Methods of localizing and / or treating diseased cells using surface-expressible activatable epitopes

Abstract

The present application relates to engineered polypeptides wherein the engineered polypeptides comprise an extracellular targeting domain comprising 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 detecting, imaging, and treating cancer are also described.

Description

[0001] Cross-references

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 338,435, filed on May 4, 2022, entitled “METHODS USING SURFACE-EXPRESSABLE ACTIVATABLE EPITOPES TO IMAGE AND / OR TREAT DISEASECELLS,” and U.S. Provisional Application No. 63 / 429,783, filed on December 2, 2022, entitled “METHODS USING SURFACE-EXPRESSABLE ACTIVATABLE EPITOPES TO IMAGE AND / OR TREAT DISEASE CELLS,” each of which is incorporated herein by reference in its entirety. Background Art

[0003] Imaging modalities such as immunoPET allow the specificity of antibodies and protein ligands to be combined with the sensitivity of PET imaging.

[0004] Additionally, affinity-based agents, antibodies, and protein ligands that have been conjugated to radionuclide moieties, toxins, and peptide fragments can be used to induce cell damage and lead to cell death, while PET imaging is performed using antibodies and protein ligands. Summary of the invention

[0005] The combination of immunoPET (which uses affinity-based agents) with affinity-based agents conjugated with detectable or therapeutic moieties (radionuclide moieties, toxins, and peptide fragments for inducing cell damage), and inducing cancer-specific expression of markers targeted by affinity-based agents, can provide a convenient new way to detect, image, or treat cancer in situ. Described herein are methods, systems, and compositions for enhancing affinity-based cancer targeting for detection or therapy.

[0006] In some aspects, the present disclosure provides a nucleic acid encoding an engineered polypeptide, comprising: (a) an extracellular directional domain comprising an epitope capable of binding to: (i) an antibody or (ii) a peptide hormone or growth factor, wherein the extracellular directional domain does not comprise an scFv; (b) an extracellular directional polypeptide spacer domain having a length of about 15 to about 40 angstroms when folded; and (c) a transmembrane domain or a membrane affinity domain capable of associating with an extracellular membrane, 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 CD3ζ, CD137 or CD28 polypeptide. In some embodiments, the intracellular directional portion of the polypeptide comprises less than or equal to 100, 75, 50, 25, 10 or 5 residues. In some embodiments, the extracellular directional polypeptide spacer domain comprises a hinge domain. In some embodiments, (a), (b) and (c) are arranged sequentially from the N-terminus to the C-terminus of the engineered polypeptide. In some embodiments, the engineered polypeptide is capable of being displayed on the surface of a cell. In some embodiments, wherein the epitope further comprises an activatable epitope that can be selectively used to bind to an extracellular ligand in a 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 to (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, wherein the epitope comprises about 15 to about 260 consecutive residues of an extracellular domain of DLL3 having at least 80% identity to the extracellular domain of SEQ ID NO: 5 or any protein 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 NO: 4, 5, 19, 20, 64, 65, or a variant thereof. In some embodiments, the epitope is capable of binding 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 directed 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 that is identical to SEQ ID NO: 56, 57, 58, 59, 60, 61, 62, 66,In some embodiments, the transmembrane 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 derived 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 with a transmembrane domain of any one of SEQ ID NOs: 68-76 or a transmembrane domain of any protein 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 derived 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 to a membrane affinity domain of any protein described herein, or a variant thereof. In some embodiments, the engineered polypeptide further comprises the following scaffold domain: (i) located at the N-terminus of the hinge domain and the C-terminus of the epitope; or (ii) located at the N-terminus of the hinge domain and containing the epitope. In some embodiments, the engineered polypeptide further comprises a scaffold domain located at the N-terminus of the hinge domain and the C-terminus of the epitope. In some embodiments, wherein the scaffold comprises a sequence having at least 80% sequence identity to SEQ ID NO: 63 or a variant thereof. In some embodiments, wherein the engineered polypeptide further comprises a scaffold domain located at the N-terminus of 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 includes 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 an 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 relative to normal cells, or a functional fragment thereof. In some embodiments, the promoter includes a survival protein promoter (BIRC5), a CXCR4 promoter, an ATP binding cassette subfamily C member 4 (ABCC4) promoter, a front gradient 2,Protein disulfide isomerase family member (AGR2) promoter, activation-induced cytidine deaminase (AICDA) promoter, UDP-GlcNAc:βGalβ-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, tight junction protein (claudin) 3 (CLDN3) promoter, tight junction protein 4 (CLDN4) promoter, type XI collagen α1 chain (COL11A1) promoter, type I collagen α1 chain (C OL1A1) promoter, cystatin SN (CST1) promoter, denticleless E3 ubiquitin protein ligase homolog (DTL) promoter, sequence similarity family 111 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 (M UC1) promoter, phospholipase A2 group IID (PLA2G2D) promoter, G protein signaling regulator 13 (RGS13) promoter, secretory globin family 2A member 1 (SCGB2A1) promoter, topoisomerase IIα (TOP2A) promoter, ubiquitin D (UBD) promoter, ubiquitin conjugating enzyme E2C (UBE2C), USH1 protein network component coordinating protein (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, α-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; receptor promoter containing a kinase domain insertion; 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 transformation 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 E2 S (UBE2S) promoter, Aurora kinase A and ninein interacting protein (AUNIP) promoter, cell division cycle 6 (CDC6) promoter, centromere protein L (CENPL) promoter, DNA replication helicase / nuclease 2 (DNA2) promoter, DSN1 homolog, MIS12 kinetochore complex component (DSN1) promoter, deoxythymidylate kinase (DTYMK) promoter, G protein-regulated neurite outgrowth inducing factor 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, calcium cyclin binding protein (CACYBP) promoter, cell division cycle related 3 (CDCA3) promoter, Centromere protein O (CENPO) promoter, flap-like structure-specific endonuclease 1 (FEN1) promoter, forkhead box M1 (FOXM1) promoter, cell proliferation-regulated protein phosphatase 2A inhibitor (KIAA1524) promoter, kinesin family member 2C (KIF2C) promoter, nuclear transporter subunit α2 (KPNA2) promoter, MYB proto-oncogene-like 2 (MYBL2) promoter, NIMA-associated kinase 2 (NEK2) promoter, RAN junction promoter, RANBP1 promoter, small nuclear ribonucleoprotein polypeptide B and B1 (SNRPB) promoter, SPC24 / NDC80 kinetochore complex component (SPC24) promoter, transforming acidic coiled-coil 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, BLM RecQ-like helicase (BLM) promoter, chromosome 17 open reading frame 53 (C17orf53) promoter, chromosome cassette protein 3 (CBX30) promoter, cell cycle protein 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 seven-way 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 Kinetochore, minichromosome maintenance 10 replication initiation factor (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, methylenetetrahydrofolate 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 ATPase 1 (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 rhythm regulator promoter, thyroid hormone receptor interacting factor 13 (TRIP13) promoter, trophinin-associated 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 assembled from multiple elements from the above items, or fully synthetic promoters composed of tiled transcription factor binding sites derived from any of the above items.

[0007] In some aspects, the disclosure provides a vector comprising any nucleic acid described herein. In some embodiments, the vector is a recombinant viral vector. In some embodiments, the vector is a non-viral vector.

[0008] In some aspects, the present disclosure provides a method for detecting, imaging or treating cancer cells, comprising: (a) administering to a subject a composition comprising any nucleic acid or vector described herein; and (b) administering to a subject an antibody or its antigen-binding fragment, a protein ligand or its functional fragment, or a small molecule configured to bind an epitope. In some embodiments, the administration of the composition comprising the nucleic acid in (a) to a subject and the administration of the antibody or its antigen-binding fragment in (b) to a subject are spaced at least about 8, 12, 16, 24, 36, 48, 60, 72, 84 or 96 hours. In some embodiments, the composition or antibody or its antigen-binding fragment, protein ligand or its functional fragment, or a small molecule configured to bind an epitope is administered parenterally, intramuscularly, subcutaneously, intratumorally, rectally, vaginally, transdermally or intravenously or administered to a subject via a cannula. In some embodiments, the composition comprising a nucleic acid or a vector is configured for intravenous administration. In some embodiments, the method further comprises detecting the binding of an antibody or its antigen-binding fragment, a protein ligand or its functional fragment, or a small molecule to an epitope. In some embodiments, the antibody or antigen-binding fragment thereof further comprises a radioisotope or contrast agent (e.g., MRI or X-ray contrast agent), or is chelated to a radioisotope or contrast agent. In some embodiments, the antibody or antigen-binding fragment thereof further comprises a radioisotope 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 and comprises 124 I. 68 Ga 11 C. 13 N. 15 O. 18 F. 68 Ga, 64 Cu, 52 Mn, 55 Co. 89 Zr, 82 Rb or any combination thereof. In some embodiments, the radioisotope comprises an alpha-emitting radioisotope and comprises 225 Ac, 211 At 227 Th, 224 Ra or any combination thereof. In some embodiments, the radioisotope comprises a beta-emitting radioisotope and comprises 177 Lu, 67 Cu, 131 I. 90 Y. 89 Sr. 186 Re, 165 Dy, 32 P.166 Ho, 188 Re or any combination thereof. In some embodiments, the radioisotope is a gamma-emitting radioisotope and comprises 99m Tc, 123 I or 131 I. In some embodiments, the antibody or antigen-binding fragment thereof further comprises a contrast agent or is chelated to a contrast agent. In some embodiments, the contrast 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 chelated to a pyrrolobenzodiazepine. (PBD), protein toxins, diphtheria toxin, glucagon-like peptide (GLP-1), cytotoxic immunomodulatory proteins, Fas ligand, auristatin or its analogs, maytansinoid, calicheamicin, duocarmycin or its analogs or doxorubicin or its analogs conjugated. 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 luminescent imaging performed on the subject. In some embodiments, the epitope is displayed on the 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.

[0009] In some aspects, the disclosure provides a cell comprising any polypeptide, nucleic acid, or vector described herein.

[0010] In some aspects, the present disclosure provides a composition comprising any polypeptide, nucleic acid or vector described herein and a pharmaceutically acceptable carrier. In some embodiments, the composition further comprises a transfection agent.

[0011] Incorporation by reference

[0012] 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The novel features of the present invention are particularly set forth in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by referring to the following detailed description and accompanying drawings which set forth illustrative embodiments in which the principles of the invention are utilized, in which:

[0014] Figure 1A ( Figure 1A ) depicts an example design of a cell surface expressible biomarker from N-terminus to C-terminus according to some embodiments described herein. 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, a hinge region, and an intracellular domain that directs membrane insertion.

[0015] Figure 1B ( Figure 1B ) Depicts example linear construct designs from N-terminus to C-terminus of engineered SSTR2 (top and middle) and DLL3 (bottom) constructs. pH Helix: pH-sensitive helix.

[0016] Figure 1C ( Figure 1C ) Depicts example linear construct designs from N-terminus to C-terminus of engineered DLL3 (top) and GFP (bottom) constructs. mIgK: mouse IgK. pHLIP: pH-sensitive membrane affinity protein motif.

[0017] Figure 2 ( Figure 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 into a pH-sensitive helix (e.g., any helix described herein) such that at high pH (e.g., pH>6.0), the epitope is blocked by the co-association of the pH-sensitive helix. When the cell surface biomarker is converted to a low pH (e.g., <6.0, such as in an acidic tumor microenvironment) in the disease microenvironment, the co-association of the pH-sensitive helix is ​​eliminated, and the epitope becomes bindable, for example, by binding to an antibody or antibody binding fragment configured to bind the epitope. Administration of an antibody or ligand that binds to the epitope can be used to detect, image, or bind to a newly bindable epitope, and thus assess the presence of accessible epitopes in the disease environment.

[0018] Figure 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 natural cell surface receptor has been engineered to have a cancer activatable epitope, wherein the epitope sequence is inserted between a distal pH-sensitive helix (e.g., any helix described herein) and a proximal sequence that is post-translationally modified only when the protein reaches the cell surface in a cancer microenvironment-specific manner. In the basic conformation, the cancer activating epitope is blocked at high pH (e.g., pH>6.0) and cannot be bound to an affinity-based reagent. When the cell surface biomarker is converted to a low pH value (e.g., <6.0, such as in an acidic tumor microenvironment) in the disease microenvironment, the sequence close to the surface binding protein is glycosylated, stabilizing the framework for expressing the epitope, and simultaneously destroying the association between the distal pH-sensitive helix and the cell surface receptor, making the epitope bindable, for example, by binding to an antibody or antibody binding fragment configured to bind to the epitope. Administration of antibodies or ligands that bind to the epitope can be used to detect, image, or bind to newly bindable epitopes, thereby assessing the presence of bindable epitopes in a disease setting.

[0019] Figure 4 ( Figure 4 ) depicts the function of a third cell surface biomarker containing an activatable epitope according to some embodiments described herein. In this embodiment, a natural cell surface receptor (e.g., SSTR2) is engineered with multiple pH-sensitive helices on the protein termini that co-associate and block the ligand-bound state of the receptor ("epitope") at high pH. When the receptor shifts to low pH (e.g., in a tumor microenvironment), the pH-sensitive helices dissociate, allowing the binding of a synthetic analog of the detectable natural ligand (e.g., 68 Ga-DOTATATE labeled somatostatin "DOTATATE", which can be detected by PET / CT).

[0020] Figure 5 ( Figure 5 ) depicts an example design of a secretable membrane-bound reporter as described in Example 3.

[0021] Figure 6 ( Figure 6 ) depicts the proposed functional mechanism of the secretable membrane-bound reporter as described in Example 3.

[0022] Figure 7 ( Figure 7 ) depicts the experiment described in Example 3, showing that Figure 5 and Figure 6 The secretable membrane-bound reporter in is spread through the culture medium to untransfected cells.

[0023] Fig. 8A ( Fig. 8A ) depicts schematics of engineered delta-like ligand 3 (DLL3) surface presentation constructs (top) and linear construct designs from the N-terminus to the C-terminus of NP244, NP245, NP246, NP247, NP248, and NP249 (bottom). DLL3 constructs were engineered by substituting domains of the DLL3 protein that serves as an antigen target onto a molecular scaffold, where a transmembrane anchor domain is used to tether DLL3 to the cell surface. SP indicates the presence of a signal peptide used to drive expression of the engineered DLL3 protein on the cell surface. The structure of DLL3 wild-type (WT) with SP is depicted on the far left. Two strategies to enhance surface trafficking were evaluated: exploiting surface trafficking instructions (signal peptide or transmembrane anchor domain) of 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 presentation of chimeric antigen receptors on immune cells (E1, F1, and G1). E1 includes the hIgG4 Fc domain, and F1 and G1 include the CD8a hinge as an extracellular scaffold to help present the DLL3 epitope.

[0024] Figure 8B ( Figure 8B ) depicts that the amount of DLL3 protein on the cell surface can be measured by FACS analysis after transient transfection with a DNA expression plasmid expressing an engineered DLL3 variant. A fluorescently labeled antibody against DLL3 was used as a detection agent in this experiment. Fig. 8A In A2, corresponding to A1, the H1299 cell line derived from human cancer was transfected with a formulated DNA nanoplasmid expressing wild-type DLL3 protein. Panels B2-G2 show the corresponding DLL3 levels produced on the cell surface when similar plasmids were introduced into H1299 cells. Figure 8B The data in Fig. 8A The results showed that C2-G2 matched the scaffold of DLL3 modeled in . B2 did not increase the expression level of DLL3. C2-G2 resulted in increased expression level of DLL3 relative to the wild-type variant in B2.

[0025] Figure 8C ( Figure 8C ) shows detection of engineered DLL3 on the surface of H1299 cells. Fig. 8A All engineered constructs increased DLL3-positive cells above wild-type DLL3 levels (red line represents NP116). The top two constructs NP249 and NP247 ( Fig. 8A D1 and G1 in the figure increased the positive percentage to about 80% and about 94%, respectively. NP247 ( Fig. 8AG1 in Figure 1 also showed significantly higher mean fluorescence intensity compared to DLL3 wild type or DLL3 stable cell lines, suggesting that there may be increased amounts of DLL3 protein being transported to the cell surface. H1299-DLL3: cell line stably expressing DLL3. NP116: SURV-DLL3 WT. NP127: CAG-DLL3 WT. GMFI: geometric mean fluorescence intensity.

[0026] Fig.8D ( Fig.8D ) shows an in vitro cell killing assay. H1299 cells were transfected with NP247 or NP249, and the ability of anti-DLL3 antibodies conjugated to a cytotoxic payload (PBD) to induce cytotoxicity was determined. When treated with an anti-DLL3 antibody conjugated to a PBD (anti-DLL3-PBD), cells expressing NP247 and NP249 experienced a substantial loss of viability. This suggests that cells expressing the NP247 or NP249 DLL3 constructs may be more susceptible to cell death when treated with anti-DLL3-PBD compared to cells transfected with wild-type DLL3 (e.g., NP116 or NP127).

[0027] Fig. 9A ( Fig. 9A ) depicts a bioengineered NP247 ( Fig. 8A G1 in ) or other protein changes of CD8short, which are used as parent sequences of the second design set (top) and linear construct designs from the N-terminus to the C-terminus of NP296, NP298, NP299, NP302, NP303 and NP304 (bottom). NP296, NP297 and NP298 are engineered to identify the "minimal domain" of DLL3 sufficient to interact with the antibody tracer. Importantly, NP298 is engineered to include a second epitope in the structure in an attempt to double the antibody binding capacity. NP299 is engineered to contain two DLL3 minimal epitopes inserted into the DLL3 extracellular domain. NP302, NP303, NP304 are 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 the soluble humanized nanobody structure; and NP304 does not contain an extracellular DLL3 domain.

[0028] Fig. 9B ( Fig. 9B ) depicts the expression level of DLL3 on the cell surface after transfection into H1299 cells. Figure 8BAs in Figure 2, these data demonstrate that DLL3 modeled into a molecular scaffold results in higher levels of surface DLL3 expression compared to H1299 transfected with a plasmid carrying the wild-type DLL3 protein. A fluorescently labeled antibody against DLL3 was used as a detection agent in this experiment. Refining the structure to include the presence of a humanized soluble nanobody domain in NP302 further enhanced expression compared to NP247. Importantly, the addition of two DLL3 binding domains on NP298 demonstrates that a multivalent approach can be used to improve antibody binding capacity and increase the overall level of antibody binding activity.

[0029] Fig. 9C ( Fig. 9C ) depicts the DLL3 expression levels of 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 antibodies against DLL3 were used as detection agents in this experiment.

[0030] Fig. 10A and 10B ( Fig. 10A and Fig. 10B ) depicts the amount of DLL3 that can be measured by FACS analysis on the cell surface after H1299 cells were transfected with wild-type DLL3 epitopes NP247 (expressing a single epitope in a CD8 scaffold) or NP298 (expressing two DLL3 epitopes in a CD8 scaffold). Fluorescently labeled antibodies against DLL3, phycoerythrin (PE, Fig. 10A ) or fluorescein isothiocyanate (FITC, Fig. 10B ) as the detection agent. Using the standard curve ( Fig. 10B ) to assess the approximate antibody binding per cell.

[0031] Fig. 10C ( Fig. 10C ) depicts the physical number of antibody copies bound to DLL3 expressed on the cell surface after transfection of H1299 cells with either the wild-type DLL3 epitope NP247 (expressing a single epitope in a CD8 scaffold) or NP298 (expressing two DLL3 epitopes in a CD8 scaffold). Antibody binding was estimated using a standard curve. NP298 showed a 32-fold increase in wild-type DLL3 epitope expression levels. Fluorescently labeled antibodies against DLL3 were used as detection agents in this experiment.

[0032] Fig. 10D ( Fig. 10D) depicts the geometric mean intensity of internalized DLL3 after H1299 cells were transfected with NP116 (wild-type DLL3) or NP247. Fluorescently labeled antibodies against DLL3 were used as detection agents in this experiment.

[0033] Fig.11 ( Fig.11 ) shows images acquired from positron emission tomography (PET). In this model system, a DNA plasmid expressing a copy of the somatostatin receptor 2 (SSTR2) gene was generated and then stably introduced into H1299 cells such that each cell line contained two genomic copies of the stably integrated SSTR2 construct. To determine the minimum number of cells that produced a measurable signal, different numbers of engineered cells with stably integrated SSTR2 were implanted subcutaneously in different locations of mice and then treated with a 68Ga-dotate positron emission tracer that binds to the SSTR2 protein on the cell surface. These data show that when cells are induced to express a surrogate biomarker on the cell surface, as few as 31,000 cancer cells can be detected in this mouse model.

[0034] Fig.12 ( Fig.12 ) shows images acquired from single photon emission computed tomography (SPECT). In this model system, a DNA plasmid expressing a copy of the SSTR2 gene was generated and then stably introduced into H1299 cells so that each cell line contained two genomic copies of the stably integrated SSTR2 construct. Engineered cells with stably integrated SSTR2 were implanted subcutaneously into mice and then treated with a protein that binds to the SSTR2 protein on the cell surface. 177 LU-dotate β-emitting tracers. These data suggest that high-energy tracers can be used to detect biomarkers on cells engineered to express these biomarkers. Middle: Coronal images of sections from kidney and tumor. Right: Maximum intensity projection (MIP). 4H, 24H, and 120H: 4 hours, 24 hours, and 120 hours after tracer administration, respectively.

[0035] Fig.13A ( Fig.13A ) depicts the physical number of antibody copies bound to DLL3 expressed on 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 in epitope expression levels compared to wild-type DLL3. NP298 showed a 37-fold increase in epitope expression levels compared to wild-type DLL3. Fluorescently labeled antibodies against DLL3 were used as detection agents in this experiment.

[0036] Fig. 13B ( Fig. 13B ) depicts an in vitro cell killing assay. H1299 cells were transfected with NP116, NP247, or NP298, and the ability of anti-DLL3 antibodies conjugated to a cytotoxic payload (PBD) to induce cytotoxicity was determined. H1299 cells expressing NP247 and NP298 experienced substantial loss of viability when treated with an anti-DLL3 antibody conjugated to a PBD (anti-DLL3-PBD). This suggests that cells expressing the NP247 or NP298 DLL3 constructs may be more susceptible to cell death when treated with anti-DLL3-PBD compared to cells transfected with wild-type DLL3 (NP116).

[0037] Fig.14 Images acquired from single-photon emission computed tomography (SPECT) are shown. In this model system, a DNA plasmid expressing a copy of the SSTR2 gene was generated and then stably introduced into H1299 cells using a lentiviral system. Engineered cells with stably integrated SSTR2 constructs were implanted subcutaneously into mice and treated with 200 μCi of [ 68 GA]-DOTATATE tracer treatment, and PET / CT imaging was performed 2 hours after tracer administration.

[0038] Fig.15 ( Fig.15 ) depicts a schematic diagram of an exemplary workflow of the methods described herein. A DNA (1) construct that activates expression of a cell surface protein can be introduced into a target cell population by transient transfection (2). After transport and disassembly in the nucleus, the DNA serves as a nucleic acid template for transcription (3-5) and produces an engineered reporter gene. Further protein modifications can occur in the cytoplasm (7) or on the cell surface (9) in response to the molecular disease state of the cell. Alternatively, the protein can be secreted into the extracellular environment to propagate the signal to neighboring cells (8). Once on the cell surface, the target antigen can be easily detected using affinity-based reagents that enable detection by imaging techniques involving radioactive tracers or other means. The use of high-energy emitters also provides the possibility of therapeutic applications. DETAILED DESCRIPTION

[0039] Overview

[0040] In some aspects, the disclosure relates to encodable (eg, under the control of a tumor or cancer-specific promoter) cell surface synthetic biomarkers that aid in identifying, localizing, or treating tumor cells in a subject.

[0041] In some cases, such encodable cell surface synthetic biomarkers include: (a) an extracellular domain configured to bind an affinity agent and (b) a membrane binding or transmembrane domain. When the nucleic acid is encoded under the control of a cancer-specific promoter (e.g., a promoter of a gene overexpressed in cancer cells relative to normal cells), administration of the nucleic acid to a subject suspected of having cancer drives the selective production of cell surface biomarkers in cancer cells. The extracellular domain may include a terminal activatable epitope or a ligand binding domain. Then, administration of a suitable affinity agent (e.g., a ligand that binds to a ligand binding domain or an affinity agent / antibody that binds to an epitope) to the subject can be used to treat tumor cells, selectively express encoded cell surface synthetic biomarkers (if the affinity agent is a suitable therapeutic antibody) or locate or identify cells (if the affinity agent is, for example, a labeled antibody).

[0042] Figure 2 An example configuration of such a cell surface synthetic biomarker is provided in which the binding of the affinity agent depends on cancer-related transcription and extracellular conditions. In this configuration, the cell surface synthetic biomarker includes an extracellular domain (201) attached to the membrane via a transmembrane domain, such as from any receptor described herein); the extracellular domain has a terminal epitope (210), which is flanked by two compatible portions of a pH-sensitive multimerization domain (220 and 230), which can be a pH-sensitive helix as described herein. By providing a signal peptide at the end of the extracellular domain, the proper external positioning of the extracellular domain can be driven. At high pH, ​​the self-association of the pH-sensitive multimerization domain (240) prevents the epitope (250) from binding to an affinity agent (e.g., an affinity agent applied to a subject). At low pH, the two compatible portions of the pH-sensitive multimerization domain (220 and 230) dissociate, allowing the epitope (210) to bind to an extracellular affinity agent. In this way, cell surface synthetic biomarkers can be dependent on the transcriptional state of cancer cells (e.g., by expression from cancer-specific promoters, or expression from promoters of genes that are overexpressed in cancer cells relative to normal cells) and the typical acidic extracellular environment of tumors. The combination of transcriptional and extracellular conditions for binding to affinity agents can result in improved specificity for cancers in targeted subjects.

[0043] Figure 3 Another example configuration of such a cell surface synthetic biomarker is provided in . In this case, the configuration resembles Figure 2configuration, except that the extracellular domain (301) is configured to bind to the 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 helps stabilize the activatable epitope when post-translationally modified. In this way, if the post-translational modification site is selected as a site that is selectively targeted by an extracellular enzyme expressed in cancer, the bindability of the activatable epitope can be controlled in a manner that depends on the extracellular pH and cancer-associated extracellular enzyme expression through the pH-sensitive helix. In this design, the activatable epitope (320) is excluded in the absence of post-translational modification and low pH (e.g., in a non-cancer microenvironment). In contrast, in an extracellular environment associated with cancer, the activatable epitope (350) is made bindable by dissociation of the pH-sensitive helix (360) and post-translational modification of the flanking post-translational modification site (340).

[0044] Figure 4 Another example configuration of such cell surface synthetic biomarkers is provided in, wherein the configuration of the biomarker includes a ligand binding domain to allow the use of a modified natural ligand as an affinity agent for the ligand binding domain. In this configuration, the membrane-bound ligand binding domain (301) is connected to a terminal pH-sensitive spiral (320), which prevents the binding of the ligand (330) to the ligand binding site (310) at low pH. At high pH (e.g., non-cancer microenvironment), the pH-sensitive spiral (350) dissociates and allows the ligand to bind to the ligand binding site (340) on the cell surface. In this example, the activation epitope is used to bind to the desired transcription (when the biomarker is encoded under the control of a cancer-specific promoter or a promoter of a gene overexpressed in cancer cells relative to normal cells), the combination of extracellular pH and extracellular post-translational conditions can lead to improved specificity of cancer in the targeted object.

[0045] Figure 5 Another example configuration of such a cell surface synthetic biomarker is provided in Figure 2 Receptors (501, 515, 510) of the type described in are modified to replace the signal peptide (501) with a secretion signal (520) and to replace the transmembrane domain (510) with a membrane binding domain (530) (e.g., a pH-sensitive membrane binding helix) to produce second generation cell surface synthetic biomarkers (520, 515, 530) that are capable of propagating to neighboring cells when expressed by cancer cells (e.g., under a cancer cell-specific promoter). Thus, such cell surface synthetic biomarkers are configured as Figure 6, where the secreted biomarker is secreted in the extracellular fluid (601) and does not attach to the cell if secreted in a non-acidic extracellular environment (e.g., a non-tumor environment), but attaches to the secreting cell (610) and neighboring cells (620) when at the appropriate pH to allow assembly of membrane-bound helices. The dependence of the extracellular conditions on the labeled cell combined with the ability to propagate to neighboring cells can lead to improved specificity and sensitivity of this configuration for targeting cancer.

[0046] definition

[0047] Although 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. Those skilled in the art may make many modifications, changes and substitutions without departing from the present invention. It should be understood that various alternatives to the embodiments of the present invention described herein may be employed.

[0048] Before describing the present invention in more detail, it should be understood that the present disclosure is not limited to the specific embodiments described, and therefore may of course vary. It should also be understood that the terminology used herein is only for the purpose of describing specific embodiments, and is not intended to be limiting, as the scope of the present disclosure is limited only by the appended claims.

[0049] Where a range of values ​​is provided, it is understood that every intervening value (in units of one-tenth of the lower limit) between the upper and lower limits of that range and any other specified value or intervening value in the specified range is included in 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 and are also included in the disclosure, subject to any expressly excluded limits in the specified range. When the specified range includes one or both limits, ranges excluding one or both of those included limits are also included in the disclosure.

[0050] It must be noted that the singular forms "a," "an," and "the," as used in the specification and the appended claims, 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 claims that follow, reference will be made to certain terms, which, unless a contrary intention is apparent, shall be defined to have the following meanings.

[0051] As used herein, the following terms have the meanings ascribed to them unless otherwise indicated. In the present disclosure, "comprises," "comprising," "containing," and "having," etc. may have the meanings ascribed to them in U.S. patent law, and may refer to "includes," "including," etc.; "consisting essentially of," or "consisting essentially of," etc., when applied to methods and compositions encompassed by the present disclosure, refer to compositions similar to the compositions disclosed herein, but which may contain additional structural groups, composition components, or method steps (or analogs or derivatives thereof as discussed above). However, such additional structural groups, composition components, or method steps, etc., do not substantially affect the basic and novel characteristics of the compositions or methods as compared to those corresponding compositions or methods disclosed herein.

[0052] As used herein, the term "or" is generally intended to be inclusive, covering both alternatives as well as one of them.

[0053] The present disclosure includes variants of any polypeptide described herein, which have one or more conservative amino acid substitutions. Such conservative substitutions can be carried out in the amino acid sequence of the polypeptide without destroying the three-dimensional structure or function of the polypeptide, or only have minimal damage to the function of the polypeptide. Conservative substitutions can be completed by replacing each other with amino acids with similar hydrophobicity, polarity and R chain length. Additionally or alternatively, by comparing the alignment sequences of homologous proteins from different species, without changing the basic function of the encoded protein, the amino acid residues (e.g., non-conservative residues) that have been mutated between species are located, and conservative substitutions can be identified. Such conservative substitution 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 polypeptide protein sequence described herein. In some embodiments, such conservative substitution variants are functional variants. Such functional variants may include sequences with substitutions such that the activity of one or more key active site residues or structural support residues of the polypeptide is not destroyed.

[0054] Conservative substitution tables providing functionally similar amino acids are available from various references (see, e.g., Creighton, Proteins: Structures and Molecular Properties (W H Freeman & Co.; 2nd ed. (December 1993)). The following eight groups each contain amino acids that are conservatively substituted for each other:

[0055] 1) Alanine (A), glycine (G);

[0056] 2) Aspartic acid (D), glutamic acid (E);

[0057] 3) Asparagine (N), glutamine (Q);

[0058] 4) Arginine (R), Lysine (K);

[0059] 5) Isoleucine (I), leucine (L), methionine (M), valine (V);

[0060] 6) Phenylalanine (F), tyrosine (Y), tryptophan (W);

[0061] 7) serine (S), threonine (T); and

[0062] 8) Cysteine ​​(C), Methionine (M)

[0063] The terms "sequence identity" or "percent identity" in the context of two or more nucleic acid or polypeptide sequences generally refer 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 specified percentage of amino acid residues or identical nucleotides, when compared and aligned for maximum match 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 with a word length (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix with a gap cost of 11 when present and an extension of 1, and adjusted for polypeptide sequences of more than 30 residues using a conditional composition scoring matrix; BLASTP, using parameters with a word length (W) of 2, an expectation (E) of 1000000, and the PAM30 scoring matrix with a gap cost of 9 for gap opening and a gap extension of 1 for sequences of less than 30 residues (these are the default parameters for BLASTP in the BLAST suite, available at https: / / blast.ncbi.nlm.nih.gov); CLUSTALW, with Smith-Waterman homology search algorithm parameters, matches of 2, mismatches of -1, and gaps of -1; MUSCLE, with default parameters; MAFFT, with parameters with retree of 2 and maximum iterations of 1000; Novafold, with default parameters; HMMERhmmalign, with default parameters.

[0064] The term "about" or "approximately" means within an acceptable error range for a particular value as determined by one skilled 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, according to the practice in the art, "about" can mean within one or more than one standard deviation. Alternatively, "about" can mean a range of up to 20%, up to 15%, up to 10%, up to 5%, or up to 1% of a given value.

[0065] The term "subject" may include humans or non-human animals in general. Therefore, the methods and compositions described herein are applicable to human and veterinary diseases and animal models. Preferred subjects are "patients", i.e., living humans who are receiving medical care for a disease or condition. This includes people who do not have a clear disease but are being investigated for pathological signs. Also included are people who are suspected of having or are at risk of a clear disease. In some embodiments, the subject has at least one cancer risk factor 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 BRCA mutations; is a current smoker, former smoker, or exposed to a lot of secondhand smoke; is exposed to carcinogens, excessive sun exposure, immunosuppressants, or hepatitis B, hepatitis C, or human papillomavirus; or is obese.

[0066] As used herein, "vector" 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 phage genome, or a baculovirus genome. Suitable vectors also include vectors derived from bacteriophages or plant, invertebrate or animal (including human) viruses, such as CELiD vectors, Doggybone DNA (dbDNA) vectors, closed-end linear duplex DNA vectors (e.g., wherein each end is covalently blocked by chemical modification), adeno-associated virus vectors (e.g., AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or pseudotype combinations thereof such as AAV2 / 5, AAV2 / 2, AAV-DJ, or AAV-DJ8), retroviral vectors (e.g., MLV, or self-inactivating or SIN versions thereof, or pseudotyped versions thereof), herpes viruses (e.g., based on HSV or EBV), lentiviral vectors (e.g., based on HIV, FIV or EIAV, or pseudotyped versions thereof), or adenoviral vectors (e.g., based on Ad5, including replication-deficient, replication-competent, or helper-dependent forms thereof). In some embodiments, the vector is a replication-competent virus-derived vector. In some embodiments, the vector is a replication-incompetent virus-derived vector. In some cases, the carrier may include free maintenance elements to promote replication in one or more target cell types, such as support / matrix attachment regions (S / MAR). The S / MAR elements are particularly useful for promoting replication in the context of "naked" nucleic acid carriers (such as microrings). Exemplary suitable S / MAR elements include, but are not limited to, EμMAR from immunoglobulin heavy chain loci, apoB MAR from human apolipoprotein B loci, Ch-LysMAR from chicken lysozyme loci, and huIFNβMAR from human IFNβ loci. The carrier may include a coding sequence that can be expressed in target cells. Therefore, as used herein, the terms "vector construct", "expression vector" and "gene transfer vector" may refer to any nucleic acid construct that can instruct the expression of a gene of interest and can be used to transfer a gene of interest to a target cell. Carriers as described herein may also include one or more cis-acting elements to stabilize or improve mRNA expression therein.Such cis-acting elements include, but are not limited to, any of the elements described in, for example, 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).

[0067] As a form of vector, the term "minicircle" as used herein generally refers to a small double-stranded circular DNA molecule that provides continuous, high-level expression of a sequence of interest present on the vector, which can encode a polypeptide, shRNA, antisense RNA, siRNA, etc. The sequence of interest is operably linked to a regulatory sequence present on the minicircle vector that controls its expression. Such minicircle vectors are described, for example, in the published U.S. patent application US20040214329, which is specifically incorporated herein by reference. As a different form of a vector, "nanoplasmid" refers to a vector that can contain a minimized bacterial ColE1 or R6K origin of replication (which allows such nanoplasmids to replicate in bacterial host strains), a bacterial RNA selectable marker, and a eukaryotic gene region. More examples of such elements (nanoplasmid origin and RNA-out selectable marker) are described, for example, in US9737620B2, which is incorporated herein by reference for the purpose of describing nanoplasmid sequence elements.

[0068] The overall length of the minicircle vector is sufficient to include the desired elements as described below, but not so long as to hinder or substantially inhibit the ability of the vector to enter a target cell (e.g., by systemic administration to a host containing the cell) upon contact with the cell to an unacceptable level. Thus, the minicircle vector can be generally at least about 0.3 kb long, typically at least about 1.0 kb long, while the parent vector can be up to 6 kb, 10 kb, or longer.

[0069] Minicircle vectors differ from bacterial plasmid vectors in that they lack an origin of replication or lack a natural origin of replication (e.g., they may contain a minimized synthetic bacterial origin of replication), and lack selectable markers commonly found in bacterial plasmids, such as β-lactamase, tetracycline resistance (tet), kanamycin resistance (kan), or other antibiotic selectable markers. As a result, the size of the minicircle becomes smaller, allowing for more efficient delivery. Minicircles lack the transgene expression silencing effect, which is associated with the vector backbone nucleic acid sequence of the parent plasmid from which the minicircle vector is excised. Minicircles may be substantially free of vector sequences other than the sequence of the recombinase hybridization product, as well as sequences of interest, i.e., transcriptional sequences and regulatory sequences required for expression.

[0070] The term "nanoplasmid" as used herein generally refers to a vector that can include a minimized bacterial ColE1 or R6K origin of replication (which allows such nanoplasmids to replicate in bacterial host strains), a bacterial RNA selectable marker, and a eukaryotic gene region. Some embodiments of nanoplasmids are described in, for example, US20150275221A1. In some embodiments, the nanoplasmid can include a fusion bacterial RNA selectable marker / minimized origin of replication. In some embodiments, the fusion bacterial RNA selectable marker / minimized origin of replication can be located in a synthetic intron within the eukaryotic gene region of the nanoplasmid.

[0071] RNA selectable markers are generally non-translated RNAs carried by vectors that regulate a target gene expressed by a chromosome to provide selection of the vector. This can be a plasmid-borne nonsense suppressor tRNA that regulates a selectable chromosomal target for nonsense suppression, as described in 2005 U.S. Patent No. 6,977,174 by Crouzet J and Soubrier F, which is included herein by reference. This can also be a plasmid-borne antisense repressor RNA, an RNA-OUT gene that represses RNA-IN regulatory targets, RNA I encoded by the pMB1 plasmid origin that represses RNA II regulatory targets, RNA I encoded by the IncB plasmid pMU720 origin that represses RNA II regulatory targets, the ParB locus Sok of plasmid RI that represses Hok regulatory targets, the Flm locus FlmB of the F plasmid that represses flmA regulatory targets, another natural antisense repressor RNA, such as those described in, for example, 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 a small synthetic small RNA similar to the SgrS, MicC or MicF scaffold as described by Park et al. in Nature Biotechnology volume 31, pages 170-174 (2013).

[0072] The term "transfection agent" generally includes any compound that mediates the incorporation of DNA or RNA into a host cell, such as a liposome. Suitable methods for transforming or transfecting host cells can be found in Sambrook et al. (MOLECULAR CLONING: ALABORATORY MANUAL. 2nd edition, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989), Ausubel et al., Short Protocols in Molecular Biology, 3rd edition, 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 polyethyleneimines, nanoparticles, liposomes, lipophilic particles, solid nanoparticles, amphiphilic peptides, micelles, dendrimers, polymer compositions, hydrogels, exosomes of synthetic or natural origin, virus-like particles, or any combination thereof.

[0073] As used herein, the terms "nucleic acid molecule" and "polynucleotide" generally refer to a polymeric form of nucleotides of any length, whether deoxyribonucleotides or ribonucleotides or their analogs. Polynucleotides can have any three-dimensional structure and can perform any known or unknown function. 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 can be linear or circular.

[0074] The term "promoter" generally refers to a DNA sequence that instructs polynucleotide transcription. Typically, a promoter can be located in the 5' region of a polynucleotide to be transcribed, near the transcription start site of the polynucleotide. More typically, a promoter is defined as the upstream region of the first exon; More typically, it is defined as the upstream region of the first of a plurality of transcription start sites. A promoter is often able to instruct the transcription of a gene on each complementary DNA strand located at 3' of the promoter. In other words, many promoters exhibit bidirectionality and can instruct the transcription of downstream genes when present in either direction (i.e., 5' to 3' or 3' to 5' relative to the coding region of a gene). In addition, a promoter can also include at least one control element such as an upstream element. Such elements include an upstream activation region (UAR) and optionally other DNA sequences that affect polynucleotide transcription such as synthetic upstream elements. Some promoters can be assembled by fragments of endogenous promoters (e.g., derived from the human genome).

[0075] The terms "coding sequence" and "encoding" as used herein when referring 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 under the control of appropriate regulatory sequences (or "control elements"). The boundaries of the coding sequence are generally determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxyl) terminus. The coding sequence may include, but is not limited to, cDNA from viral, prokaryotic or eukaryotic mRNA, genomic DNA sequences from viral, eukaryotic or prokaryotic DNA, and synthetic DNA sequences. A transcription termination sequence may be located 3' of the coding sequence, and a promoter may be located 5' of the coding sequence; if desired, together with additional control sequences such as enhancers, introns, polyadenylation sites, etc. The DNA sequence encoding the polypeptide may be optimized for expression in a selected cell by using codons preferred by the selected cell to represent a DNA copy of the desired polypeptide coding sequence.

[0076] As used herein, the term "operably connected" generally refers to an arrangement of elements in which the described components are configured to perform their usual functions. Thus, a given promoter operably connected to a coding sequence (e.g., a reporter expression cassette) can affect the expression of the coding sequence when there is an appropriate enzyme. A promoter or other control element need not be adjacent to the coding sequence as long as they function to direct its expression. For example, an untranslated but transcribed intermediate sequence may exist between the promoter sequence and the coding sequence, and the promoter sequence may still be considered to be "operably connected" to the coding sequence.

[0077] The term "pharmaceutically acceptable carrier" as used herein generally refers to a diluent, adjuvant, excipient or vehicle, which is used together with the probe of the present disclosure, and which is approved by a regulatory agency of a federal or state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopoeia for animals, and more specifically for humans. Such pharmaceutical carriers can be liquids, such as water and oils, including oils of petroleum, animal, plant or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Pharmaceutical carriers can be saline, gum arabic, gelatin, starch paste, talc, keratin, colloidal silicon dioxide, urea, etc. When applied to a patient, the probe and pharmaceutically acceptable carrier can be sterile. When the probe is administered intravenously, water is a useful carrier. 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, glyceryl monostearate, sodium chloride, glycerol, propylene, ethylene glycol, water, ethanol, etc. If necessary, the present composition may also contain a small amount of a wetting agent or emulsifier, or a pH buffering agent. The present composition may advantageously take the form of a solution, emulsion, sustained release formulation or any other form suitable for use.

[0078] The term "antibody" or "immunoglobulin" generally includes five different antibody classes that can be distinguished biochemically. For the IgG class of immunoglobulin molecules, which can be considered the prototype, the immunoglobulin contains two identical light polypeptide chains with a molecular weight of about 23,000 Daltons and two identical heavy chains with a molecular weight of 53,000-70,000. These four chains are joined by disulfide bonds to form a "Y" configuration, in which the light chains support the heavy chains from the mouth of the "Y" and continue through the variable domains.

[0079] The light chains of immunoglobulins can be classified as kappa or lambda (κ, λ). Each heavy chain class can be associated with a κ or λ light chain. In general, when immunoglobulins are produced by hybridomas, B cells, or genetically engineered host cells, the light and heavy chains can be covalently bonded to each other, and the "tail" portions of the two heavy chains can be bonded by covalent disulfide bonds or non-covalent bonds. In the heavy chain, the amino acid sequence extends from the N-terminus at the forked end of the Y configuration to the C-terminus at the bottom of each chain. The heavy chain can be classified as gamma, mu, alpha, delta, or epsilon (γ, μ, α, δ, ε), with some subclasses (e.g., γ1-γ4). It is the nature of this chain that determines the "class" of the antibody, which is IgG, IgM, IgA IgG, or IgE, respectively. Immunoglobulin subclasses (isotypes) such as IgG1, IgG2, IgG3, IgG4, IgA1, etc. can confer functional specificity.

[0080] Both light and heavy chains can be divided into regions of structural and functional homology. The term "region" when used to describe an immunoglobulin or related molecule generally refers to a portion or part of a single immunoglobulin (as in the case of the term "Fc region") or a single antibody chain, and includes a constant region or variable region, as well as more discrete portions or parts of the domain. For example, a light chain variable domain includes "complementarity determining regions" or "CDRs" as defined herein interspersed among "framework regions" or "FRs".

[0081] 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 each class member in the "constant region", or the significant variation in sequence within the regions of each class member in the "variable region". The terms "constant region" and "variable region" can also be used functionally. In this regard, it should be recognized that the variable regions of an immunoglobulin or antibody determine antigen recognition and specificity. In contrast, the constant regions of an immunoglobulin or antibody confer important effector functions, such as secretion, transplacental movement, Fc receptor binding, complement binding, etc.

[0082] The constant region and variable region of immunoglobulin heavy chain and light chain can be folded into domain.The term "domain" generally refers to the independently folded globular region of heavy chain or light chain polypeptide, which includes peptide loops (e.g., including 3 to 4 peptide loops), which are stabilized, for example, by β pleated sheets and / or intrachain disulfide bonds.The constant region domain on the immunoglobulin light chain can be interchangeably referred to as "light chain constant region domain", "CL region" or "CL domain".The constant region (e.g., hinge, CH1, CH2 or CH3 domain) on the heavy chain can be interchangeably referred to as "heavy chain constant region domain", "CH" region domain or "CH domain".The variable domain on the light chain can be interchangeably referred to as "light chain variable region domain", "VL region domain" or "VL domain".The variable domain on the heavy chain can be interchangeably referred to as "heavy chain variable region domain", "VH region domain" or "VH domain".

[0083] By convention, the numbering of the variable and constant region domains increases as they are farther from the antigen-binding portion or amino terminus of the immunoglobulin or antibody. At the N-terminus of each heavy and light immunoglobulin chain is the variable region, and at the C-terminus is the constant region; the CH3 and CL domains comprise the carboxyl termini of the heavy and light chains, respectively. Thus, the domains of light chain immunoglobulins are arranged in a VL-CL orientation, while the domains of heavy chains are arranged in a VH-CH1-hinge-CH2-CH3 orientation.

[0084] As used herein, the term "CDR" or "complementarity determining region" means the non-contiguous antigen binding sites found within the variable regions of heavy and light chain polypeptides. These specific regions have been described by Kabat et al., J. Biol. Chem. 252, 6609-6616 (1977) and Kabat et al., Sequences of protein of immunological interest. (1991) and by Chothia et al., J. Mol. Biol. 196: 901-917 (1987) and by MacCallum et al., J. Mol. Biol. 262: 732-745 (1996), wherein definitions include overlaps or subsets of amino acid residues when compared to each other. The amino acid residues comprising the CDRs defined in each of the above cited references are set forth for comparison. Preferably, the term "CDR" is a CDR as defined by Kabat based on sequence comparison.

[0085] As used herein, the terms "CH2 domain sequence", "CH3 domain sequence", "CH1 domain sequence" and "CL domain sequence" generally refer to polypeptide sequences of the CH2 domain, CH3 domain, CH1 domain and CL domain, respectively, derived from a native immunoglobulin molecule.

[0086] 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., "Sequences of Proteins of Immunological Interest", U. S. Pat. Health and Human Services, 5th Edition, 1991, which is incorporated herein by reference).

[0087] As used herein, the term "VH domain" generally includes the amino terminal variable domain of an immunoglobulin heavy chain, and the term "VL domain" includes the amino terminal variable domain of an immunoglobulin light chain.

[0088] As used herein, the term "CH1 domain" generally includes the first (most amino-terminal) constant region domain of an immunoglobulin heavy chain, which extends, for example, from about EU positions 118-215. The CH1 domain is adjacent to the amino terminus of the VH domain and the hinge region of the immunoglobulin heavy chain molecule, and is not considered part of the Fc region of an immunoglobulin heavy chain.

[0089] As used herein, the term "hinge region" or "hinge domain" generally includes the portion of the heavy chain molecule that joins the CH1 domain and the CH2 domain, or the domain of a non-heavy chain molecule that has sequence similarity to the portion of the heavy chain molecule that joins the CH1 domain and the CH2 domain. The hinge region is flexible, thus allowing the two N-terminal antigen binding regions to move independently. The hinge region can be subdivided into three different domains: an upper hinge domain, a middle hinge domain, and a lower hinge domain (see, e.g., Roux et al., J. Immunol. 1998, 161: 4083, which is incorporated herein by reference). The upper hinge domain generally refers to the portion of the heavy chain molecule that extends from the C-terminus of the CH1 domain to the first hinge cysteine. The middle hinge domain generally refers to the portion of the heavy chain molecule that extends from the first cysteine ​​in the hinge to the last cysteine. The lower hinge domain generally refers to the portion of the heavy chain molecule stretching from the last cysteine ​​in the hinge to Gly-237 according to EU numbering in the CH2 domain.

[0090] 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) located after the β-pleated sheet immunoglobulin V domain-like region, which is rich in proline, threonine and serine residues and has significant sequence identity with the immunoglobulin heavy chain hinge region or hinge domain.

[0091] As used herein, the term "CH2 domain" generally includes, for example, the portion of a heavy chain immunoglobulin molecule extending from about EU position 231-340. A CH2 domain is not closely paired with another domain. Instead, two N-linked branched carbohydrate chains are inserted between the two CH2 domains of a complete native IgG molecule.

[0092] As used herein, the term "CH3 domain" generally includes the portion of a heavy chain immunoglobulin molecule extending from the C-terminus of the CH2 domain by about 110 residues, e.g., from about positions 341-447 (EU numbering system). The CH3 domain typically forms the C-terminal portion of an antibody. However, in some immunoglobulins, 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).

[0093] As used herein, the term "CL domain" generally includes the first (most amino-terminal) constant region domain of an immunoglobulin light chain, for example, extending from about EU positions 108 to 214. The CL domain is adjacent to the VL domain.

[0094] As used herein, the term "Fc domain" generally refers to the portion of a single immunoglobulin heavy chain that begins just upstream of the hinge region of the papain cleavage site (i.e., residue 216 in IgG, taking the first residue of the heavy chain constant region as 118) and ends at the C-terminus of the antibody. Thus, a complete Fc domain comprises at least a hinge domain, a CH2 domain, and a CH3 domain.

[0095] 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; 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.

[0096] Single-chain Fv ("sFv" or "scFv") polypeptides generally refer to covalently linked VH-VL heterodimers expressed by a gene fusion comprising VH and VL encoding genes connected by a peptide-encoded linker (see, e.g., Huston et al., Proc. Nat. Acad. Sci. USA (1988) 85:5879-5883). A variety of methods have been described to identify and develop chemical structures (linkers) to convert naturally aggregated but chemically separated light and heavy chain polypeptide chains from antibody V regions into sFv molecules that will fold into a three-dimensional structure that is substantially similar to the structure of the antigen binding site. See, e.g., U.S. Patent Nos. 5,091,513, 5,132,405, and 4,946,778.

[0097] As used herein, the term "epitope" generally refers to the local region of the antigen to which an antibody can specifically bind. An epitope can be, for example, a continuous amino acid (linear or continuous epitope) of a polypeptide or an epitope can be, for example, gathered together by two or more discontinuous regions of a polypeptide or a polypeptide (conformation, nonlinearity, discontinuous or non-continuous epitope). In certain embodiments, the epitope to which an antibody is bound can be determined by, for example, NMR spectroscopy, X-ray diffraction crystallography, ELISA assay, hydrogen / deuterium exchange and 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 can be studied using well-known X-ray diffraction techniques and can be refined using computer software such as X-PLOR (Yale University, 1992, published by Molecular Simulations, Inc.; see, e.g., Meth Enzymol (1985) volumes 114 & 115, eds Wyckoff H W et al.; US 2004 / 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 can be accomplished using any of several documented methods, such as described in, for example, Champe M et al., (1995) J Biol Chem 270: 1388-1394 and Cunningham BC & Wells JA (1989) Science 244: 1081-1085.

[0098] As used herein, the term "connecting domain" generally refers to a sequence that connects two or more domains in a linear sequence. In some embodiments, any domain or region described herein can be interconnected by a linker domain, which may include, for example, (GGS)n or (GGGS)n.

[0099] The term "transmembrane domain" or "transmembrane region" generally includes an amino acid sequence of about 15 amino acid residues in length that spans the plasma membrane. A transmembrane domain or region may also include about at least 20, 25, 30, 35, 40 or 45 amino acid residues and span the plasma membrane. A transmembrane domain may be rich in hydrophobic residues and have an α-helical structure. In one embodiment, at least 50%, 60%, 70%, 80%, 90%, 95% or more of the amino acids in the transmembrane domain are hydrophobic, such as leucine, isoleucine, tyrosine or tryptophan. Transmembrane domains are described in, for example, Zagotta WN et al. (1996) Annual Rev. Neurosci. 19: 235-263, the contents of which are incorporated herein by reference.

[0100] Epitope

[0101] In some aspects, an engineered polypeptide is provided herein, comprising an extracellular directional domain (e.g., an extracellular domain) comprising an epitope. The extracellular domain may optionally comprise a signal peptide. In some embodiments, the engineered polypeptide sequence may comprise a sequence of an extracellular domain sequence containing an epitope and a sequence of an optional signal peptide sequence encoded by a single nucleic acid sequence. In some embodiments, the signal peptide sequence may be cut after the engineered polypeptide is expressed. In some embodiments, the engineered polypeptide sequence may be designed to comprise a signal peptide heterologous to the extracellular domain of the engineered polypeptide.

[0102] In some embodiments, the epitope can be bound to an antibody. In some embodiments, the epitope can be bound to 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 cancer, including acute myeloid leukemia, adrenocortical carcinoma, bladder urothelial carcinoma, breast ductal carcinoma, breast lobular carcinoma, cervical cancer, bile duct carcinoma, colorectal adenocarcinoma, esophageal cancer, gastric adenocarcinoma, glioblastoma multiforme, head and neck squamous cell carcinoma, hepatocellular carcinoma, renal chromophobe cell 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 adenocarcinoma, sarcoma, cutaneous melanoma, testicular germ cell carcinoma, thymoma, thyroid papillary carcinoma, uterine carcinosarcoma, uterine corpus endometrioid carcinoma, uveal melanoma, lip melanoma, spindle cell carcinoma, liposarcoma, nasal sarcoma, breast adenocarcinoma, insulinoma, osteosarcoma, mastocytoma, angiosarcoma, non-small cell lung cancer (NSCLC), marginal lymphoma, malignant melanoma or chronic lymphocytic leukemia. In some embodiments, the epitope can 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 can be derived from DLL3, PSMA, SSTR2 or any combination thereof.

[0103] Other examples of proteins from which epitopes may be derived may include, but are not limited to, exotoxin A, Epstein-Barr nuclear antigen 4, melanoma antigen 1 recognized by T cells, 65 kDa phosphoprotein, immediate early protein IE1, transactivator protein BZLF1, prostate specific antigen, cell tumor antigen p53 (UniProt: A0A0U1RQC9), envelope glycoprotein B, melanoma associated antigen 1, tyrosinase, Epstein-Barr nuclear antigen 1, protein K8.1, spike glycoprotein, latent membrane protein 1, protein Tax-1, Epstein-Barr nuclear antigen 3 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 anchor protein domain family member F, envelope glycoprotein B, protein LANA1, GTP binding protein GEM, envelope glycoprotein gp63, Epstein-Barr nuclearantigen 6 (Epstein-Barr nuclearantigen 6), latent membrane protein 2, small capsid protein 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: 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 virilizer homolog), protein E7, immunoglobulin heavy chain constant γ1 (fragment) (UniProt: A0A0A0MS08), immunoglobulin heavy chain constant γ1, immunoglobulin heavy chain constant γ4, endoplasmic reticulum molecular chaperone BiP, transferrin receptor protein 1 (UniProt: P02786), cell 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, TCRγ alternative reading frame protein, 60S ribosomal protein L28 (UniProt: P46779), glutamate carboxypeptidase 2, cell tumor antigen p53 (UniProt: E7EQX7), DNA (cytosine-5)-methyltransferase 1, catenin β-1 (Catenin β-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 α chain, non-erythroid 1 (UniProt: Q13813), HLA class II histocompatibility antigen DR β 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 70kDa 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: P62424), ot: Q8WZ42), cancer / testis antigen 1, cell tumor antigen p53 (UniProt: J3KP33), mucin-1 (UniProt: A0A0C4DGW3), type I cytoskeletal keratin 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 carrier 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 protein 1 (UniProt: Q92896), elongation factor 1-α1, elongation factor 1-α2, tyrosine protein kinase JAK1, DEK (UniProt: P35659) and nuclear pore complex protein Nup214 (UniProt: P35658), DNA-directed RNA polymerase II subunit RPB1, myeloid cytokinin (UniProt: P24158), phosphatidylinositol 5-phosphate 4-kinase type 2 alpha (UniProt: P48426), RNA helicase (UniProt: P48427), and proteasome ubiquitin receptor 1 (UniProt: Q16186), epidermal growth factor receptor, Golgi protein 1 (UniProt: Q92896), elongation factor 1-α1, elongation factor 1-α2, tyrosine protein kinase JAK1, DEK (UniProt: P35659) and nuclear pore complex protein Nup214 (UniProt: P35658), DNA-directed RNA polymerase II subunit RPB1, myeloid cytokinin (UniProt: P24158), phosphatidylinositol 5-phosphate 4-kinase type 2 alpha (UniProt: P48426), RNA helicase (UniProt: P48427). :A0A7I2V2S0), cytochrome c oxidase subunit 2, RNA binding protein NOB1 (UniProt: Q9ULX3), dermatan sulfate isomerase (UniProt: Q9UL01), dermatan sulfate isomerase (UniProt: A0A2R8YE23), squamous cell carcinoma antigen recognized by T cells 3 (UniProt: A0A499FI31), squamous cell carcinoma antigen recognized by T cells 3 (UniProt: Q15020), peptidylprolyl cis-trans isomerase B, melanoma-associated antigen 10, secretory protein BARF1, triosephosphate isomerase, 3β-hydroxysteroid dehydrogenase type 7, HLA Class II histocompatibility antigen, DPα1 chain, calreticulin (UniProt: P27797), minor capsid protein L2, filamentous protein A (Filamin-A) (UniProt: P21333), α-2-macroglobulin, telomerase reverse transcriptase, cytochrome b, HLA class II histocompatibility antigen, DRβ5 chain, chondroitin sulfate proteoglycan 4, follistatin-related protein 1, desmoglein-3, T cell receptor β 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-α, importin-8, possible G protein-coupled receptor 139, KICSTOR complex protein SZT2, ATP-citrate synthase, heterogeneous nuclear ribonucleoprotein U (UniProt: Q5RI18), Hsp90 Co-chaperone Cdc37-like 1 (UniProt: Q7L3B6), protein E7, protein E4, regulatory protein E2, possible protein E5, regulatory protein E2, minor capsid protein L2, protein E6, protein E6, 14-3-3 protein gamma, polyadenylate binding protein 1, serine / threonine protein kinase SMG1, protein transporter Sec24D (UniProt: O94855), interferon-induced double-stranded RNA-dependent protein kinase activator A, signal transducer and activator of transcription 1-α / β (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-related endogenous 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 H2B1 type-D, THO complex subunit 4 (UniProt: Q8IXJ6). t: 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, translation / transcription domain-associated protein (UniProt: H0Y4W2), sodium / potassium transporting ATPase subunit alpha-3 (UniProt: P13637), heterogeneous nuclear ribonucleoprotein L-like (fragment), neuronal membrane glycoprotein M6-b (UniProt: Q13491), von Willebrand factor, α-synuclein (UniProt: A0A669KB41), transcondensin-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 PRR C1, histone acetyltransferase p300, sterol O-acyltransferase 1, peroxiredoxin-5 (mitochondrial), 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-CoAδ isomerase 2 (UniProt: O75521), cytochrome P450 2E1, collagen α-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 β chain (UniProt: P07437), heparan sulfate 2-O-sulfotransferase 1, glycoprotein-C, ATP-dependent RNA helicase DDX3X (UniProt: O00571), unconventional myosin-Ig (UniProt: B0I1T2), tubulin β-8 chain, possibly 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 activating factor-like-3, melanoma antigen preferentially expressed in tumors, possible ubiquitin carboxyl-terminal hydrolase FAF-X, ATP-dependent RNA helicase DDX ... rot: Q92499), α-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 activating factor 5B, Rho-associated GTP-binding protein RhoC, transforming protein RhoA (UniProt: C9JX21), WD repeat-containing protein 75 (UniProt: Q8IWA0), protein containing multiple C2 and transmembrane domains 1, kinesin-like protein KIF16B (UniProt: A0A1B0GTU3), targeting protein of Xklp2, titin (Titin) (UniProt: A0A0A0MTS7), NBAS subunit of NRZ tethering complex (NBAS subunit of NRZ tethering complex), ORM1-like protein 1, protein TASOR2 (UniProt: Q5VWN6), interleukin-4 receptor subunit alpha (UniProt: P24394), asparagine endopeptidase (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), 72kDa type IV collagenase,Calreticulin (UniProt: K7EL50), Macrosialin, Fibromodulin (UniProt: Q06828), Lumican, Malate dehydrogenase, cytoplasmic (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 [carboxylating] (U niProt: Q15274), procollagen lysine, 2-oxoglutarate 5-dioxygenase 2 (UniProt: O00469), Golgi-specific brefeldin A resistance guanine nucleotide exchange factor 1 (UniProt: Q92538), E3 ubiquitin protein ligase RNF126, Abl interacting protein 2 (UniProt: Q9NYB9), protein NLRC5, ATP-dependent DNA helicase DDX11 (UniProt: Q96FC9), E3 ubiquitin protein ligase TRIM9, Tripartite containing protein 67 (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 γ (fragment) (UniProt: Q5T6K5), E3 ubiquitin protein ligase UBR1, nuclear pore complex protein Nup107 (UniProt: P57740), neurocan core protein (UniProt: P57741), protein), replication termination factor 2 (UniProt: Q9BY42), terminal uridyltransferase 4 (UniProt: Q5TAX3), hyaluronan-mediated motility receptor, CDK5 regulatory subunit-associated protein 2 (UniProt: Q96SN8), T complex protein 1 subunit ζ, 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 chromatin subfamily B member regulator 1 (UniProt: Q12824), putative tRNA (cytidine (32) / guanosine (34)-2'-O)-methyltransferase (UniProt: Q9UET6),Acyl CoA A6-desaturase, 116 kDa U5 small nuclear ribonucleoprotein component, nucleolar protein 56 (UniProt: O00567), nuclear pore complex protein 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, Golgi protein subfamily B member 1, bombesin receptor-activated protein C6orf89 (Bombesin receptor-activated protein C6orf89), chromosome structure maintenance protein (UniProt: G8JLG1), Erbin (UniProt: Q96RT1), transmembrane protein 161B (UniProt: B7Z6T3), E3 ubiquitin protein ligase RNF213 (UniProt: Q63HN8), neutral α-glucosidase AB (UniProt: Q14697), neutral α-glucosidase AB (UniProt: E9PKU7), equilibrative nucleobase transporter 1, Lengsin, protein disulfide isomerase A6, nuclear pore complex protein Nup93 (UniProt: Q8N1F7), homocysteine-responsive endoplasmic reticulum-resident ubiquitin-like domain member 1 protein, short transient receptor potential channel 4-associated protein, vacuolar protein sorting-associated protein 51 homolog, mothers against decapentaplegic homolog 5 (SMAD5) (Mothers against decapentaplegic homolog 5), Chromosome domain-helicase-DNA binding protein 4, Integrin β-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 anchoring protein 13 (UniProt: Q12802), Midasin, Ras GTPase activating protein binding protein 2 (UniProt: Q9UN86), Cyclin 27 homolog (UniProt: P30260), Clathrin heavy chain 1, Solute carrier family 2, Facilitated glucose transporter member 1, U3 small nucleolar RNA associated protein 4 homolog, Dedicator of cytokinesis protein 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, chromosome structure maintenance protein 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 short isoform (UniProt: P63092), protein Ni pSnap homolog 2, chromosome structure maintenance flexible hinge domain-containing protein 1, E3 ubiquitin protein ligase MYLIP, protein transporter Sec31A (UniProt: O94979), protein transporter Sec31A (UniProt: D6RHZ5), distribution protein (Dysferlin), Pecanex-like protein 2, transducin-like enhancer protein 2 (UniProt: Q04725), transducin-like enhancer protein 4 (UniProt: Q04727), gamma-interferon-induced 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 domain-containing protein 12, signal-induced proliferation-related 1-like protein 3, axon-associated protein (Spatacsin) (UniProt: Q96JI7), protein folding chaperone protein (Sacsin) (UniProt: Q9NZJ4), iron response element binding protein 2, E3 ubiquitin protein ligase UBR4 (UniProt: Q5T4S7), heterogeneous nuclear ribonucleoprotein Q (UniProt: A0A7I2V5Q6), γ-tubulin complex component (UniProt: F2Z2B9), γ-tubulin leukocyte complex component 2 (UniProt: Q9BSJ2), E3 ubiquitin-protein ligase MYCBP2, insulin-induced gene 1 protein, insulin-induced gene 1 protein (fragment), nuclear protein TPR, fasciculation and elongation protein ζ-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-inhibited guanine nucleotide exchange protein 2 (UniProt: Q9Y6D5), protein Mpv17 (UniProt: P39210),Septin-9, ubiquitin-conjugating enzyme E2 variant 3 (UniProt: Q8IX04), neurofibromin (UniProt: P21359), dynein axoneme intermediate chain 3 (UniProt: Q8IWG1), exportin-7 (UniProt: Q9UIA9), cytoplasmic carboxypeptidase 1, T complex protein 1 subunit delta, RNA polymerase II transcription factor subunit 13 mediator (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 Arrest-specific protein 7, M phase inducer 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), Mu skelin (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 editing enzyme 1, transcription factor Dp-2, histone-lysine N-methyltransferase 2C, cell division cycle-related 7-like protein, F-box / WD repeat-containing protein 11 (UniProt: Q9UKB1), store-operated calcium influx-related regulator Factor, histone-lysine N-methyltransferase EZH2, myeloid-related differentiation markers, 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 (nucleolar)-like (isoform CRA_b), melanoma-associated antigen F1, serine / threonine protein kinase ATR,Dynein axonemalheavy chain 17, inositol 1,4,5-triphosphate receptor type 1, possible E3 ubiquitin-protein ligase HERC1, double-strand break repair protein rad21 homolog, intercellular adhesion molecule 2, bent neck-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 anchoring protein 9 (UniProt: Q99996), ubiquitin carboxyl-terminal hydrolase 47, polyadenylate binding protein 3, zinc finger protein 280C, COP9 signalosome complex subunit 2 (UniProt: P61201), endonuclease G (mitochondrial), ribosomal L1 domain-containing protein 1, MA X gene-associated protein, mitochondrial tRNA methylsulfide transferase CDK5RAP1, possible 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 (mitochondrial) (UniProt: P14406), heparin-α-glucosaminide N-acetyltransferase (UniProt: Q68CP4), possible ATP-dependent RNA helicase DHX37, coronin-7 (UniProt: P57737), CREB binding protein, Polycomb protein EED (UniProt: P14406), heparin-α-glucosamine ... EED)(UniProt: O75530), integrin complex subunit 13, paired amphipathic helical protein Sin3a(UniProt: Q96ST3), clathrin interacting protein 1, ubiquitin-like modifier gene activating enzyme 1(UniProt: P22314), protein tyrosine phosphatase C-type receptor-associated protein, cyclin-dependent kinase 6, Ran binding protein 9, integrin α-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 protein 3 (UniProt: Q9Y6A5), eIF-2-α kinase activator GCN1, transcription adapter 1, reticulin-4 receptor (fragment) (UniProt: H7C0V4), P2X purinergic receptor (UniProt: K7EQ78), Gag protein, Aggrecan core 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), maternal anti-limb palsy homolog 4, mitogen-activated protein kinase (UniProt: K7EN18), E3 ubiquitin protein ligase TRIM23, glycerol-3-phosphate dehydrogenase (mitochondrial), casein kinase I isoform ε (UniProt: P49674), kinesin-like protein KIF2C, small plaque-type POZ protein (UniProt: O43791), U4 / U6.U5 triple snRNP-associated protein 2 (UniProt: K7EN18). ot: Q53GS9), SWI / SNF-related matrix-associated actin-dependent chromatin family A member 5 regulator, Kelch-like ECH-related protein 1, serine palmitoyltransferase 2 (fragment), protein mono ADP ribosyltransferase PARP3, filamentous enhancer factor 1 (UniProt: Q14511), sister chromatid cohesion protein PDS5 homolog A, β-1,3-galactosyl-O-glycosyl-glycoprotein β-1,6-N-acetylglucosaminyltransferase, general transcription factor 3C polypeptide 2 (UniProt: Q8WUA4), myotubularin, Citron Rho interacting kinase (UniProt:O14578), poly [ADP-ribose] polymerase (UniProt:E9PNI7), transport and Golgi organizing protein 6 homolog, protein transporter Sec24A, MMS19 nucleotide excision repair protein homolog, proteasome activation complex subunit 4 (UniProt:Q14997), myocyte-specific enhancer factor 2B (UniProt:Q02080), transient receptor potential cation channel subfamily M member 8 (UniProt:Q7Z2W7), RhoGTPase 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 (Adenomatous polyposis coli protein), Nck-related protein 1, RNA polymerase II transcription subunit 15 mediator (UniProt: G3V1P5), DNA excision repair protein ERCC-6-like (UniProt: Q2NKX8), HIV Tat specificity factor 1, negative elongation factor A (fragment) (UniProt: H0Y3X6), negative elongation factor A (UniProt: Q9H3P2), cytochrome P450 7B1, flagellar transport protein 80 homolog, CIZ1 protein, FAST kinase domain-containing protein 1 (mitochondrial),Cytochrome c oxidase subunit 4 isoform 1 (mitochondria), ATP synthase subunit g (mitochondria) (UniProt: O75964), dynein axoneme assembly factor 5 (UniProt: Q86Y56), eukaryotic translation initiation factor 2 subunit 1 (fragment), LisH domain-containing protein ARMC9 (UniProt: A0A804HK42), exosome-2 (UniProt: Q93063), dynein binding protein (UniProt: Q6XZF7), protein containing three motifs 26, protein kintoun, lipid droplet protein-2 (Perilipin-2) (UniProt: Q99541), quinone oxidoreductase, protein PAT1 homolog 1, neurobeachin-like protein 2 (Neurobeachin-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), neural Wiskott-Aldrich syndrome protein, cocaine esterase, casein kinase I isoform α(UniProt:P48729), BCL-6 co-repressor(UniProt:Q6W2J9), putative Polycomb group protein ASXL3(PutativePolycomb group protein ASXL3)(UniProt: Q9C0F0), retinoblastoma-like protein 2, Eyes absent homolog 3, Protein enabled homolog)(UniProt: Q8N8S7), integrin β-4, F-actin capping protein subunit β(UniProt: P47756), endoplasmic reticulum resident protein 29, 4F2 cell surface antigen heavy chain(UniProt: P08195), transketolase(UniProt: P29401), nuclear receptor co-repressor 1(UniProt: A0A088AWL3), RanBP-type and C3HC4-type zinc finger protein 1(UniProt: Q9BYM8), cell cycle and apoptosis regulatory protein 2(UniProt: Q8N163), B cell receptor-associated protein(fragment)(UniProt: C9JQ75), E3 ubiquitin protein ligase Praja-2, guanylate binding protein 1,Chromatin targets of PRMT1 protein (UniProt: Q9Y3Y2), engulfment and cell movement 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), helicase with zinc finger domain 2, DNA (cytosine-5)-methyltransferase 3A, mitogen-activated protein kinase kinase kinase kinase 4 (MAPK4) ... kinetochore protein Nuf2 (UniProt: Q9Y281), kinetochore protein Nuf2 (UniProt: Q9BZD4), trans-3-hydroxy-L-proline dehydratase (UniProt: Q96EM0), kinetochore protein Nuf2 (UniProt: Q9Y281), kinetochore protein Nuf2 (UniProt: Q9BZD4), trans-3-hydroxy-L-proline dehydratase (UniProt: Q96EM0), helicase with zinc finger domain 2 kinase4)(UniProt: G5E948), phospholipid transporter ATPase IF(UniProt: Q9Y2G3), coatomer subunit γ-2, MAP kinase interacting serine / threonine protein kinase 1(UniProt: A0A499FIS5), nuclear phosphoprotein(UniProt: P06748), BTB / POZ domain-containing protein 7(UniProt: Q9P203), melanoma inhibitory activity protein White 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 1, RAD50 interacting protein 1, leucine-rich repeat-containing protein 42, ATP-dependent DNA / RNA helicase DHX36, TRAF-type zinc finger domain-containing protein 1, ATP-dependent RNA helicase DDX19B , AT-rich interaction 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 γ (UniProt: Q96EK7), transmembrane protein 214 (UniProt: Q6NUQ4), E3 ubiquitin-protein ligase RNF38, protein farnesyltransferase subunit β (UniProt: Q6NUQ4), mitochondrial isomerase 1 (UniProt: Q14865 ... iProt: B4DL54), chromosome transmission fidelity protein 18 homolog (UniProt: Q8WVB6), glutathione S-transferase κ, 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 TRIP12 (UniProt: A0A6Q8PGG9), FAST kinase domain-containing protein 4,Serine / threonine protein kinase WNK2 (UniProt: Q9Y3S1), nonspecific serine / threonine protein kinase (UniProt: F8W9F9), chromosome structure maintenance protein 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), endoplasmic reticulum protein (Endoplasmin) (UniProt: A0A7P0TAY2), D mX-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, zona pellucida adhesion protein (Zonadhesin), Fanconi anemia M group protein, protein FAM3C, Alstrom syndrome protein 1 (UniProt: Q8TCU4), cyclic AMP-dependent transcription factor ATF-6β (UniProt: Q9994 1), 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), G protein pathway inhibitor 2, Matrilysin, Dual specificity protein phosphatase 3, Serine / threonine protein kinase PAK3, Damage control phosphatase ARMT1, HEAT repeat-containing protein 1 (UniProt: Q9H583), ATP synthase F(0) complex subunit C2 (mitochondria), Myeloid Matrix antigen 2, cell division cycle 5-like protein, nonsense transcript 3A regulator, 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-neurocortical protein 1, bromodomain-adjacent zinc finger domain protein 1A, neuroepithelial cell transformation gene 1 protein, calcium load-activated calcium channel (UniProt: Q9UM00), peroxisome 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 6 B, histone-lysine N-methyltransferase 2D, zinc finger protein 292 (UniProt: J3KNV1), endoplasmic reticulum transmembrane helical translocase, α-methylacyl CoA racemase (UniProt: Q9UHK6), receptor-type tyrosine protein phosphatase ζ, possible 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), endoplasmic reticulum lumen protein (ER lumen protein), retention 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), acidic leucine-rich nuclear phosphoprotein 32 family member A (UniProt: P39687), 55kDa centrosomal protein, solute carrier 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), alkylglycerophosphoprotein Acid synthase (UniProt: A0A2R8YEL0), possible phospholipid transport ATPase IIA, KIF binding protein (UniProt: Q96EK5), nuclear valine-containing protein-like (UniProt: O15381), inactive histone lysine N-methyltransferase 2E (UniProt: Q8IZD2), C-terminal binding protein 1, bromodomain adjacent zinc finger domain protein 2B (UniProt: Q9UIF8), tumor protein 63, helicase SKI2W, serine protein kinase ATM (UniProt: Q13315), STAGA complex 65 subunit γ, endoplasmic reticulum aminopeptidase 1, NPC intracellular cholesterol transporter 1, synaptojanin-2,Nonspecific serine / threonine protein kinase (UniProt: A0A804HLI0), Kalirin (UniProt: O60229), Ras inhibitory protein 1, neuronal 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: Q5J PF3), RecQ-mediated genome 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), mTOR regulatory-associated protein, phospholipid transporter ATPase VD, glutamine-fructose-6-phosphate aminotransferase [isomerization] 2, interferon regulatory factor 5, DmX-like protein 2 (UniProt: Q8TDJ6), Pecanex-like protein 3, possible ATP-dependent RNA helicase DDX31, general transcription factor IIE subunit 1, protein containing disintegrin and metalloproteinase domains 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 phosphatases 1, solute carrier family 2, facilitating glucose transporter member 5, Tau-tubulin kinase 2 (UniProt: Q6IQ55), Kelch-like protein 29, transcription enhancer factor TEF-3 (UniProt: A0A0A0MRF3), PRELI domain-containing protein 1 (line 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 seven-way G-type receptor 1(UniProt: A0A6I8PRU0), cadherin EGF LAG seven-way G-type receptor 3, cadherin EGF LAG seven-way 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), Ras GTPase-activating protein nGAP, Ryanodine receptor 2 (UniProt: Q92736), Pro-cathepsin H (UniProt: Q13162), and mitochondrial kinase (UTP) (UniProt: Q13163). H) (UniProt: A0A7I2V3T9), baculovirus IAP repeat-containing protein 5 (UniProt: H3BLT4), baculovirus IAP repeat-containing protein 5 (UniProt: A0A0B4J1S3), interleukin-1β, eukaryotic translation initiation factor 4E binding protein 1, melanoma-associated antigen C2, thioredoxin-dependent peroxiredoxin (mitochondrial), endothelin receptor non-selective type, actin-associated protein 10 (UniProt: Q9NZ32), protocadherin-8, sterile alpha motif domain-containing protein 9, ubiquitin carboxylase terminal hydrolase 37, cytochrome c oxidase assembly factor 3 homolog (mitochondria), interleukin-27 receptor subunit alpha, 3-oxoacyl-[acyl-carrier-protein] synthase (mitochondria), phosphomannose mutase 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), plasmalogen ethanolamine desaturase (Plasmanylethanolamine desaturase), homeobox protein TGIF2, zinc finger MYM-type protein 4, uncharacterized protein 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-α-carboxylate 3-dehydrogenase (decarboxylation), vacuolar protein sorting-associated protein 4B (UniProt: O75351), syncytin-1, ubiquitin-conjugating enzyme E2 D2, all-trans retinol 13,14-reductase, 28S ribosomal protein S5 (mitochondrial), 39S ribosomal protein L39 (mitochondrial), protein cysteine ​​N-palmitoyltransferase HHAT, transforming growth factor-β-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 α, isocitrate dehydrogenase [NADP] (cytoplasmic), sister chromatid cohesion protein PDS5 homolog B, protein kinase Cθ type,Isocitrate dehydrogenase [NADP] (mitochondria), extracellular sulfatase Sulf-2, nuclear protein localization protein 4 homolog (UniProt: Q8TAT6), CUB and sushi domain-containing protein 1 (UniProt: E5RIG2), GPI inositol deacylase (UniProt: Q75T13), Tenneurin-3, Mitogen-activated protein kinase kinase kinase 2 (UniProt: Q75T13), 2), transmembrane channel-like protein 8, serine / threonine protein kinase MRCKα, protein serine O-palmitoleyltransferase Porcupine, Xaa-Pro aminopeptidase 1, structure-specific endonuclease subunit SLX4, kinesin-like protein KIF26B (fragment), Y-linked neuroligin-4 (Neuroligin-4,Y-linked) (UniProt:Q8NFZ3), X-linked neuroligin-4 (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 mago nashi homolog 2 (Proteinmago nashi homolog 2)(UniProt: Q96A72), carbonic anhydrase 2, inactive hydroxysteroid dehydrogenase-like protein 1, protoporphyrinogen oxidase, dephospho-CoA kinase domain-containing protein, calcium uniporter (mitochondrial), armadillo repeat-containing protein 7, transmembrane protein 168, protein MMS22-like, mannosyl oligosaccharide 1,2-α-mannosidase IB, neuronal guidance factor 3 (UniProt: Q8IVL0), neuronal guidance factor 2 ( UniProt: A0A0A0MTL4), neuronal guidance factor 2 (UniProt: Q8IVL1), cytokinesis protein-specific protein 3, neuroblastoma breakpoint family member 11, serine / threonine protein phosphatase 4 regulatory subunit 1, γ-tubulin complex component 6 (UniProt: Q96RT7), vacuolar protein sorting-associated protein 8 homolog (UniProt: Q8N3P4), protein asteroid homolog 1, TRIO and F-actin binding protein, guanylate cyclase soluble subunit α-1, cryptochrome-1, adhesion G protein-coupled receptor A3, apoptosis regulator BAX (UniProt: Q07812), transmembrane protein 231, transmembrane protein 14B (UniProt: Q9NUH8), L-fucokinase,Phospholipid transporter ATPase ABCA1 (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 mTOR partner, integrin α-9 (UniProt: Q13797), adenylate kinase 7, Anoctamin-3, nuclear exosome regulator NRDE2, calcitonin Gene-related peptide receptor type 1, Golgin subfamily A member 2, receptor-interacting serine / threonine-protein kinase 3, Krev-interacting trap protein 1, argonaute-2 protein, RWD domain-containing protein 3, 60S ribosomal protein L5 (UniProt: A0A2R8Y4A2), X-linked protocadherin-11, cysteine- and glycine-rich protein 2, Schlafen family member 12-like, nebulin, α-1,3-mannosyl-glycoprotein 2-β-N-acetylglucosaminyltransferase, aldehyde dehydrogenase family 16 member A1 (UniProt: Q8IZ83), disintegrin and metalloproteinase with thrombospondin motif 7, unconventional myosin-Vb, MYCBP-related protein (UniProt: A0A140VK87), sodium / hydrogen exchange factor (UniProt: A0A7I2V2B0), mucin-4 (UniProt: A0A0G2JNL3), zinc finger FYVE domain-containing protein 9, possible ubiquitin carboxylase Terminal hydrolase MINDY-4, cytoplasmic protein NCK1, [pyruvate dehydrogenase (acetyltransferase)] kinase isozyme 3 (mitochondria), cyclin A1, α-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 (Dual specificitymitogen-activated protein kinase kinase4), adipocyte membrane-associated protein, CD9 antigen (UniProt: P21926), adhesion G protein-coupled receptor D1, cytoplasmic iron-sulfur assembly component 3, receptor-type tyrosine protein phosphatase β, RING finger protein 10, Myomegalin (UniProt: A0A075B749), NADH dehydrogenase [ubiquinone] flavoprotein 1 (mitochondrial) (UniProt: G3V0I5), phosphatidylinositol 3,4,5-triphosphate 3-phosphatase and dual-specificity protein phosphatase PTEN, solute carrier family 27 member 3,Amiloride-sensitive sodium channel subunit β, E3 ubiquitin-protein ligase HACE1, protease-activated receptor 3, metalloproteinase inhibitor 3, short-chain specific acyl-CoA dehydrogenase (mitochondrial), 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 4), protein FAM228B (UniProt: P0C875), Ras-related protein Rab-39B, protein 17B containing α / β hydrolase domain, protein 2 containing FAST kinase domain (mitochondrial), protein FAM83G, protein 2A containing SH3 and PX domains, mannose-6-phosphate isomerase (UniProt: P34949), triokinase / FMN cyclase, tectonin β-helical repeat protein 2, immunoglobulin white superfamily member 2, signal regulatory protein γ, segment polarity protein Dishevelled homolog DVL-2, Arf-GAP with SH3 domain, ANK repeat and PH domain protein 1 (UniProt: Q9ULH1), EF-hand calcium binding domain protein 13, phospholipid transporter ATPase IK (UniProt: O60423), splicing factor 3A subunit 2, glutamine-tRNA synthetase (fragment), poly (ADP-ribose) glycosyl hydrolase, Ephrin Type B receptor 1, Protrudin, Proline-rich protein 7, C-Jun-amino-terminal kinase interacting protein 2, Unconventional myosin-Ib (UniProt: E9PDF6), Transcription enhancer factor TEF-1 (UniProt: P28347), Protocadherin γ-C5, Zinc finger protein 407, Zinc finger protein 3, Pentraxin-4, Rho GTPase activating protein 29 (UniProt: Q52LW3), Plasma membrane calcium transporting ATPase 4, 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, Serine protease inhibitor B3 (Serpin B3), receptor protein tyrosine kinase (UniProt: Q504U8), receptor protein tyrosine kinase (UniProt: E9PFD7), neutral and basic amino acid transporter rBAT (UniProt: Q07837), protein FAM161A (fragment), leucine-rich repeat transmembrane protein CCDC168, serine protease inhibitor B4,Epithelial discoidin domain-containing receptor 1, serine / threonine protein kinase 36, collagen alpha-1 (XVIII) chain, DCN1-like protein 4 (UniProt: Q92564), possible ATP-dependent RNA helicase DDX60-like (UniProt: A0A804HKC9), glutamate receptor ionotropic, kainic acid 3, Myb-related protein B, NADH dehydrogenase [ubiquinone] iron-sulfur protein 2 (mitochondrial), threonine aspartase 1, testosterone (Testin), transcription factor SOX-10, doublecortin and CaM kinase-like 2 (isoform CRA_c), very long-chain fatty acid elongation protein 2, Pappalysin-2, forkhead box protein O1, inactive tyrosine-protein kinase PRAG1, splicing regulatory glutamine / lysine-rich protein 1 (UniProt: Q8WXA9), β-1,4-glucuronyltransferase 1, xylosyl and glucuronyltransferase LARGE1, SH3 domain binding protein 2, Liprin-β-1 (UniProt: Q86W92), 5-oxoprolylase, cancer / testis antigen 2, solute carrier 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, 55 kDa erythrocyte membrane protein, protein dopey-2, N-terminal kinase-like protein (UniProt: Q96KG9), proton inositol cotransporter (UniProt: Q96QE2), Arf-GAP with GTPase, ANK repeats and PH domain protein 3 (UniProt: Q96P47), zinc finger protein 423, phospholipid transporting ATPase (UniProt: A0A5K1VW70), roundabout homolog 1 (UniProt: Q96QE2), phospholipid transporting ATPase (UniProt: Q96QE2), phospholipid transporting ATPase (UniProt: Q96QE2), phospholipid transporter ... homolog 1)(UniProt: Q9Y6N7), spire protein homolog 1(UniProt: Q08AE8), transcription factor ETV7, small G protein signaling regulator 3, hyaluronidase-2, autism susceptibility gene 2 protein(UniProt: Q8WXX7), hormone-sensitive lipase, protein FAM50B, signal regulatory protein δ(UniProt: Q9H106), zinc finger protein 385C, tumor necrosis factor receptor superfamily member 11A, prickle-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, motile sperm domain-containing protein 1,Transmembrane protein KIAA1109 (fragment) (UniProt: H7C0G8), mitogen-activated protein kinase 13, protein containing caspase recruitment domain 16, glutamate receptor 4, receptor-type 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, elongation factor-C (Elongin-C) (UniProt: Q15369), isobutyryl-CoA dehydrogenase (mitochondrial), centromere protein L, transmembrane protease serine 6, WD repeat protein 87 (UniProt: Q6ZQQ6), focal adhesion protein (Supervillin) (UniProt: O95425), Sushi von Willebrand disease (von Willebrand factor type A, EGF and pentraxin domain-containing protein 1 (UniProt: Q4LDE5), protein BANP (UniProt: Q8N9N5), Derlin-2 (UniProt: Q9GZP9), zinc finger protein 7 (UniProt: P17097), HLA class II histocompatibility antigen (DRα chain) (UniProt: Q30118), Polycomb group RINGfinger protein 5 (UniProt: Q4LDE5), protein BANP (UniProt: Q8N9N5), Derlin-2 (UniProt: Q9GZP9), zinc finger protein 7 (UniProt: P17097), HLA class II histocompatibility antigen (DRα chain) (UniProt: Q30118), Polycomb group RINGfinger 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 nucleotidyl transferase 5D, nuclear receptor subfamily 4 group A member 1, laminin subunit alpha-5, cystic fibrosis transmembrane conductance regulator, hydrocephalus-inducing protein homolog, alpha-parvin, tubulin delta chain, tenascin-R, ankyrin and armadillo repeat-containing protein (UniProt: Q7Z5J8), MORC family CW Type 1 zinc finger protein, KAT8 regulatory NSL complex subunit 1 (UniProt: A0A0G2JNB1), PWWP domain-containing protein 2A, UPF0687 protein C20orf27, SET binding protein (UniProt: Q9Y6X0), DNA repair protein RAD51 homolog 2 (UniProt: O15315), sodium / inositol cotransporter 2, Pleckstrin homology domain-containing family A member 4, Neurofascin (UniProt: O94856), dystrophin β (Dystrobrevinβ), 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 B (0+), unconventional myosin-XVIIIa (UniProt: A0A0D9SFK2), transmembrane 4-domain subfamily A member 18 (UniProt: A0A499FJ34), protein phosphatase Slingshot homolog 1, dehydrogenase / reductase SDR family member 1, eye-deficient homolog 2, importin 13 (UniProt: O94829), Golgi reorganizing stacking protein 2, DNA-dependent metalloproteinase SPRTN, enoyl-CoA hydratase, protein plasmid WWC2, possible asparagine-tRNA ligase (mitochondria), sodium / glucose co-transporter 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), Possible 2-ketoglutarate 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 MTSS2 (UniProt: Q765P7), aldehyde dehydrogenase X (mitochondria), protein Niban2, protein FAM200A, breast cancer anti-estrogen resistance protein 1 (UniProt: P56945), amino acid transporter (UniProt: A0A087X0U3), maestro protein (UniProt: Q765P7). t: Q9BYG7), sodium- and chloride-dependent creatine transporter 1, 1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase delta-3, constitutive coactivator of PPAR-γ-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 enhancing protein 5, acid sphingomyelinase-like phosphodiesterase 3b, Junctophilin-2, tyrosine protein phosphatase non-receptor type 3, V-type proton ATPase 21kDa 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-β 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-protein kinase transmembrane receptor ROR1 (UniProt: Q01973), methionine-R-sulfoxide reductase B2 (mitochondrial), ENTPD4 protein, endothelin-converting enzyme 2, EEF1AKMT4-ECE2 readthrough transcript ( transcript), homeobox protein Meis1 (UniProt: H0YNY8), BRD4-interacting chromatin remodeling complex-associated protein-like, ubiquinone biosynthesis O-methyltransferase (mitochondrial) (UniProt: Q9NZJ6), integrin β-5, Frizzled-6 (UniProt: O60353), specific androgen-regulated gene protein, CTD nuclear envelope phosphatase 1, growth arrest-specific protein 6, DNA-guided RNA polymerase III subunit RPC7-like, Exophilin-5, fibroblast growth factor receptor 2, coiled-coil domain-containing protein 120, protein chib by homolog 2, kallikrein-6, villin-like protein (Villin-like protein) (UniProt: O15195), laminin subunit alpha-4 (UniProt: Q16363), SH3 and multiankyrin repeat domain protein 2 (UniProt: Q9UPX8), inositol bisphosphate polyphosphate phosphohydrolase 3-alpha, 131kDa centrosomal protein (UniProt: Q9UPN4), leucine zipper putative tumor suppressor 1, olfactory receptor 2T3, HCG1811249 (isoform CRA_e), solute carrier family 25 member 48, phosphodiesterase (UniProt: A1E5M1),Integrin α-7 (UniProt: Q13683), Gem-associated 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 adenosyltransferase FICD, zinc finger protein 785, metalloreductase STEAP1, mucolipin-2, kynurenine carboxamidase (UniProt: K7EK09), Protein diaphanous homolog 1 (Protein diaphanous homolog 1), 1)(fragment), SPARC, Cytospin-B (UniProt: A0A7I2YQJ3), zinc finger protein 764, serine protease hepsin, olfactory receptor 5K3, inactive phospholipase D5, solute carrier family 22 member 9, mucin-6 (UniProt: Q6W4X9), transcription factor SOX-30, killer cell immunoglobulin-like receptor 2DS4, serine / threonine protein kinase Nek10, HERV-H_2q24.3 proviral ancestor 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. 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 carrier family 5 member 4, 1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase β-4, vesicle transport protein SEC22c (UniProt: Q9BRL7), GPNMB protein, α-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), lysosomal-associated transmembrane protein 4B (fragment), Teashirt 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, Prickle planar cell polarity protein 3 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, chromosome biogenesis protein 1 in cell division, transmembrane protein 74B, E3 ubiquitin protein ligase RNF149(UniProt:F8WCD0), choline transporter-like protein 5, ATP5MF-PTCD1 readthrough, guidance protein-3G(Semaphorin-3G), olfactory receptor 5P2, transient receptor potential cation channel subfamily M member 4, putative protein FAM157B, G protein signaling regulator 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, phosphatidylinositol proteoglycan-1 (Glypican-1) (UniProt: P35052), olfactory receptor 4C6, serine protease 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 regulatory factor 1, double zinc ribbon and ankyrin repeat-containing protein 1 (UniProt: Q9NVP4), sodium-independent sulfate anion transporter, glutamate receptor ionotropic (NMDA 3A), two components: latent membrane protein 2 and latent membrane protein 2, two components: Epstein-Barr virus nuclear antigen 3 and Epstein-Barr virus nuclear antigen 3,Two components: 65 kDa phosphoprotein and 65 kDa phosphoprotein, 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: P22303), olfactory receptor 2T4, paraneoplastic antigen-like protein 8A, malonyl CoA acyl carrier protein transacylase (mitochondrial), calcium-binding mitochondrial carrier protein SCaMC-3 (fragment), nerve growth factor 1 (Netrin-1), transmembrane protein 79 (UniProt: Q 9BSE2), 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 neighboring 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, serotonin receptor 3D (UniProt: Q70Z44), angiotensin converting enzyme 2, transcription factor SOX-2, scavenger receptor rich cysteine B group protein with amino acid domain, lymphocyte antigen 6K, embryonic Fyn-related substrate, G protein signaling regulator 5 (UniProt: O15539), disintegrin and metalloproteinase with thrombospondin motif 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 fibronectin type III domain-containing protein 3, synaptic fusion Syntaxin-1A (UniProt: Q16623), adenylate cyclase type 4, DNA repair factor 3A containing PWWP domain (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), trophin (Trophinin) (UniProt: A0A087X070), short transient receptor potential channel 4, sodium-dependent dopamine 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, Igγ-2A chain C region (membrane-bound form), podoplanin, Liprin-α-4 (UniProt: O75335), signal peptide, protein containing CUB and EGF-like domains 2, tight junction protein 18, adrenocorticotropin-releasing factor binding protein (UniProt: D6RHH7), β-1,4-N-acetylgalactosamine transferase 3, solute carrier family 22 member 17 (UniProt: D6RHH7), ot:Q8WUG5), armadillo repeat protein missing in Velo-Cardio-Facial 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 (Sorting nexin-16)(UniProt:A0A0C4DGW2), distal membrane arm assembly complex protein 2(UniProt:B4DFT4), olfactory 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-acetyllactosamine 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 serine protease inhibitor A13, coiled-coil domain-containing protein 74B (fragment), keratin-related protein 11-1, blood group Rh (CE) polypeptide (UniProt: E7EQ47), protein SSX7, melatonin-related receptor, paired mesoderm homeobox protein 2A, ankyrin repeat domain-containing protein 36B, protein FAM83H (UniProt: A0A494C1T9), homeobox protein Hox-A1 (UniProt: E7ERT8),Golgi subfamily A member 6-like protein 2 (UniProt: Q8N9W4), PRAME family member 4, PRAME family member 9, leukocyte-derived chemokine-2, proprotein convertase subtilisin / kexin type 5 (UniProt: B1AMG5), olfactory receptor 5T3, olfactory receptor 4C3, phosphoinositide phospholipase C (UniProt: A0A3B3ISW9), calpain-11 (fragment), LILRA6, family with sequence similarity 53 member A (isoform CRA_b), C-type lectin domain family 18 member C, uncharacterized protein C9o rf57, 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 K group member 18 Env polyprotein, very long chain fatty acid ω-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 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: B0QYN8). t: A0A0A0MTJ9), sodium / glucose co-transporter 4, possible cysteine-tRNA ligase (mitochondrial) (fragment) (UniProt: F5H579), uncharacterized protein (UniProt: A0A494C1I1), HCG2044781, P antigen family member 4, two-component: HLA-A*02:01 chain and Wilms tumor protein (UniProt: A0A0A0MT54), two-component: immunoglobulin heavy chain constant gamma 1 and immunoglobulin heavy chain constant gamma 1, receptor tyrosine-protein kinase erbB-2, two-component: genomic polyprotein and genomic polyprotein or tight junction protein-18. ,

[0104] In some embodiments, the extracellular domain may include one or more epitopes. In some embodiments, the extracellular domain may include one or more epitopes homologous to the extracellular domain. In some embodiments, the extracellular domain may include one or more epitopes heterologous to the extracellular domain. In some embodiments, the extracellular domain may include about 1 to about 10 epitopes. In some embodiments, the extracellular domain may include at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or at least 10 epitopes. In some embodiments, the extracellular domain may include two or more copies of the same epitope. In some embodiments, the extracellular domain may include two or more epitopes, wherein each of the two or more epitopes is combined with the same antibody, peptide hormone or growth factor. For example, one or more copies of the same epitope sequence are inserted into the extracellular domain sequence that naturally includes a copy of the same epitope sequence.

[0105] In some embodiments, the extracellular domain may include two or more epitopes, wherein each of the two or more epitopes is a different epitope. In some embodiments, each of the two or more epitopes may be bound to the same antibody, peptide hormone or growth factor. In some embodiments, each of the two or more epitopes may be bound to different antibodies, peptide hormones or growth factors. In some embodiments, the extracellular domain may include two or more epitopes, wherein two or more epitopes may include a combination of the same and different epitopes. For example, the extracellular domain may include at least four epitopes, wherein two of the at least four epitopes are bound to a kind of antibody, peptide hormone or growth factor, and the other two of the at least four epitopes are bound to another kind of antibody, peptide hormone or growth factor.

[0106] In some embodiments, the epitope may comprise about 15 to about 300 consecutive residues of the extracellular domain of a protein described herein. In some embodiments, the epitope may 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 consecutive residues of the extracellular domain of a protein described herein.

[0107] In some embodiments, the epitope can comprise a sequence that is 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% identical to any one of SEQ ID NO:4, 5, 6, 18, 19, 20, 64, 65, or a variant thereof. In some embodiments, the epitope may comprise a sequence having at least 80% sequence identity to any one of SEQ ID NO: 4, 5, 6, 18, 19, 20, 64, 65, or a variant thereof. In some embodiments, the epitope may comprise a sequence having 100% sequence identity to SEQ ID NO: 4, 5, 6, 18, 19, 20, 64, 65, or a variant thereof.

[0108] In one embodiment, the epitope may be derived from DLL3 or a variant thereof. In this embodiment, the DLL3 epitope may comprise about 15 to about 260 consecutive residues of the DLL3 extracellular domain. In some embodiments, the DLL3 epitope may 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 DLL3 extracellular domain. In some embodiments, the DLL3 epitope may comprise amino acid residues 27-492 of DLL3. In some embodiments, the DLL3 epitope can comprise amino acid residues 189-209 of DLL3. In some embodiments, the DLL3 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: 5, 6, 18, 19, 20, 64, 65, or a variant thereof. In some embodiments, the DLL3 epitope may comprise a sequence having at least 80% identity to SEQ ID NO: 5 or a variant thereof. In some embodiments, the DLL3 epitope may comprise a sequence having 100% sequence identity to SEQ ID NO: 5, 6, 18, 19, 20, 64, 65 or a variant thereof.

[0109] In one embodiment, the epitope may be derived from SSTR2 or a variant thereof. In this embodiment, the SSTR2 epitope may comprise about 15 to about 280 consecutive residues of the SSTR2 extracellular domain. In some embodiments, the SSTR2 epitope may 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 consecutive residues of the SSTR2 extracellular domain. In some embodiments, the SSTR2 epitope may comprise amino acid residues 39-314 of SSTR2. In some embodiments, the SSTR2 epitope may 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 may comprise a sequence having at least 80% identity to SEQ ID NO: 4 or a variant thereof. In some embodiments, the SSTR2 epitope can comprise a sequence having 100% sequence identity to SEQ ID NO: 4 or a variant thereof.

[0110] In one embodiment, the epitope may be derived from PSMA or a variant thereof. In this embodiment, the PSMA epitope may comprise about 15 to about 300 consecutive residues of the PSMA extracellular domain. In some embodiments, the PSMA epitope may 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 consecutive residues of the PSMA extracellular domain.

[0111] In some embodiments, the epitope can bind to the antibody, peptide hormone, or growth factor with a Kd of less than or equal to 20 nM. For example, the epitope can bind to the 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 can bind to the 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.

[0112] In some embodiments, the epitope described herein may include an activatable epitope. In some embodiments, compared with the physical microenvironment inside or outside the cell that is not affected by the disease or condition, the activatable epitope may be preferentially combined with a binding agent (e.g., an antibody, peptide hormone, or growth factor) in the physical microenvironment inside or outside the cell affected by the disease or condition. In some embodiments, the activatable epitope can be selectively used for binding in a tumor microenvironment. For example, compared with the physical microenvironment inside or outside the non-tumor cell, the activatable epitope may be preferentially combined with a binding agent in the physical microenvironment inside or outside the tumor cell. In some embodiments, compared with the physical microenvironment inside or outside the cell that is not affected by autoimmune disease or neurodegenerative disease, the activatable epitope may be preferentially combined with a binding agent in the physical microenvironment inside or outside the cell affected by autoimmune disease or neurodegenerative disease. In some embodiments, the activatable epitope can be activated by the following mechanisms, including but not limited to phosphorylation, glycosylation, methylation, ubiquitination, α-acetylation, lipidation, lipidation of glycosylphosphatidylinositol, amidation, sulfonation, oxidation, or any combination thereof. For example, when one or more amino acid residues on the epitope are phosphorylated, glycosylated, methylated, ubiquitinated or α-acetylated, the epitope can bind to the binding agent. For example, when one or more amino acid residues on the epitope are modified by lipidation, lipidation of glycosylphosphatidylinositol, amidation, sulfonation or oxidation, the epitope can bind to the binding agent.

[0113] In some embodiments, the activatable epitope described herein can be combined with a binding agent through a conformational change of one or more pH-sensitive helices. In some embodiments, the extracellular domain comprising the activatable epitope described herein can also include a pH-sensitive helix. The pH-sensitive helix can be derived from a natural source or a recombinant source. For example, the pH-sensitive helix can be derived from any protein comprising a pH-sensitive helix. For example, the pH-sensitive helix can be designed and engineered from scratch (denovo) by organizing one or more amino acid residues. In some embodiments, the conformational change of the pH-sensitive helix can be driven by organizing one or more histidine residues in the hydrogen bond network in the pH-sensitive helix. In some embodiments, the pH-sensitive helix can be associated and multimerized with one or more pH-sensitive helices at physiological pH or at pH>6.5. In some embodiments, the pH-sensitive helix can undergo conformational changes at low pH, such as pH<6.5. In some embodiments, the pH-sensitive helix can undergo conformational changes at low pH by including when the histidine residues in the hydrogen bond network become protonated, due to the accumulation of electrostatic repulsion and / or steric repulsion mechanisms. Details of the pH-sensitive helix design and mechanism 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.

[0114] In some embodiments, the extracellular domain comprising an activatable epitope described herein may also comprise about 1 to about 10 copies of a pH-sensitive helix. For example, the extracellular domain comprising an activatable epitope described herein may also 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, the extracellular domain comprising an activatable epitope described herein may comprise at least two copies of a pH-sensitive helix. In this embodiment, the activatable epitope is flanked by at least two copies of a pH-sensitive helix.

[0115] In some cases, the pH-sensitive helix may include a pH-sensitive multimerization domain. In some embodiments, the pH-sensitive helix may associate or multimerize with one or more pH-sensitive helices at physiological pH or at a pH higher than 6.0. In some embodiments, when the pH-sensitive helix associates or multimerizes with one or more pH-sensitive helices, the epitope may not be able to bind to the binding agent. In some embodiments, a pH higher than 6.0 may 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.

[0116] 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, when the pH sensitive helix dissociates from one or more other pH sensitive helices, the epitope can bind to the binding agent.

[0117] In some cases, the extracellular domain comprising an activatable epitope and a pH-sensitive helix can be configured to bind to a pH-sensitive helix. In some embodiments, the pH-sensitive helix can bind to an extracellular domain comprising a pH-sensitive helix at a physiological pH or a pH higher than 6.0. In some embodiments, when the pH-sensitive helix binds to an extracellular domain comprising a pH-sensitive helix, the epitope may not bind to the binding agent. In some embodiments, a pH higher than 6.0 may 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.

[0118] 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, when the pH sensitive helix dissociates from the extracellular domain comprising the pH sensitive helix, the epitope can bind to the binding agent.

[0119] In some cases, the engineered polypeptides described herein may include an activatable epitope or ligand binding site incorporated into a cell membrane. In this embodiment, the engineered polypeptide may also include one or more pH-sensitive helices in the extracellular domain, wherein each of the one or more pH-sensitive helices may include a multimerization domain. In some embodiments, the one or more pH-sensitive helices may associate or multimerize at physiological pH or a pH higher than 6.0. In this embodiment, when the one or more pH-sensitive helices associate or multimerize, the epitope or ligand binding site may not be able to bind to a binding agent. In some embodiments, a pH above 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.

[0120] In some embodiments, one or more pH sensitive helices can dissociate from each other at low pH. In this embodiment, when one or more pH sensitive helices dissociate at low pH, an epitope or ligand binding site can bind to a binding agent. 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.

[0121] In some cases, the engineered polypeptide described herein may include a secretion signal. In some embodiments, the engineered polypeptide comprising a secretion signal may not include a signal peptide. For example, a secretion signal may replace a signal peptide in an engineered polypeptide. In some embodiments, the engineered polypeptide described herein may include a membrane-bound domain. In some embodiments, the engineered polypeptide comprising a membrane-bound domain may not include a transmembrane domain. For example, a membrane-bound domain may replace a transmembrane domain in an engineered polypeptide. In some embodiments, the engineered polypeptide described herein may include a secretion signal and a membrane-bound domain. In some embodiments, the membrane-bound domain may include a pH-sensitive membrane-bound helix. In some embodiments, the engineered polypeptide comprising a secretion signal and a pH-sensitive membrane-bound helix may be propagated to one or more neighboring cells. In one example, an engineered polypeptide comprising a secretion signal and a pH-sensitive membrane-bound helix may be secreted into an extracellular fluid or a physical microenvironment, and may not be attached to a cell when secreted in a non-acidic or non-tumor extracellular environment. In some embodiments, the non-acidic or non-tumor environment can have a pH higher than 6.5, such as 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-bound helix can be secreted into the extracellular fluid or physical microenvironment and can attach to cells including secreting cells and neighboring cells when secreted at a pH level that allows the assembly of membrane-bound helices. In some embodiments, the pH level that can allow the membrane-bound helix to assemble can include a pH equal to or less than 6.5, for example, 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, the pH level that can allow the membrane-bound helix to assemble can include pH = 6.5.

[0122] In some embodiments, the pH sensitive helix may 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: 1-3. In some embodiments, the pH sensitive helix may comprise a sequence having at least 80% identity to any one of SEQ ID NOs: 1-3. In some embodiments, the pH sensitive helix may comprise a sequence having 100% identity to any one of SEQ ID NOs: 1-3.

[0123] In some embodiments, an extracellular domain comprising an activatable epitope described herein can comprise a sequence following [helix]-[helix]-[helix]-[epitope], wherein [helix] represents a pH-sensitive helix; and [epitope] represents an activatable epitope.

[0124] In some embodiments, the extracellular domain comprising an activatable epitope described herein may comprise a sequence following [helix]-[linker]-[helix]-[linker]-[helix]-[linker]-[epitope], wherein [helix] represents a pH-sensitive helix; [linker] represents a linker; and [epitope] represents an activatable epitope. In some embodiments, the linker may comprise the sequence (GGS)n or (GGS)nFCYWKTCT(GGS)n, wherein 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.

[0125] In some embodiments, the extracellular domain comprising an activatable epitope described herein may comprise a sequence following [helix]-[linker]-[helix]-[linker]-[helix]-[epitope], wherein [helix] represents a pH-sensitive helix; [linker] represents a linker; and [epitope] represents an activatable epitope. In some embodiments, the linker may comprise the sequence (GGS)n or RLCRPRSAPSRCGPGLRPCAP, wherein 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.

[0126] In some cases, an engineered polypeptide comprising an extracellular domain (which comprises an epitope), a transmembrane or membrane affinity domain, and / or a hinge domain may also comprise a scaffold domain. In some embodiments, the scaffold domain may be located at the N-terminus or C-terminus of the hinge domain. In some embodiments, the scaffold domain may be located at the N-terminus or C-terminus of the epitope. In some embodiments, the scaffold domain may comprise an epitope. In some embodiments, the scaffold domain may be located at the N-terminus of the hinge domain and the C-terminus of the epitope. In some embodiments, the scaffold domain may be located at the N-terminus of the hinge domain and the N-terminus of the epitope. In some embodiments, the scaffold domain may comprise an epitope and may be located at the N-terminus of the hinge domain. In some embodiments, the scaffold domain may be located at the N-terminus of the mature polypeptide (e.g., after translation and cleavage of the secretion signal).

[0127] In some embodiments, the scaffold domain may include a heavy chain variable (VH) domain. In some embodiments, the scaffold domain may not include a light chain variable (VL) domain. In some embodiments, the scaffold domain may include a VH domain and not include a VL domain. In some embodiments, the scaffold domain may include a single-chain antibody (nanoantibody or VHH domain). In some embodiments, the nanoantibody domain may include a mutation in the complementary determining region (CDR1), CDR2 or CDR3 of the VH domain. In some embodiments, the nanoantibody domain may include an inactivating mutation in the CDR1, CDR2 or CDR3 of the VH domain. In some embodiments, such inactivating mutations can include exchanged CDR residues that replace at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 residues within a CDR region to exchange the charge of the substituted amino acid or to exchange a hydrophobic amino acid for a hydrophilic amino acid, or to replace one or more CDRs with (GGS)n or (GGGS)n.

[0128] In some embodiments, the scaffold domain may include an epitope inserted in the CDR1, CDR2 or CDR3 of a VH domain, a VHH domain or a nanobody domain. In some embodiments, the scaffold domain may include one or more epitopes inserted in the CDR1, CDR2 or CDR3 of the VH domain. For example, the scaffold domain may include about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 epitopes inserted in the CDR1, CDR2 or CDR3 of the VH domain. In some embodiments, the scaffold domain may include two or more epitopes inserted in the CDR1, CDR2 or CDR3 of the VH domain, wherein each of the two or more epitopes is a different epitope bound to the same binding agent. In some embodiments, the scaffold domain may include two or more epitopes inserted in the CDR1, CDR2 or CDR3 of the VH domain, wherein each of the two or more epitopes is a different epitope bound to the same binding agent. In some embodiments, the scaffold domain may comprise two or more epitopes inserted into the CDR1, CDR2, or CDR3 of the VH domain, wherein each of the two or more epitopes is a different epitope that binds to a different binding agent. In some embodiments, the scaffold domain may comprise two or more epitopes inserted into the CDR1, CDR2, or CDR3 of the VH domain, wherein the two or more epitopes comprise a combination of the same epitope and different epitopes that bind to the same binding agent. In some embodiments, the scaffold domain may comprise two or more epitopes inserted into the CDR1, CDR2, or CDR3 of the VH domain, wherein the two or more epitopes comprise 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.

[0129] In some embodiments, the scaffold domain may 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 may comprise a sequence having at least 80% sequence identity to SEQ ID NO: 63 or a variant thereof. In some embodiments, the scaffold domain may comprise a sequence having 100% sequence identity to SEQ ID NO: 63 or a variant thereof.

[0130] Transmembrane domain / membrane binding domain

[0131] In some aspects, the engineered polypeptides described herein may also include a transmembrane domain or a membrane affinity domain capable of associating with the extracellular membrane. In some embodiments, the engineered polypeptide sequence may include a sequence of an extracellular domain sequence containing an epitope and a transmembrane domain or a membrane affinity domain capable of associating with the extracellular membrane encoded by a single nucleic acid sequence. In some embodiments, the engineered polypeptide sequence may be designed to include a transmembrane domain heterologous to the extracellular domain of the engineered polypeptide or a membrane affinity domain capable of associating with the extracellular membrane.

[0132] In some embodiments, the engineered polypeptides described herein may include a transmembrane domain. In some embodiments, the transmembrane domain may include a transmembrane protein domain. In some embodiments, the transmembrane domain may include one or more hydrophobic amino acid residues. In some embodiments, the transmembrane domain may be derived from a protein comprising a single, single pass or single transmembrane α-helical domain. In some embodiments, the transmembrane domain may be derived from a protein comprising multiple, multiple passes or multi-transmembrane α-helical domains. In some embodiments, the transmembrane domain may be derived from a protein comprising a unidirectional transmembrane (monotopic) α-helical domain. In some embodiments, the transmembrane domain may be derived from a protein comprising a bidirectional transmembrane (bitopic) α-helical domain. In some embodiments, the transmembrane domain may be derived from a protein comprising a multidirectional transmembrane (polytopic) α-helical domain. In some embodiments, the transmembrane domain may be derived from a protein comprising a multidirectional transmembrane β-folded domain. In some embodiments, the transmembrane domain may include a single pass through the transmembrane domain. In some embodiments, the transmembrane domain may be derived from a glycosylphosphatidylinositol (GPI) anchored protein. In some embodiments, the GPI anchor may comprise a post-translational modification of the polypeptide with a glycolipid.

[0133] In some embodiments, the transmembrane domain may be derived from a natural source or a recombinant source. For example, the transmembrane domain may be derived from any membrane-bound or transmembrane protein. Non-limiting examples of transmembrane may include transmembrane domains derived from DLL3, PSMA, SSTR2, PD-L1, EGFR, CD28, CD4, CD8, CD8a, CD8b, ICOS, CD73, NKG2D, MUC16, ROR1, HER2, HER3, HER4, TCRγ, TCRδ, CD3ε, CD3γ, CD3δ, or CD3ζ. In some embodiments, the transmembrane domain may be derived from DLL3, PSMA, SSTR2, PD-L1, EGFR, CD28, CD4, CD8, CD8a, CD8b, ICOS, or CD73.

[0134] In some cases, other transmembrane domains can be identified based on sequence annotations from UniProtKB / Swiss-Prot. Alternatively or additionally, TMHMM, Memsat, Phobius and Eisenberg et al.'s hydrophobic moment diagram method (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) can be used to detect transmembrane regions from primary sequences. Suitable transmembrane domains can include 2-5 residues on the N-terminus and C-terminus of the predicted helix to ensure that the same membrane surface interactions are maintained.

[0135] In some embodiments, the transmembrane domain may 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 transmembrane domain of any one of SEQ ID NOs: 68-76. In some embodiments, the transmembrane domain may comprise 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.

[0136] In some embodiments, the engineered polypeptides described herein may include a membrane affinity domain that can associate with the extracellular membrane. For example, the engineered polypeptide may include a domain that associates with the extracellular membrane by the following mechanism: including but not limited to amphipathic α-helices (in-plane membrane helices) parallel to the membrane plane; interacting with hydrophobic loops; interacting with covalently bound membrane lipids; or ionic or electrostatic interactions with membrane lipids. In some embodiments, the membrane affinity domain may be derived from a protein comprising a unidirectional transmembrane α-helical domain. In some embodiments, the engineered polypeptide comprising a membrane affinity domain may be anchored to the membrane by integration into a bilayer of covalently bound lipids. In some embodiments, the membrane affinity domain that can associate with the extracellular membrane may be attached to one side of the membrane (e.g., the outside of the membrane) and may not span the entire membrane.

[0137] Non-limiting examples of membrane affinity domains can include members of the large homeodomain 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 include one or more alpha helices (e.g., ENTH domain); a combination of one or more beta strands and one or more alpha helices (e.g., C1 domain, PX domain); a beta sandwich (e.g., C2 domain); a beta barrel terminated by an alpha helix (e.g., PH domain); or a double zinc finger module (e.g., FYVE domain). Details of the membrane affinity domain 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.

[0138] In some embodiments, the membrane affinity domain may include a helical domain derived from bacteriorhodopsinin. Bacteriorhodopsinin is an integral membrane archaeal protein that includes a hexagonal lattice containing three identical protein chains, each containing seven transmembrane alpha helices and a retinal molecule. Details of bacteriorhodopsinin are described in Woolf TB, Molecular dynamics of individual alpha-helices of bacteriorhodopsinin 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.

[0139] In some embodiments, the membrane affinity domain may include a C1, C2, PH, FYVE, PX or ENTH domain, or a transmembrane helix derived from bacteriorhodopsin. In some embodiments, the membrane affinity domain may 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 any one of SEQ ID NOs: 77-81. In some embodiments, the membrane affinity domain may comprise a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 77-81. In some embodiments, the membrane affinity domain may comprise a sequence having 100% sequence identity to any one of SEQ ID NOs: 77-81.

[0140] Hinge domain

[0141] In some aspects, the engineered polypeptide described herein may further include a hinge domain. For example, the engineered polypeptide may include an extracellular directional polypeptide spacer domain, which may include a hinge domain. In some embodiments, the extracellular directional polypeptide spacer domain comprising a hinge domain may connect a transmembrane domain and an extracellular domain comprising an epitope. In some embodiments, the extracellular directional polypeptide spacer domain comprising a hinge domain may connect a membrane affinity domain associated with the extracellular membrane and an extracellular domain comprising an epitope. For example, the engineered polypeptide described herein may include an extracellular directional polypeptide spacer domain, which includes a hinge domain between a transmembrane domain or a membrane affinity domain and an extracellular domain comprising an epitope. In some embodiments, the engineered polypeptide sequence may include a sequence of an extracellular domain sequence containing an epitope, a sequence of a transmembrane domain or a membrane affinity domain capable of associating with an extracellular membrane, and a sequence of an extracellular directional polypeptide spacer domain comprising a hinge domain encoded by a single nucleic acid sequence. In some embodiments, the engineered polypeptide sequence can be designed to include a transmembrane domain that is heterologous to the extracellular domain of the engineered polypeptide or a membrane affinity domain that is capable of associating with the outer membrane of a cell.

[0142] 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 include a sequence (GGGS) n, where n is an arbitrary integer. In some embodiments, n is an integer between 1 to 10 or 1 to 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.

[0143] In some cases, additional transmembrane domains can be identified based on sequence annotations from UniProtKB / Swiss-Prot or by identifying regions with homology to the 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 variants 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 the hinge sequence of any protein described herein. In some embodiments, the hinge domain can comprise a sequence having at least 80% 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 100% 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.

[0144] Tests / Antibodies / Therapies

[0145] In some cases, the methods described herein involve administering to a subject a composition comprising a nucleic acid or a vector described herein, in combination with administering to the subject an antibody or antigen-binding fragment thereof, a protein ligand or a functional fragment thereof, or a small molecule configured to bind to the epitope.

[0146] Nucleic acids, vectors, antibodies or antigen-binding fragments thereof, protein ligands or small molecules can be administered to a subject by any suitable method, such as intravenous, subcutaneous, intraventricular, intrathecal, intraventricular, transdermal, intramuscular, oral, inhaled, nasal, rectal, intratumoral or near-tumoral administration. Near-tumoral administration can mean administration to tissues in the vicinity of a tumor or administration to an area where the tumor is expected to be accessible through the lymphatic system (e.g., adjacent lymph nodes). Intratumoral or near-tumoral approaches can involve the use of other imaging techniques, such as endoscopic ultrasonography (see, e.g., Shirley et al., Gastroenterol Res Pract. 2013; 2013: 207129) or by bronchoscopy (see, e.g., Rojas-Solano et al., J Bronchology Interv Pulmonol. 2018 Jul; 25(3): 168–175). In some embodiments, the composition is applied to at least one of the neck, upper malleolus in the humerus, supraclavicular, neck, axilla, mediastinum, trochlear, mesenteric, inguinal, thigh or popliteal lymph nodes. In some cases, the use based on lymph nodes can be used as a method for local delivery to tissue regions. In some cases, the composition including nucleic acid, carrier, antibody or its antigen-binding fragment, protein ligand or small molecule can be a pharmaceutically acceptable composition (for example, a composition comprising a pharmaceutically acceptable carrier). In some cases, the composition can be configured for intravenous administration.

[0147] In some cases, the administration of the nucleic acid or vector and the administration of the antibody or antigen-binding fragment thereof, the protein ligand or functional fragment thereof, or the small molecule configured to bind the epitope are separated by a sufficient time to allow the epitope encoded by the nucleic acid or vector to be expressed by the cells of the subject. In some embodiments, the time period includes at least about 8, 12, 16, 24, 36, 48, 60, 72, 84, or 96 hours.

[0148] In some cases, after 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 an epitope, a non-invasive imaging method can be performed on the subject. Such non-invasive imaging methods include MRI imaging, PET imaging, SPECT imaging, photoacoustic imaging, and bioluminescent imaging. MRI imaging detectable polypeptides include polypeptide contrast agents such as ferritin (or mutants thereof such as Pyrococcus furiousus ferritin mutants L55P, F57S, or F123S) or lanthanide binding proteins (or engineered fusions thereof such as 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-iodine symporter (e.g., in combination with a PET-active iodine / iodide 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 combination with positron-labeled acyclovir or a pyrimidine analog PET reporter such as [18F]FHBG, see Yaghoubi SS et al. Nat Protoc. 2006;1(6):3069-75), and a dopamine D2 receptor or a mutant thereof such as D2R80A or D2R194A (e.g., in combination with a positron-labeled D2 binder such as 3-(2'-[18F]-fluoroethyl)-spiropyridone). Polypeptides detectable by photoacoustic imaging include pigment-producing enzymes such as β-galactosidase (e.g., administered in combination with X-gal) and tyrosinase, autofluorescent proteins (e.g., GFP, mCherry, or derivatives thereof), non-fluorescent GFP-like pigment proteins (e.g., aeCP597 and cjBlue, and derivatives thereof), near-infrared fluorescent proteins based on bacterial phytochromes (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 reversibly photoswitchable proteins (e.g., Dronpa, Dronpa-M159T, and BphP1, or variants thereof). Polypeptide bioluminescent imaging includes luciferases (e.g., in combination with coelenterazine administration as described herein), including Gaussia luciferase, Renilla luciferase, and Photinus luciferase (e.g., including the engineered PpyRE8 and RE9 versions described in Branchini et al. Anal. Biochem. 396 (2010): 290-297).

[0149] In some cases, non-invasive imaging methods may involve detection with a radioisotope attached or associated with an antibody or antigen-binding fragment thereof, a protein ligand or functional fragment thereof, or a small molecule configured to bind 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 and comprises 124 I. 68 Ga, 11 C. 13 N. 15 O. 18 F. 68 Ga, 64 Cu, 52 Mn, 55 Co. 89 Zr, 82 Rb or any combination thereof. In some embodiments, the radioisotope comprises an alpha-emitting radioisotope, and comprises 225 Ac, 211 At 227 Th, 224 Ra or any combination thereof. In some embodiments, the radioisotope comprises a beta-emitting radioisotope and comprises 177 Lu, 67 Cu, 131 I. 90 Y. 89 Sr. 186 Re, 165 Dy, 32 P. 166 Ho, 188 Re or any combination thereof. In some embodiments, the radioisotope is a gamma-emitting radioisotope and comprises 99m Tc, 123 I or 131 I.

[0150] In some cases, non-invasive imaging methods may involve the use of contrast agents such as iron oxide nanoparticles (IONPs), superparamagnetic iron platinum nanoparticles, manganese (II) or gadolinium (III) linked or associated with antibodies or antigen-binding fragments thereof, protein ligands or functional fragments thereof, or small molecules configured to bind epitopes.

[0151] In some cases, the methods described herein may involve treatment with an antibody or antigen-binding fragment thereof, a protein ligand or functional fragment thereof, or a small molecule configured to bind an epitope linked or associated with a therapeutic agent. Such agents include protein toxins, diphtheria toxin, glucagon-like peptide (GLP-1), cytotoxic immunomodulatory proteins, Fas ligand, auristatin or an analog thereof, maytansinoid alkaloid, calicheamicin, duocarmycin or an analog thereof, or doxorubicin or an analog thereof.

[0152] Example Implementation

[0153] In some aspects, the present disclosure provides a method for 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 rather than in non-diseased cells, wherein the biomarker comprises an extracellular receptor configured to bind an affinity agent configured for detecting or treating the diseased cells. In some aspects, the present disclosure provides a method for 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 rather than in non-diseased cells, wherein the biomarker comprises an extracellular receptor containing multiple epitopes, wherein the multiple epitopes are configured to bind one or more affinity agents configured for detecting or treating the diseased cells. In some aspects, the present disclosure provides a method for detecting diseased cells, comprising: administering a composition to a subject, wherein the composition preferentially induces expression of a biomarker in diseased cells rather than in non-diseased cells, wherein the biomarker comprises an activatable epitope, wherein the activatable epitope is preferentially bindable in a physical microenvironment inside or outside the cell affected by the disease compared to a microenvironment inside or outside the 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 that is at least 80% identical to any one of SEQ ID NOs: 1-3. In some embodiments, the biomarker comprises a sequence that follows: [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; and [Epitope] represents the epitope. In some embodiments, the biomarker comprises a sequence that follows: [Helix]-[Linker]-[Helix]-[Linker]-[Helix]-[Epitope] wherein [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 present disclosure provides a method for detecting or treating diseased cells, comprising: administering a composition to a subject, wherein the composition preferentially induces surface expression of a biomarker comprising an activatable epitope in diseased cells compared to surface expression of the biomarker in non-diseased cells; wherein the activatable epitope is preferentially bindable in a microenvironment specific to the diseased cells compared to the microenvironment of cells not affected by the disease; wherein the biomarker comprises a membrane resident polypeptide or wherein 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 a functional fragment thereof, or a small molecule configured to bind to the epitope. In some embodiments, the method further comprises detecting the binding of the antibody or antigen binding fragment thereof to the epitope. In some aspects, the present disclosure provides a method for detecting or treating diseased cells, comprising: (a) administering a composition to a subject, wherein the composition preferentially induces surface expression of a biomarker comprising one or more epitopes in diseased cells compared to the surface expression of the biomarker in non-lesioned cells; and (b) administering to the subject an antibody or antigen binding fragment thereof, a protein ligand or a functional fragment thereof, or a small molecule configured to bind to the epitope or epitope. In some embodiments, the method further comprises detecting the binding of the antibody or antigen binding fragment, protein ligand or a functional fragment thereof, or a small molecule to the epitope. In some embodiments, the epitope is an activatable epitope, which is preferentially bindable in a microenvironment specific to the diseased cell compared to the intracellular or extracellular microenvironment of cells 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 that follows: [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; and [epitope] represents the epitope. In some embodiments, the biomarker comprises a sequence following: [Helix]-[Linker]-[Helix]-[Linker]-[Helix]-[Epitope], wherein [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 preferentially exhibits catalytic activity in a cancer microenvironment) and a scFv configured to bind to the activatable epitope.In some embodiments, the cancer-associated protease is uPA, EPCAM, MMP, cathepsin, serine protease, granzyme, furin, cysteine ​​protease, elastase or type II transmembrane serine protease, kallikrein-related peptidase, "disintegrin and metalloproteinase" (ADAM), plasminogen activator (serine protease), caspase or any combination thereof. In some embodiments, the antibody or its antigen-binding fragment further comprises a radioisotope. In some embodiments, the radioisotope comprises a positron emitting radioisotope, an α-emitting radioisotope, a β-emitting radioisotope or 99mTc. In some embodiments, the radioisotope is a positron emitting radioisotope and comprises. 124 I. 68 Ga, 11 C. 13 N. 15 O. 18 F. 68 Ga, 64 Cu, 52 Mn, 55 Co. 89 Zr, 82 Rb or any combination thereof. In some embodiments, the radioisotope comprises an α-emitting radioisotope and comprises 225 Ac, 211 At 227 Th, 224 Ra or any combination thereof. In some embodiments, the radioisotope comprises a beta-emitting radioisotope and comprises 177 Lu, 67 Cu, 131 I. 90 Y. 89 Sr. 186 Re, 165 Dy, 32 P. 166 Ho, 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 with any one of SEQ ID NO: 4-9. In some embodiments, the biomarker further comprises a hinge domain derived from IgG4, IgG1, CD8 or CD28 or a 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 also includes an intracellular domain comprising a reporter protein or an affinity tag. In some embodiments, the biomarker also includes an intracellular domain comprising a reporter protein, wherein the reporter protein is derived from GFP, luciferase, Nluc or Fluc, or any combination thereof. In some embodiments, the biomarker also includes an intracellular domain comprising an affinity tag, wherein the affinity tag includes HA, FLAG or MYC sequence, or any combination thereof. In some embodiments, the biomarker includes more than one copy of the epitope. In some embodiments, the composition includes a nucleic acid vector, which includes a coding sequence for the biomarker. In some embodiments, the coding sequence includes a promoter operably connected to a nucleotide sequence encoding the biomarker. In some embodiments, relative to the expression of the biomarker in the non-lesioned cells of the object, the promoter preferentially drives the expression of the biomarker in the diseased cells. In some embodiments, 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:βGalβ-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, tight junction protein 3 (CLDN3) promoter, tight junction protein 4 (CLDN4) promoter,Type XI collagen alpha 1 chain (COL11A1) promoter, type I collagen alpha 1 chain (COL1A1) promoter, cystatin SN (CST1) promoter, toothless E3 ubiquitin protein ligase homolog (DTL) promoter, sequence similarity family 111 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. ) promoter, cell surface-associated mucin 1 (MUC1) promoter, phospholipase A2 group IID (PLA2G2D) promoter, G protein signaling regulator 13 (RGS13) promoter, secretory globin family 2A member 1 (SCGB2A1) promoter, topoisomerase IIα (TOP2A) promoter, ubiquitin D (UBD) promoter, ubiquitin-conjugating enzyme E2C (UBE2C), USH1 protein network component coordinating protein (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 Factor promoter, tyrosinase promoter, stress-inducible grp78 / BiP promoter, interleukin-10 promoter, α-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, chorionic villus 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; receptor promoter containing an inserted kinase domain; rad51 recombinase promoter; TTF-1, urokinase-type plasminogen activator receptor promoter, ubiquitin conjugating enzyme E2T (UBE2T) promoter, checkpoint kinase 1 (CHEK1) promoter, epithelial cell transformation 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 replication helicase / nuclease 2 (DNA2) promoter, DSN1 homolog, MIS12 kinetochore complex component (DSN1) promoter, deoxythymidylate kinase (DTYMK) promoter, G protein-regulated neurite outgrowth inducing factor 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, calcium cyclin binding protein (CACYBP) promoter, cell division cycle related 3 (CDCA3) promoter, centromere protein O (CENPO) promoter, flap-specific endonuclease 1 (FEN1) promoter, forkhead box M1 (FOXM1) promoter, cell proliferation-regulated protein phosphatase 2A inhibitor (KIAA1524) promoter, kinesin family member 2C (KIF2C) promoter, nuclear transporter subunit α2 (KPNA2) promoter, MYB proto-oncogene-like 2 (MYBL2) promoter, NIMA-associated kinase 2 (NEK2) promoter, RAN binding protein 1 (RANBP1) 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, BLM RecQ-like helicase (BLM) promoter, chromosome 17 open reading frame 53 (C17orf53) promoter, chromosome cassette protein 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 related 5 (CDCA5) promoter, cyclin-dependent kinase inhibitor 3 (CDKN3) promoter, cadherin EGF LAG seven-pass 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 α1 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 initiation factor (MCM10) promoter, minichromosome maintenance complex component 2 (MCM2) promoter, minichromosome maintenance complex component MCM7 promoter, MRG domain binding protein (MRGBP) promoter, methylenetetrahydrofolate dehydrogenase (NADP+ dependent) 2, methylenetetrahydrofolate 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 ATPase 1 (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, eternal circadian rhythm regulator promoter, thyroid hormone receptor interacting factor 13 (TRIP13) promoter, trophin-associated 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 multiple elements from the above items, or fully synthetic promoters consisting of tiled 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 Doggybone 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, an exosome of synthetic or natural origin, 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 cancers include acute myeloid leukemia, adrenocortical carcinoma, bladder urothelial carcinoma, breast ductal carcinoma, breast lobular carcinoma, cervical cancer, bile duct carcinoma, colorectal adenocarcinoma, esophageal cancer, gastric adenocarcinoma, glioblastoma multiforme, head and neck squamous cell carcinoma, hepatocellular carcinoma, renal chromophobe cell 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 adenocarcinoma, sarcoma, skin melanoma, testicular germ cell carcinoma, thymoma, thyroid papillary carcinoma, uterine carcinosarcoma, uterine body endometrioid carcinoma, uveal melanoma, lip melanoma, spindle cell carcinoma, liposarcoma, nasal sarcoma, breast adenocarcinoma, insulinoma, osteosarcoma, mastocytoma, angiosarcoma, non-small cell lung cancer (NSCLC), marginal lymphoma, malignant melanoma or chronic lymphocytic leukemia. In some embodiments, the detecting the binding of the antibody or antigen-binding fragment thereof to the epitope comprises a PET imaging method or another radioisotope-based imaging method.

[0154] In some aspects, the present disclosure provides a nucleic acid comprising: a cancer-specific promoter (e.g., a promoter of a gene overexpressed in cancer cells relative 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 comprising 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 that follows: [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; and [epitope] represents the epitope. In some embodiments, the biomarker comprises the sequence: [helix]-[linker]-[helix]-[linker]-[helix]-[epitope],

[0155] wherein [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 overexpressed in cancer cells relative to normal cells or a protease that preferentially exhibits catalytic activity in a cancer microenvironment) together with a scFv configured to bind to the activatable epitope. In some embodiments, the cancer-associated protease is uPA, EPCAM, MMP, cathepsin, serine protease, granzyme, furin, cysteine ​​protease, elastase or type II transmembrane serine protease, kallikrein-related peptidase, "disintegrin and metalloproteinase" (ADAM), plasminogen activator (serine protease), 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 with any one of SEQ ID NO: 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 an affinity tag. In some embodiments, the biomarker further comprises an intracellular domain comprising a reporter protein, wherein the reporter protein is 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, wherein the affinity tag comprises a HA, FLAG or MYC sequence, or any combination thereof. In some embodiments, the promoter is selected from the group consisting of a survivin promoter (BIRC5), a CXCR4 promoter, an ATP binding cassette subfamily C member 4 (ABCC4) promoter, a front gradient 2, a protein disulfide isomerase family member (AGR2) promoter, an activation-induced cytidine deaminase (AICDA) promoter, a UDP-GlcNAc: βGalβ-1,3-N-acetylglucosaminyltransferase 3 (B3GNT3) promoter, a cadherin 3 (CDH3) promoter,CEA cell adhesion molecule 5 (CEACAM5) promoter, centromere protein F (CENPF) promoter, centrosomal protein 55 (CEP55) promoter, tight junction protein 3 (CLDN3) promoter, tight junction protein 4 (CLDN4) promoter, type XI collagen alpha 1 chain (COL11A1) promoter, type I collagen alpha 1 chain (COL1A1) promoter, cystatin SN (CST1) promoter, toothless E3 ubiquitin protein ligase homolog (DTL) promoter, sequence similarity family 111 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, secretory globin family 2A member 1 (SCGB2A1) promoter, topoisomerase IIα (TOP2A) promoter, ubiquitin D (UBD) promoter, ubiquitin-conjugating enzyme E2C (UBE2C), USH1 protein network component promoter Here, we report the promoters of the following proteins: heregulin (USH1C), V-set domain-containing T-cell activation inhibitor 1 (VTCN1), 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, α-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 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; receptor promoter containing inserted kinase domain; rad51 recombinase promoter; TTF-1, urokinase-type plasminogen activator receptor promoter, ubiquitin conjugating enzyme E2T (UBE2T) promoter, checkpoint kinase 1 (CHEK1) promoter, epithelial cell transformation 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 replication helicase / nuclease 2 (DNA2) promoter, DSN1 homolog, MIS 12 kinetochore complex component (DSN1) promoter, deoxythymidylate kinase (DTYMK) promoter, G protein-regulated neurite outgrowth inducing factor 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, Calcium cyclin binding protein (CACYBP) promoter, cell division cycle related 3 (CDCA3) promoter, centromere protein O (CENPO) promoter, flap-like structure-specific endonuclease 1 (FEN1) promoter, forkhead box M1 (FOXM1) promoter, cell proliferation-regulated protein phosphatase 2A inhibitor (KIAA1524) promoter, kinesin family member 2C (KIF2C) promoter, nuclear transporter subunit α2 (KPNA2) promoter, MYB proto-oncogene-like 2 (MYBL2) promoter, NIM A-associated kinase 2 (NEK2) promoter, RAN binding protein 1 (RANBP1) 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, BLM RecQ-like helicase (BLM) promoter, chromosome 17 open reading frame 53 (C17orf53) promoter, chromosome cassette protein 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 related 5 (CDCA5) promoter, cyclin-dependent kinase inhibitor 3 (CDKN3) promoter, cadherin EGF LAG seven-way 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 initiation factor (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, methylenetetrahydrofolate 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 ATPase 1 (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, eternal circadian rhythm regulator promoter, thyroid hormone receptor interacting factor 13 (TRIP13) promoter, trophin-associated 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 multiple elements from the above items, or fully synthetic promoters consisting of tiled transcription factor binding sites. In some embodiments, the nucleic acid comprises RNA or capped mRNA. In some embodiments, the RNA or the capped mRNA comprises a splicing signal, a synthetic intron, a miRNA binding site, a synthetic stem loop or tetranucleotide loop (tetraloop) or an aRNA domain. ,

[0156] In some aspects, the disclosure provides a vector comprising any nucleic acid 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 Doggybone DNA vector (dbDNA).

[0157] In some aspects, the disclosure provides a composition comprising any vector described herein or any nucleic acid described herein and 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, an exosome of synthetic or natural origin, a virus-like particle, or any combination thereof.

[0158] In some aspects, the disclosure provides cells comprising any of the nucleic acids or vectors described herein.

[0159] Table 1: Sequences of genes and components described herein

[0160]

[0161]

[0162]

[0163]

[0164]

[0165]

[0166]

[0167]

[0168]

[0169]

[0170]

[0171]

[0172]

[0173]

[0174]

[0175]

[0176]

[0177]

[0178]

[0179]

[0180]

[0181]

[0182]

[0183]

[0184]

[0185]

[0186]

[0187]

[0188]

[0189] Example

[0190] Example 1 .—Detection, imaging or treatment of cancer cells using the cell surface expressible biomarkers described herein

[0191] A vector (e.g., a viral vector or a nanoplasmid vector) is administered to a subject suspected of having cancer, the vector comprising a coding sequence for a cell surface expressible biomarker as described herein, the biomarker comprising an epitope under the control of a cancer-specific promoter (e.g., a promoter of a gene overexpressed in cancer cells relative to normal cells). A variety of cells can accept the vector, but expression of the biomarker in cancer cells is advantageous due to cancer-specific promoters. The bindable epitope can then be detected by an antibody, antibody fragment or derivative, or protein ligand that binds to the epitope. These antibodies, antibody fragments or derivatives, or protein ligands can be detected with a PET-active radionuclide (such as 124 I or 68 Ga) labeling enables detection and / or imaging of cancer cells. In some cases, for therapeutic purposes (e.g., therapeutic diagnostic purposes) against cancer, these antibodies, antibody fragments or derivatives or protein ligands can be labeled with high-energy radionuclide beta emitters (such as 177 Lu, 67 Cu or 90 Y) or alpha emitters such as 225 Ac, 211 At 227 Finally, these antibodies, antibody fragments or derivatives or protein ligands can be conjugated with other molecules that can have a toxic effect on tumor cells.

[0192] Example 2 .—Detection, imaging or treatment of cancer cells using the cell surface expressible post-translational modification biomarkers described herein

[0193] A vector (e.g., a viral vector or a nanoplasmid vector) comprising a coding sequence for a cell surface expressible biomarker as described herein, comprising an activatable epitope under the control of a cancer-specific promoter (e.g., a promoter of a gene that is overexpressed in cancer cells relative to normal cells) is administered to a subject suspected of having cancer. A variety of cells can accept the vector, but due to the cancer-specific promoter, it is advantageous for the biomarker to be expressed in cancer cells. If the biomarker is expressed in non-cancerous cells, the lack of an appropriate activation microenvironment inside or outside normal cells causes the activatable epitope to be 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 an environment where the activatable epitope can bind. The bindable activatable epitope can then be detected by an antibody, antibody fragment or derivative, or protein ligand that binds to the epitope. These antibodies, antibody fragments or derivatives, or protein ligands can be used with PET-active radionuclides (such as 124 I or 68 Ga) labeling enables detection and / or imaging of cancer cells. For therapeutic purposes against cancer (e.g., theranostics), these antibodies, antibody fragments or derivatives or protein ligands can be labeled with high-energy radionuclide beta emitters (such as 177 Lu, 67 Cu or 90 Y) or alpha emitters such as 225 Ac, 211 At 227 Finally, these antibodies, antibody fragments or derivatives or protein ligands can be conjugated with other molecules that can have a toxic effect on tumor cells.

[0194] Example 3. —Construction of pH-sensitive, self-propagating, secreted cell markers

[0195] It is believed that if the construct is secretable and contains a membrane affinity domain, allowing it to be secreted and inserted into the membrane of neighboring cells, then expression of Figure 1- Figure 4 Cancer cells of the constructs shown in can also label neighboring cells and amplify the signal produced by delivery to the tumor. In addition, it is believed that such constructs can be sensitive to pH (and therefore to the cancer microenvironment) if the membrane affinity domain contains a pH-sensitive protein motif.

[0196] Therefore, the design Figure 5A construct of the type described in (SEQ ID NO: 21-28) comprising a cleavable signal peptide capable of driving secretion of the construct, a reporter domain linked to the signal peptide, and a pH-sensitive membrane affinity protein motif (pHLIP). It is believed that such a construct will be secreted by cancer cells and only inserted into neighboring cells within the cancer microenvironment because the membrane affinity protein motif is only activated at low pH, converting from an unfolded soluble state to a membrane anchoring helix ( Figure 6 ).

[0197] The initial question was whether constructs designed in this way would spread to nearby cells. Therefore, experiments were performed ( Figure 7 ), wherein non-transfected cancer-derived H1299 cells were treated with medium obtained from H1299 cells engineered to express comparable amounts of a reporter construct comprising a cleavable mIgK secretion tag (SEQ ID NO: 32) fused to GFP and either a mutationally inactivated 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 was successfully inserted into or otherwise associated with the H1299 cell membrane. The WT version of the pH-sensitive membrane-binding motif also appeared to show superior results to the Var3 version of the pH-sensitive membrane-binding motif.

[0198] The experimental culture medium of cells expressing constructs with active membrane-binding motifs (mIgK-GFP-Var3 or mIgK-GFP-WT) induced an increase in the number of fluorescent cells relative to cells containing inactive membrane-binding motifs (mIgK-GFP-Var3dead), indicating that the secretion tag-reporter-membrane-binding domain construct is able to spread to untransfected cells.

[0199] Example 4. — Design and engineering of DLL3 constructs for cell surface expression I

[0200] DLL3 is a membrane protein that is known to be primarily localized in the Golgi apparatus or late endosomal membranes and to be trafficked to the cell surface in small amounts when overexpressed in certain cancers ( Fig. 8ATo engineer DLL3 with increased cell surface expression, two different approaches were used. The first approach utilized trafficking instructions, such as signal peptides (SPs) and transmembrane / membrane anchoring domains of other proteins known to be located on the cell surface of lung cancer cells. SPs can direct the transport of the extracellular domain across the membrane during translation, and transmembrane domains can anchor DLL3 to the cell membrane. Lung-specific trafficking instructions can include EGFR (SP / transmembrane helix; NP244; Fig. 8A B1 in), PD-L1 (SP / transmembrane helix; NP245; Fig. 8A C1 in ) and CD73 (SP / GPI anchor motif; NP249; Fig. 8A The second approach uses a different combination of trafficking instructions to cell surface traffic proteins to immune cells, including the GM-CSF SP / CD4 transmembrane helix (NP248; Fig. 8A E1 in the CD8a SP / CD28 transmembrane helix (NP244 and NP247; in Fig. 8A In order to further improve expression and / or surface presentation, shorter and less structurally complex DLL3 sequences were designed. For example, the native signal peptide, transmembrane helices, and intracellular domains 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 contained a further truncated extracellular domain of DLL3, amino acid residues 27-254 ( Fig. 8A The amino acid sequences of NP244-NP249 are shown in Table 1.

[0201] Each of the six engineered DLL3 constructs (NP244-NP247) and two different wild-type DLL3 constructs (NP116 and NP127) were 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. H1299 cells were then stained with fluorescently labeled anti-DLL3 antibodies and analyzed by fluorescence activated cell sorting (FACS) to detect DLL3 expression. Figure 8B Panels A2-G2 and Figure 8C NP116, NP127, NP244-NP249 and NP268 showed similar Fig. 8A Panels A1-G1 correspond to the amount of DLL3 expressed on the cell surface. Although NP244 ( Figure 8B-Figure 8C) did not increase DLL3 expression levels on the cell surface, but other constructs (NP245, NP249, NP248, NP246, and NP247) increased expression levels compared to wild-type DLL3 ( Figure 8B A2 and Figure 8C Compared with NP116 and NP127 in Figure 8B-Figure 8C Specifically, NP249 and NP247 increased the positive percentage to about 80% and about 94%, respectively ( Figure 8C ).

[0202] 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 cells treated with NP247 ( Fig. 8A G1 in), NP249( Fig. 8A Compared with cells expressing NP116 or NP127, cells expressing NP247 or NP249 showed a substantial loss of viability when treated with anti-DLL3-PBD ( Fig.8D The results suggest that cells expressing engineered DLL3 (i.e., NP247 or NP249) with increased expression levels on the cell surface 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).

[0203] Example 5. — Design and engineering of DLL3 constructs for cell surface expression II

[0204] Based on NP247( Fig. 8A Six additional DLL3 constructs, NP296, NP298, NP299, NP302, NP303, and NP304, were designed to identify the minimal DLL3 domain sufficient to interact with the DLL3 antibody tracer ( Fig. 9A). NP296 comprises amino acid residues 179-254 of DLL3. NP298 comprises amino acid residues 179-254 of DLL3 and a second DLL3 epitope, the purpose of adding this epitope is to double the antibody binding ability. NP299 comprises amino acid residues 27-254 of DLL3 and two DLL3 minimal epitopes inserted into the DLL3 C2 domain. NP302 comprises amino acid residues 179-254 of DLL3 and a soluble humanized nanobody structure that replaces the C2 domain of DLL3. NP303 comprises 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 comprises 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.

[0205] Each of NP247, NP298, NP302 and the wild-type control NP116 constructs were introduced into H1299 cells by transfection to analyze the expression of engineered DLL3. H1299 cells were then stained with fluorescently labeled anti-DLL3 antibodies and analyzed by FACS to detect DLL3 expression. Fig. 9B The amount of DLL3 expressed on the cell surface is shown. Cells expressing NP298 comprising two DLL3 binding domains or NP302 comprising a humanized soluble Nanobody domain showed increased levels of DLL3 expressed on the cell surface compared to cells expressing NP247 and wild-type control ( Fig. 9B ).

[0206] In addition, each of NP247, NP296, NP298, NP302, NP303, NP304 and control construct (isotype control and negative control) are imported into H1299 cells respectively. H1299 cells are plated in 24-well plates with 40,000 cells / well. 24 hours after the plate, NP247, NP296, NP286, NP298, NP302, NP303, NP304 and control construct are transfected into cells by lipofectamine according to the manufacturer's scheme. After transfection 48 hours, harvested cells are stained with anti-DLL3 antibodies, and then the anti-human antibody puted together with APC is detected. These samples are then read out by the flow cytometry on MACS Quant. Fig. 9C The amount of cells stained with anti-DLL3 antibody (% positive) and the GMFI of DLL3-positive cells are shown.

[0207] Next, the physical number of copies of anti-DLL3 antibodies bound to DLL3 expressed on the cell surface was assessed. H1299 cells were seeded into 96-well plates. 24 hours after seeding, H1299 cells were transfected with nanoplasmid vectors containing NP116 (wild-type control), NP247 (single DLL3 epitope in CD8 scaffold), or NP298 (two DLL3 epitopes in 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 were stained with an anti-DLL3 secondary antibody conjugated to 647 (AF647) and commercially available beads for epitope quantification. H1299 cells were then analyzed on a BD Biosciences FACSAria IIu ( Figure 10A-10C The number of epitopes was calculated by comparing the cellular expression to the standard curve of the quantitation beads ( Figure 10B-Figure 10C ). Cells expressing NP247 showed a 13-fold increase in anti-DLL3 antibody copy number compared to cells expressing NP116 ( Fig. 10C ). Cells expressing NP298 showed a 32-fold increase in anti-DLL3 antibody copy number compared to cells expressing NP116 ( Fig. 10C ).

[0208] In addition, H1299 cells stably expressing wild-type (NP116) or engineered DLL3 (NP298) were stained with an anti-DLL3 antibody conjugated to a commercially available internalization secondary reagent (pHrodo). H1299 cells were incubated at 37°C with 5% CO2 for six hours. Portions were removed at fixed time intervals and subsequently analyzed on a BD Biosciences FACSAria IIu. Fig. 10D The amount of internalized DLL3 is shown.

[0209] Example 6. —Positron emission tomography (PET) scanning studies of mouse model systems expressing cancer biomarkers

[0210] 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 lung lymph node metastasis of a human patient. The patient had received radiation therapy before tumor biopsy. The cell line is adherent and has an epithelial morphology. First, a DNA plasmid was designed 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 H1299 cells contained two genomic copies of the stably integrated SSTR2 construct. Different numbers of these engineered H1299 cells were then implanted subcutaneously in different locations of the mouse with the goal of determining the minimum number of cells that produced a measurable signal.

[0211] NOD.Cg-Prkdcscid Il2rgtm1Wjl / SzJ (NSG) mice were selected from the internal colony of Stanford University. At 6 weeks of age, mice were anesthetized (isofluorane 3% in 100% O2) before inoculation with H1299 tumors. Mice were then placed in a prone position and subcutaneously implanted with H1299 WT or H1299-SSTR tumors using 1:1 (vol / vol) PBS:GelTrex according to Table 2.

[0212] Table 2. H1299 tumor implantation

[0213]

[0214] SC: Subcutaneous administration

[0215] Then, the SSTR2 protein on the surface of H1299 cells was used to bind to 68 Ga-dotate positron emission tracer treatment of mice implanted with engineered H1299 cells. Fig.11 Images acquired from PET scans of this mouse model system are shown. These data demonstrate that when cells are induced to express a surrogate biomarker (eg, SSTR2) on the cell surface, only 31,000 cancer cells can be detected in this mouse model.

[0216] Once the tumor reaches 100-200 mm in size 3 The average size between 1 and 2 was 200 μCi of [ 177 Lu]-DOTATATE tracer (produced by Minerva, Denmark). SPECT / CT images were acquired 4 hours, 24 hours, and 120 hours after tracer administration ( Fig.12 ). These data suggest that high-energy tracers can be used in engineered biomarker expression approaches.

[0217] In addition, once the tumor size reaches 100-200 mm 3 The average size between 1 and 2 was given to mice, and the mice were given 200 μCi of [ 68 GA]-DOTATATE tracer (obtained from Cardinal Health). Fig.15 Images acquired from a PET / CT scan of this mouse model system 2 hours after tracer administration are shown.

[0218] Example 7. —Cell surface expression and cell killing of engineered DLL3

[0219] Each of the NP247 and NP298 constructs and the wild-type DLL3 construct (NP116) were introduced into H1299 cells by transfection to analyze the expression of engineered DLL3. H1299 cells were then stained with fluorescently labeled anti-DLL3 antibodies for FACS analysis. H1299 cells stably expressing wild-type or engineered DLL3 and commercially available beads for epitope quantification were stained with an anti-DLL3 secondary antibody conjugated with 647 (AF647). H1299 cells were subsequently analyzed on a BD Biosciences FACSAria IIu. The number of epitopes was calculated by comparing cell expression with a quantitative bead standard curve. The same H1299 cells stably expressing wild-type or engineered DLL3 were also stained with an anti-DLL3 antibody conjugated with a commercially available internalization secondary reagent (pHrodo). H1299 cells were incubated at 37°C with 5% CO2 for six hours. Partial samples were taken at fixed time intervals and subsequently analyzed on a BD Biosciences FACSAria IIu. Fig.13A The amount of DLL3 expressed on the cell surface (left) and the amount of internalized DLL3 (right) are shown. Cells expressing NP247 (1DLL3 epitope) showed a 21-fold increase in anti-DLL3 antibody copy number compared to cells expressing NP116 (wild-type DLL3). Cells expressing NP298 (2DLL3 epitope) showed a 37-fold increase in anti-DLL3 antibody copy number compared to cells expressing NP116 (WT DLL3).

[0220] 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 unconjugated anti-DLL3 antibodies or anti-DLL3 antibodies conjugated to PBD toxin (anti-DLL3-PBD) titrations. 72 hours after antibody treatment, cell viability was assessed using the CellTiter Glo 2.0 assay kit and analyzed on a Promega GloMax Navigator. Fig. 13B showed that cells expressing the engineered DLL3 NP247 or NP298 had increased expression levels on the cell surface compared to cells expressing NP116 (wild-type DLL3) and might be more susceptible to cell death when treated with anti-DLL3-PBD.

[0221] Although 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. Those skilled in the art will now appreciate that many variations, changes and substitutions are possible without departing from the present invention. It should be understood that various alternatives to the embodiments of the present invention described herein may be used to practice the present invention. The following claims are intended to define the scope of the present invention, and thus encompass methods and structures and their equivalents within the scope of these claims.

[0222] Implementation

[0223] The following embodiments are intended to be illustrative and not limiting in any way.

[0224] 1. A method for detecting, imaging or treating diseased cells, comprising:

[0225] A composition is administered to a subject, wherein the composition induces expression of a biomarker preferentially in diseased cells relative to non-diseased cells, wherein the biomarker comprises an extracellular receptor configured to bind an affinity agent configured for detecting or treating the diseased cells.

[0226] 2. A method according to embodiment 1, wherein the extracellular receptor further comprises an activatable epitope, wherein the activatable epitope is preferentially bindable in the physical microenvironment inside or outside the cell affected by the disease compared to the microenvironment inside or outside the cell not affected by the disease.

[0227] 3. A method for detecting, imaging or treating diseased cells, comprising:

[0228] administering a composition to a subject, wherein the composition induces expression of the biomarker preferentially in diseased cells relative to non-diseased cells,

[0229] wherein the biomarker comprises an activatable epitope,

[0230] wherein the activatable epitope is preferentially bindable in a physical microenvironment inside or outside of the cell affected by the disease compared to a microenvironment inside or outside of a cell not affected by the disease.

[0231] 4. The method according to embodiment 1 or 2, wherein the composition comprises a nucleic acid or a vector encoding the biomarker.

[0232] 5. The method of any one of embodiments 1-4, wherein the biomarker does not comprise a single-chain variable fragment (scFv).

[0233] 6. The method of embodiment 5, wherein the biomarker does not comprise a light chain variable (VL) domain.

[0234] 7. A method according to any one of embodiments 1-6, wherein the biomarker further comprises a pH sensitive helix.

[0235] 8. The method of embodiment 7, wherein the activatable epitope is flanked by at least two copies of a pH-sensitive helix.

[0236] 9. The method of embodiment 7 or 8, wherein the pH sensitive helix comprises a sequence that is at least 80% identical to any one of SEQ ID NOs: 1-3.

[0237] 10. The method of embodiment 8 or 9, wherein the biomarker comprises a sequence following:

[0238] [Helix]-[Linker]-[Helix]-[Linker]-[Helix]-[Linker]-[Epitope]

[0239] Wherein [helix] represents the pH sensitive helix;

[0240] [Linker] means a linker comprising the sequence (GGS)n or (GGS)nFCYWKTCT(GGS)n; and [Epitope] means the epitope.

[0241] 11. The method of embodiment 4 or 9, wherein the biomarker comprises a sequence following:

[0242] [Helix]-[Linker]-[Helix]-[Linker]-[Helix]-[Epitope]

[0243] Wherein [helix] represents the pH sensitive helix;

[0244] [Linker] means a linker comprising the sequence (GGS)n or RLCRPRSAPSRCGPGLRPCAP; and

[0245] [Epitope] means the epitope.

[0246] 12. A method for detecting, imaging or treating diseased cells, comprising:

[0247] administering a composition to a subject, wherein the composition preferentially induces surface expression of a biomarker comprising an activatable epitope in diseased cells compared to surface expression of the biomarker in non-diseased cells;

[0248] wherein the activatable epitope is preferentially bindable in a microenvironment specific to the diseased cell compared to the microenvironment of cells not affected by the disease; wherein the biomarker comprises a membrane resident polypeptide or wherein the biomarker comprises a polypeptide comprising a transmembrane domain.

[0249] 13. The method according to any one of embodiments 1-12 further comprises administering to the subject an antibody or an antigen-binding fragment thereof, a protein ligand or a functional fragment thereof, or a small molecule configured to bind to the epitope.

[0250] 14. The method according to embodiment 13 also includes detecting the binding of the antibody or its antigen-binding fragment to the epitope.

[0251] 15. A method for detecting, imaging or treating diseased cells, comprising

[0252] (a) administering a composition to a subject, wherein the composition preferentially induces surface expression of a biomarker comprising an epitope in diseased cells compared to surface expression of the biomarker in non-diseased cells; and

[0253] (b) administering to the subject an antibody or antigen-binding fragment thereof, a protein ligand or a functional fragment thereof, or a small molecule configured to bind to the epitope.

[0254] 16. A method according to embodiment 15, wherein the extracellular domain does not contain scFv.

[0255] 17. A method according to embodiment 16, wherein the extracellular domain does not include a VL domain.

[0256] 18. The method according to any one of embodiments 15-17 also includes detecting the binding of the antibody or antigen-binding fragment, protein ligand or its functional fragment or small molecule to the epitope.

[0257] 19. A method according to any one of embodiments 15-18, wherein the epitope is an activatable epitope that preferentially binds to an extracellular ligand, wherein the preferential binding is in a microenvironment specific to the diseased cell compared to the intracellular or extracellular microenvironment of cells not affected by the disease.

[0258] 20. The method of embodiment 19, wherein the activatable epitope is activated by phosphorylation, glycosylation, methylation, ubiquitination, α-acetylation, lipidation, lipidation of glycosylphosphatidylinositol, amidation, sulfonation, oxidation, or any combination thereof.

[0259] 21. A method according to any one of embodiments 15-20, wherein the biomarker further comprises a pH sensitive helix.

[0260] 22. The method of embodiment 21, wherein the activatable epitope is flanked by at least two copies of a pH-sensitive helix.

[0261] 23. The method of embodiment 21 or 22, wherein the pH sensitive helix comprises a sequence that is at least 80% identical to any one of SEQ ID NOs: 1-3.

[0262] 24. The method of any one of embodiments 21-23, wherein the biomarker comprises a sequence following:

[0263] [Helix]-[Linker]-[Helix]-[Linker]-[Helix]-[Linker]-[Epitope]

[0264] Wherein [helix] represents the pH sensitive helix;

[0265] [Linker] means a linker comprising the sequence (GGS)n or (GGS)nFCYWKTCT(GGS)n; and

[0266] [Epitope] means the epitope.

[0267] 25. The method of any one of embodiments 21-23, wherein the biomarker comprises a sequence following:

[0268] [Helix]-[Linker]-[Helix]-[Linker]-[Helix]-[Epitope]

[0269] Wherein [helix] represents the pH sensitive helix;

[0270] [Linker] means a linker comprising the sequence (GGS)n or RLCRPRSAPSRCGPGLRPCAP; and

[0271] [Epitope] means the epitope.

[0272] 26. The method of embodiment 16, wherein the biomarker further comprises a protease cleavage site for a cancer-specific protease together with a scFv configured to bind the activatable epitope.

[0273] 27. A method according to embodiment 26, 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, "disintegrin and metalloproteinase" (ADAM), plasminogen activator, serine protease plasminogen activator, caspase or any combination thereof.

[0274] 28. A method according to any one of embodiments 13-27, wherein the antibody or antigen-binding fragment thereof further comprises a radioactive isotope.

[0275] 29. The method of embodiment 20, wherein the radioactive isotope comprises a positron emitting radioisotope, an α-emitting radioisotope, a β-emitting radioisotope or 99mTc.

[0276] 30. The method of embodiment 29, wherein the radioisotope is a positron emitting radioisotope and comprises 124 I. 68 Ga 11 C. 13 N. 15 O. 18 F. 68 Ga, 64 Cu, 52 Mn, 55 Co. 89 Zr, 82 Rb or any combination thereof.

[0277] 31. The method of embodiment 29, wherein the radioisotope comprises an alpha-emitting radioisotope and comprises 225 Ac, 211 At 227 Th, 224 Ra or any combination thereof.

[0278] 32. The method of embodiment 29, wherein the radioisotope comprises a beta-emitting radioisotope and comprises 177 Lu, 67 Cu, 131 I. 90 Y. 89 Sr.186 Re, 165 Dy, 32 P. 166 Ho, 188 Re or any combination thereof.

[0279] 33. A method according to any one of embodiments 1-32, wherein the biomarker also includes an extracellular domain comprising the epitope.

[0280] 34. A method according to embodiment 33, wherein the extracellular domain is derived from a membrane protein endogenous to the subject.

[0281] 35. The method according to 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.

[0282] 36. A method according to embodiment 35, wherein the extracellular domain is derived from CD8a, CD4, TGFR1, IGF1R, PD-L1, EGFR, CD73, IgK or any combination thereof.

[0283] 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.

[0284] 38. A method according to any one of embodiments 1-37, wherein the biomarker comprises an extracellular domain, which comprises a sequence that is at least 80% identical to any one of SEQ ID NOs: 4-9, a functional fragment thereof, or a variant thereof.

[0285] 39. A method according to any one of embodiments 1-38, wherein the biomarker further comprises a hinge domain derived from IgG4, IgG1, CD8 or CD28 or the sequence (GGGS)n.

[0286] 40. The method of any one of embodiments 1-39, wherein the biomarker further comprises a transmembrane domain derived from DLL3, PSMA, SSTR2, PD-L1, EGFR, CD28, CD4, CD8a, ICOS, or CD73.

[0287] 41. A method according to any one of embodiments 1-40, wherein the biomarker further comprises an intracellular domain comprising a reporter protein or an affinity tag.

[0288] 42. A method according to embodiment 41, wherein the biomarker also includes an intracellular domain comprising a reporter protein, wherein the reporter protein is derived from GFP, luciferase, Nluc or Fluc, or any combination thereof.

[0289] 43. A method according to embodiment 41, wherein the biomarker also includes an intracellular domain comprising an affinity tag, wherein the affinity tag includes an HA, FLAG or MYC sequence, or any combination thereof.

[0290] 44. A method according to any one of embodiments 1-43, wherein the biomarker comprises more than one copy of the epitope.

[0291] 45. A method according to any one of embodiments 1-44, wherein the composition comprises a nucleic acid vector comprising a coding sequence for the biomarker.

[0292] 46. ​​A method according to embodiment 45, wherein the coding sequence comprises a promoter operably linked to a nucleotide sequence encoding the biomarker.

[0293] 47. A method according to embodiment 46, wherein the promoter preferentially drives the expression of the biomarker in the diseased cells relative to the expression of the biomarker in the non-diseased cells of the subject.

[0294] 48. A method according to embodiment 46 or 47, wherein the promoter comprises a promoter of a gene that is selectively overexpressed in diseased cells relative to non-diseased cells, or a functional fragment thereof.

[0295] 49. A method according to any one of embodiments 46-48, wherein the promoter is selected from the group consisting of survivin promoter (BIRC5), CXCR4 promoter, ATP binding cassette subfamily C member 4 (ABCC4) promoter, pre-gradient 2, protein disulfide isomerase family member (AGR2) promoter, activation-induced cytidine deaminase (AICDA) promoter, UDP-GlcNAc:βGalβ-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. , tight junction protein 3 (CLDN3) promoter, tight junction protein 4 (CLDN4) promoter, type XI collagen alpha 1 chain (COL11A1) promoter, type I collagen alpha 1 chain (COL1A1) promoter, cystatin SN (CST1) promoter, toothless E3 ubiquitin protein ligase homolog (DTL) promoter, sequence similarity family 111 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 (MM P12) 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, secretory globin family 2A member 1 (SCGB2A1) promoter, topoisomerase IIα (TOP2A) promoter, ubiquitin D (UBD) promoter, ubiquitin-conjugating enzyme E2C (UBE2C), USH1 protein network component coordinating protein (USH1C), V-set domain-containing T cell activation inhibitor 1 (VTCN1) promoter, hexokinase type II promoter, TRPM4 promoter promoter, stromelysin 3 promoter, surfactant protein A promoter, secretory leukocyte protease inhibitor promoter, tyrosinase promoter, stress-inducible grp78 / BiP promoter, interleukin-10 promoter, α-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, chorionic villus 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; receptor promoter containing inserted kinase domain; rad51 recombinase promoter; TTF-1, urokinase-type plasminogen activator receptor promoter, ubiquitin conjugating enzyme E2T (UBE2T) promoter, checkpoint kinase 1 (CHEK1) promoter, epithelial cell transformation 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 replication helicase / nuclease 2 (DNA2) promoter, DSN1 homolog, MIS12 kinetochore complex component (DSN1) promoter, deoxythymidylate kinase (DTYMK) promoter, G protein-regulated neurite outgrowth inducing factor 1 (GPRIN1) promoter, mitochondrial fission regulator 2 (MTFR2) promoter, RAD51-related 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, calcium cyclin binding protein (CACYBP) promoter, cell division cycle-related 3 (CDCA3) promoter, centromere protein O (CENPO) promoter, flap-like structure-specific endonuclease 1 (FEN1) promoter, forkhead box M1 (FOXM1) promoter, cell proliferation-regulated protein phosphatase 2A inhibitor (KIAA1524) promoter, kinesin family member 2C (KIF2C) promoter Promoter, nuclear transport protein subunit alpha 2 (KPNA2) promoter, MYB proto-oncogene-like 2 (MYBL2) promoter, NIMA-associated kinase 2 (NEK2) promoter, RAN binding protein 1 (RANBP1) promoter, small nuclear ribonucleoprotein polypeptide B and B1 (SNRPB) promoter, SPC24 / NDC80 kinetochore complex component (SPC24) promoter, transforming acidic coiled-coil 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, BLM RecQ-like helicase (BLM) promoter, chromosome 17 open reading frame 53 (C17orf53) promoter,Chromosome box protein 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 related 5 (CDCA5) promoter, cyclin-dependent kinase inhibitor 3 (CDKN3) promoter, cadherin EGF LAG seven-way 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 initiation factor (MCM10) promoter, minichromosome maintenance complex component 2 (MCM2) promoter, minichromosome maintenance complex component 7 (MCM7) promoter, MRG domain binding promoter, methylenetetrahydrofolate dehydrogenase (NADP+ dependent) 2, methylenetetrahydrofolate cyclohydrolase (MTHFD2), non-SMC condensin I complex subunit H (NCAPH), kinetochore complex component (NDC80), nudix hydrolase 1 (NUDT1), ribonuclease H2 subunit A (RNASEH2A), RuvB-like AAAATPase 1 (RUVBL1), serologically defined breast cancer antigen NY-BR-85 (SGOL1), SHC binding and spindle-associated 1 (SHCBP1), small nuclear ribonucleoprotein polypeptide G (SNRPG), timeless circadian regulator, thyroid hormone receptor interacting factor 13 (TRIP13), trophoprotein-associated protein (TROAP), 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 multiple elements from the above items, or fully synthetic promoters composed of tiled transcription factor binding sites.

[0296] 50. The method of any one of embodiments 45-49, wherein the vector is a recombinant viral vector.

[0297] 51. The method of any one of embodiments 45-49, wherein the vector is a non-viral vector.

[0298] 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 Doggybone DNA vector (dbDNA).

[0299] 53. The method of any one of embodiments 1-52, wherein the composition further comprises a transfection agent.

[0300] 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, an exosome of synthetic or natural origin, a virus-like particle, or any combination thereof.

[0301] 55. The method of any one of embodiments 1-54, wherein the disease is cancer, an autoimmune disease, or a neurodegenerative disease.

[0302] 56. The method of embodiment 55, wherein the disease is cancer.

[0303] 57. The method according to embodiment 56, wherein the cancer comprises acute myeloid leukemia, adrenocortical carcinoma, bladder urothelial carcinoma, breast ductal carcinoma, breast lobular carcinoma, cervical cancer, bile duct carcinoma, colorectal adenocarcinoma, esophageal cancer, gastric adenocarcinoma, glioblastoma multiforme, head and neck squamous cell carcinoma, hepatocellular carcinoma, renal chromophobe cell 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 adenocarcinoma, sarcoma, cutaneous melanoma, testicular germ cell carcinoma, thymoma, papillary thyroid carcinoma, uterine carcinosarcoma, uterine corpus endometrioid carcinoma, uveal melanoma, lip melanoma, spindle cell carcinoma, liposarcoma, nasal sarcoma, breast adenocarcinoma, insulinoma, osteosarcoma, mastocytoma, angiosarcoma, non-small cell lung cancer (NSCLC), marginal lymphoma, malignant melanoma, or chronic lymphocytic leukemia.

[0304] 58. A method according to any one of embodiments 14-57, wherein the detection of binding of the antibody or its antigen-binding fragment to the epitope comprises a PET imaging method or another radioisotope-based imaging method.

[0305] 59. A nucleic acid comprising:

[0306] A cancer-specific promoter operably linked to a nucleotide sequence encoding a membrane-resident polypeptide biomarker comprising an activatable epitope.

[0307] 60. A nucleic acid according to embodiment 59, wherein the extracellular domain does not contain a scFv.

[0308] 61. A nucleic acid according to embodiment 60, wherein the extracellular domain does not include a VL domain.

[0309] 62. A nucleic acid according to any one of embodiments 59-61, wherein the activatable epitope is activated by phosphorylation, glycosylation, methylation, ubiquitination, A-acetylation, lipidation (e.g., GPI), amidation, sulfonation, oxidation, or any combination thereof.

[0310] 63. A nucleic acid according to any one of embodiments 59-62, wherein the biomarker also includes an extracellular domain and a transmembrane domain comprising the epitope.

[0311] 64. A nucleic acid according to any one of embodiments 59-63, wherein the biomarker also includes a pH sensitive helix.

[0312] 65. The nucleic acid of any one of embodiments 59-64, wherein the activatable epitope is flanked by at least two copies of a pH sensitive helix.

[0313] 66. The nucleic acid of any one of embodiments 59-65, wherein the pH sensitive helix comprises a sequence that is at least 80% identical to any one of SEQ ID NOs: 1-3.

[0314] 67. The nucleic acid of any one of embodiments 59-66, wherein the biomarker comprises a sequence following:

[0315] [Helix]-[Linker]-[Helix]-[Linker]-[Helix]-[Linker]-[Epitope]

[0316] Wherein [helix] represents the pH sensitive helix;

[0317] [Linker] means a linker comprising the sequence (GGS)n or (GGS)nFCYWKTCT(GGS)n; and

[0318] [Epitope] means the epitope.

[0319] 68. The nucleic acid of any one of embodiments 59-66, wherein the biomarker comprises a sequence following:

[0320] [Helix]-[Linker]-[Helix]-[Linker]-[Helix]-[Epitope]

[0321] Wherein [helix] represents the pH sensitive helix;

[0322] [Linker] means a linker comprising the sequence (GGS)n or RLCRPRSAPSRCGPGLRPCAP; and

[0323] [Epitope] means the epitope.

[0324] 69. The nucleic acid of any one of embodiments 59-68, wherein the biomarker further comprises a protease cleavage site for a cancer-specific protease together with a scFv configured to bind the activatable epitope.

[0325] 70. A nucleic acid according to 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, "disintegrin and metalloproteinase" (ADAM), plasminogen activator (serine protease), caspase, or any combination thereof.

[0326] 71. A nucleic acid according to any one of embodiments 60-70, wherein the extracellular domain is derived from a membrane protein endogenous to the subject.

[0327] 72. The nucleic acid of any one of embodiments 60-71, 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.

[0328] 73. A nucleic acid according to embodiment 72, wherein the extracellular domain is derived from CD8a, CD4, TGFR1, IGF1R, PD-L1, EGFR, CD73, IgK, or any combination thereof.

[0329] 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.

[0330] 75. A nucleic acid according to any one of embodiments 60-74, wherein the biomarker comprises an extracellular domain, which comprises a sequence that is at least 80% identical to any one of SEQ ID NOs: 4-9, a functional fragment thereof, or a variant thereof.

[0331] 76. The nucleic acid of any one of embodiments 59-75, wherein the biomarker further comprises a hinge domain derived from an Ig-superfamily receptor or comprises the sequence (GGGS)n.

[0332] 77. The nucleic acid of embodiment 76, wherein the biomarker further comprises the hinge domain, and the hinge domain is derived from IgG4, IgG1, CD8 or CD28.

[0333] 78. The nucleic acid of any one of embodiments 60-77, wherein the biomarker further comprises a transmembrane domain derived from DLL3, PSMA, SSTR2, PD-L1, EGFR, CD28, CD4, CD8a, ICOS, or CD73.

[0334] 79. A nucleic acid according to any one of embodiments 59-78, wherein the biomarker further comprises an intracellular domain comprising a reporter protein or an affinity tag.

[0335] 80. The nucleic acid of embodiment 79, wherein the biomarker further comprises an intracellular domain comprising a reporter protein, wherein the reporter protein is derived from GFP, luciferase, Nluc or Fluc, or any combination thereof.

[0336] 81. The nucleic acid of embodiment 79, wherein the biomarker further comprises an intracellular domain comprising an affinity tag, wherein the affinity tag comprises an HA, FLAG or MYC sequence, or any combination thereof.

[0337] 82. A nucleic acid according to any one of embodiments 59-81, wherein the promoter is selected from the group consisting of survivin promoter (BIRC5), CXCR4 promoter, ATP binding cassette subfamily C member 4 (ABCC4) promoter, pre-gradient 2, protein disulfide isomerase family member (AGR2) promoter, activation-induced cytidine deaminase (AICDA) promoter, UDP-GlcNAc:βGalβ-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. , tight junction protein 3 (CLDN3) promoter, tight junction protein 4 (CLDN4) promoter, type XI collagen alpha 1 chain (COL11A1) promoter, type I collagen alpha 1 chain (COL1A1) promoter, cystatin SN (CST1) promoter, toothless E3 ubiquitin protein ligase homolog (DTL) promoter, sequence similarity family 111 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 (MM P12) 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, secretory globin family 2A member 1 (SCGB2A1) promoter, topoisomerase IIα (TOP2A) promoter, ubiquitin D (UBD) promoter, ubiquitin-conjugating enzyme E2C (UBE2C), USH1 protein network component coordinating protein (USH1C), V-set domain-containing T cell activation inhibitor 1 (VTCN1) promoter, hexokinase type II promoter, TRPM4 promoter promoter, stromelysin 3 promoter, surfactant protein A promoter, secretory leukocyte protease inhibitor promoter, tyrosinase promoter, stress-inducible grp78 / BiP promoter, interleukin-10 promoter, α-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, chorionic villus 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; receptor promoter containing inserted kinase domain; rad51 recombinase promoter; TTF-1, urokinase-type plasminogen activator receptor promoter, ubiquitin conjugating enzyme E2T (UBE2T) promoter, checkpoint kinase 1 (CHEK1) promoter, epithelial cell transformation 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 replication helicase / nuclease 2 (DNA2) promoter, DSN1 homolog, MIS12 kinetochore complex component (DSN1) promoter, deoxythymidylate kinase (DTYMK) promoter, G protein-regulated neurite outgrowth inducing factor 1 (GPRIN1) promoter, mitochondrial fission regulator 2 (MTFR2) promoter, RAD51-related 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, calcium cyclin binding protein (CACYBP) promoter, cell division cycle-related 3 (CDCA3) promoter, centromere protein O (CENPO) promoter, flap-like structure-specific endonuclease 1 (FEN1) promoter, forkhead box M1 (FOXM1) promoter, cell proliferation-regulated protein phosphatase 2A inhibitor (KIAA1524) promoter, kinesin family member 2C (KIF2C) promoter Promoter, nuclear transport protein subunit alpha 2 (KPNA2) promoter, MYB proto-oncogene-like 2 (MYBL2) promoter, NIMA-associated kinase 2 (NEK2) promoter, RAN binding protein 1 (RANBP1) promoter, small nuclear ribonucleoprotein polypeptide B and B1 (SNRPB) promoter, SPC24 / NDC80 kinetochore complex component (SPC24) promoter, transforming acidic coiled-coil 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, BLM RecQ-like helicase (BLM) promoter, chromosome 17 open reading frame 53 (C17orf53) promoter,Chromosome box protein 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 related 5 (CDCA5) promoter, cyclin-dependent kinase inhibitor 3 (CDKN3) promoter, cadherin EGF LAG seven-way 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 initiation factor (MCM10) promoter, minichromosome maintenance complex component 2 (MCM2) promoter, minichromosome maintenance complex component 7 (MCM7) promoter, MRG domain binding promoter, methylenetetrahydrofolate dehydrogenase (NADP+ dependent) 2, methylenetetrahydrofolate cyclohydrolase (MTHFD2), non-SMC condensin I complex subunit H (NCAPH), kinetochore complex component (NDC80), nudix hydrolase 1 (NUDT1), ribonuclease H2 subunit A (RNASEH2A), RuvB-like AAAATPase 1 (RUVBL1), serologically defined breast cancer antigen NY-BR-85 (SGOL1), SHC binding and spindle-associated 1 (SHCBP1), small nuclear ribonucleoprotein polypeptide G (SNRPG), timeless circadian regulator, thyroid hormone receptor interacting factor 13 (TRIP13), trophoprotein-associated protein (TROAP), 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 multiple elements from the above items, or fully synthetic promoters composed of tiled transcription factor binding sites.

[0338] 83. The nucleic acid of any one of embodiments 59-82, wherein the nucleic acid comprises RNA or capped mRNA.

[0339] 84. The nucleic acid of embodiment 83, wherein the RNA or the capped mRNA comprises a splicing signal, a synthetic intron, a miRNA binding site, a synthetic stem-loop or tetranucleotide loop, or an aRNA domain.

[0340] 85. A vector comprising the nucleic acid according to any one of embodiments 59-84.

[0341] 86. The vector of embodiment 85, wherein the vector is a recombinant viral vector.

[0342] 87. The vector of embodiment 85, wherein the vector is a non-viral vector.

[0343] 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 Doggybone DNA vector (dbDNA).

[0344] 89. A composition comprising the vector of any one of embodiments 83-88 or the nucleic acid of any one of embodiments 59-84 and a transfection agent.

[0345] 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, an exosome of synthetic or natural origin, a virus-like particle, or any combination thereof.

[0346] 91. A cell comprising the nucleic acid of any one of embodiments 59-84 or the vector of any one of embodiments 84-88.

[0347] 92. A method for detecting, imaging or treating diseased cells, comprising:

[0348] administering to a subject a composition that preferentially induces expression of a secretable biomarker in diseased cells relative to non-diseased cells, wherein the biomarker comprises:

[0349] (a) an extracellular domain configured to bind an affinity agent configured for detecting or treating the diseased cell; and

[0350] (b) A membrane affinity domain capable of associating with, inserting into or binding to the outer membrane of a cell.

[0351] 93. A method according to embodiment 92, wherein the extracellular domain does not contain a scFv.

[0352] 94. A method according to embodiment 93, wherein the extracellular domain does not include a VL domain.

[0353] 95. The method of any one of embodiments 92-94, wherein the composition comprises a nucleic acid or a vector, wherein the nucleic acid or vector encodes the secretable biomarker.

[0354] 96. A method according to any one of embodiments 92-95, wherein the biomarkers comprise (a) and (b) in N- to C-terminal order.

[0355] 97. A method according to 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.

[0356] 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.

[0357] 99. A method according to any one of embodiments 92-98, wherein the extracellular domain comprises a sequence having at least 80% sequence identity with any one of SEQ ID NOs: 4-6 or 17-19, a functional fragment thereof, or a variant thereof.

[0358] 100. The method of any one of embodiments 92-99, wherein the biomarker is encoded by an open reading frame encoding an N-terminal cleavable signal peptide.

[0359] 101. A method according to any one of embodiments 92-100, wherein the membrane affinity domain is configured to bind to the cell membrane at low pH or in the presence of a cancer-specific extracellular protease.

[0360] 102. A method according to 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 with any one of SEQ ID NOs: 10-16 or a variant thereof.

[0361] 103. The method of any one of embodiments 92-102, wherein the membrane affinity domain comprises a C1, C2, PH, FYVE, PX or ENTH domain, or a transmembrane helix derived from bacteriorhodopsin.

[0362] 104. The method of any one of embodiments 92-103, further comprising (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.

[0363] 105. The method according to embodiment 104 further includes (d) detecting the 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.

[0364] 106. A method according to embodiment 105, wherein detecting the 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.

[0365] 107. The method of any one of embodiments 93-106, wherein the composition comprises the vector, wherein the vector is a recombinant viral vector.

[0366] 108. The method of any one of embodiments 93-107, wherein the vector is a non-viral vector.

[0367] 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 doggybone DNA vector (dbDNA).

[0368] 110. The method of any one of embodiments 92-109, wherein the disease is cancer.

[0369] 111. A nucleic acid comprising an open reading frame (ORF) encoding a polypeptide, wherein the polypeptide comprises:

[0370] (a) N-terminal cleavable signal peptide;

[0371] (b) an extracellular domain configured to bind an affinity agent, wherein the extracellular domain comprises an extracellular domain of an extracellular receptor, an epitope thereof, or a ligand binding domain thereof; and

[0372] (c) A membrane affinity domain capable of associating with, inserting into or binding to the outer membrane of a cell.

[0373] 112. A nucleic acid according to embodiment 111, wherein the extracellular domain does not contain a scFv.

[0374] 113. A nucleic acid according to embodiment 112, wherein the extracellular domain does not include a VL domain.

[0375] 114. A nucleic acid according to any one of embodiments 111-113, wherein the extracellular receptor is DLL3, PSMA, SSTR2, CD8a, CD4, TGFR1, IGF1R, PD-L1, EGFR, CD73, IgK or IL-6, or any combination thereof.

[0376] 115. A nucleic acid according to any one of embodiments 111-114, wherein the membrane affinity domain comprises a C1, C2, PH, FYVE, PX or ENTH domain, or a transmembrane helix derived from bacteriorhodopsin.

[0377] 116. A nucleic acid comprising an open reading frame (ORF) encoding a polypeptide, wherein the polypeptide comprises:

[0378] (a) an extracellular domain configured to bind an affinity agent, wherein the extracellular domain comprises an extracellular domain of an extracellular receptor, an epitope thereof, or a ligand binding domain thereof; wherein the extracellular receptor is DLL3, PSMA, SSTR2, CD8a, CD4, TGFR1, CD73, or IgK, or any combination thereof;

[0379] and

[0380] (b) A membrane affinity domain capable of associating with, inserting into or binding to the outer membrane of a cell.

[0381] 117. A nucleic acid according to embodiment 116, wherein the extracellular domain does not contain a scFv.

[0382] 118. A nucleic acid according to embodiment 117, wherein the extracellular domain does not include a VL domain.

[0383] 119. A nucleic acid according to any one of embodiments 116-118, wherein the membrane affinity domain comprises a C1, C2, PH, FYVE, PX or ENTH domain, or a transmembrane helix derived from bacteriorhodopsin.

[0384] 120. A nucleic acid comprising an open reading frame (ORF) encoding a polypeptide, wherein the polypeptide comprises:

[0385] (a) an extracellular domain configured to bind an affinity agent, wherein the extracellular domain comprises an extracellular domain of an extracellular receptor, an epitope thereof, or a ligand binding domain thereof; and

[0386] (b) a membrane affinity domain capable of associating with or binding to the outer membrane of a cell, wherein the membrane affinity domain comprises a C1, C2, PH, FYVE, PX or ENTH domain.

[0387] 121. A nucleic acid according to embodiment 120, wherein the extracellular domain does not contain a scFv.

[0388] 122. A nucleic acid according to embodiment 121, wherein the extracellular domain does not include a VL domain.

[0389] 123. A nucleic acid according to any one of embodiments 120-122, wherein the extracellular receptor is DLL3, PSMA, SSTR2, CD8a, CD4, TGFR1, IGF1R, PD-L1, EGFR, CD73, IgK or IL-6, or any combination thereof.

[0390] 124. A nucleic acid according to any one of embodiments 111-121, wherein the extracellular domain comprises a sequence having at least 80% sequence identity with any one of SEQ ID NOs: 4-6 or 17-19, a functional fragment thereof, or a variant thereof.

[0391] 125. A nucleic acid according to any one of embodiments 116-124, wherein the polypeptide includes an N-terminal cleavable signal peptide.

[0392] 126. A nucleic acid according to any one of embodiments 111-125, wherein the membrane affinity domain is configured to bind to the cell membrane at low pH or in the presence of a cancer-specific extracellular protease.

[0393] 127. A nucleic acid according to 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 with any one of SEQ ID NOs: 10-16 or a variant thereof.

[0394] 128. A nucleic acid according to any one of embodiments 111-127, wherein the polypeptide further comprises a hinge domain derived from an Ig-superfamily receptor between the extracellular domain and the membrane affinity domain.

[0395] 129. A nucleic acid according to any one of embodiments 111-127, wherein the ORF is codon optimized for expression in mammalian cells.

[0396] 130. A nucleic acid according to any one of embodiments 111-129, further comprising a cancer-specific promoter, wherein the ORF is operably linked to the cancer-specific promoter.

[0397] 131. A nucleic acid according to embodiment 130, wherein the cancer-specific promoter is a promoter of a gene that is overexpressed in cancer cells relative to normal cells, or a functional fragment thereof.

[0398] 132. The nucleic acid of embodiment 130 or 131, wherein the promoter comprises a survivin promoter (BIRC5), a CXCR4 promoter, an ATP-binding cassette subfamily C member 4 (ABCC4) promoter, an anterior gradient 2, a protein disulfide isomerase family member (AGR2) promoter, an activation-induced cytidine deaminase (AICDA) promoter, a UDP-GlcNAc:βGalβ-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 tight Connexin 3 (CLDN3) promoter, tight junction protein 4 (CLDN4) promoter, type XI collagen alpha 1 chain (COL11A1) promoter, type I collagen alpha 1 chain (COL1A1) promoter, cystatin SN (CST1) promoter, toothless E3 ubiquitin protein ligase homolog (DTL) promoter, sequence similarity family 111 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 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, secretory globin family 2A member 1 (SCGB2A1) promoter, topoisomerase IIα (TOP2A) promoter, ubiquitin D (UBD) promoter, ubiquitin-conjugating enzyme E2C (UBE2C), USH1 protein network component coordinating protein (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, α-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, chorionic villus 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; receptor promoter containing inserted kinase domain; rad51 recombinase promoter; TTF-1, urokinase-type plasminogen activator receptor promoter, ubiquitin conjugating enzyme E2T (UBE2T) promoter, checkpoint kinase 1 (CHEK1) promoter, epithelial cell transformation 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 replication helicase / nuclease 2 (DNA2) promoter, DSN1 homolog, MIS12 kinetochore complex component (DSN1) promoter, deoxythymidylate kinase (DTYMK) promoter, G protein-regulated neurite outgrowth inducing factor 1 (GPRIN1) promoter, mitochondrial fission regulator 2 (MTFR2) promoter, RAD51-associated protein 1 (RAD 51AP1) promoter, small nuclear ribonucleoprotein polypeptide A' (SNRPA1) promoter, ATPase family, AAA domain-containing 2 (ATAD2) promoter, BUB1 mitotic checkpoint serine / threonine kinase (BUB1) promoter, calcium cyclin binding protein (CACYBP) promoter, cell division cycle-related 3 (CDCA3) promoter, centromere protein O (CENPO) promoter, flap-like structure-specific endonuclease 1 (FEN1) promoter, forkhead box M1 (FOXM1) promoter, cell proliferation-regulated protein phosphatase 2A inhibitor (KIAA1524) promoter, kinesin family member 2C (KIF2C) promoter , nuclear transport protein subunit alpha 2 (KPNA2) promoter, MYB proto-oncogene-like 2 (MYBL2) promoter, NIMA-associated kinase 2 (NEK2) promoter, RAN binding protein 1 (RANBP1) promoter, small nuclear ribonucleoprotein polypeptide B and B1 (SNRPB) promoter, SPC24 / NDC80 kinetochore complex component (SPC24) promoter, transforming acidic coiled-coil 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, BLM RecQ-like helicase (BLM) promoter, chromosome 17 open reading frame 53 (C17orf53) promoter, chromosome cassette protein 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 seven-way 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 initiation factor (MCM10) promoter, minichromosome maintenance complex component 2 (MCM2) promoter, minichromosome maintenance complex component 7 (MCM7) promoter, MRG domain binding promoter, methylenetetrahydrofolate dehydrogenase (NADP+ dependent) 2, methylenetetrahydrofolate cyclohydrolase (MTHFD2), non-SMC condensin I complex subunit H (NCAPH), kinetochore complex component (NDC80), nudix hydrolase 1 (NUDT1), ribonuclease H2 subunit A (RNASEH2A), RuvB-like AAAATPase 1 (RUVBL1), serologically defined breast cancer antigen NY-BR-85 (SGOL1), SHC binding and spindle-associated 1 (SHCBP1), small nuclear ribonucleoprotein polypeptide G (SNRPG), timeless circadian regulator, thyroid hormone receptor interacting factor 13 (TRIP13), trophoprotein-associated protein (TROAP), 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, a chimeric promoter assembled from multiple elements from the above items, or a fully synthetic promoter composed of tiled transcription factor binding sites derived from any of the above items.

[0399] 133. A vector comprising the nucleic acid according to any one of embodiments 111-132.

[0400] 134. The vector of embodiment 133, wherein the vector is a recombinant viral vector.

[0401] 135. The vector of embodiment 133, wherein the vector is a non-viral vector.

[0402] 136. An engineered polypeptide comprising:

[0403] (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 a scFv;

[0404] (b) an extracellular directed polypeptide spacer domain that is about 15 to about 40 angstroms in length when folded; and

[0405] (c) a transmembrane domain or a 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.

[0406] 137. An engineered polypeptide according to embodiment 136, wherein the polypeptide does not comprise a light chain variable (VL) domain.

[0407] 138. The engineered polypeptide of embodiment 136 or 137, wherein the engineered polypeptide does not comprise an intracellular signaling domain.

[0408] 139. An engineered polypeptide according to any one of embodiments 136-138, wherein the engineered polypeptide does not comprise the intracellular portion of the CD3ζ, CD137 or CD28 polypeptide.

[0409] 140. An engineered polypeptide according to 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.

[0410] 141. An engineered polypeptide according to any one of embodiments 136-140, wherein the extracellular directed polypeptide spacer domain comprises a hinge domain.

[0411] 142. An engineered polypeptide according to any one of embodiments 136-141, wherein (a), (b) and (c) are arranged in sequence from the N-terminus to the C-terminus of the engineered polypeptide.

[0412] 143. An engineered polypeptide according to any one of embodiments 136-142, wherein the engineered polypeptide is capable of being displayed on the surface of a cell.

[0413] 144. An engineered polypeptide according to any one of embodiments 136-143, wherein the epitope further comprises an activatable epitope that is selectively available for binding in a tumor microenvironment.

[0414] 145. An engineered polypeptide according to embodiment 144, wherein the activatable epitope is flanked by at least two copies of a pH-sensitive helix.

[0415] 146. An engineered polypeptide according to any one of embodiments 136-145, wherein the epitope is capable of binding: (i) the antibody or (ii) the peptide hormone or growth factor derived from DLL3, PSMA, SSTR2 or any combination thereof.

[0416] 147. An engineered polypeptide according to embodiment 146, wherein the epitope is derived from DLL3, wherein the epitope comprises about 15 to about 260 consecutive residues of the extracellular domain of DLL3 that is at least 80% identical to SEQ ID NO:5 or a variant thereof.

[0417] 148. An engineered polypeptide according to embodiment 146 or 147, wherein the epitope comprises a sequence having at least 80% sequence identity to any one of SEQ ID NO: 4, 5, 19, 20, 64, 65 or a variant thereof.

[0418] 149. An engineered polypeptide according to 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 20nM, 15nM, 10nM, 9nM, 8nM, 7nM, 6nM, 5nM, 4nM, 3nM, 2nM or 1nM.

[0419] 150. An engineered polypeptide according to 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.

[0420] 151. An engineered polypeptide according to embodiment 150, wherein the extracellular directed polypeptide spacer domain comprises a hinge sequence having at least 80% sequence identity with the hinge sequence of any one of SEQ ID NOs: 56, 57, 58, 59, 60, 61, 62, 66, 67 or a variant thereof.

[0421] 152. An engineered polypeptide according to any one of embodiments 136-151, wherein the transmembrane domain or the membrane affinity domain comprises a transmembrane domain.

[0422] 153. An engineered polypeptide according to embodiment 152, wherein the transmembrane domain comprises a single-pass transmembrane domain.

[0423] 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.

[0424] 155. An engineered polypeptide according to 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.

[0425] 156. An engineered polypeptide according to any one of embodiments 136-151, wherein the transmembrane domain or the membrane affinity domain comprises a membrane affinity domain.

[0426] 157. An engineered polypeptide according to embodiment 156, wherein the membrane affinity domain comprises a C1, C2, PH, FYVE, PX or ENTH domain, or a transmembrane helix derived from bacteriorhodopsin.

[0427] 158. The engineered polypeptide of 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: 77-82.

[0428] 159. An engineered polypeptide according to any one of embodiments 136-158, wherein the engineered polypeptide further comprises a scaffold domain, which is (i) located at the N-terminus of the hinge domain and the C-terminus of the epitope; or (ii) located at the N-terminus of the hinge domain and contains the epitope.

[0429] 160. An engineered polypeptide according to embodiment 159, wherein the engineered polypeptide further comprises a scaffold domain located at the N-terminus of the hinge domain and at the C-terminus of the epitope.

[0430] 161. An engineered polypeptide according to 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.

[0431] 162. An engineered polypeptide according to embodiment 159 or 161, wherein the engineered polypeptide further comprises a scaffold domain located at the N-terminus of the hinge domain and containing the epitope.

[0432] 163. An engineered polypeptide according to any one of embodiments 159-162, wherein the scaffold includes a heavy chain variable (VH) domain and does not include a light chain variable (VL) domain.

[0433] 164. An engineered polypeptide according to embodiment 163, wherein the VH domain comprises an inactivating mutation in the CDR1, CDR2 or CDR3 region of the VH domain.

[0434] 165. An engineered polypeptide according to embodiment 163, wherein the scaffold comprises the epitope inserted into the CDR1, CDR2 or CDR3 region of the VH domain.

[0435] 166. A nucleic acid comprising an ORF encoding a polypeptide or a functional fragment thereof according to any one of embodiments 136-165.

[0436] 167. A nucleic acid according to embodiment 166, which also includes a promoter operably linked to the ORF.

[0437] 168. A nucleic acid according to embodiment 167, wherein the promoter is not a T cell-specific promoter or a TCRA, TCRB, CMV, EF-1, hPGK, CD3 or RPBSA promoter.

[0438] 169. A nucleic acid according to any one of embodiments 167-168, wherein the promoter is a cancer-specific promoter.

[0439] 170. A nucleic acid according to any one of embodiments 167-169, wherein the promoter is a promoter of a gene overexpressed in cancer cells relative to normal cells, or a functional fragment thereof.

[0440] 171. The nucleic acid of any one of embodiments 167-170, wherein the promoter comprises a survivin promoter (BIRC5), a CXCR4 promoter, an ATP-binding cassette subfamily C member 4 (ABCC4) promoter, an anterior gradient 2, a protein disulfide isomerase family member (AGR2) promoter, an activation-induced cytidine deaminase (AICDA) promoter, a UDP-GlcNAc:βGalβ-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. promoter, tight junction protein 3 (CLDN3) promoter, tight junction protein 4 (CLDN4) promoter, type XI collagen alpha 1 chain (COL11A1) promoter, type I collagen alpha 1 chain (COL1A1) promoter, cystatin SN (CST1) promoter, toothless E3 ubiquitin protein ligase homolog (DTL) promoter, sequence similarity family 111 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, secretory globin family 2A member 1 (SCGB2A1) promoter, topoisomerase IIα (TOP2A) promoter, ubiquitin D (UBD) promoter, ubiquitin-conjugating enzyme E2C (UBE2C), USH1 protein network component coordinating protein (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, α-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, chorionic villus 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; receptor promoter containing inserted kinase domain; rad51 recombinase promoter; TTF-1, urokinase-type plasminogen activator receptor promoter, ubiquitin conjugating enzyme E2T (UBE2T) promoter, checkpoint kinase 1 (CHEK1) promoter, epithelial cell transformation 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 Sex 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 replication helicase / nuclease 2 (DNA2) promoter, DSN1 homolog, MIS12 kinetochore complex component (DSN1) promoter, deoxythymidylate kinase (DTYMK) promoter, G protein-regulated neurite outgrowth inducing factor 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, calcium cyclin binding protein (CACYBP) promoter, cell division cycle-related 3 (CDCA3) promoter, centromere protein O (CENPO) promoter, flap-like structure-specific endonuclease 1 (FEN1) promoter, forkhead box M1 (FOXM1) promoter, cell proliferation-regulated protein phosphatase 2A inhibitor (KIAA1524) promoter, kinesin family member 2C (KIF2C) promoter, nuclear transporter subunit alpha 2 (KPNA2) promoter, MYB proto-oncogene-like 2 (MYBL2) promoter, NIMA-associated kinase 2 (NEK2) promoter, RAN binding protein 1 (RANBP1) promoter, small nuclear ribonucleoprotein polypeptide B and B1 (SNRPB) promoter, SPC24 / NDC80 kinetochore complex component (SPC24) promoter, transforming acidic coiled-coil 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,Chromosome box protein 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 related 5 (CDCA5) promoter, cyclin-dependent kinase inhibitor 3 (CDKN3) promoter, cadherin EGF LAG seven-way 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 Kinetochore, minichromosome maintenance 10 replication initiation factor (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, methylenetetrahydrofolate 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 ATPase 1 (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 rhythm regulator promoter, thyroid hormone receptor interacting factor 13 (TRIP13) promoter, trophin-associated 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 assembled from multiple elements from the above items, or fully synthetic promoters composed of tiled transcription factor binding sites derived from any of the above items.

[0441] 172. A vector comprising the nucleic acid of any one of embodiments 166-171.

[0442] 173. The vector of embodiment 172, wherein the vector is a recombinant viral vector.

[0443] 174. The vector of embodiment 172, wherein the vector is a non-viral vector.

[0444] 175. A method of detecting, imaging or treating cancer cells, comprising:

[0445] (a) administering to a subject a composition comprising a nucleic acid according to any one of embodiments 166-171 or a vector according to any one of embodiments 172-174;

[0446] (b) administering to the subject an antibody or antigen-binding fragment thereof, a protein ligand or a functional fragment thereof, or a small molecule configured to bind to the epitope.

[0447] 176. The method of embodiment 175, wherein the administration of the composition comprising the nucleic acid in (a) to the subject and the administration of the antibody or antigen-binding fragment thereof in (b) to the subject are separated by at least about 8, 12, 16, 24, 36, 48, 60, 72, 84, or 96 hours.

[0448] 177. A method according to 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 parenterally, intramuscularly, subcutaneously, intratumorally, rectally, vaginally, transdermally or intravenously or via a cannula.

[0449] 178. The m...

Claims

1. A nucleic acid encoding 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 a scFv; (b) an extracellular directed polypeptide spacer domain that is about 15 to about 40 angstroms in length when folded; and (c) a transmembrane domain or a 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.

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

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

4. The nucleic acid of any one of claims 1-3, wherein the engineered polypeptide does not comprise an intracellular portion of a CD3ζ, CD137 or CD28 polypeptide.

5. The nucleic acid of any one of claims 1-4, 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 of any one of claims 1-5, wherein the extracellular targeting polypeptide spacer domain comprises a hinge domain.

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

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

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

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

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

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

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 NO: 4, 5, 19, 20, 64, 65 or a variant thereof.

14. The nucleic acid of any one of claims 1-13, 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 1-14, 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 targeting polypeptide spacer domain comprises a hinge sequence having at least 80% 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.

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

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

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 of any one of claims 17-19, 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.

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

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

24. A nucleic acid according to any one of claims 1-23, wherein the engineered polypeptide further comprises a scaffold domain, which (i) is located at the N-terminus of the hinge domain and the C-terminus of the epitope; or (ii) is located at the N-terminus of the hinge domain and contains the epitope.

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

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

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

28. The nucleic acid of any one of claims 24-27, wherein the scaffold comprises a heavy chain variable (VH) domain and excludes a 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. 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. The nucleic acid of any one of claims 1-30, further comprising an ORF encoding the engineered polypeptide.

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

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 of any one of claims 32-33, wherein the promoter is a cancer-specific promoter.

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

36. A nucleic acid according to any one of claims 32-35, wherein the promoter comprises 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:βGalβ-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. , tight junction protein 3 (CLDN3) promoter, tight junction protein 4 (CLDN4) promoter, type XI collagen alpha 1 chain (COL11A1) promoter, type I collagen alpha 1 chain (COL1A1) promoter, cystatin SN (CST1) promoter, toothless E3 ubiquitin protein ligase homolog (DTL) promoter, sequence similarity family 111 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 (MM P12) 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, secretory globin family 2A member 1 (SCGB2A1) promoter, topoisomerase IIα (TOP2A) promoter, ubiquitin D (UBD) promoter, ubiquitin-conjugating enzyme E2C (UBE2C), USH1 protein network component coordinating protein (USH1C), V-set domain-containing T cell activation inhibitor 1 (VTCN1) promoter, hexokinase type II promoter, TRPM4 promoter promoter, stromelysin 3 promoter, surfactant protein A promoter, secretory leukocyte protease inhibitor promoter, tyrosinase promoter, stress-inducible grp78 / BiP promoter, interleukin-10 promoter, α-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, chorionic villus 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; receptor promoter containing inserted kinase domain; rad51 recombinase promoter; TTF-1, urokinase-type plasminogen activator receptor promoter, ubiquitin conjugating enzyme E2T (UBE2T) promoter, checkpoint kinase 1 (CHEK1) promoter, epithelial cell transformation 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 replication helicase / nuclease 2 (DNA2) promoter, DSN1 homolog, MIS12 kinetochore complex component (DSN1) promoter, deoxythymidylate kinase (DTYMK) promoter, G protein-regulated neurite outgrowth inducing factor 1 (GPRIN1) promoter, mitochondrial fission regulator 2 (MTFR2) promoter, RAD51-related 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, calcium cyclin binding protein (CACYBP) promoter, cell division cycle-related 3 (CDCA3) promoter, centromere protein O (CENPO) promoter, flap-like structure-specific endonuclease 1 (FEN1) promoter, forkhead box M1 (FOXM1) promoter, cell proliferation-regulated protein phosphatase 2A inhibitor (KIAA1524) promoter, kinesin family member 2C (KIF2C) promoter Promoter, nuclear transport protein subunit alpha 2 (KPNA2) promoter, MYB proto-oncogene-like 2 (MYBL2) promoter, NIMA-associated kinase 2 (NEK2) promoter, RAN binding protein 1 (RANBP1) promoter, small nuclear ribonucleoprotein polypeptide B and B1 (SNRPB) promoter, SPC24 / NDC80 kinetochore complex component (SPC24) promoter, transforming acidic coiled-coil 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, BLM RecQ-like helicase (BLM) promoter, chromosome 17 open reading frame 53 (C17orf53) promoter,Chromosome box protein 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 related 5 (CDCA5) promoter, cyclin-dependent kinase inhibitor 3 (CDKN3) promoter, cadherin EGF LAG seven-way 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 initiation factor (MCM10) promoter, minichromosome maintenance complex component 2 (MCM2) promoter, minichromosome maintenance complex component 7 (MCM7) promoter, MRG domain binding promoter, methylenetetrahydrofolate dehydrogenase (NADP+ dependent) 2, methylenetetrahydrofolate cyclohydrolase (MTHFD2), non-SMC condensin I complex subunit H (NCAPH), kinetochore complex component (NDC80), nudix hydrolase 1 (NUDT1), ribonuclease H2 subunit A (RNASEH2A), RuvB-like AAAATPase 1 (RUVBL1), serologically defined breast cancer antigen NY-BR-85 (SGOL1), SHC binding and spindle-associated 1 (SHCBP1), small nuclear ribonucleoprotein polypeptide G (SNRPG), timeless circadian regulator, thyroid hormone receptor interacting factor 13 (TRIP13), trophoprotein-associated protein (TROAP), 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, a chimeric promoter assembled from multiple elements from the above items, or a fully synthetic promoter composed of tiled transcription factor binding sites derived from any of the above items.

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

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. 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 a functional fragment thereof, or a small molecule configured to bind to the epitope.

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

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 parenterally, intramuscularly, subcutaneously, intratumorally, rectally, vaginally, transdermally or intravenously or via a cannula.

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

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

45. The method of any one of claims 40-44, 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. ​​The method of claim 45, wherein the antibody or antigen-binding fragment thereof further comprises or is chelated to a radioactive isotope.

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

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

49. The method of any one of claims 40-47, wherein the radioisotope comprises an alpha-emitting radioisotope and comprises 225 Ac, 211 At 227 Th, 224 Ra or any combination thereof.

50. The method of any one of claims 40-47, wherein the radioisotope comprises a beta-emitting radioisotope and comprises 177 Lu, 67 Cu, 131 I. 90 Y. 89 Sr. 186 Re, 165 Dy, 32 P. 166 Ho, 188 Re or any combination thereof.

51. The method of any one of claims 40-47, wherein the radioisotope is a gamma-emitting radioisotope and comprises 99m Tc, 123 I or 131 I.

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

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 40-44, wherein the antibody or antigen-binding fragment thereof further comprises an antibody drug conjugate (ADC).

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

56. The method according to any one of claims 40-55, 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 luminescent imaging performed on the subject.

57. The method of claim 56, wherein the epitope is displayed on a tumor cell in the subject.

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 the nucleic acid of any one of claims 1-36 or the vector of any one of claims 37-39.

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

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