WO2025056739A1 - Enhancement of car-t cell efficacy by inhibiting nr2f6 - Google Patents

Abstract

The invention relates to a modified immune cells for use in the treatment of a solid tumor in a subject, wherein the modified immune cell comprises one or more exogenous nucleic acid molecules encoding a transgenic construct targeting an antigen expressed in a cancerous cell of said solid tumor, in said immune cell, Nuclear Receptor Subfamily 2 Group F Member 6 (NR2F6) activity is inhibited (in comparison to a control immune cell), and binding of the immune cell to the antigen is associated with death of said cancerous cell expressing the antigen, and inducing a secondary immune reaction in the subject against cancerous cells of the solid tumor, wherein said secondary immune reaction is non-specific to the antigen targeted by the transgenic construct (epitope spreading). The invention relates further to a modified immune cell comprising one or more exogenous nucleic acid molecules encoding a transgenic construct targeting an antigen expressed in a cancerous cell of a solid tumor, wherein in said cell, NR2F6 activity and Casitas B-lineage lymphoma proto-oncogene-b (CBLB) activity is inhibited (compared to a control immune cell). The invention relates further to a pharmaceutical composition comprising the modified immune, suitable for the treatment of a solid tumor, comprising additionally a pharmaceutically acceptable carrier, and to an in vitro method for enhancing the cytolytic activity of a modified immune cell.

Description

ENHANCEMENT OF CAR-T CELL EFFICACY BY INHIBITING NR2F6

DESCRIPTION

The invention relates to the field of cellular therapeutic agents, in particular to means for reducing the exhaustion and/or for enhancing the cytolytic activity of therapeutic immune cells in the treatment of solid tumors.

The invention relates to a modified immune cell for use in the treatment of a solid tumor in a subject, wherein the modified immune cell comprises one or more exogenous nucleic acid molecules encoding a transgenic construct targeting an antigen expressed in a cancerous cell of said solid tumor, wherein in said immune cell, Nuclear Receptor Subfamily 2 Group F Member 6 (NR2F6) activity is inhibited (in comparison to a control immune cell), and binding of said immune cell to the antigen during treatment is associated with death of said cancerous cell expressing the antigen, and inducing a secondary immune reaction in the subject against cancerous cells of the solid tumor, wherein said secondary immune reaction is non-specific to the antigen targeted by the transgenic construct (epitope spreading).

In embodiments, the invention relates to the medical use of a CAR-expressing cytotoxic immune cell, in which NR2F6 activity is inhibited or removed, in the treatment of a solid tumor, wherein said treatment comprises inducing a non-antigen-specific secondary immune reaction in the subject against cancerous cells of the tumor.

The invention relates further to a modified immune cell comprising one or more exogenous nucleic acid molecules encoding a transgenic construct targeting an antigen expressed in a cancerous cell of a solid tumor, wherein in said cell, NR2F6 activity and Casitas B-lineage lymphoma proto-oncogene-b (CBLB) activity is inhibited (compared to a control immune cell).

The invention relates further to a pharmaceutical composition comprising the modified immune cell, suitable for the treatment of a solid tumor, comprising additionally a pharmaceutically acceptable carrier, and to an in vitro method for reducing the exhaustion and/or enhancing the cytolytic activity of a modified immune cell.

BACKGROUND OF THE INVENTION

Cancer immunotherapy is a promising approach for tumor treatment, which is strictly dependent on understanding the immune system (Guha et al, 2022). There are multiple types of immunotherapies commonly used in cancer treatment. Immune checkpoint inhibitors (ICIs) are the most common type of cancer immunotherapy consisting of monoclonal antibodies that target tumor antigens to induce an immune response. Therapeutic cancer vaccines are another type of immunotherapy that generates an endogenous immune response against tumor antigens by presenting their antigens with cell-, peptide-, virus- or gene-based formulations. By only relying on the endogenous immune response, the list of immune-related adverse events (irAEs) for this therapy is smaller than for ICIs. Nonetheless, neither ICIs nor cancer vaccines overcome tumor antigen heterogeneity, which is particularly observed in solid tumors, hampering their therapeutic efficiency in solid tumors (Guha et al., 2022). Within cancer immunotherapies, chimeric antigen receptor T cell (CAR-T cell) therapies are an advancing technology that has gained recognition over the past decade (Yan et al., 2023). CAR-T cell therapy promises to advance the efficacy of cancer therapy in a more targeted manner by combining the antigen recognition ability of antibodies with the cytotoxic effector function of immune cells such as CD8⁺ T cells.

However, current CAR-T cell therapy, despite its remarkable success in blood cancer patients, has not yet reached its full potential. It is until now costly and only suitable for a limited number of cancer types, which are mainly hematologic cancers. In 2018, the European Medicines Agency (EMA) approved the first CAR-T cell-based immunotherapies, i.e. , Kymriah (tisagenlecleucel) and Yescarta (axicabtagene ciloleucel), after 2017, the first CAR-T cell products were approved in the US. To date the Food and Drug Administration (FDA) has already approved six different CAR-T cell products, which are directed to two different targets, namely CD19 and B-cell maturation antigen (BCMA), which are predominantly found on cells that lead to different types of blood cancers.

However, to date these therapeutic approaches are only available for patients with relapsed or refractory cancer. More recently, the EMA has approved Tecartus (Brexucabtagene autoleucel), for example, for the treatment of mantle cell lymphoma (MCL), so that three products are now available on the European market. These clinically approved CAR-T cell therapies target antigens of the B-cell lineage, e.g., in leukemia and lymphoma (ECIS - European Cancer Information System; Dine et al., 2017; Zhuang et al., 2012; Marin-Avedo et al., 2018; Fleischer et al., 2019; Holstein et al., 2020 and Depil et al., 2020). However, although up to 50% of patients with such hematological malignancies respond to CAR-T cell therapy, treatment success is often limited by the fact that some patients only respond in the short term and treatment has to be repeated.

Overall, CAR-T cells have been transformative in the treatment of hematological diseases and are rightly regarded as one of the major breakthroughs in cancer immunotherapy. However, the approved CAR-T cell therapy currently on the market are all 1) targeting cancer of liquid tissues, e.g., hematological cancers and 2) based on autologous cells (i.e., the cells are obtained from the patients). Despite the active research field, a breakthrough for solid cancer still has to be found and there is no CAR-T cell therapy directed to solid tumors on the market despite the urgent medical need for effective treatment concepts against solid cancers.

Recent studies directed to the development of efficient CAR-T cell therapies for solid tumors are focused on improving the solid tumor specificity of the CAR by identifying new antigens (i.e., tumor neoantigens) with higher immunogenic effects and lower off-tumor toxicity (Yan et al., 2023). Novel targets tested for lung cancer include e.g., Mesothelin (MSLN), carcino-embryonic antigen (CEA), Receptor Tyrosine Kinase Like Orphan Receptor 1 (ROR1), Epidermal growth factor receptor variant III (EGFRvlll), B7 homolog 3 protein (B7-H3 also known as CD276), Mucin 1 (MUC1), Tumor necrosis factor receptor superfamily member 4 (TNFRSF4, also known as CD134 or 0X040), Claudin 6 (CLDN6), oncofetal chondroitin sulphate (ofCS) (Maalej et al., 2023). However, while tumor specificity reduces the immune-related adverse events (irAEs), it does not solve several of the challenges that CAR-T cell therapies for solid tumors still face (Guha et al., 2022).

The shortcomings of current CAR-T cell therapies in solid tumors are multifactorial and include either limited settlement and penetration of non-inflamed tumors or terminal T cell dysfunction within the immunosuppressive tumor immune microenvironment (TIME) and subsequent treatment failure. Another inherent weakness of current CAR-T treatment approaches is that the therapeutically targeted surface antigens on malignant cells (i) are heterogeneously expressed within solid tumors and (ii) are not entirely restricted to tumors but are also present in other non- cancerous tissues. The main causes of failure of current therapies in the treatment of solid tumors are thus the following.

Solid tumors have a strong tumor immune microenvironment (TIME) barrier, especially in the lung, that physically, e.g., due to the extracellular matrix, and biochemically, e.g., by the presence of cytokines or due to cell-mediated immunosuppression, hampers the ability of CAR-T cells to infiltrate the tumor and results in low persistence due to exhaustion and functional inactivity of CAR-T cells. This results in the inability of the CAR-T cells to efficiently kill the tumor cells (Mamdani et al., 2022; Hosseinkhani et al., 2020). CAR-T cells have been reported to be almost completely metabolically dysfunctional at the solid tumor site and are usually exhausted by solid tumor-mediated immunosuppression, inhibiting their cytotoxic functions. Further, the immunosuppressive properties of TIME, such as particularly high TGFbeta levels, lead to a substantial decrease in granzyme secretion and subsequently to a progressive loss of effective anti-tumor cytotoxicity.

Further, solid tumors such as lung tumors show a high heterogeneity of tumor antigens (primary resistance). Due to this heterogeneity solid tumors can often not be effectively eliminated by CAR-T cells targeting one or even two tumor surface antigens. Non-small cell lung cancer (NSCLC) is one such example of a highly incurable cancer with inhomogeneous tumor surface antigen expression that has not yet responded to CAR-T therapies (Porter et al., 2015). Additionally, a downregulation of the CAR-T target antigen can result in tumor cell immune evasion (acquired resistance) from CAR-T cell therapy (Mamdani et al., 2022; Zhong et al., 2020; Fuchsl et al., 2022). Further, it must be noted that tumors often express antigens that are also present in non-cancerous tissue (e.g., CD19 in acute lymphatic leukemia (ALL)), rendering it difficult to specifically target the tumor cells and resulting in immune-related adverse events (irAEs).

Several approaches for CAR-T cell therapies are under investigation to overcome these hurdles in treating solid tumors by, for instance, increasing the T cell cytotoxic function (Guha et al., 2022; Majzner et al., 2018). Examples include studies that combine CAR-T cells with plug-in technology, such as the secretion of cytokines to stimulate anti-cancer effect (e.g., KJ-C2113 drug candidate by Carsgen Therapeutics in preclinical testing) or the modification of immunosuppressive pathways (e.g., PD1 gene editing) (Maalej et al., 2023; Chen et al., 2018).

Kumar et al. (“Deletion of Cblb-b inhibits CD8⁺ T cell exhaustion and promotes CAR-T cell function”, Journal for immunotherapy of cancer, 2020) analyzes the role of Casitas B-lineage lymphoma proto-oncogene-b (CBLB) T cell exhaustion. They show that CBLB depletion inhibits CAR-T cell exhaustion in the Rag1 knockout mouse model. This is a mouse line model that completely lacks an endogenous adaptive immune system. Therefore, and due to the lack of an endogenous T cell compartment in these Rag1 knockout mice used by Kumar et al, it was not possible to investigate cross-priming of tumor antigens. Therefore, Kumar et al., by using Rag1 knockout mice, have no evidence in their study to suggest that an effective secondary and polyclonal immune response can be induced by CBLB gene-edited CAR-T treatment of antigenically heterogeneous solid tumors. Another approach is the inhibition of Nuclear Receptor Subfamily 2 Group F Member 6 (NR2F6) as immune check point in T cells or other cells, such as cord blood cells, in order to enhance antitumor activity of these cells. Such approaches are for example disclosed in Hermann-Kleiter et al. (2015), showing that NR2F6-modified T cells show a significant tumor rejection advantage in the therapeutic adoptive transfer of T cells in vivo in a state-of-the-art tumor mouse model (B16). Further, Klepsch et al. (“Nucelar receptor NR2F6 inhibition potentiates responses to PD-L1/PD-1 cancer immune checkpoint blockade”, Nature Communications, 2018) evaluates the role of NR2F6 as intracellular immune checkpoint. It was shown that inhibition of NR2F6 and PD-L1 showed a synergism in anti-tumor treatment. Klepsch et al. (“Targeting the orphan nuclear receptor NR2F6 in T cells primes tumors for immune checkpoint therapy”, Cell communication and Signaling, 2020) discloses that NR2F6 gene ablation in primary mouse T cells potentiates established PD-L1 and CTLA-4-blockade anticancer therapies. However, no modification or effect of the inhibition of NR2F6 on CAR-T cells targeting an antigenically heterogeneous solid tumor, is disclosed in these documents.

Regen BioPharma have disclosed that inhibiting NR2F6 in CAR-T cells reduces T cell exhaustion (Regen Biopharma Inc., “Regen Biopharma Inc. Beginning Experiments Validating Its Proprietary CAR-T cell Therapy”, 2022; Regen BioPharma Inc., “Regen BioPharma, Inc., Progresses Its DuraCar Therapeutic”, 2022). This is shown for anti-CD19 CAR-T cells, directed against B cell associated cancers such as B-cell lymphomas. Further approaches are disclosed in US 2017/0304418 and US 2021/0317180. However, there is no suggestion in these documents that this approach overcomes the hurdles of solid tumor therapy by CAR-T cells. In particular, there is no suggestion of an effective secondary and polyclonal immune response in the treatment of an antigen heterogenous solid tumor.

Most of these studies are still in early clinical trial testing without curative solutions or a clear market leader. The lack of understanding of the exact tumor rejection mechanisms triggered by CAR-T within the endogenous T cell compartment of the patient's body is hampering the development of these therapies. This is demonstrated by the fact that none of the CAR-T approaches are effective or show a durable response against tumors with a high antigen heterogeneity such as most solid tumors.

In light of the disadvantages outlined above and the inherent difficulties in developing cellular therapeutics that show high avidity and cytotoxic activity towards malignant cells in solid tumors, the field is in urgent need of novel means for improving the activity and ultimately efficacy of cellular therapies such as CAR-T cells, in order to overcome difficulties in solid tumor treatment due to CAR-T cell exhaustion, metabolic dysfunction and, subsequently, functional inactivity as well as solid tumor antigen heterogeneity.

SUMMARY OF THE INVENTION

In light of the prior art the technical problem underlying the invention was the provision of alternative or improved means for enhancing the cytolytic activity of therapeutic immune cells against solid tumors, in particular for immune cells with antigen-specific targeting constructs that direct the immune cell to particular tumor targets. A further object of the invention was the provision of alternative or improved means for reducing the sensitivity of therapeutic immune cells to exhaustion and inactivation by the microenvironment of a solid tumor.

A further object of the invention was to provide means for improving immune cell therapies such as CAR-T or TCR T cell therapies for the treatment of solid tumors.

Another object of the invention was the provision of immune cell therapies for the treatment of solid tumors that can be produced in a time- and cost-efficient and simple manner.

Another object of the invention was the provision of immune cell therapies for the treatment of solid tumors that show low systemic cytotoxicity and immune-related side effects in a subject.

A further object of the invention was the provision of immune cell therapies for the treatment of solid tumors that result in a durable immune response and do not require repeated administration of said cell therapy in a subject.

These problems are solved by the features of the independent claims. Preferred embodiments of the present invention are provided in the dependent claims.

In one aspect, the present invention relates to a modified immune cell for use in the treatment of a solid tumor in a subject, wherein the modified immune cell comprises one or more exogenous nucleic acid molecules encoding a transgenic construct targeting an antigen expressed in a cancerous cell of said solid tumor, in said immune cell, Nuclear Receptor Subfamily 2 Group F Member 6 (NR2F6) activity is inhibited (in comparison to a control immune cell), and binding of the immune cell to the antigen is associated with death of said cancerous cell expressing the antigen, and inducing a secondary immune reaction in the subject against cancerous cells of the solid tumor, wherein said secondary immune reaction is nonspecific to the antigen targeted by the transgenic construct (epitope spreading).

The modified immune cells of the present invention are surprisingly effective and specific in the treatment of solid tumors. Thereby, the inventors have discovered that the high efficiency and specificity of the inventive immune cells (for example NR2F6-modified CAR-T cells) in comparison to the immune cell therapies of the prior art is mainly related to (1) reduced sensitivity to exhaustion and metabolic inactivation by chronic tumor antigen stimulation and the tumor immune micro environment (TIME) resulting in efficient killing of tumor cells expressing the antigen targeted by the transgenic construct, e.g., a CAR, and (2) the subsequent induction of a secondary, polyclonal and persistent immune reaction due to the initial killing of the tumor cells expressing the targeted antigen. The induction of this secondary immune reaction advantageously results in efficient killing and eradication particularly of heterogenous tumors comprising tumor cells not expressing the antigen targeted by the transgenic construct, which are usually not susceptible to immune cell therapy, thereby allowing a tumor relapse-free survival of the patients.

In contrast to the therapeutic immune cells of the prior art, the inhibition of NR2F6 maintains a robust tumor cell-killing activity of the inventive immune cells albeit the harsh tumor microenvironment (TIME) of the solid tumor and chronic tumor antigen stimulation, usually exhausting and inactivating immune cells of the prior art such as CAR-T cells. The inhibition of NR2F6 hinders exhaustion-mediated immune cell dysfunction and maintains the long-lasting cytotoxic effector function of the inventive immune cells. This allows efficient killing and induction of the secondary immune reaction. Further, NR2F6 is a very localised and inducible "exhaustion factor" selectively activated in immune cells within the tumor microenvironment (TIME), thus, the cytotoxic activity of the immune cells of the present invention is advantageously restricted to the tumor site, reducing the chance of immune related adverse effects. The inventive immune cells, in which NR2F6 is inhibited, activate a tumor-focused immune response that allows recognition of a plethora of tumor antigens of the given solid cancer type. Thereby, self-antigens which are also expressed in healthy cells, do not trigger a robust and durable activation of the patient's own immune system. The secondary immune reaction is thus specific to the solid tumor of the patient, advantageously resulting in reduced immune related adverse events (irAE).

These beneficial effects are also demonstrated in the examples below. As shown in Fig.8, only in immunocompetent wildtype recipient mice (but not in a Rag1 knockout mouse line that completely lacks an endogenous adaptive immune system) inhibition of e.g. NR2F6 in CAR-T cells lead to superior anti-tumor immunity against EpCAM-antigen heterogeneous PanC-02 tumor loads. The data presented in Fig.8 clearly demonstrate the requirement of the endogenous immune system for the observed tumor growth advantage based on epitope spreading. Only epitope spreading leads to a durable secondary immune response by the endogenous adaptive immune system. Further, as demonstrated in Fig. 10 complete NR2F6-modified CAR-T responders have developed a secondary and polyclonal anti-tumor memory for tumor control that can be transferred to EpCAM-negative WT tumor recipients. These results provide proof of concept for a long-term immunological memory of the endogenous T cell compartment induced secondary to the NR2F6-modified CAR-T therapy of the invention for host-protective tumor control of EpCAM- negative tumors (in a tumor antigen-agnostic effect).

There is no mention in the prior art of an effective and secondary and polyclonal immune response by the patient's own immune system that can be generated by NR2F6 gene-edited CAR-T treatment. However, this tumor epitope spreading is the key mechanistic basis for a durable immune memory response promoted by the proposed NR2F6-modified CAR-T treatment of an antigenically heterogeneous solid tumor. This is the underlying mechanism for the present invention. The prior art such as Klepsch et al. or the documents by RegenBio Pharma present no evidence to suggest that an effective secondary and polyclonal immune response by the endogenous T cell compartment is induced by NR2F6 gene-edited CAR-T treatment of antigenically heterogeneous solid tumors. This is however, is the key mechanistic basis of a durable immune memory response promoted by the NR2F6-modified CAR-T treatment regimens of a priori antigenically heterogeneous solid tumors

Further advantages to the present invention include the finding that the inventive immune cells enable the treatment of highly heterogenous solid tumors such as NSCLC. Solid tumors are often antigenically diverse and cannot be effectively eliminated by immune cell therapies of the prior art, e.g., CAR-T cells designed to target one or even two tumor surface antigens. One of the most challenging design aspects for solid tumor immunotherapies is thus to provide a specific therapy that targets the tumor cells but overcomes antigen loss or heterogeneity of antigens within the tumor. The modified immune cells of the present invention harnessing the TIME-inducible NR2F6 immune checkpoint surprisingly ensure tumor-specificity while inducing a secondary immune reaction resulting therein that tumor cells not expressing the antigen targeted by the immune cells of the present invention become more easily visible to the patient’s immune system by cross- priming (epitope spreading). The responsiveness of solid tumor patients to CAR-T cell therapies of the prior art is usually low and highly variable due to intrinsic and/or acquired tumor resistance to current CAR-T regimens. In contrast, the self-perpetuation and expansion of a systemic secondary tumor immunity triggered by the immune cells of the present invention make the majority of solid tumor patients receptive to immune cell therapy. Thus, this invention advantageously transforms immune cell therapy such as CAR-T and TCR-therapy for solid tumors from non-curative to curative by providing a gene-editing plug-in technology that targets the NR2F6 immune checkpoint in the immune cells.

Unlike immune cell therapies of the prior art, the immune cells of the present invention effectively kill cancer cells carrying the target antigen in the immune microenvironment (TIME) of the solid tumor due to their reduced sensitivity to exhaustion and metabolic inactivation within the TIME. Dying cancer cells now expose an abundance of tumor cell antigens for cross-priming (epitope spreading) so that the body's T cells recognize a broad variety of non-self tumor antigens (both on the surface and intracellularly expressed in the patient's tumor cells) and subsequently kill all tumor cells, including those that do not (or no longer due to acquired resistance by immune evasion) express the antigen targeted by the inventive immune cells. Therefore, the modified immune cells of the present invention surprisingly provide a "tumor antigen-agnostic immune cell therapy" that results in depletion of all cancerous cells of the solid tumor, including those that do not express the antigen targeted by the transgenic construct, e.g., a CAR. Due to the initiation of a generation of long-lasting secondary rather than temporary/episodic immunoreactivity against solid tumors by such inventive cell therapies further the need for repeated treatments is reduced, resulting in lower costs and less burden for the patients.

Further, in contrast to immune cell therapies of the prior art the immune cells of the present invention surprisingly do not require leukocyte depletion prior to administration to a subject, as the endogenous immune system is substantially contributing to the secondary, durable immune reaction. Thus, the inventive approach further reduces patient suffering due to additional treatments prior to immune cell therapy and treatment costs.

The present invention can advantageously be applied to any state-of-the-art immune cell therapy, such as CAR-T cell therapy. The inventive concept of NR2F6 inhibition in modified immune cells can e.g., be applied to any state-of-the-art CAR for the given cancer entity as a plug-in technology. This advantageously allows for the personalisation of CAR-T cell therapies by complementing current strategies (e.g., targeting tumor neoantigens). For therapy personalisation a patient's biopsy can be used to identify the neoantigen expressed in the solid tumor of the patient. Subsequently, CAR-T cells expressing the chosen neoantigen-specific CAR can be generated from autologous T cells in which NR2F6 is inhibited, e.g., by gene silencing or an inhibitor of NR2F6, e.g., a small molecule inhibitor. This concept thus improves the therapeutic efficiency of existing and emerging CAR-T cell therapies in the treatment of solid tumors.

In addition, the inventive concept is surprisingly also suited for application to the emerging off-the- shelf allogenic CAR-T cell therapy. T cells for allogenic CAR-T cell therapies are collected from healthy donors and can be stably engineered in advance to be ready for use in patients. Herein, following biopsy, the allogenic CAR-expressing the best neoantigen for the patient is selected. Within the cells expressing this CAR NR2F6 is inhibited prior to administration to the patient or by simultaneous or sequential administration of the cells and an inhibitor of NR2F6 to the patient. The inventive concept can thus be easily and broadly applied to existing and emerging immune cell therapies and substantially improves the efficiency of such therapies. The present invention is therefore defined by a combination of features that represent a novel approach over earlier technologies of the prior art. For example, in embodiments, the present invention comprises (a) administering a modified immune cell in which NR2F6 activity is inhibited, disrupted or removed, in combination with (b) inducing a secondary non-antigen-specific immune reaction against cancerous cells (epitope spreading). The present invention is therefore defined by one or more novel technical or medical effects, that were not previously evident in or derivable from the disclosures of the prior art. Importantly, the identification of this novel medical effect, in particular the relationship between (a) the modified immune cell in which NR2F6 activity is inhibited, in combination with (b) the secondary non-antigen-specific immune reaction against cancerous cells (epitope spreading), also enables its practical implementation in treating additional patients, previously thought not to be effectively treatable, and enhancing the efficacy of therapeutic cell products, thus positively influencing the treatment, dosage and administration of the therapeutic cells. Further, the invention advantageously transforms immune cell therapy such as CAR-T and TCR-therapy for solid tumors from non-curative to curative by providing a gene-editing plug-in technology that targets the NR2F6 immune checkpoint in the immune cells.

The identification of this previously unknown and beneficial technical effect, of inducing or enhancing epitope spreading by employing a cytotoxic immune cell in which NR2F6 activity is inhibited, leads to a novel clinical situation. Through the identification of this novel effect, patient groups not previously treatable (such as those with solid tumors and/or tumors with diverse cancer antigen heterogeneity) may now be effectively treated. Additionally, due to the increase in efficacy expected through the present approach, reduced numbers of therapeutic cells are expected to be necessary to induce the desired medical effects. Thus, the identification of the novel mechanism of epitope spreading in the context of e.g., CAR-expressing immune cells, and enhancement of this effect using NR2F6 inhibition in said immune cells, represents a novel combination of features, sufficient to distinguish the invention from the prior art. A novel clinical situation is created with direct implications for the treatment of novel patient groups and/or for administration regimes.

In one embodiment, the secondary immune reaction is directed to one or more antigens expressed by the cancerous cell.

In one embodiment the one or more antigens expressed by the cancerous cell are non-self-tumor antigens.

In one embodiment the one or more antigens expressed by the cancerous cell are expressed intracellularly and/or extracellularly.

In one embodiment the secondary immune reaction is a T cell mediated immune reaction, preferably a polyclonal T cell mediated immune reaction.

In one embodiment the inhibition of NR2F6 is associated with a resistance and/or a reduced sensitivity of said cell to inhibition of cytolytic activity by the tumor immune microenvironment (TIME) of the solid tumor.

In one embodiment, the immune cells modified with a transgenic antigen targeting construct in which NR2F6 activity is inhibited, are characterized by an increase in cytolytic activity of 50% or more, preferably 65% or more, more preferably 80% or more, In one embodiment, the immune cells modified with a transgenic antigen targeting construct in which NR2F6 activity is inhibited, are characterized by an increase in cytolytic activity of 50% or more, such as 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100%, 150%, 200%, or 300% or more, within the tumor microenvironment (TIME), when compared to a control immune cell without NR2F6 inhibition.

Functional assays for determining quantitatively, or semi-quantitatively the increase in cytolytic activity in the immune cells in which NR2F6 and optionally CBLB is inhibited, are available to a skilled person, some examples of which are described herein. For example, the in vitro assays described in the examples, may be applied in order to determine an increase in cytolytic activity over control immune cells, i.e., immune cells in which NR2F6 and optionally CBLB is not inhibited.

In one embodiment the inhibition of NR2F6 is associated with a resistance and/or reduced sensitivity of said cell to exhaustion by chronic tumor antigen stimulation within the tumor immune microenvironment (TIME).

In one embodiment, the immune cells modified with a transgenic antigen targeting construct in which NR2F6 activity is inhibited, are characterized by an increase in metabolic activity of 50% or more, preferably 75% or more, more preferably 100% or more. In one embodiment, the immune cells modified with a transgenic antigen targeting construct in which NR2F6 activity is inhibited, are characterized by an increase in metabolic activity of 50% or more, such as 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100%, 150%, 200%, or 300% or more, when compared to a control immune cell without NR2F6 inhibition.

Functional assays for determining quantitatively, or semi-quantitatively the increase in metabolic activity in the immune cells in which NR2F6 and optionally CBLB is inhibited, are available to a skilled person, some examples of which are described herein. For example, the in vitro assays described in the examples, may be applied in order to determine an increase in metabolic activity over control immune cells, i.e., immune cells in which NR2F6 and optionally CBLB is not inhibited.

In one embodiment in said cell additionally Casitas B-lineage lymphoma proto-oncogene-b (CBLB) activity is inhibited (compared to a control immune cell).

Surprisingly the simultaneous inhibition of NR2F6 and CBLB in the immune cells of the present invention results in a synergistic effect on the effector activity of said immune cells in the treatment of a solid tumor and further synergistically reduces the sensitivity of said cells to exhaustion and inactivation by chronic tumor antigen stimulation and the tumor microenvironment (TIME). Without being bound to theory this synergistic effect can be attributed to NR2F6 inhibition resulting in a metabolic shift of said immune cells in the TIME and CBLB resulting in resistance of said cells to exhaustion by the TIME, in particular resistance to TGFbeta. Such a synergistic effect of inhibiting NR2F6 and CBLB would not have been expected by a skilled person in light of the prior art.

In one embodiment the transgenic construct is expressed transiently in said immune cell.

The inventors surprisingly discovered that the immune cells of the present invention can be modified to express a transgenic construct (e.g., a CAR or a TCR) transiently, by using mRNA encoding the CAR or TCR. Thereby, such transiently transfected cells surprisingly maintain their efficiency and specificity in the treatment of a solid tumor when compared to immune cells permanently expressing the transgenic construct. This can be attributed to the immune cells of the present invention inducing a secondary reaction of the patient’s immune system, which is persistent and independent of the antigen targeted by the modified immune cells. In contrast to permanent expression, transient expression of the transgenic construct in said inventive immune cells does not require the use of specialized and time-consuming gene editing techniques. The manufacturing of transiently modified immune cells thus does not require highly trained personnel and specialized GMP facilities and is further less time consuming. The immune cells according to the present invention can thus be produced in a more efficient, cost-effective and faster manner (e.g., shortening vein-to-vein times (the time between collection of T cells to CAR-T infusion) from 2/3 weeks to 1 day), making cell-based therapies more available for solid cancer patients, and avoiding the need for bridging therapies to compensate the waiting time until the immune cells for therapy are prepared.

In one embodiment the transgenic construct is a T cell receptor (TCR).

In one embodiment the transgenic construct is a chimeric antigen receptor (CAR).

The particular type or form of antigen-targeting construct is not intended as a limiting feature of the invention. The inventive concept is based on an immune cell-intrinsic NR2F6- and optionally CBLB-in hibition mediated increase in their cytolytic activity and decrease in their exhaustion sensitivity by the TIME of a solid tumor. The enhanced activity of the inventive modified immune cells is therefore not dependent on the antigen-targeting construct, which is merely considered as a means for bringing the modified immune cell into proximity with the tumor cell.

Tumor associated antigens targeted via the transgenic construct, e.g., CAR or TCR construct, may be, without limitation, selected from Glypican-3 (GPC3), human epidermal growth factor receptor 2 (HER2), tumor-associated ganglioside GD2 (GD2), epidermal growth factor receptor (EGFR), EGFR variant III (EGFR vl 11), oncofetal chondroitin sufate (ofCS), EGFR806, carcinoembryonic antigen (CEA), prostate-specific membrane antigen (PSMA), folate receptor alpha (FRa), epithelial cell adhesion molecule (EPCAM), mucine 1 (MUC1), receptor tyrosine kinase-like orphan receptor 1 (ROR1), MUCI16eto, vascular endothelial growth factor receptor 2 (VEGFR2), neural cell adhesion molecule L1 (CD171), prostate stem cell antigen (PSCA), erythropoietin-producing hepatocellular carcinoma A2 (EphA2), fibroblast activation protein (FAP), carbonic anhydrase 9 (CAIX), hepatocyte growth factor receptor (c-MET), neural cell adhesion molecule L1 (L1-CAM), Mesothelin (MSLN), programmed Cell Death 1 Ligand 1 (PD- L1), Wilms-tumor-protein (WT1), New York esophageal squamous cell carcinoma-1 (NY-ESO-1), melanoma-associated antigen-encoding gene A1 (MAGE-A1), melanoma-associated antigenencoding gene A1 (MAGE-A2), melanoma-associated antigen-encoding gene A1 (MAGE-A4), Claudin 18.2, Alpha-Fetoprotein (AFP), Nectin4/FAP, Lewis Y, MUC16, AXL receptor tyrosine kinase (AXL), CD20, CD80/86, Delta-like ligand 3 (DLL-3), death receptor 5 (DR5), glycoprotein 100 (gp100), latent membrane protein (LMP-1), killer cell lectin-like receptor K1 (NKG2D), guanylate cyclase-C (GUCY2C), tumor associated glycoprotein-72 (TA-72), CD46, Anthrax toxin receptor 1 (ANTXR1), mucin 3A (MUC3A), trophoblast cell-surface antigen 2 (Trop2), Integrin avp6 (avp6), CD47, chondroitin sulfate proteoglycan 4 (GSPG4), Glypican 2 (GP 2), B7 Homolog 3 (B7-H3 also termed CD276), prostate specific antigen (PSA), anti-Prostatic Acid Phosphatase (PAP), CD32A, C133 and Interleukin 13 receptor alpha 2 (IL13Ra2).

A detailed summary of the published clinical trials of chimeric antigen receptor T cells (CAR-T) and TCR-transduced T cells (TCR-T) is disclosed in Mo et al (Journal of Cancer, 2017; 8(9): 1690-1703), Hartmann et al. (EMBO Molecular Medicine, 2017; 9 (9): 1183-1197), Townsend et al. (Journal of Experimental & Clinical Cancer Research, 2018, 37:163), Marofi et al. (Stem Cell Research & Therapy, 2021 , 12, 81), Sorkhabi et al. (Frontiers in Immunology, 2023, 14, 1113882) and Drougkas et al. (Journal of Cancer Research and Clinical Oncology, 2023, 149(6), 2709- 2734).

In embodiments, combined approaches, based on antigen targeting constructs directed to two or more of the above antigens, may also be employed, for example by using one or more targeting constructs targeting for example mesothelin (MSLN), Epidermal growth factor receptor variant III (EGFRvlll), B7 homolog 3 protein (B7-H3 also known as CD276), Mucin 1 (MUC1) and/or oncofetal chondroitin sulphate (ofCS).

Not only are tumor-type specific and diversified antigens expressed in one kind of cancer targeted, but also one antigen is expressed in multiple kinds of cancers. These antigens may also be targeted. For example, the NY-ESO-1 is highly expressed in melanoma, multiple myeloma, NSCLC, synovial sarcoma, breast cancer, renal cell cancer, hepatocellular cancer, esophageal cancer, ovarian cancer and bladder cancer. Similarly, mesothelin is highly expressed in mesothelioma and breast cancer, cervical cancer, pancreatic cancer, ovarian cancer, lung cancer and endometrial cancer.

In further embodiments, the transgenic constructs such as CAR constructs to be employed can be exchanged easily, therefore allowing a modular composition of clinically applicable CARs. The antigen-specificity of the CAR is variable and not limiting to the present invention.

The specific antigens described herein are of exemplary nature and represent preferred nonlimiting embodiments of the invention. The inventive concept of NR2F6 and optionally of CBLB inhibition can be applied to any given modified immune cell regardless of antigen specificity of the immune cell.

In one embodiment in said cell the activity of NR2F6 and optionally of CBLB is inhibited by at least 50% in comparison to a control immune cell, preferably by at least 60%, more preferably by at least 70% or a removal of NR2F6 and optionally of CBLB activity. In one embodiment in said cell the activity of NR2F6 and optionally of CBLB is inhibited by at least 50% (in comparison to a control immune cell), such as 50, 55, 60, 65, 70, 75, 80, 85, 90 or 100% (in comparison to a control immune cell).

By way of example, a control immune cell is the same type of cell as the cell in which said inhibition is present, although the measures taken to inhibit the activity of NR2F6 have not been carried out in the control cell. For example, in the context of a therapeutic CAR-T cell, a control cell may be considered as a T cell, or CAR-T cell, in which no measure has been taken to inhibit the activity of NR2F6, and the cell in which NR2F6 has been inhibited is compared to the control cell.

Functional assays for determining quantitatively or semi-quantitatively the inhibition of NR2F6 and optionally of CBLB in the immune cells are available to a skilled person, some examples of which are described herein. For example, the in vitro assays described in the examples, may be applied in order to determine the inhibition of NR2F6 and CBLB.

In one embodiment, inhibition of NR2F6 activity and optionally of CBLB activity is obtained by disrupting the expression and/or sequence of a NR2F6 gene and optionally a CBLB gene, preferably by CRISPR-Cas, zinc finger nucleases (ZFNs), integrases, site specific recombinases, meganucleases, homing endonucleases, or TALENs, more preferably by CRISPR/Cas9, prior to administration of said cells in a subject. In one embodiment, the inhibition of NR2F6 activity and optionally of CBLB activity is obtained by knock-down of NR2F6 and optionally of CBLB, preferably by RNA interference of NR2F6 expression and optionally of CBLB expression, such as by small interfering RNA (siRNA), short hairpin RNA (shRNA), micro-RNA (miRNA), morpholinos and/or antisense oligonucleotides (ASO).

RNA interference approaches for NR2F6 and optionally for CBLB inhibition (i.e. “silencing” or “knock-down”) are preferred for a number of reasons due to their inherent advantages in biological systems. By not interfering with the genome structure or integrity, the RNAi technology has an excellent safety profile compared to manipulated genomes.

A skilled person is capable of designing effective RNA targeting sequences based on the target sequence and common knowledge in the art. Software for such approaches is commonly available, with which a skilled person can design sequences used e.g., as siRNAs, shRNAs, miRNAs or ASOs to interfere with the expression of NR2F6 and optionally of CBLB. For example, the program BLOCK-iT RNAi from Thermo Fisher may be employed. Alternative software can also be identified and used.

In one embodiment, the inhibition of NR2F6 activity and optionally of CBLB activity is obtained by treatment of said cells with a NR2F6 antagonist and optionally a CBLB antagonist, such as a small molecule inhibitor of NR2F6 and optionally of CBLB.

A non-limiting example for a small molecule inhibitor of NR2F6 is TES-4207 developed by TESPharma, Peruggia/ltaly. Further non-limiting examples of small molecule inhibitors of NR2F6 are disclosed in WC2019/104199, WO2019/104201 and US2019/0358224. A non-limiting example for a small molecule inhibitor of CBLB is NX-1607 developed by Nurix/USA. Further nonlimiting examples of small molecule inhibitors of CBLB are disclosed in W02020/210508, WO2020/236654, WO2020/264398, WO2019/148005 and WO2022/272248,

In one embodiment, the inhibition of NR2F6 activity and optionally of CBLB activity is obtained prior to administration of said cells to a subject and/or by simultaneous or sequential administration of said cells and a NR2F6 antagonist and optionally a CBLB antagonist to a subject.

In one embodiment, the cell is a T cell, preferably a CD4+ or a CD8⁺ T cell.

In one embodiment, the cell is a cytotoxic T cell, preferably a CD4+ or a CD8⁺ T cell.

In one embodiment, the cell is a natural killer (NK) cell.

In one embodiment, the solid tumor is selected from the group consisting of glioblastoma, lung carcinoma, breast carcinoma, kidney carcinoma, pancreatic carcinoma, melanoma, intestinal carcinoma, ovarian carcinoma, prostate carcinoma and colon carcinoma.

In one aspect, the invention relates to a modified immune cell comprising one or more exogenous nucleic acid molecules encoding a transgenic construct targeting an antigen expressed in a cancerous cell of a solid tumor, wherein in said cell, NR2F6 activity is inhibited (compared to a control immune cell).

In one aspect, the invention relates to a modified immune cell comprising one or more exogenous nucleic acid molecules encoding a transgenic construct targeting an antigen expressed in a cancerous cell of a solid tumor, wherein in said cell, NR2F6 activity and Casitas B-lineage lymphoma proto-oncogene-b (CBLB) activity is inhibited (compared to a control immune cell).

In one aspect, the invention relates to a pharmaceutical composition comprising a modified immune cell according to present invention, suitable for the treatment of a solid tumor, comprising additionally a pharmaceutically acceptable carrier.

In one aspect, the invention relates to an in vitro method for enhancing the cytolytic activity of a modified immune cell, said immune cell comprising one or more exogenous nucleic acid molecules encoding a transgenic construct targeting an antigen expressed in a cancerous cell of a solid tumor, the method comprising inhibiting in said immune cell the activity Nuclear Receptor Subfamily 2 Group F Member 6 (NR2F6) and optionally the activity of Casitas B-lineage lymphoma proto-oncogene-b (CBLB), wherein inhibiting NR2F6 activity and optionally CBLB activity preferably comprises: a. genetic modification of the T cell genome by disrupting the expression and/or sequence of a NR2F6 gene and optionally a CBLB gene, preferably by CRISPR-Cas, zinc finger nucleases (ZFNs), integrases, site specific recombinases, meganucleases, homing endonucleases, or TALENs, b. knock-down of NR2F6 and optionally of CBLB, preferably by RNA interference of NR2F6 expression and optionally of CBLB expression, such as by small interfering RNA (siRNA), short hairpin RNA (shRNA), micro-RNA (miRNA), morpholinos and/or antisense oligonucleotides (ASO), or c. treatment of said cells with a NR2F6 antagonist and optionally a CBLB antagonist, such as a small molecule inhibitor of NR2F6 and optionally of CBLB.

All suitable methods for transferring the genetic information/nucleic acid molecule for the transgenic antigen targeting construct, e.g., a CAR or TCR, and NR2F6 and optionally CBLB inhibition into the cell are encompassed by the present invention, and a suitable method may be selected by a skilled person when carrying out the invention. For example, multiple methods of transfecting immune cells are known in the art, including any given viral-based gene transfer method, such as those based on modified Retroviridae, and non-viral methods such as lipid nanoparticles and cationic polymers, DNA-based transposons, episomal cDNA vectors and direct transfer of mRNA by electroporation or lipid nanoparticles.

All features described in the present specification may be employed to define any other embodiment or aspect of the invention, for example, features used to describe the immune cell may be used to describe the immune cell for use in the treatment of a solid tumor, the pharmaceutical composition, or the method for enhancing the cytolytic activity of a modified immune cell, and vice versa. Similarly, features used to describe the methods of the invention may be used to describe the cells or compositions, and vice versa.

DETAILED DESCRIPTION

As described in detail herein, the invention relates to modified immune cells, in which NR2F6 and optionally CBLB is inhibited, thereby enhancing the cytolytic function and reducing the sensitivity to exhaustion and inactivation of said cells by the microenvironment of a solid tumor. The enhancement of cytolytic function and reduction of exhaustion and inactivation leads to a higher efficiency and induction of a secondary immune reaction, resulting in a higher efficiency of said cells against tumors.

Genetically modified immune cells

The present invention contemplates, in particular embodiments, immune cells genetically modified to express an antigen-specific targeting construct, targeting an antigen expressed in a cancerous cell of a solid tumor, wherein in said immune cells NR2F6 is inhibited. These immune cells are intended for use in the treatment of a solid tumor, said treatment comprising the induction of a secondary immune response (epitope spreading).

As used herein, the term "genetically engineered" or "genetically modified" or “modified” refers to the addition of extra genetic material in the form of DNA or RNA into the total genetic material in a cell. The terms, "genetically modified cells", "modified cells", and "redirected cells" are used interchangeably. As used herein, the term "gene therapy" or “modification” refers to the introduction-permanently or transiently- of extra genetic material in the form of DNA or RNA into the total genetic material in a cell that restores, corrects, or modifies expression of a gene, or for the purpose of expressing a transgenic construct targeting an antigen expressed in a cancerous cell of a solid tumor, e.g., a CAR or TCR. The expression of said transgenic construct can be “transiently” or “stable”. Transient expression relates to the temporary expression of the construct by introducing an exogenous nucleic acid into the cell without integration of said nucleic acid into the genome of the cell. Stable expression refers to the long-term expression of the construct by introducing an exogenous nucleic acid into the cell, which is integrated into the genome of the cell.

An "immune cell" or "immune effector cell" is any cell of the immune system that has one or more effector functions (e.g., cytotoxic cell killing activity, secretion of cytokines, induction of antibodydependent cellular cytotoxicity (ADCC) and/or complement-depending cytotoxicity (CDC)). Immune cells, such as T cells and NK cells of the invention, can be autologous/autogeneic ("self) or non-autologous ("non-self," e.g., allogeneic, syngeneic or xenogeneic). "Autologous", as used herein, refers to cells from the same subject, and represent a preferred embodiment of the invention. "Allogeneic", as used herein, refers to cells of the same species that differ genetically to the cell in comparison. "Syngeneic", as used herein, refers to cells of a different subject that are genetically identical to the cell in comparison. "Xenogeneic", as used herein, refers to cells of a different species to the cell in comparison. In preferred embodiments, the cells of the invention are autologous or allogeneic.

A "T cell" also termed "T lymphocyte" is an immune cell belonging to the group of lymphocytes. A T cell can be a thymocyte, immature T lymphocyte, mature T lymphocyte, resting T lymphocyte, cytokine-induced killer cell (CIK cell), activated T lymphocyte or tumor infiltrating lymphocyte (TIL). T cells originate from the bone marrow and migrate via the blood stream to the thymus, where they generate T cell receptors (TCR) and undergo a positive and negative selection in which the cells that show high affinity to endogenous proteins are degraded. T cells may be a T helper (Th; CD4+ T cell, CD4 T cell) cell, for example a T helper (Th) cell, such as a TH1 , TH2, TH3, TH 17, TH9 or TFH cell. The T cell can be a cytotoxic T cell (CTL; CD8+ T cell, CD8 T cell) or a CD4+CD8+ T cell or any other subset of T cells such as a cytokine-induced killer (CIK) cell which is typically a CD3- and CD56-positive, non-major histocompatibility complex (MHC)- restricted, natural killer (NK)-like T lymphocyte. The T cell can be a naive, effector, memory, effector memory, central memory, memory stem T cell. The T cell can be an umbilical cord blood cell. The T cell can be a peripheral lymphocyte. A T cell can be derived and expanded from peripheral mononuclear blood cells (PBMC). The T cell may be autologous with respect to an individual to whom it is to be administered. The T cell may be allogeneic with respect to an individual to whom it is to be administered.

A cytotoxic T cell (also known as TC, cytotoxic T lymphocyte, CTL, T-killer cell, cytolytic T cell, T cell or killer T cell, and as may be used interchangeably herein) is a T cell (a type of white blood cell) that has cytolytic activity against e.g., cancer cells. In some embodiments, the cytolytic activity can be associated with a CD8+ and/or a CD4+ T cell. Both T cell subpopulations can generate and release lytic granule content, that is cytolytic enzymes such as granzymes. The term "cytolytic" or “cytolytic activity” refers to an immune cell such as a T cell's capacity to kill target cells, e.g., by the release of lytic granule content, the latter are also referred to as secretory lysosomes. The terms CD8 and CD8⁺ cell both refer to the same type of cell. The terms CD4 and CD4⁺ cell both refer to the same type of cell.

As a central element of the adaptive immune response, T cells are capable of eliminating infections and transformed tumor cells. CD8⁺ T cells can mature into cytotoxic T lymphocytes (CTLs) and are primarily involved in the destruction of infected or transformed cells by releasing cytolytic granules into the immunological synapse. These granules include perforin and granzymes that are released in the Ca²⁺-dependent regulated secretion pathway and induce apoptosis within the target cells. As soon as the CTL recognizes and binds its target cell, secretory lysosomes move and cluster around the microtubule organizing center. After membrane fusion, perforin and granzymes are released into the immunological synapse. Perforin is a poreforming molecule capable of membrane permeabilization that is important for the entry of granzymes into the target cell cytosol. Within the target cell, programmed cell death pathways are initiated by granzymes.

Natural killer cells, also known as NK cells or large granular lymphocytes (LGL), are a type of cytotoxic lymphocyte critical to the innate immune system that belong to the rapidly expanding family of known innate lymphoid cells (ILC) and represent 5-20% of all circulating lymphocytes in humans. The role of NK cells is analogous to that of cytotoxic T cells in the vertebrate adaptive immune response. NK cells provide rapid responses to virus-infected cell and other intracellular pathogens acting at around 3 days after infection, and respond to tumor formation. Typically, immune cells detect the major histocompatibility complex (MHC) presented on infected cell surfaces, triggering cytokine release, causing the death of the infected cell by lysis or apoptosis. NK cells are unique, however, as they have the ability to recognize and kill stressed cells in the absence of antibodies and MHC, allowing for a much faster immune reaction. NK cells can be identified by the presence of CD56 and the absence of CD3 (CD56+, CD3-).

The present invention provides methods for modifying immune cells which express a transgenic construct targeting an antigen expressed in a cancerous cell of a solid tumor described herein. In one embodiment, the method comprises transfecting or transducing immune cells isolated from an individual such that the immune cells express one or more antigen specific constructs (e.g, CAR or TCR) as described herein. In certain embodiments, the immune cells are isolated from an individual and genetically modified without further manipulation in vitro. Such cells can then be directly re-administered into the individual. In further embodiments, the immune cells are first activated and stimulated to proliferate in vitro prior to being genetically modified to express a transgenic construct such as a CAR or TCR, potentially together with an inhibitor of NR2F6 and optionally of CBLB such as a small molecule inhibitor, a CRISPR-Cas system or an RNAi system. In this regard, the immune cells may be cultured before and/or after being genetically modified.

In particular embodiments, prior to modification of the immune effector cells described herein, the source of cells is obtained from a subject. In particular embodiments, the modified immune effector cells comprise T cells. T cells can be obtained from a number of sources including, but not limited to, peripheral blood mononuclear cells, bone marrow, lymph nodes tissue, cord blood, thymus issue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In certain embodiments, T cells can be obtained from a unit of blood collected from a subject using any number of techniques known to the skilled person, such as sedimentation, e.g., FICOLL™ separation, antibody-conjugated bead-based methods such as MACS™ separation (Miltenyi). In one embodiment, cells from the circulating blood of an individual are obtained by apheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocyte, B cells, other nucleated white blood cells, red blood cells, and platelets. In one embodiment, the cells collected by apheresis may be washed to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing. The cells can be washed with PBS or with another suitable solution that lacks calcium, magnesium, and most, if not all other, divalent cations. As would be appreciated by those of ordinary skill in the art, a washing step may be accomplished by methods known to those in the art, such as by using a semiautomated flow through centrifuge. For example, the Cobe 2991 cell processor, the Baxter CytoMate, or the like. After washing, the cells may be resuspended in a variety of biocompatible buffers or other saline solution with or without buffer. In certain embodiments, the undesirable components of the apheresis sample may be removed in the cell directly resuspended culture media.

In certain embodiments, T cells are isolated from peripheral blood mononuclear cells (PBMC) by lysing the red blood cells and depleting the monocytes, for example, by centrifugation through a PERCOLL™ gradient. A specific subpopulation of T cells can be further isolated by positive or negative selection techniques. One method for use herein is cell sorting and/or selection via negative magnetic immune-adherence or flow cytometry that uses a cocktail of monoclonal antibodies directed to cell surface markers present on the cells negatively selected.

PBMC may be directly genetically modified to express a transgenic construct targeting an antigen expressed in a tumor cell of a solid tumor using methods contemplated herein. In certain embodiments, after isolation of PBMC, T lymphocytes are further isolated and in certain embodiments, both cytotoxic and helper T lymphocytes can be sorted into naive, memory, and effector T cell subpopulations either before or after genetic modification and/or expansion. CD8⁺ cells can be obtained by using standard methods. In some embodiments, CD8⁺ cells are further sorted into naive, central memory, and effector cells by identifying cell surface antigens that are associated with each of those types of CD8⁺ cells.

In some embodiments, the immune cell of the present invention, for example the T cells described herein, can be obtained from inducible pluripotent stem cells (iPSCs) using methods known to a skilled person.

Accepted approaches for producing modified cells, e.g., expressing a CAR, rely on the genetic modification and expansion of mature circulating T cells. Such processes utilize autologous T cells and reduce risk of graft-versus- host (GvHD) disease from allogeneic T cells through endogenous TCR expression as well as rejection through MHC incompatibility. As an alternative, direct in vitro differentiation of engineered T cells from pluripotent stem cells, such as inducible pluripotent stem cells, provides an essentially unlimited source of cells that can be genetically modified to express the CAR of the present invention. In some embodiments, a so-called master iPSC line can be maintained, which represents a renewable source for consistently and repeatedly manufacturing homogeneous cell products. In some embodiments, the transformation of a master iPSC cell line with the CAR encoding nucleic acid is contemplated, prior to expansion and differentiation to the desired immune cell, preferably T cell or NK cell. T lymphocytes can for example be generated from iPSCs, such that iPSCs could be modified with the transgenic construct encoding nucleic acids and subsequently expanded and differentiated to T cells for administration to the patient. Differentiation to the appropriate immune cell, such a T cell, could also be conducted from the iPSCs before transformation with the transgenic construct encoding nucleic acids and expansion prior to administration. All possible combinations of iPSC expansion, genetic modification and expansion to provide suitable numbers of cells for administration are contemplated in the invention.

The immune effector cells, such as T cells, NK cells or CIK cells, can be genetically modified following isolation using known methods, or the immune effector cells can be activated and expanded (or differentiated in the case of progenitors) in vitro prior to being genetically modified.

The T cells can be genetically modified following isolation using known methods, or the T cells can be activated and expanded (or differentiated in the case of progenitors) in vitro prior to being genetically modified. In a particular embodiment, the T cells are genetically modified with the transgenic construct contemplated herein (e.g., transduced with a viral vector comprising a nucleic acid encoding a CAR) and then are activated and expanded in vitro. In various embodiments, T cells can be activated and expanded before or after genetic modification to express a transgenic construct, using methods as described, for example, in U.S. Patents 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681 ; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041 ; and U.S. Patent Application Publication No. 2006/0121005. In a further embodiment, a mixture of, e.g., one, two, three, four, five or more, different expression vectors can be used in genetically modifying a donor population of T cells wherein each vector encodes a different antigen targeting construct.

In one embodiment, the invention provides a method of storing genetically modified immune cells which exhibit NR2F6 and optionally CBLB inhibition, comprising cryopreserving the immune cells such that the cells remain viable upon thawing. A fraction of the immune effector cells can be cryopreserved by methods known in the art to provide a permanent source of such cells for the future treatment of patients afflicted with the condition to be treated. When needed, the cryopreserved cells can be thawed, grown and expanded for more such cells.

NR2F6 and its role in the exhaustion of cells

Nuclear Receptor Subfamily 2 Group F Member 6 (also termed NR2F6, Ear2 or V-erbA-related protein 2) is an intracellularly expressed nuclear receptor that has been characterized as an intracellular immune checkpoint in immune cells such as effector T cells, potentially controlling tumor development and growth. Mechanistically, NR2F6 acts as a negative-regulatory signaling intermediate "downstream" of the antigen receptor and determines the threshold of TCR/CD28 activation-induced effector functions by acting as a transcriptional repressor that antagonizes the DNA accessibility of activation-induced NFAT/AP-1 transcription factors at cytokine gene loci. Casitas B-lineage lymphoma proto-oncogene-b (CBLB) is an ubiquitin ligase that is an intracellular checkpoint in the negative regulation of T cell activation. CBLB expression in T cells causes ligand-induced T cell receptor down-modulation, controlling the activation degree of T cells during antigen presentation.

The tumor immune microenvironment (TIME) is the environment within tumor, including the tumor cells, surrounding blood vessels, immune cells, fibroblasts, signaling molecules and the extracellular matrix (ECM) of the tumor. Tumors can influence the microenvironment by releasing extracellular signals, promoting tumor angiogenesis and inducing peripheral immune tolerance, while the immune cells in the microenvironment can affect the growth and evolution of cancerous cells. Immune cells infiltrate into the tumor microenvironment, interact with each other and tumor cells, and then harbor an immunosuppressive phenotype that is responsible for the immune escape of tumor cells and the following tumor progression. These immunosuppressive cells include MDSCs, M2-macrophages, Tregs, N2-TANs, mast cells, Bregs, dendritic cells. They secrete cytokines like IL-2, IL-10, and TGF-p, growth factors like VEGF, the checkpoints ligands like PD-L1 or express checkpoints on the cell surface like PD-1 , TIM-3 on Tregs, that negatively regulate the anti-tumor immune response, remodel the extracellular matrix, and promote the angiogenesis. As a result, these immunosuppressive cells and their interaction generate an immunosuppressive microenvironment (TIME) and promote the proliferation, evasion, and migration of tumor cells. Further, chronic stimulation (also termed persistent stimulation) of effector immune cells such as CD8+ T cells by tumor antigens present within the TIME results in exhaustion of these cells. The exhausted immune cells within the TIME are characterized by increased expression of multiple co-inhibitory receptors such as NR2F6, loss of effector function, poor proliferation and self-renewal capacity, and dysregulated metabolic activity. In embodiments the modified immune cells of the present invention, in which NR2F6 and optionally CBLB is inhibited show a resistance and/or reduced sensitivity to exhaustion and/or inhibition by the TIME. These cells thus do not or not completely lose their effector function against the solid tumor, such as their cytolytic activity.

The term “secondary immune reaction” refers to an immune reaction that is induced by immunological cell death (ICD), e.g., by killing of a tumor cell by the immune cells of the present invention. During the death of cancer cells by ICD, the release of DAMPs ("damage-associated molecular patterns", molecular structures that occur during cell damage), chemokines and cytokines is induced in connection with the processing, presentation and release of intra- and extracellular tumor antigens of the killed cancer cell, which attract antigen-presenting cells such as dendritic cells (DCs). After phagocytosis by the DCs (termed effectorcytosis), they migrate to the lymph nodes where they present the processed tumor antigens to NK and T lymphocytes. Combined with the effect of co-stimulatory factors, the lymphocytes are prepared and activated in the process of cross-presentation. The now active and tumor antigen-specific T cells in turn proliferate and migrate to the tumor, where they recognize the tumor cells by the antigen epitopes and induce their killing. T umor-specific effector CD8⁺ T cells are thus recruited into the tumor microenvironment to maintain a T cell mediated and polyclonal immune response directed to a multitude of cancer antigens (epitope spreading).

As used herein, the term "epitope spreading" has its ordinary meaning in the art. For example, epitope spreading is the development of immune responses to endogenous epitopes secondary to the release of self-antigens during an immune response. For example, epitope spreading is the development of a secondary immune response, i.e. to tumor epitopes, based on the release of non-self tumor antigens during immunogenic cell death (ICD) of tumor cells, promoted by a first effective anti-tumor immune response. In other words, the term epitope spreading refers to the broadening and diversification of tumor epitope specificity following an initial focused, epitopespecific immune response (i.e. , here, the killing of a tumor cell by the tumor antigen-specific immune cells using, for example, therapeutic adoptive transfer of tumor antigen-specific CAR- or TCR-encoding T cells as in the present invention).

For example, after the primary antigen-specific immune response (triggered by adoptive transfer of tumor antigen-specific CAR- or TCR-encoding T cells), the secondary immune response, preferably based on epitope spreading, involves an extension of the immune response to other antigens released by the primary immune response that are unique to tumour cells and not expressed, or at least much less expressed or expressed at negligible levels, on normal cells. For example, such tumour antigens presented to the endogenous immune system include tumour neoantigens and tumour-associated antigens generated and/or upregulated by tumour cells as a result of various tumour-specific alterations.

For example, after the primary tumor antigen-specific immune response such as by CAR-T that promotes ICD of tumor cells, additional tumor antigens (advantageously now expressed either on the surface or intracellularly in tumor cells) are exposed to the endogenous immune system, preferably involving epitope spreading to promote endogenous T cell activation. This polyclonal immune response involves the extension of the immune response to non-self tumor epitopes in a tumor antigen-agnostic secondary memory immune response against multiple tumor antigens. Essentially, to the best knowledge of the inventors within the present invention it is the first time that an adoptive immune cell therapy such as i.e. CAR-T (designed to target only a few and strictly defined tumor surface antigens) is able to overcome the limitation of the a priori heterogeneity of tumor antigens and thus promote the rejection of i.e. CAR-targeted antigennegative tumors, which are frequently found in antigen-heterogeneous solid tumors such as e.g. in NSCLC.

The principle of this secondary immune reaction induced by ICD is disclosed for example in WO 2014/011993. Methods for determining a secondary immune reaction induced by ICD, such as the modified immune cells of the present invention, can be derived from this document and are further disclosed in the figures and examples below (see Fig. 7-12). These examples clearly demonstrate in a mouse model that only in an immunocompetent mouse model, the inhibition of NR2F6 in the immune cells of the present invention leads to the induction of a secondary immune reaction against the solid tumor (see Fig. 8).

Antigen-targeting construct:

As used herein, the term “antigen targeting construct”, “targeting construct” or “transgenic construct targeting an antigen” refers to a transgenic molecule (encoded by an exogenous nucleic acid molecule) capable of directing an immune cell such as a T cell to a particular antigen, or group of antigens such as an antigen expressed in a cancerous cell of a solid tumor.

An antigen target construct is therefore preferably a chimeric antigen receptor (CAR) or a T cell receptor (TCR). Engineered immune cells have emerged as a new stage in precision cancer therapy, through forced expression of these antigen targeting molecules on preferably autologous or donor immune cells, they result in specifically recognizing tumor antigens and enhance their therapeutic specificity and efficacy.

Chimeric Antigen Receptors: According to the present invention, a chimeric antigen receptor polypeptide (CAR), comprises an extracellular antigen-binding domain, comprising an antibody or antibody fragment that binds a target antigen, a transmembrane domain, and an intracellular domain. CARs are typically described as comprising an extracellular ectodomain (antigen-binding domain) derived from an antibody and an endodomain comprising signaling modules derived from T cell signaling proteins.

In a preferred embodiment, the ectodomain preferably comprises variable regions from the heavy and light chains of an immunoglobulin configured as a single-chain variable fragment (scFv). The scFv is preferably attached to a hinge region that provides flexibility and transduces signals through an anchoring transmembrane moiety to an intracellular signaling domain. The transmembrane domains originate preferably from either CD8a or CD28. In the first generation of CARs the signaling domain consists of the CD3 zeta chain of the TCR complex. The term “generation” refers to the structure of the intracellular signaling domains. Second generation CARs are equipped with a single costimulatory domain originated from CD28 or 4-1 BB. Third generation CARs already include two costimulatory domains, e.g. CD28, 4-1 BB, ICOS or 0X40, CD3 zeta. The present invention preferably relates to a second or third generation CAR.

In various embodiments, genetically engineered receptors that redirect cytotoxicity of immune effector cells toward cancerous cells of solid tumors are provided. These genetically engineered receptors referred to herein as chimeric antigen receptors (CARs). CARs are molecules that combine antibody-based specificity for a desired antigen with a T cell receptor-activating intracellular domain to generate a chimeric protein that exhibits an antigen specific cellular immune activity. As used herein, the term, "chimeric," describes being composed of parts of different proteins or DNAs from different origins.

CARs contemplated herein, comprise an extracellular domain (also referred to as a binding domain or antigen-binding domain) that binds to a target antigen, a transmembrane domain, and an intracellular domain, or intracellular signaling domain. Engagement of the antigen binding domain of the CAR on the surface of a target cell results in clustering of the CAR and delivers an activation stimulus to the CAR-containing cell. The main characteristics of CARs are their ability to redirect immune effector cell specificity, thereby triggering proliferation, cytokine production, phagocytosis or production of molecules that can mediate cell death of the target antigen expressing cell in a major histocompatibility complex (MHC) independent manner, exploiting the cell specific targeting abilities of monoclonal antibodies, soluble ligands or cell specific coreceptors.

In various embodiments, a CAR comprises an extracellular binding domain that comprises a humanized antigen-specific binding domain; a transmembrane domain; one or more intracellular signaling domains. In particular embodiments, a CAR comprises an extracellular binding domain that comprises an antigen binding fragment thereof; one or more spacer domains; a transmembrane domain; one or more intracellular signaling domains.

The "extracellular antigen-binding domain" or "extracellular binding domain" are used interchangeably and provide a CAR with the ability to specifically bind to the target antigen of interest. The binding domain may be derived either from a natural, synthetic, semi-synthetic, or recombinant source. Preferred are scFv domains.

“Specific binding” is to be understood as via one skilled in the art, whereby the skilled person is clearly aware of various experimental procedures that can be used to test binding and binding specificity. Methods for determining equilibrium association or equilibrium dissociation constants are known in the art. Some cross-reaction or background binding may be inevitable in many protein-protein interactions; this is not to detract from the “specificity” of the binding between CAR and epitope. “Specific binding” describes binding of an antibody or antigen binding fragment thereof (or a CAR comprising the same) to a target antigen at greater binding affinity than background binding. The term “directed against” is also applicable when considering the term “specificity” in understanding the interaction between antibody and epitope.

An "antigen (Ag)" refers to a compound, composition, or substance that can stimulate the production of antibodies or a T cell response in an animal. In particular embodiments, the target antigen is an epitope of a desired polypeptide. An "epitope" refers to the region of an antigen to which a binding agent binds. Epitopes can be formed both from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of a protein.

Tumor associated antigens targeted via the transgenic construct, e.g., CAR or TCR construct, include without limitation GPC3, HER2, GD2, EGFR variant III (EGFR vl 11), EGFR, EGFR806, CEA, PSMA, FRa, EPCAM, MUC1 , ROR1 , MUCI16eto, VEGFR2, CD171 , PSCA, EphA2, FAP, CAIX, c-MET, L1-CAM, Mesothelin, PD-L1 , WT1 , NY-ESO-1 , MAG E-A 1/3/4, Claudin 18.2, VEGFR2, AFP, Nectin4/FAP, Lewis Y, Glypican-3, MUC16, AFP, AXL, CD20, CD80/86, DLL-3, DR5, EpHA2, FR-a, gp100, LMP-1 , NKG2D, GUCY2C, TA-72, CD46, ANTXR1 , MUC3A, Trop2, av 6, CD47, GSPG4, Glypican 2, B7-H3, PSA, PAP, Mage-A1 , CD32A, ROR, C133, 0X040, CLDN6 and IL13Ra2 .

The term “non-self-antigen” or “non-self-tumor antigen” refers to an antigen that is expressed by a tumor cell (intra-or extracellularly) and not or only to a negligible amount also present in non- cancerous cells. Examples for “non-self-tumor antigen” also referred to as neoantigens include without limitation mesothelin (MSLN), carcino-embryonic antigen (CEA), Receptor Tyrosine Kinase Like Orphan Receptor 1 (ROR1), Epidermal growth factor receptor variant III (EGFRvlll), B7 homolog 3 protein (B7-H3 also known as CD276), Mucin 1 (MUC1), Tumor necrosis factor receptor superfamily member 4 (TNFRSF4, also known as CD134 or 0X040), Claudin 6 (CLDN6) and oncofetal chondroitin sulphate (ofCS).

"Single-chain Fv" or "scFv" antibody fragments comprise the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain and in either orientation {e.g., VL-VH or VH-VL). Generally, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding. In preferred embodiments, a CAR contemplated herein comprises antigen-specific binding domain that is an scFv and may be a murine, human or humanized scFv. Single chain antibodies may be cloned from the V region genes of a hybridoma specific for a desired target. scFv can be also obtained from phage display libraries, thus bypassing the traditional hybridoma technology. In particular embodiments, the antigen-specific binding domain that is a humanized scFv that binds a human target antigen polypeptide.

Antibodies and antibody fragments:

Typically, a CAR comprises an extracellular antigen-binding domain, comprising an antibody or antibody fragment that binds a target polypeptide. Antibodies or antibody fragments of the invention therefore include, but are not limited to polyclonal, monoclonal, bispecific, human, humanized or chimeric antibodies, single chain fragments (scFv), single variable fragments (ssFv), single domain antibodies (such as VHH fragments from nanobodies), Fab fragments, F(ab')2 fragments, fragments produced by a Fab expression library, anti-idiotypic antibodies and epitope-binding fragments or combinations thereof of any of the above, provided that they retain similar binding properties of the CAR described herein, preferably comprising the corresponding CDRs, or VH and VL regions as described herein. Also mini-antibodies and multivalent antibodies such as diabodies, triabodies, tetravalent antibodies and peptabodies can be used in a method of the invention. The immunoglobulin molecules of the invention can be of any class (i.e. IgG, IgE, IgM, IgD and IgA) or subclass of immunoglobulin molecules. Thus, the term antibody, as used herein, also includes antibodies and antibody fragments comprised by the CAR of the invention, either produced by the modification of whole antibodies or synthesized de novo using recombinant DNA methodologies.

As used herein, an "antibody" generally refers to a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes or fragments of immunoglobulin genes. Where the term “antibody” is used, the term “antibody fragment” may also be considered to be referred to. The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as the myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD, and IgE, respectively. The basic immunoglobulin (antibody) structural unit is known to comprise a tetramer or dimer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" (L) (about 25 kD) and one "heavy" (H) chain (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids, primarily responsible for antigen recognition. The terms "variable light chain" and "variable heavy chain" refer to these variable regions of the light and heavy chains respectively. Optionally, the antibody or the immunological portion of the antibody, can be chemically conjugated to, or expressed as, a fusion protein with other proteins.

The CARs of the invention are intended to bind against mammalian, in particular human, protein targets. The use of protein names may correspond to either mouse or human versions of a protein.

Affinities of binding domain polypeptides and CAR proteins according to the present disclosure can be readily determined using conventional techniques, e.g., by competitive ELISA (enzyme- linked immunosorbent assay), or by binding association, or displacement assays using labeled ligands, or using a surface-plasmon resonance device such as the Biacore.

Humanized antibodies comprising one or more CDRs of antibodies of the invention or one or more CDRs derived from said antibodies can be made using any methods known in the art. For example, four general steps may be used to humanize a monoclonal antibody. These are: (1) determining the nucleotide and predicted amino acid sequence of the starting antibody light and heavy variable domains (2) designing the humanized antibody, i.e., deciding which antibody framework region to use during the humanizing process (3) the actual humanizing methodologies/techniques and (4) the transfection and expression of the humanized antibody. See, for example, U.S. Pat. Nos. 4,816,567; 5,807,715; 5,866,692; 6,331 ,415; 5,530,101 ; 5,693,761 ; 5,693,762; 5,585,089; 6,180,370; 5,225,539; 6,548,640.

The term humanized antibody means that at least a portion of the framework regions, and optionally a portion of CDR regions or other regions involved in binding, of an immunoglobulin is derived from or adjusted to human immunoglobulin sequences. The humanized, chimeric or partially humanized versions of the mouse monoclonal antibodies can, for example, be made by means of recombinant DNA technology, departing from the mouse and/or human genomic DNA sequences coding for H and L chains or from cDNA clones coding for H and L chains. Humanized forms of mouse antibodies can be generated by linking the CDR regions of non-human antibodies to human constant regions by recombinant DNA techniques (Queen et al., 1989; WO 90/07861). Alternatively, the monoclonal antibodies used in the method of the invention may be human monoclonal antibodies. Human antibodies can be obtained, for example, using phagedisplay methods (WO 91/17271 ; WO 92/01047).

As used herein, humanized antibodies refer also to forms of non-human (e.g. murine, camel, llama, shark) antibodies that are specific chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences of antibodies) that contain minimal sequence derived from non-human immunoglobulin.

As used herein, human or humanized antibody or antibody fragment means an antibody having an amino acid sequence corresponding to that of an antibody produced by a human and/or has been made using any of the techniques for making human antibodies known in the art or disclosed herein. Human antibodies or fragments thereof can be selected by competitive binding experiments, or otherwise, to have the same epitope specificity as a particular mouse antibody. The humanized antibodies of the present invention surprisingly share the useful functional properties of the mouse antibodies to a large extent. Human polyclonal antibodies can also be provided in the form of serum from humans immunized with an immunogenic agent. Optionally, such polyclonal antibodies can be concentrated by affinity purification using amyloid fibrillar and/or non-fi brillar polypeptides or fragments thereof as an affinity reagent. Monoclonal antibodies can be obtained from serum according to the technique described in WO 99/60846.

Variable Regions and CDRs

A variable region of an antibody refers to the variable region of the antibody light chain or the variable region of the antibody heavy chain, either alone or in combination. The variable regions of the heavy and light chain each consist of four framework regions (FR) connected by three complementarity determining regions (CDRs) also known as hypervariable regions. The CDRs in each chain are held together in close proximity by the FRs and, with the CDRs from the other chain, contribute to the formation of the antigen-binding site of antibodies.

There are a number of techniques available for determining CDRs, such as an approach based on cross-species sequence variability (i.e., Kabat et al. Sequences of Proteins of Immunological Interest, (5th ed., 1991 , National Institutes of Health, Bethesda Md.)); and an approach based on crystallographic studies of antigen-antibody complexes (Al-Lazikani et al. (1997) J. Molec. Biol. 273:927-948). Alternative approaches include the IMGT international ImMunoGeneTics information system, (Marie-Paule Lefranc). The Kabat definition is based on sequence variability and is the most commonly used method. The Chothia definition is based on the location of the structural loop regions, wherein the AbM definition is a compromise between the two used by Oxford Molecular's AbM antibody modelling software (refer www.bioinf.org.uk : Dr. Andrew C.R. Martin's Group). As used herein, a CDR may refer to CDRs defined by one or more approach, or by a combination of these approaches.

Additional components of the CAR

In certain embodiments, the CARs may comprise linker residues between the various domains, added for appropriate spacing and conformation of the molecule, for example a linker comprising an amino acid sequence that connects the VH and VL domains and provides a spacer function compatible with interaction of the two sub-binding domains so that the resulting polypeptide retains a specific binding affinity to the same target molecule as an antibody that comprises the same light and heavy chain variable regions. CARs contemplated herein, may comprise one, two, three, four, or five or more linkers. In particular embodiments, the length of a linker is about 1 to about 25 amino acids, about 5 to about 20 amino acids, or about 10 to about 20 amino acids, or any intervening length of amino acids.

Illustrative examples of linkers include glycine polymers; glycine-serine polymers; glycine-alanine polymers; alanine-serine polymers; and other flexible linkers known in the art, such as the Whitlow linker. Glycine and glycine-serine polymers are relatively unstructured, and therefore may be able to serve as a neutral tether between domains of fusion proteins such as the CARs described herein.

In particular embodiments, the binding domain of the CAR is followed by one or more "spacers” or “spacer polypeptides," which refers to the region that moves the antigen binding domain away from the effector cell surface to enable proper cell/cell contact, antigen binding and activation. In certain embodiments, a spacer domain is a portion of an immunoglobulin, including, but not limited to, one or more heavy chain constant regions, e.g., CH2 and CH3. The spacer domain can include the amino acid sequence of a naturally occurring immunoglobulin hinge region or an altered immunoglobulin hinge region. In one embodiment, the spacer domain comprises the CH2 and CH3 domains of IgG 1 or lgG4. In one embodiment the Fc-binding domain of such a spacer/hinge region is mutated in a manner that prevents binding of the CAR-To Fc-receptors expressed on macrophages and other innate immune cells.

The binding domain of the CAR may in some embodiments be followed by one or more "hinge domains," which play a role in positioning the antigen binding domain away from the effector cell surface to enable proper cell/cell contact, antigen binding and activation. A CAR may comprise one or more hinge domains between the binding domain and the transmembrane domain (TM). The hinge domain may be derived either from a natural, synthetic, semi-synthetic, or recombinant source. The hinge domain can include the amino acid sequence of a naturally occurring immunoglobulin hinge region or an altered immunoglobulin hinge region. Illustrative hinge domains suitable for use in the CARs described herein include the hinge region derived from the extracellular regions of type 1 membrane proteins such as CD8 alpha, CD4, CD28, PD1 , CD152, and CD7, which may be wildtype hinge regions from these molecules or may be altered. In another embodiment, the hinge domain comprises a PD1 , CD152, or CD8 alpha hinge region.

The "transmembrane domain" is the portion of the CAR-That fuses the extracellular binding portion and intracellular signaling domain and anchors the CAR-To the plasma membrane of the immune effector cell. The TM domain may be derived either from a natural, synthetic, semisynthetic, or recombinant source. The TM domain may be derived from the alpha, beta or zeta chain of the T cell receptor, CD3E, CD3 , CD4, CD5, CD8 alpha, CD9, CD 16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, and PD1. In one embodiment, the CARs contemplated herein comprise a TM domain derived from CD8 alpha or CD28

In particular embodiments, CARs contemplated herein comprise an intracellular signaling domain. An "intracellular signaling domain," refers to the part of a CAR-That participates in transducing the message of effective binding to a human target polypeptide into the interior of the immune effector cell to elicit effector cell function, e.g., activation, cytokine production, proliferation and cytotoxic activity, including the release of cytotoxic factors to the CAR-bound target cell, or other cellular responses elicited with antigen binding to the extracellular CAR domain. The term "effector function" refers to a specialized function of an immune effector cell. Effector function of the T cell, for example, may be cytolytic activity or help or activity including the secretion of a cytokine. Thus, the term "intracellular signaling domain" refers to the portion of a protein which transduces the effector function signal and that directs the cell to perform a specialized function. CARs contemplated herein comprise one or more co-stimulatory signaling domains to enhance the efficacy, expansion and/or memory formation of T cells expressing CAR receptors. As used herein, the term, "co-stimulatory signaling domain" refers to an intracellular signaling domain of a co-stimulatory molecule. Co-stimulatory molecules are cell surface molecules other than antigen receptors or Fc receptors that provide a second signal required for efficient activation and function of T lymphocytes upon binding to antigen.

In one embodiment, the CAR comprises an intracellular domain, which comprises a co- stimulatory domain and a signalling (activation) domain. The CAR construct may therefore include an intracellular signaling domain (CD3 zeta) of the native T cell receptor complex and one or more co-stimulatory domains that provide a second signal to stimulate full T cell activation. Co- stimulatory domains are thought to increase CAR-T cell cytokine production and facilitate T cell replication and T cell persistence. Co-stimulatory domains have also been shown to potentially prevent CAR-T cell exhaustion, increase T cell anti-tumor activity, and enhance survival of CAR-T cells in patients. As a non-limiting example, CAR constructs with the 4-1 BB co-stimulatory domain have been associated with gradual, sustained expansion and effector function, increased persistence, and enriched central memory cells (TCM) in the T cell subset composition in preclinical studies. 4-1 BB is a member of the tumor necrosis factor (TNF) superfamily, and it is an inducible glycoprotein receptor in vivo that is primarily expressed on antigen-activated CD4 and CD8⁺ T cells. As a non-limiting example, CD28 is member of the immunoglobulin (Ig) superfamily. It is constitutively expressed on resting and activated CD4⁺ and CD8⁺ T cells and plays a critical role in T cell activation by stimulating the PI3K-AKT signal transduction pathway. In one embodiment, the intracellular domain comprises both 4-1 BB and CD28 co-stimulatory domains. Other co-stimulatory domains comprise ICOS and 0X40 that can be combined with the CD3 zeta signalling (activation) domain.

The T cell receptor, or TOR, is a molecule typically found on the surface of T cells, or T lymphocytes that is responsible for recognizing fragments of antigen as peptides bound to major histocompatibility complex (MHO) molecules. The TOR is composed of two different protein chains. In humans, in 95% of T cells the TCR consists of an alpha (a) chain and a beta (P) chain (encoded by TRA and TRB, respectively), whereas in 5% of T cells the TCR consists of gamma and delta (y/<5) chains (encoded by TRG and TRD, respectively). Each chain is composed of two extracellular domains: Variable (V) region and a Constant (C) region, both of Immunoglobulin superfamily (IgSF) domain forming antiparallel p-sheets. The Constant region is proximal to the cell membrane, followed by a transmembrane region and a short cytoplasmic tail, while the Variable region binds to the peptide/MHC complex.

The variable domain of both the TCR a-chain and p-chain each have three hypervariable or complementarity determining regions (CDRs). There is also an additional area of hypervariability on the p-chain (HV4) that does not normally contact antigen and, therefore, is not considered a CDR.

The residues in these variable domains are located in two regions of the TCR, at the interface of the a- and p-chains and in the p-chain framework region that is thought to be in proximity to the CD3 signal-transduction complex. CDR3 is the main CDR responsible for recognizing processed antigen, although CDR1 of the alpha chain has also been shown to interact with the N-terminal part of the antigenic peptide, whereas CDR1 of the p-chain interacts with the C-terminal part of the peptide.

Recombinant TCRs have been previously transfected into therapeutic T cells intended for the treatment of proliferative disease. For example, TCR-T cells are engineered by transducing preferably autologous alpha-beta or gamma-delta cells with a retroviral or lentiviral vector encoding TCR (typically an alpha chain non-covalently bound with a beta chain) that recognizes peptides of interest and CD3z genes. When the engineered T cells recognize peptides bound to the major histocompatibility complex (MHC) on the surface of antigen-presenting or tumor cells, they become activated and start expanding. The first TCR-T cell therapy was used in clinical trial for metastatic melanoma, whose TCR recognizing an HLA-A2-restricted peptide from a melanocytic differentiation antigen, melanoma antigen recognized by T cells 1 (MART-1).

"Peptide" "polypeptide”, “polypeptide fragment" and "protein" are used interchangeably, unless specified to the contrary, and according to conventional meaning, i.e., as a sequence of amino acids. Polypeptides are not limited to a specific length, e.g., they may comprise a full-length protein sequence or a fragment of a full length protein, and may include post-translational modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations and the like, as well as other modifications known in the art, both naturally occurring and non-naturally occurring.

In various embodiments, the CAR polypeptides contemplated herein comprise a signal (or leader) sequence at the N-terminal end of the protein, which co-translationally or post-translationally directs transfer of the protein. Polypeptides can be prepared using any of a variety of well-known recombinant and/or synthetic techniques. Polypeptides contemplated herein specifically encompass the CARs of the present disclosure, or sequences that have deletions from, additions to, and/or substitutions of one or more amino acid of a CAR as disclosed herein.

An "isolated peptide" or an "isolated polypeptide" and the like, as used herein, refer to in vitro isolation and/or purification of a peptide or polypeptide molecule from a cellular environment, and from association with other components of the cell, i.e., it is not significantly associated with in vivo substances. Similarly, an "isolated cell" refers to a cell that has been obtained from an in vivo tissue or organ and is substantially free of extracellular matrix.

Nucleic acids

As used herein, the terms "polynucleotide" or "nucleic acid molecule" refers without limitation to ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or any mimetic or structurally modified nucleic acid thereof, including, without limitation DNA, messenger RNA (mRNA), RNA, genomic RNA (gRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), microRNA (miRNA), small interfering RNA (siRNA), single guide RNA (sgRNA), short hairpin RNA (shRNA), piwi-interacting RNA (piRNA), small nuclear RNA (snRNA), plus strand RNA (RNA(+)), minus strand RNA (RNA(-)), genomic DNA (gDNA), complementary DNA (cDNA), antisense oligonucleotides (ASO), recombinant polynucleotides, branched polynucleotides, plasmids, nucleic acid probes and primers. Polynucleotides include single and double stranded polynucleotides. Preferably, polynucleotides of the invention include polynucleotides or variants having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of the reference sequences described herein, typically where the variant maintains at least one biological activity of the reference sequence. In various illustrative embodiments, the present invention contemplates, in part, polynucleotides comprising expression vectors, viral vectors, and transfer plasmids, and compositions, and cells comprising the same.

Polynucleotides can be prepared, manipulated and/or expressed using any of a variety of well- established techniques known and available in the art. In order to express a desired polypeptide, a nucleotide sequence encoding the polypeptide, can be inserted into appropriate vector. Examples of vectors are plasmid, autonomously replicating sequences, and transposable elements. Additional exemplary vectors include, without limitation, plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC), or Pl-derived artificial chromosome (PAC), bacteriophages such as lambda phage or Ml 3 phage, and animal viruses. Examples of categories of animal viruses useful as vectors include, without limitation, retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpesvirus {e.g., herpes simplex virus), poxvirus, baculovirus, papillomavirus, and papovavirus {e.g., SV40). Examples of expression vectors are pCIneo vectors (Promega) for expression in mammalian cells; pLenti4/V5-DEST™, pLenti6/V5-DEST™, and pLenti6.2/V5- GW/lacZ (Invitrogen) for lentivirus-mediated gene transfer and expression in mammalian cells. In particular embodiments, the coding sequences of the chimeric proteins disclosed herein can be ligated into such expression vectors for the expression of the chimeric protein in mammalian cells. The "control elements" or "regulatory sequences" present in an expression vector are those non-translated regions of the vector - origin of replication, selection cassettes, promoters, enhancers, translation initiation signals (Shine Dalgarno sequence or Kozak sequence) introns, a polyadenylation sequence, 5' and 3' untranslated regions - which interact with host cellular proteins to carry out transcription and translation. Such elements may vary in their strength and specificity. Depending on the vector system and host utilized, any number of suitable transcription and translation elements, including ubiquitous promoters and inducible promoters may be used.

An “exogenous nucleic acid”, “exogenous genetic element”, or “transgenic” nucleic acid or construct relates to any nucleic acid introduced into the cell, which is not a component of the cells “original” or “natural” genome or pool of nucleic acids found “naturally” in an unmodified T cell. Exogenous nucleic acids may be integrated or non-integrated in the genetic material of the target T cell or relate to stably transduced nucleic acids. Delivery of an exogenous nucleic acid may lead to genetic modification of the initial cell through permanent integration of the exogenous nucleic acid molecule in the initial cell. However, delivery of the exogenous nucleic acid may also be transient, meaning that the delivered genetic material for provision of the one or more TF disappears from the cell after a certain time. Nucleic acid molecule delivery and potentially genetic modification of a biological cell, i.e. a T cell, can be performed and determined by a skilled person using commonly available techniques. For example, for detecting genetic modification sequencing of the genome or parts thereof of a cell is possible, thereby identifying if exogenous nucleic acids are present. Alternatively, other molecular biological techniques may be applied, such as the polymerase chain reaction (PCR), to identify/amplify exogenous genetic material. Exogenous nucleic acids may be detected by vector sequences, or parts of vector sequences, e.g. those remaining at the site of genetic modification. In cases where vector sequences (for example vector sequences flanking a therapeutic transgene) can be removed from the genome or do not remain after modification, for example by CRISPR technology, the addition of a transgene may still be detected by sequencing efforts by detecting sequences comprising an exogenous sequence at a “non-natural” position in the genome.

Embodiments of the invention relate to genetically modified immune cells comprising one or more exogenous nucleic acid molecules encoding a transgenic antigen-targeting construct. In embodiments of the invention, the exogenous nucleic acid represents a nucleic acid sequence not found naturally in an immune cell, based on for example comparisons with an unmodified human genome sequence. In embodiments of the invention, the transgenic antigen-targeting construct is a nucleic acid sequence coding an antigen targeting construct, for which the coding sequence is not found naturally in an immune cell, based on for example comparisons with an unmodified human genome sequence. In some embodiments, the sequence is present at a “nonnatural location” of the genome. In some embodiments, the targeting construct comprises or consists of a non-naturally occurring sequence, i.e. a synthetic sequence designed and created using recombinant or other molecular biological techniques. According to the invention, NR2F6 and optionally CBLB activity is inhibited (compared to a control immune cell).

Inhibition of NR2F6 and CBLB

According to the present invention NR2F6 and optionally CBLB can be inhibited by gene silencing or gene editing, e.g., by disrupting the expression and/or sequence of a NR2F6 and optionally a CBLB gene.

“Gene editing” also termed “genetic modification” is a type of genetic engineering in which DNA is inserted, deleted or replaced in the genome of a living organism by manipulated nucleases, or "molecular scissors". These nucleases generate site-specific double-strand breaks (DSBs) at desired sites in the genome. The induced double-strand breaks are repaired by non-homologous end compound (HEJ) or homologous recombination (HR), resulting in targeted mutations ("edits"). Examples of manipulated nucleases that can be used for gene editing include meganucleases, integrases, site specific recombinases, homing endonucleases, zinc finger nuclei (ZFNs), transcription activator-like effector-based nucleases (TALENs), Mega-TALENs, and the CRISPR-Cas system.

Methods for introducing exogenous molecules, e.g., nucleic acid sequences, into cells are well known to experts and include lipid-mediated transfer (i.e., liposomes, including neutral and cationic lipids), electroporation, direct injection, cell fusion, particle bombardment, biopolymer nanoparticles, calcium phosphate co-precipitation, DEAE-dextran-mediated transfer and viral vector-mediated transfer. “Gene transfer”, “nucleic acid transfer”, gene sequence transfer”, “transgene transfer” are used interchangeably. In embodiments, nucleic acid constructs are preferably transferred into cells by non-viral vectors and methods such as electroporation.

In some embodiments, inhibiting NR2F6 and optionally CBLB comprises genetic modification of the immune cell genome by disrupting the expression and/or sequence of the NR2F6 and optionally CBLB gene by CRISPR-Cas. In further embodiments of the invention, CRISPR- mediated insertion of the nucleic acid encoding for a transgenic antigen targeting construct, e.g., CAR or TCR encoding may be employed. CRISPR is an abbreviation of Clustered Regularly Interspaced Short Palindromic Repeats and is a family of DNA sequences in bacteria. The sequences contain snippets of DNA from viruses that have attacked the bacterium. These snippets are used by the bacterium to detect and destroy DNA from further attacks by similar viruses. These sequences play a key role in a bacterial defense system and form the basis of a technology known as CRISPR/Cas that effectively and specifically changes genes within organisms.

Sequences of the CRISPR loci are transcribed and processed into CRISPR RNAs (crRNAs) which, together with a trans-activating crRNAs (tracrRNAs), complex with CRISPR-associated (Cas) proteins to dictate specificity of DNA cleavage by Cas nucleases through Watson-Crick base pairing between nucleic acids (Wiedenheft, B et al (2012). Nature 482: 331-338; Horvath, P et al (2010). Science 327: 167-170; Fineran, PC et a. (2012). Virology 434: 202-209).

It was shown that the three components required for the type II CRISPR nuclease system are the Cas9 protein, the mature crRNA and the tracrRNA, which can be reduced to two components by fusion of the crRNA and tracrRNA into a single guide RNA (sgRNA) and that re-targeting of the Cas9/sgRNA complex to new sites could be accomplished by altering the sequence of a short portion of the gRNA (Garneau, JE et al (2010). Nature 468: 67-71 ; Deltcheva, E et al. (2011). Nature 471 : 602-607, Jinek, M et al (2012) Science 337: 816-821).

CRISPR-Cas systems are RNA-guided adaptive immune systems of bacteria and archaea that provide sequence-specific resistance against viruses or other invading genetic material. This immune-like response has been divided into two classes on the basis of the architecture of the effector module responsible for target recognition and the cleavage of the invading nucleic acid (Makarova KS et al. Nat Rev Microbiol. 2015 Nov; 13(11):722-36.). Class 1 comprises multisubunit Cas protein effectors and Class 2 consists of a single large effector protein. Both Class 1 and 2 use CRISPR RNAs (crRNAs) to guide a Cas nuclease component to its target site where it cleaves the invading nucleic acids. Due to their simplicity, Class 2 CRISPR-Cas systems are the most studied and widely applied for genome editing. The most widely used CRISPR-Cas system is CRISPR-Cas9. It was demonstrated that the CRISPR/Cas9 system could be engineered for efficient genetic modification in mammalian cells.

In some embodiments of the invention, an RNA guided DNA endonuclease is employed. In the context of the present invention, the term “RNA guided DNA endonuclease” refers to DNA endonucleases that interact with at least one RNA-Molecule. DNA endonucleases are enzymes that cleave the phosphodiester bond within a DNA polynucleotide chain. In case of RNA guided DNA endonuclease the interacting RNA-Molecule may guide the RNA guided DNA endonuclease to the site or location in a DNA where the endonuclease becomes active. In particular, the term RNA guided DNA endonuclease refers to naturally occurring or genetically modified Cas nuclease components or CRISPR-Cas systems, which include, without limitation, multi-subunit Cas protein effectors of class 1 CRISPR-Cas systems as well as single large effector Cas proteins of class 2 systems.

Details of the technical application of CRISPR/Cas systems and suitable RNA guided endonuclease are known to the skilled person and have been described in detail in the literature, as for example by Barrangou R et al. (Nat Biotechnol. 2016 Sep 8;34(9):933-941), Maeder ML et al. (Mol Ther. 2016 Mar;24(3):430-46) and Cebrian-Serrano A et al. (Mamm Genome. 2017; 28(7): 247-261). The present invention is not limited to the use of specific RNA guided endonucleases and therefore comprises the use of any given RNA guided endonucleases in the sense of the present invention suitable for use in the method described herein.

Any RNA guided DNA endonuclease known in the art may be employed in accordance with the present invention. RNA guided DNA endonuclease comprise, without limitation, Cas proteins of class 1 CRISPR-Cas systems, such as Cas3, Cas8a, Cas5, Cas8b, Cas8c, Cas10d, Cse1 , Cse2, Csy1 , Csy2, Csy3, GSU0054, Cas10, Csm2, Cmr5, Csx11 , Csx10 and Csf1 ; Cas proteins of class 2 CRISPR-Cas systems, such as Cas9, Csn2, Cas4, Cpf1 , C2c1 , C2c3 and C2c2; corresponding orthologous enzymes/CRISPR effectors from various bacterial and archeal species; engineered CRISPR effectors with for example novel PAM specificities, increased fidelity, such as SpCas9-HF1/eSpCas9, or altered functions, such as nickases. Particularly preferred RNA guided DNA endonuclease of the present invention are Streptococcus pyogenes Cas9 (SpCas9), Staphylococcus aureus Cas9, Streptococcus thermophilus Cas9, Neisseria meningitidis Cas9 (NmCas9), Francisella novicida Cas9 (FnCas9), Campylobacter jejuni Cas9 (CjCas9), Cas12a (Cpf1) and Cas13a (C2C2) (Makarova KS et al. (November 2015). Nature Reviews Microbiology. 13 (11): 722-36).

The definition and explanations provided herein are mainly focused on the SpCas9 Crispr/Cas system. However, the person skilled in the art is aware of how to use alternative Crispr/Cas systems as well as tools and methods that provide or allow the gain of information on the details of such alternative systems.

In accordance with the method of the invention, the RNA guided DNA endonuclease may be introduced as a protein, but alternatively the RNA guided DNA endonuclease may also be introduced in form of a nucleic acid molecule encoding said protein. It will be appreciated that the nucleic acid molecule encodes said RNA guided DNA endonuclease in expressible form such that expression in the cell results in a functional RNA guided DNA endonuclease protein such as Cas9 protein. Means and methods to ensure expression of a functional polypeptide are well known in the art. For example, the coding sequences for the endonuclease may be comprised in a vector, such as for example a plasmid, cosmid, virus, bacteriophage or another vector used conventionally e.g., in genetic engineering.

Furthermore, the method of the present invention comprises introducing into the cell at least one guide RNA. In the context of the present invention, a “guide RNA” refers to RNA molecules interacting with RNA guided DNA endonuclease leading to the recognition of the target sequence to be cleaved by the RNA guided DNA endonuclease. According to the present invention, the term “guide RNA” therefore comprises, without limitation, target sequence specific CRISPR RNAs (crRNA), trans-activating crRNAs (tracrRNA) and chimeric single guide RNAs (sgRNA).

According to the present invention NR2F6 and optionally CBLB can be inhibited by knock down of NR2F6 expression and optionally of CBLB expression, e.g., by RNA interference (RNAi).

RNAi is a post-transcriptionally mediated gene silencing mechanism that is triggered by doublestranded RNA (dsRNA) to induce sequence-specific translational repression or mRNA degradation. Historically, RNAi was known by other names, including co-suppression, post- transcriptional gene silencing (PTGS), and quelling.

In the nucleus, the micro RNA (miRNA) genes are transcribed into 500-3000 nucleotide pri- miRNAs by action of the RNA polymerase II. These pri-miRNAs are capped and polyadenylated. In addition, pri-miRNA contain one or multiple stem-loop sequences and are cleaved by the Drosha-DGCR8 complex to 60-100 nucleotide double-stranded pre-miRNA hairpin structures. Ran GTPase and Exportin-5 mediate the export of pre-miRNAs from the nucleus into the cytoplasm. There, they are further processed by an RNase III enzyme called Dicer to an imperfect duplex structure of 22 nucleotides. One of the strands resembles the mature miRNA that binds to Argonaut (Ago) proteins and is incorporated in the RNA-induced silencing complex (RISC). As a consequence of RISC binding, mRNA degradation or repression of protein translation is induced. The fate of the target mRNA molecules depends on the grade of complementarity between the target mRNA molecule and miRNA but is also affected by the incorporated Ago protein. While incorporation of Ago 2 leads to direct cleavage of the target mRNA, the other Ago proteins negatively impact mRNA stability or attenuate translation.

For the engineered knockdown of specific targets, several dsRNA molecules can be used that enter the RNAi pathway at different points. Transfection with small interfering RNA (siRNA) molecules that enter the RNAi pathway in the cytosol only leads to transient protein knockdown. For long-term manipulation of gene expression, it is necessary to deliver dsRNA molecules by integrating gene transfer vectors. Therefore, short hairpin RNA (shRNA) or miRNA molecules can be applied.

Both enter the RNAi pathway in the nucleus and are then processed to siRNA-like molecules. shRNAs mimic the pre-miRNA stem-loop structure. Their expression is driven by the strong RNA polymerase III promoters that lead to high-level expression and stable gene knockdown.

A person skilled in the art is capable of designing suitable small interfering RNA (siRNA) short hairpin RNA (shRNA) or miRNA molecules. Examples, e.g., for siRNA molecules for NR2F6 inhibitor are disclosed in WO2010/004051 A1 .

Another possibility is the application of artificial miRNAs to mediate a stable knockdown within primary T cells. These artificial miRNAs are analogous to the pri-miRNA and, therefore, are a step further towards mimicking natural miRNA biology. This has several advantages for potential clinical applications. Most importantly, using the endogenous miRNA processing machinery does not trigger cellular self-defense mechanisms such as interferon induction.

In addition, artificial miRNAs are transcribed by RNA polymerase II promoters comparable to most of the natural miRNAs. These promoters mediate regulated and tissue-specific expression and further enable the simultaneous expression of selector or therapeutic transgenes. Moreover, it is possible to combine multiple miRNAs in one expression cassette to target regions in the same or different mRNAs, therefore, gaining an additive effect in target downregulation. Such target down-regulation is referred to herein as e.g., knock-down, silencing, or RNA interference.

Further morpholinos can be employed for knock down of NR2F6 and optionally of CBLB expression. A morpholino also termed “morpholino oligomer” or“phosphorodiamidate morpholino oligomer (PMO)” is an oligomer comprising DNA bases attached to a backbone of methylenemorpholine rings linked through phosphorodiamidate groups. Morpholinos block access of other molecules to small (approx. 25 base) specific sequences of the base-pairing surfaces of ribonucleic acid (RNA), e.g., of mRNA or pre-mRNA, thereby depending on their sequence blocking for example translation or splicing of the RNA molecules. Morpholinos do not triggering the degradation of said RNA molecules. A person skilled in the art is capable of designing and synthesizing morpholinos for knock down of NR2F6 and optionally of CBLB expression. Further antisense oligonucleotides (ASOs) can be employed for knock down of NR2F6 and optionally of CBLB expression. An antisense oligonucleotide is a short, synthetic, single-stranded oligodeoxynucleotide that can alter RNA and reduce, restore, or modify protein expression through several distinct mechanisms. These mechanisms include modulating pre-mRNA by redirecting polyadenylation, altering splicing or cleaving internucleotide bonds and modulation mRNA by hindering translation or cleavage. A person skilled in the art is capable of designing and synthesizing ASOs for knock down of NR2F6 and optionally of CBLB expression.

According to the present invention NR2F6 and optionally CBLB can be inhibited by treatment of the immune cells of the present invention with a NR2F6 antagonist and optionally a CBLB antagonist. Antagonists of NR2F6 and CBLB are preferably small molecule inhibitors. Examples for such small molecule inhibitors are TES-4207 (NR2F6 inhibitor) and NX-1607 (CBLB inhibitor). Further examples for such inhibitors are disclosed in WO2019/104199, WO2019/104201 , US2019/0358224, WC2020/210508, WO2020/236654, WO2020/264398, WC2019/148005 and WO2022/272248,

Compositions and Formulations

The compositions contemplated herein may comprise one or more polypeptides, polynucleotides, vectors comprising same, genetically modified immune cells, etc., as contemplated herein. Compositions include but are not limited to pharmaceutical compositions. A "pharmaceutical composition" refers to a composition formulated in pharmaceutically-acceptable or physiologically-acceptable solutions for administration to a cell or an animal, either alone, or in combination with one or more other modalities of therapy. It will also be understood that, if desired, the compositions of the invention may be administered in combination with other agents as well, such as, e.g., cytokines, growth factors, hormones, small molecules, chemotherapeutics, pro-drugs, drugs, antibodies, or other various pharmaceutically-active agents. There is virtually no limit to other components that may also be included in the compositions, provided that the additional agents do not adversely affect the ability of the composition to deliver the intended therapy.

A "pharmaceutical composition" refers to a composition formulated in pharmaceutically- acceptable or physiologically- acceptable solutions for administration to a cell or an animal, either alone, or in combination with one or more other modalities of therapy. It will also be understood that, if desired, the compositions of the invention may be administered in combination with other agents as well, such as, e.g., cytokines, growth factors, hormones, small molecules, chemotherapeutics, pro-drugs, drugs, antibodies, or other various pharmaceutically-active agents. There is virtually no limit to other components that may also be included in the compositions, provided that the additional agents do not adversely affect the ability of the composition to deliver the intended therapy.

The term "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

As used herein "pharmaceutically acceptable carrier, diluent or excipient" includes without limitation any adjuvant, carrier, excipient, glidant, sweetening agent, diluent, preservative, dye/colorant, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, surfactant, or emulsifier which has been approved by the United States Food and Drug Administration as being acceptable for use in humans or domestic animals. Exemplary pharmaceutically acceptable carriers include, but are not limited to pyrogen- free water; isotonic saline; Ringer's solution; phosphate buffer solutions; and any other compatible substances employed in pharmaceutical formulations.

In particular embodiments, compositions of the present invention comprise an amount of immune cells contemplated herein. As used herein, the term "amount" refers to "an amount effective" or "an effective amount" of a genetically modified therapeutic cell, e.g., T cell, to achieve a beneficial or desired prophylactic or therapeutic result, including clinical results.

A "prophylactically effective amount" refers to an amount of a genetically modified therapeutic cell effective to achieve the desired prophylactic result. Typically, but not necessarily, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount is less than the therapeutically effective amount. The term prophylactic does not necessarily refer to a complete prohibition or prevention of a particular medical disorder. The term prophylactic also refers to the reduction of risk of a certain medical disorder occurring or worsening in its symptoms.

A "therapeutically effective amount" of a genetically modified therapeutic cell may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the stem and progenitor cells to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the virus or transduced therapeutic cells are outweighed by the therapeutically beneficial effects. The term "therapeutically effective amount" includes an amount that is effective to "treat" a subject (e.g., a patient). When a therapeutic amount is indicated, the precise amount of the compositions of the present invention to be administered can be determined by a physician with consideration of individual differences in age, weight, tumor size, extent of infection or metastasis, and condition of the patient (subject).

It can generally be stated that a pharmaceutical composition comprising the immune cells described herein may be administered at a dosage of 10² to 10¹⁰ cells/kg body weight, preferably 10⁵ to 10⁷ cells/kg body weight, including all integer values within those ranges. The number of cells will depend upon the ultimate use for which the composition is intended as will the type of cells included therein. For uses provided herein, the cells are generally in a volume of a liter or less, can be 500 mis or less, even 250 mis or 100 mis or less. Hence the density of the desired cells is typically greater than 10⁶ cells/ml and generally is greater than 10⁷ cells/ml, generally 10⁸ cells/ml or greater. The clinically relevant number of cells can be apportioned into multiple infusions that cumulatively equal or exceed 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, or 10¹² cells. In some aspects of the present invention, particularly when all the infused cells are redirected to a particular target antigen, lower numbers of cells may be administered. Cell compositions may be administered multiple times at dosages within these ranges. The cells may be allogeneic, syngeneic, xenogeneic, or autologous to the patient undergoing therapy.

Generally, compositions comprising the cells activated and expanded as described herein may be utilized in the treatment and prevention of diseases that arise in individuals who are immunocompromised. In particular, compositions comprising the modified immune cells contemplated herein are used in the treatment of solid tumors. The modified immune cells of the present invention may be administered either alone, or as a pharmaceutical composition in combination with carriers, diluents, excipients, and/or with other components such as IL-2 or other cytokines or cell populations. In particular embodiments, pharmaceutical compositions contemplated herein comprise an amount of genetically modified immune cells, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. In particular embodiments, pharmaceutical compositions contemplated herein comprise an amount of genetically modified immune cells, in combination with an inhibitor of NR2F6 and optionally an inhibitor of CBLB.

Pharmaceutical compositions of the present invention comprising an immune cell may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. Compositions of the present invention are preferably formulated for parenteral administration, e.g., intravascular (intravenous or intraarterial), intraperitoneal or intramuscular administration.

The liquid pharmaceutical compositions, whether they be solutions, suspensions or other like form, may include one or more of the following: sterile diluents such as water for injection, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils such as synthetic mono or diglycerides which may serve as the solvent or suspending medium, polyethylene glycols, glycerin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. The parenteral preparation can be enclosed in ampoules, disposable syringes, multiple dose vials or bags made of glass or plastic. An injectable pharmaceutical composition is preferably sterile.

In a particular embodiment, compositions contemplated herein comprise an effective amount of immune cells, alone or in combination with one or more therapeutic agents. Thus, the immune cell compositions may be administered alone or in combination with other known cancer treatments, such as radiation therapy, chemotherapy, transplantation, immunotherapy, hormone therapy, photodynamic therapy, etc. The compositions may also be administered in combination with antibiotics. Such therapeutic agents may be accepted in the art as a standard treatment for a particular disease state as described herein, such as a particular cancer. Exemplary therapeutic agents contemplated include cytokines, growth factors, steroids, NSAIDs, DMARDs, antiinflammatories, chemotherapeutics, radiotherapeutics, therapeutic antibodies, or other active and ancillary agents.

The immune cell product can be stored frozen in dimethyl sulfoxide (DMSO)Zhuman serum albumin (10% 190% vol/vol) in the gas phase of liquid nitrogen till the conditioning treatment of the patient has been administered. Such storage does not impede the viability and functionality of the T cell product.

Therapeutic Methods

The genetically modified cells contemplated herein provide improved methods of adoptive immunotherapy for use in the treatment of a solid tumor.

In the context of the present invention the term solid tumor refers without limitation to glioblastoma, lung carcinoma, breast carcinoma, kidney carcinoma, pancreatic carcinoma, skin carcinoma such as melanoma, intestinal carcinoma, ovarian carcinoma, prostate carcinoma, colon carcinoma, and sarcoma.

Sarcomas as defined in the context of the present invention include, but are not limited to a chondrosarcoma, fibrosarcoma, lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma, Abernethy's sarcoma, adipose sarcoma, liposarcoma, alveolar soft part sarcoma, ameloblastic sarcoma, botryoid sarcoma, chloroma sarcoma, chorio carcinoma, embryonal sarcoma, Wilms' tumor sarcoma, endometrial sarcoma, stromal sarcoma, Ewing's sarcoma, fascial sarcoma, fibroblastic sarcoma, giant cell sarcoma, granulocytic sarcoma, Hodgkin's sarcoma, idiopathic multiple pigmented hemorrhagic sarcoma, immunoblastic sarcoma of B cells, lymphoma, immunoblastic sarcoma of T cells, Jensen's sarcoma, Kaposi's sarcoma, Kupffer cell sarcoma, angiosarcoma, leukosarcoma, malignant mesenchymoma sarcoma, parosteal sarcoma, reticulocytic sarcoma, Rous sarcoma, serocystic sarcoma, synovial sarcoma, and telangiectaltic sarcoma.

Melanomas according to the present invention include, but are not limited to include, for example, acral-lentiginous melanoma, amelanotic melanoma, benign juvenile melanoma, Cloudman's melanoma, S91 melanoma, Harding-Passey melanoma, juvenile melanoma, lentigo maligna melanoma, malignant melanoma, nodular melanoma, subungal melanoma, and superficial spreading melanoma.

In the context of the present invention the term solid tumor further refers without limitation to acinar carcinoma, acinous carcinoma, adenocystic carcinoma, adenoid cystic carcinoma, carcinoma adenomatosum, carcinoma of adrenal cortex, alveolar carcinoma, alveolar cell carcinoma, basal cell carcinoma, carcinoma basocellulare, basaloid carcinoma, basosquamous cell carcinoma, bronchioalveolar carcinoma, bronchiolar carcinoma, bronchogenic carcinoma, cerebriform carcinoma, cholangiocellular carcinoma, chorionic carcinoma, colloid carcinoma, comedo carcinoma, corpus carcinoma, cribriform carcinoma, carcinoma en cuirasse, carcinoma cutaneum, cylindrical carcinoma, cylindrical cell carcinoma, duct carcinoma, carcinoma durum, embryonal carcinoma, encephaloid carcinoma, epiermoid carcinoma, carcinoma epitheliale adenoides, exophytic carcinoma, carcinoma exulcere, carcinoma fibrosum, gelatiniform carcinoma, gelatinous carcinoma, giant cell carcinoma, carcinoma gigantocellulare, glandular carcinoma, granulosa cell carcinoma, hair-matrix carcinoma, hematoid carcinoma, hepatocellular carcinoma, Hurthle cell carcinoma, hyaline carcinoma, hypernephroid carcinoma, infantile embryonal carcinoma, carcinoma in situ, intraepidermal carcinoma, intraepithelial carcinoma, Krompecher's carcinoma, Kulchitzky-cell carcinoma, large-cell carcinoma, lenticular carcinoma, carcinoma lenticulare, lipomatous carcinoma, lymphoepithelial carcinoma, carcinoma medullare, medullary carcinoma, melanotic carcinoma, carcinoma molle, mucinous carcinoma, carcinoma muciparum, carcinoma mucocellulare, mucoepidermoid carcinoma, carcinoma mucosum, mucous carcinoma, carcinoma myxomatodes, nasopharyngeal carcinoma, oat cell carcinoma, carcinoma ossificans, osteoid carcinoma, papillary carcinoma, periportal carcinoma, preinvasive carcinoma, prickle cell carcinoma, pultaceous carcinoma, renal cell carcinoma of kidney, reserve cell carcinoma, carcinoma sarcomatodes, Schneiderian carcinoma, scirrhous carcinoma, carcinoma scroti, signet-ring cell carcinoma, carcinoma simplex, small-cell carcinoma, solanoid carcinoma, spheroidal cell carcinoma, spindle cell carcinoma, carcinoma spongiosum, squamous carcinoma, squamous cell carcinoma, string carcinoma, carcinoma telangiectaticurn, carcinoma telangiectodes, transitional cell carcinoma, carcinoma tuberosum, tuberous carcinoma, verrucous carcinoma, and carcinoma villosum. As used herein, the terms "individual", "subject" and “patient” are often used interchangeably and refer to any animal that exhibits a symptom of a solid tumor that can be treated with the cellbased therapeutics, and methods disclosed elsewhere herein. Suitable subjects include laboratory animals (such as mouse, rat, rabbit, or guinea pig), farm animals, and domestic animals or pets (such as a cat or dog). Non-human primates and, preferably, human patients, are included. Typical subjects include human patients that have a solid cancer, have been diagnosed with a solid cancer, or are at risk or having a solid cancer.

As used herein "treatment" or "treating" includes any beneficial or desirable effect on the symptoms or pathology of a disease or pathological condition and may include even minimal reductions in one or more measurable markers of the disease or condition being treated.

T reatment can involve optionally either the reduction or amelioration of symptoms of the disease or condition, or the delaying of the progression of the disease or condition. "Treatment" does not necessarily indicate complete eradication or cure of the disease or condition, or associated symptoms thereof.

As used herein, "prevent" and similar words such as "prevented", "preventing" or “prophylactic” etc., indicate an approach for preventing, inhibiting, or reducing the likelihood of the occurrence or recurrence of, a disease or condition. It also refers to delaying the onset or recurrence of a disease or condition or delaying the occurrence or recurrence of the symptoms of a disease or condition. As used herein, "prevention" and similar words also includes reducing the intensity, effect, symptoms and/or burden of a disease or condition prior to onset or recurrence of the disease or condition.

FIGURES

The invention is further described by the following figures. These are not intended to limit the scope of the invention but represent preferred embodiments of aspects of the invention provided for greater illustration.

Fig.1 : T cell-intrinsic NR2F6 directly antagonizes metabolic fitness in vivo and in vitro. Schematic outline of NR2F6 as an inducible exhaustion factor in tumor immune responses.

Fig.2 : NR2F6 acts as an inducible and highly confined "immune checkpoint " within the solid tumor immune microenvironment (TIME). This allows the inventive NR2F6-modified CAR-T cells to trigger a mostly “compartmentalized immunogenic cell death therapy outcome”, and, subsequently, cross-priming of tumor antigens to the endogenous polyclonal T cells only in close proximity to the solid tumor site. As a direct consequence, this reduces systemic toxicity/irAE of the inventive NR2F6-modified CAR-T therapy regimens.

Fig.3 : Loss of NR2F6 leads to metabolic shifting and increases metabolic fitness of CAR -T cells during chronic stimulation.

Fig.4 : NR2F6 depletion boosts cytotoxic effector function of CAR-T cells in vitro.

Fig.5 : NR2F6 is upregulated during chronic stimulation and NR2F6 depletion boosts cytotoxic effector function of CAR-T cells and promote ICD of tumor cells in co-cultures in vitro. Fig.6 : NR2F6 knockout CD8⁺ T cells maintain a gene signature of both (A) an activated T cell phenotype and (B) an intact killing machinery despite repetitive stimulation that leads to exhaustion.

Fig.7 : Acute depletion of NR2F6 in CAR-T cells improves anti-tumor activity and significantly prolongs survival in immunocompetent mice. NR2F6 acute gene editing in CAR-T cells (when compared to CAR-T cells of the prior art) sustains effector functions against antigenically heterogeneous PanC-02 tumor loads allowing durable tumor growth inhibition in fully immunocompetent mice in vivo. Of note and additionally, no obvious irAE have been observed in the NR2F6-modified CAR-T therapy group.

Fig.8: Efficiency of NR2F6-modified CAR-T therapy requires a profound secondary adaptive immune response. Only in immunocompetent wildtype mice (see Fig.7A-C above), but not in Rag1-/- mice (that completely lack an endogenous adaptive immune system), inhibition of NR2F6 in CAR-T cells leads to superior anti-tumor immunity against EpCAM antigen heterogenous PanC-02 tumor loads, implying the strict requirement of the endogenous immune system for the NR2F6-modified CAR-T tumor growth benefit (epitope spreading, leading to a secondary immune response by the endogenous adaptive immune system present only in immunocompetent wildtype mice).

Fig.9 : Albeit providing a robust and long-term tumor growth benefit (see Fig. 7A-C above), CAR-T GFP+ are rejected by the host immune system of immunocompetent recipients within the first week after infusion. This can be explained by ICD-mediated epitope spreading that occurred within this first week, which is selectively promoted by the NR2F6-modified CAR-T therapy, but not, or at least to a much lesser extent, by the conventional WT CAR-T therapy. The finding validates a time-boxed strategy employing the inventive NR2F6-modified CAR-T cells as transient trigger of a secondary immune response by the endogenous T cell compartment to promote durable host-protective anti-tumor immune response as an innovative strategy.

Fig.10: Complete NR2F6-modified CAR-T responders reject PanC02 antigen negative tumors upon rechallenge after 5 and 10 months due to epitope spreading. Remission-free survivor mice (similar to the EpCAM-positive tumors) show efficient EpCAM-negative tumor detection and clearance upon rechallenge, validating that an immunogenic tumor cell death (ICD) has been induced by the transient CAR-T therapy (see Fig. 9 above demonstrating the transient nature of the NR2F6-modified CAR-T therapy), that has triggered a secondary immune response by effective tumor antigen cross-priming (epitope spreading). Furthermore, complete NR2F6- modified CAR-T responders demonstrate anti-tumor memory, that can be transferred to EpCAM- negative tumor-bearing WT recipient: POC of immunological memory for host protective tumor control of EpCAM negative tumors (tumor-antigen agnostic effect).

Fig.11 : NR2F6-modified CAR-T treated tumor bearing mice show enhanced anti-tumor activity of the endogenous immune system. “Immunogenic Cell Death Phenomenon” mediated by the inventive NR2F6-modified CAR-T cells activate innate immune cells at the solid tumor microenvironment as a crucial and essential prerequisite for triggering robust cross-priming of multiple tumor antigens. Proof of concept for tumor antigen cross-priming: NR2F6-modified (but not WT) CAR-T therapy can effectively “warm up” a deserted "cold" TIME as a prerequisite for tumor antigen cross-priming. NR2F6^crisP^r'^/'.sgO3 (but not control WT) CAR-T treated mice, capable of enhanced anti-tumor activity, effectively prime the endogenous T cell compartment against multiple PanC02 tumor antigens within 3 weeks of NR2F6-modified CAR-T therapy in vivo. Fig.12: A//?2A6-modified CAR-T-treated complete responders mount a superior immune response against EpCAM negative tumors as determined by scRNAseq of CD45+ immune cells.

Fig.13: Sustained TCF1 DNA binding in NR2F6^c,'^sp’-¹- CAR-T cells during chronic stimulation. Human NR2F6 directly blocks DNA binding and transactivation of human TCF-1 . TCF-1 is the established key regulator of clonotypic memory differentiation but also and exhaustion resistance in CD8⁺ T cells. This biochemical mechanism may provide a mechanistic explanation for the observed metabolic changes and, subsequently, the exhaustion-resistance of NR2F6-/- CAR-T cells.

Fig.14: When stimulated by the antigen receptor, human NR2F6 gene-edited primary T cells show significantly enhanced effector responses that can be further potentiated by combinatorial inhibition of CBLB. Combinatorial NR2F6 and CBLB inhibition in primary human T cells cooperate in boosting antigen-receptor signaling in primary human T cells, validating the inventive combinatorial CAR-T therapy regimen concept.

Fig.15: Schematic cartoon of the “Immunogenic Cell Death Phenomenon” mediated by the inventive NR2F6-modified CAR-T cells that activate innate immune cells at the solid tumor immune microenvironment, triggering robust cross-priming of the endogenous immune system by epitope spreading. Subsequently, this promotes a secondary and polyclonal T cell-based rejection also of CAR-targeted antigen-negative tumors, frequently present in antigen- heterologous solid tumors.

Fig.1 : T cell-intrinsic NR2F6 directly antagonizes metabolic fitness in vivo and in vitro. Schematic outline of NR2F6 as an inducible exhaustion factor in the solid tumor immune responses. The NR2F6 high CD8⁺ T cells are more exhausted when compared to NR2F6 low CD8⁺ cells thereby representing the terminal state of CD8⁺ T cell exhaustion. This validates a central role of NR2F6 in terminal T cell exhaustion, offering strategies to increase responses to CAR-T immunotherapy by NR2F6-modified CAR-T cells. Nutrient deprivation in the TIME antagonizes metabolic fitness of TILs and, together with persistent tumor antigen exposure, results in a dysfunctional T cell differentiation state termed ..exhaustion". Loss of NR2F6 leads to metabolic shifting and increases metabolic fitness of CAR-T cells during chronic stimulation. Without being bound to theory, it is concluded that NR2F6 signaling axis that is massively upregulated at the solid tumor site is a dominant driver of the T cell metabolic dysfunction in cancerous tissue.

Fig.2 : NR2F6 acts as an inducible and highly confined "immune checkpoint "at the solid tumor immune microenvironment (TIME). A) Massive NR2F6 gene induction in T cells at the solid tumor site: Inflammatory signals localized to the murine TIME lead to >500% NR2F6 upregulation in Tumor-infiltrating lymphocytes (TILs). As a consequence, NR2F6 is highly localized to the solid tumor site mainly impairing effector activity of TILs but not peripheral T cells. This allows the inventive NR2F6-modified CAR-T cells to trigger a mostly “compartmentalized immunogenic cell death therapy outcome”, and, subsequently, cross-priming of tumor antigens to the endogenous polyclonal T cells only in close proximity to the tumor site. As a direct consequence, this reduces systemic toxicity/irAE of the inventive NR2F6-modified CAR-T therapy regimens. B) Selective upregulation (>300%) of NR2F6 in primary T cells during repeatedly antigen-stimulated in vitro (WT exhausted) but not in single antigen-stimulated cells (WT not exhausted). C) Mechanistically, i.e. concomitant RAR and/or RXR activation as an example of inflammatory signals within the TIME further boosts NR2F6 expression in repeatedly antigen-stimulated T cells, leading to an additional >300% NR2F6 upregulation.

Fig.3 : Loss of NR2F6 leads to metabolic shifting and increases metabolic fitness of CAR-T cells during chronic stimulation. A-C) NR2F6 gene-edited T cells show a superior stress test profile indicating that NR2F6-modified T cells exhibit a higher OXPHOS activity in an exhausted and non-exhausted state. D) Similarly, our data demonstrate a benefit in extracellular acidification rate (EACR) of NR2F6-/- CD8+ T cells. In summary T cell-intrinsic NR2F6 directly antagonizes metabolic fitness. B-D) Metabolic rate as measured by Seahorse Cell Mito Stress Test analysis of oxygen consumption rate (OCR, see B&C) and extracellular acidification rate (ECAR, see D) of control or NR2F6-deficient CAR-T cells under resting and challenge conditions on day 4 of chronic stimulation. Data are shown for n = 6 donors in duplicates from two independent experiments. FCCP=Carbonyl cyanide p-trifluoro-methoxyphenyl hydrazone, AA+Rot=Antimycin A+Rotenone; two-way ANOVA [B+D], two-tailed unpaired Student's t-test [C], E) PCA blot from bulk RNAseq data on d4 of chronic stimulation. F) Gene ontoloty (GO) terms of normalized RNAseq data showing significantly enriched pathways in NR2F6'^/' CAR-T cells compared to wildtype control CAR-T cells. G) GSEA of NR2F6-deficient CAR-T cells compared with control cells after 4 days of serial killing using the hallmark gene collection. Normalized enrichment scores (NES) and p values are shown. A positive NES indicates that the gene set was enriched in NR2F6-deficient cells. Data shown as mean±SEM *p<0.05 **p<0.01 ***p<0.001

Fig.4 : NR2F6 depletion boosts effector function and delays exhaustion formation during chronic stimulation. (A) NR2F6 knockout in CAR-T cells sustains cytotoxic effector function during serial killing of PanC02 tumor cells in vitro. At different effector-to-target (E:T) ratios as indicated, the effector profiles of T cells can be significantly enhanced by NR2F6 gene editing: (B) NR2F6 gene editing rescues at least 80% of the cytotoxic effector function CAR-T cells compared to not gene edited CAR-T cells. (Measurements done in serial killing rounds as indicated). C) CAR structure, scFv = single chain variable fragment, h = hinge, TM = transmembrane; D) CAR transduction efficiency is comparable in wildtype and NR2F6'^/_ CAR-T cells E) Experimental setup of serial killing. F) IFNy production after 24-hour co-culture with tumor cells 5 days after T cell activation. Control and NR2F6-deficient EpCAM-28^ CAR-T cells were stimulated 5:1 with PanC02-EpCAM. Data are mean ± SEM from n = 5 replicates, derived from two independent experiments. Two- tailed unpaired Student's t-test. G) The cytotoxicity of control and NR2F6-deficient EpCAM-28^ CAR-T cells against GFP+ PanC02-EpCAM cells was assessed following serial stimulation, beginning five days after T cell activation at a 5:1 ratio of T cells to tumor cells at 48-hour intervals in media without cytokines. The data presented are the mean ± SEM of n = 9 replicates from four independent experiments using two way Anova. H) IFNy (left) and Granzyme B (right) release in the supernatant after each consecutive re-stimulation round (every 48h) with GFP+ PanC02- EpCAM tumor cells beginning 5 days after T cell activation using Bioplex. Data are mean ± SD using multiple unpaired Student’s t test from n = 6 replicates, derived from two independent experiments. I) Flow cytometry analysis of Tcm-like CD62L+ CD44+ CAR-T cells on day four of serial killing; n= 6 from two independent experiments; unpaired Student’s test .J) Representative image (left) and ratio (right) of progenitor (Tim3-Ly108+) and terminally (Tim3+Ly108-) exhausted T cells during serial killing assay (d4 = 2^nd round and d8 = 4^th round after serial killing start) of wildtype and NR2F6'^/_ CAR-T cells. Data are mean ± SEM of n = 6 donors. Mann-Whitney test. Tpex, progenitor exhausted T cells; Ttex, terminally exhausted T cells; Tex transient exhausted T cells. K) Flow cytometry analysis (left) and geometric mean fluorescent intensity (gMFI) of TCF7 (right) in control and NR2F6-deficient CAR-T cells 9 days after T cell activation, 4 days after serial killing start. L) PCA blot from RNAseq data obtained from d8 of serial killing / d13 after T cell activation. M) Gene ontoloty (GO) terms of normalized RNAseq data showing significantly enriched pathways in NR2F6'^/' CAR-T cells compared to wildtype control CAR-T cells 8 days after serial killing start / d13 after T cell activation. Data shown as mean ± SEM *p<0.05 **p<0.01 ***p<0.001.

Fig.5 : NR2F6 mRNA is upregulated during CAR-T cell production and chronic stimulation in vitro. A) NR2F6 expression is induced during CD8⁺ CAR-T cell production in vitro (n = 6). B) CAR-T cell expansion during production on day 5 after T cell isolation, however, is comparable between the two genotypes. C) NR2F6 expression is induced upon chronic antigen-specific stimulation with PanC02-EpCAM tumor cells (n=3; dO of chronic stimulation = d5 of CAR production). D) No altered expansion phenotype between wildtype and NR2F6-deficient CAR-T cells during chronic stimulation has been observed. E) Quantification of Gasdermin E (GSDME) clipping during coculture of tumor cells and NR2F6-deficient or wildtype CAR-T cells harvested after 4 days of chronic stimulation and re-stimulated for 6 hours with PanC02-EpCAM tumor cells (biological replicates, n=6, two independent experiments). *p<0.05 **p<0.01 ***p<0.001. As a remarkable result, NR2F6-deficient CAR-T cells induce pyroptosis in the co-cultered tumor cells; pyroptosis represents the major form of ICD. Mechanistically, as demonstrated lege artis by tumor-intrinsic GSDME clipping, increased production of GRZB and IFNy leads to the desired qualitative switch from apoptosis to pyroptosis. These data represent a functional validation of the NR2F6 pathway for the induction of immunogenic cell death (ICD) of tumor cells by NR2F6-modified CAR-T cells.

Fig.6 : NR2F6 knockout CD8⁺ T cells maintain a gene signature of both (A) an activated T cell phenotype and (B) and intact killing machinery despite repetitive stimulation that leads to exhaustion. (A) RNA-seq analysis reveals distinct differences between NR2F6-/- and wildtype CD8⁺ T cells and a skewing towards a less exhausted and more cytotoxic phenotype. (B) Upregulated granzyme genes GzmA, GzmC and GzmD, all established to exert key role in cytotoxic killing of tumor cells, are separately depicted.

Fig.7 : Acute depletion of NR2F6 in CAR-T cells improves anti-tumor activity and significantly prolongs survival in immunocompetent mice. NR2F6 acute gene editing in CAR-T cells sustains effector functions allowing durable tumor growth inhibition of heterogenous PanC-02 tumor loads (EpCAM+/EpCAM- at a ratio of approx. 75:25) allowing durable tumor growth inhibition in fully immunocompetent mice in vivo. Additionally, no obvious irAE have been observed in the NR2F6- modified CAR-T therapy group. (A) Analysis of tumor clearance and survival. Tumor area of wildtype mice injected subcutaneously with PanC02-EpCAM cells and treated 2 days later with genetically modified CAR-T cells using CRISPR/Cas9 (NTC vs NR2F6^{crispr sg04}) compared to the non-CAR polyclonal gene modified CD8⁺ T cell receiving as well as no therapy group. Tumor area was measured by caliper. Two-way ANOVA test with Dunnett’s multiple comparison test, Log-Rank test, n = 10 mice pooled from two independent experiments. Data shown as mean±SEM *p<0.05 **p<0.01 ***p<0.001. Remission is achieved in a preclinical in vivo mouse model of solid tumors: NR2F6 CRISPR/Cas9 gene-edited anti-EpCAM CAR-T cell therapy inhibits tumor growth by at least 80% (compared to non-edited CAR-T cells of the prior art) and leads to overall survival in at least 50% of the mice tested. (B) T umor growth curves of single mice within the therapy groups. Of note, after treatment with NR2F6 gene-edited anti-EpCAM CAR-T cells, no significant irAEs are observed. C) Overall survival analysis of PanC02-EpCAM tumor-bearing mice treated with NTC or NR2F6^crispr'^/_ CAR-T cells using a non-sequence- overlapping independent NR2F6 targeting sgRNA; n=5 from one independent experiment; D) CAR-T cell frequency in the peripheral blood of CAR-T treated tumor-bearing mice on day 6 and 13 after ACT ; n=3 from one independent experiment; multiple unpaired Student’s t test. Of note and albeit providing a robust and long-term tumor growth benefit, CAR-T are completely rejected by the host immune system of immunocompetent recipients. E) Editing efficiency of NR2F6- targeting sgRNAs determined by tracking of indel by decomposition (TIDE) analysis; n=3 from 3 independent experiments F) As a specificity control for the investigation EpCAM CAR-T system, killing assay have been performed using PanC02 EpCAM positive and negative, tumor cells respectively.

Fig.8: Efficiency of NR2F6-modified CAR-T therapy requires an intact secondary adaptive immune response. Analysis of tumor clearance (A) and survival (B) of Ragl'⁷' mice injected subcutaneously with 5 x 10⁵ PanC02-EpCAM cells and treated 2 days later with genetically modified CAR-T cells (NTC vs NR2F6^crisP^r'⁷'^s9°⁴ or NR2F6^crisP^r'⁷'^s9°³) compared to the no therapy group that received PBS. Tumor volume was measured by caliper. Two-way ANOVA test with Dunnett’s multiple comparison test (A), Log-Rank test (Bj. n = 6 mice, representative of two independent experiments. Only in immunocompetent wildtype mice (but not in Rag1-/- mice that completely lack an endogenous adaptive immune system), depletion of NR2F6 in CAR-T cells leads to superior anti-tumor immunity against PanC-02 tumor loads, implying the strict requirement of the endogenous adaptive immune system for the NR2F6-modified CAR-T tumor growth benefit (i.e. ICD-mediated epitope spreading, leading to a secondary immune response by the endogenous adaptive immune system present only in immunocompetent wildtype mice).

Fig.9 : CAR-T GFP+ are rejected by the host immune system of immunocompetent recipients within the first week after infusion. Albeit providing a robust and long-term tumor growth benefit (see Fig. 6A), CAR-T GFP+ are rejected by the host immune system of immunocompetent recipients within the first week after infusion. This can be explained by ICD-mediated epitope spreading, which is selectively promoted by the NR2F6-modified CAR-T therapy, but not, or at least to a much lesser extent, by the conventional WT CAR-T therapy. The finding validates a time-boxed transient strategy employing the inventive NR2F6-modified CAR-T cells as trigger of a secondary immune response and improve polyclonality of the endogenous T cell compartment. This underlying key mechanism allows a durable immunotherapy outcome leading to host- protective anti-tumor immune response as an innovative strategy.

Fig.10: Complete NR2F6-modified CAR-T responders reject PanC02 antigen negative tumors upon rechallenge after 5 and 10 months due to epitope spreading. A) Experimental setup and timeline. B and C) analysis of tumor clearance. Remission-free NR2F6-modified CAR-T cells treated complete responders (see Fig. 7A&B above) show similarly efficient tumor clearance upon rechallenge of both EpCAM-positive and EpCAM-negative PanC02 tumor loads, validating that an immunogenic tumor cell death has been induced by the time-boxed transient CAR-T therapy (see Fig. 8), that has triggered a secondary and polyclonal immune response by effective tumor antigen cross-priming (epitope spreading). Furthermore, complete NR2F6-modified CAR-T responders demonstrate anti-tumor memory, that can be transferred to EpCAM-negative tumorbearing WT recipient: proof of concept (POC) of a durable immunological memory for host protective tumor control of EpCAM negative tumors (epitope spreading as a tumor-antigen agnostic therapy outcome). Tumor growth (B) and survival (C) of NTC- and NR2F6-modified CAR-T-treated complete responders (CR) from the initial induction of PanC02-EpCAM+ tumor cells (see Fig.7A&B above) rechallenged 150 days later with PanC02-EpCAM-positive (left flank) and PanC02-EpCAM-negative (right flank) tumor cells (1^st rechallenge). Naive age-matched C57BL6 mice were used as controls (naive wildtype). D and E) As all NR2F6^crisP^r'⁷' CAR-T cell treated mice survived the 1^st rechallenge, they again received a 2^nd rechallenge 150 days later (300 days after initial tumor induction) with 1 PanC02-EpCAM- tumor cells. Naive age-matched C57BL6 mice were used as controls (as PanC02 tumor antigen-naive wildtype controls). Tumor growth (D) and survival (E) of naive PanC02-EpCAM^negative tumor bearing wildtype mice receiving intravenously splenocytes from either NR2F6-modified CAR-T-treated complete responders (CR) euthanized on d305 or splenocytes from PanC-naive control mice. F) IFNy release after 24-hour co-culture of splenocytes with tumor cells. Naive and 2^nd rechallenge CR endogenous T cells were stimulated 5:1 with PanC02-EpCAM+ tumor cells. Data are mean ± SEM from n = 4. Data shown as mean±SEM *p<0.05 **p<0.01 ***p<0.001. Thus, a notable finding is that complete NR2F6-modified CAR-T responders have developed a secondary and polyclonal anti-tumor memory for tumor control that can be transferred to EpCAM-negative WT tumor recipients. These results provide proof of concept for a long-term immunological memory of the endogenous T cell compartment induced secondary to the NR2F6-modified CAR-T therapy of the invention for host- protective tumor control of EpCAM-negative tumors (in a tumor antigen-agnostic effect).

Fig.11 : NR2F6-modified CAR-T treated tumor bearing mice show enhanced anti-tumor activity of the endogenous immune system. “Immunogenic Cell Death Phenomenon” mediated by the inventive NR2F6-modified CAR-T cells activate innate immune cells as a crucial and essential prerequisite for triggering robust cross-priming of multiple tumor antigens. Proof of concept for tumor antigen cross-priming: NR2F6-modified (but not WT) CAR-T therapy can effectively warm up a deserted "cold" TIME as a prerequisite for tumor antigen cross-priming. NR2F6^crispr'^z' (but not control WT) CAR-T treated mice, capable of enhanced anti-tumor activity, effectively prime the endogenous T cell compartment against multiple PanC02 tumor antigens within 3 weeks of NR2F6-modified CAR-T therapy in vivo. A) Experimental setup and timeline. Ly5.1 mice were used as recipients to track Ly5.2 CAR-T cells from Cas9 transgenic mice. B-E) UMAP and FLOWsome analysis of spleens from NTC' and NR2F6^crispr'^z'^sg03 CAR-T cell treated wildtype tumor-bearing mice on d8 after tumor inoculation. Expression of 16 markers was analyzed by full spectrum flow (Cytek Aurora). NTC' and NR2F6^crispr'^/' groups concatenated leading to 7 divergent populations (C), and assigned by marker intensity (D). Genotype specific UMAPs of spleens from NTC' and NR2F6^crispr'^z' CAR-T treated wildtype tumor bearing mice. E) Data are pooled from three spleens. F) Manual gating of CD11 c+ CD8⁺ XCR1 + cDC1 of multicolor flow data G) Granzyme B ELISPOT. Wild type mice bearing PanC02-EpCAM+ tumors (n=5) treated without CAR-T cells (PBS) or with CD8⁺ CAR-T cells (NTC' and NR2F6^crispr'^/ . Data shown as mean±SEM *p<0.05 **p<0.01 ***p<0.001

Fig.12: NR2F6-modified CAR-T-treated complete responders mount a superior immune response against EpCAM negative tumors determined by scRNAseq. A) CD45⁺ TILs from tumor-bearing NR2F6-modified CAR-T-treated complete responders (CR) (2^nd rechallange) and naive mice were used for scRNA-seq and scTCR-seq. Cell annotation and cluster formation showing naive vs. CR. B) Stacked charts showing proportions of each cell cluster. Asterisks mark significant differences between groups. C) Dotplots showing differential expression of signature genes of proliferating and exhausted CD8⁺ T cells comparing CR vs. naive. D) ORA terms showing enriched pathways in intratumoral proliferating CD8⁺, exhausted CD8⁺, macrophages and cDC1 cells. E) TCR scRNA-seq; Gini index and Shannon entropy of TCR clonality were compared in naive and CR tumor bearing mice, n = 4. *p<0.05 **p<0.01 ***p<0.001 .

Fig.13: NR2F6 directly blocks DNA binding and transactivation of TCF-1. Sustained TCF1 DNA binding in NR2F6^crispr'^z' CAR-T cells during chronic stimulation. TCF-1 is the established key regulator of clonotypic memory and exhaustion resistance in CD8⁺ T cells. This biochemical mechanism may, at least in part, provide a mechanistic explanation for the observed metabolic changes and, subsequently, the exhaustion-resistance of NR2F6-/- CAR-T cells. A) NTC' and NR2F6^crispr'^/'^mouse CAR-T cells were kept in culture with IL7 and IL15 or co-cultured with PanC02- EpCAM for 4 days in vitro (chronic stimulation). Nuclear extracts were isolated, and electromobility assay (EMSA) was performed for TCFI . DNA binding is strongly enhanced in stimulated NR2F6^crisP^r'^/' CAR-T cells compared to control. Equal loading of nuclear cell extracts was controlled by immunoblotting (WB) of HDAC. One representative experiment out of three is shown. B) EMSA quantification of TCF1 DNA binding in PanC co-cultured CAR-T cells. C&D) Consistently, a functional cross-talk between human NR2F6 and human TCF1 at the transcriptional level was confirmed by promoter reporter assays of TCF-1 enhancer driven TOPFIash promoter reporter (TOPFIash is a luciferase reporter that contains a minimal promoter coupled to TCF1 -binding sites upstream of the firefly luciferase gene) using the human leukaemic cell line Jurkat, ectopically expressing human NR2F6 or human TCF1 WT or mutant proteins. This is shown by both EMSA (C) and transcriptional activity (D) readouts. Taken together, this NR2F6:TCF1 cross-talk appears to be centrally involved in regulating the metabolic fitness and exhaustion resilience of CD8⁺ T cells. Mechanistically, NR2F6 acts as a cofactor and/or modulator of TCF1 and is specifically involved in modulating TCF1 DNA binding during chronic antigen stimulation.

Fig.14: When stimulated by the antigen receptor, human NR2F6 gene-edited primary T cells show significantly enhanced effector responses that can be further potentiated by combinatorial inhibition of CBLB. Combinatorial NR2F6 and CBLB inhibition in primary human T cells acts synergistically in boosting antigen-receptor signaling in primary human T cells, validating the inventive combinatorial CAR-T therapy regimen concept. (A) Scheme of experimental set-up. Knockout of NR2F6 via lentiviral integration of Cas9, sgRNA and puromycin resistance gene in human leukaemic Jurkat cells (B) Confirmation of NR2F6 knockout by western blot (n = 5 replicates from 4 passages). (C) Scheme of experimental set-up. Knockout of NR2F6 via lentivirus in primary human T cells (D) Analysis of CD69, IL-2, IFNy and GrzB expression by flow cytometry upon 4h restimulation with 0,3125 ug/mL anti-CD3 and 1 ug/mL anti-CD28 antibodies in the presence of golgi stop/plug. n = 5 donors. Data are represented as mean ± SEM with individual data points. *p<0.05; **p<0.01 ; ***p<0.001 ; ****p<0.0001 ; n.s. =not significant; two- tailed paired t-test).

Fig.15: Schematic illustration of the "immunogenic cell death (ICD) effects" mediated by the inventive NR2F6-modified CAR-T cells, which activate innate immune cells in the immune microenvironment of solid tumours, triggering robust cross-priming of the endogenous T cell compartment, tumour epitope spreading and, subsequently, immune cell infiltration and rejection also of CAR-targeted antigen-negative tumours, which are frequently present in antigen- heterologous solid tumours. As a result, ICD mediated by the inventive NR2F6-modified CAR-T cells enhances cross-priming of multiple tumour antigens (advantageously tumour antigens expressed either on the surface or intracellularly in tumour cells), which significantly broadens the TCR repertoire of the endogenous immune system's anti-tumour T cells (polyclonality), thereby orchestrating a secondary and more robust systemic immune response. This demonstrates the ability of the inventive NR2F6-modified CAR-T cells to induce a tumour antigen-specific polyclonal immune memory response, thereby providing a durable immunotherapeutic outcome to enable efficient clearance of heterogeneous solid tumour cells.

EXAMPLES The invention is demonstrated through the examples disclosed herein. The examples provided represent particular embodiments and are not intended to limit the scope of the invention. The examples are to be considered as providing a non-limiting illustration and technical support for carrying out the invention.

Methods

Animals

Female C57BL/6, B6(C)-Gt(ROSA)26Sorem1 .1 (CAG-cas9*,-EGFP)Rsky/J , Ly5.1 and NR2F6-⁷- mice were housed in cages up to 5 or 10 mice under specific pathogen free (SPF) conditions and bred in-house at the animal facility in Innsbruck. Animals were controlled frequently by the stuff of the animal facility and us. All animal experimentation was performed according to European guidelines and approved by the Austrian federal ministry of education, science and research (2023-0.203.973).

Cell lines

The PanC02-EpCAM cell line, originating from a mouse pancreatic ductal adenocarcinoma, was a kind gift of Sebastian Kobold from the LMU (Munich). PanC02-EpCAM negative were enriched using flow sorting. PanC02-EpCAM expressing the fluorescent protein GFP were generated by retroviral transduction using the pMP71-GFP (Sebastian Kobold) construct. GFP+ PanC cells were enriched using flow sorting. Mouse tumor cell lines were maintained in DMEM + 10% fetal calf serum (FCS) + 2mM L-Glutamine (L-Glut) + 100U/mL PenStrep, further referred to as DMEM+++. PlatinumE (PlatE) cells were obtained from Cell Biolabs, Inc and cultured in DMEM+++ supplemented with 1 g/mL Puromycin (Merck) and 10pg/mL Blasticidin (Sigma) to ensure transgene expression, further referred to as PlatE medium.AII cell lines were generally cultured in T175 tissue culture flasks (Sarstedt) and passaged every 2 days and routinely checked for mycoplasma infection.

Cell line passaging and harvest

Adherent cell lines were passaged as follows: cell cuture vessel, typically a T 175cm tissue culture flask, was washed once with 6mL warm 1x phosphate buffer saline (PBS) and then dissociated using 3mL T rypLE Express (Thermofisher) for a few minutes at 37°C until cells start to float. Reaction was stopped by adding twice the amount of cell culture medium. Cell suspension was transferred to a Falcon tube and centrifuged at 1250rpm for 5 minutes at room-temperature (RT). Cells were then used for assays, in vivo experiments or resuspended in 1 mL cell culture medium and splitted accordingly: tumor cells typically 1 :10 and PlatE 1 :5 depending on application.

Primary mouse CD8⁺ T cells isolation

Primary mouse CD8⁺ T cells were isolated by negative selection using the CD8a⁺ T cell isolation kit (Miltenyi). In brief, mice were sacrificed and spleen as well as lymph nodes were excised. After pressing organs through a sieve to achieve a single cell suspension, cells were resuspended in 3mL Erylysis-buffer per spleen and incubated for 5min at RT. Reaction was stopped by adding twice the amount of buffer C (PBS supplemented with 0.5% bovine serum albumin [BSA] and 2mM ethylenediaminetetraacetic acid [EDTA]). Splenocytes were counted and incubated with antibodies and magnetic beads according to manufacturer’s instructions. CD8⁺ T cells were separated from the rest of the cells by applying the cell suspension on LS columns (Miltenyi) placed onto a strong magnet. Columns were flushed twice with 3mL of buffer C and flow through was collected. Enriched CD8⁺ T cells were then counted with LUNA automated cell counter (Logos Biosystems) and used for downstream applications. Enriched T cells were frequently checked for purity using FACS analysis.

Plasmids and constructs

For retroviral production, the pMP71 backbone was used for all transfections. The second generation ocmEpCAM chimeric antigen receptor (CAR) consists of a single-chain variable fragment (scFv) targeting mouse EpCAM protein, fused to a CD8 hinge and a CD28 transmembrane region. Intracellularly, it consists of a CD28 co- and a CD3zeta stimulatory domain. For detection, a myc-tag was inserted between the scFv and the hinge region. The pMP71-GFP plasmid was used as a control and to transduce tumor cell lines. All plasmids were visualized and generated on https://benchling.com.

Transfection of PlatE cells and virus production

For optimal viral production, PlatE cells were split three times a week and cultured not more than 12 weeks after thawing as recommended by the supplier. The day prior to transfection, 8x10⁵ PlatE cells were seeded into tissue-culture treated 6-well plates in PlatE medium and incubated overnight at 37°C 5% CO2 to reach a confluency of around 70%. On the day of transfection, PlatE medium was discarded and 3mL DMEM supplemented with 10% FCS and 2mM L-Glut without antibiotics was added. Transfection was carried out using the calcium precipitation method: per well, 15pL calcium chloride (2.5M) was mixed with 18pg of transfer plasmid (pMP71-CAR or pMP71-CAR-GFP) and ddH20 to a final volume of 150pL. This was slowly added into 150pL transfection buffer (1 ,6g NaCI, 74mg KCI, 50mg Na2HPO4, 1g HEPES [all Sigma Aldrich], in 100mL H2O, pH 7.1) in a dropewise manner while vortexing constantly. The mix was incubated for 30min at room temperature. 300pl of the transfection mix was then added onto PlatE cells and incubated for 6 hours at 37°C 5% CO2. After incubation, medium was discarded and 3mL DMEM +++ were added and plates were put for at 37°C 5% CO2 for 42 hours.

CD8⁺ T cell activation and culturing for CAR transduction

12-well plates were incubated with 5pg/mL ocCD3 antibody (BioXCell) solution in PBS overnight at 4°C. After negative selection on day 0, CD8⁺ T cells were resuspended at 3*10⁶/mL in CAR RPMI (RPMI 1640 + 10% FCS + 100UI/mL penicillin/streptomycin + 2mM L-Glutamine + 1 mM sodium pyruvate + 1 mM HEPES + 50pM 2-mercaptoethanol). Coated plates were washed twice with 1x PBS before seeding 3x10⁶ T cells into pre-coated wells in CAR RPMI supplemented with 1 g/mL ocCD28 and 10ng/mL IL-2. Plates were incubated overnight at 37°C 5% CO2.

Retroviral transduction of CD8⁺ T cells and CAR detection

In general, CAR-T cells used for in vivo experiments were transduced with a CAR together with a GFP reporter, whereas CAR-T cells for in vitro experiments were transduced with a CAR only.

For viral transduction, non-tissue culture treated 24-well plates (Corning) were coated with 12.5pg/mL Retronection solution (Takara) in 1x PBS overnight at 4°C. The next day, plates were blocked with 2% BSA fraction V solution in ddH2O for 30min at 37°C. In the meanwhile, supernatant containing CAR viral particles was harvested from PlatE cells and filtered through a 0.45pm cellulose-acetate syringe filter (Sartorius). 2mL fresh DMEM+++ was added to PlatE cells to harvest supernatant again after 24 hours. After blocking, plates were washed with 1x PBS supplemented with 25mM HEPES and finally 2mL of viral supernatant was added to each well of the RN-coated plate and spun at 3000xg for 2 hours at 4°C.

One hour before the end of the spin, CD8⁺ T cells were prepared. Approximately 22 hours after plate-bound activation, T cells were harvested and counted using LUNA automated cell counter. T cells were adjusted to 1x10⁶/mL in CAR RPMI supplemented 10ng/mL IL-2 and mouse ocCD23/CD28 activation beads (Gibco) at a ratio of one bead per T cell (25pl bead solution/mL). After the spin, viral supernatant was discarded and 1 mL T cell suspension was added per well and spun at 800xg for 30min at 32°C in a table-top centrifuge. Plates were then put at 37°C 5% CO2 overnight. The next day, viral supernatant was harvest again from PlatE cells and filtered through a 0.45pm cellulose-acetate syringe filter (Sartorius). 0.5mL of the viral particle containing supernatant was mixed with 0.5mL CAR RPMI and 1 ml of the mix was added directly to T cells. Plates were then spun a second time at 800xg for 30min at 32°C and incubated for another 5 hours. After this last incubation, T cells were harvested, activation beads were removed and T cells were counted. Cells were then adjusted to 1 .5x10⁶/mL in CAR RPMI supplemented with 10ng/mL interleukin 15 (IL-15) as well as interleukin 7 (IL-7) and seeded into 24- or 12-well plates. Cultures were maintained at 1.5x10⁶/mL in IL-15 and IL-7 containing medium.

Transduction efficiency was assessed either by using an amyc-FITC antibody (Miltenyi; 1 :400) or GFP by flow cytometry.

For in vitro experiments CAR-T were used on the 5^th day after isolation, whereas for in vivo experiments CAR-Ts from the 7^th day were used.

Genome editing of CAR-T cells using CRISPR/Cas9

For acute depletion of NR2F6 in CD8⁺ (CAR) T cells, the CRISPR/Cas9 system was applied. In this case, (CAR) T cells from Cas9 transgenic mice were electroporated with synthetic NR2F6 targeting single guide (sg)RNAs (Horizon Discovery) 48 hours after T cell activation using the Amaxa mouse T cell nucleofactor kit on the nucleofector 2b device (both Lonza Biosciences). In this study, different kinds of guides with different cut sites in the NR2F6 gene were used. sgRNA 3 (sg03) was used directly, whereas crispr (cr)RNA 4 (crRNA 4) and the non-targeting control (NTC) crRNA were aligned with a corresponding tracer RNA to yield a fully competent sgRNA. Therefore, 200pM crRNA and 200pM tracer RNA solution in a ratio of 1 :1 were heated to 95°C in a thermocycler and left to cool at RT to allow sufficient complexing.

Five hours after the second spin transduction, transduced T cells were harvested and counted. 7x10⁶ to 1x10⁷ T cells were pelleted and transfected with 3.1 pM NR2F6 or NTC sgRNA solution. T cells were electroporated using the X-001 program on a Nucleofector 2b device (Lonza) and immediately recovered in transfection medium supplemented with 10% FCS, 2mM L-Glut, 100IU/mL Pen/Strep and medium component A and B in a 12 well plate according to manufacturer’s protocol. After a 1 -hour rest, 1 mL CAR RPMI containing 20ng/mL IL-7 and IL-15 was added per 1 mL T cells. T cells were passaged the next day.

NR2F6-crRNA4 5’ CTCAAGAAGTGCTTCCGGGT 3’ (SEQ ID No.1);

NR2F6-sgRNA3 5’ CCGCAATCTCAGCTACACCT 3’ U(SEQ ID No.2 )

Assessment of gene editing efficiency

On day 5 after electroporation, one million bulk transduced gene edited T cells were used for genomic (g)DNA isolation using the NucleoSpin Tissue kit (Macherey-Nagel) according to manufacturer’s instructions. NR2F6 sgRNA-targeted loci were PCR amplified using 100ng gDNA template from NR2F6 as well as NTC sgRNA treated samples in a 20pl reaction using the Phusion Flash High-Fidelity PCR Master Mix (Thermofisher). Cycling conditions were as follows: 10 seconds 98°C, 30 cycles of 10 seconds 66°C, 12 or 13 seconds 72°C follow by a final elongation step for 60 seconds at 72°C. Annealing temperatures were assessed online using the T_m calculator from Thermofisher (Thermo F. Tm calculator [Online tool] [Available from: https://www.thermofisher.com/at/en/home/brands/thermo-scientific/molecular-biology/molecular- biology-learning-center/molecular-biology-resource-library/thermo-scientific-web-tools/tm- calculator.html). Extension times were calculated according to manufacturer’s protocol (15 seconds per 1 kilobase). PCR products were cleaned up using the PCR Clean Up kit according to manufacturer’s instructions (New England Biolabs). Sanger sequencing was performed with purified DNA fragments by Eurofins Genomics. Data was analyzed using the Tracking of Indels by Decomposition (TIDE) algorithm (Brinkmann et al, 2014). sgRNA3 forward primer GGTGAGCCACTAAGTTGGCC (SEQ ID No.3), sgRNA3 reverse primer AGCACCTGCACGCATGTATC (SEQ ID No. 4). cRNA4 forward primer ATGGGGCTGGTGTTCTCAGA (SEQ ID NO. 5), crRNA4 reverse primer CTCTGAGTGGCTGCCTCCAGGT (SEQ IS NO. 6)

Tumor inoculation and ACT with gene-edited CAR-T cells

PanC02-EpCAM were cultured as described above and were checked for EpCAM expression by flow cytometry prior to injection. The day before, tumor cells were split 1 :2 to ensure sufficient engraftment in vivo. Mice used for in vivo experiments were shaved a few days before the injection of tumor cells to mitigate stress. To mimic the clinical situation, one million PanC02- EpCAM tumor cells were injected subcutaneously (s.c) into the right flank of 8- to 12-week-old immunocompetent female mice. Two days later when tumors reached an approximate size of 50mm³, tumor bearing mice were randomly assigned into different treatment groups based on their tumor size and injected intravenously (i.v.) with 3.6*10⁶ NTC or NR2F6 CRISPR/Cas9 gene- edited CAR-T cells in 50pl PBS. As a control, NTC as well as NR2F6 depleted polyclonal T cells were injected into tumor bearing mice. Tumor growth and mouse weight were assessed three times per week using a digital caliper and a scale, respectively, and tumor size was calculated with the following equation: !/₂ ^x (length x width²). Tumor measurement was performed in a blinded fashion. Mice were sacrificed when tumors reached over 1500mm³, a 20 percent weight loss was observed or when animals were multimorbid. For survival analysis, tumor sizes were collected in a Kaplan-Meier plot.

For experiments using immunodeficient RAGT^/_ mice, animals were inoculated with 5x10⁵ PanC02-EpCAM and were treated with 1x10⁶ gene edited CAR-T cells on day two after tumor injection.

Detection of CAR-T cells and analysis of innate immune cells in tumor bearing mice using multicolor flow cytometry

Ly5.1 recipient mice were challenged with PanC02-EpCAM tumors and treated with NTC or NR2F6 depleted CAR-T cells two or three days later as stated above.

For CAR-T cell detection over time, blood was collected from tumor bearing mice using a lancet and an EDTA containing vessel on day 6 and 13 after tumor injection. Red blood cell (RBC) lysis was performed using 40pL blood and in-house Erylysis buffer. Cells were washed thoroughly with buffer C before FcR-block (BD) was added to prevent unspecific binding. Cells were then stained for viability using fixable viability stain (FVS) 780 (1 :2000) and the following surface antibodies: CD45.1 Pb (Biolegend; 1 :200), CD45.2 PE (eBiosciences; 1 :100), CD4 V500 (BD Biosciences; 1 :400), CD8 APC (Thermo Scientific; 1 :200), CD3 PeCy7 (eBiosciences; 1 :200). Samples were then run on a BD FACS Canto II on medium flow rate. CAR-T cells were identified by CD45.1- CD45.2+ GFP+ staining in the FlowJo software (v10.9.0).

For analysis of the innate immune cell compartment, we used a multicolor FACS panel.

Therefore, mice were sacrificed on day 8 after tumor injection. Spleens were harvested and meshed through a sieve to yield a single cell suspension. After RBC lysis, splenocytes were counted, 2x10⁶ were transferred to a 96 round bottom plate and first stained for viability using FVS440 according to manufacturer’s instructions (1 :1000; BD Biosciences). Next, FcR-block was added followed by the antibody mix in a total volume of 50 L.

Following antibodies were used:

# antibody company dilution # antibody company dilution

Of note, brilliant stain buffer (1 :10; BD Biosciences) was added to the antibody mix to prevent clogging of brilliant dyes. After 20min of incubation, cells were washed twice and acquired on a Cytek Aurora machine (5 laser configuration). Unmixing was performed using a mix of cell and bead samples, respectively, and checked using single stained cell samples. Populations were verified using fluorescence minus one (FMO) controls. Data was analyzed in FlowJo (v10.9.0).

Interferony blocking experiments

PanC02-EpCAM tumor bearing mice were treated with NTC orNR2F6 edited CAR-T cells as stated above. Starting on the day of ACT, animals were injected every second day with 200pg/mL ocIFNy or IgG solution (BioXcell) intraperitonally (ip) for 4 times in total. Tumor growth and weight were monitored frequently and mice were sacrificed if abort criterions were met.

Tumor rechallenge of remission-free

Prior to rechallenge, PanC02-EpCAM tumor cells were enriched for EpCAM positive and EpCAM negative cells using flow sorting and maintained in DMEM+++. Thirteen weeks after first tumor challenge, mice, which showed complete remission (CR) after treatment with NTC and NR2F6 acute depleted CAR-T cells, were inspected by the veterinarian of the animal facility of Innsbruck before being injected with 2.5x10⁶ EpCAM positive and 1x10⁶ EpCAM negative PanC tumor cells into the right and left flank, respectively. “PanC-naTve” female C57BL/6 mice were used as controls and injected with an identical tumor load. Monitoring of tumor bearing mice was performed as described above.

Retroviral transduction of tumor cell lines

Retrovirus containing supernatant was produced as described above. For PanC02-EpCAM-GFP: PlatE cells were transfected with 18pg of transfer plasmid (pMP71-GFP) to produce viral particles. The day prior transduction, PanC were seeded into 6-well plates and incubated overnight at 37°C 5% CO2. The next day, medium was replaced by 2mL DMEM+++ supplemented with 8pg/ml polybrene. Then 2mL of filtered viral supernatant was added directly and the plates were spun at 800g for 2 hours at 32°C. Transduction efficiency was assessed by flow cytometry and yielded 70% being GFP positive. GFP+ cells were enriched by flow sorting.

Incucyte serial killing assay

To ensure sufficient target expression, PanC02-EpCAM-GFP were subjected to flow sorting the week prior to the assay. Tumor cells were stained with aEpCAM-APC (Miltenyi; 1 :400) and an EpCAM high GFP positive population was sorted directly in DMEM+++ on a FACS Aria (Beckton and Dickinson) machine at the FACS Core facility of the Medical University Innsbruck (Dr. Sopper). Flow sorted tumor cells were cultured at 1x10⁶ cells per T175 tissue culture flask and then expanded to be used in the serial killing assay. On the first day of the assay, 6x10⁴ PanC- EpCAM^hi-GFP cells were seeded into flat bottom 96 well plates in CAR RPMI and left to adhere for a few hours.

After CAR-T production and assessment of transduction efficiency (~70%), bulk transduced T cells were counted and adjusted to 3x10⁶ CAR+/mL. 3x10⁵ CAR-T cells were then added at an effectontarget ratio (E:T) of 5:1 to tumor cells in CAR RPMI without cytokines. Live cell imaging was performed in an Incucyte® live cell analysis system (Sartorius) using the 10x objective. Fluorescence pictures were taken every two hours for up to 10 days.

Every 48h, co-cultures were taken from the Incucyte machine and CAR-T cells were restimulated with new target cells. For this, PanC-EpCAM^hi-GFP cells were harvested from T175 tissue culture flasks and resuspended at 6x10⁵/mL in CAR RPMI. 10OpI of medium was removed from the cocultures and stored at -20°C for cytokines assessment. Then, 1 OOpI tumor cell suspension was added and plates were put back into the Incucyte. It was made sure that at least one hour lies between restimulation and the next scan to allow sedimentation of tumor cells.

Cytotoxicity was determined by decreasing GFP fluorescence and plotted relative to the first scan after each restimulation (green integrated intensity per mm² relative to first scan after restimulation). Analysis was performed using the built-in analysis software (Sartorius). CAR-T cells from single mice in duplicates were considered as biological replicates.

Surface and intracellular antibody staining of mouse CAR-T cells

Staining was performed with up to 1x10⁶ (CAR) T cells in a 96 round bottom plate (Thermo Fisher). For viability assessment, T cells were washed twice with Hanks buffered salt solution (HBSS) containing Magnesium and Calcium. Then, 1 OOpI HBSS supplemented with a fixable viability dye (BD Bioscience) at a final dilution of 1 :2000 was added and incubated for 10min at RT in the dark. After the incubation, cells were washed with buffer C and subjected to surface antibody staining. Surface antigens were stained for 20 minutes at 4°C in a 40pL reaction in buffer C using the following antibodies: myc-FITC (Miltenyi, 1 :400), Ly108-Pb (Biolegend, 1 :200), PD-1-bv510 (Biolegend, 1 :200), Tim3-PerCp-Cy5.5 (Biolegend, 1 :200), Lag3-Pe-Cy7 (Biolegend, 1 :200), CD39-PE (Biolegend, 1 :400), CD45-APC (Biolegend, 1 :200), CD44-PeCy7 (Biolegend; 1 :200), CD62L-APC (Biolegend; 1 :200).

For intracellular (IC) cytokine staining, CAR-T cells were transferred to 96 round bottom plate and stained with surface antibodies as stated above. Cells were fixed using the fixation kit from Biolegend for 20 minutes. After fixation, cells were permeabilized by washing twice with the kits Perm/Wash buffer. Intracellular proteins were stained for at least 30min to one hour at 4°C in Perm/Wash buffer using the following antibodies: IFNy-PeCy7 (Biolegend; 1.200), TNFa- PerCpCy5.5 (Biolegend; 1 :200), IL2-APC (Biolegend; 1 :200), GzmB-PE (Biolegend; 1 :200).

For intracellular transcription factor staining, CAR-T cells were chronically stimulated with PanC- EpCAM^hi-GFP. On day four of co-culture, wells were harvested and transferred to a 96 round bottom plate and stained for surface antigens. Finally, cells were fixed using the Foxp3 fixation kit (eBiosciences) for 30min at 4°C. Cells were permeabilized twice with Perm/Wash and stained with IC antibodies for one hour at 4°C: TCF7-Pb (Cell Signaling; 1 :100) and TOX-APC (Miltenyi; 1 :100).

After antibody staining cells were wash twice and resuspended in 200p I buffer C before acquisition on a FACS Canto II (BD Biosciences).

For absolute cell counts, precision count beads (Biolegend) were applied. Therefore, cells were stained directly in culture medium for 20min at 4°C. Plates were spun and cells were resuspended in 100pL buffer C. 1 OOpI precision count beads were added per sample and acquired on a FACS Canto II. Total cell counts were calculated according to the manufacturer’s recommendations.

Data was analyzed using FlowJo software (BD Biosciences, version 10.9.0).

Cytokine production assay

On day 5 of CAR-T production, 6x10⁴ PanC-EpCAM^hi-GFP cells were seeded into 96 flat bottom well plates and incubated for a few hours to allow attachment. Then, 3x10⁵ CAR+ T cells were added at an effectontarget (E:T) ratio of 5:1 and incubated overnight in CAR RPMI without cytokines. The next day, Golgi Plug and Stop (1 :250 and 1 :500; BD Bioscience) was added for 6 hours before cells were subjected to antibody staining and IC FACS analysis.

Gasdermin E clipping assay

1.5x10⁶ CAR-T cells were co-cultured with PanC-EpCAM^hi-GFP in a 24-well plate at an E:T of 5:1 . CAR-T cells were restimulated every second day with 3x10⁵ fresh tumor cells. On day 4, wells were harvested by trypsinization 6 hours after restimulation. Briefly, cell culture medium was collected in a 15mL Falcon tube, wells were wash with 1x PBS and 400pL TrypLE Express was added per well. When adherent cells started to detach, 800pL CAR RPMI was added to stop reaction and medium was transferred to 15mL tube. After a wash step with ice-cold 1x PBS, cells were resuspended in 30pL RIPA buffer supplemented with EDTA and protease inhibitor cocktail (Sigma) and incubated for 30min on ice while vortexing frequently. Then, lysates were spun for 15min at 15000xg at 4°C. Supernatant was transferred to a new 1.5mL Eppendorf tube, supplemented with 5pL Pierce™ Lane Marker Reducing Sample Buffer (Thermo Fisher Scientific) and frozen at -80°C. Samples were thawn, heated to 95°C and subjected to immunoblotting using the following antibodies: Gasdermin E (Abeam, 1 :1000), Actin (Santa Cruz; 1 :1000) Bands were quantified using Imaged and visualized using GraphPad Prism.

UMAP and FlowSOM analysis

Unmixed files from our multicolor FACS panel were gated for viable CD3- CD19- NK1.1- cells and populations were down sampled using the DownSample (v3.3.1) plugin in FlowJo to yield an even representation. Down sampled populations from 3 independent mice per treatment regimen were concatenated and uniform manifold approximation and projection (UMAP) analysis (v4.1 .1) was applied using following parameters: CD11 b, CD11c, XCR1 , CD8, CD4, Ly6c, F4/80, MHCII, CD86. Clusters were defined using the FlowSOM plugin (v4.1.0) in FlowJo (v10.9.0).

Cytokine detection in supernatant

Cytokine release was measured in supernatants using the Luminex xMAP technology. In brief, frozen supernatants were thawed and diluted in CAR RPMI. Assays for INFy (Biorad) and GzmB (ThermoFisher) detection were performed according to manufacturer’s instruction on a Bio-Plex 200 machine (Biorad). Absolute concentrations were determined using a standard curve and plotted in GraphPad Prism (v9.5.1).

RNA isolation and qRT-PCR

To assess NR2F6 induction during CAR-T production, 1-2x10⁵ T cells were harvested, washed once with 1x PBS and resuspended in 350pL RLT buffer (Qiagen) and stored at -80°C.

For co-culture samples, cells were harvested and live (CAR) T cells were enriched by density gradient centrifugation. Briefly, cells were resuspended in an 80% Percoll solution (Sigma Aldrich) in HBSS. A 40% Percoll layer was loaded on top and cells were spun at 250g for 30min without a brake. After the spin, the interface layer was harvested and subjected to CAR positive selection using aBiotin microbeads (Miltenyi) and amyc-Biotin (Biolegend; 1 pg/mL) according to manufacturer’s instructions. 1-2x10⁵ CAR+ T cells were then resuspended in 350pL RLT buffer and stored at -80°C.

For Quantitative real-time PCR (qRT-PCR), total RNA was isolated using the RNeasy Mini kit (Qiagen) according to manufacturer’s instruction and subjected to cDNA synthesis using Omniscript RT kit (Qiagen) in a 20pL reaction using random hexamer as well as poly-A primers. qRT-PCR was performed in duplicates using the LUNA mastermix (New England Biolabs) and pre-designed NR2F6 Taqman primers (Fisher Scientific) on a 7500 Real-Time PCR machine (Applied Biosystems). Target gene expression was normalized to Rlp13a using the AAct method. Ct values over 32 were considered as not expressed. Primers used for qPCR: Mm01340321_m1 , MmO1612986_gH

Seahorse assay

For analysis of oxidative phosphorylation (OxPhos), 2*10⁵ (CAR) T cells were seeded into polylysine pre-coated Seahorse culture plates in XF RPMI without phenol red supplemented with 20mM glucose, 2mM glutamine and 1 mM pyruvate (all Agilent). Assay was performed using the Cell Mito Stress Test Kit on a Seahorse XFp device (Agilent). Briefly, Seahorse plates were put in a CC>2-free incubator for one hour to allow equilibration. Mitochondrial respiration was analyzed first by assessing the basal respiration, then injecting 1 ,5pM of the ATP synthase inhibitor oligomycin, 1 M of the uncoupler carbonyl cyanide-4-(trifluoromethoxy)-phenylhydrazone (FCCP) and finally 0.5pM of rotenone/antimycin A. Results were analyzed using the Seahorse Wave Software (Agilent) and visualized in Graph Pad Prism.

Flow sorting of tumor and CAR-T cells

PanC02-EpCAM tumor cells were harvested as described above. Cells were transferred to FACS tube and up to 1x10⁷ cells were stained in 10OpI buffer C containing ocEpCAM-APC (Miltenyi, 1 :400) antibody for 20 minutes at 4°C. After incubation, PanC were washed twice with buffer C and finally resuspend in ice-cold DMEM+++ supplemented with 2mM Ethylenediaminetetraacetic acid (EDTA) at 1x10⁷/mL. CAR-T cells from co-culture were harvested and transferred to a FACS tube for antibody staining. Up to 10⁷ cells were resuspended in 10OpI buffer C containing amyc FITC (Miltenyi, 1 :400), aCD45 APC-Cy7 (Biolegend, 1 :200) and ocEpCAM APC (Miltenyi, 1 :400) and incubated for 20 minutes at 4°C. Then, cells were washed twice with buffer C and resuspended at 1x10⁷/mL ice-cold CAR RPMI supplemented with 2mM EDTA. Cells were flow sorted using a BD FACS Aria (Beckton and Dickinson) at the FACS Core Facility at the Medical University Innsbruck. For viability staining, 10pl 4',6-diamidino-2-phenylindole (DAPI) was added to 1 ml of cell suspension straight before acquisition. Viable EpCAM positive tumor cells were directly sorted into DMEM+++ and seeded into T175 flasks immediately after sort. 1x10⁵ viable CD45+ CAR+ T cells were directly sorted into 200pl RLT buffer (Qiagen) supplemented with 5pL 14.3M 2-mercaptoethanol (BME) and 2.5pL RNAsin (Promega) in a RNAse free 0.5mL Eppendorf tube to be subjected to bulk RNA sequencing (RNAseq).

Bulk RNAseq

For bulk RNA sequencing (RNAseq), total RNA was isolated using the RNeasy Micro kit (Qiagen) according to manufacturer’s instructions. RNA was eluted in the kits elution buffer and stored at - 80°C before quality control (QC). Quality control and library preparation and sequencing was performed by the MultiOmics Core Facility at the Medical University of Innsbruck.

Statistical analysis

Statistical analysis was performed using GraphPad Prism software (v9.5.1). Data are represented as mean ± standard error of the mean (SEM) unless otherwise stated. Comparisons between two groups were calculated using two-tailed unpaired Student’s t-test or non-parametric Mann- Whitney test as indicated in the figure legends. Comparisons between two or more groups with different variables were analyzed performing two-way ANOVA with the Geisser-Greenhouse correction. In case of multiple comparison, the Sidak method was used. Bulk RNAseq and scRNAseq data were analyzed as stated above. Survival data was collected in a Kaplan-Meier curve and tested using log-rank tests. P-values <0.05 were considered significant. *p < 0.05, **p < 0.01 , ***p < 0.001 , ****p < 0.0001

Culture of human cellsJ urkat-TAg T cells were cultured in RPMI-1640 supplemented with 10%FCS, 2 mM L-Glut, 100 U/mL penicillin, 100 pg/mL streptomycin (all PAN Biotech), 1 mM sodium pyruvate and a 1x mix of non-essential amino acids (Sigma) (from here on cRPMI). Primary human T cells cells (CD3+, sorted from PBMCs; Biolegend, 480131) were cultured in cRPMI with 10 ng/mL IL-2 (Biolegend; 589106).

Combinatorial NR2F6 and CBLB inhibition in primary human T cells:

NR2F6 knockout was achieved by lentivirus-mediated stable integration of hEF1a-driven puromycin resistance gene and Cas9 together with U6-driven sgRNA (Dharmacon; GSGH11935- 247620068 and GSGH11935-247640844 targeting NR2F6; DH-GSGC11963 for non-targeting- control). Lentiviral particles were produced using LentiArt™ Virus Packaging Kit (Creative Biolabs; CART-027CL) and concentrated using Lenti-X™ Concentrator (Takara, 631232) according to manufacturer’s instructions. Transduction was performed in the presence of 8 pg/mL polybrene (Sigma; TR-1003-G) - in case of primary T cells 1 day upon pre-stimulation with T- Activator Dynabeads (Thermo Fisher; 11131 D) at a 1 :1 ratio of cells and beads. Transduced cells were enriched via puromycin treatment (1 pg/mL for Jurkat cells during whole culture time; 2,5 pg/mL for primary T cells for 3 days). Immunoblot analysis of NR2F6 levels in Jurkat cells was performed with anti-NR2F6 antibody from Proteintech; 60117-2-lg. NR2F6-deficient primary human T cells were stimulated via 0,3125 pg/mL plate-coated anti-CD3 antibodies and 1 pg/mL soluble anti-CD28 antibodies (BioXcell; BE0001-2 & BE0248) in the presence of either DMSO or 1 pM Nurix and analyzed by flow cytometry as described earlier (addition of brefeldin A and monensin (BD BioSciences; 555029 & 554724) after 45 min; intracellular staining). Following antibodies were used: CD4 Pb (317429); CD8 BV510 (301048); CD25 PerCP-Cy5.5 (356112); CD69 A647 (310918); IL-2 PE (500307); IFNy PE-Cy7 (502528); grzB FITC (515403) (all from Biolegend).

Example 1 (Figure 2):

Reverse transcription polymerase chain reaction (RT-PCR) analysis of tumor-infiltrating lymphocytes (TILs) versus lymph nodes (LN, draining and non-draining) and spleen provides valuable insights into the gene expression profiles and immune responses associated with these different cells. Tissue Collection: Tissue samples from the tumor, draining lymph nodes, nondraining lymph nodes, and spleen were collected. These tissues represent different microenvironments with varying immune cell populations and activities, thus defining how the tumor microenvironment influences NR2F6 gene expression in infiltrating lymphocytes. RNA Extraction: Total RNA is extracted from each tissue sample. This RNA includes both messenger RNA (mRNA) and non-coding RNA, which can be analyzed to understand gene expression patterns. Reverse Transcription: The extracted RNA is then reverse transcribed into complementary DNA (cDNA) using reverse transcriptase enzymes. This step converts RNA into a form that can be analyzed via PCR. RT-PCR Analysis: RT-PCR is performed on the cDNA samples for each sample. This technique amplifies and quantifies the selected NR2F6 mRNA levels. Results of quantitative PCR (qPCR) shown has revealed the NR2F6 gene expression levels. Data Analysis: The results of the RT-qPCR analysis are analyzed statistically to compare the gene expression profiles among the different samples. The findings from the RT-qPCR analysis have provided insights into how NR2F6 gene expression patterns in TILs compare to those in lymph nodes (draining and non-draining) and the spleen, revealing that NR2F6 as immune-related gene is strongly upregulated in response to the solid tumor microenvironment.

Example 2 (Figure 3): Metabolic analysis using a Seahorse XFp Analyzer, is a powerful technique for studying cellular metabolism. The Seahorse XFp Analyzer measures the rate at which cells consume oxygen (OCR) and the rate of lactate production (extracellular acidification rate, ECAR). These parameters are indicative of different aspects of cellular metabolism.

Cell Seeding: Cells of interest are seeded onto a specialized microplate, typically containing wells with sensors. These sensors allow for real-time measurements of oxygen and pH. Loading

Reagents: Prior to the experiment, specific compounds are loaded into the microplate wells. For OCR measurements, mitochondrial inhibitors like oligomycin, FCCP, and antimycin A are used. For ECAR measurements, glycolysis modulators are applied.

Measurement: The XFp Analyzer injects these compounds into the wells at specific experimental settings during the experiment. The Seahorse software analyzes the data generated from the OCR and ECAR measurements, allowing us to derive key metabolic parameters, including basal respiration, ATP production, proton leak, spare respiratory capacity, glycolysis rate. Using these experimental conditions, genetic manipulation of NR2F6 has defined how NR2F6 negatively impacts cellular metabolism of primary T cells. This data uncovered how NR2F6-modified T cells better utilize energy, better adapt to different environments, and better respond to metabolic stressors. In summary, Seahorse stress test kits are valuable tool for metabolic analyses, providing mechanistic insights into cellular energy metabolism of NR2F6-modified cells. To maintain their killing capabilities, especially in the harsh TIME, CD8⁺ cytotoxic T cells depend on a high metabolic activity. Therefore, we next assessed the metabolic profile of our CAR-T cells during chronic stimulation using the Seahorse technology. Therefore, we enriched co-cultures for CAR+ T cells using positive magnetic separation and decided to subject them to the Seahorse Cell Mito Stress Test on day four of co-culture where we have seen the biggest difference in Tpex/Ttex ratio. Analysis revealed that NR2F6-modified CAR-T cells showed a superior stress test profile with a higher basal as well as maximum oxygen consumption rate (OCR) when compared to wildtype CAR-T cells. Further, knockout CAR-T cells exhibit a significant higher extracellular acidification rate (ECAR) owing to an increased glycolytic potential. Gene ontology of bulk RNAseq data again from day 4 of chronic stimulation also showed enrichment in various metabolic pathways. GSEA analysis between knockout and wildtype CAR-T cells showed that gene sets associated with OXPHOS, glycolysis and fatty acid oxidation (FAO) were significantly upregulated in NR2F6-modified CAR-T cells. Our results indicate that NR2F6 knockout in CAR-T alters the performance by increasing and maintaining effector cytokine production and prevents terminally exhaustion and differentiation, respectively, by preserving a pool of Tpex cells in vitro. Mechanistically, loss of the transcription factor NR2F6 leads to metabolic reprogramming with i.e. a shift to OXPHOS and glycolysis meeting the energetic needs during repetitive stimulation.

Example 3 (Figure 4):

Performing a CAR-T cell tumor co-culture and serial killing assay with IncuCyte involves using live-cell imaging technology to monitor the interaction between CAR-T cells and tumor cells over time. Cell Preparation: CAR-T Cells: T cells are isolated, cultured, and engineered to express chimeric antigen receptors (CARs) specific to the EpCAM tumor antigen. Tumor Cells: EpCAM- positive PanC02 tumor cells are cultured, and labeled with a fluorescent GFP for tracking. Coculture Setup: CAR-T cells and EpCAM-positive PanC02 tumor cells are mixed together in a culture dish or microplate to initiate the co-culture. The ratio of CAR-T cells to tumor cells is controlled, typically at different effector-to-target (E:T) ratios, to assess varying levels of cytotoxicity. Plating: The co-culture is plated in wells of a microplate suitable for IncuCyte imaging. These plates have transparent bottoms for live-cell imaging. Incubation: The plated coculture is placed inside the IncuCyte system within a cell culture incubator. The system maintains controlled temperature, humidity, and CO2 levels. Imaging and Analysis: The IncuCyte system captures images of the co-culture at defined time intervals over several hours or days. The images can be analyzed using IncuCyte's software to track various parameters, such as cell confluence, tumor cell death, and CAR-T cell activity. Serial killing refers to the observation of multiple rounds of tumor cell killing by CAR-T cells over time. The IncuCyte system generates quantitative data, including kinetic profiles of CAR-T cell cytotoxicity, tumor cell death, and other relevant parameters. This assay provides the data to assess the effectiveness of CAR-T cell- mediated tumor killing at different E:T ratios and time points. In summary, the IncuCyte live-cell imaging system allows for real-time monitoring and quantification of CAR-T cell-mediated tumor cell killing, providing valuable information for optimizing CAR-T cell therapies and understanding their mechanisms of action.

Example 4 (Figure 5):

Based on the data obtained from scRNAseq analysis, we investigated NR2F6 depletion in the context of synthetic immunotherapy using chimeric antigen receptor (CAR) T cells. To test our hypothesis, we used an investigational second-generation CAR directed against murine EpCAM. The CAR consists of a scFv region fused to an intracellular CD28 as well as CD3 domain via a CD8 hinge and a CD28 transmembrane region. We used the pancreatic ductal adenocarcinoma PanC02 cell line, which was engineered to express mouse EpCAM, as a target. Our lab previously has shown that NR2F6 is a negative regulator of T cells, which is induced upon TCR stimulation via aCD3/CD28. We now determine NR2F6 induction in WT NR2F6-proficient T cells during CAR-T production. We observed a vast increase in NR2F6 mRNA levels up to tenfold, which led us to test the consequences of NR2F6 depletion in CAR-T cells. We could not observe any differences in CAR transduction efficiency or expansion of NR2F6'^/~ or wildtype CD8⁺ T cells during CAR-T production. Investigating the quality of tumor cell death induction, we quantified of Gasdermin E (GSDME) clipping during co-culture of tumor cells and NR2F6-deficient or wildtype CAR-T cells harvested after chronic stimulation and re-stimulated for 6 hours with PanC02- EpCAM tumor cells (biological replicates, n=6, two independent experiments). As a remarkable result, NR2F6-deficient CAR-T cells induce pyroptosis in the co-cultered tumor cells. Pyroptosis represents the major form of ICD. Mechanistically, as demonstrated by tumor-intrinsic GSDME clipping, increased production of GRZB and IFNy leads to the desired qualitative switch of ICD and pyroptosis. These data represent a functional validation of the NR2F6 pathway for the induction of immunogenic cell death (ICD) of tumor cells by NR2F6-modified CAR-T cells. Taken together, NR2F6-modified CAR-T cells were shown to primarily kill their targets in a Perforin- granzyme B dependent manner, which in turn leads to activation of apoptotic pathways via Caspase 3 but also to cleavage of gasdermine E (GsdmE) in the tumor target cells ultimately resulting in tumor cell death. Of note, gasdermins (Gsdms) are activated by proteolytic removal of autoinhibitory carboxy- terminal domains, producing an active amino-terminal fragment, thereby converting noninflammatory apoptosis to pyroptosis, a highly immunogenic form of programmed cell death (Mamonkin et al., 2015; Zhan et al., 2020). To further validate the high quality killing phenotype observed with NR2F6'^/_ CAR-T cells on target tumor cell level, we assessed GsdmE clipping on day four of chronic stimulation in PanC-EpCAM using western blot. Quantification revealed that tumor cells co-cultured with NR2F6-modified CAR-T cells showed a significant increased GsdmE clipping activity of full-length GsdmE to N-GsdmE when compared to wildtype CAR-T cells.

Example 5 (Figure 6):

Bulk RNA sequencing (RNA-seq) analysis of gene signatures during T cell exhaustion is a technique to gain insights into the molecular mechanisms and signaling pathways involved in this process. T Cell Isolation: T cells, particularly CD8⁺ cytotoxic T cells, are isolated from murine tissue or blood samples. RNA Extraction and Library Preparation: Total RNA is extracted from the isolated T cells. This RNA includes mRNA transcripts that provide information about gene expression. The extracted RNA is converted into cDNA libraries, using reverse transcription. These cDNA libraries represent the transcripts present in the T cells. RNA Sequencing: The cDNA libraries are subjected to high-throughput RNA sequencing, which generates millions of short sequence-reads corresponding to the RNA molecules present in the sample. The sequencing data undergoes quality control to remove low-quality reads and ensure the data's reliability. Alignment: The sequence reads are aligned or mapped to the reference genome or transcriptome to determine which genes are expressed and at what levels. Differential Expression Analysis: By comparing gene expression levels between exhausted T cells and non-exhausted T cells (e.g., WT or NR2F6-deficeint mice after single or repetitive stimulation, respectively), we identify differentially expressed genes associated with T cell activation versus exhaustion. Gene Signature Identification: Researchers have defined gene signatures by selecting a set of genes that are consistently upregulated or downregulated in activated versus exhausted T cells. These signatures can be used to characterize the exhaustion phenotype. Functional Enrichment Analysis: Gene ontology (GO) analysis or pathway enrichment analysis is performed to understand the biological processes, molecular functions, and pathways associated with the identified gene signatures. Biological Insights: These results allow to gain insights into the molecular processes and regulatory networks contributing to T cell activation versus exhaustion. In summary, RNAseq analysis of gene signatures during T cell exhaustion provides a comprehensive view of gene expression changes in exhausted T cells, helping us to understand the underlying biology and potential therapeutic targets for immune-related disorders.

Granzyme A, Granzyme C and Granzyme D are enzymes found in cytotoxic immune cells, particularly natural killer (NK) cells and cytotoxic T lymphocytes (CTLs). These enzymes play essential roles in the immune system's defense against tumor cells by inducing cell death in target tumor cells. These granzymes are typically expressed and released into the immunological synapse when CTLs recognize and engage with the target tumor cells. Once inside the target cell, granzymes initiate a series of proteolytic events that lead to tumor cell death. Thus, the process is crucial for eliminating cancer cells. Understanding the expression of these granzymes is important in the context of immunology and cancer research, as it can inform the development of therapies that augment the anti-tumor immune responses.

Upon acute stimulation of CAR-T cells with PanC02-EpCAM at an effector to target (E:T) ratio of 5:1 overnight, NR2F6'^/' CAR-T cells produced significantly more effector cytokine IFNy assessed by flow cytometry. Using FACS analysis, we set out to determine the phenotypic characteristics of our CAR-T cells during chronic stimulation. We observed that loss of NR2F6 led to an attenuated terminally differentiation status assessed by CD62L and CD44 co-staining. Chronically stimulated knockout CAR-T showed significantly more CD62L CD44 double positive cells tending towards a skewing to a central memory (CM) like phenotype. We next wondered if this increase in double positive CAR-T cells with sustained cytokine secretion also leads to a difference in exhaustion manifestation. Indeed when we looked at PD1+ CAR-T cells, we noticed a significant difference in the progenitor exhausted T cell (Tpex, Ly108+ Tim3-) to terminally exhausted T cells (Ttex, Ly108- Tim3+) (Tpex/Ttex) ratio as underlying mechanism of the benefits of NR2F6-modified CAR-T cells. Moreover, FACS analysis revealed that NR2F6-/- CAR-T cells expressed more TCF7 protein on day 4 of co-culture resembling previously published data showing that Tpex cells remain TCF7 positive (Utzschneider et al., 2020). Finally, we subjected CAR-T cells prior and during chronic stimulation to bulk RNA sequencing (RNAseq) to elucidate transcriptional differences. Gene ontology (GO) analysis using DEGs revealed e.g. the promotion/augmentation of T cell trafficking as well as cell killing/cytotoxic pathways supporting the superior killing phenotype robustly observed in NR2F6-modified CAR-T cells (Fig4L+M).

Example 6 (Figure 7):

Studying tumor growth inhibition in mice using subcutaneous tumor models and CAR-T cell therapy is a common approach in cancer research. Tumor Model Selection: We select a specific tumor cell line PanC02 expressing EpCAM and inject these cells subcutaneously into mice. This creates a localized solid tumor in the mice. CAR-T Cell Therapy: Chimeric Antigen Receptor T (CAR-T) cells are specialized immune cells engineered to target specific tumor antigens. We used a CAR-T to recognize EpCAM present on the surface of the PanC02 tumor cells being studied. These CAR-T cells are then produced in the laboratory. Treatment Administration: Once the tumor reaches a certain size, the mice are divided into experimental groups. One group receives the NR2F6-modified CAR-T cell therapy, while the control group receive a CAR-T cells of the prior art or no treatment. Monitoring T umor Growth: Over time, we regularly measure and record the size of the tumors in both the treatment and control groups. This is typically done using calipers to measure the dimensions of the subcutaneous tumors, allowing to quantify the rate of tumor growth in the treated mice compared to the control group. Tumor growth inhibition is determined by comparing the size and growth rate of tumors in the treated group to those in the control group. Data Interpretation: Data collected from these experiments provide information about the efficacy of CAR-T cell therapy in inhibiting tumor growth, allowing us to assess whether the therapy leads to tumor regression, stabilization, or delayed growth compared to the control group. In summary, this preclinical mouse model approach firmly validated the superior effectiveness of the inventive NR2F6-modified CAR-T cell therapy and provided strong experimental data that support the development of future clinical trials in human patients.

Based on our remarkable results obtained from in vitro assays we tested the anti-tumor efficacy of NR2F6-modified CAR-T cells in a solid mouse tumor model in vivo. For translational reasons, we decided to switch from germ line knockout to a more clinically relevant approach to employ NR2F6 gene editing in primary T cells using CRISPR/Cas9. Hence, we used Cas9 transgenic (Cas9tg) CD8⁺ T cells and delivered synthetic single guide (sg)RNAs targeting the NR2F6 locus by electroporation. For in vivo experiments, we inoculated fully immunocompetent mice with one million PanC-EpCAM cells subcutaneously. Due to lack of an antibody recognizing native NR2F6 in primary mouse T cells on western blot level, we determined the knockout efficiency using TIDE analysis. sgRNA (sg)03 frequently yielded an efficiency over 90%, whereas sg04 was less efficient with 40%. Treating tumor-bearing mice with NR2F6^crisP^r'^/' CAR-T cells led to reduced tumor growth and prolonged overall survival compared to animals treated with NTC~ CAR-T cells. Of note, in the knockout treated group 40% (6/15) of animals showed a complete response after tumor challenge in contrast to 7% (1/14) in the NTC treated one. In vivo experiments using an independent and sequence non-overlapping sgRNA showed similar results indicating that the knockout of NR2F6 has been responsible for the superior anti-tumor response. NR2F6 depleted CD8⁺ T cells, however, failed to induce an anti-tumor immune response suggesting that a CAR is required. Several clinical studies highlighted the importance of persistence of CAR-T cells regarding clinical outcome (Melenhorst et al., 2022; Shiqi et al., 2023). Therefore, we challenged Ly5.1 mice with PanC-EpCAM tumors and treated them with CAR-T cells like stated above. On day 6 after adoptive cell transfer (ACT), we could not observe any differences in CAR-T cell numbers in the spleen between NR2F6 depleted and NTC' CAR-T treated animals. Of note, however, CAR-T cells were again undetectable in the spleen in all animals irrespective of therapy regimen, when analyzed seven days later (see Fig.9).

Example 7 (Figure 8):

Creating a research model involving Rag1 knockout mice, which lack an adaptive immune system, and studying CAR-T cell therapy in the context of subcutaneous tumor load provides valuable insights into the role of the adaptive immune system in an anti-tumor immune response. Rag1 knockout mice, which lack mature B and T lymphocytes due to a disruption in the Rag1 gene, are chosen as the experimental model, because they have a severely compromised adaptive immune system.

Searching for the underlying mechanism of tumor rejection in NR2F6-modified CAR-T cells, we reasoned that CAR treatment in this therapy group must have led to immunogenic cell death (ICD) known to promote priming and activation of the endogenous immune system ultimately resulting in a polyclonal and durable tumor cell clearance. To test this ICD-mediated tumor epitope spreading (ES) hypothesis as a secondary event of NR2F6-modified CAR-T therapy regimen, we injected immunodeficient RAG1'⁷' recipients, lacking an endogenous T cell/adaptive immune compartment, with PanC-EpCAM and subsequent treatment two days later with NR2F6^crisP^r'^/'or NTC' CAR-T cells. In strict contrast to the results obtained with fully immunocompetent WT recipient mice, the NR2F6-modified CAR-T treatment cohort no longer showed tumor growth and survival benefits. The loss of therapeutic benefit in the RAG1-/- recipient groups argues that an endogenous T cell response is required for the superior tumor control achieved by the NR2F6-modified CAR-T cell regimen. As previously published, NR2F6 deficient CAR-T cells produce more IFNy, which is crucial in amplifying an immune response (Schorder et al., 2004).

Subcutaneous tumor cells (e.g., PanC02 cell lines expressing a specific EpCAM antigen) are injected into the flank of Rag1 knockout mice to establish subcutaneous solid tumors. Chimeric Antigen Receptor (CAR) T cells are engineered to express CARs specific to the EpCAM tumor antigen, and infused into Rag1 knockout mice with established subcutaneous solid PanC02 tumors. This step aims to study the impact of CAR-T cell therapy in the absence of an adaptive endogenous immune response. Tumor growth in the mice is regularly monitored by measuring the size of subcutaneous tumors as in Fig. 7A&B above. Whether CAR-T cell therapy can lead to subcutaneous tumor regression or delay tumor growth in the absence of adaptive immunity provides insights into the indirect effect of the adaptive endogenous immune system. Defining CAR-T cell therapy's failure in the absence of adaptive immune responses firmly validates the contributions of a secondary immune response in the context of the inventive NR2F6-modified CAR-T cell therapy by epitope spreading, the latter strictly depending on the endogenous adaptive immune system. In summary, this study provides strong validation that CAR-T cells alone cannot exert anti-tumor effects against EpCAM tumor antigen heterogenous PanC-02 tumor loads in the absence of endogenous adaptive immune cells, a key finding that has implications for our inventive NR2F6-modified CAR-T cell therapy strategy. Example 8 (Figure 9):

We observe that immunogenic GFP-positive CAR-T cells are rejected by the host immune system within one week. This highlights the immunogenicity of the CAR-T Cells, engineered to express a foreign protein, such as GFP (Green Fluorescent Protein), that can be recognized as foreign by the host's immune system, triggering an immune response against the CAR-T cells themselves. Because the host's immune system identifies the CAR-T cells as foreign and mounts a robust immune response, it let to the rapid elimination of the CAR-T cells within a short period of less than one week. In summary, the rapid rejection of immunogenic CAR-T cells applied by the host immune system underscores a transient time-boxed (less than 1 week) therapy approach, triggered by the inventive NR2F6-modified CAR-T cells, to be sufficient to induce a secondary immune response by the endogenous immune system (epitope spreading), while maximizing their effectiveness in targeting heterologous tumor loads.

Example 9 (Figure 10):

Rechallenging NR2F6-modified CAR-T-treated complete responders with EpCAM antigenpositive and antigen-negative tumors is a common approach to study antigen cross-priming and epitope spreading in the context of cancer immunotherapy. Primary Tumor Challenge: Initially, mice are injected with EpCAM antigen-positive tumor cells to establish a primary tumor. Thereafter anti-EpCAM CAR-T cell therapy was applied to stimulate an immune response against the tumor. Survival Analysis: Mice that respond to immunotherapy and survive without visible signs of the primary tumor are identified (see Fig. 7A). Rechallenge Phase with EpCAM antigenpositive or antigen-negative tumors: NR2F6-modified CAR-T-treated complete responder mice are divided into groups. One group is rechallenged with the same EpCAM antigen-positive tumor cells that were used in the primary tumor challenge. Rechallenge with EpCAM antigen-negative tumor: Another group is rechallenged with EpCAM antigen-negative tumor cells. These tumor cells do not express the specific EpCAM antigen targeted by CAR-T cells in the primary tumor challenge. The growth of tumors in the rechallenged mice is monitored over time in order to assess whether the immune response generated during the primary challenge provides protection against tumor rechallenge not only for EpCAM antigen-positive but also for EpCAM antigen-negative tumors, when directly compared to naive mice (epitope spreading).

As result, therapy with NR2F6 knockout CAR-T cells establishes long-lasting high quality memory responses triggered by epitope spreading (ES). To ultimately verify if NR2F6^crisP^r'^/' CAR-T cell therapy promotes ES and, subsequently, the activation of the endogenous immune system, we challenged long-term complete responders simultaneously with EpCAM positive and EpCAM negative PanC tumors injected in the right and left flank, respectively. Confirming our hypothesis, each NR2F6-modified CAR-T complete responder rapidly and identically eradicated both EpCAM-positive and EpCAM-negative tumors and showed superior survival compared to untreated control mice. Importantly, this occurred regardless of whether EpCAM was expressed on the PanC02 tumor cell used.

Therefore, as an underlying mechanism of action, antigen cross-priming/epitope spreading has occurred, because the mice previously exposed to the EpCAM antigen-positive tumor similarly exhibit a delayed growth for the EpCAM antigen-negative tumor. This data validate that the immune response initiated during the primary challenge has primed a secondary response by the endogenous T cells capable of recognizing other tumor antigens (epitope spreading). Epitope spreading occurs when the immune response diversifies to target additional tumor-specific antigens beyond the one initially targeted such as in our case EpCAM. Because the mice show clear evidence of epitope spreading, it also implies a broader and more robust polyclonal antitumor immune response by the endogenous immune compartment. The observed successful protection against tumor rechallenge also suggests the development of immune memory, where the immune system "remembers" the diverse antigens of the given tumor and can respond more effectively upon rechallenge. In summary, these key experimental findings help us to understand the dynamics of the immune response to tumor cells, and particularly confirm the therapy benefit of effective antigen cross-priming and epitope spreading, the key aspect of our inventive NR2F6- modified CAR-T immunotherapy. Achieving curative outcomes with CAR-T cell therapy for solid cancers by the inventive NR2F6-modified CAR-T, allows to determine the optimal CAR-T cell dose and time frame for the infusion schedule to maximize efficacy. Furthermore, developing a time-boxed transient therapy regimen of the inventive NR2F6-modified CAR-T formulation mitigates severe side effects of CAR-T cell therapy, making the treatment safer and more tolerable.

Example 10 (Figure 11):

To elucidate how NR2F6^crisP^r'^/' CAR-T cells boost the endogenous immune system, we applied a multi-color FACS panel focusing on the innate immune compartment and ELISPOT to evaluate tumor antigen specific T cell responses. The experimental procedure is outlined in Fig. 11 A. On day 8 after tumor injection, we isolated splenocytes from NR2F6^crispr'^/_ and A/TC receiving animals and subjected them to multi-color flow cytometry. Uniform manifold approximation and projection (UMAP) and FLOWSOM analysis with our flow data (viable CD3- CD19- NK1.1-) revealed seven populations. As result, WT fully immuno-competent mice treated with NR2F6^crisP^r'^/' CAR-T cells showed an enrichment in population 5 characterized by the expression of CD11c, MHC-II, CD8, XCR1 as marker genes typically expressed by conventional dendritic cells typel (cDC1). Furthermore, manual gating revealed a significant difference in cDC1 numbers in NR2F6- modified CAR-T treated animals. cDC1 were proven to be indispensable for anti-tumor immunity primarily by priming and supporting the differentiation of cytotoxic T lymphocytes (CTLs) (Hildner et al., 2008). Using ELISPOT assay with isolated primary CD3+ T cells in the presence of DCs loaded either with primary PanC02 tumor, PanC02 cell line lysate or with peptide pools of previously published PanC02 neoantigens (Kinkead et al., 2018), we assessed polyclonal T cell activation responses in the presence or absence of the CAR-targeted tumor antigens. As result, we are able to confirm cDC1 -mediated ES via T cell priming: Treatment with NR2F6^crisP^r'^/' CAR-T therapy (but not or much less NTC~ CAR-T therapy as well as control CAR-T untreated mice) resulted in an augmented endogenous T cell response as shown by enhanced GzmB production similarly triggered by CAR-targeted as well as non-CAR-targeted tumor antigens recall challenge. The efficient tumor clearance in the complete absence of CAR-T cells can be explained by this underlying mechanism. Taken together, our data indicate that NR2F6-modified (but not NTC) CAR-T cell therapy induces a robust increase in cDC1 cellu larity and differentiation required for their T cell priming capability, which apparently results in a robust secondary and polyclonal immune response by the endogenous T cell compartment in this specific cohort.

Example 11 (Figure 12):

Therapy with NR2F6 knockout CAR-T cells establishes long-lasting high quality memory responses triggered by epitope spreading (ES, see Fig.10). Assuming that cDC1 -mediated ES might have taken place in the NR2F6crispr-/- CAR-T-treated complete responder cohort as the mechanistic basis for the efficient rejection of the EpCAM-negative PanC02 tumors, we repeated the challenge experiments with EpCAM-negative PanC02 tumors and evaluated the characteristics of the endogenous immune response by scRNA and scTCR-seq of tumor infiltrating CD45+ cells. In parallel, ACT of spleen cells transferred from the complete responder cohort into PanC02 tumor antigen-naive mice was performed, followed by ex vivo assays with endogenous CD8⁺ T cells on day 5 after tumor injection. Testing whether one could confer anti- PanC02 tumor immune protection to tumor bearing wildtype mice via anti-tumor treatment (ACT) using NR2F6-modified CAR-T complete responder splenocytes. Indeed, mice treated with these splenocytes were able to control tumor growth efficiently and showed a survival benefit when directly compared to animals treated with splenocytes from PanC antigen naive mice. To gain a mechanistic insight of tumor infiltrating leucocytes in NR2F6-modified CAR-T complete responder cohort, we performed scRNA- and scTCR-seq using CD45+ tumor infiltrating cells. Based on their expression profile, we identified different clusters representing prominent immune cells types (Fig 12A). Strikingly, we observed a pronounced difference in cell composition between NR2F6- modified CAR-T complete responder and PanC antigen naive mice. NR2F6-modified CAR-T complete responder mice harbored decreased numbers of exhausted CD8⁺ and regulatory T cells, and among other changes particularly showed an increase in CD8⁺ proliferating T cells as well as cDC1s. Of note, we did not find any CAR+ T cells among tumor infiltrating cells in the NR2F6-modified CAR-T complete responder cohort excluding that they did contribute to tumor clearance. When we looked at CD8⁺ T cells subsets, we did observe differences in gene expression when compared to control tumor-infiltrating cells in CD8⁺ proliferating T cells (Fig 12C). Consistent with our working hypothesis, GO analysis of DEGs using immune cells types, major alterations in cellularity between groups have been found in CD8⁺ proliferating and exhausted T cells and cDC1s. Along this line of argumentation, the T cell subsets were found to have upregulated distinct pathways well established for positive regulation of T cell function, whereas cDC1 s showed upregulation of pathways associated with migration, chemotaxis, T cell cytokine production, activation and differentiation (Fig 12D). To further validate if ES has taken place, we determined at T cell receptor (TCR) clonality as well as diversity by scTCR-seq of TILs. As result, NR2F6-modified CAR-T complete responder cohort, when compared to control mice, showed a reduction in clonality with a concomitant increase in diversity indicating that T cells were primed towards many epitopes without one being prominent. Taken together, the results show that NR2F6^crisP^r'^/' CAR-T therapy resulted in an establishment of a polyclonal and long- lasting memory immune response characterized by particularly lower CD8 exhaustion of TILs as well as an increased cellularity of cDC1s, explaining both the increased TCR diversity and superior anti-tumor activity of the endogenous CD8⁺ T cell compartment. Together with an ICD- mediated epitope spreading (ES), this likely is promoting the robustly augmented T cell priming as a secondary effect selectively observed in the NR2F6-modified CAR-T treatment cohort,

Example 12 (Figure 13):

TCF1 is the established key regulator of clonotypic memory and exhaustion resistance in CD8⁺ T cells. To investigate a functional interaction of NR2F6 with TCF1 , DNA binding and transactivation analyses were performed using CRISR/Cas9-mediated acute NR2F6 knockout technology and TCF1 DNA binding studies in NTC or NR2F6crispr-/- murine CAR-T cells under defined stimulation conditions. Complementarily, in human leukaemic Jurkat T cells ectopically expressing NR2F6 or TCFI WT or mutant proteins, promoter transactivation assays of TCF1 enhancer driven luciferase reporter containing a minimal promoter coupled to TCF1 enhancer sites upstream of a firefly luciferase gene as well as DNA binding analyses using electromobility assay (EMSA) of TCF1 in nuclear extracts were performed. Mechanistically, NR2F6 acts as a co- factor and/or modulator of TCF1 and is specifically involved in the modulation of TCF1 DNA binding during chronic antigen stimulation of T cells. Collectively, these findings demonstrate that there is a TCF1 :NR2F6 cross-talk that may have an essential and non-redundant regulatory function in regulating T cell exhaustion. This functional interaction may be a promising target for improving cancer immunotherapy response rates.

Example13 (Figure 14):

Lentiviral integration of Cas9, sgRNA and the puromycin resistance gene into primary human T cells was used to knockout human NR2F6. Subsequently, in the presence of DMSO or Nurix (an established and selective small molecule inhibitor of CBLB), effector responses were analysed by flow cytometry as described above. NR2F6 gene-edited primary human T cells, which already have significantly enhanced effector responses upon antigen receptor stimulation, can be further enhanced by combinatorial inhibition of CBLB. As a result, the combinatorial inhibition of NR2F6 and CBLB in primary human T cells in vitro significantly cooperates to enhance the antigen receptor signalling in human T cells, thus validating the concept of such a combinatorial immunotherapy regimen.

Summary (Figure 15):

Figure 15 shows a schematic cartoon of the “Immunogenic Cell Death Phenomenon” mediated by the inventive NR2F6-modified CAR-T cells that activate innate immune cells at the solid tumor microenvironment. In strict contrast of CAR-T cells of the prior art, the inventive NR2F6-modified CAR-T are allowing cross-priming, epitope spreading and, subsequently, immune cell infiltration and rejection also of CAR-targeted-negative tumors. The experimental findings validate the development of a time-boxed transient strategy employing the inventive NR2F6-modified CAR-T cells to trigger a secondary immune response and improve polyclonality by the endogenous T cell compartment of the given anti-tumor immune response as an innovative strategy able to transform non-curative adoptive T cell therapy of the prior art into a curative treatment for antigen heterologous solid cancers.

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Claims

1 . A modified immune cell for use in the treatment of a solid tumor in a subject, wherein the modified immune cell comprises one or more exogenous nucleic acid molecules encoding a transgenic construct targeting an antigen expressed in a cancerous cell of said solid tumor, in said immune cell, Nuclear Receptor Subfamily 2 Group F Member 6 (NR2F6) activity is inhibited (in comparison to a control immune cell), and binding of the immune cell to the antigen is associated with death of said cancerous cell expressing the antigen, and inducing a secondary immune reaction in the subject against cancerous cells of the solid tumor, wherein said secondary immune reaction is non-specific to the antigen targeted by the transgenic construct (epitope spreading).

2. The modified immune cell for use according to claim 1 , wherein the secondary immune reaction is directed to one or more antigens expressed by the cancerous cell, preferably to one or more antigens expressed by the cancerous cell intracellularly and/or extra cellularly.

3. The modified immune cell for use according to the preceding claim, wherein the one or more antigens expressed by the cancerous cell are non-self-tumor antigens.

4. The modified immune cell for use according to any one of the preceding claims, wherein the secondary immune reaction is a T cell mediated immune reaction, preferably a polyclonal T cell mediated immune reaction.

5. The modified immune cell for use according to any one of the preceding claims, wherein the inhibition of NR2F6 is associated with a resistance and/or a reduced sensitivity of said cell to inhibition of cytolytic activity by the tumor immune microenvironment (TIME) of the solid tumor.

6. The modified immune cell for use according to any one of the preceding claims, wherein the inhibition of NR2F6 is associated with a resistance and/or reduced sensitivity of said cell to exhaustion by chronic tumor antigen stimulation within the tumor immune microenvironment (TIME).

7. The modified immune cell for use according to any one of the preceding claims, wherein in said cell additionally Casitas B-lineage lymphoma proto-oncogene-b (CBLB) activity is inhibited (compared to a control immune cell).

8. The modified immune cell for use according to any one of the preceding claims, wherein the transgenic construct is expressed transiently in said immune cell.

9. The modified immune cell for use according to any one of the preceding claims, wherein the transgenic construct is a chimeric antigen receptor (CAR) and/or wherein the cell is a T cell, preferably a CD4⁺ or a CD8⁺ T cell.

10. The modified immune cell for use according to any one of the preceding claims, wherein in said cell the activity of NR2F6 and optionally of CBLB is inhibited by at least 50% (in comparison to a control immune cell), preferably by at least 60%, more preferably by at least 70% or a removal of NR2F6 and optionally of CBLB activity.

11 . The modified immune cell for use according to any one of the preceding claims, wherein the inhibition of NR2F6 activity and optionally of CBLB activity is obtained by disrupting the expression and/or sequence of a NR2F6 gene and optionally a CBLB gene, preferably by CRISPR-Cas, zinc finger nucleases (ZFNs), integrases, site specific recombinases, meganucleases, homing endonucleases, or TALENs, more preferably by CRISPR/Cas9, prior to administration of said cells in a subject.

12. The modified immune cell for use according to any one of the preceding claims, wherein the inhibition of NR2F6 activity and optionally of CBLB activity is obtained by knock-down of NR2F6 and optionally of CBLB, preferably by RNA interference of NR2F6 expression and optionally of CBLB expression, such as by small interfering RNA (siRNA), short hairpin RNA (shRNA), micro-RNA (miRNA), morpholinos and/or antisense oligonucleotides (ASO).

13. The modified immune cell for use according to any one of the preceding claims, wherein the inhibition of NR2F6 activity and optionally of CBLB activity is obtained by treatment of said cells with a NR2F6 antagonist and optionally a CBLB antagonist, such as a small molecule inhibitor of NR2F6 and optionally of CBLB.

14. The modified immune cell for use according to any one of the preceding claims, wherein the inhibition of NR2F6 activity and optionally of CBLB activity is obtained prior to administration of said cells to a subject and/or by simultaneous or sequential administration of said cells and a NR2F6 antagonist and optionally a CBLB antagonist to a subject.

15. The modified immune cell for use according to any one of the preceding claims, wherein the solid tumor is selected from the group consisting of glioblastoma, lung carcinoma, breast carcinoma, kidney carcinoma, pancreatic carcinoma, melanoma, intestinal carcinoma, ovarian carcinoma, prostate carcinoma and colon carcinoma.

16. A modified immune cell comprising one or more exogenous nucleic acid molecules encoding a transgenic construct targeting an antigen expressed in a cancerous cell of a solid tumor, wherein in said cell, NR2F6 activity and Casitas B-lineage lymphoma proto- oncogene-b (CBLB) activity is inhibited (compared to a control immune cell).

17. A pharmaceutical composition comprising a modified immune cell according to the preceding claim, suitable for the treatment of a solid tumor, comprising additionally a pharmaceutically acceptable carrier.

18. An in vitro method for enhancing the cytolytic activity of a modified immune cell, said immune cell comprising one or more exogenous nucleic acid molecules encoding a transgenic construct targeting an antigen expressed in a cancerous cell of a solid tumor, the method comprising inhibiting in said immune cell the activity of Nuclear Receptor Subfamily 2 Group F Member 6 (NR2F6) and optionally the activity of Casitas B-lineage lymphoma proto-oncogene-b (CBLB), wherein inhibiting NR2F6 activity and optionally CBLB activity preferably comprises: a. genetic modification of the T cell genome by disrupting the expression and/or sequence of a NR2F6 gene and optionally a CBLB gene, preferably by CRISPR- Cas, zinc finger nucleases (ZFNs), integrases, site specific recombinases, meganucleases, homing endonucleases, or TALENs, b. knock-down of NR2F6 and optionally of CBLB, preferably by RNA interference of NR2F6 expression and optionally of CBLB expression, such as by small interfering RNA (siRNA), short hairpin RNA (shRNA), micro-RNA (miRNA), morpholinos and/or antisense oligonucleotides (ASO), or c. treatment of said cells with a NR2F6 antagonist and optionally a CBLB antagonist, such as a small molecule inhibitor of NR2F6 and optionally of CBLB.