Reporter systems for radionuclide imaging

Abstract

The present invention provides: (i) a gene expression construct for expressing a reporter gene in a cell, wherein the reporter gene encodes a fusion protein comprising a transmembrane domain fused in-frame to a reporter domain, wherein the transmembrane domain, upon insertion of the fusion protein into the cell membrane, anchors the fusion protein to the cell membrane and simultaneously expresses the reporter domain on the cell surface; and (ii) a reporter system comprising a radiolabeled reporter peptide, wherein the reporter domain comprises a large polypeptide subunit of a split luciferase and the reporter peptide comprises a small peptide subunit of the split luciferase, and both subunits associate by complementation to form a luciferase complex.

Description

[Technical Field]

[0001] This invention belongs to the field of medicine, particularly gene therapy and cell therapy. In particular, this invention provides artifacts and methods for tracking cells, such as T cells, after their infusion in chimeric antigen receptor (CAR) T cell therapy. Furthermore, this invention also provides artifacts and methods for use, as a whole, in oncolytic virus therapy and other gene / cell therapies. [Background technology]

[0002] The preclinical and early clinical development of novel therapies such as T-cell therapy or oncolytic virus therapy greatly benefits from reliable follow-up methods for cells or viruses after they have been infused into subjects for therapeutic purposes. Imaging of molecular and cell therapies is essential for understanding variability in therapeutic responses, the effectiveness of new therapeutic strategies, and patient safety monitoring. In vivo imaging has been shown to have several unique characteristics that make it an ideal approach for tracking primary immune responses to cancer in experimental systems and for translating results obtained from small animals into patients. In vivo imaging is non-invasive, provides systemic information, offers dynamic information through dynamic imaging, and allows for standardization.

[0003] One successful tracking method involves immunopositron emission tomography (PET), also known as mAb imaging, in which cells are monitored using monoclonal antibodies labeled with radionuclides that target specific cell surface markers or receptors. For example, immunoPET can be used to enable in vivo visualization of CD8-positive tumor-infiltrating lymphocytes (TILs) in a patient's body. In vivo imaging studies have shown that such CD8+ TILs provide predictive value for T-cell therapy in preclinical solid tumor models.

[0004] Another tracking method may utilize endogenous or heterologously expressed reporter genes. The human sodium-iodine cotransporter (hNIS) (2.2Kb) can be used as a reporter gene in the clinical monitoring of CAR T cell therapy, where cotransporter activity in transduced T cells is detected by SPECT imaging, specifically technetium-99m pertechnetinate. 99m It is visualized as intracellular accumulation of the TcO4- probe. PET imaging is also possible. 124 This method is supported when using an I-probe.

[0005] While the NIS reporter gene system has advantages due to its low immunogenicity, its use is limited. For example, it is naturally expressed in thyroid, stomach, salivary gland, mammary gland, and sometimes mammary gland cells. Furthermore, tracer probes may leak out uncaptured, resulting in a shorter imaging timeframe. Most importantly, NIS reporter genes are relatively long, thus hindering easy cloning into therapeutic cells or viruses. [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] WO2016 / 040835 [Patent Document 2] WO2014151736A1 [Patent Document 3] U.S. Patent No. 5,871,986 [Patent Document 4] U.S. Patent No. 4,879,236 [Patent Document 5] U.S. Patent No. 4,683,202 [Patent Document 6] U.S. Patent No. 5,928,906 [Patent Document 7] U.S. Patent No. 5,925,565 [Patent Document 8] U.S. Patent No. 5,935,819 [Patent Document 9] U.S. Patent No. 5,994,624 [Patent Document 10] U.S. Patent No. 5,981,274 [Patent Document 11] U.S. Patent No. 5,945,100 [Patent Document 12] U.S. Patent No. 5,780,448 [Patent Document 13] U.S. Patent No. 5,736,524 [Patent Document 14] U.S. Patent No. 5,702,932 [Patent Document 15] U.S. Patent No. 5,656,610 [Patent Document 16] U.S. Patent No. 5,589,466 [Patent Document 17] U.S. Patent No. 5,580,859 [Patent Document 18] U.S. Patent No. 5,789,215 [Patent Document 19] U.S. Patent No. 5,384,253 [Patent Document 20] U.S. Patent No. 4,684,611 [Patent Document 21] U.S. Patent No. 4,952,500 [Patent Document 22] U.S. Patent No. 6,410,319 [Non-patent literature]

[0007] [Non-Patent Document 1] Hall et al. (ACS Chem. Biol. July 2012, pp. 1848-1857) [Non-Patent Document 2] Dixon et al. (ACS Chem. Biol. 2016, Vol. 11: pp. 400-408) [Non-Patent Document 3] Botta et al. (J Biol Chem. 2019, Vol. 294 (45): pp. 16587-16603) [Non-Patent Document 4] Paulmurugan, Proc Natl Acad Sci USA. 2002; 99: 15608-15613. [Non-licensed Document 5] Deng, J Virol Methods. September 2011; 176(1-2): 108-111 [Non-licensed Document 6] Dixon, ACS Chem. Biol. November 2016, pp. 400-408 [Non-licensed Document 7] Zorzi 2019 (Med. Chem. Commun. 10, 1068 pages) [Non-licensed Document 8] Maniatis, Molecular Cloning, A Laboratory Manual (Cold Spring Harbor, 1990) [Non-licensed Document 9] Ausubel, 1994, Current Protocols In Molecular Biology (John Wiley & Sons, 1996) [Non-licensed Document 10] Pelletier and Sonenberg, Nature, 334:320~325 pages (1988) [Non-licensed Document 11] Macejak and Sarnow, Nature, 353:90~94 pages (1991) [Non-licensed Document 12] Carbonelli, FEMS Microbiol. Lett., 172(1): pages 75~82 (1999) [Non-licensed Document 13] Levenson, Hum. Gene Ther. 9(8):1233~1236 pages (1998) [Non-licensed Document 14] Cocea, Biotechniques, 23(5): pages 814~816 (1997) [Non-licensed Document 15] Wilson, Science, 244:1344~1346 pages (1989) [Non-licensed Document 16] Nabel, Science, 244:1342~1344 pages (1989) [Non-licensed Document 17] Harlan and Weintraub, J. Cell Biol., 101(3): 1094-1099 (1985) [Non-licensed Document 18] Tur-Kaspa, Mol. Cell Biol., 6:716~718 pages (1986) [Non-licensed Document 19] Potter, Proc. Natl. Acad. Sci. USA, 81:7161~7165 pages (1984) [Non-licensed Document 20] Graham and Van Der Eb, Virology, 52:456~467 pages (1973) [Non-licensed Document 21] Chen and Okayama, Mol. Cell Biol., 7(8):2745~2752 (1987) [Non-licensed Document 22] Rippe, Mol. Cell Biol., 10:689~695 pages (1990) [Non-licensed Document 23] Gopal, Mol. Cell Biol., 5:1188-190 (1985) [Non-licensed Document 24] Fechheimer, Proc. Natl. Acad. Sci. USA, 89(17): pages 8463~8467 (1987) [Non-licensed Document 25] Nicolau and Sene, Biochem. & Biophys. Acta., 721:185~190 pages (1982) [Non-licensed Document 26] Fraley, Proc. Natl. Acad. Sci. USA, 76:3348~3352 pages (1979) [Non-licensed Document 27] Nicolau, Meth. Enzym., 149: 157-176 (1987) [Non-Patent Document 28] Wong et al., Gene, 10: pp. 879-894 (1980) [Non-Patent Document 29] Kaneda et al., Science, 243: pp. 375-378 (1989) [Non-Patent Document 30] Kato et al., J. Biol. Chem., 266:3361-3364 (1991) [Non-Patent Document 31] Wu and Wu, J. Biol. Chem., 262: pp. 4429-4432 (1987) [Non-Patent Document 32] Omirulleh et al., Plant Mol. Biol., 21(3):415-428 (1987) [Non-Patent Document 33] Potrykus et al., Mol. Gen. Genet., 199(2): pp. 169-177 (1985) [Non-Patent Document 34] Remington's Pharmaceutical Sciences, 16th edition, edited by Mack (1980) [Overview of the project] [Problems that the invention aims to solve]

[0008] Therefore, the demand for smaller, easily cloneable, and more specific PET / SPECT reporters that can be expressed in cells remains unmet. [Means for solving the problem]

[0009] The present invention provides a construct and method for nuclear imaging based on reporter gene expression, wherein the reporter gene specifically binds to a nuclear probe with high affinity, and advantageously, the reporter gene is short and can be easily cloned into therapeutic viral vectors used in gene therapy or into cells used in cell therapy for the purpose of monitoring these therapies using PET / SPECT.

[0010] The artifacts and methods described herein provide an indirect imaging system in which cellular and molecular processes are investigated and linked to the expression of a reporter gene. Compared to currently available PET / SPECT reporter genes and other indirect cell labeling methods, the present invention is unique and highly specific.

[0011] One unique advantage of the present invention is that the reporter gene supports bioluminescence (BL) imaging in addition to PET / SPECT imaging. Therefore, the hybrid BL / PET / SPECT reporter gene can be applied as either a (companion) diagnostic or therapeutic agent in cell tracking or therapeutic methods in diagnostic / prognostic situations.

[0012] The present invention then, in a first embodiment, is described below: - A gene expression construct for expressing a reporter gene within a cell, wherein the reporter gene encodes a fusion protein containing a transmembrane domain fused in-frame to a reporter domain, and the transmembrane domain, upon insertion of the fusion protein into the cell membrane, fixes the fusion protein to the cell membrane and simultaneously expresses the reporter domain on the cell surface; - A reporter peptide labeled with a radioactive labeling material; The reporter domain comprises, preferably, a large polypeptide subunit of a splitruciferase, and the reporter peptide comprises, preferably, a small peptide subunit of the splitruciferase, wherein both subunits associate complementaryly to form a (preferably luminescent) luciferase complex, thereby providing a reporter system.

[0013] In a preferred embodiment of the system of the present invention, the reporter domain consists of a large polypeptide subunit of the splitrusiferase, and the reporter peptide consists of a small peptide subunit of the splitrusiferase.

[0014] In another preferred embodiment of the system of the present invention, a small peptide subunit has high affinity for the large polypeptide subunit of the split luciferase. As used herein, the term “high affinity” means an intermolecular interaction between two entities that is strong enough to cause the formation of a complex detectable under physiological or assay conditions. As used herein, the term “high affinity” means that the two subunits associate at a Kd of less than 0.1 μM, more preferably less than 10 nM, even more preferably less than 1 nM, even more preferably between 0.1 nM and 1 nM, and even more preferably between 0.5 nM and 1 nM.

[0015] In another preferred embodiment of the system of the present invention, the split luciferase may be selected from firefly (Photinus pyralis) luciferase (FLuc), click beetle (e.g., Pyrophorus plagiophthalamus) luciferase, Gaussia (e.g., Gaussia princeps) luciferase (GLuc), sea urchin (Renilla) (e.g., Renilla reniformis) luciferase (RLuc), Oplophorus (e.g., Oplophorus gracilirostris) luciferase (OLuc; NanoLuc), and bacterial luciferase (Lux). Most preferably, the split luciferase is NanoLuc.

[0016] In yet another preferred embodiment of the system of the present invention, the reporter peptide has a length of 9 to 30 amino acid residues. Preferably, the length of the reporter peptide is between 10 and 25 amino acids. For example, 11 to 22 amino acids. Preferably, the reporter peptide is not cleaved in the blood.

[0017] In yet another preferred embodiment of the system of the present invention, the large polypeptide subunit comprises the amino acid sequence of SEQ ID NO: 48 or an amino acid sequence having at least 90%, preferably at least 95%, sequence identity with respect to the entire length of the amino acid sequence, wherein the amino acid sequence having at least 90%, preferably at least 95%, sequence identity with respect to SEQ ID NO: 48 preferably binds to the reporter peptide with high binding affinity, and the dissociation constant Kd is less than 0.1 μM, more preferably less than 10 nM, even more preferably less than 1 nM, even more preferably between 0.1 nM and 1 nM, and even more preferably between 0.5 nM and 1 nM.

[0018] In yet another preferred embodiment of the system of the present invention, the small peptide subunit comprises, and preferably consists of, any of the amino acid sequences of SEQ ID NOs. 28 to 46.

[0019] In yet another preferred embodiment of the system of the present invention, the reporter domain is fused to the transmembrane domain at its C-terminus.

[0020] In yet another preferred embodiment of the system of the present invention, the transmembrane domain is selected from the transmembrane domains of the proteins PDGFR, CD8, B7, TLR4, CD4, neurexin 3b, Notch receptor polypeptide, CD28, CD137(41BB), CD3C, and other shortened human type I and type II transmembrane proteins, and is optionally combined with an intraplasmic domain of the protein (which may act to enhance surface expression), and preferably the transmembrane domain comprises a sequence selected from the group consisting of SEQ ID NOs: 1 to 11.

[0021] In yet another preferred embodiment of the system of the present invention, the fusion protein preferably further comprises a leader peptide fused in-frame to the transmembrane domain at the N-terminus. The leader sequence preferably comprises, or is, a signal peptide (which can function to direct the fusion protein to the cell membrane). The leader sequence in the reporter gene is preferably located immediately upstream (5') of the start codon of the transmembrane domain sequence or may include the start codon. The leader sequence preferably encodes a signal peptide (which can function to direct the fusion protein to the cell membrane). The leader sequence is preferably selected from the group consisting of the leader sequences of human or mouse IgK, CD8, OSM, IgG2 H, BM40, Secrecon, IgKVIII, CD33, tPA, chymotrypsinogen, trypsinogen-2, IL-2, albumin (HSA), and insulin, and preferably, the leader sequence comprises a sequence selected from the group consisting of SEQ ID NOs: 12-26.

[0022] In yet another preferred embodiment of the system of the present invention, the gene expression construct further comprises regulatory elements such as a promoter or a polyA sequence.

[0023] In yet another preferred embodiment of the system of the present invention, the reporter peptide preferably comprises a radiolabeled substance bound to the reporter peptide via a chelating agent. Preferably, the chelating agent is bound to the reporter peptide via a linker.

[0024] In a preferred embodiment of the system of the present invention, the radiolabeled substance is 51 Cr, 52 Fe, 52m Mn, 62 Cu, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 72 As, 77 As, 89 Zr, 90 Y,97 Ru, 99 Tc(for example) 99m Tc), 105 Rh, 109 Pd, 111 In, 111 Ag, 113m In, 121 Sn, 124 I, 127 Te, 142 Pr, 143 Pr, 149 PM, 151 PM, 153 Sm, 157 Gd, 159 Gd, 161 Tb, 165 Dy, 166 Ho, 169 Er, 169 Yb, 172 Tm, 175 Yb, 177 Lu, 186 Re, 188 Re, 198 Au, 199 Au, 203 Pb, 11 C, 18 F, 15 O, and 13 Selected from N.

[0025] In a particularly preferred embodiment of the system of the present invention, the radiolabeled material is 111 It is In.

[0026] In another particularly preferred embodiment of the system of the present invention, the chelating agent is 1,4,7,10-tetraazacyclododecane-N,N,N",N'"-tetraacetic acid (DOTA).

[0027] In a particularly preferred embodiment of the system of the present invention, the linker is 6-aminohexanoic acid (6ahx).

[0028] In a preferred embodiment of the system of the present invention, the linker is linked to the reporter peptide via a valine residue.

[0029] In a preferred embodiment of the system of the present invention, the fusion protein is manipulated to present a reporter domain on the cell surface.

[0030] In a preferred embodiment of the system of the present invention, the gene expression construct is contained in a vector. The vector preferably includes a promoter operably ligated to a transcription unit encoding a reporter gene as described herein, preferably the reporter gene being operably ligated to a eukaryotic signal sequence.

[0031] In a more preferred embodiment of the system of the present invention, the vector is contained in recombinant cells, preferably T cells.

[0032] In alternative preferred embodiments of the system of the present invention, the vector is contained in a viral genome, and examples of viral genomes include genomes of oncolytic viruses such as adenovirus, reovirus, measles virus, herpes simplex virus, Newcastle disease virus, vaccinia virus, seneca virus, enterovirus RIGVIR, semliki forest virus, vesicular stomatitis virus, and poliovirus, or genomes of viruses for cell transformation such as retrovirus or lentivirus.

[0033] In another embodiment, the present invention provides a reporter peptide comprising a small peptide subunit of a splitruciferase, wherein the small peptide subunit associates complementarily with a large polypeptide subunit of the splitruciferase to form a luciferase complex, the small peptide subunit has high affinity for the large polypeptide subunit, the reporter peptide has a length of 9 to 30 amino acid residues, and the reporter peptide is preferably labeled with a radionuclide suitable for use in PET or SPECT, wherein the radionuclide is preferably bound to the reporter peptide via a chelating agent and a linker.

[0034] The split luciferase is preferably selected from firefly (Photinus pyralis) luciferase (FLuc), click beetle (e.g., Pyrophorus plagiophthalmus) luciferase, Gaussia (e.g., Gaussia princeps) luciferase (GLuc), sea urchin (e.g., Renilla reniformis) luciferase (RLuc), Oplophorus (e.g., Oplophorus gracilirostris) luciferase (OLuc; NanoLuc), and bacterial luciferase (Lux), with the split luciferase preferably being NanoLuc.

[0035] In a preferred embodiment of the reporter peptide of the present invention, the length of the reporter peptide is between 10 and 25 amino acids, for example, 11 to 22 amino acids. Preferably, the reporter peptide is not cleaved in the blood.

[0036] In a preferred embodiment of the reporter peptide of the present invention, the radiolabeled substance is 51 Cr, 52 Fe, 52m Mn, 62 Cu, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 72 As, 77 As,89 Zr, 90 Y, 97 Ru, 99 Tc (for example 99m Tc), 105 Rh, 109 Pd, 111 In, 111 Ag, 113m In, 121 Sn, 124 I, 127 Te, 142 Pr, 143 Pr, 149 Pm, 151 Pm, 153 Sm, 157 Gd, 159 Gd, 161 Tb, 165 Dy, 166 Ho, 169 Er, 169 Yb, 172 Tm, 175 Yb, 177 Lu, 186 Re, 188 Re, 198 Au, 199 Au, 203 Pb, 11 C, 18 F, 15 O, and 13 N is selected from. Preferably, the radioactive labeling substance is 111 In.

[0037] In a preferred embodiment of the reporter peptide of the present invention, the chelating agent is 1,4,7,10-tetraazacyclododecane-N,N,N",N'"-tetraacetic acid (DOTA).

[0038] In a preferred embodiment of the reporter peptide of the present invention, the linker is 6-aminohexanoic acid (6ahx).

[0039] In a preferred embodiment of the reporter peptide of the present invention, the reporter peptide contains, preferably consists of, any one of the sequences of SEQ ID NOs: 28 to 46.

[0040] In another embodiment, the present invention provides a pharmaceutical composition for infusion into a subject's body, comprising the reporter peptide of the present invention.

[0041] In another embodiment, the present invention provides a pharmaceutical composition for infusion into a subject's body, comprising a vector or recombinant cells containing a gene expression construct of the reporter system of the present invention.

[0042] In another embodiment, the present invention is as follows: - A pharmaceutical composition for infusion into a target body, comprising a vector or recombinant cells containing the gene expression construct of the reporter system of the present invention, and - Pharmaceutical composition containing the reporter peptide of the present invention The present invention provides a pharmaceutical combination for simultaneous, separate, or sequential infusion into the body of a target.

[0043] In another embodiment, the present invention is provided.

[0044] In this specification, the term "pharmaceutical composition" in the embodiments of the present invention described above may also be expressed as "diagnostic composition," which suggests that the purpose of this composition is diagnostic rather than therapeutic.

[0045] The present invention also provides a method for treating a disease or monitoring disease treatment, comprising the step of administering a therapeutically or diagnostically effective amount of a reporter system or a pharmaceutical combination of the present invention to a subject in need thereof.

[0046] Examples of disease candidates that can be treated or monitored using the reporter system of the present invention include cancer therapies using oncolytic viruses or (CAR) T cells.

[0047] In another embodiment, the present invention provides cells transduced by a reporter-system gene expression construct according to the present invention, preferably human cells or animal cells, more preferably non-human mammalian cells. [Brief explanation of the drawing]

[0048] [Figure 1] This figure shows the photosignals collected after the reaction of HiBiT peptides at various concentrations with TMLgBiT expressed in the membrane of HEK-293 cells. [Figure 2] This figure shows the results of calculating the Kd for the reaction between LgBiT protein and HiBiT peptide using a single site-specific binding function. The calculated Kd for HiBiT is 6.8 nM. The calculated Kd for HiBiT-DOTA is 1.3 nM. The calculated Kd for HiBiT-6ahx-DOTA is 0.7 nM. [Figure 3] This figure shows the CPM obtained by a gamma-ray counter on TMLgBiT-expressing cells and control cells after the addition of 1 nmol of radioactive [111In]-DOTA-6ahx-HiBiT peptide. [Figure 4] This figure shows SPECT images of live mice injected with cells expressing the TMLgBiT reporter (right flank) and cells not expressing the TMLgBiT reporter (left flank) after infusion with radiolabeled HiBiT peptide. Highly distinctive signals are detected. [Figure 5] This figure shows the nucleic acid sequence of the pBiT1.1-C[TK / LgBiT] vector of Example 1. Reference sites of the vector sequence: base pair: 3865; HSV-TK promoter: 27~779; MCS: 815~865; LgBiT: 903~1376; SV40 late poly(A) signal: 1410~1631; plasmid replication origin from ColE1: 1956~1992; β-lactamase (Ampr) coding region: 2747~3607 (reverse). [Figure 6] This figure shows the in vivo distribution of the radiolabeled HiBiT-6ahx-Dota peptide one hour after administration, as described in Example 1. This figure shows that the peptide is widely and rapidly eliminated by the kidneys, and that there is no nonspecific accumulation in other tissues. [Figure 7]This figure shows SPECT images of live mice with PC3-TMLgBiT tumors that were injected with DOTA-6ahx-HiBiT (left) and [111In]-DOTA-6ahx-HiBiT (right). The left image is a BL image taken 30 minutes after injection of 1 nM DOTA-6ahx-HiBiT and intraperitoneal injection of NanoLuc substrate. The right image is a dynamic SPECT scan performed over 1 hour after injection of 0.13 nM [111In]DOTA-6ahx-HiBiT. In both cases, a unique signal originating from the tissue is visible. [Figure 8] This figure shows the in vivo distribution of [111In]-DOTA-6ahx-HiBiT and DOTA-6ahx-HiBiT in 100-fold excess (in the case of interference tests) in mice with PC3-TMLgBiT tumors. Effective signal interference is achieved in the organs. [Modes for carrying out the invention]

[0049] The term “reporter gene,” as used herein, means a gene that encodes a reporter protein whose expression can be readily quantified or observed using a “probe.” Since gene regulation typically occurs at the transcriptional stage, transcriptional regulation and promoter activity are often evaluated based on the quantification of gene products. In embodiments of the present invention, the reporter gene encodes, among other things, a protein domain (reporter domain) presented on the cell surface that is recognized by an externally supplied probe, and the externally supplied probe consists of a labeled peptide (reporter peptide) that binds to the reporter domain of the reporter protein by high-affinity protein fragment complementation.

[0050] Protein-fragment complementation assays, such as the NanoLuc luciferase complementation assay, are commonly used to infer protein-protein interactions based on the weak association of LgBiT polypeptide fragments and SmBiT peptide fragments. The basic principle of this assay lies in the split complementation assay, where a bioluminescent enzyme is split into two distinct components that reassemble to form a functional bioluminescent complex. The strengths of these assays lie not only in the surprisingly strong tendency of bioluminescent proteins to form their native and active structures, but also in their applicability to imaging and in vivo studies.

[0051] In an embodiment of the present invention, the reporter gene encodes a reporter protein, which corresponds to a large fragment of a split bioluminescent protein (e.g., LgBiT, the large fragment of NanoLuc) that is expressed and immobilized on the cell membrane via a transmembrane domain fused to the reporter protein. The probe used for detecting the reporter protein is formed by a small peptide (e.g., the HiBiT peptide of NanoBiT) that corresponds to a small complementary fragment of the split bioluminescent protein and has high specific affinity for the large fragment (mainly the N-terminus) of the split bioluminescent protein, meaning that these two fragments have nanomolar affinity for each other. The reporter gene is expressed in cells (e.g., in transduced T cells in vitro), and these cells can therefore be tracked in vivo in the target by PET / SPECT imaging after the probe is added to the target blood circulation.

[0052] As an experimental demonstration, the inventors have shown that HEK-293 cells transfected in vitro expressing the reporter gene TMLgBiT (TM stands for transmembrane) reacted when subcutaneously injected into mice. 111 This study demonstrates that the in-DOTA-6ax-HiBiT peptide could be detected in vivo by PET / SPECT imaging one hour after injection.

[0053] This new reporter system will enable diagnostic and prognostic imaging for cell therapies (particularly T-cell therapy).

[0054] The luminescent protein NanoLuc® (Nluc), discovered and further developed by Hall et al. (ACS Chem. Biol. July 2012, pp. 1848-1857), was artificially created from deep-sea shrimp (Oplophorus gracilirostris) luciferase using directed evolution. The enzyme's luminescence was optimized using the synthesis of coelenterazine analogs and the identification of novel substrates obtained through screening. The Nluc protein is a 19.1 kDa monomer, highly soluble, and stable ATP-independent enzyme. The optimal substrate, flimazine, produces glow-type luminescence (half-life > 2h) with a specific activity higher than that of firefly luciferase (Fluc) or sea urchin luciferase (Rluc). Due to its higher luminescence intensity, higher solubility, and smaller size compared to Fluc (61 kDa) or Rluc (36 kDa), Nluc is considered a promising tool for in vitro protein-protein interaction assays.

[0055] In conventional luciferase complementation assays (LCAs) for detecting protein-protein interactions (PPIs) in living cells using bioluminescence, the complementary DNA (cDNA) of luciferase is first split into N-terminal and C-terminal fragments, which are then fused to the cDNA of the protein pair of interest. The cells of interest are transformed or transfected with the resulting recombinant cDNA so that the recombinant protein pair is expressed in the cells. When the recombinant proteins interact with each other, the enzymatic activity of the split luciferase is restored. Compared to other assays for detecting protein-protein interactions in living cells, these assays have a wide dynamic range for interaction signals due to the extremely low background signal in the sample.

[0056] One such luciferase complementation assay using Nluc was described by Dixon et al. (ACS Chem. Biol. 2016, Vol. 11: pp. 400-408). This system, called NanoLuc® Binary Technology (NanoBiT), is a two-subunit system using NanoLuc® luciferase that can be applied to the intracellular detection of PPIs. The large BiT (LgBiT; 18kDa, SEQ ID NOs. 47 and 48) subunit and the small BiT (SmBiT; 11-amino acid peptide) subunit are expressed as fusions to the protein of interest, where PPIs facilitate subunit complementation, resulting in a brightly luminescent enzyme. Unlike related approaches where the enzyme or protein is simply split, LgBiT was optimized alone for structural stability, and SmBiT was selected from a peptide library specifically for PPI applications. As a result, a subunit pair is formed that associates weakly (Kd = 190 μM) but still maintains 30% of the activity of full-length NanoLuc at saturation. In contrast to many split systems, the LgBiT:SmBiT interaction is reversible, allowing for the detection of rapidly dissociating proteins. A NanoBiT reporter assay was recently developed by Botta et al. (J Biol Chem. 2019, Vol. 294 (45): pp. 16587-16603). This technique has been used in essence to study PPIs in membrane protein complexes.

[0057] The term “split luciferase” is used herein in the art-recognized form of a luciferase protein, which is split into an N-terminal and a C-terminal domain, and is non-functional in that neither of the two non-functional halves emits luminescence when used individually, but whose luciferase activity is restored by domain completion or rearrangement when the two non-functional halves are brought close together. Functional enzymes exhibit light emission upon the addition of a suitable substrate such as flimazine. Split luciferase is a well-known term in relation to protein fragment completion assay (PCA) techniques, particularly bimolecule fluorescence completion assays for the confirmation and quantification of protein-protein interactions (PPIs), such as the split luciferase completion assay (SLCA) (Paulmurugan et al., Proc Natl Acad Sci USA. 2002; 99: pp. 15608-15613; Deng et al., J Virol Methods. September 2011; 176(1-2): pp. 108-111).

[0058] While the low affinity of the SmBiT small peptide to LgBiT (Kd > 100 μM) in this split lycopherase complementation assay may be beneficial for PPI testing, the present invention is based on the use of a small peptide having high affinity to the large polypeptide fragment of the split lycopherase. This high-affinity peptide may be referred to herein as a reporter peptide and elsewhere as a complementary reporter. In the case of the HiBiT peptide (VSGWRLFKKIS[w / o Met] or MVSGWRLFKKIS[w / Met]; Dixon et al., ACS Chem. Biol. November 2016, pp. 400-408; WO2016 / 040835) (1.3 kDa), its Kd for binding to LgBiT is 0.7 nM.

[0059] Other peptides that have been shown to have high affinity for NLuc include NVSGWRLFKKISN (NLpep78; Kd=3.4nM, Dixon et al., 2016), NVTGYRLFKKISN (NLpep79; Kd=8.5nM, Dixon et al., 2016), VSGWRLFKKISN (NLpep80, Dixon et al., 2016), and those shown in WO2014151736A1 (see below) and WO2016040835A1, such as NLpep83(w / Met)MNVSGWRLFKKIS. Binding affinity can be measured, in particular, as disclosed in WO2014151736A1 (e.g., Example 26). In this case, NanoGlo luciferase assay reagent (Promega) or PBS + 0.1% Prionex® protein stabilizer and flimazine were added to the binding pair (non-luminescent polypeptide and non-luminescent peptide) whose affinity was to be measured, and the mixture was shaken at room temperature for 10 minutes. Luminescence was then detected using GloMax with an integration time of 0.5 seconds, and the Kd value was determined using Graphpad Prism, 1-site specific binding. The dissociation constant can be measured under various buffer conditions (e.g., PBS for complementation, then NanoGlo buffer for detection; PBS for complementation and detection; or NanoGlo buffer for complementation and detection), preferably in PBS.

[0060] [Table 1]

[0061] In embodiments of the present invention, a preferred reporter peptide is an 11-amino acid HiBiT peptide having the sequence VSGWRLFKKIS (SEQ ID NO: 25). HiBiT and LgBiT efficiently form a stable complex that acts as an active binding pair for detecting radionuclides by in vivo imaging.

[0062] In a remarkably preferred embodiment of the present invention, a beneficial aspect of the NanoBiT reporter assay is used. In some embodiments, the present invention may involve ligating a radiolabel to a HiBiT subunit, thereby providing a method for tracking immune cells. This method is superior to tracking luciferase activity, which is not readily measurable in vivo. Therefore, the inventors propose the use of radiolabeled HiBiT peptides in some embodiments of the present invention. This is because the inventors have surprisingly discovered that HiBiT and LgBiT can be used in the reporter gene system proposed herein, since the protein affinity between LgBiT and HiBiT is essentially maintained. The advantage is that the LgBiT subunit is small (only 0.8 kb) and easy to clone, and when combined with a radiolabeled HiBiT subunit, it enables multimodal and highly specific imaging. It has now been shown that T cells expressing transmembrane LgBiT can be generated, and that T cells can be tracked in vivo simply by adding radiolabeled SmBit.

[0063] Therefore, the present invention provides methods for diagnostic and prognostic imaging that are useful, for example, when tracking T cells in T cell therapy. Tracking cells or oncolytic viruses after infusion is also applicable to animal studies, such as those used in preclinical research.

[0064] The method of the present invention enables non-invasive in vivo imaging and provides whole-body information. PET imaging, for example, 124 It may also be performed using an I-tracer, while single-photon emission computed tomography (SPECT) is, for example, 99m The procedure may also be carried out using TcO4. The bioluminescence mode of the peptide reporter reconstituted together with the reporter domain of the fusion protein may also be used simultaneously.

[0065] The BL / PET / SPECT reporter genes proposed by this invention are advantageous because they are small, easy to clone, and enable multimodal and highly specific imaging. SPECT can be used for diagnostic / prognostic imaging of cell therapies, particularly T-cell therapy, oncolytic virus therapy, and general gene therapies (e.g., nanoparticles). The inventors have found that novel reporter genes can serve as specific reporter genes for SPECT / PET imaging when a high-affinity reporter peptide (possibly linked to a chelating agent) is used as a radiolabeled tracer. As described in one embodiment herein, exemplary TMLgBiT and DOTA-linker-HiBiT peptides represent one embodiment of a novel system for in vivo SPECT / PET imaging and correspond to preferred embodiments in the embodiments of this invention.

[0066] As envisioned by the inventors, alternative embodiments of the present invention may be based on various types of luciferases commonly used to detect protein-protein interactions, for example, firefly (Photinus pyralis) luciferase known as FLuc, sea slug (Renira reniformis) luciferase known as RLuc, copepod (Gaussia princeps) luciferase known as GLuc, and click beetle (P. plagiophthalma) and Cratomorphus discintus (Cratomorphus) luciferase known as CBR and ELuc, respectively. Examples include luciferase from the deep-sea shrimp (Oplophorus gracilirostris), known as distinctus luciferase, or NanoLuc or NLuc. The present invention is not based on the enzymatic characteristics of luciferase. More precisely, the essential element of the present invention relates to the presence of a small peptide subunit of luciferase that complements the main subunit of luciferase, as illustrated herein in the case of NanoLuc. For example, luciferase complementation subunits derived from other luciferases may be used, including a large peptide expressed in cells, preferably as a transmembrane peptide, and a small subunit peptide that can be radiolabeled. The small subunit peptide may be longer than 20 amino acids, for example, 22 to 25 amino acids. Such combinations, which are alternatives to LgBiT and HiBiT as described herein, are conceived as embodiments of the present invention, in which the radiolabeled reporter peptide is preferably about 30 amino acids or less and preferably not cleaved in the blood.

[0067] The reporter of the present invention is expressed as a transmembrane fusion protein, in which the reporter domain is the extracellular portion, and any transmembrane domain is used to present the reporter domain to the outside of the cell. The transmembrane domain of the fusion protein enables the retention of the reporter domain on the cell surface. The term “transmembrane domain,” as used herein, means any transmembrane protein domain as a region of the polypeptide chain of a protein that is self-stable and folds independently of the rest. The term “transmembrane domain,” as used herein, includes any portion of a cell transmembrane protein. Transmembrane domains of proteins may share common structural features, such as an α-helix stretch consisting of 21 to 26 hydrophobic amino acids, such as isoleucine, valine, phenylalanine, tryptophan, or methionine. The term “transmembrane,” as used herein, means a protein (also called a polypeptide herein) that binds to the cell's plasma membrane and extends from an intracellular domain or cytoplasmic domain to an extracellular domain or extracellular domain. Therefore, the reporter domain is preferably fused to the transmembrane domain at its C-terminus.

[0068] The transmembrane domain may originate from either a natural or synthetic source. If the source is natural, the domain may originate from any membrane-bound or transmembrane protein. Transmembrane regions particularly useful for the purposes of this specification may originate from members selected from the following group (i.e., including at least these transmembrane regions): the α, β, or ζ chain of the T cell receptor; CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154. Alternatively, the transmembrane domain may be synthetic, in which case it is thought to mainly consist of hydrophobic residues such as leucine and valine. In another embodiment, the transmembrane domain contains a triplet of phenylalanine, tryptophan, and valine at each end of the synthetic transmembrane domain.

[0069] In a preferred embodiment of the present invention, the transmembrane portion of the fusion protein may include, for example, a PDGFR transmembrane domain. In an alternative preferred embodiment of the present invention, the reporter domain may be fused to a transmembrane domain of CD8, for example, corresponding to AA183-203 of CD8, or to a transmembrane domain of CD4, neurexin 3b, Notch receptor polypeptide, CD28, CD137(41BB), CD8α, or CD3C, as the transmembrane domain of the fusion protein of the present invention. Various other transmembrane carrier peptides or carrier proteins may be used as the transmembrane domain of the fusion protein for extracellular expression of the reporter domain, as long as the reporter domain is capable of interacting extracellularly with an extracellularly radiolabeled reporter peptide.

[0070] In a preferred embodiment, the fusion protein further comprises a leader sequence, preferably fused at the N-terminus of the transmembrane domain in the fusion protein. Such a leader sequence can be selected from any suitable leader sequence that causes membrane-directed expression of the fusion product. Suitable leader sequences include, for example, a mouse IgK chain leader sequence fused to the N-terminus of the LgBiT peptide subunit. Alternatively, a human CD8 leader sequence may be used, which is also preferably fused to the N-terminus of the LgBiT peptide subunit. Other alternative leader sequences located at the N-terminus of the LgBiT peptide subunit may be selected from the group consisting of, but are not limited to, human OSM, mouse IgK, human IgG2 H, BM40, Secrecon, human IgKVIII, CD33, tPA, human chymotrypsinogen, human trypsinogen-2, human IL-2, albumin (HSA), and human insulin leader sequences.

[0071] The reporter peptide in the embodiments of the present invention is preferably not expressed in cells. The reporter peptide in the embodiments of the present invention is preferably administered in vivo to a subject by recombinant cells expressing the fusion protein described herein.

[0072] The reporter peptide in an embodiment of the present invention is preferably radiolabeled. The term “radiolabeled material,” as used herein, includes reference to radionuclides. In certain embodiments, the label may be “radiopaque” labeling, such as labeling that is readily visible using X-rays. Radiopaque materials are well known to those skilled in the art. The most common radiopaque materials include iodide salts, bromide salts, or barium salts. Other radiopaque materials are also known, but are not limited to, organobismuth derivatives, radiopaque polyurethanes, organobismuth composites, radiopaque barium polymer composites, etc. Preferred radiolabeled materials include, for example, radiolabeling and / or labels detectable by MRI, NMR, PET, etc. Notably preferred radiolabeled materials include, but are not limited to, 51 Cr, 52 Fe, 52m Mn, 62 Cu, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 72 As, 77 As, 89 Zr, 90 Y, 97 Ru, 99 Tc, 105 Rh, 109 Pd, 111 In, 111 Ag, 113m In, 121 Sn, 127 Te, 142 Pr, 143 Pr, 149 PM, 151 PM, 153 Sm, 157 Gd, 159 Gd,161 Tb, 165 Dy, 166 Ho, 169 Er, 169 Yb, 172 Tm, 175 Yb, 177 Lu, 186 Re, 188 Re, 198 Au, 199 Au, and 203 Pb is one example. While not limited to specific useful PET labels, 11 C, 18 F, 15 O, 13 Examples include N. Common labels used in MRI include, but are not limited to, gadolinium chelates and various surface-modified iron oxide nanoparticles or microparticles. Gadolinium chelates such as gadopentetate dimeglumine are the most widely used paramagnetic contrast agents. Iron oxide particles are part of the class of superparamagnetic MRI contrast agents. These compounds typically consist of a magnetite (iron oxide) core, coated with dextran or siloxane, encapsulated with polymers, or further modified.

[0073] The radiolabeled substance is preferably bound to the reporter peptide using a chelating agent. To this end, the reporter peptide in the embodiment of the present invention is preferably bound to a chelating agent (via the formation of a coordination bond) to enable the binding of the radiolabeled substance to the reporter peptide. Chelating groups are well known to those skilled in the art. In certain embodiments, the chelating group is derived from ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), cyclohexyl 1,2-diaminetetraacetic acid (CDTA), ethylene glycol-O,O'-bis(2-aminoethyl)-N,N,N',N'-tetraacetic acid (EGTA), N,N-bis(hydroxybenzyl)-ethylenediamine-N,N'-diacetic acid (HBED), triethylenetetraminehexaacetic acid (TTHA), 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid (DOTA), hydroxyethyldiaminetriacetic acid (HEDTA), 1,4,8,11-tetra-azacyclotetradecane-N,N',N",N"'-tetraacetic acid (TETA), substituted DTPA, substituted EDTA, etc. Some examples of preferred chelating agents include unsubstituted or substituted 2-iminothiolanes and 2-iminothiacyclohexane, particularly 2-imino-4-mercaptomethyltilan. One chelating agent, namely 1,4,7,10-tetraazacyclododecane-N,N,N",N'"-tetraacetic acid (DOTA), is particularly preferred because it has the ability to chelate several diagnostically and therapeutically important metals, such as radionuclides and radiolabeled materials. Examples of particularly preferred chelating agents include DOTA and its derivatives, cb-do2a, tcmc, TETA, CB-TE2A, CB-TE1A1P, DIAMSAR, NOTA and its derivatives, NETA, NETA-monoamide, TACN™, DOTAGA, NODAGA, DTPA, CHX-A-DTPA, TRAP, AAZTA, H2dedpa, h4octapa, h2decapa, H2azapa, HBED, SHBED, BPCA, CP256, DFO, PCTA, p-SCN-Bn-DFO296, p-SCN-Bn-H6phospa, HEHA, and PEPA.

[0074] The chelating agent may then be directly bound to the reporter peptide, but preferably, it is bound to the reporter peptide using a linker. “Linker” or “conjugate,” as used herein, is a molecule used to link two or more molecules together, and is sometimes called a spacer. In certain embodiments, linkers typically have the ability to form a covalent bond with both molecules. Conjugates are well known to those skilled in the art. In preferred embodiments of the present invention, linkers include, but are not limited to, 6-aminohexanoic acid (6ahx), 4-aminobutyric acid (GABA), (2-aminoethoxy)acetic acid (AEA), PEG2 spacer (8-amino-3,6-dioxaoctanoic acid), PEG3 spacer (12-amino-4,7,10-trioxadodecanoic acid), PEG4 spacer (15-amino-4,7,10,13-tetraoxapentadecanoic acid), 5-aminovaleric acid (Ava), β-alanine, and Ttds (trioxatridecane-succinamic acid). When directly bound or when 6ahx is used as a linker, the affinity between the (fusion) peptides is increased, resulting in very favorable outcomes. The linker is preferably bound to a valine residue of the reporter peptide, because this binding has the least effect on the interaction (reconstitution) between the reporter peptide and the reporter domain of the fusion protein.

[0075] Alternatively, the linker between the chelating agent and the reporter peptide may contain, or consist of, a non-covalent albumin-binding ligand. Such a linker can extend the circulating half-life of the radiolabeled reporter peptide. Non-limiting examples of such ligands are provided, in particular, in Zorzi et al. 2019 (Med. Chem. Commun. 10, p. 1068), specifically the albumin-binding molecules in Tables 1-3, and the albumin-binding small organic compounds, albumin-binding peptide ligands, and albumin-binding protein ligands in Figures 2-4 of the literature, all of which are incorporated herein by reference.

[0076] Next, a preferably chelated and radiolabeled reporter peptide is preferably injected into a living subject as a tracer to interact with cells expressing the fusion protein described herein.

[0077] A particularly preferred embodiment of the radiolabeled reporter peptide in the present invention is [ 111 In]-DOTA-6ahx-HiBiT is the correct command.

[0078] The proper reconstitution of the fusion protein's reporter domain and reporter peptide can be confirmed by in vitro testing for luminescence generation using a suitable luciferin-based luminescent substrate, namely d-luciferin (FLuc, CBR, and ELuc), coelenterazine, or its derivatives or analogs, such as flimazine (RLuc, GLuc, and NanoLuc).

[0079] The present invention is particularly applicable to cell therapies (e.g., CAR T cells or TCR T cells). Chimeric antigen receptor (CAR) T cells are T cells that have been genetically engineered to produce artificial T cell receptors for use in immunotherapy. CARs are receptor proteins engineered to give T cells a new ability to target specific proteins. This receptor is chimeric in that it combines both antigen-binding and T cell-activating functions into a single receptor. The premise of CAR-T immunotherapy is to modify T cells to recognize cancer cells in order to more effectively target and destroy cancer cells.

[0080] In CAR T-cell therapy, T cells are collected from the patient and transformed to express a specific CAR, which is programmed to target antigens present on the tumor surface. CAR-T cells may be derived from the patient's own T cells in their blood (autologous) or from T cells of another healthy donor (allogeneic). Subsequently, the transformed T cells are infused into the patient to attack their tumor. After infusion, CAR-T cells act as "living drugs" against cancer cells. When CAR-T cells come into contact with target antigens on cells, they bind to them, become activated, and then begin to proliferate and become cytotoxic. CAR-T cells destroy cells through several mechanisms, including the rapid proliferation of stimulated cells, increased toxicity (cytotoxicity) of CAR-T cells to other living cells, and increased secretion of factors that can affect other cells, such as cytokines, interleukins, and growth factors.

[0081] The inventors hereby propose to include, in these CAR T cells or within the cells of other therapeutic cell therapies, a reporter gene of the present invention that enables in vivo radionuclide imaging of therapeutic cells. The term "radionuclide imaging," as used herein, means a non-invasive technique for inferring the distribution of radionuclides within a subject's body (tissue) by detecting photons emitted due to the decay of tracers introduced into the subject's body using a (gamma) radiation detector placed outside the subject under study.

[0082] One form of radionuclide imaging envisioned by the inventors includes positron emission tomography (PET). When used herein, the terms “positron emission tomography” or “PET” refer to one of the two major nuclear imaging techniques currently in widespread use, in which the three-dimensional distribution of tracers labeled with a positron emitter is measured. Capture is performed using a set of detectors arranged around the target. The positron emitter is a radioisotope (e.g., 11 C,13 N, 15 O, 18 F) is the answer.

[0083] Another form of radionuclide imaging envisioned by the inventors includes single-photon emission computed tomography (SPECT). When used herein, the terms “single-photon emission computed tomography” or “SPECT” refer to one of the two major nuclear imaging techniques currently in widespread use, in which imaging is performed by acquiring images from multiple angles using gamma rays. For the heart, a 180-degree arc is preferred. Cross-sectional images are created for all axial locations covered by the field of view (FOV) of the gamma camera, resulting in a stack of 2D images forming a 3D dataset. This technique requires the delivery of a gamma-ray emitting radioisotope (radionic nuclide) into the patient's body, for example, by injection into the bloodstream. In embodiments of the present invention, the radioisotope is bound to a small peptide to provide a tracer molecule. Examples of radionuclides for use in SPECT include, for example, 99 Y or 111 Examples include [mention specific examples]. Other examples are provided below in this specification. The term “radionuclide imaging,” as used herein, also includes references to hybrid imaging systems (e.g., PET / CT, SPECT / CT, and PET / MR).

[0084] Further details of the present invention, including specific methods and techniques for creating and using its subject matter, are described below.

[0085] Expression constructs and transformations The term “vector” is used to mean a carrier nucleic acid molecule into which a nucleic acid sequence can be inserted for introduction into a cell that can replicate the nucleic acid sequence. The nucleic acid sequence may be “exogenous,” where “exogenous” means that the sequence is foreign to the cell into which the vector is introduced, or that the sequence is homologous to a sequence in the cell but is located in a position within the host cell nucleic acid where the sequence is not normally present. Examples of vectors include plasmids, cosmids, viruses (bacteriophages, animal viruses, and plant viruses), and artificial chromosomes (e.g., YACs). Those skilled in the art will be well capable of constructing vectors using standard recombination techniques (see, for example, Maniatis et al., Molecular Cloning, A Laboratory Manual (Cold Spring Harbor, 1990) and Ausubel et al., 1994, Current Protocols In Molecular Biology (John Wiley & Sons, 1996), both of which are incorporated herein by reference).

[0086] The term “expression vector” refers to any type of gene construct containing a transcribed nucleic acid that codes for RNA. In some cases, the RNA molecule is then translated into a protein, polypeptide, or peptide. In other cases, for example, when producing an antisense molecule or ribozyme, these sequences are not translated. An expression vector may contain various “regulatory sequences,” where “regulatory sequences” refer to nucleic acid sequences necessary for the operably linked coding sequence to be transcribed and, in some cases, translated in a particular host cell. In addition to regulatory sequences that govern transcription and translation, vectors and expression vectors may also contain nucleotide sequences that perform other functions.

[0087] In certain embodiments, plasmid vectors are intended for use in cloning and gene transfer. Generally, plasmid vectors containing replicons and regulatory sequences derived from a species compatible with host cells are used with these hosts. Typically, the vector has replication sites, as well as marking sequences that enable phenotypic selection in transformed cells. In a non-limiting example, Escherichia coli (E. coli) is often transformed using derivatives of pBR322, a plasmid derived from the E. coli species. pBR322 contains genes for ampicillin and tetracycline resistance and therefore provides a convenient means for identifying transformed cells. The pBR plasmid, or other microbial plasmids or phages, must also contain, or be modified to contain, a promoter that the microorganism can use to express its own proteins, for example.

[0088] Furthermore, phage vectors containing replicons and regulatory sequences compatible with host microorganisms can be used as transformation vectors together with these hosts. For example, phagelambda GEM(trademark)-1 may be used when preparing recombinant phage vectors, which can then be used to transform host cells such as E. coli LE392.

[0089] Bacterial host cells containing the expression vector, such as E. coli, are grown in one of several suitable media, e.g., LB. Expression of recombinant proteins in certain vectors can be induced by contacting the host cells with a promoter-specific active agent, e.g., by adding IPTG to the medium, or by switching the incubation to a higher temperature, as will be understood by those skilled in the art. After culturing the bacteria for a further period, usually between 2 and 24 hours, the cells are harvested by centrifugation and washed to remove any remaining medium.

[0090] Many prokaryotic vectors can also be used to transform eukaryotic host cells. However, it may be desirable to select vectors modified for the specific purpose of expressing proteins in eukaryotic host cells. Expression systems are designed for regulated and / or high levels of expression in such cells. For example, insect cell / baculovirus systems can result in high levels of protein expression of heterologous nucleic acid segments, as described in U.S. Patents 5,871,986 and 4,879,236, both incorporated herein by reference, and are available, for example, from INVITROGEN® under the name MAXBAC® 2.0 and from CLONTECH® under the name BACPACK® baculovirus expression system.

[0091] Other examples of expression systems include STRATAGENE®'s COMPLETE CONTROL® inducible mammalian expression system, which uses a synthetic ecdysone-inducible receptor, or its pET expression system, which is an E. coli expression system. Another example of an inducible expression system is available from INVITROGEN®, which has the T-REX® (tetracycline-regulated expression) system, an inducible mammalian expression system using a full-length CMV promoter. INVITROGEN® also offers a yeast expression system called the Pichia methanolica expression system, designed for high-level production of recombinant proteins in the methylotrope yeast Pichia methanolica. Those skilled in the art will be familiar with methods of expressing vectors, such as expression constructs, for the purpose of producing nucleic acid sequences or polypeptides, proteins, or peptides of the same family.

[0092] Regulatory markers The construct may include additional 5' and / or 3' elements such as promoters and polyA sequences. These elements may originate from the host cell, i.e., be homogeneous with the host, or they may originate from another source, i.e., heterogeneous.

[0093] A "promoter" is a regulatory sequence, which is a region within a nucleic acid sequence, where the initiation and rate of transcription are controlled. A promoter may include a gene element to which regulatory proteins and molecules, such as RNA polymerase and other transcription factors, can bind to initiate specific transcription of the nucleic acid sequence. The expressions "operationally positioned," "operationally linked," "controlled," and "transcriptionally regulated" mean that the promoter is located in the correct functional position and / or orientation relative to a given nucleic acid sequence in order to control its transcription initiation and / or expression.

[0094] Typically, promoters contain sequences that function to locate the start site of RNA synthesis. The best-known example of this is the TATA box, but in some promoters that lack a TATA box, such as the promoter of the mammalian terminal deoxynucleotidyltransferase gene and the promoter of the SV40 late gene, a separate element that overlaps the start site itself helps to determine the start location. Additional promoter elements regulate the frequency of transcription initiation. Typically, these are located in the 30–110 bp region upstream of the start site, but some promoters have been shown to also contain functional elements downstream of the start site. To place the coding sequence "under the control" of the promoter, the 5' end of the transcription start site of the transcription reading frame is positioned "downstream" (i.e., 3') of the selected promoter. The "upstream" promoter promotes DNA transcription and facilitates the expression of the encoded RNA.

[0095] The spacing between promoter elements is often flexible, and therefore, promoter function is maintained even if the positions of the elements are reversed or moved relative to each other. In tk promoters, the spacing between promoter elements can be increased to a distance of 50 bp, beyond which activity begins to decrease. Promoters appear to allow individual elements to function cooperatively or independently to activate transcription. Promoters may or may not be used with "enhancers," where "enhancers" refer to cis-acting regulatory elements involved in the transcriptional activation of nucleic acid sequences.

[0096] Promoter may be naturally associated with the nucleic acid molecule and can be obtained by isolating a 5' non-coding sequence located upstream of the coding segment and / or exon. Such promoters may be called “endogenous.” Similarly, enhancers may also be naturally associated with the nucleic acid molecule and are located downstream or upstream of its sequence. Alternatively, several advantages can be obtained by placing the coding nucleic acid segment under the control of a recombinant or heterologous promoter, which means a promoter that is not normally associated with the nucleic acid molecule in its natural environment. A recombinant or heterologous enhancer means an enhancer that is not normally associated with the nucleic acid molecule in its natural environment. Examples of such promoters or enhancers include promoters or enhancers of other genes, promoters or enhancers isolated from any other virus or prokaryotic or eukaryotic cell, and promoters or enhancers that are not “naturally occurring,” i.e., those containing mutations that alter the expression of various elements and / or various transcriptional regulatory regions. For example, the most commonly used promoters in recombinant DNA construction include β-lactamase (penicillinase), lactose, and tryptophan (trp) promoter systems. In addition to generating promoter and enhancer nucleic acid sequences by synthesis, sequences may also be prepared using nucleic acid amplification techniques, including recombinant cloning and / or PCR®, together with the compositions disclosed herein (see U.S. Patents 4,683,202 and 5,928,906, respectively, incorporated herein by reference). Furthermore, it is intended that regulatory sequences that direct the transcription and / or expression of sequences in non-nuclear organelles such as mitochondria and chloroplasts may also be used.

[0097] Naturally, it is considered important to use promoters and / or enhancers that effectively direct the expression of the DNA segment in a selected organelle, cell type, tissue, organ, or organism for expression. Those skilled in the field of molecular biology are usually familiar with the use of promoter, enhancer, and cell type combinations for protein expression (see, for example, Sambrook et al., 1989, incorporated herein by reference). The promoter used may be useful under appropriate conditions that lead to high levels of expression of the introduced DNA segment, such as being constitutive, tissue-specific, inducible, and / or advantageous for the large-scale production of recombinant proteins and / or recombinant peptides. The promoter may be heterologous or endogenous.

[0098] Furthermore, any promoter / enhancer combination (e.g., according to the Eukaryotic Promoter Database EPDB) can also be used to promote expression. The use of T3, T7, or SP6 cytoplasmic expression systems is another feasible embodiment. Eukaryotic cells can boost cytoplasmic transcription from specific bacterial promoters when appropriate bacterial polymerases are provided as part of a delivery complex or as additional gene expression constructs.

[0099] For efficient translation of coding sequences, specific start signals may also be required. These signals may include ATG start codons or adjacent sequences. Exogenous translational regulatory signals, including ATG start codons, may need to be provided. Those skilled in the art will be able to easily determine this and provide the necessary signals. It is well known that start codons must be "in-frame" with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational regulatory signals and start codons may be natural or synthetic. Expression efficiency can be improved by including appropriate transcriptional enhancer elements.

[0100] In certain embodiments of the present invention, the use of intra-sequence ribosome entry site (IRES) elements is used to create multi-gene or polycistronic messages. IRES elements can bypass the ribosome scanning model of 5' methylation cap-dependent translation and initiate translation at an internal site (Pelletier and Sonenberg, Nature, 334:320-325 (1988)). IRES elements derived from two members of the Picornaviridae family (polio and encephalomyocarditis) (Pelletier and Sonenberg, hereafter) and IRES derived from mammalian messages (Macejak and Sarnow, Nature, 353:90-94 (1991)) are described. All are useful in embodiments of the present invention. IRES elements can be ligated to heterologous open reading frames. Multiple open reading frames, each separated by an IRES, can be transcribed together to create a polycistronic message. Thanks to the IRES element, each open reading frame can approach the ribosome for efficient translation. Multiple genes can be efficiently expressed using a single promoter / enhancer to transcribe a single message (see U.S. Patents 5,925,565 and 5,935,819, respectively, incorporated herein by reference). The stoichiometry of the simultaneous expression of multiple factors or multiple units of complex proteins or multiple genes cloned into a single vector (i.e., a polycistronic vector) can be improved by the use of 2A "self-cleaving" peptides, which are 18-22 amino acid-length viral oligopeptides that result in polypeptide "cleavage" during translation in eukaryotic cells.Suitable examples for use in embodiments of the present invention include, for example, 2A sequences / peptides derived from foot-and-mouth disease virus (F2A), equine rhinitis A virus (E2A), porcine teschovirus-1 (P2A), or Thosea asigna virus (T2A), which may have a GSG linker at the N-terminus, and the corresponding coding sequence may be inserted in-frame between two coding DNA sequences.

[0101] Suitable promoters for use in embodiments of the present invention include, but are not limited to, the CMV promoter, EF1-α, PGK1, SV40, CAGGS, UBC, human B-actin constitutive promoter, tissue-specific promoters (e.g., CD2, CD8, CD3, TCF-1 promoters), inductive promoters (e.g., NFAT, NFkb, TOX, TOX2, BATF3), and chemically inductive promoters (e.g., tetracycline or doxycycline-regulated transcriptional activation [Tet-On / Tet-Off] systems).

[0102] other vector array elements A vector may contain a multi-cloning site (MCS), which is a nucleic acid region containing multiple restriction enzyme sites, and the vector can be digested using any of these restriction enzyme sites in conjunction with standard recombination techniques (see, for example, Carbonelli et al., FEMS Microbiol. Lett., 172(1):75-82 (1999), Levenson et al., Hum. Gene Ther. 9(8):1233-1236 (1998), and Cocea, Biotechniques, 23(5):814-816 (1997), incorporated herein by reference). "Restriction enzyme digestion" means the catalytic cleavage of a nucleic acid molecule by an enzyme that functions only at a specific site within the nucleic acid molecule. Many of these restriction enzymes are commercially available. The use of such enzymes is widely understood by those skilled in the art. Often, vectors are linearized or fragmented using restriction enzymes that cleave within the MCS so that an exogenous sequence can be linked to the vector. "Lucmination" refers to the process of forming a phosphodiester bond between two nucleic acid fragments, which may or may not be adjacent to each other. Techniques using restriction enzymes and luciation reactions are well known to those skilled in the art of recombinant technology.

[0103] Most transcribed eukaryotic RNA molecules undergo RNA splicing, which removes introns from the primary transcript. Vectors containing eukaryotic genome sequences may require donor splicing sites and / or acceptor splicing sites to ensure proper processing of the transcript for protein expression (see, for example, Chandler et al., 1997, incorporated herein by reference).

[0104] Typically, the vectors or constructs of the present invention include at least one termination signal. The “termination signal” or “terminator” includes a DNA sequence involved in the specific termination of an RNA transcript by RNA polymerase. Thus, in certain embodiments, a termination signal is intended to end the production of an RNA transcript. The terminator may be required in vivo to achieve a desired message level.

[0105] In eukaryotic systems, the terminator region may also contain a specific DNA sequence that enables site-specific cleavage of a new transcript to expose a polyadenylation site. This signals a specific endogenous polymerase that adds a stretch (poly-A) consisting of approximately 200 adenosine residues to the 3' end of the transcript. RNA molecules modified with this poly-A tail appear to be more stable and translated more efficiently. Therefore, in other embodiments using eukaryotes, it is preferable that the terminator contains a signal for RNA cleavage, and more preferably that the terminator signal promotes polyadenylation of the message. The terminator element and / or polyadenylation site element can serve to enhance the message level and minimize read-through from the cassette to other sequences.

[0106] Terminators intended for use in the present invention include, but are not limited to, any known transcriptional terminators described herein or known to those skilled in the art, including gene termination sequences such as bovine growth hormone terminators and viral termination sequences such as SV40 terminators. In certain embodiments, the termination signal may be the absence of a transcribable or translatable sequence, such as an untranslatable / untranscribeable sequence resulting from sequence cleavage.

[0107] In expression, particularly in eukaryotic expression, it is common practice to include a polyadenylation signal to ensure proper polyadenylation of the transcript. The nature of the polyadenylation signal is not considered critical to the success of the invention, and any such sequence may be used. Preferred embodiments include the SV40 polyadenylation signal or the bovine growth hormone polyadenylation signal, both of which are simple, readily available, and known to function well in a variety of target cells. Polyadenylation can enhance the stability of the transcript or promote cytoplasmic transport.

[0108] To propagate the vector in host cells, the vector may contain one or more origin sites, which are specific nucleotide sequences at which replication begins. Alternatively, if the host cell is yeast, an ARS (arranged replication sequence) may be used.

[0109] Transformation method Suitable methods for nucleic acid delivery to be used in conjunction with the present invention include substantially any method by which nucleic acid molecules (e.g., DNA) can be introduced into cells, as described herein or as may be known to those skilled in the art. Such methods include, but are not limited to, the following: direct delivery of DNA, for example, by ex vivo transfection (Wilson et al., Science, 244:1344-1346 (1989), Nabel et al., Science, 244:1342-1344 (1989)), by injection (U.S. Patents Nos. 5,994,624, 5,981,274, 5,945,100, 5,780,448, 5,736,524, 5,702,932, 5,656,610, 5,589,466, and 5,580,859, respectively, incorporated herein by reference), and microinjection (Harlan and Weintraub, J. Cell, incorporated herein by reference). Methods including Biol., 101(3):1094-1099 (1985); U.S. Patent No. 5,789,215; electroporation method (incorporated herein by reference, U.S. Patent No. 5,384,253, Tur-Kaspa et al., Mol. Cell Biol., 6:716-718 (1986); Potter et al., Proc. Natl. Acad. Sci. USA, 81:7161-7165 (1984)); calcium phosphate precipitation method (Graham and Van Der Eb, Virology, 52:456-467 (1973); Chen and Okayama, Mol. Cell Biol., 7(8):2745-2752 (1987); Rippe et al., Mol. Cell Biol., 10: pp. 689-695 (1990); Method using DEAE-dextran followed by polyethylene glycol (Gopal, Mol. Cell Biol., 5: pp. 1188-190 (1985)); Method using direct ultrasonic loading (Fechheimer et al., Proc. Natl. Acad. Sci.USA, 89(17):pp. 8463-8467 (1987)); liposome-mediated transfection (Nicolau and Sene, Biochem. & Biophys. Acta., 721:pp. 185-190 (1982); Fraley et al., Proc. Natl. Acad. Sci. USA, 76:pp. 3348-3352 (1979); Nicolau et al., Meth. Enzym., 149:pp. 157-176 (1987); Wong et al., Gene, 10:pp. 879-894 (1980); Kaneda et al., Science, 243:pp. 375-378 (1989); Kato et al., J. Biol. Chem., 266:pp. 3361-3364 (1991)) and receptor-mediated transfection (Wu and Wu, J. Biol. Methods by Chem., 262:4429-4432 (1987); Wu and Wu, 1988); methods by PEG-mediated protoplast transformation (incorporated herein by reference, Omirulleh et al., Plant Mol. Biol., 21(3):415-428 (1987); U.S. Patents No. 4,684,611 and 4,952,500, respectively); DNA incorporation via drying / inhibition (Potrykus et al., Mol. Gen. Genet., 199(2):169-177 (1985)); and any combination of such methods.

[0110] Transduction of T cells can be performed, for example, using a virus or a non-viral gene transfer method. Examples of virus-based methods include lentiviral gene transfer and retroviral gene transfer. The gene expression construct for expressing the reporter gene in cells can be expressed in cells such as T cells or natural killer (NK) cells. The gene expression construct for expressing the reporter gene in cells can be expressed as integrated nucleic acid (e.g., DNA integrated into the host genome using transposases / transposons) or as non-integrated nucleic acid (e.g., mRNA delivered by a viral vector such as a lentivirus or retrovirus). Subsequently, T cells or NK cells expressing the gene expression construct for expressing the reporter gene in cells can be administered to human patients or other subjects in addition to a pharmaceutical preparation or excipient to treat or prevent diseases (e.g., cancer, fungal infections, bacterial infections, or viral infections). In some embodiments, naked DNA or a suitable vector encoding a gene expression construct for expressing a reporter gene in cells may be introduced into target T cells (e.g., T cells obtained from a human patient with cancer or another disease). Methods for stably transfecting T cells by electroporation using naked DNA are known in the art. See, for example, U.S. Patent No. 6,410,319. Typically, naked DNA means DNA encoding a gene expression construct for expressing the reporter gene of the present invention in cells, which is contained in a plasmid expression vector in an orientation appropriate for expression. In some embodiments, using naked DNA can reduce the time required to produce T cells expressing the gene expression construct for expressing the reporter gene in cells, which are produced using the method of the present invention. Alternatively, a viral vector (e.g., a retroviral vector, adenovirus vector, adeno-associated virus vector, or lentiviral vector) may be used to introduce the gene expression construct into T cells. Typically, a vector encoding a gene expression construct used to transfect T cells derived from a target is used. It is desirable that the vector does not replicate in the target T cells. Numerous virus-based vectors are known in which the number of viral copies maintained in the cell is small enough to maintain the cell's viability. Exemplary vectors include pFB-neo-vectors (STRATAGENE®) and vectors based on HIV, SV40, EBV, HSV, or BPV. Alternatively, transposon systems such as Sleeping Beauty or PiggyBac represent a non-viral method for gene transfer and provide a cost-effective alternative to the high-cost production of viruses in accordance with Good Manufacturing Practice (GMP) standards for the manufacture and quality control of pharmaceuticals intended for clinical use. Those skilled in the art will readily understand how to apply DNA plasmid systems, including transposon systems consisting of transposases and transposons such as Sleeping Beauty or PiggyBac, which are now frequently used for genetic manipulation of human cells for therapeutic purposes.

[0111] Once it is confirmed that transfected or transduced T cells can perform the desired regulation and express the gene expression construct as a surface membrane protein at the desired level, it can be determined whether the reporter functions in host cells to provide the desired binding to a complementary HiBiT peptide labeled with a radionuclide. Subsequently, the transduced T cells may be reintroduced or administered to a target to activate an antitumor response in the target. To facilitate administration, the transduced T cells may be prepared into a pharmaceutical composition, preferably using a suitable pharmaceutically acceptable carrier or diluent, or into an implantation suitable for in vivo administration. Means for preparing such compositions or implantations have been described in the Art (see, for example, Remington's Pharmaceutical Sciences, 16th edition, Mack (ed.) (1980)). If necessary, the transduced T cells expressing the reporter gene may be formulated into semi-solid or liquid formulations, such as capsules, solutions, injections, inhalations, or aerosols, by conventional methods for each of their administration routes. By means known in the art, the release and absorption of a composition can be prevented or minimized until it reaches a target tissue or organ, or sustained release of the composition can be ensured. Typically, a pharmaceutically acceptable form that does not inactivate cells expressing the reporter gene is preferred. Therefore, preferably, transduced T cells can be processed into a pharmaceutical composition containing an equilibrium salt solution such as Hanks equilibrium salt solution or physiological saline. [Examples]

[0112] (Example 1) Chimeric transmembrane LgBiT A transmembrane LgBiT sequence was created by cleaving an LgBiT sequence from pBiT1.1-C[TK / LgBiT] (see Figure 5; Promega, Madison, WI, USA) using restriction enzymes BglII and SalI, and inserting it into the cloning site of the pDisplay® vector (Thermos Fisher Scientific, Waltham, Ma, USA), a mammalian expression vector for cell surface protein presentation. The recombinant protein is fused at its N-terminus to a mouse Ig κ chain leader sequence for secretory pathway processing, and at its C-terminus to a platelet-derived growth factor receptor (PDGFR) transmembrane domain for immobilizing the protein on the plasma membrane and enabling extracellular presentation. In this way, a construct containing the coding sequence of a chimeric or fusion protein is created, comprising the IgGκ chain leader sequence-LgBiT sequence-PDGFR transmembrane domain sequence (from 5' to 3' of the nucleic acid and from the N-terminus to the C-terminus of the expressed fusion protein). Next, using BamHI and NotI, the complete sequence for the chimeric protein was excised from the pDisplay® vector and inserted into the multi-cloning site of the pCDH-EF1-MCS lentiviral vector (System Biosciences, Palo Alto, CA, USA) to create the plasmid pCDH-EF1-LgBiT. The chimeric protein was under the control of the EF1-α promoter. Lentiviral particles were generated by transfection of HEK293 cells using the packaging plasmid and plasmid pCDH-EF1-LgBiT. Virus quantification was performed by antigen capture ELISA (ZeptoMetrix, NY, USA) to measure HIV p24 levels. For transduction, PC3 cells (human prostate cancer cell line) were resuspended in culture medium. Pseudotyped viral particles containing the chimeric transmembrane LgBiT construct were added to the cells (1 × 10⁻⁶). 5 (40 ng of virus per cell was used). Transduced PC3 cells were selected by serial dilution.

[0113] HiBiT synthesis The HiBiT peptide VSGWRLFKKIS was synthesized using an Nα-Fmoc solid-phase peptide synthesis strategy. The Fmoc-protected sequence (Val-Ser(tBu)-Gly-Trp(Boc)-Arg(Pbf)-Leu-Phe-Lys(Boc)-Lys(Boc)-Ile-Ser(tBu)) was coupled to a 2-chlorotrityl chloride resin in dimethylformamide (DMF) for 45 minutes using hexafluorophosphate azabenzotriazole tetramethyluronium (HATU) (3.8 equivalents) and N,N-diisopropylethylamine (DIPEA) (7.8 equivalents). Fmoc deprotection was performed by treating the resin with a 20% solution of piperidine dissolved in DMF. Amide formation and Fmoc deprotection were monitored by the Kaiser test. If the reaction was incomplete, double coupling or Fmoc deprotection was performed. Peptide synthesis was initiated by loading Fmoc-L-Ser(tBu)-OH (1.6 mmol, 4 equivalents) onto a solid support (0.25 g, load capacity: 1.6 mmol / g). This resin was shaken at room temperature for 90 minutes. The resin was capped with dichloromethane / methanol / N,N-diisopropylethylamine (DCM / MeOH / DIPEA) (10 mL, 80:15:5, v / v / v) at room temperature for 15 minutes. Subsequent deprotection of Fmoc and coupling with Fmoc-L-Ile-OH, Fmoc-L-Lys(Boc)-OH, Fmoc-L-Lys(Boc)-OH, Fmoc-L-Phe-OH, Fmoc-L-Leu-OH, Fmoc-L-Arg(Pbf)-OH, Fmoc-L-Trp(Boc)-OH, Fmoc-Gly-OH, Fmoc-L-Ser(tBu)-OH, and Fmoc-L-Val-OH were carried out using 4 equivalents of each protected amino acid according to the protocol described above.

[0114] DOTA-6ahx-HiBiT synthesis Linker attachment to the N-terminal valine residue was carried out using Fmoc-6ahx-OH (2 equivalents), HATU (3.8 equivalents), and DIPEA (7.8 equivalents) dissolved in DMF. The resin was stirred at room temperature for 2 hours. The Fmoc protecting group was then removed by washing the resin twice with DMF and treating the resin with a 20% solution of piperidine dissolved in DMF. DOTA-tris(tBu) ester (3 equivalents) was attached to the peptide in the presence of benzotriazole-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBoP) (3 equivalents), DIPEA (6 equivalents), and DMF. The reaction was carried out overnight at room temperature. Peptide cleavage and simultaneous deprotection from the solid support were carried out by treating the resin with a solution of trifluoroacetic acid / water / triisopropylsilane (TFA / H2O / TIPS) (95:2.5:2.5, v / v / v) at room temperature for 6 hours. The filtrate was collected and the solvent was evaporated under reduced pressure. The residue was triturated with cold diethyl ether to obtain the final crude product, which was purified by HPLC to obtain pure DOTA-6ahx-HiBiT as a white solid (12 mg, 16.8%). ESI-MS: m / z 910.80 [M+2H] 2+ , 922.21[M+Na+H] 2+ , and 930.19[M+2Na] 2+ .

[0115] Radioactive labeling of DOTA-6ahx-HiBiT A mixture of DOTA-6ahx-HiBiT (1 nmol), ascorbic acid / gentisic acid (10 μL, 50 mM), sodium acetate (1 μL, 2.5 M), and H2O (29.7 μL) 111 InCl3 (93.3 μL, 150 MBq) was added. This mixture was incubated at 90°C for 20 minutes. The reaction was monitored by elution with sodium citrate solution (0.1 M, pH 5.0) using instant thin-layer chromatography (iTLC) with glass fiber sheets impregnated with silica gel. The reaction mixture was cooled for 5 minutes, and diethylenetriaminepentaacetic acid (DTPA) (5 μL) was added to remove any remaining free indium. -111 [111 The radiochemical yield and molar activity of In]-DOTA-6ahx-HiBiT were measured to be 93% and 150 MBq / nmol, respectively.

[0116] result The above-described examples provide embodiments of a novel reporter gene for PET / SPECT imaging according to the present invention.

[0117] The reporter gene produced in this example contains a membrane expression protein based on the LgBiT portion of NanoLuc, and the tracer is a HiBiT peptide chemically modified by the addition of a chelating agent and a linker.

[0118] First, the inventors evaluated the functionality of a novel membrane-expressed LgBiT moiety. Cells expressing transmembrane LgBiT (TMLgBiT) were seeded, and HiBiT peptide was added along with the bioluminescent substrate flimazine. If complementarity occurs between the TMLgBiT peptide and the HiBiT peptide, light is generated after the addition of the substrate. Figure 1 shows the light signals collected after the reaction of various concentrations of HiBiT peptide with TMLgBiT expressed in the membrane of HEK-293 cells.

[0119] Next, using various types of linkers, HiBiT peptides were linked to DOTA chelating agents, and the affinity of the newly generated DOTA-HiBiT peptides for LgBiT proteins was evaluated in vitro.

[0120] The equilibrium dissociation constant (Kd) was calculated as follows. An Opti-MEM solution containing 10% fetal bovine serum (FBS) (Thermo Fisher Scientific) was prepared, and LgBiT protein was diluted in Opti-MEM with 10% FBS from a starting concentration of 200 nM to a final concentration of 2 nM (500 μL each). A 3-fold dilution series of synthesized peptides (DOTA-6ahx-HiBiT, DOTA-HiBiT) and natural HiBiT was prepared and diluted as follows: 350 μL of Opti-MEM with 10% FBS was added to 150 μL of peptide solution. 90 μL of the above prepared solution was added triplicately to a white assay plate (Costar 3600), and 10 μL of 2 nM LgBiT solution (final concentration 0.2 nM) was added to the well containing the peptide solution. The mixture was incubated at 600 RPM for 30 minutes using an orbital shaker. A solution of flimazine (Promega) and 1 mM DTT (Thermo Fisher Scientific) was prepared by dissolving them in Opti-MEM with 10% FBS, and 10 μL was added to each well. After adding the solution, the plate was incubated on an orbital shaker at 600 RPM for 5 minutes. Luminescence was measured using a GloMax Multi illuminometer (Promega) with an integration time of 0.5 seconds per well. The Kd value was calculated using GraphPad Prism and single-site specific binding.

[0121] These results indicated an improved affinity of the HiBiT peptide for LgBiT, with a decrease in Kd to approximately one-tenth, suggesting that the in vivo reaction was not impaired at all. Due to its high stability, the HiBiT-6ahx-DOTA peptide (HiBiT-linker-DOTA) was selected for subsequent tests. Figure 2 shows the calculated Kd values ​​for the reaction between LgBiT protein and HiBiT peptide, given using a single-site specific binding function. The calculated Kd values ​​were 6.8 nM for HiBiT, 1.3 nM for HiBiT DOTA, and 0.7 for HiBiT-6ahx-DOTA.

[0122] Next, HiBiT-6ahx-DOTA for SPECT imaging 111 We evaluated the complementation response in living cells when radiolabeled with indium (150 mBq / nanomol).

[0123] [ 111 The In]-DOTA-6ahx-HiBiT peptide (1 nM) was added to cells expressing the reporter gene TMLgBiT, and cells not expressing TMLgBiT were used as a negative control (20,000 cells / well). The cells were washed three times, and radioactivity was measured using a gamma-ray counter. A clear difference in radioactive signal (2000-fold) was observed between TMLgBiT-expressing cells and cells not expressing the reporter gene. Figure 3 shows the radioactivity of 1 nmol [ 111 Radiation data (measured in units of CPM using a gamma-ray counter) for TMLgBiT-expressing cells and control cells after the addition of the In]-DOTA-6ahx-HiBiT peptide is provided.

[0124] Finally, in vivo experiments showed that cells expressing the novel reporter gene TMLgBiT were exposed to a dose of 20 mBq [ 111 It is shown that it can be specifically detected using the In]-DOTA-6ahx-HiBiT peptide. In Figure 4, [ 111 The signal generated from subcutaneously injected TMLgBiT-expressing cells one hour after injection of the In]-DOTA-6ahx-HiBiT peptide is shown. Since the signal is also generated from the bladder, it is suggested that the peptide is rapidly excreted through the kidneys and urine. Body distribution data one hour after injection was recorded, and the radioactivity concentration in various tissues was expressed as a percentage (%) of the injection dose per gram of tissue. As shown in Figure 6, [ 111 In]-DOTA-6ahx-HiBiT (as a whole peptide) is broadly and rapidly eliminated by the kidney, with no significant nonspecific accumulation in other tissues.

[0125] In summary, these data demonstrate that a novel reporter gene system based on the TMLgBiT protein acts as a specific reporter gene for SPECT / PET imaging when the HiBiT peptide is linked to a chelating agent and radiolabeled. The combination of transmembrane (TM)LgBiT and DOTA-linker-HiBiT peptide represents a novel system for in vivo SPECT / PET imaging. Figure 4 provides SPECT images of live mice injected with cells expressing TMLgBiT (right flank) and cells not expressing the reporter (left flank). Highly distinctive signals are detected.

[0126] Further experiments were conducted. The results are shown in Figures 7 and 8. In these experiments, 8-week-old male BALB / C nude mice (n=12) were given 5 × 10⁻¹⁰⁻¹ 6 Nine PC-3-TM-LgBiT-expressing cells were injected (n=8). The cells for injection were prepared in a 50:50 ratio solution of PBS (Sigma-Aldrich) and Matrigel (Corning), and the final injection volume was 50 μL. After tumor cell transplantation, the tumors were allowed to grow for approximately 3-4 weeks. To clarify the functionality of the imaging system, dynamic whole-body SPECT / CT scans (VECT / CT Milabs) were performed. Mice were anesthetized with 1-2% isoflurane / O2, and their body temperature was maintained at 37°C during the imaging period (1 hour) by using a warmed bed aperture. In the case of the PC-3 tumor model, [ 111 A 1-hour dynamic SPECT / CT scan was performed immediately after tail vein injection of In-DOTA-6ahx-HiBiT (labeled with 20 MBq at 0.13 nmol in 200 μL PBS). To measure the inhibitory effect on PC-3 tumor uptake, 13 nmol of DOTA-6ahx-HiBiT (approximately 100-fold excess) was administered to [ 111 It was administered simultaneously with In]-DOTA-6ahx-HiBiT (n=4). 111Immediately after injection of In-DOTA-6ahx-HiBiT, dynamic scans were acquired over a total of 1 hour using 30 time frames. The acquired images were reconstructed using SR-OSEM on a 36×36×35 mm matrix with 0.80×0.80 mm isotropic voxels, with 9 approximation iterations and a subset of 128 images. The images are shown in Figure 7.

[0127] The in vivo distribution was evaluated by ex vivo analysis. In this case, mice were euthanized and approximately 20 MBq of [ 111 Autopsies were performed 1 hour after tail vein injection of the In]-DOTA-6ahx-HiBiT peptide (n=4 per treatment group / control). Organs (blood, heart, skin, lungs, liver, spleen, stomach, small intestine, colon, tail, muscle, brain, tumor, kidney, and bone) were weighed, and radioactivity uptake in tumors and other organs was measured and expressed as the injection dose ratio per gram of tissue (%ID / g). Tumors and organs were counted using a gamma-ray counter (PerkinElmer). The counting time was 60 seconds per sample, using the energy window specific to the isotope and a counting error not exceeding 5%. After counting, tumors were frozen in liquid nitrogen for further analysis.

[0128] [Table 2A]

[0129] [Table 2B]

[0130] [Table 2C]

Claims

1. (i) A gene expression construct for expressing a reporter gene in a cell, wherein the reporter gene encodes a fusion protein containing a transmembrane domain fused in-frame to a reporter domain, and the transmembrane domain, upon insertion of the fusion protein into the cell membrane, fixes the fusion protein to the cell membrane and simultaneously expresses the reporter domain on the cell surface; and (ii) A reporter peptide labeled with a radiolabeled substance; The reporter system comprises a reporter domain containing a large polypeptide subunit of split luciferase, and a reporter peptide containing a small peptide subunit of split luciferase, wherein both subunits associate complementaryly to form a luciferase complex.

2. The reporter system according to claim 1, wherein the reporter domain consists of a large polypeptide subunit of splitruciferase, and the reporter peptide consists of a small peptide subunit of splitruciferase.

3. The reporter system according to claim 2, wherein the split luciferase is selected from firefly (Photinus pyralis) luciferase (FLuc), click beetle (e.g., Pyrophorus plagiophthalmus) luciferase, Gaussia (e.g., Gaussia princeps) luciferase (GLuc), sea cucumber (e.g., Renilla reniformis) luciferase (RLuc), Oplophorus (e.g., Oplophorus gracilirostris) luciferase (OLuc; NanoLuc), and bacterial luciferase (Lux).

4. The reporter system according to claim 3, wherein the split ciferase is NanoLuc.

5. The reporter system according to any one of claims 1 to 4, wherein the small peptide subunit has high affinity for the large polypeptide subunit of the splitrusiferase.

6. The reporter system according to any one of claims 1 to 5, wherein the two subunits are associated at a Kd of less than 0.1 μM.

7. The reporter system according to any one of claims 1 to 6, wherein the reporter peptide has a length of 9 to 30 amino acid residues.

8. The reporter system according to any one of claims 1 to 7, wherein the reporter peptide has a length of 11 to 22 amino acid residues.

9. The reporter system according to any one of claims 1 to 8, wherein the large polypeptide subunit includes a sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 48 or a sequence that binds to the small peptide subunit at a Kd of less than 0.1 μM.

10. The reporter system according to any one of claims 1 to 9, wherein the small peptide subunit has the amino acid sequence of any of SEQ ID NOs. 28 to 46.

11. The reporter system according to any one of claims 1 to 10, wherein the transmembrane domain is selected from the transmembrane domains of the protein PDGFR, CD8, B7 protein, TLR4, CD4, neurexin 3b, Notch receptor polypeptide, CD28, CD137 (41BB), CD3C, and other shortened human type I and type II transmembrane proteins.

12. The reporter system according to any one of claims 1 to 11, wherein the transmembrane domain comprises a sequence selected from the group consisting of SEQ ID NOs: 1 to 11.

13. The reporter system according to any one of claims 1 to 12, further comprising a sequence of a leader peptide fused in-frame to a transmembrane domain of the fusion protein.

14. The reporter system according to claim 13, wherein the leader sequence is selected from the group consisting of human or mouse leader sequences of IgK, CD8, OSM, IgG2 H, BM40, Secrecon, IgKVIII, CD33, tPA, chymotrypsinogen, trypsinogen-2, IL-2, albumin (HSA), and insulin.

15. The reporter system according to claim 13 or 14, wherein the leader sequence includes a sequence selected from the group consisting of sequence numbers 12 to 26.

16. The reporter peptide is 51 Cr, 52 Fe, 52m Mn, 62 Cu, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 72 As, 77 As, 89 Zr, 90 Y, 97 Ru, 99 Tc, 99m Tc, 105 Rh, 109 Pd, 111 In, 111 Ag, 113m In, 121 Sn, 124 I, 127 Te, 142 Pr, 143 Pr, 149 Pm, 151 Pm, 153 Sm, 157 Gd, 159 Gd, 161 Tb, 165 Dy, 166 Ho, 169 Er, 169 Yb, 172 Tm, 175 Yb, 177 Lu, 186 Re, 188 Re, 198 Au, 199 Au, 203 Pb, 11 C, 18 F, 15 O, and 13 N, and a reporter system according to any one of claims 1 to 15, comprising a radiolabel selected therefrom.

17. A reporter system according to any one of claims 1 to 16, wherein the gene expression construct is contained in the vector, the vector is contained in recombinant cells; or the vector is contained in a viral genome, the viral genome being an oncolytic virus genome including adenovirus, reovirus, measles virus, herpes simplex virus, Newcastle disease virus, vaccinia virus, seneca virus, enterovirus RIGVIR, Semryki forest virus, vesicular stomatitis virus, and poliovirus, or a virus genome for cell transformation including retrovirus or lentivirus.

18. The reporter system according to any one of claims 1 to 17, wherein the recombinant cell is a T cell.

19. A reporter peptide comprising a small peptide subunit of a split luciferase, wherein the small peptide subunit associates complementarily with a large polypeptide subunit of the split luciferase to form a luciferase complex, the small peptide subunit has high affinity for the large polypeptide subunit, the reporter peptide has a length of 9 to 30 amino acid residues, and the reporter peptide is labeled with a radionuclide suitable for use in PET or SPECT, the radionuclide being bound to the reporter peptide via a chelating agent and a linker.

20. The reporter peptide according to claim 19, wherein the split luciferase is selected from firefly (Photinus pyralis) luciferase (FLuc), click beetle (e.g., Pyrophorus plagiophthalmus) luciferase, Gaussia (e.g., Gaussia princeps) luciferase (GLuc), sea cucumber (e.g., Renilla reniformis) luciferase (RLuc), Oplophorus (e.g., Oplophorus gracilirostris) luciferase (OLuc; NanoLuc), and bacterial luciferase (Lux).

21. The reporter peptide has a length of 9 to 30 amino acid residues, contains, or consists of, any one of the sequences of SEQ ID NO: 28 to 46, and the radiolabeled substance is 51 Cr, 52 Fe, 52m Mn, 62 Cu, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 72 As, 77 As, 89 Zr, 90 Y, 97 Ru, 99 Tc, 105 Rh, 109 Pd, 111 In, 111 Ag, 113m In, 121 Sn, 124 I, 127 Te, 142 Pr, 143 Pr, 149 Pm, 151 Pm, 153 Sm, 157 Gd, 159 Gd, 161 Tb, 165 Dy, 166 Ho, 169 Er, 169 Yb, 172 Tm, 175 Yb, 177 Lu, 186 Re, 188 Re, 198 Au, 199 Au, 203 Pb, 11 C, 18 F, 15 O, and 13 N, and is the reporter peptide according to claim 19 or 20.

22. a) A pharmaceutical composition for infusion into a subject's body, comprising a vector or recombinant cell containing a reporter gene expression construct according to any one of claims 1 to 18, and b) A pharmaceutical composition comprising the reporter peptide described in any one of claims 19 to 21. A pharmaceutical combination containing the above, to be administered simultaneously, separately, or sequentially into the body of a target.

Images