Immunologically engineered cells expressing successive truncated functional domains of gm-csf and uses thereof

Abstract

The application belongs to the technical field of biology and particularly relates to an immune engineering cell expressing a continuous truncated functional region of GM-CSF and application. The application first provides a CAR-NK cell expressing a continuous truncated functional region of GM-CSF, which expresses a novel chimeric antigen receptor structure; the chimeric antigen receptor structure comprises a continuous truncated functional region fragment of GM-CSF protein and a chimeric antigen receptor; the continuous truncated functional region fragment of GM-CSF protein comprises an A helix fragment, an exon 1 fragment and a C helix fragment. The application also provides a CAR-T cell also expressing a continuous truncated functional region of GM-CSF.

Description

[0001] Priority Application

[0002] This application claims priority to Chinese Invention Patent Application

CN2023115954083

CN2023115817245

[0003] The present application belongs to the field of biotechnology, and specifically relates to an immune engineering cell expressing a continuous truncated functional region of GM-CSF and application thereof. BACKGROUND

[0004] For tumors, traditional surgical removal, chemotherapy, and radiotherapy methods have not been able to meet the effective monitoring and treatment purposes, and effective alternative therapies are urgently needed to tackle this problem. In recent years, with the emergence of immunotherapy, revolutionary progress has been made in the field of tumor treatment, such as immune checkpoint inhibitors, bispecific antibodies, and CAR-T cell therapy, which have achieved remarkable achievements.

[0005] Chimeric antigen receptor (CAR) is an artificial receptor molecule manufactured by genetic engineering technology, which can endow immune effector cells (such as T cells, NK cells) with specificity for a certain target antigen epitope, thereby enhancing the function of lymphocytes in recognizing antigen signals and activation. The first generation of CAR has only CD3 ξ (containing 3 ITAM) and FcRγ (containing 2 ITAM) signaling domains in the intracellular, which can only provide the first signal for T cell activation. Early clinical trials of the first generation of CAR-T showed that the first generation of CAR-T could not effectively maintain the proliferation and activation of T cells, and did not achieve satisfactory results, indicating that the first generation of CAR-T cells may lack sufficient activation signals to maintain T cell proliferation and effective anti-tumor effect. Researchers have developed the second generation of CAR-T based on the intracellular signaling sequence of the first generation of CAR-T. The second generation of CAR-T cells showed enhanced in vivo expansion and persistence, and its effect has been confirmed in clinical trials.

[0006] After that, CAR-T related technologies have been continuously innovated, but among the 939 immune cell therapy trials initiated since 1993, only about half are for solid tumors. However, 90% of global cancer incidence is solid tumors. Unfortunately, compared to the good effect shown on hematological tumors, the effect of most reported CAR-T cell treatments for solid tumors has always been difficult to effectively break through at some key stages. Therefore, technical innovation is still needed in this field.

[0007] NK cells are important members of the innate immune system, primarily found in the blood and lymphoid organs. They require no prior sensitization and are not restricted by the major histocompatibility complex (MHC) complex. They can rapidly and directly kill target cells, demonstrating a broad spectrum of anti-tumor activities. NK cells kill target cells in a variety of ways. Once the balance between inhibitory and activating signals within the NK cell shifts toward activation, the NK cell forms a synapse with the target cell, releasing effector granules to lyse the target cell and produce effector cytokines. Expressing chimeric antigen receptors (CARs) on the NK cell surface can significantly enhance the anti-cancer efficacy of these immune cells. Currently, the scientific community believes that CAR-NK cells offer several significant advantages over CAR-T cells. However, the development of CAR-NK cells with tumor-killing efficacy presents challenges, primarily due to the inherent antiviral properties of NK cells, which make genetic modification difficult.

[0008] Patent publication number CN114934071A, entitled "A CAR Vector Expressing Immunomodulatory Factors and Its Application," discloses a CAR-T cell expressing a virally transduced chimeric antigen receptor and an immunomodulatory factor, the full-length granulocyte-macrophage colony-stimulating factor (GM-CSF). NK cell-activating receptors include natural cytotoxicity receptors (NCRs), such as NKG2D, CD16 (FcgRIIIa), FasL, and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), as well as costimulatory receptors such as LFA-1, CD244 (2B4), and CD137 (41BB). These receptors differ significantly from the intracellular signaling activation domains of CAR-T cells. Therefore, conventional CAR molecule designs that work well for CAR-T cells may not necessarily be effective in activating NK cells.

[0009] Therefore, it is necessary to make improvements to the existing technology. Summary of the Invention

[0010] In view of this, the purpose of the present invention is to provide an immune engineering cell expressing the first continuously truncated functional region of GM-CSF and its application. The present invention specifically adopts the following technical solutions.

[0011] One aspect of the present invention is to provide a CAR-NK cell expressing the first continuously truncated functional region of GM-CSF.

[0012] A CAR-NK cell expressing continuously truncated functional regions of GM-CSF, wherein the CAR-NK cell expresses a novel chimeric antigen receptor structure; the chimeric antigen receptor structure comprises continuously truncated functional region fragments of the GM-CSF protein and a chimeric antigen receptor; the continuously truncated functional region fragments of the GM-CSF protein include an A helix fragment, an exon 1 fragment, and a Chelix fragment; the continuously truncated functional region fragments of the GM-CSF protein are connected to the chimeric antigen receptor via a P2A sequence; the chimeric antigen receptor comprises a CD8 signal peptide, an antigen recognition region, a hinge region, a human CD8 transmembrane region, a human 4-1BB costimulatory signal region, and a human CD3 ζ signaling domain.

[0013] Furthermore, the nucleotide sequence of the continuously truncated functional region fragments of the GM-CSF protein is shown in SEQ ID NO.1.

[0014] Furthermore, the novel chimeric antigen receptor structure is also connected to the EF1-α promoter sequence.

[0015] Furthermore, the nucleotide sequence of the chimeric antigen receptor expressed by the CAR-NK cell is shown in SEQ ID NO.3.

[0016] Another aspect of the present invention is to provide a CAR-T cell expressing the first continuously truncated functional region of GM-CSF.

[0017] A CAR-T cell expressing continuously truncated functional regions of GM-CSF, wherein the CAR-T cell expresses a novel chimeric antigen receptor structure; the novel chimeric antigen receptor structure comprises continuously truncated functional region fragments of the GM-CSF protein and a chimeric antigen receptor; the continuously truncated functional region fragments of the GM-CSF protein include an A helix fragment, an exon 1 fragment, and a Chelix fragment; the continuously truncated functional region fragments of the GM-CSF protein are connected to the chimeric antigen receptor via a P2A sequence; the chimeric antigen receptor comprises a CD8 signal peptide, an antigen recognition region, a hinge region, a human CD8 transmembrane region, a human 4-1BB costimulatory signal region, and a human CD3 ζ signaling domain.

[0018] Furthermore, the nucleotide sequence of the continuously truncated functional region fragments of the GM-CSF protein is shown in SEQ ID NO.1.

[0019] Furthermore, the novel chimeric antigen receptor structure is also connected to the EF1-α promoter sequence.

[0020] Furthermore, the nucleotide sequence of the chimeric antigen receptor expressed by the CAR-T cell is shown in SEQ ID NO.3.

[0021] The application further provides a lentiviral vector of a CAR expression vector, which comprises a nucleic acid encoding a chimeric antigen receptor and a nucleic acid encoding a continuous truncated functional region of GM-CSF; the nucleic acid encoding the chimeric antigen receptor is connected with the nucleic acid encoding the continuous truncated functional region of GM-CSF through a sequence encoding P2A; the fragment of the continuous truncated functional region of GM-CSF comprises an A helix fragment, an exon 1 fragment and a C helix fragment; the chimeric antigen receptor comprises a CD8 signal peptide, an antigen recognition region, a hinge region, a human CD8 transmembrane region, a human 4-1BB costimulatory signal region and a human CD3 zeta signal domain; and the lentivirus comprises pWPXLD, psPAX2 and pMD2.G.

[0022] The antigen binding domain in the CAR provided by the application can target CD19, BCMA, HER2, Claudin18.2, Mesothelin, GPC3, GD2, etc.

[0023] The CAR-NK cell described above is used for preparing a TNF-alpha promoter.

[0024] The CAR-NK cell can promote the expression of TNF-alpha in tumor tissues or cells.

[0025] The CAR-NK cell described above is used for preparing an IFN-gamma promoter.

[0026] The CAR-NK cell can promote the expression of IFN-gamma in tumor tissues or cells.

[0027] The CAR-T cell described above is used for preparing a drug for treating solid tumors.

[0028] Further, the tumor comprises a hematological tumor, liver cancer, breast cancer, lung cancer, esophageal cancer, gastric cancer, ovarian cancer, glioma, pancreatic cancer, sarcoma or glioblastoma.

[0029] Beneficial technical effects:

[0030] In one aspect, the application constructs an immune engineering cell expressing a continuous truncated functional region of GM-CSF, and the immune engineering cell expresses a novel chimeric antigen receptor structure. Compared with a GM-CSF full-length protein, the novel chimeric antigen receptor structure of the application only comprises a partial continuous truncated functional region of the GM-CSF full-length protein, reduces the sequence length of the overexpressed gene, but does not affect the expression and function of the CAR, on the contrary, since the continuous truncated functional region does not contain a signal peptide, it can function intracellularly, thereby enhancing the expression of the CAR and improving the proliferation ability of the CAR-T cell.

[0031] And, the experiments of the present application further prove that under the condition of all continuous truncated functional regions, different number of continuous truncated functional regions will also cause different technical effects. Specifically, the proliferation ability of the CAR-T cells (CAR-X5-T) of the present application which express 3 continuous truncated functional regions of GM-CSF is significantly better than that of the conventional CAR-T cells and the CAR-T cells (CAR-GM-T) prepared from the full-length GM-CSF, and the comparative analysis shows that the proliferation effect of the CAR-X5-T cells is obviously better than that of the CAR-T cells (CAR-X2-T) which express 2 continuous truncated functional regions of GM-CSF. It is shown that under the condition of all continuous truncated functional regions, further shortening the number of continuous truncated functional regions does not necessarily make the proliferation effect of CAR-T cells better. Obviously, the CAR-X5-T cells of the present application have higher yield and lower production cost when used for industrial production, and have greater industrial popularization value.

[0032] On the other hand, the experiments of the present application prove that the CAR-NK cells (CAR-X5-NK) which express the continuous truncated functional regions of GM-CSF have strong cytokine secretion ability, and the secreted TNF-α and IFN-γ are significantly higher than those of the conventional CAR-NK cells. Therefore, the CAR-X5-NK of the present application is suitable for preparing TNF-α promoters and IFN-γ promoters, and has greater industrial production value.

[0033] In summary, due to the difference between NK cell modification and T cell modification, the CAR structure expressed and functioning on CAR-T cells does not necessarily have the same effect on CAR-NK cells. In addition, unlike the guess of those skilled in the art, it is not necessarily that the smaller the length of the expressed factor, the stronger the function of the CAR, and further experiments are needed to verify. BRIEF DESCRIPTION OF DRAWINGS

[0034] In order to more clearly illustrate the technical solutions of the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiment or prior art description. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, each element or part is not necessarily drawn according to the actual proportion. Obviously, the drawings described below are some embodiments of the present application, and those skilled in the art can obtain other drawings according to these drawings without paying creative labor.

[0035] Figure 1 The construction diagram of the new CAR structure expression vector of the present application;

[0036] Figure 2 The structure schematic diagram of the full-length GM-CSF protein functional region;

[0037] Figure 3 This is a graph showing the results of the CAR positivity test of the CAR-X5-T cells constructed in one embodiment of the present invention;

[0038] Figure 4 This is a graph showing the proliferation of CAR-X5-T cells constructed in one embodiment of the present invention;

[0039] Figure 5 This is a graph showing the proliferation of CAR-X2-T cells in the control group constructed in one of the embodiments of the present invention;

[0040] Figure 6 This is a graph showing the killing effect of CAR-X5-T cells on tumor cells constructed in one embodiment of the present invention;

[0041] Figure 7 Figure 2 shows the detection results of TNF-α and IFN-γ after co-culture of CAR-X5-NK cells and tumor cells when the effector-target cell ratio was 5:1;

[0042] Figure 8 This is a graph showing the killing effect of CAR-X5-NK cells on tumor cells constructed in one embodiment of the present invention. DETAILED DESCRIPTION

[0043] To make the purpose, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0044] As used herein, "and / or" includes any and all combinations of one or more of the associated listed items.

[0045] Herein, "plurality" means two or more than two, ie, it includes two, three, four, five, etc.

[0046] As used in this specification, the term "about" typically means + / - 5% of the stated value, more typically + / - 4% of the stated value, more typically + / - 3% of the stated value, more typically + / - 2% of the stated value, even more typically + / - 1% of the stated value, and even more typically + / - 0.5% of the stated value.

[0047] In this specification, certain embodiments may be disclosed in a format that is within a range. It should be understood that this description of "within a range" is merely for convenience and brevity and should not be interpreted as a rigid limitation on the disclosed range. Therefore, the description of a range should be considered to have specifically disclosed all possible subranges and individual numerical values ​​within this range. For example, the description of a range of 1-6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numbers within this range, such as 1, 2, 3, 4, 5, and 6. Regardless of the breadth of the range, the above rules apply.

[0048] Definition of noun:

[0049] The "novel chimeric antigen receptor structure" described in the present invention refers to a chimeric antigen receptor (CAR) and a continuously truncated functional region fragment (X5) of the GM-CSF protein connected to the chimeric antigen receptor via P2A. The chimeric antigen receptor includes a CD8 signal peptide, an antigen recognition region, a hinge region, a human CD8 transmembrane region, a human 4-1BB costimulatory signaling region, and a human CD3 ζ signaling domain.

[0050] The "GM-CSF truncated region" of the present invention refers to a truncated region obtained by modifying the functional region fragments of the full-length GM-CSF protein by truncation or deletion; wherein the functional region fragments include signal peptide fragments, A helix fragments, exon 1 fragments, C helix fragments and exon 2 fragments. The full-length structure of the GM-CSF protein is as follows Figure 2 shown.

[0051] Based on the above modification methods, the full-length GM-CSF protein was prepared into five short peptides containing different truncated fragments, including:

[0052] GM-CSF first truncation region: contains only the A helix fragment region of GM-CSF.

[0053] GM-CSF second truncation region: contains the exon 1 fragment region and the Chelix fragment region of GM-CSF.

[0054] GM-CSF third truncation region: contains only the exon 2 fragment of GM-CSF.

[0055] GM-CSF 4th truncation region: contains the A helix, C helix and exon 2 fragments of GM-CSF.

[0056] GM-CSF 5th truncation region: contains the A helix, exon 1 and C helix fragment regions of GM-CSF.

[0057] On the one hand, the purpose of the present invention is to explore the improvement of CAR function by truncating the full-length GM-CSF protein.

[0058] Another invention is to explore the effect of different numbers of functional regions on CAR function under the premise of continuous truncation of functional regions.

[0059] In the following examples, for easy distinction, the short peptide containing three consecutive truncated functional regions is called the first consecutive truncated functional region, represented by X5; the short peptide containing two consecutive truncated functional regions is called the second consecutive truncated functional region, represented by X2.

[0060] The sequence information involved in the present invention is shown in Table 1.

[0061] Table 1

[0062]

[0063] Example 1

[0064] Construction of novel CAR expression vector and viral packaging.

[0065] 1.1 Construction of lentiviral expression vector for novel CAR vector.

[0066] The molecular cloning method was used to construct a lentiviral expression vector pWPXLD-CAR-P2A-X5 expressing a novel CAR. The CAR fragment contains a CD8 signal peptide, an antigen recognition region (SCFV), a hinge region (hinge), a human CD8 transmembrane region (TM), a human 4-1BB costimulatory signal region, a human CD3ζ signaling domain, P2A, and a fragment (X5). Figure 1 shown.

[0067] 1.2 Packaging of novel CAR lentivirus.

[0068] Take 293T cells in good growth state, digest the cells with trypsin, and 6 Cells were passaged at a density of 100 cells / dish. When the cell confluence reached 80%, transfection was performed using calcium phosphate transfection with the CAR vector as the core plasmid and psPAX2 and pMD2.G as helper plasmids. Eight hours after transfection, the culture medium containing the calcium phosphate precipitate was discarded. 48 and 72 hours after transfection, the cell culture supernatant was collected as the viral stock solution and centrifuged at 2000 rpm for 10 minutes. The cell pellet was removed, and cell debris was removed using a 0.22μm disposable syringe filter (PES membrane).

[0069] Example 2

[0070] CAR positivity rate detection.

[0071] T cells were transduced with the lentivirus described in Example 1 to obtain novel CAR-X5-T cells. The His-tagged target protein was used as the primary antibody, and a His antibody conjugated to fluorescein APC was used as the secondary antibody to detect the CAR positivity rate. Conventional CAR refers to traditional second-generation CAR-T cells, CAR-GM refers to CAR-T cells containing full-length GM, and CAR-X5 refers to CAR-T cells containing fragment X5.

[0072] The results are as follows Figure 3 The CAR-positive rate in CAR-X5-T cells was 61.83%, which was higher than that in conventional CAR-T cells and CAR-GM-T cells, indicating that only part of the GM-CSF functional region was contained, and the functional region was a continuous truncated region, which could enhance the expression of CAR.

[0073] Example 3

[0074] This example detects the proliferation ability of the prepared CAR-X5-T cells.

[0075] The same number of T cells were transduced with the lentivirus in Example 1 to obtain novel CAR-X5-T cells (experimental group). Three days later, the number of CAR-X5-T cells was detected using a cell counter and the expansion fold was calculated. The results are as follows: Figure 4 As shown in the results, the proliferation ability of CAR-X5-T cells was significantly better than that of conventional CAR-T cells and CAR-GM-T cells, indicating that reducing the length of the GM-CSF functional region improved the proliferation ability of CAR-T cells, suggesting that CAR-T cells modified with the "first continuous truncated functional region" containing GM-CSF have better proliferation effect.

[0076] Furthermore, using a similar method to Example 1, lentivirus was used to transduce T cells to obtain CAR-X2-T cells (control group), and their proliferation was regularly detected. The results were as follows: Figure 5 As shown in the figure, through comparative analysis, it was found that the proliferation ability of CAR-X5-T cells was significantly better than that of CAR-X2-T cells, suggesting that under the premise that both have continuously truncated functional regions, further shortening the number of continuously truncated functional regions of GM-CSF (i.e., the "second continuously truncated functional region") may not necessarily have a better effect on promoting the proliferation of CAR-T cells.

[0077] The above experimental results show that containing a specific number of continuously truncated functional regions of GM-CSF protein (i.e., the "first continuously truncated functional region") can not only promote the expression of CAR, but also enhance the proliferation ability of CAR-T cells, and its proliferation ability is significantly stronger than that of CAR-T prepared with full-length GM-CSF and CAR-T expressing the "second continuously truncated functional region" of GM-CSF.

[0078] In the CAR-X2-T cells involved in this embodiment, the nucleotide sequence of X2 is as follows.

[0079] Table 2

[0080]

[0081] Example 4

[0082] This example provides verification of CAR-X5-T cells killing tumor cells.

[0083] CAR target-positive SK-OV3 cells (human ovarian cancer cells) were used as target cells, and the CAR-X5-T cells obtained in Example 3 were used as effector cells. The two cell types were co-cultured at an effector-target ratio of 5:1, and the killing effect of CAR-X5-T cells on SK-OV3 cells was monitored.

[0084] The results are as follows Figure 6 As shown in the figure, starting from approximately 10 hours, CAR-X5-T cells showed significantly higher tumor cell killing ability than conventional CAR-T cells. Analysis of the killing efficacy of the killing curve for 40 hours showed that the killing efficiency of CAR-X5-T cells was similar to that of CAR-GM-T cells, but significantly higher than that of conventional CAR-T cells, and the killing effect was also initiated earlier than that of conventional CAR-T cells.

[0085] The above experimental results show that appropriately reducing the length of the GM-CSF functional region can enhance the proliferation ability of CAR-T cells without significantly weakening their killing effect on tumor cells.

[0086] Example 5

[0087] This example detects the cytokine secretion ability of CAR-X5-NK cells.

[0088] The lentivirus in Example 1 was used to transduce NK cells to obtain novel CAR-X5-NK cells. The supernatants of CAR-NK cells and tumor cells were collected after 24 hours of co-culture, and the IFN-γ and TNF-α in the supernatants were analyzed. The IFN-γ and TNF-α in the supernatants of CAR-X5-NK cells and target cells were significantly increased ( Figure 7 ), the specific results are shown in Table 2.

[0089] Table 3 Detection results of cytokines secreted by CAR-NK cells.

[0090]

[0091] The results showed that compared with conventional CAR, CAR-NK cells modified with the "first continuously truncated functional region" containing GM-CSF secreted 76.9 times more TNF-α and 1.9 times more IFN-γ than conventional CAR-NK cells, suggesting that CAR-NK cells expressing an appropriate number of continuously truncated functional regions of GM-CSF protein can significantly enhance the cytokine secretion ability of CAR-NK cells.

[0092] Example 6

[0093] This example further demonstrates the ability of CAR-X5-NK cells to kill tumor cells based on Example 5.

[0094] CAR target-positive SK-OV3 cells were used as target cells, and the CAR-X5-NK cells in Example 5 were used as effector cells. The two cells were co-cultured at an effector-target ratio of 5:1, and the killing effect of CAR-X5-NK cells on SK-OV3 cells was monitored. The results are shown in Figure 5. Figure 8 As shown in the figure, the killing efficacy of the killing curve for 30 hours was analyzed, which generally showed that the killing ability of CAR-X5-NK cells against tumor cells was slightly weaker than that of conventional CAR-NK cells.

[0095] The above results indicate that although CAR-NK cells modified with an appropriate number of continuously truncated functional regions of the GM-CSF protein (i.e., the "first continuously truncated functional region") have stronger cytokine secretion capabilities than conventional CAR-NK cells, their tumor cell killing effect is not necessarily better than that of conventional CAR-NK cells.

[0096] It should be noted that, in this document, the terms "comprises," "includes," or any other variations thereof are intended to encompass non-exclusive inclusion, such that a process, method, article, or apparatus comprising a series of elements includes not only those elements but also other elements not explicitly listed, or elements inherent to such process, method, article, or apparatus. In the absence of further limitations, an element defined by the phrase "comprising a ..." does not exclude the presence of other identical elements in the process, method, article, or apparatus comprising the element.

[0097] The embodiments of the present application are described above with reference to the accompanying drawings, but the present application is not limited to the above-described specific embodiments, and the above-described specific embodiments are merely illustrative, but not restrictive, and a person of ordinary skill in the art can make many forms under the inspiration of the present application without departing from the purpose of the present application and the scope protected by the claims, and these all belong to the protection of the present application.

Claims

1. A CAR-NK cell expressing serially truncated functional regions of GM-CSF, characterized in that: The CAR-NK cell expresses a novel chimeric antigen receptor structure; the chimeric antigen receptor structure comprises a continuously truncated functional region fragment of the GM-CSF protein and a chimeric antigen receptor; the continuously truncated functional region fragment of the GM-CSF protein comprises an A helix fragment, an exon 1 fragment, and a Chelix fragment; the continuously truncated functional region fragment of the GM-CSF protein is connected to the chimeric antigen receptor via a P2A sequence; the chimeric antigen receptor comprises a CD8 signal peptide, an antigen recognition region, a hinge region, a human CD8 transmembrane region, a human 4-1BB costimulatory signal region, and a human CD3 ζ signaling domain; the nucleotide sequence of the continuously truncated functional region fragment of the GM-CSF protein is shown in SEQ ID NO.1; the nucleotide sequence of the chimeric antigen receptor expressed by the CAR-NK cell is shown in SEQ ID NO.

3.

2. The CAR-NK cell according to claim 1, wherein The novel chimeric antigen receptor structure is also connected to the EF1-α promoter sequence.

3. Use of the CAR-NK cells of claim 1 or 2 in the preparation of a TNF-α promoter; or use of the CAR-NK cells of claim 1 or 2 in the preparation of an IFN-γ promoter.

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