (UniProt:K7EK09), protein clear homolog 1 (fragment), SPARC, cytospin-B (UniProt:A0A7I2YQJ3), zinc finger protein 764, serine protease hepsin, olfactory receptor 5K3, inactive phospholipase D5, solute transporter family 22 member 9, mucin-6 (UniProt: Prot:Q6W4X9), transcription factor SOX-30, killer cell immunoglobulin-like receptor 2DS4, serine / threonine-protein kinase Nek10, HERV-H_2q24.3 proviral ancestral Env polyprotein, FH1 / FH2 domain-containing protein 3 (fragment), WD repeat-containing protein 91 (UniProt:A4D1P6), cleavage and polyadenylation specificity factor subunit 6 (UniProt:Q16630), transmembrane protein 104 (UniProt:Q8NE00), programmed cell death 1 ligand 1, and tandem repeat kinase 1 (UniProt:Q16630). Protein ABHD12B, aquaporin-7 (UniProt:B7Z4U2), growth / differentiation factor 5, photoreceptor ciliary actin regulator, membrane-associated guanylate kinase, WW and PDZ domain-containing protein 2 (UniProt:E7EWI0), solute transporter family 5 member 4, 1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase beta-4, endoplasmic reticulum transport protein SEC22c (UniProt:Q9BRL7), GPNMB protein, alpha-fetoprotein, glycosaminoglycan xylosyl kinase,Ras-related protein Rab-38, RING-type E3 ubiquitin transferase (UniProt:D6RAZ0), ATP-binding cassette subfamily B member 5, C-type lectin domain family 10 member A (UniProt:Q8IUN9), complement component C7, tissue factor, adhesion G protein-coupled receptor G6, DNA excision repair protein ERCC-8 (UniProt:A0A6Q8PH55), oligodendrocyte transcription factor 2, lactadherin (fragment), anoctamin (UniProt:A0A2R8Y532), angiotensin-converting enzyme (UniProt:F6X3S4), lysosome-associated transmembrane protein 4B (fragment), and Teashi rt homolog 3, C-Jun-amino-terminal kinase interacting protein 3 (UniProt:E9PFH7), protogenin (UniProt:Q2VWP7), ankyrin repeat domain-containing protein 20B, ATP-sensitive inward rectifier potassium channel 10, sulfhydryl oxidase 2, spiny planar cell polarity protein 3 (UniProt:O43900), cyclin-Y-like protein 1, transcription factor COE4 (UniProt:Q9BQW3), T-box transcription factor TBX4, ribokinase, protein phosphatase 1F, zinc finger protein GLIS3, insulinoma-associated protein 2, DPCR1, Bioorientation of chromosomes in cell division protein 1, transmembrane protein 74B, E3 ubiquitin-protein ligase RNF149 (UniProt:F8WCD0), choline transporter-like protein 5, ATP5MF-PTCD1 read-through, semaphorin-3G, olfactory receptor 5P2, transient receptor potential cation channel subfamily M member 4, putative protein FAM157B, G protein-signaling modulator 1 (UniProt:Q86YR5), patatin-like phospholipase domain-containing protein 4, chloride channel protein ClC-Ka, Rho GTPase-activating protein 28 (UniProt:Q9P2N2), mitochondrial thiamine pyrophosphate carrier, leptin receptor, zinc finger protein ZIC 4, glypican-1 (UniProt:P35052), olfactory receptor 4C6, serpin I2, mast cell / stem cell growth factor receptor Kit, olfactory receptor 52N1, NLR family CARD domain-containing protein 4, E3 ubiquitin-protein ligase RNF43, NADPH oxidase 4 (UniProt:Q9NPH5), phosphoinositide 3-kinase regulatory subunit 5 (UniProt:X6R3K3), PPP2R1A-PPP2R2A interacting phosphatase regulator 1, double zinc ribbon and ankyrin repeat-containing protein 1 (UniProt:Q9NVP4), sodium-independent sulfate anion transporter, glutamate receptor ionotropic type, NMDA 3A, two components: latent membrane protein 2 and latent membrane protein 2, two components: Epstein-Barr nuclear antigen 3 and Epstein-Barr nuclear antigen 3, two components: 65 kDa phosphorylated protein and 65 kDa phosphorylated protein, two components: latent membrane protein 1 and latent membrane protein 1, parathyroid hormone-related protein (fragment), fallopian tube-specific glycoprotein, hepatitis A virus cell receptor 2 (UniProt: Q8TDQ0), anion exchange transporter, cell adhesion molecule 2, cadherin-24, ubiquitin thioesterase OTU1, acetylcholinesterase (UniProt: P2230 3), olfactory receptor 2T4, paraneoplastic antigen-like protein 8A, malonyl-CoA acyl carrier protein transacylase, mitochondria, calcium-binding mitochondrial carrier protein SCaMC-3 (fragment), Netrin-1, transmembrane protein 79 (UniProt:Q9BSE2), potassium voltage-gated channel subfamily A member 10, solute carrier family 22 member 16 (fragment), Vang-like protein 1, roundabout homolog 3 (UniProt:Q96MS0), trypsin-3 (UniProt:P35030), fragile X mental retardation 1 neighbor protein, T cell surface glycoprotein CD1c,Potassium voltage-gated channel subfamily H member 1 (UniProt:O95259), putative gap junction epsilon-1 protein, SLIT and NTRK-like protein 1, uncharacterized protein KIAA0408, 5-hydroxytryptamine receptor 3D (UniProt:Q70Z44), angiotensin-converting enzyme 2, transcription factor SOX-2, scavenger receptor cysteine-rich domain-containing group B protein, lymphocyte antigen 6K, embryonic Fyn-related substrate, regulator of G protein signaling 5 (UniProt: ot:O15539), a disintegrin and metalloproteinase with thrombospondin motifs 2 (UniProt:A0A1B0GTY3), fatty acid-binding protein, brain, cilia- and flagella-associated protein 65, coiled-coil domain-containing protein 39 (UniProt:Q9UFE4), zinc finger protein 521 (UniProt:Q96K83), parathyroid hormone-related protein, Rho guanine nucleotide exchange factor 4 (UniProt:E7EV07), leucine-rich repeat and fibroneclase Syntaxin type III domain-containing protein 3, syntaxin-1A (UniProt:Q16623), adenylate cyclase type 4, PWWP domain-containing DNA repair factor 3A (fragment), IQ domain-containing protein F5, sodium channel protein (UniProt:E9PG18), oocyte secretory protein 2, Rho guanine nucleotide exchange factor 10-like protein (UniProt:Q9HCE6), trophinin (UniProt:A0A087X070), short transient receptor potential channel 4, sodium-dependent dopamine tran transporter, unidentified protein, brain-specific serine protease 4, transmembrane protease serine 4 (UniProt:Q9NRS4), choline O-acetyltransferase, serine palmitoyltransferase small subunit B, large T antigen, Ig gamma-2 A chain C region, membrane-bound form, podoplanin, liprin-alpha-4 (UniProt:O75335), signal peptide, CUB and EGF-like domain-containing protein 2, claudin-18, corticotropin-releasing factor binding protein (UniProt:D6RHH7),beta-1,4-N-acetylgalactosaminyltransferase 3, solute transporter family 22 member 17 (UniProt:Q8WUG5), armadillo repeat protein deletion in palatocardiofacial syndrome (UniProt:E9PDC3), selenoprotein V, transmembrane protein 255A, small T antigen sT, nephrin, titin (UniProt:Q8WZ42), ADAM9 protein, retinitis pigmentosa 1-like 1 protein, transmembrane and coiled-coil domain-containing protein 2, protein crumbs homolog 1, sorting nexin-16 (UniProt:A0A0C4DGW2), distal membrane-arm assembly complex protein 2 (UniProt:B4DFT4), olfactory receptor receptor 4C11, PH and SEC7 domain-containing protein 1, olfactory receptor 51E1, contactin-6, COL11A2, POTE ankyrin domain family member H, keratin, type I cytoskeleton 24, uncharacterized serine / threonine-protein kinase SBK3, arylsulfatase I, tubulin polyglutamylase TTLL11, aquaporin-12B, calcitonin, neuroblastoma breakpoint family member 14 (UniProt:A0A087WVU4), inactive N-acetyllactosaminide alpha-1,3-galactosyltransferase (UniProt:Q4G0N0), serine protease 33 (fragment), 7-methylguanosine phosphate specific 5'-nucleotidase (fragment), carcinoembryonic antigen-related cell adhesion molecule 18, trypsin-3 (UniProt: A0A7P0MNE9), leukocyte immunoglobulin-like receptor subfamily B member 3 (UniProt: A0A0G2JMM1), uncharacterized protein C5orf60, leucine-rich repeat-containing protein 37A3 (UniProt: O60309), putative serpin A13, coiled-coil domain-containing protein 74B (fragment), keratin-associated protein 11-1, blood group Rh (CE) polypeptide (UniProt: E7EQ47), protein SSX7, melatonin-related receptor, paired mesodermal homeobox protein 2A, ankyrin repeat domain-containing protein 36B, protein FAM83H (UniProt: A0A494C1T9),Homeobox protein Hox-A1 (UniProt:E7ERT8), golgin subfamily A member 6-like protein 2 (UniProt:Q8N9W4), PRAME family member 4, PRAME family member 9, leukocyte-derived chemotaxin-2, proprotein convertase subtilisin / kexin type 5 (UniProt:B1AMG5), olfactory receptor 5T3, olfactory receptor 4C3, phosphoinositide phospholipase C (UniProt:A0A3B3ISW9), calpain-11 (fragment), LILRA6, sequence Similar to family 53, member A, isoform CRA_b, C-type lectin domain family 18 member C, uncharacterized protein C9orf57, homeobox protein Hox-B1, two components: insulin, isoform 2 and endoplasmic reticulum chaperone BiP, two components: insulin (UniProt: P01308) and endoplasmic reticulum chaperone BiP, prostate stem cell antigen, interleukin-22, BTB / POZ domain-containing protein 16, olfactory receptor 51B4, endogenous retrovirus group K member 18 Env polyprotein, very long chain fatty acid omega-hydroxylase, hyaluronan and proteoglycan linking protein 3 (UniProt:H3BTH8), kallikrein-4, two components: spike glycoprotein and spike glycoprotein, two components: major capsid protein L1 and major capsid protein L1, ADAMTS-like protein 1 (UniProt:A2A343), putative uncharacterized protein GAFA-1, SMAD5 antisense gene protein 1, putative uncharacterized protein encoded by LINC00615, B lymphocyte antigen CD20 (UniProt:E9PKH8), endogenous retrovirus group K member 113 Gag polyprotein, endogenous retrovirus group K member 24 Gag polyprotein, neuropeptide B / W receptor type 1, cadherin-18 (UniProt:D6RIH8), SUMO-conjugating enzyme UBC9 (UniProt:B0QYN7), neutral cholesterol ester hydrolase 1 (UniProt:A0A0A0MTJ9), sodium / glucose cotransporter 4, high-potency cysteine-tRNA ligase,Mitochondria (fragment) (UniProt:F5H579), unknown protein (UniProt:A0A494C1I1), HCG2044781, P antigen family member 4, 2 components: HLA-A, * 02:01 chain and Wilms tumor protein (UniProt: A0A0A0MT54), two components: immunoglobulin heavy constant gamma 1 and immunoglobulin heavy constant gamma 1, receptor tyrosine-protein kinase erbB-2, two components: genomic polyprotein and genomic polyprotein, or claudin-18 may be mentioned.

[0099] In some embodiments, the extracellular domain can include one or more epitopes. In some embodiments, the extracellular domain can include one or more epitopes that are homologous to the extracellular domain. In some embodiments, the extracellular domain can include one or more epitopes that are heterologous to the extracellular domain. In some embodiments, the extracellular domain can include about 1 to about 10 epitopes. In some embodiments, the extracellular domain can include at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or at least 10 epitopes. In some embodiments, the extracellular domain can include two or more copies of the same epitope. In some embodiments, the extracellular domain can include two or more epitopes, where each of the two or more epitopes binds to the same antibody, peptide hormone, or growth factor. For example, one or more copies of the same epitope sequence are inserted into an extracellular domain sequence that naturally includes one copy of the same epitope sequence.

[0100] In some embodiments, the extracellular domain can include two or more epitopes, where each of the two or more epitopes is a different epitope. In some embodiments, each of the two or more epitopes can bind to the same antibody, peptide hormone, or growth factor. In some embodiments, each of the two or more epitopes can bind to a different antibody, peptide hormone, or growth factor. In some embodiments, the extracellular domain can include two or more epitopes, where the two or more epitopes can include a combination of the same and different epitopes. For example, the extracellular domain can include at least four epitopes, where two of the at least four epitopes bind to one antibody, peptide hormone, or growth factor, and the other two of the at least four epitopes bind to another antibody, peptide hormone, or growth factor.

[0101] In some embodiments, an epitope can comprise about 15 to about 300 contiguous residues of an extracellular domain of a protein described herein. In some embodiments, an epitope can comprise at least about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or at least about 300 contiguous residues of an extracellular domain of a protein described herein.

[0102] In some embodiments, the epitope can comprise a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or at least 99.9% identity to any one of SEQ ID NOs: 4, 5, 6, 18, 19, 20, 64, 65, or a variant thereof. In some embodiments, the epitope can comprise a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 4, 5, 6, 18, 19, 20, 64, 65, or a variant thereof. In some embodiments, the epitope can comprise a sequence having 100% sequence identity to SEQ ID NOs: 4, 5, 6, 18, 19, 20, 64, 65, or a variant thereof.

[0103] In one embodiment, the epitope can be derived from DLL3 or a variant thereof. In this embodiment, the DLL3 epitope can comprise about 15 to about 260 consecutive residues of the extracellular domain of DLL3. In some embodiments, the DLL3 epitope can comprise at least about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, or at least about 260 consecutive residues of the extracellular domain of DLL3. In some embodiments, the DLL3 epitope can include DLL3 amino acid residues 27 to 492. In some embodiments, the DLL3 epitope can include DLL3 amino acid residues 189 to 209. In some embodiments, the DLL3 epitope can include a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or at least 99.9% identity to SEQ ID NO: 5, 6, 18, 19, 20, 64, 65, or a variant thereof. In some embodiments, the DLL3 epitope can include a sequence having at least 80% identity to SEQ ID NO: 5, or a variant thereof. In some embodiments, the DLL3 epitope can include a sequence having 100% sequence identity to SEQ ID NO: 5, 6, 18, 19, 20, 64, 65, or a variant thereof.

[0104] In one embodiment, the epitope can be derived from SSTR2 or a variant thereof. In this embodiment, the SSTR2 epitope can include about 15 to about 280 contiguous residues of the extracellular domain of SSTR2. In some embodiments, an SSTR2 epitope can have at least about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or at least about 300 contiguous residues of the extracellular domain of SSTR2. In some embodiments, an SSTR2 epitope can include amino acid residues 39-314 of SSTR2. In some embodiments, the SSTR2 epitope can comprise a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or at least 99.9% identity to SEQ ID NO: 4, or a variant thereof. In some embodiments, the SSTR2 epitope can comprise a sequence having at least 80% identity to SEQ ID NO: 4, or a variant thereof. In some embodiments, the SSTR2 epitope may comprise a sequence having 100% sequence identity to SEQ ID NO:4, or a variant thereof.

[0105] In one embodiment, the epitope can be derived from PSMA or a variant thereof, in this embodiment, the PSMA epitope can include about 15 to about 300 contiguous residues of the extracellular domain of PSMA. In some embodiments, a PSMA epitope can have at least about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or at least about 300 contiguous residues of the extracellular domain of PSMA.

[0106] In some embodiments, the epitope can bind to an antibody, peptide hormone, or growth factor with a Kd of less than or equal to 20 nM. For example, the epitope can bind to an antibody, peptide hormone, or growth factor with a Kd of less than or equal to 20 nM, 19 nM, 18 nM, 17 nM, 16 nM, 15 nM, 14 nM, 13 nM, 12 nM, 11 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, or 0.1 nM. In some embodiments, the epitope is capable of binding to an antibody, peptide, or growth hormone with a Kd of less than or equal to 100 pM, 90 pM, 80 pM, 70 pM, 60 pM, 50 pM, 40 pM, 30 pM, 20 pM, 10 pM, 5 pM, or 1 pM.

[0107] In some embodiments, the epitopes described herein can include activatable epitopes. In some embodiments, the activatable epitopes can be preferentially accessible to binding agents (e.g., antibodies, peptide hormones, or growth factors) in the internal or external physical microenvironment of cells affected by a disease or condition, compared to the internal or external physical microenvironment of cells not affected by the disease or condition. In some embodiments, the activatable epitopes can be selectively available for binding in tumor microenvironments. For example, the activatable epitopes can be preferentially accessible to binding agents in the internal or external physical microenvironment of tumor cells, compared to the internal or external physical microenvironment of non-tumor cells. In some embodiments, the activatable epitopes can be preferentially accessible to binding agents in the internal or external physical microenvironment of cells affected by an autoimmune disease or neurodegenerative disease, compared to the internal or external physical microenvironment of cells not affected by the autoimmune disease or neurodegenerative disease. In some embodiments, the activatable epitope can be activated by a mechanism including, but not limited to, phosphorylation, glycosylation, methylation, ubiquitination, α-acetylation, lipidation, glycosylphosphatidylinositol lipidation, amidation, sulfonation, oxidation, or any combination thereof.For example, the epitope can be accessible to the binding agent when one or more amino acid residues on the epitope are phosphorylated, glycosylated, methylated, ubiquitination, or α-acetylation.For example, the epitope can be accessible to the binding agent when one or more amino acid residues on the epitope are modified by lipidation, glycosylphosphatidylinositol lipidation, amidation, sulfonation, or oxidation.

[0108] In some embodiments, the activatable epitopes described herein may be accessible to the binding agent by a conformational change of one or more pH-sensitive helices. In some embodiments, the extracellular domains comprising the activatable epitopes described herein may further comprise a pH-sensitive helix. The pH-sensitive helix may be derived from natural or recombinant sources. For example, the pH-sensitive helix may be derived from any protein that comprises a pH-sensitive helix. For example, the pH-sensitive helix may be designed and engineered de novo by organizing one or more amino acid residues. In some embodiments, the conformational change of the pH-sensitive helix may be driven by the organization of one or more histidine residues in a hydrogen bond network in the pH-sensitive helix. In some embodiments, the pH-sensitive helix may associate and multimerize with one or more pH-sensitive helices at physiological pH or at pH>6.5. In some embodiments, the pH-sensitive helix may undergo a conformational change at low pH, e.g., pH<6.5. In some embodiments, the pH-sensitive helix can undergo conformational changes at low pH through a mechanism involving electrostatic and / or steric repulsion organized as histidine residues in a hydrogen bond network that are protonated. Details of the design and mechanism of the pH-sensitive helix are described in Boyken SE, et al. De novo design of tunable, pH-driven conformational changes. Science. 2019 May 17;364(6441):658-664. doi: 10.1126 / science.aav7897. PMID: 31097662; PMCID: PMC7072037.

[0109] In some embodiments, an extracellular domain comprising an activatable epitope as described herein can further comprise about 1 to about 10 copies of a pH-sensitive helix. For example, an extracellular domain comprising an activatable epitope as described herein can further comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or at least 10 copies of a pH-sensitive helix. In some embodiments, an extracellular domain comprising an activatable epitope as described herein can comprise at least two copies of a pH-sensitive helix. In this embodiment, the activatable epitope can be flanked by at least two copies of a pH-sensitive helix.

[0110] In some cases, the pH-sensitive helix can include a pH-sensitive multimerization domain. In some embodiments, the pH-sensitive helix can associate and multimerize with one or more pH-sensitive helices at bodily pH or a pH higher than 6.0. In some embodiments, the epitope may not be accessible to the binding agent when the pH-sensitive helix associates and multimerizes with one or more pH-sensitive helices. In some embodiments, a pH greater than 6.0 can include pH>6.1, pH>6.2, pH>6.3, pH>6.4, pH>6.5, pH>6.6, pH>6.7, pH>6.8, pH>6.9, pH>7.0, pH>7.1, pH>7.2, pH>7.3, pH>7.4, pH>7.5, pH>7.6, pH>7.7, pH>7.8, pH>7.9, pH>8.0, pH>8.5, pH>9.0, pH>9.5, or pH>10.0.

[0111] In some embodiments, the pH-sensitive helix can dissociate from one or more other pH-sensitive helices at low pH. For example, the pH-sensitive helix can dissociate from one or more other pH-sensitive helices at pH<6.0, pH<5.9, pH<5.8, pH<5.7, pH<5.6, pH<5.5, pH<5.4, pH<5.3, pH<5.2, pH<5.1, pH<5.0, pH<4.9, pH<4.8, pH<4.7, pH<4.6, pH<4.5, pH<4.4, pH<4.3, pH<4.2, pH<4.1, pH<4.0, pH<3.5, pH<3.0, pH<2.5, pH<2.0, pH<1.5, or pH<1.0. In some embodiments, the epitope can be accessible to the binding agent when the pH-sensitive helix dissociates from one or more other pH-sensitive helices.

[0112] In some cases, the extracellular domain comprising an activatable epitope and a pH-sensitive helix may be configured to bind to the pH-sensitive helix. In some embodiments, the pH-sensitive helix may bind to the extracellular domain comprising the pH-sensitive helix at bodily pH or at a pH higher than 6.0. In some embodiments, the epitope may not be accessible to the binding agent when the pH-sensitive helix binds to the extracellular domain comprising the pH-sensitive helix. In some embodiments, a pH greater than 6.0 can include pH>6.1, pH>6.2, pH>6.3, pH>6.4, pH>6.5, pH>6.6, pH>6.7, pH>6.8, pH>6.9, pH>7.0, pH>7.1, pH>7.2, pH>7.3, pH>7.4, pH>7.5, pH>7.6, pH>7.7, pH>7.8, pH>7.9, pH>8.0, pH>8.5, pH>9.0, pH>9.5, or pH>10.0.

[0113] In some embodiments, the pH-sensitive helix can dissociate from the extracellular domain comprising the pH-sensitive helix at low pH. For example, the pH-sensitive helix can dissociate from the extracellular domain comprising the pH-sensitive helix at pH<6.0, pH<5.9, pH<5.8, pH<5.7, pH<5.6, pH<5.5, pH<5.4, pH<5.3, pH<5.2, pH<5.1, pH<5.0, pH<4.9, pH<4.8, pH<4.7, pH<4.6, pH<4.5, pH<4.4, pH<4.3, pH<4.2, pH<4.1, pH<4.0, pH<3.5, pH<3.0, pH<2.5, pH<2.0, pH<1.5, or pH<1.0. In some embodiments, the epitope can be accessible to the binding agent when the pH-sensitive helix dissociates from the extracellular domain comprising the pH-sensitive helix.

[0114] In some cases, the engineered polypeptide described herein can include an activatable epitope or ligand binding site integrated into the cell membrane. In this embodiment, the engineered polypeptide can further include one or more pH-sensitive helices in the extracellular domain, where each of the one or more pH-sensitive helices can include a multimerization domain. In some embodiments, the one or more pH-sensitive helices can associate or multimerize at physiological pH or at a pH higher than 6.0. In this embodiment, the epitope or ligand binding site may not be accessible to the binder when the one or more pH-sensitive helices associate or multimerize. In some embodiments, a pH greater than 6.0 can include pH>6.1, pH>6.2, pH>6.3, pH>6.4, pH>6.5, pH>6.6, pH>6.7, pH>6.8, pH>6.9, pH>7.0, pH>7.1, pH>7.2, pH>7.3, pH>7.4, pH>7.5, pH>7.6, pH>7.7, pH>7.8, pH>7.9, pH>8.0, pH>8.5, pH>9.0, pH>9.5, or pH>10.0.

[0115] In some embodiments, one or more pH-sensitive helices can dissociate from each other at low pH. In this embodiment, the epitope or ligand binding site may be accessible to the binder when one or more pH-sensitive helices dissociate at low pH. In some embodiments, low pH can include pH<6.0, pH<5.9, pH<5.8, pH<5.7, pH<5.6, pH<5.5, pH<5.4, pH<5.3, pH<5.2, pH<5.1, pH<5.0, pH<4.9, pH<4.8, pH<4.7, pH<4.6, pH<4.5, pH<4.4, pH<4.3, pH<4.2, pH<4.1, pH<4.0, pH<3.5, pH<3.0, pH<2.5, pH<2.0, pH<1.5, or pH<1.0.

[0116] In some cases, the engineered polypeptides described herein can include a secretion signal. In some embodiments, the engineered polypeptides that include a secretion signal may not include a signal peptide. For example, the secretion signal may replace the signal peptide in the engineered polypeptide. In some embodiments, the engineered polypeptides described herein can include a membrane binding domain. In some embodiments, the engineered polypeptides that include a membrane binding domain may not include a transmembrane domain. For example, the membrane binding domain may replace the transmembrane domain in the engineered polypeptide. In some embodiments, the engineered polypeptides described herein can include a secretion signal and a membrane binding domain. In some embodiments, the membrane binding domain can include a pH-sensitive membrane binding helix. In some embodiments, the engineered polypeptides that include a secretion signal and a pH-sensitive membrane binding helix can propagate to one or more neighboring cells. In one example, the engineered polypeptides that include a secretion signal and a pH-sensitive membrane binding helix can be secreted into an extracellular fluid or physical microenvironment and may not attach to cells when secreted into a non-acidic or non-tumor extracellular environment. In some embodiments, the non-acidic or non-tumor environment can have a pH greater than 6.5, e.g., pH>6.6, pH>6.7, pH>6.8, pH>6.9, pH>7.0, pH>7.1, pH>7.2, pH>7.3, pH>7.4, pH>7.5, pH>7.6, pH>7.7, pH>7.8, pH>7.9, pH>8.0, pH>8.5, pH>9.0, pH>9.5, or pH>10.0. In some embodiments, the non-acidic or non-tumor environment can have a pH=7.2. In another example, an engineered polypeptide comprising a secretion signal and a pH-sensitive membrane-associated helix can be secreted into an extracellular fluid or physical microenvironment and can attach to cells, including the secreting cell and neighboring cells, when secreted at a pH level that can allow assembly of the membrane-associated helix.In some embodiments, pH levels that may allow assembly of membrane-bound helices may include a pH equal to or less than 6.5, e.g., pH<6.4, pH<6.3, pH<6.2, pH<6.1, pH<6.0, pH<5.9, pH<5.8, pH<5.7, pH<5.6, pH<5.5. In some embodiments, pH levels that may allow assembly of membrane-bound helices may include pH=6.5.

[0117] In some embodiments, the pH sensitive helix can include a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or at least 99.9% identity to any one of SEQ ID NOs: 1-3. In some embodiments, the pH sensitive helix can include a sequence having at least 80% identity to any one of SEQ ID NOs: 1-3. In some embodiments, the pH sensitive helix can include a sequence having 100% identity to any one of SEQ ID NOs: 1-3.

[0118] In some embodiments, an extracellular domain that comprises an activatable epitope described herein can comprise a sequence set forth as [helix]-[helix]-[helix]-[epitope], where [helix] represents a pH-sensitive helix; and [epitope] represents an activatable epitope.

[0119] In some embodiments, an extracellular domain comprising an activatable epitope as described herein can comprise the sequence set forth as: [helix]-[linker]-[helix]-[linker]-[helix]-[linker]-[epitope], where [helix] represents a pH sensitive helix; [linker] represents a linker; and [epitope] represents an activatable epitope. In some embodiments, the linker can comprise the sequence (GGS)n or (GGS)nFCYWKTCT(GGS)n, where n is an integer between 1 and 10. For example, n=1, n=2, n=3, n=4, n=5, n=6, n=7, n=8, n=9, or n=10.

[0120] In some embodiments, an extracellular domain comprising an activatable epitope as described herein can comprise the sequence set forth as: [helix]-[linker]-[helix]-[linker]-[helix]-[epitope], where [helix] represents a pH sensitive helix; [linker] represents a linker; and [epitope] represents an activatable epitope. In some embodiments, the linker can comprise the sequence (GGS)n or RLCRPRSAPSRCGPGLRPCAP, where n is an integer between 1 and 10. For example, n=1, n=2, n=3, n=4, n=5, n=6, n=7, n=8, n=9, or n=10.

[0121] In some cases, an engineered polypeptide comprising an extracellular domain comprising an epitope, a transmembrane or membrane affinity domain, and / or a hinge domain can further comprise a scaffold domain. In some embodiments, the scaffold domain can be located N-terminal or C-terminal to the hinge domain. In some embodiments, the scaffold domain can be located N-terminal or C-terminal to the epitope. In some embodiments, the scaffold domain can comprise an epitope. In some embodiments, the scaffold domain can be located N-terminal to the hinge domain and C-terminal to the epitope. In some embodiments, the scaffold domain can be located N-terminal to the hinge domain and N-terminal to the epitope. In some embodiments, the scaffold domain can comprise an epitope and can be located N-terminal to the hinge domain. In some embodiments, the scaffold domain can be located at the N-terminus of the mature polypeptide (e.g., after translation and cleavage of the secretion signal).

[0122] In some embodiments, the scaffold domain can comprise a heavy chain variable (VH) domain. In some embodiments, the scaffold domain may not comprise a light chain variable (VL) domain. In some embodiments, the scaffold domain can comprise a VH domain and does not comprise a VL domain. In some embodiments, the scaffold domain can comprise a single chain antibody (nanobody or VHH domain). In some embodiments, the nanobody domain can comprise a mutation in a complementarity determining region (CDR1), CDR2, or CDR3 of the VH domain. In some embodiments, the nanobody domain can comprise an inactivating mutation in a CDR1, CDR2, or CDR3 of the VH domain. In some embodiments, such inactivating mutations can include replacing at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 residues in the CDR regions, where the charge of the replaced amino acid has been swapped or a hydrophobic amino acid has been swapped for a hydrophilic amino acid, or one or more of the CDRs have been replaced with (GGS)n or (GGGS)n.

[0123] In some embodiments, the scaffold domain can include an epitope inserted into the CDR1, CDR2, or CDR3 of the VH domain, VHH domain, or nanobody domain. In some embodiments, the scaffold domain can include one or more epitopes inserted into the CDR1, CDR2, or CDR3 of the VH domain. For example, the scaffold domain can include about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 epitopes inserted into the CDR1, CDR2, or CDR3 of the VH domain. In some embodiments, the scaffold domain can include two or more epitopes inserted into the CDR1, CDR2, or CDR3 of the VH domain, where each of the two or more epitopes is the same epitope that binds to the same binding agent. In some embodiments, the scaffold domain can include two or more epitopes inserted into the CDR1, CDR2, or CDR3 of the VH domain, where each of the two or more epitopes is a different epitope that binds to the same binding agent. In some embodiments, the scaffold domain can include two or more epitopes inserted into the CDR1, CDR2, or CDR3 of the VH domain, where each of the two or more epitopes is a different epitope that binds to a different binding agent. In some embodiments, the scaffold domain can include two or more epitopes inserted into the CDR1, CDR2, or CDR3 of the VH domain, where each of the two or more epitopes includes a combination of the same epitope and different epitopes that bind to the same binding agent. In some embodiments, the scaffold domain can comprise two or more epitopes inserted into the CDR1, CDR2, or CDR3 of the VH domain, where the two or more epitopes include a combination of the same epitope that binds to the same binding agent and different epitopes that bind to the same or different binding agents.

[0124] In some embodiments, the scaffold domain can comprise a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or at least 99.9% sequence identity to SEQ ID NO: 63, or a variant thereof. In some embodiments, the scaffold domain can comprise a sequence having at least 80% sequence identity to SEQ ID NO: 63, or a variant thereof. In some embodiments, the scaffold domain can comprise a sequence having 100% sequence identity to SEQ ID NO:63, or a variant thereof. Transmembrane / Membrane Binding Domains

[0125] In some aspects, the engineered polypeptides described herein can further comprise a transmembrane domain or a membrane-affinity domain that can associate with the outer membrane of a cell.In some embodiments, the engineered polypeptide sequence can comprise a sequence for an extracellular domain sequence that contains an epitope and a sequence for a transmembrane domain or a membrane-affinity domain that can associate with the outer membrane of a cell that is encoded by a single nucleic acid sequence.In some embodiments, the engineered polypeptide sequence can be designed to include a transmembrane domain or a membrane-affinity domain that can associate with the outer membrane of a cell that is heterologous to the extracellular domain of the engineered polypeptide.

[0126] In some embodiments, the engineered polypeptides described herein can include a transmembrane domain. In some embodiments, the transmembrane domain can include a membrane-spanning protein domain. In some embodiments, the transmembrane domain can include one or more hydrophobic amino acid residues. In some embodiments, the transmembrane domain can be derived from a protein that includes a single, one-time, or single-span transmembrane α-helical domain. In some embodiments, the transmembrane domain can be derived from a protein that includes multiple, multiple-time, or multiple-span transmembrane α-helical domains. In some embodiments, the transmembrane domain can be derived from a protein that includes a monotopic transmembrane α-helical domain. In some embodiments, the transmembrane domain can be derived from a protein that includes a bitopic transmembrane α-helical domain. In some embodiments, the transmembrane domain can be derived from a protein that includes a polytopic transmembrane α-helical domain. In some embodiments, the transmembrane can be derived from a protein that includes a polytopic transmembrane β-sheet domain. In some embodiments, the transmembrane domain can include a single-span transmembrane domain. In some embodiments, the transmembrane domain can be derived from a glycosylphosphatidylinositol (GPI)-tethered protein. In some embodiments, the GPI anchor can include a glycolipid post-translational modification of the polypeptide.

[0127] In some embodiments, the transmembrane domain can be derived from natural or recombinant sources. For example, the transmembrane domain can be derived from any membrane-bound or transmembrane protein. Non-limiting examples of transmembrane can include transmembrane domains derived from DLL3, PSMA, SSTR2, PD-L1, EGFR, CD28, CD4, CD8, CD8a, CD8b, ICOS, CD73, NKG2D, MUC16, ROR1, HER2, HER3, HER4, TCR gamma, TCR delta, CD3 epsilon, CD3 gamma, CD3 delta, or CD3 zeta. In some embodiments, the transmembrane domain can be derived from DLL3, PSMA, SSTR2, PD-L1, EGFR, CD28, CD4, CD8, CD8a, CD8b, ICOS, or CD73.

[0128] In some cases, other transmembrane domains may be identical based on sequence annotation from UniProtKB / Swiss-Prot. Alternatively or additionally, transmembrane regions can be detected from primary sequences using the hydrophobic moment plot method of TMHMM, Memsat, Phobius and Eisenberg et al. (Eisenberg et al. J Mol Biol. 1984 Oct 15;179(1):125-42. doi: 10.1016 / 0022-2836(84)90309-7, which is incorporated herein by reference in its entirety). A suitable transmembrane domain can include 2-5 residues at the N- and C-termini of the predicted helices to ensure maintenance of the same membrane surface interactions.

[0129] In some embodiments, the transmembrane domain can include a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or at least 99.9% sequence identity to the transmembrane domain of any one of SEQ ID NOs: 68-76. In some embodiments, the transmembrane domain can include a sequence having at least 80% sequence identity to the transmembrane domain of any one of SEQ ID NOs: 68-76. In some embodiments, the transmembrane domain can comprise a sequence having 100% sequence identity to the transmembrane domain of any one of SEQ ID NOs:68-76.

[0130] In some embodiments, the engineered polypeptides described herein can include a membrane-affinity domain that can associate with the outer membrane of a cell. For example, the engineered polypeptides can include a domain that associates with the outer membrane of a cell by mechanisms including, but not limited to, interactions with amphipathic α-helices parallel to the membrane plane (in-plane membrane helices); interactions with hydrophobic loops; interactions with covalently attached membrane lipids; or ionic or electrostatic interactions with membrane lipids. In some embodiments, the membrane-affinity domain can be derived from a protein that includes a monotopic α-helical domain. In some embodiments, the engineered polypeptides that include a membrane-affinity domain can be tethered to the membrane by incorporation of covalently attached lipids into the bilayer. In some embodiments, the membrane-affinity domain that can associate with the outer membrane of a cell can be attached to one side of the membrane (e.g., the outside of the membrane) and may not span the entire membrane.

[0131] Non-limiting examples of membrane affinity domains include members of the large homology domain family, including the pleckstrin homology (PH) domain superfamily; Fab1, YTOB, Vac1, EEA1 (FYVE) domains; phagocyte oxidase or Phox homology (PX) domains; C1 and C2 domains (defined by homology to regions in protein kinase C); epsin N-terminal homology (ENTH) domains; band 4.1, ezrin, radixin, moesin (FERM) domains; and Bin, amphiphysin, Rvs (BAR) domains. In some embodiments, the membrane affinity domain can comprise one or more alpha helices (e.g., an ENTH domain); a combination of one or more beta strands and one or more alpha helices (e.g., a C1 domain, a PX domain); a beta sandwich (e.g., a C2 domain); a beta barrel capped at one end by an alpha helix (e.g., a PH domain); or a dual zinc finger module (e.g., a FYVE domain, details of membrane affinity domains are described in Hurley JH. Membrane binding domains. Biochim Biophys Acta. 2006 Aug;1761(8):805-11. doi: 10.1016 / j.bbalip.2006.02.020. Epub 2006 Mar 24. PMID: 16616874; PMCID: PMC2049088).

[0132] In some embodiments, the membrane affinity domain can comprise a helical domain derived from bacteriorhodopsin. Bacteriorhodopsin is an integral membrane archaeal protein that comprises a hexagonal lattice that comprises seven transmembrane alpha helices and three identical protein chains, each of which contains one molecule of retinal. Details of bacteriorhodopsin are described in Woolf TB. Molecular dynamics of individual alpha-helices of bacteriorhodopsin in dimyristol phosphatidylocholine. I. Structure and dynamics. Biophys J. 1997 Nov;73(5):2376-92. doi: 10.1016 / S0006-3495(97)78267-5. PMID: 9370432; PMCID: PMC1181140.

[0133] In some embodiments, the membrane affinity domain can include a C1, C2, PH, FYVE, PX, or ENTH domain, or a transmembrane helix from bacteriorhodopsin. In some embodiments, the membrane affinity domain can include a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or at least 99.9% sequence identity to any one of SEQ ID NOs: 77-81. In some embodiments, the membraneophilic domain can comprise a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 77 to 81. In some embodiments, the membraneophilic domain can comprise a sequence having 100% sequence identity to any one of SEQ ID NOs: 77 to 81. Hinge Domain

[0134] In some aspects, the engineered polypeptides described herein can further comprise a hinge domain. For example, the engineered polypeptide can comprise an extracellular-directed polypeptide spacer domain that can comprise a hinge domain. In some embodiments, the extracellular-directed polypeptide spacer domain that comprises a hinge domain can connect an extracellular domain that comprises a transmembrane domain and an epitope. In some embodiments, the extracellular-directed polypeptide spacer domain that comprises a hinge domain can connect an extracellular domain that comprises a membrane affinity domain that is associated with the outer membrane of a cell and an epitope. For example, the engineered polypeptides described herein can comprise n extracellular-directed polypeptide spacer domains that comprise a hinge domain between a transmembrane domain or an extracellular domain that comprises a membrane affinity domain and an epitope. In some embodiments, the engineered polypeptide sequence can comprise a sequence for an extracellular domain sequence that contains an epitope, a sequence for a transmembrane domain or a membrane affinity domain that can be associated with the outer membrane of a cell, and a sequence for an extracellular-directed polypeptide spacer domain that comprises a hinge domain that is encoded by a single nucleic acid sequence. In some embodiments, the engineered polypeptide sequence can be designed to include a transmembrane domain or a membraneophilic domain capable of associating with the outer membrane of a cell that is heterologous to the extracellular domain of the engineered polypeptide.

[0135] In some embodiments, the hinge domain can be derived from an Ig superfamily receptor. For example, the hinge domain can be derived from CD4, IgG1, IgG2, IgG3, IgG4, or IgK, or any combination thereof. In some embodiments, the hinge domain can comprise the sequence (GGGS)n, where n is any integer. In some embodiments, n is an integer between 1 and 10 or between 1 and 20. For example, n=1, n=2, n=3, n=4, n=5, n=6, n=7, n=8, n=9, or n=10. In some embodiments, the hinge domain can be derived from CD4, CD8, CD8a, CD8b, CD28, or any combination thereof.

[0136] In some cases, other transmembrane domains can be identified based on sequence annotation from UniProtKB / Swiss-Prot or by identifying regions with homology to CD4, IgG1, IgG2, IgG3, IgG4, IgK, CD8, CD8a, CD8b, or CD28 hinge regions. In some embodiments, the hinge domain can comprise a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or at least 99.9% sequence identity to the hinge sequence of any one of SEQ ID NOs: 56, 57, 58, 59, 60, 61, 62, 66, 67, or a variant thereof. In some embodiments, the hinge domain can comprise a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or at least 99.9% sequence identity to a hinge sequence of any of the proteins described herein. In some embodiments, the hinge domain can comprise a sequence having at least 80% sequence identity to any one of the hinge sequences of SEQ ID NOs: 56, 57, 58, 59, 60, 61, 62, 66, 67, or a variant thereof. In some embodiments, the hinge domain can comprise a sequence having 100% sequence identity to any one of the hinge sequences of SEQ ID NOs: 56, 57, 58, 59, 60, 61, 62, 66, 67, or a variant thereof. Detection methods / antibodies / treatments

[0137] In some cases, the methods described herein include administering to the subject an antibody or antigen-binding fragment thereof, a protein ligand or functional fragment thereof, or a small molecule configured to bind to the epitope in conjunction with administering to the subject a composition comprising a nucleic acid or vector described herein.

[0138] The nucleic acid, vector, antibody or antigen-binding fragment thereof, protein ligand, or small molecule can be administered to the subject by any suitable method, such as intravenous, subcutaneous, intracerebroventricular, intrathecal, intraventricular, transdermal, intramuscular, oral, inhalation, nasal, rectal, intratumoral, or tumor-proximal. Tumor-proximal may refer to administration to tissues near the tumor, or administration to an area that is expected to be accessible to the tumor by the lymphatic system (e.g., adjacent lymph nodes). Intratumoral or tumor-proximal approaches may include the use of additional imaging techniques, such as, for example, endoscopic ultrasound (see, e.g., Shirley et al. Gastroenterol Res Pract. 2013; 2013: 207129) or bronchoscopy (see, e.g., Rojas-Solano et al. J Bronchology Interv Pulmonol. 2018 Jul; 25(3): 168-175). In some embodiments, the composition is administered to at least one of cervical, epitrochlear, supraclavicular, cervical, axillary, mediastinal, supraclavicular, mesenteric, inguinal, femoral, or popliteal lymph nodes. In some cases, lymph node-based administration can serve as a method of focused local delivery to tissue regions. In some cases, the composition comprising the nucleic acid, vector, antibody or antigen-binding fragment thereof, protein ligand, or small molecule can be a pharma- ceutically acceptable composition (e.g., one that comprises a pharma-ceutically acceptable carrier). In some cases, the composition can be configured for intravenous administration.

[0139] In some cases, the steps of administering the nucleic acid or vector and administering the antibody or antigen-binding fragment thereof, protein ligand or functional fragment thereof, or small molecule configured to bind to the epitope are separated by a sufficient time to allow the epitope encoded by the nucleic acid or vector to be expressed by cells of the subject, which in some embodiments includes at least about 8, 12, 16, 24, 36, 48, 60, 72, 84, or 96 hours.

[0140] In some cases, non-invasive imaging can be performed on a subject after the antibody or its antigen-binding fragment, protein ligand or its functional fragment, or small molecule that is configured to bind to an epitope is administered to the subject.Such non-invasive imaging can include MRI imaging, PET imaging, SPECT imaging, photoacoustic imaging, and bioluminescence imaging.The polypeptide that can be detected by MRI imaging can include polypeptide contrast agents such as ferritin (or its mutants, such as Pyrococcus furiousus ferritin mutants L55P, F57S, or F123S), or lanthanide binding protein (or its engineered fusions, such as the LBT-ubiquitin fusions described in Daughtry et al. ChemBioChem 2012, 13, 2567-2574). Synthetic biomarkers detectable by PET or SPECT imaging include the human sodium iodide symporter (e.g., in conjunction with administration of a PET active iodine / iodine isotope, see, e.g., Penheiter et al. Curr Gene Ther. 2012 Feb; 12(1): 33-47), HSV-tk or a mutant thereof, such as HSV-sr39tk (e.g., in conjunction with administration of a positron-labeled acycloguanosine or pyrimidine analog PET reporter, such as [18F]FHBG, see, Yaghoubi SS et al. Nat Protoc. 2006; 1(6): 3069-75), and dopamine D2 receptor or a mutant thereof, such as D2R80A or D2R194A (e.g., in conjunction with administration of a positron-labeled D2 binder, such as 3-(2'-[18F]-fluoroethyl)-spiperone).Polypeptides detectable by photoacoustic imaging include chromogenic enzymes such as β-galactosidase (e.g., in combination with administration of X-gal) and tyrosinase, autofluorescent proteins (e.g., GFP, mCherry, or derivatives thereof), non-fluorescent GFP-like chromoproteins (e.g., aeCP597 and cjBlue, and derivatives thereof), bacteriophytochrome-based near-infrared fluorescent proteins (e.g., IFP1.4, Wi-Phy, IFP1.4rev, IFP2.0, iRFP713, iRFP720, iRFP713 / V256C, iRFP682, iRFP702, iRFP670, mIFP, iBlueberry, GAF-FP, BphP1-FP / C20S, or AphB variants), and reversible photoswitch proteins (e.g., Dronpa, Dronpa-M159T, and BphP1, or variants thereof). Polypeptide bioluminescence imaging includes luciferases (e.g., in combination with administration of coelenterazine as described herein), including Gaussian luciferase, Renilla luciferase, and firefly luciferase (including, e.g., the engineered Ppy RE8 and RE9 versions described in Branchini et al. Anal. Biochem. 396(2010):290-297).

[0141] In some cases, the non-invasive imaging method can include detection by a radioisotope linked to or associated with an antibody or antigen-binding fragment thereof, a protein ligand or functional fragment thereof, or a small molecule configured to bind to an epitope. In some embodiments, the radioisotope comprises a positron-emitting radioisotope, an alpha-emitting radioisotope, a beta-emitting radioisotope, or a gamma-emitting radioisotope. In some embodiments, the radioisotope is a positron-emitting radioisotope, including 124I, 68Ga, 11C, 13N, 15O, 18F, 68Ga, 64Cu, 52Mn, 55Co, 89Zr, 82Rb, or any combination thereof. In some embodiments, the radioisotope comprises an alpha-emitting radioisotope, including 225Ac, 211At, 227Th, 224Ra, or any combination thereof. In some embodiments, the radioisotope comprises a beta emitting radioisotope, including 177Lu, 67Cu, 131I, 90Y, 89Sr, 186Re, 165Dy, 32P, 166Ho, 188Re, or any combination thereof. In some embodiments, the radioisotope is a gamma emitting radioisotope, including 99mTc, 123I, or 131I.

[0142] In some cases, non-invasive imaging methods can include the use of an imaging agent linked to or associated with an antibody or antigen-binding fragment thereof, a protein ligand or functional fragment thereof, or a small molecule configured to bind to an epitope, such as iron oxide nanoparticles (IONPs), superparamagnetic iron platinum nanoparticles, manganese (II), or gadolinium (III).

[0143] In some cases, the methods described herein can include treatment with an antibody or antigen-binding fragment thereof, a protein ligand or functional fragment thereof, or a small molecule configured to bind to an epitope linked to or associated with a therapeutic agent, such as a protein toxin, diphtheria toxin, glucagon-like peptide (GLP-1), a cytotoxic immunomodulatory protein, Fas ligand, an auristatin or analog thereof, a maytansinoid, a calicheamicin, a duocarmycin or analog thereof, or a doxorubicin or analog thereof.

[0144] Example of an embodiment

[0145] In some aspects, the disclosure provides a method of detecting, imaging, or treating diseased cells, comprising administering a composition to a subject, wherein the composition preferentially induces expression of a biomarker in diseased cells relative to non-disease cells, the biomarker comprising an extracellular receptor configured to bind to an affinity reagent configured for detection or treatment of the diseased cells. In some aspects, the disclosure provides a method of detecting, imaging, or treating diseased cells, comprising administering a composition to a subject, wherein the composition preferentially induces expression of a biomarker in diseased cells relative to non-disease cells, the biomarker comprising an extracellular receptor containing multiple epitopes configured to bind to one or more affinity reagents configured for detection or treatment of the diseased cells. In some aspects, the disclosure provides a method of detecting diseased cells, comprising administering a composition to a subject, the composition preferentially inducing expression of a biomarker in diseased cells compared to non-diseased cells, the biomarker comprising an activatable epitope, the activatable epitope being preferentially accessible for binding in an internal or external physical microenvironment of the cell affected by the disease compared to an internal or external microenvironment of a cell not affected by the disease. In some embodiments, the biomarker further comprises a pH-sensitive helix. In some embodiments, the activatable epitope is flanked by two copies of a pH-sensitive helix. In some embodiments, the pH-sensitive helix comprises a sequence having at least 80% identity to any one of SEQ ID NOs: 1-3. In some embodiments, the biomarker comprises the sequence: [helix]-[linker]-[helix]-[linker]-[helix]-[linker]-[epitope], where [helix] represents the pH sensitive helix; [linker] represents a linker comprising the sequence (GGS)n or (GGS)nFCYWKTCT(GGS)n; and [epitope] represents the epitope.In some embodiments, the biomarker comprises the sequence of: [helix]-[linker]-[helix]-[linker]-[helix]-[epitope], where [helix] represents the pH sensitive helix; [linker] represents a linker comprising the sequence (GGS)n or RLCRPRSAPSRCGPGLRPCAP; and [epitope] represents the epitope. In some aspects, the disclosure provides a method of detecting or treating diseased cells, comprising administering a composition to a subject, wherein the composition induces surface expression of a biomarker comprising an activatable epitope in diseased cells preferentially relative to surface expression of the biomarker in non-disease cells; the activatable epitope is preferentially accessible for binding in a microenvironment specific to the diseased cells compared to a microenvironment of cells not affected by the disease; or the biomarker comprises a membrane resident polypeptide or the biomarker comprises a polypeptide comprising a transmembrane domain. In some embodiments, the method further comprises administering to the subject an antibody or antigen-binding fragment thereof, a protein ligand or functional fragment thereof configured to bind to the epitope, or a small molecule. In some embodiments, the method further comprises detecting binding of the antibody or antigen-binding fragment thereof to the epitope. In some aspects, the present disclosure provides a method of detecting or treating disease cells, comprising: (a) administering to the subject a composition, wherein the composition induces surface expression of a biomarker comprising one or more epitopes in disease cells preferentially relative to surface expression of the biomarker in non-disease cells; and (b) administering to the subject an antibody or antigen-binding fragment thereof, a protein ligand or functional fragment thereof, or a small molecule configured to bind to the one or more epitopes. In some embodiments, the method further comprises detecting binding of the antibody or antigen-binding fragment thereof, a protein ligand or functional fragment thereof, or a small molecule to the epitope.In some embodiments, the epitope is an activatable epitope that is preferentially accessible for binding in a microenvironment specific to the diseased cell compared to an intracellular or extracellular microenvironment of a cell not affected by the disease. In some embodiments, the activatable epitope is activated by phosphorylation, glycosylation, methylation, ubiquitination, A-acetylation, lipidation (e.g., GPI), amidation, sulfonation, oxidation, or any combination thereof. In some embodiments, the biomarker further comprises a pH-sensitive helix. In some embodiments, the activatable epitope is flanked by two copies of a pH-sensitive helix. In some embodiments, the pH-sensitive helix comprises a sequence having at least 80% identity to any one of SEQ ID NOs: 1-3. In some embodiments, the biomarker comprises a sequence set forth in: [helix]-[linker]-[helix]-[linker]-[helix]-[linker]-[epitope], where [helix] represents the pH sensitive helix; [linker] represents a linker comprising the sequence (GGS)n or (GGS)nFCYWKTCT(GGS)n; and [epitope] represents the epitope. In some embodiments, the biomarker comprises a sequence set forth in: [helix]-[linker]-[helix]-[linker]-[helix]-[epitope], where [helix] represents the pH sensitive helix; [linker] represents a linker comprising the sequence (GGS)n or RLCRPRSAPSRCGPGLRPCAP; and [epitope] represents the epitope. In some embodiments, the biomarker further comprises a protease cleavage site for a cancer-associated protease (e.g., a protease that is overexpressed in cancer cells relative to normal cells, or a protease that exhibits preferential catalytic activity in the cancer microenvironment) along with an scFv configured to bind to the activatable epitope.In some embodiments, the cancer-associated protease is uPA, EPCAM, an MMP, a cathepsin, a serine protease, a granzyme, a furin, a cysteine ​​protease, an elastase, or a type II transmembrane serine protease, a kallikrein-related peptidase, a "disintegrin and metalloproteinase" (ADAM), a plasminogen activator (a serine protease), a caspase, or any combination thereof. In some embodiments, the antibody or antigen-binding fragment thereof further comprises a radioisotope. In some embodiments, the radioisotope comprises a positron-emitting radioisotope, an alpha-emitting radioisotope, a beta-emitting radioisotope, or 99mTc. In some embodiments, the radioisotope is a positron-emitting radioisotope. 124 I, 68In some embodiments, the radioisotope comprises an alpha emitting radioisotope, including 225Ac, 211At, 227Th, 224Ra, or any combination thereof. In some embodiments, the radioisotope comprises a beta emitting radioisotope, including 177Lu, 67Cu, 131I, 90Y, 89Sr, 186Re, 165Dy, 32P, 166Ho, 188Re, or any combination thereof. In some embodiments, the biomarker further comprises an extracellular domain comprising the epitope. In some embodiments, the extracellular domain is derived from a membrane protein endogenous to the subject. In some embodiments, the extracellular domain is derived from DLL3, PSMA, SSTR2, CD8a, CD4, TGFR1, IGF1R, PD-L1, EGFR, CD73, IgK, or IL-6. In some embodiments, the biomarker comprises an extracellular domain comprising a sequence having at least 80% identity to any one of SEQ ID NOs: 4-9. In some embodiments, the biomarker further comprises a hinge domain derived from IgG4, IgG1, CD8, or CD28, or the sequence (GGGS)n. In some embodiments, the biomarker further comprises a transmembrane domain derived from DLL3, PSMA, SSTR2, PD-L1, EGFR, CD28, CD4, CD8a, ICOS, or CD73. In some embodiments, the biomarker further comprises an intracellular domain comprising a reporter protein or affinity tag. In some embodiments, the biomarker further comprises an intracellular domain comprising a reporter protein, the reporter protein being derived from GFP, luciferase, Nluc, or Fluc, or any combination thereof. In some embodiments, the biomarker further comprises an intracellular domain comprising an affinity tag, the affinity tag comprising an HA, FLAG, or MYC sequence, or any combination thereof. In some embodiments, the biomarker comprises two or more copies of the epitope. In some embodiments, the composition comprises a nucleic acid vector comprising a coding sequence for the biomarker.In some embodiments, the coding sequence comprises a promoter operably linked to a nucleotide sequence encoding a biomarker, hi some embodiments, the promoter drives expression of the biomarker preferentially in diseased cells relative to expression of the biomarker in non-diseased cells of the subject. In some embodiments, the promoter is a survivin promoter (BIRC5), CXCR4 promoter, ATP-binding cassette subfamily C member 4 (ABCC4) promoter, anterior gradient 2, protein disulfide isomerase family member (AGR2) promoter, activation-induced cytidine deaminase (AICDA) promoter, UDP-GlcNAc:beta Gal beta-1,3-N-acetylglucosaminyltransferase 3 (B3GNT3) promoter, cadherin 3 (CDH3) promoter, CEA cell adhesion molecule 5 (CEACAM5) promoter, centromere protein F (CENPF) promoter, centrosomal protein 55 (CEP55) promoter, claudin 3 (CLDN3) promoter, claudin 4 (CLDN4) promoter, type XI collagen alpha 1 chain (COL11A1) promoter, type I collagen alpha 1 chain (COL1A1) promoter, cystatin SN (CST1) promoter, dentate-less E3 ubiquitin tin protein ligase homolog (DTL) promoter, family with sequence similarity member B (FAM111B) promoter, forkhead box A1 (FOXA1) promoter, kinesin family member 20A (KIF20A), laminin subunit gamma 2 (LAMC2) promoter, mitotic spindle positioning (MISP) promoter, matrix metallopeptidase 1 (MMP1) promoter, matrix metallopeptidase 12 (MMP12) promoter, Matrix metallopeptidase 13 (MMP13) promoter, mesothelin (MSLN) promoter, cell surface-associated mucin 1 (MUC1) promoter, phospholipase A2 group IID (PLA2G2D) promoter, regulator of G protein signaling 13 (RGS13) promoter, secretoglobin family 2A member 1 (SCGB2A1) promoter, topoisomerase II alpha (TOP2A) promoter, ubiquitin D (UBD) promoter, ubiquitin-conjugating enzyme E2 (U2A) promoter,C (UBE2C), USH1 protein network component harmonin (USH1C), V-set domain-containing T cell activation inhibitor 1 (VTCN1) promoter, hexokinase type II promoter, TRPM4 promoter, stromelysin 3 promoter, surfactant protein A promoter, secretory leukocyte protease inhibitor promoter, tyrosinase promoter, stress-inducible grp78 / BiP promoter, interleukin-10 promoter, alpha-B-crystallin / heat shock protein 27 promoter, epidermal growth factor receptor promoter, mucin-like glycoprotein promoter, mts1 promoter, NSE promoter, somatostatin tin receptor promoter, c-erbB-3 promoter, c-erbB-2 promoter, c-erbB4 promoter, thyroglobulin promoter, α-fetoprotein promoter, villin promoter, albumin promoter, glycoprotein A33 promoter, B cell specific Moloney leukemia virus insertion site 1 promoter, cyclooxygenase-2 promoter, fibroblast growth factor promoter; human epidermal growth factor receptor 2, human telomerase reverse transcriptase promoter; kinase domain insertion-containing receptor promoter; rad51 recombinase promoter; TTF-1, urokinase-type plasminogen activator receptor promoter, ubiquitin-conjugating enzyme E2 T (UBE2T) promoter, checkpoint kinase 1 (CHEK1) promoter, epithelial cell transforming 2 promoter (ECT2), BCL2-like 12 (BCL2L12) promoter, centromere protein I (CENPI) promoter, E2F transcription factor 1 (E2F1) promoter, flavin adenine dinucleotide synthetase 1 (FLAD1) promoter, protein phosphatase, Mg2+ / Mn2+-dependent 1G (PPM1G) promoter, ubiquitin-conjugating enzyme E2S (UBE2S) promoter, aurora kinase A and ninein interacting protein (AUNIP) promoter, cell division cycle 6 (CDC6) promoter, centromere protein L (CENPL) promoter, DNA replicative helicase / nuclease 2 (DNA2) promoter, DSN1 homolog, MIS12 kinetochore complex component (DSN1) promoter, deoxythymidylate kinase (DTYMK) promoter, G protein-regulated inducer of neurite outgrowth 1 (GPRIN1) promoter, mitochondrial fission regulator 2 (MTFR2) promoter, RAD51-associated protein 1 (RAD51AP1) promoter, small nuclear ribonucleoprotein polypeptide A' (SNRPA1) promoter, ATPase family, AAA domain containing 2 (ATAD2) promoter, BUB1 mitotic checkpoint serine / threonine kinase (BUB1) promoter, calcyclin-binding protein (CACYBP) promoter, cell division cycle associated 3 (CDCA3) promoter, centromere protein O (CENPO) promoter promoter, flap structure-specific endonuclease 1 (FEN1) promoter, forkhead box M1 (FOXM1) promoter, cell proliferation-regulated inhibitor of protein phosphatase 2A (KIAA1524) promoter, kinesin family member 2C (KIF2C) promoter, karyopherin subunit alpha 2 (KPNA2) promoter, MYB proto-oncogene-like 2 (MYBL2) promoter, NIMA-related kinase 2 (NEK2) promoter, RAN-binding protein 1 (RANBP1) promoter, small nuclear ribonucleoprotein polypeptides B and B1 (SNRPB) promoter, SPC24 / NDC80 kinetochore complex component (SPC24) promoter, transforming acidic coiled-coil-containing protein 3 (TACC3) promoter, TBC1 domain family member 31 (TBC1D31) promoter, thymidine kinase 1 (TK1) promoter, zinc finger protein 695 (ZNF695) promoter, aurora kinase A (AURKA) promoter, BLMRecQ-like helicase (BLM) promoter, chromosome 17 open reading frame 53 (C17orf53) promoter, chromobox 3 (CBX30) promoter, cyclin B1 (CCNB1) promoter, cyclin E1 (CCNE1) promoter, cyclin F (CCNF), cell division cycle 20 (CDC20) promoter, cell division cycle 45 (CDC45) promoter, cell division cycle associated 5 (CDCA5) promoter, cyclin-dependent kinase inhibitor 3 (CDKN3) promoter, cadherin EGF LAG 7-transmembrane G-type receptor 3 (CELSR3) promoter, centromere protein A (CENPA) promoter, centrosomal protein 72 (CEP72) promoter, CDC28 protein kinase regulatory subunit 2 (CKS2) promoter, type X collagen alpha 1 chain (COL10A1) promoter, chromosome segregation 1-like (CSE1L) promoter, DBF4 zinc finger promoter, GINS complex subunit 1 (GINS1) promoter, G protein-coupled receptor 19 (GPR19) promoter, kinesin family member 18A (KIF18A) promoter, kinesin family member 4A (KIF4A) promoter, kinesin family member C1 (KIF C1) promoter, minichromosome maintenance 10 replication initiator (MCM10) promoter, minichromosome maintenance complex component 2 (MCM2) promoter, minichromosome maintenance complex component 7 (MCM7) promoter, MRG domain binding protein (MRGBP) promoter, methylenetetrahydrofolate dehydrogenase (NADP+-dependent) 2, methenyltetrahydrofolate cyclohydrolase (MTHFD2) promoter, non-SMC condensin I complex subunit H (NCAPH) promoter, NDC80, kinetochore complex component (NDC80) promoter, nudix hydrolase 1 (NUDT1) promoter, ribonuclease H2 subunit A (RNASEH2A) promoter, RuvB-like AAA (RcA) promoter,ATPase1 (RUVBL1) promoter, serologically defined breast cancer antigen NY-BR-85 (SGOL1) promoter, SHC-binding and spindle-associated 1 (SHCBP1) promoter, small nuclear ribonucleoprotein polypeptide G (SNRPG) promoter, timeless circadian regulator promoter, thyroid hormone receptor interactor 13 (TRIP13) promoter, trophinin-related protein (TROAP) promoter, ubiquitin-conjugating enzyme E2C(UBE2C) promoter, WD repeat and HMG box DNA binding protein 1 (WDHD1) promoter, alphafetoprotein (AFP) promoter, fragments thereof, any combination thereof, chimeric promoters compiled from multiple elements from the foregoing, or fully synthetic promoters composed of tile-like transcription factor binding sites. In some embodiments, the vector is a recombinant viral vector. In some embodiments, the vector is a non-viral vector. In some embodiments, the non-viral vector is a nanoplasmid, a plasmid, a minicircle, a closed-end linear duplex (CELiD), or a dog-bone DNA vector (dbDNA). In some embodiments, the composition further comprises a transfection agent. In some embodiments, the transfection agent is a linear or branched polyethyleneimine, a nanoparticle, a lipophilic particle, a solid nanoparticle, a peptide, a micelle, a dendrimer, a polymer composition, a hydrogel, a synthetic or naturally derived exosome, a virus-like particle, or any combination thereof. In some embodiments, the disease is cancer, an autoimmune disease, or a neurodegenerative disease. In some embodiments, the disease is cancer. In some embodiments, the cancer is acute myeloid leukemia, adrenocortical carcinoma, bladder urothelial carcinoma, breast ductal carcinoma, breast lobular carcinoma, cervical carcinoma, cholangiocarcinoma, colorectal adenocarcinoma, esophageal carcinoma, gastric adenocarcinoma, glioblastoma multiforme, head and neck squamous cell carcinoma, hepatocellular carcinoma, renal chromophobe carcinoma, renal clear cell carcinoma, papillary renal cell carcinoma, low grade glioma, lung adenocarcinoma, lung squamous cell carcinoma, mesothelioma, ovarian serous adenocarcinoma, pancreatic ductal adenocarcinoma, paraganglioma and pheochromocytoma, prostate adenocarcinoma, sarcoma, cutaneous melanoma, testicular germ cell carcinoma, thymoma, papillary thyroid carcinoma, uterine carcinosarcoma, uterine endometrioid carcinoma, uveal melanoma, melanoma of the lip, spindle cell carcinoma, liposarcoma, nasal sarcoma, breast adenocarcinoma, insulinoma, osteosarcoma, mast cell tumor, angiosarcoma, non-small cell lung cancer (NSCLC), marginal zone lymphoma, malignant melanoma, or chronic lymphocytic leukemia. In some embodiments, the detecting binding of the antibody or antigen-binding fragment thereof to the epitope comprises PET imaging or another radioisotope-based imaging method.

[0146] In some aspects, the disclosure provides a nucleic acid comprising a cancer-specific promoter (e.g., a promoter of a gene that is overexpressed in cancer cells compared to normal cells) operably linked to a nucleotide sequence encoding a membrane-resident polypeptide biomarker comprising an activatable epitope. In some embodiments, the activatable epitope is activated by phosphorylation, glycosylation, methylation, ubiquitination, A-acetylation, lipidation (e.g., GPI), amidation, sulfonation, oxidation, or any combination thereof. In some embodiments, the biomarker further comprises an extracellular domain and a transmembrane domain that comprise the epitope. In some embodiments, the biomarker further comprises a pH-sensitive helix. In some embodiments, the activatable epitope is flanked by two copies of a pH-sensitive helix. In some embodiments, the pH-sensitive helix comprises a sequence having at least 80% identity to any one of SEQ ID NOs: 1-3. In some embodiments, the biomarker comprises a sequence set forth in: [helix]-[linker]-[helix]-[linker]-[helix]-[linker]-[epitope], where [helix] represents the pH sensitive helix; [linker] represents a linker comprising the sequence (GGS)n or (GGS)nFCYWKTCT(GGS)n; and [epitope] represents the epitope. In some embodiments, the biomarker comprises a sequence set forth in: [helix]-[linker]-[helix]-[linker]-[helix]-[epitope], where [helix] represents the pH sensitive helix; [linker] represents a linker comprising the sequence (GGS)n or RLCRPRSAPSRCGPGLRPCAP; and [epitope] represents the epitope. In some embodiments, the biomarker further comprises a protease cleavage site for a cancer-associated protease (e.g., a protease that is overexpressed in cancer cells relative to normal cells or that exhibits preferential catalytic activity in the cancer microenvironment) together with an scFv configured to bind to the activatable epitope. In some embodiments, the cancer-associated protease is uPA, EPCAM, MMP, cathepsin,The biomarker is a serine protease, a granzyme, a furin, a cysteine ​​protease, an elastase, or a type II transmembrane serine protease, a kallikrein-related peptidase, a "disintegrin and metalloproteinase" (ADAM), a plasminogen activator (serine protease), a caspase, or any combination thereof. In some embodiments, the extracellular domain is derived from a membrane protein endogenous to the subject. In some embodiments, the extracellular domain is derived from DLL3, PSMA, SSTR2, CD8a, CD4, TGFR1, IGF1R, PD-L1, EGFR, CD73, IgK, or IL-6. In some embodiments, the biomarker comprises an extracellular domain comprising a sequence having at least 80% identity to any one of SEQ ID NOs: 4-9. In some embodiments, the biomarker further comprises a hinge domain derived from IgG4, IgG1, CD8, or CD28, or the sequence (GGGS)n. In some embodiments, the biomarker further comprises a transmembrane domain derived from DLL3, PSMA, SSTR2, PD-L1, EGFR, CD28, CD4, CD8a, ICOS, or CD73. In some embodiments, the biomarker further comprises an intracellular domain comprising a reporter protein or affinity tag. In some embodiments, the biomarker further comprises an intracellular domain comprising a reporter protein, the reporter protein being derived from GFP, luciferase, Nluc, or Fluc, or any combination thereof. In some embodiments, the biomarker further comprises an intracellular domain comprising an affinity tag, the affinity tag comprising an HA, FLAG, or MYC sequence, or any combination thereof. In some embodiments, the promoter is a survivin promoter (BIRC5), a CXCR4 promoter, an ATP-binding cassette subfamily C member 4 (ABCC4) promoter, anterior gradient 2, a protein disulfide isomerase family member (AGR2) promoter, an activation-induced cytidine deaminase (AICDA) promoter, a UDP-GlcNAc:beta Gal beta-1,3-N-acetylglucosaminyltransferase 3 (B3GNT3) promoter,Cadherin 3 (CDH3) promoter, CEA cell adhesion molecule 5 (CEACAM5) promoter, Centromeric protein F (CENPF) promoter, Centromeric protein 55 (CEP55) promoter, Claudin 3 (CLDN3) promoter, Claudin 4 (CLDN4) promoter, Collagen type XI alpha 1 chain (COL11A1) promoter, Collagen type I alpha 1 chain (COL1A1) promoter, Cystatin SN (CST1) promoter, E3 ubiquitin protein ligase homolog without dentate (DTL) promoter, Family 111 member B with sequence similarity (FAM111B) promoter, Forkhead box A1 (FOXA1) promoter, Kinesin family member 20A (KIF20A) promoter , laminin subunit gamma 2 (LAMC2) promoter, mitotic spindle positioning (MISP) promoter, matrix metallopeptidase 1 (MMP1) promoter, matrix metallopeptidase 12 (MMP12) promoter, matrix metallopeptidase 13 (MMP13) promoter, mesothelin (MSLN) promoter, cell surface-associated mucin 1 (MUC1) promoter, phospholipase A2 group IID (PLA2G2D) promoter, regulator of G-protein signaling 13 (RGS13) promoter, secretoglobin family 2A member 1 (SCGB2A1) promoter, topoisomerase II alpha (TOP2A) promoter, ubiquitin D (UBD) promoter, ubiquitin-conjugating enzyme E2 C(UBE2C), USH1 protein network component harmonin (USH1C), V-set domain-containing T cell activation inhibitor 1 (VTCN1) promoter, hexokinase type II promoter, TRPM4 promoter, stromelysin 3 promoter, surfactant protein A promoter, secretory leukocyte protease inhibitor promoter, tyrosinase promoter, stress-inducible grp78 / BiP promoter, interleukin-10 promoter, alpha-B-crystallin / heat shock protein 27 promoter, epidermal growth factor receptor promoter, mucin-like glycoprotein promoter, mts1 promoter, NSE promoter,somatostatin receptor promoter, c-erbB-3 promoter, c-erbB-2 promoter, c-erbB4 promoter, thyroglobulin promoter, α-fetoprotein promoter, villin promoter, albumin promoter, glycoprotein A33 promoter, B cell specific Moloney leukemia virus insertion site 1 promoter, cyclooxygenase-2 promoter, fibroblast growth factor promoter; human epidermal growth factor receptor 2, human telomerase reverse transcriptase promoter; kinase domain insertion containing receptor promoter; rad51 recombinase promoter; TTF-1, urokinase type plasminogen activator receptor promoter, ubiquitin conjugating enzyme E2 T (UBE2T) promoter, checkpoint kinase 1 (CHEK1) promoter, epithelial cell transforming 2 promoter (ECT2), BCL2-like 12 (BCL2L12) promoter, centromere protein I (CENPI) promoter, E2F transcription factor 1 (E2F1) promoter, Flavin adenine dinucleotide synthetase 1 (FLAD1) promoter, protein phosphatase, Mg2+ / Mn2+-dependent 1G (PPM1G) promoter, ubiquitin-conjugating enzyme E2S (UBE2S) promoter, aurora kinase A and ninein interacting protein (AUNIP) promoter, cell division cycle 6 (CDC6) promoter, centromere protein L (CENPL) promoter, DNA replicative helicase / nuclease 2 (DNA2) promoter, DSN1 homolog, MIS12 kinetochore complex component (DSN1) promoter, deoxythymidylate kinase (DTYMK) promoter, G protein-regulated inducer of neurite outgrowth 1 (GPRIN1) promoter, mitochondrial fission regulator 2 (MTFR2) promoter, RAD51-associated protein 1 (RAD51AP1) promoter, small nuclear ribonucleoprotein polypeptide A' (SNRPA1) promoter, ATPase family, AAA domain containing 2 (ATAD2) promoter, BUB1 mitotic checkpoint serine / threonine kinase (BUB1) promoter, calcyclin-binding protein (CACYBP) promoter, cell division cycle associated 3 (CDCA3) promoter, centromere protein O (CENPO) promoter promoter, flap structure-specific endonuclease 1 (FEN1) promoter, forkhead box M1 (FOXM1) promoter, cell proliferation-regulated inhibitor of protein phosphatase 2A (KIAA1524) promoter, kinesin family member 2C (KIF2C) promoter, karyopherin subunit alpha 2 (KPNA2) promoter, MYB proto-oncogene-like 2 (MYBL2) promoter, NIMA-related kinase 2 (NEK2) promoter, RAN-binding protein 1 (RANBP1) promoter, small nuclear ribonucleoprotein polypeptides B and B1 (SNRPB) promoter, SPC24 / NDC80 kinetochore complex component (SPC24) promoter, transforming acidic coiled-coil-containing protein 3 (TACC3) promoter, TBC1 domain family member 31 (TBC1D31) promoter, thymidine kinase 1 (TK1) promoter, zinc finger protein 695 (ZNF695) promoter, aurora kinase A (AURKA) promoter, BLMRecQ-like helicase (BLM) promoter, chromosome 17 open reading frame 53 (C17orf53) promoter, chromobox 3 (CBX30) promoter, cyclin B1 (CCNB1) promoter, cyclin E1 (CCNE1) promoter, cyclin F (CCNF), cell division cycle 20 (CDC20) promoter, cell division cycle 45 (CDC45) promoter, cell division cycle associated 5 (CDCA5) promoter, cyclin-dependent kinase inhibitor 3 (CDKN3) promoter, cadherin EGF LAG 7-transmembrane G-type receptor 3 (CELSR3) promoter, centromere protein A (CENPA) promoter, centrosomal protein 72 (CEP72) promoter, CDC28 protein kinase regulatory subunit 2 (CKS2) promoter, type X collagen alpha 1 chain (COL10A1) promoter, chromosome segregation 1-like (CSE1L) promoter, DBF4 zinc finger promoter, GINS complex subunit 1 (GINS1) promoter, G protein-coupled receptor 19 (GPR19) promoter, kinesin family member 18A (KIF18A) promoter, kinesin family member 4A (KIF4A) promoter, kinesin family member C1 (KIF C1) promoter, minichromosome maintenance 10 replication initiator (MCM10) promoter, minichromosome maintenance complex component 2 (MCM2) promoter, minichromosome maintenance complex component 7 (MCM7) promoter, MRG domain binding protein (MRGBP) promoter, methylenetetrahydrofolate dehydrogenase (NADP+-dependent) 2, methenyltetrahydrofolate cyclohydrolase (MTHFD2) promoter, non-SMC condensin I complex subunit H (NCAPH) promoter, NDC80, kinetochore complex component (NDC80) promoter, nudix hydrolase 1 (NUDT1) promoter, ribonuclease H2 subunit A (RNASEH2A) promoter, RuvB-like AAA (RcA) promoter,The nucleic acid is selected from the group consisting of ATPase1 (RUVBL1) promoter, serologically defined breast cancer antigen NY-BR-85 (SGOL1) promoter, SHC-binding and spindle-associated 1 (SHCBP1) promoter, small nuclear ribonucleoprotein polypeptide G (SNRPG) promoter, timeless circadian regulator promoter, thyroid hormone receptor interactor 13 (TRIP13) promoter, trophinin-related protein (TROAP) promoter, ubiquitin-conjugating enzyme E2 C (UBE2C) promoter, WD repeat and HMG box DNA-binding protein 1 (WDHD1) promoter, alpha fetoprotein (AFP) promoter, a fragment thereof, any combination thereof, a chimeric promoter compiled from multiple elements from the foregoing, or a completely synthetic promoter composed of tiled transcription factor binding sites. In some embodiments, the nucleic acid comprises an RNA or a capped mRNA. In some embodiments, the RNA or the capped mRNA comprises a splice signal, a synthetic intron, a miRNA binding site, a synthetic stem-loop or tetraloop, or an aRNA domain.

[0147] In some aspects, the present disclosure provides a vector comprising any of the nucleic acids described herein. In some embodiments, the vector is a recombinant viral vector. In some embodiments, the vector is a non-viral vector. In some embodiments, the non-viral vector is a nanoplasmid, a plasmid, a minicircle, a closed-end linear duplex (CELiD), or a dog-bone DNA vector (dbDNA).

[0148] In some aspects, the disclosure provides a composition comprising any of the vectors described herein or any of the nucleic acids described herein and a transfection agent, in some embodiments, said transfection agent is a linear or branched polyethyleneimine, a nanoparticle, a lipophilic particle, a solid nanoparticle, a peptide, a micelle, a dendrimer, a polymer composition, a hydrogel, a synthetic or naturally derived exosome, a virus-like particle, or any combination thereof.

[0149] In some aspects, the disclosure provides a cell comprising any of the nucleic acids or vectors described herein.

[0150] [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9] [Table 1-10] [Table 1-11] [Table 1-12] [Table 1-13] [Table 1-14] [Table 1-15] [Table 1-16] [Table 1-17] [Table 1-18] [Table 1-19] [Table 1-20] [Table 1-21] [Table 1-22] [Table 1-23] [Table 1-24] [Table 1-25] [Table 1-26] [Table 1-27] [Table 1-28] [Table 1-29] EXAMPLES

[0151] Example 1 DETECTION, IMAGING, OR TREATMENT OF CANCER CELLS USING THE CELL SURFACE-EXPRESSABLE BIOMARKERS DESCRIBED HEREIN A vector (e.g., a viral vector or a nanoplasmid vector) containing a coding sequence for a cell surface expressible biomarker described herein that includes an epitope under the control of a cancer-specific promoter (e.g., a promoter of a gene that is overexpressed in cancer cells compared to normal cells) is administered to a subject suspected of having cancer. Although a variety of cells may receive the vector, expression of the biomarker is favored in cancerous cells due to the cancer-specific promoter. The accessible epitopes can then be detected by antibodies, antibody fragments or derivatives, or protein ligands that bind to the epitopes. These antibodies, antibody fragments or derivatives, or protein ligands can be coupled to PET-active radionuclides, e.g., PET-activated radionuclides, that allow for detection and / or imaging of cancer cells. 124 I or 68 In some cases, for therapeutic purposes against cancer (e.g., theranostic purposes), these antibodies, antibody fragments or derivatives, or protein ligands can be labeled with tumor-killing high-energy radionuclide beta-emitters (e.g., 177 Lu, 67 Cu, or 90 Y) or alpha emitters (e.g. 225 Ac, 211 At, 227 Finally, these antibodies, antibody fragments or derivatives, or protein ligands can be conjugated to other molecules that may exert a toxic effect on tumor cells. Example 2 DETECTION, IMAGING, OR TREATMENT OF CANCER CELLS USING THE CELL SURFACE-EXPRESSABLE POST-TRANSLATIONALLY MODIFIED BIOMARKERS DESCRIBED HEREIN

[0152] A vector (e.g., a viral vector or a nanoplasmid vector) containing a coding sequence for a cell surface expressible biomarker described herein that contains an activatable epitope under the control of a cancer-specific promoter (e.g., a promoter of a gene that is overexpressed in cancer cells compared to normal cells) is administered to a subject suspected of having cancer. Although a variety of cells may receive the vector, expression of the biomarker is favored in cancerous cells due to the cancer-specific promoter. In the event that the biomarker is expressed in a non-cancerous cell, the absence of a proper activation microenvironment inside or outside a normal cell leaves the activatable epitope blocked and undetectable. When a biomarker containing an activatable epitope is expressed in the tumor microenvironment or inside a tumor cell, the activatable epitope is revealed on the surface of the cancer cell, and in this context, the activatable epitope is accessible for binding. The accessible activatable epitope can then be detected by an antibody, an antibody fragment or derivative, or a protein ligand that binds to the epitope. These antibodies, antibody fragments or derivatives, or protein ligands can be coupled to PET active radionuclides that allow for the detection and / or imaging of cancer cells, e.g. 124 I or 68 For therapeutic purposes against cancer (e.g., theranostic), these antibodies, antibody fragments or derivatives, or protein ligands can be coupled to tumor-killing high-energy radionuclide beta-emitters, e.g., 177 Lu, 67 Cu, or 90 Y, or alpha emitters, e.g. 225 Ac, 211 At, 227 Finally, these antibodies, antibody fragments or derivatives, or protein ligands can be conjugated to other molecules that may exert a toxic effect on tumor cells. Example 3 Construction of a pH-sensitive self-propagating secretory cell marker.

[0153] It was believed that if the construct was secretable and contained a membrane-philic domain such that it was secreted and inserted into the membrane of neighboring cells, cancer cells expressing the constructs shown in Figures 1-4 would also be able to label neighboring cells and amplify the signal generated by delivery to the tumor. Such constructs were further believed to be pH-sensitive (and therefore cancer microenvironment-sensitive) if the membrane-philic domain contained a pH-sensitive protein motif.

[0154] Therefore, we designed a construct of the type described in Figure 5, which contains a cleavable signal peptide that can drive secretion of the construct, a reporter domain linked to the signal peptide, and a pH-sensitive membrane affinity protein motif (pHLIP) (SEQ ID NOs: 21-28). Because the membrane affinity protein motif is only activated at low pH and transitions from an unfolded soluble state to a membrane-tethered helix, it was envisioned that such a construct would be secreted by cancer cells and inserted only into neighboring cells within the cancer microenvironment (Figure 6).

[0155] The first question was whether constructs designed in this manner would propagate to nearby cells. Therefore, an experiment (Figure 7) was performed in which untransfected cancer-derived H1299 cells were treated with media obtained from H1299 cells engineered to express equivalent amounts of a reporter construct containing GFP and a cleavable mIgK secretion tag (SEQ ID NO: 32) fused to either a mutated inactive membrane-binding helix (mIgK-GFP-Var3dead) or an active pH-sensitive membrane-binding helix (mIgK-GFP-Var3 or mIgK-GFP-WT). Since the H1299 cells themselves did not contain the expression construct, any GFP fluorescence was interpreted as coming from a GFP-helix polypeptide that had successfully inserted into, or otherwise associated with, the membrane of the H1299 cells. It appeared that the WT version of the pH-sensitive membrane-binding motif also performed better than the Var3 version of the pH-sensitive membrane-binding motif.

[0156] Experimental media from cells expressing constructs with an active membrane-binding motif (mIgK-GFP-Var3 or mIgK-GFP-WT) induced an increased number of fluorescent cells compared with cells containing an inactive membrane-binding motif (mIgK-GFP-Var3dead), indicating that the secretion tag-reporter-membrane-binding domain construct was able to propagate to untransfected cells. Example 4 Design and engineering of DLL3 constructs for cell surface expression I

[0157] DLL3 is a membrane protein known to be predominantly located in the Golgi or late endosomal membrane and to be weakly trafficked to the cell surface when overexpressed in certain cancers (A1 in FIG. 8A). Two different approaches were used to engineer DLL3 with increased cell surface expression. The first approach utilized trafficking instructions such as signal peptides (SPs) and transmembrane / membrane tethering domains from other proteins known to reside at the cell surface of lung cancer cells. SPs can direct trafficking of the extracellular domain across the membrane during translation, and transmembrane domains can tether DLL3 to the cell membrane. Lung-specific trafficking instructions can include EGFR (SP / transmembrane helix; NP244; B1 in FIG. 8A), PD-L1 (SP / transmembrane helix; NP245; C1 in FIG. 8A), and CD73 (SP / GPI-anchor motif; NP249; D1 in FIG. 8A). The second approach utilized different combinations of trafficking directives for cell surface transport of proteins on immune cells, including the GM-CSF SP / CD4 transmembrane helix (NP248; E1 in Figure 8A) or the CD8a SP / CD28 transmembrane helix (NP244 and NP247; F1 and G1 in Figure 8A, respectively). To further improve expression and / or surface presentation, shorter DLL3 sequences with less structural complexity were designed. For example, the native signal peptide, transmembrane helix, and intracellular domain were removed from DLL3. NP244, NP245, NP249, NP248, and NP246 (A1-F1) constructs were designed to contain amino acid residues 27-492 of DLL3. NP247 contains a further truncated extracellular domain of DLL3 from amino acid residues 27-254 (G1 in Figure 8A). The amino acid sequences of NP244-NP249 are shown in Table 1.

[0158] Each of the six engineered DLL3 constructs (NP244-NP247) and two different wild-type DLL3 constructs (NP116 and NP127) was separately introduced into human non-small cell lung cancer H1299 cells (also known as NCI-H1299 or CRL-5803) by transfection to analyze the expression of engineered DLL3. The H1299 cells were then stained with a fluorescently labeled anti-DLL3 antibody and analyzed by fluorescence activated cell sorting (FACS) to detect DLL3 expression. Panels A2-G2 of Figure 8B, and NP116, NP127, NP244-NP249, and NP268 of Figure 8C show the amount of DLL3 expressed on the cell surface corresponding to panels A1-G1 of Figure 8A. NP244 (Figures 8B-8C) did not increase the expression level of DLL3 on the cell surface, but the other constructs NP245, NP249, NP248, NP246, and NP247 increased the expression level of DLL3 compared to wild-type DLL3 (A2 in Figure 8B and NP116 and NP127 in Figure 8C) (Figures 8B-8C). Specifically, NP249 and NP247 increased the positive percentage to about 80% and about 94%, respectively (Figure 8C).

[0159] Next, an in vitro cell killing assay was performed to determine the ability of anti-DLL3 antibodies conjugated with a cytotoxic payload (anti-DLL3-PBD) to induce cytotoxicity. Anti-DLL3-PBD was introduced into H1299 cells transfected with NP247 (G1 in FIG. 8A), NP249 (D1 in FIG. 8A), NP116 (wild-type control), or NP127 (wild-type control). Compared to cells expressing NP116 or NP127, cells expressing NP247 or NP249 showed a substantial decrease in viability when treated with anti-DLL3-PBD (FIG. 8D). This result suggests that cells expressing engineered DLL3 with increased expression levels at the cell surface (i.e., NP247 or NP249) may be more susceptible to cell death when treated with anti-DLL3-PBD compared to cells expressing wild-type DLL3 (i.e., NP116 or NP127). Example 5 Design and engineering of DLL3 constructs for cell surface expression II

[0160] Six additional DLL3 constructs, NP296, NP298, NP299, NP302, NP303, and NP304, were designed based on NP247 (G1 in Figure 8A) to identify the minimal DLL3 domain that is sufficient for interaction with the DLL3 antibody tracer (Figure 9A). NP296 contains amino acid residues 179-254 of DLL3. NP298 contains amino acid residues 179-254 of DLL3 and a second DLL3 epitope added with the goal of doubling antibody binding capacity. NP299 contains amino acid residues 27-254 of DLL3 and two DLL3 minimal epitopes inserted into the DLL3 C2 domain. NP302 contains amino acid residues 179-254 of DLL3 and a soluble humanized nanobody structure that replaces the C2 domain of DLL3. NP303 contains amino acid residues 179-254 of DLL3, a soluble humanized nanobody structure that replaces the C2 domain of DLL3, and two DLL3 minimal epitopes inserted into the nanobody structure. NP304 contains a soluble humanized nanobody structure, and two DLL3 minimal epitopes inserted into the nanobody structure. The amino acid sequences of NP296-NP299 and NP302-NP304 are shown in Table 1.

[0161] Each of the NP247, NP298, NP302, and wild-type control NP116 constructs was separately introduced into H1299 cells by transfection to analyze the expression of engineered DLL3. H1299 cells were then stained with fluorescently labeled anti-DLL3 antibody and analyzed by FACS to detect DLL3 expression. Figure 9B shows the amount of DLL3 expressed on the cell surface. Cells expressing NP298, which contains two DLL3 binding domains, or NP302, which contains a humanized soluble nanobody domain, showed increased detection levels of DLL3 expressed on the cell surface compared to cells expressing NP247 and the wild-type control (Figure 9B).

[0162] In addition, NP247, NP296, NP298, NP302, NP303, NP304, and control constructs (isotype control and negative control) were each introduced into H1299 cells separately. H1299 cells were plated in 24-well plates at 40,000 cells / well. 24 hours after plating, NP247, NP296, NP286, NP298, NP302, NP303, NP304, and control constructs were transfected into cells by lipofectamine according to the manufacturer's protocol. 48 hours after transfection, cells were harvested and stained with anti-DLL3 antibody followed by APC-conjugated anti-human antibody for detection. These samples were then read by flow cytometry on a MACS Quant. Figure 9C shows the amount of cells stained with anti-DLL3 antibody (positive %) and the GMFI of DLL3-positive cells.

[0163] Next, the physical number of copies of anti-DLL3 antibody bound to DLL3 expressed on the cell surface was evaluated. H1299 cells were seeded in 96-well plates. 24 hours after seeding, H1299 cells were transfected with nanoplasmid vectors containing NP116 (wild type control), NP247 (with a single DLL3 epitope on the CD8 scaffold), or NP298 (with two DLL3 epitopes on the CD8 scaffold) by lipofectamine according to the manufacturer's protocol. H1299 cells were then stained with fluorescently labeled anti-DLL3 antibodies for FACS analysis. Cells and commercial beads for epitope quantification were then stained with anti-DLL3 secondary antibodies conjugated to Alexa Fluor® 647 (AF647). H1299 cells were then analyzed on a BD Biosciences FACSAria IIu (Figures 10A-10C). The number of epitopes was calculated by comparing the expression of cells to a quantification bead standard curve (Figure 10B-10C). Cells expressing NP247 showed a 13-fold increase in copy number of anti-DLL3 antibodies compared to cells expressing NP116 (Figure 10C). Cells expressing NP298 showed a 32-fold increase in copy number of anti-DLL3 antibodies compared to cells expressing NP116 (Figure 10C).

[0164] In addition, H1299 cells stably expressing wild type (NP116) or engineered DLL3 (NP298) were also stained with anti-DLL3 antibody conjugated to a commercial internalization secondary reagent (pHrodo). H1299 cells were incubated at 37°C with 5% CO2 for 6 hours. Portions of the samples were removed at regular time intervals and then analyzed on a BD Biosciences FACSAria IIu. Figure 10D shows the amount of internalized DLL3. Example 6 Positron Emission Tomography (PET) Scanning Study of Mouse Model Systems Expressing Cancer Biomarkers

[0165] A mouse model system was developed using engineered H1299 cells that stably express somatostatin receptor 2 (SSTR2). The H1299 cell line was established from a pulmonary lymph node metastasis from a human patient. The patient underwent radiation therapy prior to tumor biopsy. The cell line is adherent and has an epithelial morphology. First, a DNA plasmid was engineered to express a copy of the SSTR2 gene. Next, the H1299 cells were transfected with the plasmid using a third generation lentiviral system (System Biosciences; LL410PA-1) so that the cells contained two genomic copies of the SSTR2 construct stably integrated. Different numbers of these engineered H1299 cells were then subcutaneously implanted into mice at different locations with the goal of determining the minimum number of cells that would produce a measurable signal.

[0166] NOD.Cg-Prkdcscid Il2rgtm1Wjl / SzJ (NSG) mice were selected from an in-house colony at Stanford University. At 6 weeks of age, mice were anesthetized (isoflurane 3% in 100% O2) prior to H1299 tumor inoculation. Mice were then placed in a prone position and H1299 WT or H1299-SSTR2 tumors were implanted subcutaneously using 1:1 (vol / vol) PBS:GelTrex according to Table 2.

[0167] [Table 2]

[0168] Mice implanted with engineered H1299 cells are then subjected to immunoprecipitation to determine whether the engineered H1299 cells bind to the SSTR2 protein on the H1299 cell surface. 68 The mouse model was treated with Ga-dotate positron-emitting tracer. Figure 11 shows images obtained from PET scans of this mouse model system. These data illustrate that even a small number of cancer cells, as few as 31,000, can be detected in this mouse model when cells are induced to express a surrogate biomarker (e.g., SSTR2) on the cell surface.

[0169] Tumor size is 100-200mm 3 Once the cells reached an average size between 100 and 150 μCi of [ 177 Lu]-DOTATATE tracer (Minerva, Denmark). SPECT / CT images were obtained 4 hours, 24 hours, and 120 hours after tracer administration (Figure 12). These data indicate that high-energy tracers can be used for engineered biomarker expression approaches.

[0170] In addition, the tumor size is 100-200 mm 3 When the cells reached an average size between 100 and 150 μCi of 100 μg / mL, the mice were injected with 200 μCi of [ 68 GA]-DOTATATE tracer (obtained from Cardinal Health) was administered. Figure 15 shows images obtained from a PET / CT scan of this mouse model system 2 hours after tracer administration. Example 7 Engineered cell surface expression of DLL3 and cell killing

[0171] Each of the NP247 and NP298 constructs and the wild-type DLL3 construct (NP116) was introduced into H1299 cells by transfection separately to analyze the expression of engineered DLL3.Then, H1299 cells were stained with fluorescently labeled anti-DLL3 antibody for FACS analysis.Then, H1299 cells stably expressing wild-type or engineered DLL3 and commercial beads for epitope quantification were stained with anti-DLL3 secondary antibody conjugated to Alexa Fluor® 647 (AF647).Then, H1299 cells were analyzed on BD Biosciences FACSAria IIu.The number of epitopes was calculated by comparing the expression of cells with the quantification bead standard curve.The same H1299 cells stably expressing wild-type or engineered DLL3 were also stained with anti-DLL3 antibody conjugated to commercial internalization secondary reagent (pHrodo). H1299 cells were incubated at 37°C with 5% CO2 for 6 hours. Sample aliquots were removed at regular time intervals and then analyzed on a BD Biosciences FACSAria IIu. Figure 13A shows the amount of DLL3 expressed on the cell surface (left) and the amount of internalized DLL3 (right). Cells expressing NP247 (one DLL3 epitope) showed a 21-fold increase in the copy number of anti-DLL3 antibodies compared to cells expressing NP116 (wild type DLL3). Cells expressing NP298 (two DLL3 epitopes) showed a 37-fold increase in the copy number of anti-DLL3 antibodies compared to cells expressing NP116 (WT DLL3).

[0172] Next, H1299 cells stably expressing WT or engineered DLL3 were seeded at 5,000 cells / well in 96-well plates. 24 hours after seeding, H1299 cells were treated with titrations of either unconjugated anti-DLL3 antibody or anti-DLL3 antibody conjugated to PBD toxin (anti-DLL3-PBD). 72 hours after antibody treatment, cell viability was assessed using CellTiter Glo 2.0 assay kit and analyzed in Promega GloMax Navigator. Figure 13B shows that cells expressing engineered DLL3, NP247 or NP298, which have increased expression levels at the cell surface, may be more susceptible to cell death when treated with anti-DLL3-PBD compared to cells expressing NP116 (wild type DLL3).

[0173] While preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will occur to those skilled in the art without departing from the invention herein. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in the practice of the invention. It is intended that the following claims define the scope of the invention, and that methods and structures within the scope of these claims, and their equivalents, be covered thereby. Embodiment

[0174] The following embodiments are intended to be illustrative and not limiting in any way. 1. A method for detecting, imaging, or treating diseased cells, comprising: administering to a subject a composition, said composition inducing expression of a biomarker preferentially in diseased cells relative to non-diseased cells, said biomarker comprising an extracellular receptor configured to bind to an affinity reagent configured for detection or treatment of said diseased cells; A method comprising: 2. The method of embodiment 1, wherein the extracellular receptor further comprises an activatable epitope, which is preferentially accessible for binding in an internal or external physical microenvironment of the cell affected by the disease compared to an internal or external microenvironment of a cell not affected by the disease. 3. A method of detecting, imaging, or treating diseased cells, comprising: administering a composition to a subject, wherein the composition induces expression of a biomarker preferentially in diseased cells relative to non-diseased cells. Including, the biomarker comprises an activatable epitope; the activatable epitope is preferentially accessible for binding in the internal or external physical microenvironment of the cell affected by the disease compared to the internal or external microenvironment of a cell not affected by the disease; method. 4. The method of embodiment 1 or 2, wherein the composition comprises a nucleic acid or vector encoding the biomarker. 5. The method of any one of embodiments 1 to 4, wherein said biomarker does not comprise a single chain variable fragment (scFv). 6. The method of embodiment 5, wherein said biomarker does not comprise a variable light chain (VL) domain. 7. The method of any one of embodiments 1-6, wherein the biomarker further comprises a pH-sensitive helix. 8. The method of embodiment 7, wherein said activatable epitope is flanked by at least two copies of a pH-sensitive helix. 9. The method of embodiment 7 or 8, wherein the pH-sensitive helix comprises a sequence having at least 80% identity to any one of SEQ ID NOs: 1-3. 10. The biomarker is [helix]-[linker]-[helix]-[linker]-[helix]-[linker]-[epitope] (Wherein, [helix] represents the pH-sensitive helix; [Linker] represents a linker comprising the sequence (GGS)n or (GGS)nFCYWKTCT(GGS)n; [Epitope] represents the above-mentioned epitope. 10. The method of embodiment 8 or 9, comprising the sequence set forth in 11. The biomarker is [helix]-[linker]-[helix]-[linker]-[helix]-[epitope] (Wherein, [helix] represents the pH-sensitive helix; [Linker] represents a linker comprising the sequence (GGS)n or RLCRPRSAPSRCGPGLRPCAP; [Epitope] represents the above-mentioned epitope. 10. The method of embodiment 4 or 9, comprising the sequence set forth in 12. A method for detecting, imaging, or treating diseased cells, comprising: administering a composition to a subject, wherein said composition induces surface expression of a biomarker that comprises an activatable epitope on diseased cells preferentially relative to surface expression of said biomarker on non-diseased cells. Includes; the activatable epitope is preferentially accessible for binding in a microenvironment specific to the diseased cell compared to a microenvironment of a cell not affected by the disease; the biomarker comprises a membrane-resident polypeptide or the biomarker comprises a polypeptide that comprises a transmembrane domain. method. 13. The method of any one of embodiments 1 to 12, further comprising administering to the subject an antibody or antigen-binding fragment thereof, a protein ligand or functional fragment thereof, or a small molecule configured to bind to the epitope. 14. The method of embodiment 13, further comprising detecting binding of said antibody or antigen-binding fragment thereof to said epitope. 15. A method for detecting, imaging, or treating diseased cells, comprising: (a) administering to a subject a composition, wherein the composition preferentially induces surface expression of a biomarker that comprises an epitope on diseased cells relative to surface expression of the biomarker on non-diseased cells; and (b) administering to the subject an antibody or antigen-binding fragment thereof, a protein ligand or functional fragment thereof, or a small molecule configured to bind to the epitope. A method comprising: 16. The method of embodiment 15, wherein the extracellular domain does not comprise an scFv. 17. The method of embodiment 16, wherein the extracellular domain does not comprise a VL domain. 18. The method of any one of embodiments 15 to 17, further comprising detecting binding of said antibody or antigen-binding fragment, protein ligand or functional fragment thereof, or small molecule to said epitope. 19. The method of any one of embodiments 15-18, wherein the epitope is an activatable epitope preferentially accessible for binding to an extracellular ligand, and the preferential accessibility for binding is in a microenvironment specific to the diseased cells compared to the intracellular or extracellular microenvironment of cells not affected by the disease. 20. The method of embodiment 19, wherein the activatable epitope is activated by phosphorylation, glycosylation, methylation, ubiquitination, alpha-acetylation, lipidation, lipidation with glycosylphosphatidylinositol, amidation, sulfonation, oxidation, or any combination thereof. 21. The method of any one of embodiments 15 to 20, wherein the biomarker further comprises a pH-sensitive helix. 22. The method of embodiment 21, wherein the activatable epitope is flanked by at least two copies of a pH-sensitive helix. 23. The method of embodiment 21 or 22, wherein the pH-sensitive helix comprises a sequence having at least 80% identity to any one of SEQ ID NOs: 1-3. 24. The biomarker is [helix]-[linker]-[helix]-[linker]-[helix]-[linker]-[epitope] (Wherein, [helix] represents the pH-sensitive helix; [Linker] represents a linker comprising the sequence (GGS)n or (GGS)nFCYWKTCT(GGS)n; [Epitope] represents the above-mentioned epitope. 24. The method according to any one of embodiments 21 to 23, comprising the sequence described in 25. The biomarker is [helix]-[linker]-[helix]-[linker]-[helix]-[epitope] (Wherein, [helix] represents the pH-sensitive helix; [Linker] represents a linker comprising the sequence (GGS)n or RLCRPRSAPSRCGPGLRPCAP; [Epitope] represents the above-mentioned epitope. 24. The method according to any one of embodiments 21 to 23, comprising the sequence described in 26. The method of embodiment 16, wherein the biomarker further comprises a protease cleavage site for a cancer-specific protease together with an scFv configured to bind to the activatable epitope. 27. The method of embodiment 26, wherein said cancer-specific protease is uPA, EPCAM, MMP, cathepsin, serine protease, granzyme, furin, cysteine ​​protease, elastase, or type II transmembrane serine protease, kallikrein-related peptidase, a "disintegrin and metalloproteinase" (ADAM), plasminogen activator, serine protease plasminogen activator, caspase, or any combination thereof. 28. The method of any one of embodiments 13 to 27, wherein the antibody or antigen-binding fragment thereof further comprises a radioisotope. 29. The method of embodiment 20, wherein the radioisotope comprises a positron-emitting radioisotope, an alpha-emitting radioisotope, a beta-emitting radioisotope, or 99mTc. 30. The radioisotope is a positron-emitting radioisotope; 124 I, 68 Ga, 11 C. 13 N, 15 O. 18 F, 68 Ga, 64 Cu, 52 Mn, 55 Co, 89 Zr, 82 30. The method of embodiment 29, comprising administering to a subject, a mammal, a human, a humanized mammal, a medicament, a humanized human ... 31. The radioisotope comprises an alpha-emitting radioisotope; 225 Ac, 211 At, 227 Th, 224 30. The method of embodiment 29, comprising administering to a subject a subject, comprising administering to a subject a subject, a subject, a group comprising a subject, a group including ... 32. The radioisotope comprises a beta-emitting radioisotope; 177 Lu, 67 Cu, 131 I, 90 Y, 89 Sr, 186 Re, 165 Dy, 32 P, 166 Ho, 188 30. The method of embodiment 29, comprising: 33. The method of any one of embodiments 1 to 32, wherein the biomarker further comprises an extracellular domain comprising the epitope. 34. The method of embodiment 33, wherein the extracellular domain is derived from a membrane protein endogenous to the subject. 35. The method of embodiment 33, wherein the extracellular domain is derived from 158, PSMA, SSTR2, CD8a, CD4, TGFR1, IGF1R, PD-L1, EGFR, CD73, IL-6, or any combination thereof. 36. The method of embodiment 35, wherein the extracellular domain is derived from CD8a, CD4, TGFR1, IGF1R, PD-L1, EGFR, CD73, IgK, or any combination thereof. 37. The method of embodiment 35 or 36, wherein the extracellular domain further comprises an activatable epitope derived from DLL3, PSMA, SSTR2, or any combination thereof. 38. The method of any one of embodiments 1 to 37, wherein the biomarker comprises an extracellular domain comprising a sequence having at least 80% identity to any one of SEQ ID NOs: 4 to 9, a functional fragment thereof, or a variant thereof. 39. The method of any one of embodiments 1 to 38, wherein the biomarker further comprises a hinge domain derived from IgG4, IgG1, CD8, or CD28, or the sequence (GGGS)n. 40. The method of any one of embodiments 1 to 39, wherein the biomarker further comprises a transmembrane domain derived from DLL3, PSMA, SSTR2, PD-L1, EGFR, CD28, CD4, CD8a, ICOS, or CD73. 41. The method of any one of embodiments 1 to 40, wherein the biomarker further comprises an intracellular domain comprising a reporter protein or an affinity tag. 42. The method of embodiment 41, wherein the biomarker further comprises an intracellular domain comprising a reporter protein, the reporter protein being derived from GFP, luciferase, Nluc, or Fluc, or any combination thereof. 43. The method of embodiment 41, wherein the biomarker further comprises an intracellular domain comprising an affinity tag, the affinity tag comprising an HA, FLAG, or MYC sequence, or any combination thereof. 44. The method of any one of embodiments 1 to 43, wherein the biomarker comprises two or more copies of the epitope. 45. The method of any one of embodiments 1 to 44, wherein the composition comprises a nucleic acid vector comprising a coding sequence for the biomarker. 46. ​​The method of embodiment 45, wherein the coding sequence comprises a promoter operably linked to a nucleotide sequence encoding a biomarker. 47. The method of embodiment 46, wherein the promoter preferentially drives expression of the biomarker in diseased cells relative to expression of the biomarker in non-diseased cells of the subject. 48. The method of embodiment 46 or 47, wherein the promoter comprises a promoter of a gene that is selectively overexpressed in diseased cells compared to non-diseased cells, or a functional fragment thereof. 49. The promoter is selected from the group consisting of survivin promoter (BIRC5), CXCR4 promoter, ATP-binding cassette subfamily C member 4 (ABCC4) promoter, anterior gradient 2, protein disulfide isomerase family member (AGR2) promoter, activation-induced cytidine deaminase (AICDA) promoter, UDP-GlcNAc:beta Gal beta-1,3-N-acetylglucosaminyltransferase 3 (B3GNT3) promoter, cadherin 3 (CDH3) promoter, CEA cell adhesion molecule 5 (CEACAM5) promoter, centromere protein F (CENPF) promoter, centrosomal protein 55 (CEP55) promoter, claudin 3 (CLDN3) promoter, claudin 4 (CLDN4) promoter, type XI collagen alpha 1 chain (COL11A1) promoter, type I collagen alpha 1 chain (COL1A1) promoter, cystatin SN (CST1) promoter, and dentate-less E3 ubiquitin tan. Protein ligase homolog (DTL) promoter, family 111 member B with sequence similarity (FAM111B) promoter, forkhead box A1 (FOXA1) promoter, kinesin family member 20A (KIF20A), laminin subunit gamma 2 (LAMC2) promoter, mitotic spindle positioning (MISP) promoter, matrix metallopeptidase 1 (MMP1) promoter, matrix metallopeptidase 12 (MMP12) promoter, and matrix metallopeptidase 13 (MMP13) promoter, mesothelin (MSLN) promoter, cell surface-associated mucin 1 (MUC1) promoter, phospholipase A2 group IID (PLA2G2D) promoter, regulator of G protein signaling 13 (RGS13) promoter, secretoglobin family 2A member 1 (SCGB2A1) promoter, topoisomerase II alpha (TOP2A) promoter, ubiquitin D (UBD) promoter, ubiquitin-conjugating enzyme E2 (U2) promoter,C (UBE2C), USH1 protein network component harmonin (USH1C), V-set domain-containing T cell activation inhibitor 1 (VTCN1) promoter, hexokinase type II promoter, TRPM4 promoter, stromelysin 3 promoter, surfactant protein A promoter, secretory leukocyte protease inhibitor promoter, tyrosinase promoter, stress-inducible grp78 / BiP promoter, interleukin-10 promoter, alpha-B-crystallin / heat shock protein 27 promoter, epidermal growth factor receptor promoter, mucin-like glycoprotein promoter, mts1 promoter, NSE promoter, somatostatin tin receptor promoter, c-erbB-3 promoter, c-erbB-2 promoter, c-erbB4 promoter, thyroglobulin promoter, α-fetoprotein promoter, villin promoter, albumin promoter, glycoprotein A33 promoter, B cell specific Moloney leukemia virus insertion site 1 promoter, cyclooxygenase-2 promoter, fibroblast growth factor promoter; human epidermal growth factor receptor 2, human telomerase reverse transcriptase promoter; kinase domain insertion-containing receptor promoter; rad51 recombinase promoter; TTF-1, urokinase-type plasminogen activator receptor promoter, ubiquitin-conjugating enzyme E2 T (UBE2T) promoter, checkpoint kinase 1 (CHEK1) promoter, epithelial cell transforming 2 promoter (ECT2), BCL2-like 12 (BCL2L12) promoter, centromere protein I (CENPI) promoter, E2F transcription factor 1 (E2F1) promoter, flavin adenine dinucleotide synthetase 1 (FLAD1) promoter, protein phosphatase, Mg2+ / Mn2+-dependent 1G (PPM1G) promoter, ubiquitin-conjugating enzyme E2S (UBE2S) promoter, aurora kinase A and ninein interacting protein (AUNIP) promoter, cell division cycle 6 (CDC6) promoter, centromere protein L (CENPL) promoter, DNA replicative helicase / nuclease 2 (DNA2) promoter, DSN1 homolog, MIS12 kinetochore complex component (DSN1) promoter, deoxythymidylate kinase (DTYMK) promoter, G protein-regulated inducer of neurite outgrowth 1 (GPRIN1) promoter, mitochondrial fission regulator 2 (MTFR2) promoter, RAD51-associated protein 1 (RAD51AP1) promoter, small nuclear ribonucleoprotein polypeptide A' (SNRPA1) promoter, ATPase family, AAA domain containing 2 (ATAD2) promoter, BUB1 mitotic checkpoint serine / threonine kinase (BUB1) promoter, calcyclin-binding protein (CACYBP) promoter, cell division cycle associated 3 (CDCA3) promoter, centromere protein O (CENPO) promoter promoter, flap structure-specific endonuclease 1 (FEN1) promoter, forkhead box M1 (FOXM1) promoter, cell proliferation-regulated inhibitor of protein phosphatase 2A (KIAA1524) promoter, kinesin family member 2C (KIF2C) promoter, karyopherin subunit alpha 2 (KPNA2) promoter, MYB proto-oncogene-like 2 (MYBL2) promoter, NIMA-related kinase 2 (NEK2) promoter, RAN-binding protein 1 (RANBP1) promoter, small nuclear ribonucleoprotein polypeptides B and B1 (SNRPB) promoter, SPC24 / NDC80 kinetochore complex component (SPC24) promoter, transforming acidic coiled-coil-containing protein 3 (TACC3) promoter, TBC1 domain family member 31 (TBC1D31) promoter, thymidine kinase 1 (TK1) promoter, zinc finger protein 695 (ZNF695) promoter, aurora kinase A (AURKA) promoter, BLMRecQ-like helicase (BLM) promoter, chromosome 17 open reading frame 53 (C17orf53) promoter, chromobox 3 (CBX30) promoter, cyclin B1 (CCNB1) promoter, cyclin E1 (CCNE1) promoter, cyclin F (CCNF), cell division cycle 20 (CDC20) promoter, cell division cycle 45 (CDC45) promoter, cell division cycle associated 5 (CDCA5) promoter, cyclin-dependent kinase inhibitor 3 (CDKN3) promoter, cadherin EGF LAG 7-transmembrane G-type receptor 3 (CELSR3) promoter, centromere protein A (CENPA) promoter, centrosomal protein 72 (CEP72) promoter, CDC28 protein kinase regulatory subunit 2 (CKS2) promoter, type X collagen alpha 1 chain (COL10A1) promoter, chromosome segregation 1-like (CSE1L) promoter, DBF4 zinc finger promoter, GINS complex subunit 1 (GINS1) promoter, G protein-coupled receptor 19 (GPR19) promoter, kinesin family member 18A (KIF18A) promoter, kinesin family member 4A (KIF4A) promoter, kinesin family member C1 ( KIFC1) promoter, minichromosome maintenance 10 replication initiator (MCM10) promoter, minichromosome maintenance complex component 2 (MCM2) promoter, minichromosome maintenance complex component 7 (MCM7) promoter, MRG domain binding protein (MRGBP) promoter, methylenetetrahydrofolate dehydrogenase (NADP+-dependent) 2, methenyltetrahydrofolate cyclohydrolase (MTHFD2) promoter, non-SMC condensin I complex subunit H (NCAPH) promoter, kinetochore complex component (NDC80) promoter, nudix hydrolase 1 (NUDT1) promoter, ribonuclease H2 subunit A (RNASEH2A) promoter, RuvB-like AAA (RcA) promoter,ATPase1 (RUVBL1) promoter, serologically defined breast cancer antigen NY-BR-85 (SGOL1) promoter, SHC-binding and spindle-associated 1 (SHCBP1) promoter, small nuclear ribonucleoprotein polypeptide G (SNRPG) promoter, timeless circadian regulator promoter, thyroid hormone receptor interactor 13 (TRIP13) promoter, trophinin-related protein (TROAP) promoter, ubiquitin-conjugating enzyme E2 C (UBE2C) promoter, WD repeat and HMG box DNA-binding protein 1 (WDHD1) promoter, alpha fetoprotein (AFP) promoter, fragments thereof, any combination thereof, a chimeric promoter compiled from a plurality of elements from the above, or a completely synthetic promoter composed of tile-like transcription factor binding sites. 50. The method of any one of embodiments 45 to 49, wherein the vector is a recombinant viral vector. 51. The method of any one of embodiments 45 to 49, wherein the vector is a non-viral vector. 52. The method of embodiment 51, wherein the non-viral vector is a nanoplasmid, a plasmid, a minicircle, a closed-end linear duplex (CELiD), or a dog-bone DNA vector (dbDNA). 53. The method of any one of embodiments 1 to 52, wherein the composition further comprises a transfection agent. 54. The method of embodiment 53, wherein the transfection agent is a linear or branched polyethyleneimine, a nanoparticle, a lipophilic particle, a solid nanoparticle, a peptide, a micelle, a dendrimer, a polymer composition, a hydrogel, a synthetic or naturally derived exosome, a virus-like particle, or any combination thereof. 55. The method of any one of embodiments 1 to 54, wherein the disease is cancer, an autoimmune disease, or a neurodegenerative disease. 56. The method of embodiment 55, wherein the disease is cancer. 57.The cancer is acute myeloid leukemia, adrenocortical carcinoma, bladder urothelial carcinoma, ductal carcinoma, breast lobular carcinoma, cervical cancer, cholangiocellular carcinoma, colorectal adenocarcinoma, esophageal cancer, gastric adenocarcinoma, glioblastoma multiforme, head and neck squamous epithelium. Cell carcinoma, hepatocellular carcinoma, renal chromophobe carcinoma, renal clear cell carcinoma, renal papillary cell carcinoma, low-grade glioma, lung adenocarcinoma, lung squamous cell carcinoma, mesothelioma, ovarian serous adenocarcinoma, pancreatic ductal adenocarcinoma, paraganglioma and pheochromocytoma, pre- 57. The method of embodiment 56, comprising prostate adenocarcinoma, sarcoma, cutaneous melanoma, testicular germ cell carcinoma, thymoma, papillary thyroid carcinoma, uterine carcinosarcoma, uterine endometrioid carcinoma, uveal melanoma, melanoma of the lip, spindle cell carcinoma, liposarcoma, nasal sarcoma, breast adenocarcinoma, insulinoma, osteosarcoma, mast cell tumor, angiosarcoma, non-small cell lung cancer (NSCLC), marginal zone lymphoma, malignant melanoma, or chronic lymphocytic leukemia. 58. The method of any one of embodiments 14 to 57, wherein the step of detecting binding of the antibody or antigen-binding fragment thereof to the epitope comprises a PET imaging method or another radioisotope-based imaging method. 59. A nucleic acid comprising a cancer-specific promoter operably linked to a nucleotide sequence encoding a membrane-resident polypeptide biomarker that comprises an activatable epitope. 60. The method of embodiment 59, wherein the extracellular domain does not comprise an scFv. 61. The method of embodiment 60, wherein the extracellular domain does not comprise a VL domain. 62. The nucleic acid of any one of embodiments 59 to 61, wherein the activatable epitope is activated by phosphorylation, glycosylation, methylation, ubiquitination, A-acetylation, lipidation (e.g., GPI), amidation, sulfonation, oxidation, or any combination thereof. 63. The nucleic acid of any one of embodiments 59 to 62, wherein the biomarker further comprises an extracellular domain comprising the epitope and a transmembrane domain. 64. The nucleic acid of any one of embodiments 59 to 63, wherein the biomarker further comprises a pH-sensitive helix. 65. The nucleic acid of any one of embodiments 59 to 64, wherein the activatable epitope is flanked by at least two copies of a pH-sensitive helix. 66. The nucleic acid according to any one of embodiments 59 to 65, wherein the pH-sensitive helix comprises a sequence having at least 80% identity to any one of SEQ ID NOs: 1 to 3. 67. The biomarker is: [helix]-[linker]-[helix]-[linker]-[helix]-[linker]-[epitope] (Wherein, [helix] represents the pH-sensitive helix; [Linker] represents a linker comprising the sequence (GGS)n or (GGS)nFCYWKTCT(GGS)n; [Epitope] represents the above-mentioned epitope. 67. The nucleic acid according to any one of embodiments 59 to 66, comprising a sequence according to the invention. 68. The biomarker is: [helix]-[linker]-[helix]-[linker]-[helix]-[epitope] (Wherein, [helix] represents the pH-sensitive helix; [Linker] represents a linker comprising the sequence (GGS)n or RLCRPRSAPSRCGPGLRPCAP; [Epitope] represents the above-mentioned epitope. 67. The nucleic acid according to any one of embodiments 59 to 66, comprising a sequence according to the invention. 69. The nucleic acid of any one of embodiments 59 to 68, wherein the biomarker further comprises a protease cleavage site for a cancer-specific protease together with an scFv configured to bind to the activatable epitope. 70. The nucleic acid of embodiment 67, wherein the cancer-specific protease is uPA, EPCAM, MMP, cathepsin, serine protease, granzyme, furin, cysteine ​​protease, elastase, or type II transmembrane serine protease, kallikrein-related peptidase, a "disintegrin and metalloproteinase" (ADAM), plasminogen activator (serine protease), caspase, or any combination thereof. 71. The nucleic acid of any one of embodiments 60 to 70, wherein the extracellular domain is derived from a membrane protein endogenous to the subject. 72. The nucleic acid of any one of embodiments 60 to 71, wherein the extracellular domain is derived from DLL3, PSMA, SSTR2, CD8a, CD4, TGFR1, IGF1R, PD-L1, EGFR, CD73, IgK, or IL-6, or any combination thereof. 73. The nucleic acid of embodiment 72, wherein the extracellular domain is derived from CD8a, CD4, TGFR1, IGF1R, PD-L1, EGFR, CD73, IgK, or any combination thereof. 74. The nucleic acid of embodiment 72 or 73, wherein the extracellular domain further comprises an activatable epitope derived from DLL3, PSMA, SSTR2, or any combination thereof. 75. The nucleic acid of any one of embodiments 60-74, wherein the biomarker comprises an extracellular domain comprising a sequence having at least 80% identity to any one of SEQ ID NOs: 4-9, a functional fragment thereof, or a variant thereof. 76. The nucleic acid of any one of embodiments 59 to 75, wherein the biomarker further comprises a hinge domain derived from an Ig superfamily receptor or comprises the sequence (GGGS)n. 77. The nucleic acid of embodiment 76, wherein the biomarker further comprises the hinge domain, the hinge domain being derived from IgG4, IgG1, CD8, or CD28. 78. The nucleic acid of any one of embodiments 60 to 77, wherein the biomarker further comprises a transmembrane domain derived from DLL3, PSMA, SSTR2, PD-L1, EGFR, CD28, CD4, CD8a, ICOS, or CD73. 79. The nucleic acid of any one of embodiments 59 to 78, wherein the biomarker further comprises an intracellular domain comprising a reporter protein or affinity tag. 80. The nucleic acid of embodiment 79, wherein the biomarker further comprises an intracellular domain comprising a reporter protein, the reporter protein being derived from GFP, luciferase, Nluc, or Fluc, or any combination thereof. 81. The nucleic acid of embodiment 79, wherein the biomarker further comprises an intracellular domain comprising an affinity tag, the affinity tag comprising an HA, FLAG, or MYC sequence, or any combination thereof. 82. The promoter is selected from the group consisting of survivin promoter (BIRC5), CXCR4 promoter, ATP-binding cassette subfamily C member 4 (ABCC4) promoter, anterior gradient 2, protein disulfide isomerase family member (AGR2) promoter, activation-induced cytidine deaminase (AICDA) promoter, UDP-GlcNAc:beta Gal beta-1,3-N-acetylglucosaminyltransferase 3 (B3GNT3) promoter, cadherin 3 (CDH3) promoter, CEA cell adhesion molecule 5 (CEACAM5) promoter, centromere protein F (CENPF) promoter, centrosomal protein 55 (CEP55) promoter, claudin 3 (CLDN3) promoter, claudin 4 (CLDN4) promoter, type XI collagen alpha 1 chain (COL11A1) promoter, type I collagen alpha 1 chain (COL1A1) promoter, cystatin SN (CST1) promoter, and dentate-less E3 ubiquitin tan. Protein ligase homolog (DTL) promoter, family 111 member B with sequence similarity (FAM111B) promoter, forkhead box A1 (FOXA1) promoter, kinesin family member 20A (KIF20A), laminin subunit gamma 2 (LAMC2) promoter, mitotic spindle positioning (MISP) promoter, matrix metallopeptidase 1 (MMP1) promoter, matrix metallopeptidase 12 (MMP12) promoter, and matrix metallopeptidase 13 (MMP13) promoter, mesothelin (MSLN) promoter, cell surface-associated mucin 1 (MUC1) promoter, phospholipase A2 group IID (PLA2G2D) promoter, regulator of G protein signaling 13 (RGS13) promoter, secretoglobin family 2A member 1 (SCGB2A1) promoter, topoisomerase II alpha (TOP2A) promoter, ubiquitin D (UBD) promoter, ubiquitin-conjugating enzyme E2 (U2) promoter,C (UBE2C), USH1 protein network component harmonin (USH1C), V-set domain-containing T cell activation inhibitor 1 (VTCN1) promoter, hexokinase type II promoter, TRPM4 promoter, stromelysin 3 promoter, surfactant protein A promoter, secretory leukocyte protease inhibitor promoter, tyrosinase promoter, stress-inducible grp78 / BiP promoter, interleukin-10 promoter, alpha-B-crystallin / heat shock protein 27 promoter, epidermal growth factor receptor promoter, mucin-like glycoprotein promoter, mts1 promoter, NSE promoter, somatostatin tin receptor promoter, c-erbB-3 promoter, c-erbB-2 promoter, c-erbB4 promoter, thyroglobulin promoter, α-fetoprotein promoter, villin promoter, albumin promoter, glycoprotein A33 promoter, B cell specific Moloney leukemia virus insertion site 1 promoter, cyclooxygenase-2 promoter, fibroblast growth factor promoter; human epidermal growth factor receptor 2, human telomerase reverse transcriptase promoter; kinase domain insertion-containing receptor promoter; rad51 recombinase promoter; TTF-1, urokinase-type plasminogen activator receptor promoter, ubiquitin-conjugating enzyme E2 T (UBE2T) promoter, checkpoint kinase 1 (CHEK1) promoter, epithelial cell transforming 2 promoter (ECT2), BCL2-like 12 (BCL2L12) promoter, centromere protein I (CENPI) promoter, E2F transcription factor 1 (E2F1) promoter, flavin adenine dinucleotide synthetase 1 (FLAD1) promoter, protein phosphatase, Mg2+ / Mn2+-dependent 1G (PPM1G) promoter, ubiquitin-conjugating enzyme E2S (UBE2S) promoter, aurora kinase A and ninein interacting protein (AUNIP) promoter, cell division cycle 6 (CDC6) promoter, centromere protein L (CENPL) promoter, DNA replicative helicase / nuclease 2 (DNA2) promoter, DSN1 homolog, MIS12 kinetochore complex component (DSN1) promoter, deoxythymidylate kinase (DTYMK) promoter, G protein-regulated inducer of neurite outgrowth 1 (GPRIN1) promoter, mitochondrial fission regulator 2 (MTFR2) promoter, RAD51-associated protein 1 (RAD51AP1) promoter, small nuclear ribonucleoprotein polypeptide A' (SNRPA1) promoter, ATPase family, AAA domain containing 2 (ATAD2) promoter, BUB1 mitotic checkpoint serine / threonine kinase (BUB1) promoter, calcyclin-binding protein (CACYBP) promoter, cell division cycle associated 3 (CDCA3) promoter, centromere protein O (CENPO) promoter promoter, flap structure-specific endonuclease 1 (FEN1) promoter, forkhead box M1 (FOXM1) promoter, cell proliferation-regulated inhibitor of protein phosphatase 2A (KIAA1524) promoter, kinesin family member 2C (KIF2C) promoter, karyopherin subunit alpha 2 (KPNA2) promoter, MYB proto-oncogene-like 2 (MYBL2) promoter, NIMA-related kinase 2 (NEK2) promoter, RAN-binding protein 1 (RANBP1) promoter, small nuclear ribonucleoprotein polypeptides B and B1 (SNRPB) promoter, SPC24 / NDC80 kinetochore complex component (SPC24) promoter, transforming acidic coiled-coil-containing protein 3 (TACC3) promoter, TBC1 domain family member 31 (TBC1D31) promoter, thymidine kinase 1 (TK1) promoter, zinc finger protein 695 (ZNF695) promoter, aurora kinase A (AURKA) promoter, BLMRecQ-like helicase (BLM) promoter, chromosome 17 open reading frame 53 (C17orf53) promoter, chromobox 3 (CBX30) promoter, cyclin B1 (CCNB1) promoter, cyclin E1 (CCNE1) promoter, cyclin F (CCNF), cell division cycle 20 (CDC20) promoter, cell division cycle 45 (CDC45) promoter, cell division cycle associated 5 (CDCA5) promoter, cyclin-dependent kinase inhibitor 3 (CDKN3) promoter, cadherin EGF LAG 7-transmembrane G-type receptor 3 (CELSR3) promoter, centromere protein A (CENPA) promoter, centrosomal protein 72 (CEP72) promoter, CDC28 protein kinase regulatory subunit 2 (CKS2) promoter, type X collagen alpha 1 chain (COL10A1) promoter, chromosome segregation 1-like (CSE1L) promoter, DBF4 zinc finger promoter, GINS complex subunit 1 (GINS1) promoter, G protein-coupled receptor 19 (GPR19) promoter, kinesin family member 18A (KIF18A) promoter, kinesin family member 4A (KIF4A) promoter, kinesin family member C1 ( KIFC1) promoter, minichromosome maintenance 10 replication initiator (MCM10) promoter, minichromosome maintenance complex component 2 (MCM2) promoter, minichromosome maintenance complex component 7 (MCM7) promoter, MRG domain binding protein (MRGBP) promoter, methylenetetrahydrofolate dehydrogenase (NADP+-dependent) 2, methenyltetrahydrofolate cyclohydrolase (MTHFD2) promoter, non-SMC condensin I complex subunit H (NCAPH) promoter, kinetochore complex component (NDC80) promoter, nudix hydrolase 1 (NUDT1) promoter, ribonuclease H2 subunit A (RNASEH2A) promoter, RuvB-like AAA (RcA) promoter,ATPase1 (RUVBL1) promoter, serologically defined breast cancer antigen NY-BR-85 (SGOL1) promoter, SHC-binding and spindle-associated 1 (SHCBP1) promoter, small nuclear ribonucleoprotein polypeptide G (SNRPG) promoter, timeless circadian regulator promoter, thyroid hormone receptor interactor 13 (TRIP13) promoter, trophinin-related protein (TROAP) promoter, ubiquitin-conjugating enzyme E2 C (UBE2C) promoter, WD repeat and HMG box DNA-binding protein 1 (WDHD1) promoter, alpha fetoprotein (AFP) promoter, fragments thereof, any combination thereof, chimeric promoters compiled from a plurality of elements from the above, or a completely synthetic promoter composed of tile-like transcription factor binding sites. 83. The nucleic acid of any one of embodiments 59 to 82, wherein the nucleic acid comprises RNA or capped mRNA. 84. The nucleic acid of embodiment 83, wherein the RNA or the capped mRNA comprises a splice signal, a synthetic intron, an miRNA binding site, a synthetic stem-loop or tetraloop, or an aRNA domain. 85. A vector comprising a nucleic acid according to any one of embodiments 59 to 84. 86. The vector described in embodiment 85, wherein the vector is a recombinant viral vector. 87. The vector described in embodiment 85, wherein the vector is a non-viral vector. 88. The vector of embodiment 87, wherein the non-viral vector is a nanoplasmid, a plasmid, a minicircle, a closed-end linear duplex (CELiD), or a dog-bone DNA vector (dbDNA). 89. A composition comprising a vector according to any one of embodiments 83 to 88 or a nucleic acid according to any one of embodiments 59 to 84, and a transfection agent. 90. The composition of embodiment 89, wherein the transfection agent is a linear or branched polyethyleneimine, a nanoparticle, a lipophilic particle, a solid nanoparticle, a peptide, a micelle, a dendrimer, a polymer composition, a hydrogel, a synthetic or naturally derived exosome, a virus-like particle, or any combination thereof. 91. A cell comprising a nucleic acid according to any one of embodiments 59 to 84 or a vector according to any one of embodiments 84 to 88. 92. A method for detecting, imaging, or treating diseased cells, comprising: administering to a subject a composition that induces expression of a secretable biomarker preferentially in diseased cells relative to non-diseased cells, said biomarker comprising: (a) an extracellular domain configured to bind to an affinity reagent configured for detection or treatment of the diseased cell; and (b) a membrane-affinity domain capable of associating with, inserting into, or binding to the outer membrane of a cell Including steps A method comprising: 93. The method of embodiment 92, wherein the extracellular domain does not comprise an scFv. 94. The method of embodiment 93, wherein the extracellular domain does not comprise a VL domain. 95. The method of any one of embodiments 92 to 94, wherein the composition comprises a nucleic acid or vector, the nucleic acid or vector encoding the secretable biomarker. 96. The method of any one of embodiments 92-95, wherein the biomarker comprises, in order from N-terminus to C-terminus, (a) and (b). 97. The method of any one of embodiments 92-96, wherein the extracellular domain comprises the extracellular domain of an extracellular receptor, an epitope thereof, or a ligand-binding domain thereof. 98. The method of any one of embodiments 92-97, wherein the extracellular receptor is DLL3, PSMA, SSTR2, CD8a, CD4, TGFR1, IGF1R, PD-L1, EGFR, CD73, IgK, or IL-6, or any combination thereof. 99. The method of any one of embodiments 92 to 98, wherein the extracellular domain comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 4 to 6 or 17 to 19, a functional fragment thereof, or a variant thereof. 100. The method of any one of embodiments 92 to 99, wherein the biomarker is encoded by an open reading frame encoding an N-terminal cleavable signal peptide. 101. The method of any one of embodiments 92 to 100, wherein the membrane affinity domain is configured to bind to a cell membrane at low pH or in the presence of a cancer-specific extracellular protease. 102. The method of embodiment 101, wherein the membrane affinity domain is configured to bind to a cell membrane at low pH, and the affinity domain comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 10 to 16, or a variant thereof. 103. The method of any one of embodiments 92 to 102, wherein the membrane affinity domain comprises a C1, C2, PH, FYVE, PX, or ENTH domain, or a transmembrane helix derived from bacteriorhodopsin. 104. (c) administering to the subject an antibody or antigen-binding derivative or fragment thereof, a protein ligand or functional fragment thereof, or a small molecule configured to bind to the extracellular domain. The method of any one of embodiments 92 to 103, further comprising: 105. (d) detecting binding of said antibody or an antigen-binding derivative or fragment thereof, said protein ligand or a functional fragment thereof, or said small molecule to said extracellular domain. 105. The method of embodiment 104, further comprising: 106. The method of embodiment 105, wherein the step of detecting binding of the antibody or antigen-binding derivative or fragment thereof, the protein ligand or functional fragment thereof, or the small molecule to the extracellular domain comprises a PET imaging method or another radioisotope-based imaging method. 107. The method of any one of embodiments 93 to 106, wherein the composition comprises the vector, and the vector is a recombinant viral vector. 108. The method of any one of embodiments 93 to 107, wherein the vector is a non-viral vector. 109. The method of embodiment 108, wherein the non-viral vector is a nanoplasmid, a plasmid, a minicircle, a closed-end linear duplex (CELiD), or a dog-bone DNA vector (dbDNA). 110. The method of any one of embodiments 92 to 109, wherein the disease is cancer. 111. A nucleic acid comprising an open reading frame (ORF) encoding a polypeptide, the polypeptide comprising: (a) an N-terminal cleavable signal peptide; (b) an extracellular domain configured to bind to an affinity reagent, the extracellular domain comprising an extracellular domain of an extracellular receptor, an epitope thereof, or a ligand-binding domain thereof; and (c) a membrane-affinity domain capable of associating with, inserting into, or binding to the outer membrane of a cell A nucleic acid comprising: 112. The nucleic acid of embodiment 111, wherein the extracellular domain does not comprise an scFv. 113. The nucleic acid of embodiment 112, wherein the extracellular domain does not comprise a VL domain. 114. The nucleic acid of any one of embodiments 111 to 113, wherein the extracellular receptor is DLL3, PSMA, SSTR2, CD8a, CD4, TGFR1, IGF1R, PD-L1, EGFR, CD73, IgK, or IL-6, or any combination thereof. 115. The nucleic acid according to any one of embodiments 111 to 114, wherein the membrane-affinity domain comprises a C1, C2, PH, FYVE, PX, or ENTH domain, or a transmembrane helix derived from bacteriorhodopsin. 116. A nucleic acid comprising an open reading frame (ORF) encoding a polypeptide, the polypeptide comprising: (a) an extracellular domain configured to bind to an affinity reagent, said extracellular domain comprising an extracellular domain of an extracellular receptor, an epitope thereof, or a ligand binding domain thereof; said extracellular receptor is DLL3, PSMA, SSTR2, CD8a, CD4, TGFR1, CD73, or IgK, or any combination thereof; and (b) a membrane-affinity domain capable of associating with, inserting into, or binding to the outer membrane of a cell A nucleic acid comprising: 117. The nucleic acid of embodiment 116, wherein the extracellular domain does not comprise an scFv. 118. The nucleic acid of embodiment 117, wherein the extracellular domain does not comprise a VL domain. 119. The nucleic acid according to any one of embodiments 116 to 118, wherein the membrane-affinity domain comprises a C1, C2, PH, FYVE, PX, or ENTH domain, or a transmembrane helix derived from bacteriorhodopsin. 120. A nucleic acid comprising an open reading frame (ORF) encoding a polypeptide, the polypeptide comprising: (a) an extracellular domain configured to bind to an affinity reagent, the extracellular domain comprising the extracellular domain of an extracellular receptor, an epitope thereof, or a ligand-binding domain thereof; and (b) a membrane-affinity domain capable of associating with or binding to the outer membrane of a cell, said membrane-affinity domain comprising a C1, C2, PH, FYVE, PX, or ENTH domain. A nucleic acid comprising: 121. The nucleic acid of embodiment 120, wherein the extracellular domain does not comprise an scFv. 122. The nucleic acid of embodiment 121, wherein the extracellular domain does not comprise a VL domain. 123. The nucleic acid of any one of embodiments 120 to 122, wherein the extracellular receptor is DLL3, PSMA, SSTR2, CD8a, CD4, TGFR1, IGF1R, PD-L1, EGFR, CD73, IgK, or IL-6, or any combination thereof. 124. The nucleic acid according to any one of embodiments 111 to 121, wherein the extracellular domain comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 4 to 6 or 17 to 19, a functional fragment thereof, or a variant thereof. 125. The nucleic acid according to any one of embodiments 116 to 124, wherein the polypeptide comprises an N-terminal cleavable signal peptide. 126. The nucleic acid according to any one of embodiments 111 to 125, wherein the membrane affinity domain is configured to bind to a cell membrane at low pH or in the presence of a cancer-specific extracellular protease. 127. The nucleic acid of embodiment 126, wherein the membrane affinity domain is configured to bind to a cell membrane at low pH, and the affinity domain comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 10 to 16, or a variant thereof. 128. The nucleic acid according to any one of embodiments 111 to 127, wherein the polypeptide further comprises a hinge domain derived from an Ig superfamily receptor between the extracellular domain and the membrane-affinic domain. 129. The nucleic acid of any one of embodiments 111 to 127, wherein the ORF is codon-optimized for expression in mammalian cells. 130. The nucleic acid according to any one of embodiments 111 to 129, further comprising a cancer-specific promoter, wherein said ORF is operably linked to said cancer-specific promoter. 131. The nucleic acid of embodiment 130, wherein the cancer-specific promoter is a promoter of a gene that is overexpressed in cancer cells compared to normal cells, or a functional fragment thereof. 132. The promoter is selected from the group consisting of survivin promoter (BIRC5), CXCR4 promoter, ATP-binding cassette subfamily C member 4 (ABCC4) promoter, anterior gradient 2, protein disulfide isomerase family member (AGR2) promoter, activation-induced cytidine deaminase (AICDA) promoter, UDP-GlcNAc:beta Gal beta-1,3-N-acetylglucosaminyltransferase 3 (B3GNT3) promoter, cadherin 3 (CDH3) promoter, CEA cell adhesion molecule 5 (CEACAM5) promoter, centromere protein F (CENPF) promoter, centrosomal protein 55 (CEP55) promoter, claudin 3 (CLDN3) promoter, claudin 4 (CLDN4) promoter, type XI collagen alpha 1 chain (COL11A1) promoter, type I collagen alpha 1 chain (COL1A1) promoter, cystatin SN (CST1) promoter, and dentate-less E3 ubiquitin tachykinin (DTT) promoter. Protein ligase homolog (DTL) promoter, family 111 member B with sequence similarity (FAM111B) promoter, forkhead box A1 (FOXA1) promoter, kinesin family member 20A (KIF20A), laminin subunit gamma 2 (LAMC2) promoter, mitotic spindle positioning (MISP) promoter, matrix metallopeptidase 1 (MMP1) promoter, matrix metallopeptidase 12 (MMP12) promoter, and matrix metallopeptidase 13 (MMP13) promoter, mesothelin (MSLN) promoter, cell surface-associated mucin 1 (MUC1) promoter, phospholipase A2 group IID (PLA2G2D) promoter, regulator of G protein signaling 13 (RGS13) promoter, secretoglobin family 2A member 1 (SCGB2A1) promoter, topoisomerase II alpha (TOP2A) promoter, ubiquitin D (UBD) promoter, ubiquitin-conjugating enzyme E2 (U2) promoter,C (UBE2C), USH1 protein network component harmonin (USH1C), V-set domain-containing T cell activation inhibitor 1 (VTCN1) promoter, hexokinase type II promoter, TRPM4 promoter, stromelysin 3 promoter, surfactant protein A promoter, secretory leukocyte protease inhibitor promoter, tyrosinase promoter, stress-inducible grp78 / BiP promoter, interleukin-10 promoter, alpha-B-crystallin / heat shock protein 27 promoter, epidermal growth factor receptor promoter, mucin-like glycoprotein promoter, mts1 promoter, NSE promoter, somatostatin tin receptor promoter, c-erbB-3 promoter, c-erbB-2 promoter, c-erbB4 promoter, thyroglobulin promoter, α-fetoprotein promoter, villin promoter, albumin promoter, glycoprotein A33 promoter, B cell specific Moloney leukemia virus insertion site 1 promoter, cyclooxygenase-2 promoter, fibroblast growth factor promoter; human epidermal growth factor receptor 2, human telomerase reverse transcriptase promoter; kinase domain insertion-containing receptor promoter; rad51 recombinase promoter; TTF-1, urokinase-type plasminogen activator receptor promoter, ubiquitin-conjugating enzyme E2 T (UBE2T) promoter, checkpoint kinase 1 (CHEK1) promoter, epithelial cell transforming 2 promoter (ECT2), BCL2-like 12 (BCL2L12) promoter, centromere protein I (CENPI) promoter, E2F transcription factor 1 (E2F1) promoter, flavin adenine dinucleotide synthetase 1 (FLAD1) promoter, protein phosphatase, Mg2+ / Mn2+-dependent 1G (PPM1G) promoter, ubiquitin-conjugating enzyme E2S (UBE2S) promoter, aurora kinase A and ninein interacting protein (AUNIP) promoter, cell division cycle 6 (CDC6) promoter, centromere protein L (CENPL) promoter, DNA replicative helicase / nuclease 2 (DNA2) promoter, DSN1 homolog, MIS12 kinetochore complex component (DSN1) promoter, deoxythymidylate kinase (DTYMK) promoter, G protein-regulated inducer of neurite outgrowth 1 (GPRIN1) promoter, mitochondrial fission regulator 2 (MTFR2) promoter, RAD51-associated protein 1 (RAD51AP1) promoter, small nuclear ribonucleoprotein polypeptide A' (SNRPA1) promoter, ATPase family, AAA domain containing 2 (ATAD2) promoter, BUB1 mitotic checkpoint serine / threonine kinase (BUB1) promoter, calcyclin-binding protein (CACYBP) promoter, cell division cycle associated 3 (CDCA3) promoter, centromere protein O (CENPO) promoter promoter, flap structure-specific endonuclease 1 (FEN1) promoter, forkhead box M1 (FOXM1) promoter, cell proliferation-regulated inhibitor of protein phosphatase 2A (KIAA1524) promoter, kinesin family member 2C (KIF2C) promoter, karyopherin subunit alpha 2 (KPNA2) promoter, MYB proto-oncogene-like 2 (MYBL2) promoter, NIMA-related kinase 2 (NEK2) promoter, RAN-binding protein 1 (RANBP1) promoter, small nuclear ribonucleoprotein polypeptides B and B1 (SNRPB) promoter, SPC24 / NDC80 kinetochore complex component (SPC24) promoter, transforming acidic coiled-coil-containing protein 3 (TACC3) promoter, TBC1 domain family member 31 (TBC1D31) promoter, thymidine kinase 1 (TK1) promoter, zinc finger protein 695 (ZNF695) promoter, aurora kinase A (AURKA) promoter, BLMRecQ-like helicase (BLM) promoter, chromosome 17 open reading frame 53 (C17orf53) promoter, chromobox 3 (CBX30) promoter, cyclin B1 (CCNB1) promoter, cyclin E1 (CCNE1) promoter, cyclin F (CCNF), cell division cycle 20 (CDC20) promoter, cell division cycle 45 (CDC45) promoter, cell division cycle associated 5 (CDCA5) promoter, cyclin-dependent kinase inhibitor 3 (CDKN3) promoter, cadherin EGF LAG 7-transmembrane G-type receptor 3 (CELSR3) promoter, centromere protein A (CENPA) promoter, centrosomal protein 72 (CEP72) promoter, CDC28 protein kinase regulatory subunit 2 (CKS2) promoter, type X collagen alpha 1 chain (COL10A1) promoter, chromosome segregation 1-like (CSE1L) promoter, DBF4 zinc finger promoter, GINS complex subunit 1 (GINS1) promoter, G protein-coupled receptor 19 (GPR19) promoter, kinesin family member 18A (KIF18A) promoter, kinesin family member 4A (KIF4A) promoter, kinesin family member C1 ( KIFC1) promoter, minichromosome maintenance 10 replication initiator (MCM10) promoter, minichromosome maintenance complex component 2 (MCM2) promoter, minichromosome maintenance complex component 7 (MCM7) promoter, MRG domain binding protein (MRGBP) promoter, methylenetetrahydrofolate dehydrogenase (NADP+-dependent) 2, methenyltetrahydrofolate cyclohydrolase (MTHFD2) promoter, non-SMC condensin I complex subunit H (NCAPH) promoter, kinetochore complex component (NDC80) promoter, nudix hydrolase 1 (NUDT1) promoter, ribonuclease H2 subunit A (RNASEH2A) promoter, RuvB-like AAA (RcA) promoter,132. The nucleic acid of embodiment 130 or 131, which is a ATPase1 (RUVBL1) promoter, a serologically defined breast cancer antigen NY-BR-85 (SGOL1) promoter, a SHC-binding and spindle-associated 1 (SHCBP1) promoter, a small nuclear ribonucleoprotein polypeptide G (SNRPG) promoter, a timeless circadian regulator promoter, a thyroid hormone receptor interactor 13 (TRIP13) promoter, a trophinin-related protein (TROAP) promoter, a ubiquitin-conjugating enzyme E2 C (UBE2C) promoter, a WD-repeat and HMG box DNA-binding protein 1 (WDHD1) promoter, an alpha-fetoprotein (AFP) promoter, a functional fragment thereof, any combination thereof, a chimeric promoter compiled from multiple elements from the foregoing, or a completely synthetic promoter composed of tiled transcription factor binding sites derived from any of the foregoing. 133. A vector comprising a nucleic acid according to any one of embodiments 111 to 132. 134. The vector described in embodiment 133, wherein the vector is a recombinant viral vector. 135. The vector described in embodiment 133, wherein the vector is a non-viral vector. 136. An engineered polypeptide comprising: (a) an extracellular-targeting domain comprising an epitope capable of binding to (i) an antibody or (ii) a peptide hormone or growth factor, wherein the extracellular-targeting domain does not comprise an scFv; (b) an extracellular-directed polypeptide spacer domain having a length of about 15 to about 40 angstroms when folded; and (c) a transmembrane domain or a membrane-affinic domain capable of associating with the outer membrane of a cell Including, At least two of (a), (b), and (c) are heterologous to one another; Engineered Polypeptides 137. The engineered polypeptide of embodiment 136, wherein the polypeptide does not comprise a light chain variable (VL) domain. 138. The engineered polypeptide of embodiment 136 or 137, wherein the engineered polypeptide does not contain an intracellular signaling domain. 139. The engineered polypeptide of any one of embodiments 136 to 138, wherein the engineered polypeptide does not comprise an intracellular portion of a CD3 zeta, CD137, or CD28 polypeptide. 140. The engineered polypeptide of any one of embodiments 136-139, wherein the intracellular targeting portion of the polypeptide comprises less than or equal to 100, 75, 50, 25, 10, or 5 residues. 141. The engineered polypeptide of any one of embodiments 136-140, wherein the extracellular-directed polypeptide spacer domain comprises a hinge domain. 142. The engineered polypeptide of any one of embodiments 136-141, wherein (a), (b), and (c) are in the order from the N-terminus to the C-terminus of the engineered polypeptide. 143. The engineered polypeptide of any one of embodiments 136-142, wherein the engineered polypeptide is capable of being displayed on the cell surface. 144. The engineered polypeptide of any one of embodiments 136 to 143, wherein the epitope further comprises an activatable epitope selectively available for binding in the tumor microenvironment. 145. The engineered polypeptide of embodiment 144, wherein the activatable epitope is flanked by at least two copies of a pH-sensitive helix. 146. The engineered polypeptide according to any one of embodiments 136 to 145, wherein (i) the antibody or (ii) the epitope capable of binding to the peptide hormone or growth factor is derived from DLL3, PSMA, SSTR2, or any combination thereof. 147. The engineered polypeptide of embodiment 146, wherein the epitope is derived from DLL3, and the epitope comprises about 15 to about 260 contiguous residues of the extracellular domain of DLL3 having at least 80% identity to SEQ ID NO:5, or a variant thereof. 148. The engineered polypeptide of embodiment 146 or 147, wherein the epitope comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 4, 5, 19, 20, 64, 65, or a variant thereof. 149. The engineered polypeptide of any one of embodiments 136-148, wherein the epitope is capable of binding to (i) the antibody or (ii) the peptide hormone or growth factor with a Kd of less than or equal to 20 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM. 150. The engineered polypeptide of any one of embodiments 136-149, wherein the extracellular-directed polypeptide spacer domain comprises a hinge sequence from CD8, CD8a, CD8b, IgG4, IgG1, IgG2, IgG3, IgK, CD4, or CD28, or any combination thereof. 151. The engineered polypeptide of embodiment 150, wherein the extracellular-directed polypeptide spacer domain comprises a hinge sequence having at least 80% sequence identity to any one of the hinge sequences of SEQ ID NOs: 56, 57, 58, 59, 60, 61, 62, 66, 67, or a variant thereof. 152. The engineered polypeptide according to any one of embodiments 136 to 151, wherein the transmembrane domain or the membrane affinity domain comprises a transmembrane domain. 153. The engineered polypeptide of embodiment 152, wherein the transmembrane domain comprises a single-spanning transmembrane domain. 154. The engineered polypeptide of embodiment 152 or 153, wherein the transmembrane domain comprises a transmembrane domain derived from DLL3, PSMA, SSTR2, PD-L1, EGFR, CD28, CD4, CD8, CD8a, CD8b, ICOS, or CD73. 155. The engineered polypeptide of any one of embodiments 152-154, wherein the transmembrane domain comprises a sequence having at least 80% sequence identity to the transmembrane domain of any one of SEQ ID NOs: 68-76. 156. The engineered polypeptide according to any one of embodiments 136 to 151, wherein the transmembrane domain or the membraneophilic domain comprises a membraneophilic domain. 157. The engineered polypeptide of embodiment 156, wherein the membrane affinity domain comprises a C1, C2, PH, FYVE, PX, or ENTH domain, or a transmembrane helix derived from bacteriorhodopsin. 158. The engineered polypeptide according to embodiment 156 or 157, wherein the membrane affinity domain comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 77 to 82. 159. The engineered polypeptide of any one of embodiments 136-158, wherein the engineered polypeptide further comprises: (i) a scaffold domain that is N-terminal to the hinge domain and C-terminal to the epitope; or (ii) a scaffold domain that is N-terminal to the hinge domain and contains the epitope. 160. The engineered polypeptide of embodiment 159, wherein the engineered polypeptide further comprises a scaffold domain N-terminal to the hinge domain and C-terminal to the epitope. 161. The engineered polypeptide of any one of embodiments 159-160, wherein the scaffold comprises a sequence having at least 80% sequence identity to SEQ ID NO: 63, or a variant thereof. 162. The engineered polypeptide of embodiment 159 or 161, wherein the engineered polypeptide further comprises a scaffold domain N-terminal to the hinge domain and containing the epitope. 163. The engineered polypeptide of any one of embodiments 159-162, wherein the scaffold comprises a heavy chain variable (VH) domain and no light chain variable (VL) domain. 164. The engineered polypeptide of embodiment 163, wherein the VH domain comprises an inactivating mutation in the CDR1, CDR2, or CDR3 region of the VH domain. 165. The engineered polypeptide of embodiment 163, wherein the scaffold comprises the epitope inserted into the CDR1, CDR2, or CDR3 region of the VH domain. 166. A nucleic acid comprising an ORF encoding a polypeptide or a functional fragment thereof according to any one of embodiments 136 to 165. 167. The nucleic acid of embodiment 166, further comprising a promoter operably linked to the ORF. 168. The nucleic acid of embodiment 167, wherein the promoter is not a T cell specific promoter or a TCRA, TCRB, CMV, EF-1, hPGK, CD3, or RPBSA promoter. 169. The nucleic acid according to any one of embodiments 167 to 168, wherein the promoter is a cancer-specific promoter. 170. The nucleic acid according to any one of embodiments 167 to 169, wherein the promoter is a promoter of a gene that is overexpressed in cancer cells compared to normal cells, or a functional fragment thereof. 171. The promoter is selected from the group consisting of survivin promoter (BIRC5), CXCR4 promoter, ATP-binding cassette subfamily C member 4 (ABCC4) promoter, anterior gradient 2, protein disulfide isomerase family member (AGR2) promoter, activation-induced cytidine deaminase (AICDA) promoter, UDP-GlcNAc:beta Gal beta-1,3-N-acetylglucosaminyltransferase 3 (B3GNT3) promoter, cadherin 3 (CDH3) promoter, CEA cell adhesion molecule 5 (CEACAM5) promoter, centromere protein F (CENPF) promoter, centrosomal protein 55 (CEP55) promoter, claudin 3 (CLDN3) promoter, claudin 4 (CLDN4) promoter, type XI collagen alpha 1 chain (COL11A1) promoter, type I collagen alpha 1 chain (COL1A1) promoter, cystatin SN (CST1) promoter, and dentate-less E3 ubiquitin tachykinin (DTT) promoter. Protein ligase homolog (DTL) promoter, family 111 member B with sequence similarity (FAM111B) promoter, forkhead box A1 (FOXA1) promoter, kinesin family member 20A (KIF20A), laminin subunit gamma 2 (LAMC2) promoter, mitotic spindle positioning (MISP) promoter, matrix metallopeptidase 1 (MMP1) promoter, matrix metallopeptidase 12 (MMP12) promoter, and matrix metallopeptidase 13 (MMP13) promoter, mesothelin (MSLN) promoter, cell surface-associated mucin 1 (MUC1) promoter, phospholipase A2 group IID (PLA2G2D) promoter, regulator of G protein signaling 13 (RGS13) promoter, secretoglobin family 2A member 1 (SCGB2A1) promoter, topoisomerase II alpha (TOP2A) promoter, ubiquitin D (UBD) promoter, ubiquitin-conjugating enzyme E2 (U2) promoter,C (UBE2C), USH1 protein network component harmonin (USH1C), V-set domain-containing T cell activation inhibitor 1 (VTCN1) promoter, hexokinase type II promoter, TRPM4 promoter, stromelysin 3 promoter, surfactant protein A promoter, secretory leukocyte protease inhibitor promoter, tyrosinase promoter, stress-inducible grp78 / BiP promoter, interleukin-10 promoter, alpha-B-crystallin / heat shock protein 27 promoter, epidermal growth factor receptor promoter, mucin-like glycoprotein promoter, mts1 promoter, NSE promoter, somatostatin tin receptor promoter, c-erbB-3 promoter, c-erbB-2 promoter, c-erbB4 promoter, thyroglobulin promoter, α-fetoprotein promoter, villin promoter, albumin promoter, glycoprotein A33 promoter, B cell specific Moloney leukemia virus insertion site 1 promoter, cyclooxygenase-2 promoter, fibroblast growth factor promoter; human epidermal growth factor receptor 2, human telomerase reverse transcriptase promoter; kinase domain insertion-containing receptor promoter; rad51 recombinase promoter; TTF-1, urokinase-type plasminogen activator receptor promoter, ubiquitin-conjugating enzyme E2 T (UBE2T) promoter, checkpoint kinase 1 (CHEK1) promoter, epithelial cell transforming 2 promoter (ECT2), BCL2-like 12 (BCL2L12) promoter, centromere protein I (CENPI) promoter, E2F transcription factor 1 (E2F1) promoter, flavin adenine dinucleotide synthetase 1 (FLAD1) promoter, protein phosphatase, Mg2+ / Mn2+-dependent 1G (PPM1G) promoter, ubiquitin-conjugating enzyme E2S (UBE2S) promoter, aurora kinase A and ninein interacting protein (AUNIP) promoter, cell division cycle 6 (CDC6) promoter, centromere protein L (CENPL) promoter, DNA replicative helicase / nuclease 2 (DNA2) promoter, DSN1 homolog, MIS12 kinetochore complex component (DSN1) promoter, deoxythymidylate kinase (DTYMK) promoter, G protein-regulated inducer of neurite outgrowth 1 (GPRIN1) promoter, mitochondrial fission regulator 2 (MTFR2) promoter, RAD51-associated protein 1 (RAD51AP1) promoter, small nuclear ribonucleoprotein polypeptide A' (SNRPA1) promoter, ATPase family, AAA domain containing 2 (ATAD2) promoter, BUB1 mitotic checkpoint serine / threonine kinase (BUB1) promoter, calcyclin-binding protein (CACYBP) promoter, cell division cycle associated 3 (CDCA3) promoter, centromere protein O (CENPO) promoter promoter, flap structure-specific endonuclease 1 (FEN1) promoter, forkhead box M1 (FOXM1) promoter, cell proliferation-regulated inhibitor of protein phosphatase 2A (KIAA1524) promoter, kinesin family member 2C (KIF2C) promoter, karyopherin subunit alpha 2 (KPNA2) promoter, MYB proto-oncogene-like 2 (MYBL2) promoter, NIMA-related kinase 2 (NEK2) promoter, RAN-binding protein 1 (RANBP1) promoter, small nuclear ribonucleoprotein polypeptides B and B1 (SNRPB) promoter, SPC24 / NDC80 kinetochore complex component (SPC24) promoter, transforming acidic coiled-coil-containing protein 3 (TACC3) promoter, TBC1 domain family member 31 (TBC1D31) promoter, thymidine kinase 1 (TK1) promoter, zinc finger protein 695 (ZNF695) promoter, aurora kinase A (AURKA) promoter, BLMRecQ-like helicase (BLM) promoter, chromosome 17 open reading frame 53 (C17orf53) promoter, chromobox 3 (CBX30) promoter, cyclin B1 (CCNB1) promoter, cyclin E1 (CCNE1) promoter, cyclin F (CCNF), cell division cycle 20 (CDC20) promoter, cell division cycle 45 (CDC45) promoter, cell division cycle associated 5 (CDCA5) promoter, cyclin-dependent kinase inhibitor 3 (CDKN3) promoter, cadherin EGF LAG 7-transmembrane G-type receptor 3 (CELSR3) promoter, centromere protein A (CENPA) promoter, centrosomal protein 72 (CEP72) promoter, CDC28 protein kinase regulatory subunit 2 (CKS2) promoter, type X collagen alpha 1 chain (COL10A1) promoter, chromosome segregation 1-like (CSE1L) promoter, DBF4 zinc finger promoter, GINS complex subunit 1 (GINS1) promoter, G protein-coupled receptor 19 (GPR19) promoter, kinesin family member 18A (KIF18A) promoter, kinesin family member 4A (KIF4A) promoter, kinesin family member C1 ( KIFC1) promoter, minichromosome maintenance 10 replication initiator (MCM10) promoter, minichromosome maintenance complex component 2 (MCM2) promoter, minichromosome maintenance complex component 7 (MCM7) promoter, MRG domain binding protein (MRGBP) promoter, methylenetetrahydrofolate dehydrogenase (NADP+-dependent) 2, methenyltetrahydrofolate cyclohydrolase (MTHFD2) promoter, non-SMC condensin I complex subunit H (NCAPH) promoter, kinetochore complex component (NDC80) promoter, nudix hydrolase 1 (NUDT1) promoter, ribonuclease H2 subunit A (RNASEH2A) promoter, RuvB-like AAA (RcA) promoter,The nucleic acid of any one of embodiments 167-170, comprising a ATPase1 (RUVBL1) promoter, a serologically defined breast cancer antigen NY-BR-85 (SGOL1) promoter, a SHC-binding and spindle-associated 1 (SHCBP1) promoter, a small nuclear ribonucleoprotein polypeptide G (SNRPG) promoter, a timeless circadian regulator promoter, a thyroid hormone receptor interactor 13 (TRIP13) promoter, a trophinin-related protein (TROAP) promoter, a ubiquitin-conjugating enzyme E2 C (UBE2C) promoter, a WD-repeat and HMG box DNA-binding protein 1 (WDHD1) promoter, an alpha-fetoprotein (AFP) promoter, a functional fragment thereof, any combination thereof, a chimeric promoter compiled from a plurality of elements from the foregoing, or a completely synthetic promoter composed of tile-like transcription factor binding sites from any of the foregoing. 172. A vector comprising a nucleic acid according to any one of embodiments 166 to 171. 173. The vector described in embodiment 172, wherein the vector is a recombinant viral vector. 174. The vector described in embodiment 172, wherein the vector is a non-viral vector. 175. A method for detecting, imaging, or treating cancer cells, comprising: (a) administering to a subject a composition comprising a nucleic acid according to any one of embodiments 166 to 171 or a vector according to any one of embodiments 172 to 174; (b) administering to the subject an antibody or antigen-binding fragment thereof, a protein ligand or functional fragment thereof, or a small molecule configured to bind to the epitope. A method comprising: 176. The method of embodiment 175, wherein the steps of administering to the subject the composition comprising the nucleic acid in (a) and administering to the subject the antibody or antigen-binding fragment thereof in (b) are separated by at least about 8, 12, 16, 24, 36, 48, 60, 72, 84, or 96 hours. 177. The method of embodiment 175, wherein the composition, or the antibody or antigen-binding fragment thereof, protein ligand or functional fragment thereof, or small molecule configured to bind to the epitope, is administered to the subject by parenteral, intramuscular, subcutaneous, intratumoral, rectal, vaginal, transdermal, or intravenous administration, or by cannula. 178. The method of any one of embodiments 175 to 177, wherein the composition comprising the nucleic acid or the vector is configured for intravenous administration. 179. The method of any one of embodiments 175 to 178, further comprising detecting binding of the antibody or antigen-binding fragment, protein ligand or functional fragment thereof, or small molecule to the epitope. 180. The method of any one of embodiments 175-179, wherein the antibody or antigen-binding fragment thereof further comprises or is chelated to a radioisotope or a magnetic resonance imaging (MRI) contrast agent. 181. The method of embodiment 180, wherein the antibody or antigen-binding fragment thereof further comprises a radioisotope or is chelated to a radioisotope. 182. The method of any one of embodiments 180-181, wherein the radioisotope comprises a positron-emitting radioisotope, an alpha-emitting radioisotope, a beta-emitting radioisotope, or a gamma-emitting radioisotope. 183. The radioisotope is a positron-emitting radioisotope; 124 I, 68 Ga, 11 C. 13 N, 15 O. 18 F, 68 Ga, 64 Cu, 52 Mn, 55 Co, 89 Zr, 82 The method of any one of embodiments 180-182, comprising administering to a patient a compound comprising: Rb, Rb, or any combination thereof. 184. The radioisotope comprises an alpha-emitting radioisotope;225 Ac, 211 At, 227 Th, 224 The method of any one of embodiments 180 to 182, comprising: 185. The radioisotope comprises a beta-emitting radioisotope; 177 Lu, 67 Cu, 131 I, 90 Y, 89 Sr, 186 Re, 165 Dy, 32 P, 166 Ho, 188 The method of any one of embodiments 180 to 182, comprising: 186. The radioisotope is a gamma-ray emitting radioisotope; 99m Tc, 123 I, or 131 The method of any one of embodiments 180 to 182, comprising I. 187. The method of embodiment 180, wherein the antibody or antigen-binding fragment thereof further comprises an MRI contrast agent or is chelated to an MRI contrast agent. 188. The method of embodiment 187, wherein the MRI contrast agent comprises iron oxide nanoparticles (IONPs), superparamagnetic iron platinum nanoparticles, manganese(II), or gadolinium(III). 189. The method of any one of embodiments 175-179, wherein the antibody or antigen-binding fragment thereof further comprises an antibody drug conjugate (ADC). 190. The method of embodiment 189, wherein the antibody drug conjugate is conjugated to a pyrrolobenzodiazepine (PBD), a protein toxin, a diphtheria toxin, a glucagon-like peptide (GLP-1), a cytotoxic immunomodulatory protein, a Fas ligand, an auristatin or an analog thereof, a maytansinoid, a calicheamicin, a duocarmycin or an analog thereof, or a doxorubicin or an analog thereof. 191. The method of any one of embodiments 179 to 190, further comprising detecting the binding of the antibody or antigen-binding fragment, protein ligand or functional fragment thereof, or small molecule to the epitope by MRI imaging, PET imaging, SPECT imaging, photoacoustic imaging, or luminescence imaging performed on the subject. 192. The m...

Claims

1. A nucleic acid encoding an engineered polypeptide, said polypeptide comprising: (a) an extracellular-targeting domain comprising an epitope capable of binding (i) an antibody or (ii) a peptide hormone or growth factor, wherein the extracellular-targeting domain does not comprise an scFv; (b) an extracellular-directing polypeptide spacer domain having a length, when folded, of about 15 to about 40 angstroms; and (c) a transmembrane domain or a membrane-affinic domain capable of associating with the outer membrane of a cell. Including, At least two of (a), (b), and (c) are heterologous to one another; Nucleic acid.

2. The nucleic acid of claim 1 , wherein the polypeptide does not include a variable light (VL) domain.

3. The nucleic acid of claim 1 or 2, wherein the engineered polypeptide does not contain an intracellular signaling domain.

4. The nucleic acid of any one of claims 1 to 3, wherein the engineered polypeptide does not comprise the intracellular portion of a CD3 zeta, CD137, or CD28 polypeptide.

5. The nucleic acid of any one of claims 1 to 4, wherein the intracellular targeting portion of the polypeptide comprises less than or equal to 100, 75, 50, 25, 10, or 5 residues.

6. The nucleic acid according to any one of claims 1 to 5, wherein the extracellular-directed polypeptide spacer domain comprises a hinge domain.

7. 7. The nucleic acid of any one of claims 1 to 6, wherein (a), (b), and (c) are in the order from the N-terminus to the C-terminus of the engineered polypeptide.

8. The nucleic acid of any one of claims 1 to 7, wherein the engineered polypeptide is capable of being displayed on the cell surface.

9. The nucleic acid of any one of claims 1 to 8, wherein the epitope further comprises an activatable epitope selectively available for binding to an extracellular ligand in the tumor microenvironment.

10. 10. The nucleic acid of claim 9, wherein the activatable epitope is flanked by at least two copies of a pH-sensitive helix.

11. 11. The nucleic acid of any one of claims 1 to 10, wherein the epitope capable of binding (i) the antibody or (ii) the peptide hormone or growth factor is derived from DLL3, PSMA, SSTR2, or any combination thereof.

12. 12. The nucleic acid of claim 11, wherein the epitope is derived from DLL3, and the epitope comprises from about 15 to about 260 contiguous residues of the extracellular domain of DLL3 having at least 80% identity to SEQ ID NO:5, or a variant thereof.

13. 13. The nucleic acid of claim 11 or 12, wherein the epitope comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 4, 5, 19, 20, 64, 65, or a variant thereof.

14. 14. The nucleic acid of any one of claims 1 to 13, wherein the epitope is capable of binding to (i) the antibody or (ii) the peptide hormone or growth factor with a Kd of less than or equal to 20 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM.

15. The nucleic acid of any one of claims 1 to 14, wherein the extracellular-directed polypeptide spacer domain comprises a hinge sequence from CD8, CD8a, CD8b, IgG4, IgG1, IgG2, IgG3, IgK, CD4, or CD28, or any combination thereof.

16. The nucleic acid of claim 15, wherein the extracellular-directed polypeptide spacer domain comprises a hinge sequence having at least 80% sequence identity to any one of the hinge sequences of SEQ ID NOs: 56, 57, 58, 59, 60, 61, 62, 66, 67, or a variant thereof.

17. The nucleic acid according to any one of claims 1 to 16, wherein the transmembrane domain or the membrane-affinic domain comprises a transmembrane domain.

18. The nucleic acid of claim 17 , wherein the transmembrane domain comprises a single-spanning transmembrane domain.

19. 19. The nucleic acid of claim 17 or 18, wherein the transmembrane domain comprises a transmembrane domain derived from DLL3, PSMA, SSTR2, PD-L1, EGFR, CD28, CD4, CD8, CD8a, CD8b, ICOS, or CD73.

20. The nucleic acid according to any one of claims 17 to 19, wherein the transmembrane domain comprises a sequence having at least 80% sequence identity with the transmembrane domain of any one of SEQ ID NOs: 68 to 76.

21. The nucleic acid according to any one of claims 1 to 20, wherein the transmembrane domain or the membrane-affinic domain comprises a membrane-affinic domain.

22. 22. The nucleic acid of claim 21, wherein the membrane affinity domain comprises a C1, C2, PH, FYVE, PX, or ENTH domain, or a transmembrane helix derived from bacteriorhodopsin.

23. The nucleic acid according to claim 21 or 22, wherein the membrane affinity domain comprises a sequence having at least 80% sequence identity with any one of SEQ ID NOs: 77 to 82.

24. 24. The nucleic acid of any one of claims 1-23, wherein the engineered polypeptide further comprises: (i) a scaffold domain that is N-terminal to the hinge domain and C-terminal to the epitope; or (ii) a scaffold domain that is N-terminal to the hinge domain and contains the epitope.

25. 25. The nucleic acid of claim 24, wherein the engineered polypeptide further comprises a scaffold domain N-terminal to the hinge domain and C-terminal to the epitope.

26. The nucleic acid of any one of claims 24 to 25, wherein the scaffold comprises a sequence having at least 80% sequence identity to SEQ ID NO: 63, or a variant thereof.

27. 27. The nucleic acid of claim 24 or 26, wherein the engineered polypeptide further comprises a scaffold domain N-terminal to the hinge domain and containing the epitope.

28. 28. The nucleic acid of any one of claims 24 to 27, wherein the scaffold comprises a heavy chain variable (VH) domain and no light chain variable (VL) domain.

29. The nucleic acid of claim 28, wherein the VH domain comprises an inactivating mutation in the CDR1, CDR2, or CDR3 region of the VH domain.

30. 29. The nucleic acid of claim 28, wherein the scaffold comprises the epitope inserted into the CDR1, CDR2, or CDR3 region of the VH domain.

31. 31. The nucleic acid of any one of claims 1 to 30, further comprising an ORF encoding the engineered polypeptide.

32. 32. The nucleic acid of claim 31, further comprising a promoter operably linked to the ORF.

33. 33. The nucleic acid of claim 32, wherein the promoter is not a T cell specific promoter or a TCRA, TCRB, CMV, EF-1, hPGK, CD3, or RPBSA promoter.

34. The nucleic acid according to any one of claims 32 to 33, wherein the promoter is a cancer-specific promoter.

35. The nucleic acid according to any one of claims 32 to 34, wherein the promoter is a promoter of a gene that is overexpressed in cancer cells compared to normal cells, or a functional fragment thereof.

36. The promoter is selected from the group consisting of survivin promoter (BIRC5), CXCR4 promoter, ATP-binding cassette subfamily C member 4 (ABCC4) promoter, anterior gradient 2, protein disulfide isomerase family member (AGR2) promoter, activation-induced cytidine deaminase (AICDA) promoter, UDP-GlcNAc:beta Gal beta-1,3-N-acetylglucosaminyltransferase 3 (B3GNT3) promoter, cadherin 3 (CDH3) promoter, CEA cell adhesion molecule 5 (CEACAM5) promoter, centromere protein F (CENPF) promoter, centrosomal protein 55 (CEP55) promoter, claudin 3 (CLDN3) promoter, claudin 4 (CLDN4) promoter, type XI collagen alpha 1 chain (COL11A1) promoter, type I collagen alpha 1 chain (COL1A1) promoter, cystatin SN (CST1) promoter, dentate-less E3 ubiquitin protein Deoxyribonuclease ligase homolog (DTL) promoter, family 111 member B with sequence similarity (FAM111B) promoter, forkhead box A1 (FOXA1) promoter, kinesin family member 20A (KIF20A), laminin subunit gamma 2 (LAMC2) promoter, mitotic spindle positioning (MISP) promoter, matrix metallopeptidase 1 (MMP1) promoter, matrix metallopeptidase 12 (MMP12) promoter, and matrix metallopeptidase 13 (MMP14) promoter. metallopeptidase 13 (MMP13) promoter, mesothelin (MSLN) promoter, cell surface-associated mucin 1 (MUC1) promoter, phospholipase A2 group IID (PLA2G2D) promoter, regulator of G protein signaling 13 (RGS13) promoter, secretoglobin family 2A member 1 (SCGB2A1) promoter, topoisomerase II alpha (TOP2A) promoter, ubiquitin D (UBD) promoter, ubiquitin-conjugating enzyme E2C (UBE2C), USH1 protein network component harmonin (USH1C), V-set domain-containing T cell activation inhibitor 1 (VTCN1) promoter, hexokinase type II promoter, TRPM4 promoter, stromelysin 3 promoter, surfactant protein A promoter, secretory leukocyte protease inhibitor promoter, tyrosinase promoter, stress-inducible grp78 / BiP promoter, interleukin-10 promoter, alpha-B-crystallin / heat shock protein 27 promoter, epidermal growth factor receptor promoter, mucin-like glycoprotein promoter, mts1 promoter, NSE promoter, somatostatin tin receptor promoter, c-erbB-3 promoter, c-erbB-2 promoter, c-erbB4 promoter, thyroglobulin promoter, α-fetoprotein promoter, villin promoter, albumin promoter, glycoprotein A33 promoter, B cell specific Moloney leukemia virus insertion site 1 promoter, cyclooxygenase-2 promoter, fibroblast growth factor promoter; human epidermal growth factor receptor 2, human telomerase reverse transcriptase promoter; kinase domain insertion containing receptor promoter; rad51 recombinase promoter; TTF-1, urokinase type plasminogen activator receptor promoter, ubiquitin conjugating enzyme E2 T (UBE2T) promoter, checkpoint kinase 1 (CHEK1) promoter, epithelial cell transforming 2 promoter (ECT2), BCL2-like 12 (BCL2L12) promoter, centromere protein I (CENPI) promoter, E2F transcription factor 1 (E2F1) promoter, flavin adenine dinucleotide synthetase 1 (FLAD1) promoter, protein phosphatase, Mg2+ / Mn2+-dependent 1G (PPM1G) promoter, ubiquitin-conjugating enzyme E2S (UBE2S) promoter, aurora kinase A and ninein interacting protein (AUNIP) promoter, cell division cycle 6 (CDC6) promoter, centromere protein L (CENPL) promoter, DNA replicative helicase / nuclease 2 (DNA2) promoter, DSN1 homolog, MIS12 kinetochore complex component (DSN1) promoter, deoxythymidylate kinase (DTYMK) promoter, G protein-regulated inducer of neurite outgrowth 1 (GPRIN1) promoter, mitochondrial Mitosis Regulatory Factor 2 (MTFR2) promoter, RAD51-associated protein 1 (RAD51AP1) promoter, small nuclear ribonucleoprotein polypeptide A' (SNRPA1) promoter, ATPase family, AAA domain-containing 2 (ATAD2) promoter, BUB1 mitotic checkpoint serine / threonine kinase (BUB1) promoter, calcyclin-binding protein (CACYBP) promoter, cell division cycle-associated 3 (CDCA3) promoter, centromere protein O (CENPO) promoter, flap structure-specific endonuclease 1 (FEN1) promoter, forkhead box M1 (FOXM1) promoter, cell proliferation-regulated inhibitor of protein phosphatase 2A (KIAA1524) promoter, kinesin family member 2C (KIF2C) promoter, karyopherin subunit alpha 2 (KPNA2) promoter, MYB proto-oncogene-like 2 (MYBL2) promoter, NIMA-related kinase 2 (NEK2) promoter, RAN-binding protein 1 (RAN BP1) promoter, small nuclear ribonucleoprotein polypeptide B and B1 (SNRPB) promoter, SPC24 / NDC80 kinetochore complex component (SPC24) promoter, transforming acidic coiled-coil-containing protein 3 (TACC3) promoter, TBC1 domain family member 31 (TBC1D31) promoter, thymidine kinase 1 (TK1) promoter, zinc finger protein 695 (ZNF695) promoter, aurora kinase A (AURKA) promoter, BLMRecQ-like helicase (BLM) promoter, chromosome 17 open reading frame 53 (C17orf53) promoter, chromobox 3 (CBX30) promoter, cyclin B1 (CCNB1) promoter, cyclin E1 (CCNE1) promoter, cyclin F (CCNF), cell division cycle 20 (CDC20) promoter, cell division cycle 45 (CDC45) promoter, cell division cycle associated 5 (CDCA5) promoter, cyclin-dependent kinase inhibitor 3 (CDKN3) promoter, cadherin EGF LAG 7-transmembrane G-type receptor 3 (CELSR3) promoter, centromere protein A (CENPA) promoter, centrosomal protein 72 (CEP72) promoter, CDC28 protein kinase regulatory subunit 2 (CKS2) promoter, type X collagen alpha 1 chain (COL10A1) promoter, chromosome segregation 1-like (CSE1L) promoter, DBF4 zinc finger promoter, GINS complex subunit 1 (GINS1) promoter, G protein-coupled receptor 19 (GPR19) promoter, kinesin family member 18A (KIF18A) promoter, kinesin family member 4A (KIF4A) promoter, kinesin family member C1 ( KIFC1) promoter, minichromosome maintenance 10 replication initiator (MCM10) promoter, minichromosome maintenance complex component 2 (MCM2) promoter, minichromosome maintenance complex component 7 (MCM7) promoter, MRG domain binding protein (MRGBP) promoter, methylenetetrahydrofolate dehydrogenase (NADP+-dependent) 2, methenyltetrahydrofolate cyclohydrolase (MTHFD2) promoter, non-SMC condensin I complex subunit H (NCAPH) promoter, kinetochore complex component (NDC80) promoter, nudix hydrolase 1 (NUDT1) promoter, ribonuclease H2 subunit A (RNASEH2A) promoter, RuvB-like AAA36. The nucleic acid of any one of claims 32-35, comprising an ATPase 1 (RUVBL1) promoter, a serologically defined breast cancer antigen NY-BR-85 (SGOL1) promoter, a SHC-binding and spindle associated 1 (SHCBP1) promoter, a small nuclear ribonucleoprotein polypeptide G (SNRPG) promoter, a timeless circadian regulator promoter, a thyroid hormone receptor interactor 13 (TRIP13) promoter, a trophinin-related protein (TROAP) promoter, a ubiquitin-conjugating enzyme E2 C (UBE2C) promoter, a WD repeat and HMG box DNA-binding protein 1 (WDHD1) promoter, an alpha-fetoprotein (AFP) promoter, a functional fragment thereof, any combination thereof, a chimeric promoter compiled from multiple elements from the foregoing, or a completely synthetic promoter composed of tiled transcription factor binding sites from any of the foregoing.

37. A vector comprising the nucleic acid according to any one of claims 1 to 36.

38. The vector of claim 37 , wherein the vector is a recombinant viral vector.

39. The vector of claim 37 , wherein the vector is a non-viral vector.

40. 1. A method of detecting, imaging, or treating cancer cells, comprising: (a) administering to a subject a composition comprising a nucleic acid according to any one of claims 1 to 36 or a vector according to any one of claims 37 to 39; (b) administering to the subject an antibody or antigen-binding fragment thereof, a protein ligand or functional fragment thereof, or a small molecule configured to bind to the epitope. A method comprising:

41. 41. The method of claim 40, wherein the steps of administering the composition comprising the nucleic acid in (a) to the subject and administering the antibody or antigen-binding fragment thereof in (b) to the subject are at least about 8, 12, 16, 24, 36, 48, 60, 72, 84, or 96 hours apart.

42. The method of claim 40 or 41, wherein the composition, or the antibody or antigen-binding fragment thereof, protein ligand or functional fragment thereof, or small molecule configured to bind to the epitope, is administered to the subject by parenteral, intramuscular, subcutaneous, intratumoral, rectal, vaginal, transdermal, or intravenous administration, or by cannula.

43. The method of any one of claims 40 to 42, wherein the composition comprising the nucleic acid or vector is adapted for intravenous administration.

44. The method of any one of claims 40 to 43, further comprising detecting binding of said antibody or antigen-binding fragment, protein ligand or functional fragment thereof, or small molecule to said epitope.

45. The method of any one of claims 40 to 44, wherein the antibody or antigen-binding fragment thereof further comprises or is chelated to a radioisotope or a magnetic resonance imaging (MRI) contrast agent.

46. 46. ​​The method of claim 45, wherein the antibody or antigen-binding fragment thereof further comprises a radioisotope or is chelated to a radioisotope.

47. 47. The method of any one of claims 40 to 46, wherein the radioisotope comprises a positron emitting radioisotope, an alpha emitting radioisotope, a beta emitting radioisotope, or a gamma emitting radioisotope.

48. the radioisotope is a positron-emitting radioisotope, １２４ I, ６８ G.A. １１ C. １３ N. １５ O. １８ F. ６８ G.A. ６４ Cu, ５２ Mn, ５５ Co, ８９ Zr, ８２ 48. The method of any one of claims 40 to 47, comprising Rb, Rb, or any combination thereof.

49. the radioisotope comprises an alpha-emitting radioisotope; ２２５ A c, ２１１ At, ２２７ Th, ２２４ The method of any one of claims 40 to 47, comprising:

50. the radioisotope comprises a beta-emitting radioisotope; １７７ Lu, ６７ Cu, １３１ I, ９０ Y. ８９ Sr, １８６ Re, １６５ Dy, ３２ P. １６６ Ho, １８８ 48. The method of any one of claims 40 to 47, comprising:

51. the radioisotope is a gamma-ray emitting radioisotope; ９９ｍ Tc, １２３ I, or １３１ The method of any one of claims 40 to 47, comprising I.

52. 45. The method of claim 44, wherein the antibody or antigen-binding fragment thereof further comprises an MRI contrast agent or is chelated to an MRI contrast agent.

53. 53. The method of claim 52, wherein the MRI contrast agent comprises iron oxide nanoparticles (IONPs), superparamagnetic iron platinum nanoparticles, manganese (II), or gadolinium (III).

54. The method of any one of claims 40 to 44, wherein the antibody or antigen-binding fragment thereof further comprises an antibody drug conjugate (ADC).

55. 55. The method of claim 54, wherein the antibody drug conjugate is conjugated to a pyrrolobenzodiazepine (PBD), a protein toxin, diphtheria toxin, glucagon-like peptide (GLP-1), a cytotoxic immunomodulatory protein, Fas ligand, an auristatin or analog thereof, a maytansinoid, a calicheamicin, a duocarmycin or analog thereof, or doxorubicin or analog thereof.

56. 56. The method of any one of claims 40 to 55, further comprising detecting the binding of the antibody or antigen-binding fragment, protein ligand or functional fragment thereof, or small molecule to the epitope by MRI imaging, PET imaging, SPECT imaging, photoacoustic imaging, or luminescence imaging performed on the subject.

57. 57. The method of claim 56, wherein the epitope is presented in tumor cells of the subject.

58. 58. The method of claim 57, wherein the tumor cell is a liver, ovarian, pancreatic, breast, lung, smooth muscle, bladder, kidney, skin, prostate, or bone tumor cell.

59. A cell comprising a nucleic acid according to any one of claims 1 to 36 or a vector according to any one of claims 37 to 39.

60. A pharma- ceutically acceptable composition comprising a nucleic acid according to any one of claims 1 to 36 or a vector according to any one of claims 37 to 39, and a pharma- ceutically acceptable carrier.

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