IFN gamma biosensor

Abstract

The present invention provides an Interferon-gamma (IFN-γ) sensor and a corresponding IFN-γ reporter cell. Furthermore, it provides the use of the IFN-γ reporter cell for measuring IFN-γ levels. Also encompassed are the corresponding vectors comprising the IFN-γ sensor.

Description

[0001] IFN Gamma Biosensor

[0002] FIELD OF THE INVENTION

[0003] The present invention provides an Interferon-gamma (IFN-y) sensor and a corresponding IFN-y reporter cell. Furthermore, it provides the use of the IFN-y reporter cell for measuring IFN-y levels. Also encompassed are the corresponding vectors comprising the IFN-y sensor.

[0004] BACKGROUND

[0005] IFN-y is a versatile cytokine known for its antiviral, antitumor, and immune-modulating functions. The type II interferon is inter alia secreted by T cells (cytotoxic and Th1) and natural killer (NK) cells and promotes activation of the cellular immune response. So far IFN-y was measured by ELISA assay which has drawbacks since it is a pure endpoint detection assay and time consuming.

[0006] Thus, there is a need for an efficient assay for monitoring IFN-y which allows to measure IFN-y in cell culture over time.

[0007] SUMMARY

[0008] The present invention solves the above-mentioned problem by providing an IFN-y sensor and a corresponding IFN-y reporter cell line as biosensor for detecting and measuring levels of IFN-y.

[0009] Accordingly, one aspect refers to an IFN-y sensor comprising a nucleotide sequence comprising the following elements at least one gamma interferon activation site (GAS), a promotor linked to the gamma interferon activation site, and a nucleotide sequence encoding at least one fluorescent protein under the control of the promotor.

[0010] A further aspect refers to an IFN-y reporter cell comprising an IFN-y sensor comprising a nucleotide sequence encoding an IFN-y sensor comprising the following elements at least one gamma interferon activation site (GAS), a promotor linked to the gamma interferon activation site, and a nucleotide sequence encoding at least one fluorescent protein under the control of the promotor.

[0011] In preferred embodiments the IFN-y reporter cell comprises an exogenous nucleotide sequence encoding a signal transducer or activator of transcription.

[0012] The exogenous expression of a signal transducer has the surprising effect of an effective transduction of the INF-gamma signal to the reporter. As shown in Figure 13 significantly higher fluorescent read out could be measured with exogenous STAT-1 expression. Hence, the inventors provide a highly sensitive and precise measurement for IFN-y activity and IFN-y concentrations which are independent of the endogenous level of the respective signal transducer or activator of transcription and not hampered by low levels of signal transducer or activator of transcription.

[0013] The fluorescent protein, such as enhanced green fluorescent protein EGFP, allows for an easy readout, since there is no need to add a further substrate for detection, unlike luciferase-based biosensors or secreted alkaline phosphatase (SEAP)-based biosensors, which necessitate substrates such as luciferin and 1 ,2-dioxetane Disodium 3-(4-methoxyspiro {1 ,2-dioxetane-3,2'- (5'-chloro)tricyclo [3.3.1.13 7]decan}-4-yl)phenyl phosphate(1 ,2-dioxetane CSPD), respectively. Thus, measurements can be carried out at multiple time points within the same sample, since the addition of a substrate deterring the environment of the IFN-y secreting cells to be measured is not necessary. The readout of the fluorescent protein can be carried out during culture of the IFN-y secreting cells (typically at one or more time points during culture) without influencing the function of the IFN-y secreting cells.

[0014] A more specific embodiment refers to an IFN-y reporter cell comprising a nucleotide sequence encoding an IFN-y sensor comprising the following elements at least one gamma interferon activation site (GAS), a promotor linked to the gamma interferon activation site, and a nucleotide sequence encoding at least one fluorescent protein under the control of the promotor; a signal transducer or activator of transcription, and an IFN-y receptor.

[0015] IFN-y binding to the IFN-y receptor activates a signal transducer or activator of transcription (such as STAT-1 , a pivotal compound of the type II IFN signaling pathway) which in turn activates the GAS. The GAS is linked to a promotor which regulates expression of a gene encoding a light sensor protein, such as a fluorescent protein. Therefore, an increasing IFN-y concentration triggers an increase in reporter EGFP expression, providing a dynamic and responsive method for monitoring IFN-y activity. This configuration ensures the precision of the biosensor rendering it sensitive and precise in detecting IFN-y activity and precise measuring of IFN-y concentrations.

[0016] Accordingly, in one embodiment the IFN-y reporter cell comprises an exogenous nucleotide sequence encoding a signal transducer or activator of transcription. In one embodiment the signal transducer or activator of transcription is STAT-1 . The signal transducer or activator of transcription may be endogenous or exogenous. Typically, the signal transducer or activator of transcription is exogenous and under control of a constitutive promotor.

[0017] The promotor linked to the GAS may be an IFN-stimulated gene (ISG) promotor, such as ICAM- The IFN-y reporter cell works for any solution containing IFN-y, in particular for cell culture of any cell line secreting IFN-y or cell culture supernatant to be measured for its IFN-y content. The system works with any other IFN-y producing cells in any stimulatory setting and is therefore very versatile.

[0018] In one embodiment, the IFN-y reporter cell is an antigen presenting cell. The selection of antigen presenting cells as the host for the IFN-y sensor is advantageous, especially when intended for use as bystanders in a co-culture of for example CD8+ T cells and peptide-loaded antigen presenting cells (APCs).

[0019] The assay can be extended to any cell line that expresses IFN-y receptors either naturally or after engineering and is thus not limited to specific cell types.

[0020] The invention allows the detection of IFN-y in a dose dependent manner with substantially no sensitivity for human type I interferon.

[0021] Hence, the invention provides a versatile reporter system for specifically detecting IFN-y in an efficient way that also allows for multiple timepoint measurements in the same sample.

[0022] A further aspect refers to a nucleic acid encoding such IFN-y sensor.

[0023] Accordingly, the invention refers to the use of the IFN-y reporter cell, the IFN-y sensor and the nucleic acid encoding such IFN-y sensor for use of measuring IFN-y levels.

[0024] The invention enables precise measurement of IFN-y concentration. For example, it enables precise measurement of IFN-y concentration in the supernatant culture medium of the co-culture of immune cells with APCs. This replaces the conventional ELISA assay, streamlining and enhancing the experimental process.

[0025] Accordingly, in some embodiments the IFN-y reporter cells also function as bystanders, reporting the concentration of IFN-y released into the culture medium supernatant of immune cells and APCs, as third party cells sensing the IFN-y produced by the immune cells after activation by APCs. Moreover, these reporter cells may serve in some embodiments in reporting the strength of different activating receptors when co-cultured with peptide-loaded APCs, or any other APCs like tumor cells, or the functionality of T cell / NK cell engagers via detecting released IFN-y in the culture medium supernatant.

[0026] In this way, reporter cells replace the need for ELISA and reduce the overall timeline of experiments, providing a more efficient and streamlined approach to determine IFN-y concentrations.

[0027] In contrast to an ELISA assay, which is a pure endpoint detection assay, the IFN-y reporter cells allow monitoring of the IFN-y concentration in the culture medium supernatant at different timepoints during the co-culture without changing the co-culture conditions because neither extraction of supernatant nor the addition of a substrate for detecting and readout of the IFN-y is necessary. In one embodiment, the IFN-y reporter cell is of the same cell type as the used APC. This setting is particular advantageous since there is no additional different component added to the cell culture, such as a different cell line which may have an impact on the cell culture. In addition, this setting allows for immediate readout at any time point during the co-culture. In other words, once the IFN-y reporter cell is co-cultured with the APC and maintained in coculture for at least 6 hours, the read-out can occur at any time point during co-culture, since the IFN-y reporter cell is induced at least 6 hours after starting the co-culture. Thus, about 6 hours after starting the co-culture, the readout can be carried out immediately at any time without further assay induction or incubation time.

[0028] In one embodiment the IFN-y reporter cell: IFN-y secreting cell ratio is 0.1 -5 : 1 , 0.4-2 : 1 , 0.2- 1.5 : 1 , 0.5-1 :1 , 0.6-0.9 : 1 , such as 0.8:1 .

[0029] FIGURE LEGENDS

[0030] Figure 1 : Illustration of the construction of the antigen-presenting (K562) Interferon gamma biosensor cell line. The K562 antigen-presenting IFN-y biosensor cell line is created by incorporating a construct containing eight gamma interferon activation site (GAS) sequence repeats, positioned in cis before the promoter of the genes ICAM1 & EGFP, and as a separate construct STAT-1 (under control of constant active E1 A promoter together with a CMV enhancer). The transduced biosensor cell line has undergone screening for IFN-y sensitivity across various parameters, including a high contrast-to-noise ratio (lower background signal), higher dynamic range of EGFP, and IFN-y titration sensitivity. Upon IFN-y binding to the IFN-y receptor (IFNGR), a signaling cascade is initiated, leading to the binding of STAT-1 to the GAS sequences and the subsequent induction of expression of the reporter gene EGFP positioned downstream.

[0031] Figure 2: Dose-response of K562 IFN-y biosensor cells to human IFN-y. A) Cells were stimulated with increasing concentrations of recombinant human IFN-y ranging from 0 pg / ml to 4000 pg / ml. After overnight incubation, the EGFP signal was recorded using flow cytometry. The EGFP mean fluorescent intensity (MFI) is shown as normalized to the EGFP positive population frequency (mean ± SD). The inset represents the lower concentration 0 pg / ml to 1000pg / ml) and corresponding normalized EGFP MFI, B) Illustrates the concentrations of IFN-y detected in the culture medium supernatants (SN) of co-cultures involving NY-ESO-1 -specific TCR transduced CD8+ cells and T2 cells loaded with the corresponding peptide. The IFN-y concentration was determined by employing a standard curve generated using a range of functional human IFN-y, assessed with the biosensor cells.

[0032] Figure 3: Comparison of Biosensor cell line & ELISA assay. 144 CD8+ T cell clones were tested for IFN-y release when co-cultured with specific peptide-loaded or unloaded APCs. A),C),& E) show the optical density as a read out for IFN-y concentration measured in an ELISA assay. B), D),& F) show the MFI of EGFP as a read out for IFN-y concentration measured using the IFN-y Biosensor cell line. A) to F), “+’ represent co-cultures of CD8+ T cell clones having unique TCRs recognizing relevant peptide-loaded APCs, represent co-cultures of CD8+ T cell clones having unique TCRs with unloaded APCs cells. Figure 4: Detection of IFN-y using biosensor cells as bystander cells in a co-culture experiment. A) Illustrates the standard curve plotting IFN-y against EGFP mean fluorescent intensity (MFI); B) Displays the IFN-y concentration calculated using the standard curve generated in Figure A; C) Conducts an ELISA assay to evaluate IFN-y levels and examines the influence of biosensor cells as bystanders in the co-culture experiment. The graph exhibits IFN-y concentrations obtained through ELISA in co-cultures with or without biosensor cells. MFI - Mean Fluorescent Intensity, NY-ESO-1 TCR - CD8+ cells transduced with NY-ESO-1 specific TCR, PMA / lono - PMA & lonomycin treated, APC (K562-A2 cells, i.e. K562 cell transduced with the gene for HLA-A*02:01 and stably expressing the HLA-A*02:01 protein on the cell surface) loaded with relevant peptide (K562_A2_ld_Pep) or without relevant peptide-loaded (K562_A2_unl).

[0033] Figure 5: Biosensor Cell Line Stability Assessment. A) Concentration-Response

[0034] Relationship Over 10 Weeks: The graph depicts the relationship between IFN-y concentrations (IFN-y) and their corresponding EGFP Mean Fluorescent Intensity (MFI) for each week over a 10-week period. The data is fitted using a polynomial 3rd order equation to capture the dynamic response of the biosensor cell line, B) Quality of Fit Analysis: To evaluate the quality of fit, the calculated IFN-y concentrations (determined using the generated polynomial equation) are compared against the theoretical IFN-y concentrations. This analysis provides insights into the accuracy and reliability of the biosensor cell line's response over the experimental duration.

[0035] Figure 6: Sensitivity of biosensor cells to Type I (IFN-y) and Type II (IFNa and IFNp). Mean Fluorescence Intensity (MFI) of EGFP in biosensor cells after a 16-hour incubation with various concentrations of IFN-y (ranging from 0 to 4000 pg / ml) or a fixed concentration of 4000 pg / ml of IFNa or IFNp.

[0036] Figure 7: Sensitivity of the assay using IFN-y Biosensors as bystander cells in co-culture experiments. A) IFN-y Biosensor cells were co-cultured with antigen presenting cells and untransduced CD8+ T cells, and stimulated with increasing concentrations of recombinant human IFN-y concentration ranging from Opg / ml to 4000pg / ml. eGFP fluorescense intensity (RFU) was analysed over time (for 12 hours) using a Envision fluorescence reader. The inset represent response at 6 hours upon co-culture. B) Table displays values of eGFP fluorescence intensity for a condition with 0 pg / ml IFN-y (neg sample) and stimulated with 62.25 pg / ml IFN-y concentration (pos. sample) over 12 hours of co-culture experiment. Mean values of pos. and neg. samples, as well as standard deviation of neg. sample were calculated based on triplicates. Sensitivity was defined as the minimum concentration at which the assay can distinguish the pos. from neg. sample fulfilling following formula: mean positive > mean negative+3 x standard deviation.

[0037] Figure 8: Accuracy of the assay using IFN-y Biosensors as bystander cells in co-culture experiment. A) IFN-y Biosensor cells irradiated with 50 Gy, in order to prevent their proliferation, were co-cultured with antigen presenting cells (K562_A2) and untransduced CD8+ T cells to be simulated in bystander experiment. For activation of IFN-y Biosensor cells, a range of IFN-y concentrations from Opg / ml to 4000pg / ml was added. eGFP fluorescence was analysed after 24 hrs of co-culture using an Envision fluorescence reader. Data were fitted using a polynomial 3rd order equation. B) Based on the fitted standard curve, calculated concentrations were assessed. The accepted accuracy of the calculated concentrations of IFN-y was considered 80%-120% (100%±20%) of the theoretical concentrations.

[0038] Figure 9: Defining optimal effector : target ratio in bystander experiments. IFN-y Biosensor cells were co-cultured with target T2 (A) or K562_A11 (B) cells and titrated numbers of CD8+ T cells, either transduced with NY-ESO-1 TCR (A) or KRAS G12V TCR (B) or left untransduced. Target cells were unloaded or loaded with 10-5M concentration of relevant NY-ESO-1 peptide (A) or relevant KRAS G12V peptide or WT irrelevant peptide (B). eGFP fluorescence was analysed at 16 hrs using an Envision fluorescence plate reader. Supernatants of co-cultures were collected for IFN-y ELISA.

[0039] Figure 10: Testing an optimal number of Biosensor cells. Titrated numbers (5x104-0.5x104) of IFN-y Biosensor cells were co-cultured with (2.5x104) antigen presenting cells (K562_A2) and (2x104) untransduced CD8+ T cells. For activation of IFN-y Biosensor cells, a range of IFN-y concentrations from Opg / ml to 2000pg / ml was added. eGFP fluorescence was analysed after 16 hrs of co-culture using an Envision fluorescence reader. Data were fitted using a polynomial 3rd order equation.

[0040] Figure 11 : Improvement of the resolution of the assay. IFN-y Biosensor cells non-irradiated (A) or irradiated 50 Gy (B) were co-cultured with APCs (K562_A2) and NY-ESO-1 TCR transduced CD8+ T cells, and stimulated with increasing concentrations of recombinant human IFN-y ranging from 0 pg / ml to 4000 pg / ml. eGFP fluorescence intensity was analysed every hour in the range of 14-24 hours using an Envision fluorescence reader.

[0041] Figure 12: Monitoring of T cell activation and killing over time. IFN-y Biosensor cells (nonirradiated or irradiated 50 Gy) were co-cultured with tumor cells Mel624.38_NLR (A) or DAN- G_A11_NLR (B) expressing red fluorescence marker nuclight red (NLR) and CD8+ T cells transduced with NY-ESO-1 TCR (A) or KRAS G12V TCR (B) or left untransduced. eGFP fluorescence was analysed over time using an Envision fluorescence plate reader or Incucyte life-cell imaging system. NLR fluorescence was analysed with Incucyte life-cell imaging system.

[0042] Figure 13: Superior effect of exogenous STAT1 expression on the assay with IFN-y Biosensor cell line expressing four gamma Interferon activation site (GAS) elements. A) Two K562 IFN-y Biosensor cell lines were created by incorporating a construct containing four GAS sequence repeats, positioned in cis before the ICAM1 promoter and the reporter gene EGFP, with or without exogenous STAT1 expression. Both Biosensor cell lines underwent bulk selection for low basal EGFP background expression and then high level of EGFP expression upon stimulation with 20 ng / ml of IFN-y. Subsequently, the cells were cultured for 1 week without IFN-y. Generated cell lines were stimulated with increasing concentrations of recombinant human IFN-y ranging from 0 pg / ml to 4000 pg / ml. EGFP fluorescence intensity (RFU) was analyzed after 16 hours of incubation with fluorescence reader. B) Exogenous STAT1 expression was confirmed on mRNA level by qPCR in relation to the reference genes (GUSB / TBP). DETAILED DESCRIPTION OF THE INVENTION

[0043] Before the invention is described in detail with respect to some of its preferred embodiments, the following general definitions are provided.

[0044] The present invention as illustratively described in the following may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein.

[0045] The present invention will be described with respect to particular embodiments and with reference to certain figures, but the invention is not limited thereto but only by the claims.

[0046] Where the term “comprising” is used in the present description and claims, it does not exclude other elements. For the purposes of the present invention, the term “consisting of” is considered to be a preferred embodiment of the term “comprising of’. If hereinafter a group is defined to comprise at least a certain number of embodiments, this is also to be understood to disclose a group which preferably consists only of these embodiments.

[0047] For the purposes of the present invention, the term “obtained” is considered to be a preferred embodiment of the term “obtainable”. If hereinafter e.g. a compound is defined to be obtainable from a specific source, this is also to be understood to disclose a compound which is obtained from this source.

[0048] Where an indefinite or definite article is used when referring to a singular noun, e.g. “a”, “an” or “the”, this includes a plural of that noun unless something else is specifically stated. The terms “about” or “approximately” in the context of the present invention denote an interval of accuracy that the person skilled in the art will understand to still ensure the technical effect of the feature in question. The term typically indicates deviation from the indicated numerical value of ±10%, and preferably of ±5%.

[0049] “Sequence identity” or “percentage identity” and “sequence similarity” can be determined by alignment of two peptide or two nucleotide sequences using global or local alignment algorithms. For example, sequence similarity or identity can be determined by searching against databases such as FASTA, BLAST, etc., but hits should be retrieved and aligned pairwise to compare sequence identity. Two proteins or two protein domains, or two nucleic acid sequences that described herein as at least 80% identical to a reference sequence, include peptide sequences and nucleic sequences which are at least 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% preferably at least 90%, 95%, 98%, 99% identical to the reference sequence.

[0050] “Nucleic acid" generally means a polymer of DNA or RNA, which can be single-stranded or double-stranded, synthesized or obtained (e.g., isolated and / or purified) from natural sources, which can contain natural, non-natural or altered nucleotides, and which can contain a natural, non-natural or altered internucleotide linkage, such as a phosphoroamidate linkage or a phosphorothioate linkage, instead of the phosphodiester found between the nucleotides of an unmodified oligonucleotide. Preferably, the nucleic acids described herein are recombinant. As used herein, the term "recombinant" refers to (i) molecules that are constructed outside living cells by joining natural or synthetic nucleic acid segments to nucleic acid molecules that can replicate in a living cell, or (ii) molecules that result from the replication of those described in (i) above. For purposes herein, the replication can be in vitro replication or in vivo replication. The nucleic acids can be constructed based on chemical synthesis and / or enzymatic ligation reactions using procedures known in the art or commercially available (e.g. from Genscript, Thermo Fisher and similar companies). See, for example Sambrook et al., a nucleic acid can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed upon hybridization (e.g., phosphorothioate derivatives and acridine substituted nucleotides).

[0051] The term “exogenous” refers to molecules that have been transferred by any of the genetic engineering techniques into a respective cell.

[0052] Technical terms are used by their common sense. If a specific meaning is conveyed to certain terms, definitions of terms will be given in the following in the context of which the terms are used.

[0053] A first aspect of the invention refers to an interferon-gamma (IFN-y) sensor comprising a nucleotide sequence comprising the following elements at least one gamma interferon activation site (GAS), a promotor linked to the GAS, and a nucleotide sequence encoding at least one light sensor protein under the control of the promotor.

[0054] The term “light sensor protein” refers to fluorescent proteins but also includes light absorbing proteins, such as melanin, flavonoids, carotinoids, anthocyans. In a specific embodiment the light sensor protein is a fluorescent protein.

[0055] Accordingly, one embodiment of the invention refers to an interferon-gamma (IFN-y) sensor comprising a nucleotide sequence comprising the following elements at least one gamma interferon activation site (GAS), a promotor linked to the GAS, and a nucleotide sequence encoding at least one fluorescent protein under the control of the promotor.

[0056] The IFN-y sensor can detect IFN-y when upon binding of IFN-y receptor the GAS is activated triggering the expression of the fluorescent protein via the promotor controlling the expression of the fluorescent protein which is linked to the GAS. Typically, IFN-y binds to an IFN-y receptor. Downstream of the IFN-y receptor a signal transducer or activator of transcription (such as STAT-1) activates the GAS.

[0057] The term “gamma interferon activation site”, abbreviated as “GAS”, refers to a nucleotide sequence, preferably a DNA sequence, that induces transcription in response to IFN-y. The GAS may have a length of 5 to 10 nucleotides. In one embodiment, the GAS may have a length of 6 to 15 nucleotides. In one embodiment, the GAS may have a length 7 to 12 nucleotides. In one embodiment, the GAS may have a length of 8 to 11 nucleotides. The GAS may have a sequence as set out in SEQ ID NO: 1 (TTCN(2-4)GAA, such as TTCNNNNGAA) or a sequence which is at least 80% identical thereto. In particular, the GAS may have a sequence as set out in SEQ ID NO: 2 (TTCCGGGAA) or a sequence which is at least 80% identical thereto.

[0058] The GAS is linked to a promotor which controls the nucleotide sequence encoding a fluorescent protein. Thereby the IFN-y levels are translated into fluorescent intensities.

[0059] In one embodiment the nucleotide sequence may comprise at least two GAS, such as at least three GAS, at least 4 GAS, at least 5 GAS, at least 6 GAS, at least 7 GAS or at least 8 GAS. In a preferred embodiment the nucleotide sequence may comprise at least 8 GAS. In one embodiment the nucleotide sequence encoding an IFN-y sensor comprises 8 GAS.

[0060] The GAS sequences may be separated by a nucleotide spacer. The nucleotide spacer may have the sequence as set out in SEQ ID NO: 3 or a sequence which is at least 80% identical thereto. Alternatively, the nucleotide spacer may have a sequence as set out in SEQ ID NO: 4 or a sequence which is at least 80% identical thereto. Between the GAS different spacers or the same spacers could be used.

[0061] An exemplary sequence comprising 8 GAS, which are separated by the nucleotide spacers set out in SEQ ID NO: 3 and SEQ ID NO: 4 in alteration, is set out in SEQ ID NO: 11 .

[0062] Typically, the last GAS is followed by a spacer, hence an exemplary sequence comprising 8 GAS including the spacer after the last GAS is set out in SEQ ID NO: 12.

[0063] Accordingly, an exemplary sequence including 4 GAS, each followed by a spacer, wherein the last spacer is followed by a the ICAM-1 promoter sequence, is set out in SEQ ID NO: 13.

[0064] Accordingly, an exemplary sequence including 8 GAS, each followed by a spacer, wherein the last spacer is followed by a the ICAM-1 promoter sequence, is set out in SEQ ID NO: 6.

[0065] The promotor linked to the GAS is a nucleotide sequence, preferably a DNA sequence. Typically, the promotor linked to the GAS is an IFN-stimulated gene (ISG) promotor, such as ICAM-1 . In one embodiment the promotor has the sequence as set out in SEQ ID NO: 5 or a sequence which is at least 80% identical thereto.

[0066] The fluorescent protein may be any fluorescent protein. The fluorescent protein may be for example GFP or any derivate thereof. In a preferred embodiment the fluorescent protein is EGFP.

[0067] A further aspect refers to an IFN-y reporter cell comprising the IFN-y sensor as defined herein. Thus, in particular, the IFN-y reporter cell comprises an IFN-y sensor comprising a nucleotide sequence comprising the following elements at least one GAS, a promotor linked to the GAS, and a nucleotide sequence encoding at least one fluorescent protein under the control of the promotor.

[0068] More particularly, the IFN-y reporter cell may comprise an IFN-y sensor comprising a nucleotide sequence comprising the following elements at least one GAS, a promotor linked to the gamma interferon activation site, and a nucleotide sequence encoding at least one fluorescent protein under the control of the promotor a signal transducer or activator of transcription, and an IFN-y receptor.

[0069] A specific embodiment refers to an IFN-y reporter cell comprising an IFN-y sensor comprising a nucleotide sequence comprising the following elements

[0070] - 8 GAS, a promotor linked to the gamma interferon activation site, and a nucleotide sequence encoding EGFP,

[0071] - STAT- 1 , and an IFN-y receptor.

[0072] The IFN-y receptor may be naturally in the cell and / or the cell may be engineered to express the IFN-y receptor encoded by a nucleotide sequence introduced into the cell.

[0073] When it is referred to “an IFN-y receptor” it means that the cell comprises at least one IFN-y receptor. Typically the cell comprises several IFN-y receptors.

[0074] Typically, the IFN-y reporter cell comprises an exogenous nucleotide sequence encoding a signal transducer or activator of transcription. In one embodiment the signal transducer or activator of transcription is STAT-1 . Typically, the signal transducer or activator of transcription is under control of a constitutive promotor. The amino acid sequence of STAT-1 may have sequence identical or at least 80% identical to the sequence as set out in SEQ ID NO: 7. The nucleotide sequence encoding STAT- 1 may have a sequence identical or at least 70% identical to the sequence as set out in SEQ ID NO: 8.

[0075] The term “comprising an exogenous nucleotide sequence encoding a signal transducer or activator of transcription” as used herein means that the IFN-y reporter cell expresses an exogenous nucleotide sequence encoding a signal transducer or activator of transcription, typically under a constitutive promotor. Accordingly, one embodiment refers to a cell comprising a plasmid containing a nucleotide sequence encoding a signal transducer or activator of transcription.

[0076] For example, comprising STAT-1 , means that the cell expresses STAT-1 . One embodiment thus refers to a cell comprising a plasmid encoding STAT-1 under the control of a constitutive promotor. In other wors, one embodiment refers to a cell comprising a plasmid comprising a nucleotide sequence encoding STAT-1 under the control of a constitutive promotor. The term “IFN-y reporter cell” also termed “biosensor cell” herein, refers to a cell which is capable of sensing extracellular IFN-y typically by a moiety capable of binding to IFN-y, such as an IFN-y receptor which is integrated into the plasma membrane of the IFN-y reporter cell. The IFN-y reporter cell may be any cell that naturally expresses an IFN-y receptor or in which a nucleotide sequence which encodes an IFN-y receptor and for expressing an IFN-y receptor at its surface is introduced.

[0077] In one embodiment the IFN-y reporter cell is an antigen presenting cell. The term “antigen presenting cell” refers to a cell that displays an antigen bound by a major histocompatibility complex (MHC). The antigen presenting cell may comprise an MHC I and / or an MHC II complex. In one embodiment the cell is a lymphoblast cell, e.g. a K562 cell.

[0078] In one embodiment the IFN-y reporter cell is used in co-culture with a T cell and an APC and is of same cell type as the APC. Accordingly, one aspect refers to a composition comprising an IFN-y reporter cell, a T cell and an APC, wherein the APC and the IFN-y reporter cell are of the same cell type. For example, the APC is a K562 cells and accordingly the IFN-y reporter cell is a K562 cells, as well.

[0079] The IFN-y reporter cell may be a bystander cell. Bystander T cell activation refers to the activation of T cells without antigen recognition. In other words, the bystander cell is an antigen presenting cell which does not present the antigen MHC complex to which the antigen receptor of the IFN-y secreting cell binds. Preferably the bystander cell is of the same cell type the as the peptide loaded APC.

[0080] In one embodiment the IFN-y reporter cell is irradiated.

[0081] In one embodiment, the IFN-y reporter cell is treated to prevent proliferation. In one embodiment the IFN-y reporter cell is irradiated to prevent proliferation, e.g. is irradiated for example with 50 Gy.

[0082] A further aspect refers to the use of the IFN-y sensor as described herein, the IFN-y reporter cell as described herein for / n vitro measuring IFN-y levels from an IFN-y secreting cell.

[0083] In one embodiment, the IFN-y secreting cell is in co-culture with the IFN-y reporter cell.

[0084] In another embodiment, the IFN-y secreting cell is cultured in a different vessel as the IFN-y reporter cell and the IFN-y reporter cell is contacted with the cell culture supernatant of an IFN-y secreting cell. In this setting no alteration of the established cell culture protocol is necessary.

[0085] The measurement of the IFN-y levels by the IFN-y sensor as described herein may occur at one time point or at several time points. As explained above, the assay is feasible for measuring IFN- y levels at several time points within the same sample, since the IFN-y sensor and in particular the IFN-y reporter cell does not harm the cell culture of the IFN-y secreting cells. Thus, measurement of IFN-y levels by the IFN-y sensor as described herein may be carried out at least at two time points. In one embodiment, the IFN-y secreting cell is an immune cell.

[0086] The term immune cell includes NK cells, T cells, cytokine induced killer (CIK) cells, lymphokine- activated killer (LAK) cells, tumor infiltrating lymphocytes (TILs) and / or cells comprising a chimeric antigen receptor. In a specific embodiment the immune cell is selected from the group consisting of NK cell or T cell.

[0087] The term “T cell” comprises but is not limited to helper T cells also named CD4+ T cells, cytotoxic T cells also named CD8+ T cell, memory T cells, regulatory T cells, natural killer T cells, and gamma delta T cells.

[0088] In a specific embodiment, the T cell is selected from the group consisting of CD4+ T cell and CD8+ T cell. In one embodiment, the immune cells, in particular the T cells, are in co-culture with the corresponding antigen presenting cells.

[0089] Another aspect refers to a method for in vitro measuring IFN-y levels comprising the steps: contacting the IFN-y reporter cell as described herein with a medium, measuring the fluorescence signal of the IFN-y reporter cell; wherein the fluorescence signal is indicative for the IFN-y levels in the medium.

[0090] Typically, a higher fluorescence signal indicates a higher IFN-y concentration in the medium and lower fluorescence signal indicates a lower IFN-y concentration.

[0091] In one embodiment, the medium comprises the supernatant of the IFN-y secreting cell.

[0092] In another embodiment, the medium comprises at least one IFN-y secreting cell. In other words, the IFN-y reporter cell and the IFN-y secreting cell are co-cultured in one vessel.

[0093] Thus, one embodiment refers to a method for in vitro measuring IFN-y levels from an IFN-y secreting cell comprising the steps: providing an IFN-y secreting cell in co-culture with an IFN-y reporter cell, measuring the fluorescence signal of the IFN-y reporter cell.

[0094] Another embodiment refers to a method for / n vitro measuring IFN-y levels from an IFN-y secreting cell comprising the steps: contacting an IFN-y reporter cell with the cell culture supernatant of a IFN-y secreting cell, measuring the fluorescence signal of the IFN-y reporter cell.

[0095] Another aspect refers to a composition comprising the IFN-y sensor as defined herein, a nucleotide sequence encoding signal transducer or activator of transcription as defined herein. Another aspect refers to a vector comprising the IFN-y sensor as defined herein.

[0096] Accordingly, one general embodiment refers to a vector comprising an IFN-y sensor comprising a nucleotide sequence comprising the following elements at least one gamma interferon activation site (GAS), a promotor linked to the GAS, and a nucleotide sequence encoding at least one fluorescent protein under the control of the promotor.

[0097] The vector may be a plasmid, shuttle vector, phagemide, cosmid, transposon, expression vector, retroviral vector, adenoviral vector or particle and / or vector to be used in gene therapy. Preferably, the vector is an expression vector. More preferably, the vector is a retroviral, more specifically a gamma-retroviral or lentiviral vector.

[0098] A “vector” is any molecule or composition that has the ability to carry a nucleic acid sequence into a suitable host cell where synthesis of the encoded polypeptide can take place. Typically, and preferably, a vector is a nucleic acid that has been engineered, using recombinant DNA techniques that are known in the art, to incorporate a desired nucleic acid sequence (e.g. a nucleic acid of the invention). The vector may comprise DNA or RNA and / or comprise liposomes. The vector may be a plasmid, shuttle vector, phagemide, cosmid, transposon, expression vector, retroviral vector, lentiviral vector, adenoviral vector or particle and / or vector to be used in gene therapy. A vector may include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication. A vector may also include one or more selectable marker genes and other genetic elements known to those of ordinary skill in the art. A vector preferably is an expression vector that includes a nucleic acid according to the present invention operably linked to sequences allowing for the expression of said nucleic acid.

[0099] Further Embodiments

[0100] The invention is further described by the following embodiments:

[0101] 1 . An interferon-gamma (IFN-y) sensor comprising a nucleotide sequence comprising the following elements at least one gamma interferon activation site (GAS), a promotor linked to the GAS, and a nucleotide sequence encoding at least one light sensor protein, preferably fluorescent protein, under the control of the promotor.

[0102] 2. IFN-y sensor according to embodiment 1 , wherein the promotor linked to the GAS is an IFN-stimulated gene (ISG) promotor, wherein optionally the IFN-stimulated gene (ISG) promotor is ICAM-1 .

[0103] 3. IFN-y sensor according to any one of the preceding embodiments, wherein the fluorescent protein is EGFP. 4. IFN-y sensor according to any one of the preceding embodiments, wherein the nucleotide sequence encoding an IFN-y sensor comprises at least two GAS.

[0104] 5. IFN-y sensor according to any one of the preceding embodiments, wherein the IFN-y sensor comprises at least 8 GAS.

[0105] 6. IFN-y sensor according to any one of the preceding embodiments, wherein the IFN-y sensor comprises 8 GAS.

[0106] 7. IFN-y sensor according to any one of the preceding embodiments, wherein the GAS has a sequence as set out in SEQ ID NO: 1 (TTCNp^GAA).

[0107] 8. IFN-y sensor according to any one of the preceding embodiments, wherein the GAS has a sequence as set out in SEQ ID NO: 2 (TTCCGGGAA), or a sequence which is at least 80% identical thereto.

[0108] 9. IFN-y sensor according to any one of the preceding embodiments, wherein the promotor has the sequence as set out in SEQ ID NO: 5 or a sequence which is at least 80% identical thereto.

[0109] 10. IFN-y sensor according to embodiments 7 to 9, wherein the GAS are separated by a nucleotide spacer.

[0110] 11 . IFN-y sensor according to embodiments 7 to 9, wherein the GAS nucleotide spacer has a sequence as set out in SEQ ID NO: 3 or SEQ ID No: 4.

[0111] 12. IFN-y reporter cell comprising the IFN-y sensor as defined in embodiments 1 to 11.

[0112] 13. IFN-y reporter cell according to embodiment 12, further comprising an exogenous nucleotide sequence encoding signal transducer or activator of transcription.

[0113] 14. IFN-y reporter cell according to embodiment 13, wherein the signal transducer or activator of transcription is STAT-1 .

[0114] 15. IFN-y reporter cell according to embodiment 13 or 14, wherein the signal transducer or activator of transcription is under control of a constitutive promotor.

[0115] 16. IFN-y reporter cell according to embodiment 12 to 15, wherein the cell expresses an IFN-y receptor.

[0116] 17. IFN-y reporter cell according to any one embodiments 12 to 16, wherein the cell is an antigen presenting cell. 18. IFN-y reporter cell according to any one of embodiments 12 to 17, wherein the cell is capable of sensing extracellular IFN-y.

[0117] 19. Use of the IFN-y sensor according to embodiments 1 to 11 , the IFN-y reporter cell according to any one of embodiments 12 to 18 for / n vitro measuring IFN-y levels from an IFN-y secreting cell.

[0118] 20. The use according to embodiment 19, wherein the IFN-y secreting cell is in co-culture with the IFN-y reporter cell.

[0119] 21 . The use according to any one of embodiment 19, wherein the IFN-y secreting cell is cultured in a different vessel as the IFN-y reporter cell and wherein the IFN-y reporter cell is contacted with the cell culture supernatant of the IFN-y secreting cell.

[0120] 22. The use according to any one of embodiments 19 to 21 , wherein the measuring occurs at least at two time points.

[0121] 23. The use according to embodiments 19 to 22, wherein the IFNy secreting cell is an immune cell.

[0122] 24. The use according to embodiment 23, wherein the immune cell is selected from the group consisting of NK cell or T cell.

[0123] 25. The use according to embodiment 24, wherein T cell is selected from the group consisting of CD4+ T cell and CD8+ T cell.

[0124] 26. The use according to embodiment 25, wherein the T cells are in co-culture with the corresponding antigen presenting cells.

[0125] 27. The use according to embodiment 26, wherein the antigen presenting cell is of the same cell type as the IFN-y reporter cell.

[0126] 28. A composition comprising an IFN-y sensor as defined in embodiments 1 to 11 , a nucleotide sequence encoding a signal transducer or activator of transcription.

[0127] 29. A composition according to embodiment 28, wherein the signal transducer or activator of transcription is STAT-1.

[0128] 30. A composition according to embodiment 29, wherein the amino acid sequence of STAT- 1 has the sequence identical or at least 80% identical to the sequence as set out in SEQ ID NO: 7.

[0129] 31 . A composition according to any one of embodiments 28 to 30, wherein the signal transducer or activator of transcription is under control of a constitutive promotor. 32. Vector comprising the IFN-y sensor according to embodiments 1 to 11 .

[0130] The application further contains the following items:

[0131] 1 . An interferon-gamma (IFN-y) sensor comprising a nucleotide sequence comprising the following elements at least one gamma interferon activation site (GAS), a promotor linked to the GAS, and a nucleotide sequence encoding at least one fluorescent protein under the control of the promotor.

[0132] 2. IFN-y sensor according to item 1 , wherein the promotor linked to the GAS is an IFN- stimulated gene (ISG) promotor, wherein optionally the IFN-stimulated gene (ISG) promotor is ICAM-1 .

[0133] 3. IFN-y sensor according to any one of the preceding items, wherein the fluorescent protein is EGFP.

[0134] 4. IFN-y sensor according to any one of the preceding items, wherein the nucleotide sequence comprising at least one GAS comprises 8 GAS.

[0135] 5. IFN-y sensor according to any one of the preceding items, wherein GAS has a sequence as set out in SEQ ID NO: 1 (TTCN(2-4)GAA), wherein optionally the GAS has a sequence as set out in SEQ ID NO: 2 (TTCCGGGAA).

[0136] 6. IFN-y sensor according to any one of the preceding items, wherein the promotor linked to the GAS has the sequence as set out in SEQ ID NO: 5.

[0137] 7. IFN-y sensor according to any one of the preceding items, wherein the GAS sequences are separated by a nucleotide spacer.

[0138] 8. IFN-y sensor according to item 8, wherein the GAS nucleotide spacer has a sequence as set out in SEQ ID NO: 3 or SEQ ID No: 4.

[0139] 9. IFN-y reporter cell comprising IFN-y sensor as defined in items 1 to 8. 10. IFN-y reporter cell according to item 9, further comprising an exogenous nucleotide sequence encoding signal transducer or activator of transcription, optionally wherein the signal transducer or activator of transcription is STAT-1.

[0140] 11 . IFN-y reporter cell according to item 9 or 10, wherein the IFN-y reporter cell expresses an IFN-y receptor.

[0141] 12. IFN-y reporter cell according to any one of items 9 to 11 , wherein the IFN-y reporter cell is an antigen presenting cell.

[0142] 13. Use of the IFN-y sensor according to any one of items 1 to 8 and / or the IFN-y reporter cell according to any one of items 9 to 12 for in vitro measuring IFN-y levels from an IFN-y secreting cell.

[0143] 14. The use according to item 13, wherein the IFN-y secreting cell is in co-culture with the IFN-y reporter cell.

[0144] 15. The use according to item 14, wherein the measurement is carried out with a fluorescence intensity reader or a fluorescence cell imaging system.

[0145] 16. The use according to item 13, wherein the IFN-y secreting cell is cultured in a different vessel as the IFN-y reporter cell and wherein the IFN-y reporter cell is contacted with the cell culture supernatant of the IFN-y secreting cell.

[0146] 17. The use according to item 16, Wherein the measurement is carried out with a fluorescent intensity reader, a fluorescence cell imaging system or flow cytometry.

[0147] 18. The use according to items 14 to 17, wherein the measuring occurs at least at two time points.

[0148] 19. The use according to items 14 to 18 wherein the IFN-y secreting cell is selected from the group consisting of NK cell or T cell.

[0149] EXAMPLES

[0150] IFN-y sensor and IFN-y reporter cells In this example the construction of antigen-presenting (K562) IFN-y reporter cells involves the coordinated expression of STAT-1 , a pivotal component of the type II IFN signaling pathway. Simultaneously, an enhanced green fluorescent protein (EGFP) reporter gene was integrated, specifically regulated under the control of the intercellular adhesion molecule 1 (ICAM1) promoter fused to eight interferon-gamma-activated sites (GAS) (as depicted in Figure 1). This genetic configuration ensures the precision of our biosensor, enhancing its sensitivity and accuracy in detecting IFN-y activity.

[0151] To achieve this, the STAT-1 coding sequence was cloned into a gamma retroviral vector (pES12- 6). Additionally, the EGFP reporter gene, under the control of the ICAM1 promoter fused to eight GAS, was separately cloned into the same gamma retroviral vector (pES12-6). Subsequently, virus particles were generated for both the construct by transfecting HEK293 cells using Mirus transfection agent. The virus supernatant was collected after 72 hours and utilized for further transduction.

[0152] To generate the K562 IFN-y reporter cells, the K562 cells were co-transduced using the virus supernatant generated in the previous step.

[0153] K562 biosensor cell line is sensitive to human IFN-y

[0154] To assess the responsiveness of the developed biosensor cell line for its sensitivity and dynamic range to human Interferon-y (IFN-y), the cells were exposed to a spectrum of IFN-y concentrations, ranging from 0 pg / ml to 4000 pg / ml. The choice of this broad concentration range allows for a comprehensive evaluation of the biosensor's performance across a wide dynamic spectrum of IFN-y stimulation.

[0155] Following a 16 hr incubation period under cell culture conditions (38°C, 5% CO2, in RPMI 1640 (Cat # 21875-034, Life Technologies) supplemented with 10%FBS, 1 % L-Glutamin, 1 % MEM- NEAA, & 1 % Na-Pyruvat, cells were collected by centrifugation (450g, 5 min), washed one with PBS and resuspended in 1 ml PBS. The EGFP signal, serving as a readout for the activation of the IFN-y signaling pathway, was quantified using a standard flow cytometer. The EGFP mean fluorescent intensity (MFI) was then normalized with respect to the EGFP-positive population frequency, providing a normalized measure that accounts for variations in cell frequency and facilitates the comparison of responses across different IFN-y concentrations (Figure 2A).

[0156] The normalized MFI of EGFP for corresponding IFN-y concentrations follow an exponential trend and can be fit into 3rdorder polynomial function with R2=1. The data substantiates the capability of the engineered biosensor cell line to accurately report concentrations of IFN-y in the culture medium supernatant.

[0157] We evaluated the biosensor's capability to detect IFN-y levels in the supernatant medium of cocultures involving CD8+ cells with NY-ESO-1 -specific TCR and peptide-loaded APCs. In summary, we utilized serially diluted supernatants from co-cultures, alongside a range of human IFN-y, to establish a standard curve. The biosensor cells underwent a 16 Hr incubation period under cell culture conditions as described above with either culture medium supernatant ora range of human IFN-y, and the following day, the expression of EGFP was analyzed in these cells using a flow cytometer as described above. The biosensor cells demonstrated a linear relationship in detecting IFN-y concentration, as illustrated in Figure 2B, showcasing their effectiveness in accurately assessing IFN-y levels.

[0158] Comparative Analysis of the K562 Biosensor Cell Line and Traditional ELISA Assay for throughput screening of TCR

[0159] To assess the utility of the developed biosensor cell line in high-throughput screening for TCR discovery by detecting IFN-y in co-culture medium supernatants, supernatants from co-cultures involving unique CD8+ T cell clones containing distinct TCRs were employed with APCs either unloaded or loaded with relevant peptide. Simultaneously, the same supernatant medium underwent IFN-y detection using an ELISA assay.

[0160] In brief, single-cell-sorted CD8+ T cells were co-cultured overnight with APCs either unloaded or loaded with relevant peptide. The following day, 50 pl of supernatant was utilized for IFN-y detection via ELISA, while another 50 pl was employed to incubate the developed K562 IFN-y biosensor cell line for 16 hrs at 38°C, 5% CO2 in RPMI 1640 (Cat# 21875-034, Life Technologies) supplemented with 10%FBS, 1 % L-Glutamin, 1 % MEM-NEAA, & 1 % Na-Pyruvat. On the subsequent day, the EGFP signal was recorded using flow cytometry.

[0161] The difference in IFN-y levels between supernatants containing CD8+ T cell clones and peptide- loaded or unloaded APCs was evaluated. A total of 144 clones underwent testing using both ELISA assay and the biosensor cell lines. Notably, all tested clones demonstrated a heightened correlation with both ELISA and biosensor cell outcomes (see Figure 3).

[0162] IFN-y detection by biosensor as a bystander in CD8+ T cells & APCs in co-culture

[0163] To validate the utility of the engineered biosensor cells in detecting IFN-y as a bystander within a co-culture environment comprising CD8+ T cells with unique TCRs and APCs either loaded with relevant peptide or unloaded, -50,000 IFNy-K562 biosensor cells were introduced into the coculture consisting of 50,000 of NY-ESO-1 -specific TCR transduced CD8+ T cells and 25,000 APCs (also K562-A2), and incubated for 16 hours under cell culture conditions at 38°C, 5% CO2 in RPMI 1640, supplemented with 10 % FBS, 1 % L-Glut, 1 % Sodium pyruvate, 1 % MEM NEAA, and 1 % Pen / Strep. The IFN-y released by activated CD8+ T cells subsequently triggered the biosensor cells, leading to an increase in the EGFP signal in the IFNy-K562 biosensor cells, which was quantified using a flow cytometer. Additionally, the impact of biosensor cells on the co-culture was assessed by comparing the IFN-y levels in supernatants of co-cultures with or without biosensors using an ELISA assay (Figure 4C).

[0164] The biosensor effectively detected IFN-y levels as bystander cells as expected in a dose dependent manner. Notably, the analysis revealed no discernible difference in IFN-y levels in the supernatant between co-cultures with or without biosensors, confirmed by ELISA assay for IFN-y (Figure 4C). This finding underscores the biosensor's proficiency in capturing IFN-y dynamics as a bystander without influencing the overall IFN-y levels in the co-culture system.

[0165] Biosensor cells are stable for its sensitivity to human IFN-y To validate the stability of the developed biosensor cell line, a 10-week testing study was conducted, involving 20 cell splits to assess its sensitivity to human IFN-y. Weekly, after the second split, a defined portion of the cells (50 000 cells) underwent a 16-hour incubation with a range of IFN-y concentrations (0 - 4000 pg / ml) under conditions described above. The subsequent day, the cells were analyzed for EGFP expression using flow cytometry. The remaining, untreated cells were kept in culture and further propagated.

[0166] Throughout the whole propagation period that included 20 cell splits, the IFN-y biosensor cells consistently exhibited an increase in EGFP expression corresponding to the escalating concentrations of human IFN-y. The data exhibited an excellent fit with an exponential 3rdorder equation (Figure 5A), yielding an R2value of 0.999 for the majority of the weeks. While some variability in the MFI of EGFP can be observed between different weeks, the overall dynamic range per week remained consistent with minimal variation. The observed variation in MFI over weeks can be attributed to inter-day measurement inconsistencies inherent to the flow cytometer.

[0167] To further evaluate the quality of fit, a back-calculation of the IFN-y concentration was performed using the polynomial equation generated for each week. The calculated IFN-y concentrations were then plotted against theoretical IFN-y concentrations (Figure 5B). Remarkably, the calculated IFN-y concentrations displayed a very high correlation with the theoretical values, affirming the biosensor cell line's consistent ability to accurately sense IFN-y concentrations over an extended period.

[0168] K562 biosensor cells are less sensitive to human Typel interferon, IFNa and IFNp

[0169] To investigate the cross-talk between Type I and Type II interferons in the biosensor cells, a comparison was conducted using the MFI of EGFP in biosensor cells. These cells were subjected to varying concentrations of IFN-y and a fixed concentration of 4000 pg / ml of either IFNa or IFNp.

[0170] As anticipated, IFN-y demonstrated a dose-dependent activation response in the biosensor cells, leading to an observable increase in the EGFP signal. Notably, the EGFP signal generated by 4000 pg / ml of IFNa was equivalent to that produced by 318.61 pg / ml of IFN-y, while 4000 pg / ml of IFNp yielded a signal equivalent to 788.52 pg / ml of IFN-y (see Figure 6), hence showing the low sensitivity of the biosensor cells towards IFNa or IFNp.

[0171] Sensitivity of the assay using IFN-y Biosensors as bystander cells in co-culture experiment.

[0172] To assess the sensitivity of the assay using IFN-y Biosensor cell line as a bystander cells in coculture with antigen presenting (APC) cells and CD8+ T cells, 50,000 IFN-y Biosensor cells were co-cultured with 25,000 APCs (K562_A2) and 20,000 untransduced CD8+ T cells in RPMI 1640 medium, supplemented with 10 % FBS, 1 % L-Glut, 1 % Sodium pyruvate, 1 % MEM NEAA, and 1 % Pen / Strep at 38°C, 5% CO2. Additionally, increasing concentrations of recombinant human IFN-y ranging from 0 pg / ml to 4000 pg / ml were added in order to trigger activation of IFN-y Biosensors. eGFP fluorescence intensity represented as read fluorescence units (RFU) was analysed for 12 hours using an EnVision fluorescence reader (Figure 7A).

[0173] In the assay Biosensors effectively detected IFN-y levels as bystander cells in a dose dependent manner already at 6 hours of co-culture. Sensitivity of the assay was defined as the minimum concentration at which the assay can distinguish the positive (pos) from negative (neg) sample fulfilling following formula: mean pos. > mean neg. +3 x standard deviation (SD) (Figure 7B). Mean values of pos. and neg. samples, as well as standard deviation of neg. sample were calculated based on triplicates. Notably, the analysis revealed that IFN-y Biosensors used as bystander cells in co-culture can distinguish between background and a signal from 62.25 pg / ml IFN-y after 6h of stimulation.

[0174] Accuracy of the assay using IFN-y Biosensors as bystander cells in co-culture experiment.

[0175] To assess the accuracy of the assay 50,000 IFN-y Biosensor cells irradiated with 50 Gy, to prevent their proliferation, were co-cultured with 25,000 APCs (K562_A2) and 20,000 untransduced CD8+ T in RPMI 1640 medium, supplemented with 10 % FBS, 1 % L-Glut, 1 % Sodium pyruvate, 1 % MEM NEAA, and 1 % Pen / Strep at 38°C, 5% CO2. A range of defined IFN-y concentrations from 0 pg / ml to 4000 pg / ml was added for activation of Biosensor cells. The eGFP signal, serving as a read-out for the activation of the IFN-y signaling pathway, was quantified 24 hours upon co-culture using an EnVision fluorescence plate reader.

[0176] The eGFP signal for defined IFN-y concentrations followed an exponential trend and was fitted into 3rd order polynomial function with R2=1 (Figure 8 A). Based on the fitted standard curve, calculated concentrations were assessed (Figure 8B). The accepted accuracy of the calculated concentrations of IFN-y was considered 80%-120% (100%±20%) of the theoretical concentrations. The data substantiates the capability of the Biosensor cell line used as bystander cells to accurately report concentrations of IFN-y in the 250 -4000 pg / ml range.

[0177] Optimal effectortarget (E:T) ratio in bystander experiments.

[0178] To define an optimal E:T ratio in experiments with IFN-y Biosensor cells used as bystander, 50,000 Biosensors cells were co-cultured with 25,000 target T2 (Figure 9A) or K562_A11 (Figure 9B) cells and titrated numbers (50,000; 20,000; 10,000; 5,000; 2,500; 1 ,000; 500 or 100) of effector CD8+ T cells untransduced or transduced with NY-ESO-1 (Figure 9A) or KRAS G12V TCR (Figure 9B). Targets were unloaded or loaded with 10-5M concentration of relevant NY-ESO-1 peptide (Figure 9A) or relevant KRAS G12V peptide or WT irrelevant peptide (Figure 9B) for 2 hours priorto coculture and washed with culture medium. Co-cultures were set up in RPMI 1640 medium, supplemented with 10 % FBS, 1 % L-Glut, 1 % Sodium pyruvate, 1 % MEM NEAA, and 1 % Pen / Strep at 38°C, 5% CO2. eGFP fluorescence was analysed at 16 hours using an EnVision fluorescence plate reader (Figure 9A and B). Additionally, supernatants of co-cultures were collected for IFN-y ELISA (Figure 9A and B).

[0179] Notably, the Biosensor cells effectively detected specific activation of very low numbers of effector cells (100 and 500) upon co-culture with relevant peptide, confirming high sensitivity of the assay, and making it competitive to the used IFN-y ELISA. Overall, E:T ratio 0.8:1 showed an optimal signal to background ratio for read-out with fluorescence plate reader. Optimal number of Biosensor cells in bystander experiments.

[0180] Based on the pre-defined optimal E:T ratio 0.8:1 , an optimal number of Biosensor cells in the coculture assay was tested. For that purpose, titrated numbers (50,000; 25000; 10,000 and 5000) of IFN-y Biosensor cells were co-cultured with 25,000 antigen presenting cells (K562_A2) and 20,000 untransduced CD8+ T cells in RPMI 1640 medium, supplemented with 10 % FBS, 1 % L- Glut, 1 % Sodium pyruvate, 1 % MEM NEAA, and 1 % Pen / Strep at 38°C, 5% CO2. A range of defined IFN-y concentrations from 0 pg / ml to 2000 pg / ml was added for activation of Biosensor cells. eGFP fluorescence was analysed after 16 hrs of co-culture using an EnVision fluorescence reader. Data were fitted using a polynomial 3rd order equation (Figure 10) and a back-calculation of the IFN-y concentration was performed using the polynomial equation.

[0181] Considering the R2 value of the fitted curve and SD (showed with error bars on the graphs) of the back-calculated IFN-y concentrations based on the fitted curve, the analysis revealed that an optimal number of Biosensor cells in co-cultures with EnVision read-out is 50,000.

[0182] Improvement of the resolution of the assay.

[0183] To test the impact of IFN-y Biosensor irradiation on the read-out of the assay, IFN-y Biosensor cells (non-irradiated or irradiated 50 Gy) were co-cultured with APCs (K562_A2) and NY-ESO-1 TCR transduced CD8+ T cells and stimulated with increasing concentrations of recombinant human IFN-y ranging from Opg / ml to 4000pg / ml. Co-cultures were performed in RPMI 1640 medium, supplemented with 10 % FBS, 1 % L-Glut, 1 % Sodium pyruvate, 1 % MEM NEAA, and 1 % Pen / Strep at 38°C, 5% CO2. eGFP fluorescence intensity was analysed over a time range of 14-24 hours upon co-culture using an EnVision fluorescence reader (Figure 11).

[0184] The biosensor effectively detected IFN-y levels as bystander cells, as expected, in a dose dependent manner in both experimental conditions. Additional irradiation of Biosensors with 50 Gy, to prevent their proliferation, improved resolution of the assay throughout the tested IFN-y concentrations.

[0185] Monitoring of T cell activation and tumor cell killing overtime.

[0186] To test whether IFN-y Biosensor cells can be used to monitor T cell activation in co-culture with adherent tumor cells, 50,000 IFN-y Biosensor cells (for EnVision read-out) or 25,000 IFN-y Biosensor cells (for Incucyte read-out) - non-irradiated or irradiated with 50 Gy - were co-cultured with 25,000 tumor cells (Mel624.38_NLR or DAN-G_A11_NLR), both expressing red fluorescence marker nuclight red (NLR) and 20,000 CD8+ T cells, either untransduced or transduced with NY-ESO-1 TCR or KRAS G 12V TCR in RPMI 1640 medium, supplemented with 10 % FBS, 1 % L-Glut, 1 % Sodium pyruvate, 1 % MEM NEAA, and 1 % Pen / Strep at 38°C, 5% CO2. eGFP fluorescence was analysed over time using anEnvision fluorescence plate reader or Incucyte lifecell imaging system and NLR fluorescence was analysed over time with Incucyte life-cell imaging system (Figure 12 A, B).

[0187] Notably, eGFP fluorescence read out on EnVision and Incucyte showed a similar trend for both tested TCRs. Moreover, using Incucyte as a read-out, increase of eGFP signal indicating T cell activation and decrease of NLR signal representing tumor cell killing correlated together. Irradiation of IFN-y Biosensors with 50 Gy, improved long-term killing read-out on Incucyte, most likely through preventing overgrowth of Biosensors and subsequent deprivation of nutrients in the culture medium.

[0188] The findings highlight IFN-y Biosensors as a useful tool to monitor T cell activation and simultaneous detection of tumor cell killing over time in the co-culture system with TCR-transduced CD8+ T cells.

[0189] Superior effect of an exogenous STAT1 expression on the assay with IFN-y Biosensor cell line expressing four gamma Interferon activation site (GAS) elements.

[0190] To test whether exogenous expression of STAT1 improves the assay with IFN-g Biosensor cells, two K562 IFN-y Biosensor cell lines were generated as follows:

[0191] The STAT-1 coding sequence was cloned into a gamma retroviral vector (pES12-6). Additionally, the EGFP reporter gene, under the control of the ICAM1 promoter fused to four GAS elements, was separately cloned into the same gamma retroviral vector (pES12-6). To generate viral particles for transduction, the gamma retroviral vector (pES12-6) containing the desired construct (either 4GAS-EGFP or STAT1 cloned in pES12-6) was transfected into HEK293FT cells using Mirus Transfection Reagent (Cat # MIR2305). A mixture of Mirus reagent and DMEM medium supplemented with 1 % L-Glutamin, 1 % MEM-NEAA and 1 % Na-Pyruvat was prepared, and DNA for each construct was added together with helper plasmids. After a 48-hour incubation at 37°C and 5% CO2, the virus supernatant (VSN) was collected.

[0192] To generate the K562 IFN-y reporter cells, the K562 cells were either only transduced with the above described 4GAS-construct (SEQ ID NO: 13) or co-transduced with the 4GAS-construct and the ST AT1 -construct using the virus supernatant generated in the previous step.

[0193] The VSN was centrifuged to remove residual HEK cells, and 1 mL / well (24-well plate) of VSN was added to retronectin-coated wells. Subsequently, 0.25 x106K562 cells were seeded per well and incubated overnight at 37°C and 5% CO2.

[0194] Generated cell lines underwent bulk selection for low EGFP background expression and then high level of EGFP expression upon 16-hour stimulation with 20 ng / ml of IFN-y. Subsequently, the cells were expanded for 1 week without IFN-y addition. To assess the functionality of the generated cell lines, 5x104IFN-g Biosensor cells, from each cell line, were stimulated with increasing concentrations of recombinant human IFN-y ranging from 0 pg / ml to 4000 pg / ml in RPMI 1640 medium (Cat # 21875-034, Life Technologies), supplemented with 10 % FBS, 1 % L-Glut, 1 % Sodium pyruvate, 1 % MEM NEAA, and 1 % Pen / Strep at 37°C, 5% CO2. EGFP fluorescence intensity represented as read fluorescence units (RFU) was analysed after 16 hours with EnVision fluorescence reader (Figure 13A). Additionally, 1x106of cells from each generated cell line were collected for RNA extraction and subsequent qPCR analysis of exogenous STAT1 expression in relation to the expression of housekeeping genes [3-glucuronidase (GUSB) and TATA-binding protein (TBP). For a relative quantification of STAT1 mRNA expression following formula was used 2AACt, where ACt=mean Cp of (GUSB / TBP) - Cp of STAT1 (Figure 13B).

[0195] IFN-y Biosensor cell line expressing STAT1 exogenously showed significantly increased response to the stimulation with titrated concentrations of human IFN-y. The findings highlight importance of the exogenous STAT1 expression for the improvement of IFN-y biosensor cell lines response in the assay.

Claims

Claims1 . Interferon-gamma (IFN-y) reporter cell comprising a nucleotide sequence encoding an IFN-y sensor comprising the following elements at least one gamma interferon activation site (GAS), a promotor linked to the GAS, and a nucleotide sequence encoding at least one fluorescent protein under the control of the promotor; and an exogenous nucleotide sequence encoding signal transducer or activator of transcription.

2. IFN-y reporter cell according to claim 1 , wherein the promotor linked to the GAS is an IFN-stimulated gene (ISG) promotor, wherein optionally the IFN-stimulated gene (ISG) promotor is ICAM-1 .

3. IFN-y reporter cell according to any one of the preceding claims, wherein the fluorescent protein is EGFP.

4. IFN-y reporter cell according to any one of the preceding claims, wherein the nucleotide sequence comprising at least one GAS comprises 8 GAS.

5. IFN-y reporter cell according to any one of the preceding claims, wherein GAS has a sequence as set out in SEQ ID NO: 1 (TTCN(2-4)GAA), wherein optionally the GAS has a sequence as set out in SEQ ID NO: 2 (TTCCGGGAA).

6. IFN-y reporter cell according to any one of the preceding claims, wherein the promotor linked to the GAS has the sequence as set out in SEQ ID NO: 5.

7. IFN-y reporter cell according to any one of the preceding claims, wherein the GAS sequences are separated by a nucleotide spacer.

8. IFN-y reporter cell according to claim 7, wherein the GAS nucleotide spacer has a sequence as set out in SEQ ID NO: 3 or SEQ ID No: 4.

9. IFN-y reporter cell according to any one of the preceding claims, wherein the signal transducer or activator of transcription is STAT-1.1020 10. IFN-y reporter cell according to any one of the preceding claims, wherein the IFN-y reporter cell expresses an IFN-y receptor.11 . IFN-y reporter cell according to any one of the preceding claims, wherein the IFN-y reporter cell is an antigen presenting cell. 02512. Use of the IFN-y reporter cell according to any one of the preceding claims for in vitro measuring IFN-y levels.

13. Use according to claim 12, wherein the IFN-y levels of at least one IFN-y secreting 030 cell are measured.

14. Use according to claim 13, wherein the IFN-y secreting cell is in co-culture with the IFN-y reporter cell. 035 15. Use according to claim 13, wherein the IFN-y secreting cell is cultured in a different vessel as the IFN-y reporter cell and wherein the IFN-y reporter cell is contacted with the cell culture supernatant of the IFN-y secreting cell.

16. Use according to claim 13 to 15, wherein the measurement is carried out with a 040 fluorescent intensity reader, a fluorescence cell imaging system or flow cytometry.

17. Use according to any one of claims 12 to 16, wherein the measuring occurs at least at two time points. 045 18. Use according to claim 13 to 17, wherein the IFN-y secreting cell is selected from the group consisting of NK cell or T cell.

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