Genetically modified immune cells for the treatment of tumors and autoimmune diseases

Abstract

The present invention concerns genetically modified immune cells for the treatment of tumors and autoimmune diseases, wherein said modified immune cells are armed with innovative chimeric activating receptors directed to immune checkpoint (IC) ligands.

Description

[0001] Genetically modified immune cells for the treatment of tumors and autoimmune diseases

[0002] The present invention concerns genetically modified immune cells for the treatment of tumors and autoimmune diseases. In particular, the present invention concerns genetically modified immune cells, such as NK cells, for the treatment of tumors and autoimmune diseases, wherein said modified cells are armed with innovative chimeric activating receptors directed to immune checkpoint (IC) ligands.

[0003] It is well known that the fight against cancer is one of the 21stcentury challenges for researchers and clinicians. In fact, cancer is becoming the most frequent cause of morbidity and mortality in Western countries.

[0004] In this scenario, the battle against solid tumors is the one that requires greater efforts, because, in addition to defeating the tumor, it must also counteract a series of elements that constitute what is called tumor microenvironment (TME), which sustains and protects the tumor and reduces the effectiveness of immune responses and therapeutic treatments.

[0005] In the last decades, great efforts have been made to improve the outcome of oncological patients with the establishment of intense multimodal treatment approaches and a better characterization of tumor biology. Although today selected patient groups require less invasive procedures and abbreviated courses of chemotherapy, the majority of patients need intensive systemic and multimodal interventions that may cause unavoidable long-term toxicity and an increased secondary malignancy rate. Moreover, subsets of patients suffering from recurrent or resistant tumors still carry a very poor prognosis and conventional therapies cannot offer a cure in these situations. Thus, the development of new, more effective, and less toxic therapies is an urgent clinical need.

[0006] Besides novel molecular medicines, immunotherapeutic approaches represent a highly innovative and promising field in cancer treatment. The repertoire of immunotherapeutic drugs is broad and comprises different monoclonal antibodies (mAbs) to target immune or cancer cells, therapeutic vaccines to induce antitumor immune responses, oncolytic viruses, artificial proteins crosslinking tumor and immune cells, or most recently genetically engineered immune cells that engage in an enforced interaction with cancer cells. However, their success is still restricted to a small group of tumor types and, even in that context, responses are frequently heterogeneous mainly due to the fact that tumor cells develop various immune escape mechanisms capable of impairing the immunosurveillance and efficacy of the immunotherapeutic approaches. Furthermore, immune therapies are enormously expensive (several billion EUR / year) and are often accompanied by high toxicity.

[0007] Chimeric Antigen Receptor (CAR) T cells provide an innovative immunotherapeutic platform that may lead to eradicating cancer without causing chronic disabilities.

[0008] However, to obtain CAR-T cells is necessary to use the autologous patient's cells in order to not cause graft versus host disease (GvHD). Furthermore, it is important to note that after conventional anti-tumor therapy the patient is immunocompromised and therefore the quantity and the quality of autologous cells for the generation of CAR-T is complicated and often results in low effected and persistent productsFive CAR-T cell products have been so far approved for the treatment of cancers refractory to standard treatments. However, a / |3 CAR-T cells are sometimes limited in their application due to their poor scalability, long manufacturing time, excessive cost, and significant toxicity.

[0009] In addition, the present therapy using CAR-T cells or monoclonal antibodies (mAbs) is able to target antigens selectively expressed on the surface of given tumors. A limitation of these approaches is the fact that these antigens are mostly infrequent and confined to a tumor type. In addition, some of these antigens, during tumor progression, are shedded or may be lost or modified thus compromising the effect of mAbs or CAR-T cell approaches.

[0010] An innovative strategy, that may overcome such limitations, is the selection of "common" antigens expressed by different (e.g. histotype, stage, site) tumors. Excellent candidates with these characteristics are represented by the tumor ligands of inhibitory checkpoints that are commonly expressed and / or strongly upregulated by most tumors as well as from the immunosuppressive elements of TME. However, the interaction with the respective receptor (expressed on effector cells such as T or NK cells) results in a negative signal which blocks the anti-tumor effector function of these cells.

[0011] Previous studies demonstrated that the ligands of immune checkpoints (IC) are highly expressed in many tumor types not only by tumor cells but also by tumor-associated (TA) cells. Their interaction with IC may induce exhaustion of innate and adaptive effector cells and generate / recruit immunosuppressive cells including myeloid-derived suppressor cells (MDSC) and regulatory T cells (Treg). Different clinical trials have been performed based on the use of mAbs able to disrupt the interaction between IC and IC-ligands. This treatment may unleash tumorspecific T cells and NK cells allowing tumor cell killing. Cancer immunotherapy, based on this innovative therapeutic approach, witnessed major progress. Indeed, the success of IC blockade has been one of the greatest achievements in cancer therapy and has boosted research which led to the discovery of other potential checkpoints such as LAG-3 and TIM-3. However, therapies based on IC blockage may be associated with immune-related adverse events requiring clinical management and with loss of therapeutic efficacy. Indeed, although important results have been achieved for some malignancies, results for patients with relapsed / refractory (R / R) solid tumors remain largely unsatisfactory. In addition, at least for this group of patients, the rate of cancer-related mortality remains high due to the loss of tumor antigens and / or tumor heterogeneity and / or the presence of a strongly immunosuppressive Tumor microenvironment (TME).

[0012] It is also known, as revealed by several studies, that immune checkpoints are involved in the pathogenesis of various autoimmune diseases, such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), multiple sclerosis (MS), Sjogren’s syndrome (SS) and ankylosing spondylitis (AS). The noticeable hallmarks of autoimmune diseases are the breakdown of self-tolerance and the self-attack of the immune system, that is, our immune system fails to distinguish self from non-self. (PMID: 26212387).

[0013] In the light of the above, it is therefore clear the need to provide new therapies for tumors and for autoimmune diseases able to overcome the above-mentioned limitations.

[0014] According to the present invention, a new cellular product suitable for the treatment of tumors and autoimmune diseases is provided. In particular, the present invention provides an allogeneic, off-the-shelf, and ready-to-use cell product armed with innovative chimeric “activating” receptors directed to immune checkpoint (IC) ligands, wherein said ligands can be expressed by a tumor, as well as by the immunosuppressive elements of TME, or in the presence of an autoimmune disease. The genetically modified cells, in particular NK cells (gmNK), according to the invention are therefore able to be physiologically activated in the presence of the respective immune checkpoint ligand. Therefore, in the case of a tumor, NK cells of the invention are capable of simultaneously blocking some of its escape mechanisms promoting NK and T cell expansion.

[0015] NK cells are crucial components of the innate immune system and play an important role in the host response against pathogens and cancer. In particular, they have the ability to kill tumor target cells and produce soluble factors important for regulating both innate and adaptive immune responses.

[0016] Importantly, HLA-class I down-regulation, one of the most common mechanisms of tumor immune evasion from a / [3-T cells, results in NK cell triggering and activation of their functional program. However, the majority of tumor cells and TA cells maintained the expression of non-classical HLA-class I (HLA-E and HLA-G) that could inhibit NK cell anti-tumor activity. Based on this observation different mAbs directed against the specific inhibitory receptors (e.g.:NKG2A) expressed by NK cells have been developed for clinical use to overcome the inhibitory signals mediated by NKG2A with HLA-E interaction.

[0017] NK cells represent an emerging cellular therapeutic option with the capacity to infiltrate tissues and display strong cytotoxic activity against tumor cells. This occurs in the absence of specificity for given antigens and no need for multi-step priming and extensive proliferation that characterizes T cell responses. Moreover, allogeneic NK cells compared to T cells, do not cause Graft versus Host Disease and display a low incidence of cytokine release syndrome (CRS) thus representing an “off- the-shelf” product immediately available in high amounts for therapeutic use.

[0018] The product according to the present invention represents a new and unexplored adoptive-cell therapy, in particular for the treatment of R / R tumors, based on exploiting / overcoming, rather than suffering, the inhibitory effect of TME.

[0019] In fact, according to the present invention, the interaction between ICs and their ligands converts the negative signal into an activating signal inducing a strong effector function and anti-tumor response. More in detail, the nucleotide sequence provided according to the present invention encodes a) a chimeric receptor able to recognize its immune checkpoint receptor or ligand, b) a self-cleaving molecule, such as a single chain variable fragment, able to bind an inhibitory receptor and c) a self-cleaving cytokine / chemokine.

[0020] The three elements a), b) and c) according to the invention work synergistically in order to obtain a potentiate effectiveness on tumors or autoimmune diseases. In particular, the chimeric receptor a) targets an immune checkpoint ligand and activates the innate immune cell on which said chimeric receptor is expressed; at the same time molecule b) advantageously blocks a molecule able to inhibit immune cell anti-tumor activity, such as NKG2a, whereas soluble factor c) advantageously potentiate their immune cell function, survival, migration and / or proliferation as well as induce local inflammation capable to recruit other effector immune cells which can work in synergy with the proposed genetically modified cells to defeat the tumor by benefiting from the absence of two inhibition signals and the presence of a cytokine / chemokine that also allows their survival and proliferation.

[0021] Unexpectedly, according to the present invention, the combined presence of the three above-mentioned components has proven to be capable of inducing not only proliferation and survival of genetically modified NK cells, but also simultaneously blocking two of the main inhibition mechanisms used by tumors to turn off the anti-tumor response.

[0022] It is also worth noting that it is known in the prior art that the systemic administration of cytokines, even though it can be necessary in order to potentiate the effect of the immune cells, can have several toxic effects. Furthermore, systemic administration of cytokines does not ensure an adequate concentration at tissue level capable of adequately stimulating the effector cells. With the construct according to the present invention, the cytokine or chemokine is directly produced by the immune cell locally and at low and controlled doses, without any toxic effect.

[0023] In addition, the production of molecules, such as single chain or antibody fragments, specifically targeting NK cell inhibitory receptors could ameliorate the efficacy of therapy. This is possible thanks to a safe, robust, and easy genetic modification (gm) of allogenic (donor) NK cells using retroviral vectors. The vectors according to the invention, encoding for chimeric proteins bearingexIC fused to cytoplasmatic activating and co- stimulatory domains, may counteract rather than reinforce, the TME. Receptor / Ligand interaction will result in NK cell activation and cell killing when gmNK cells bind to ICs-ligands in TME and in the inflammatory microenvironment (i.e. autoimmune diseases).

[0024] In particular, as shown below, the vector produced according to the present invention encodes for chimeric proteins bearing an IC ectodomain (exIC) fused to the cytoplasmatic NKG2D and 4.1 BB co-stimulatory domains in frame with NKG2A single chain variable fragment (scfv)(in order to prevent the activation of the inhibitor signal) and a secreted IL-15 that sustain NK cell proliferation and function.

[0025] Since the above-mentioned characteristics are common to a broad range of tumor types, the product according to the present invention may be suitable for the treatment of different R / R malignancies. Moreover, since, as explained above, the immune checkpoints are involved in the pathogenesis of various autoimmune diseases, on the basis of the data reported below it is plausible that theexIC gmNK products according to the invention are also effective for the treatment of autoimmune diseases.

[0026] In addition, the chimeric receptor according to the present invention can also be expressed in other types of immune cells besides NK, such as for example gamma delta T cells, that exert their function without the necessity of specific TCR binding as alfa / beta T cells.

[0027] As can be seen from the experimental results reported below, modified gmNK cells according to the invention carrying PD-1 asexIC, also producing an antibody fragment against NK inhibitory receptor and producing IL15, were tested. The results surprisingly showed thatexPD1- NK cells displayed a higher cytolytic activity as compared to nontransduced (NT)-NK cells against PD-L1+tumor cells at different effector: target ratios. It was also demonstrated that the ability of gmNK cells to release IL15 allows them to maintain anti-tumor activity for longer time intervals. Furthermore, IL15 allows gmNK cells to release IFNg which induces PDL-1 expression on tumor cells and TA-cells, thus making them more susceptible to gmNK cell recognition and killing. GmNK cells were also able to release sigle cell variable fragment (scfv) cable to recognise NKG2A. This binding is able to prevent the activation of the inhibitor signalling in NK cells following to the binding to non-canonical class I molecules expressed by tumor cells and TA. In addition, according to the present invention it was shown in vivo thatexPD1-NK cells are highly efficient in controlling tumour growth, that they were present in mice for long time after their administration and that they are effective in the treatment of the tumor since a very low count of tumor cells in treated mice was observed after the treatment withexPD1- NK cells according to the present invention.

[0028] The gmNK cells provided according to the present invention advantageously represent a springboard to rapidly develop sustainable cellular therapies potentially able to cure a broad number of tumor types and autoimmune diseases, thus making therapy available and affordable for a much wider patient population. Indeed, their applicability could be extended to high-risk patients with relapsed / refractory (R / R) disease as well as to patients with plastic malignancies characterized by immunosuppressive TME. Moreover, the products provided according to the present invention are ready-to-use donor-derived cellular products armed with a broadly reactive - activating receptor which may allow major advances in adoptive cellular therapy. In addition, according to the present invention, the use of third-party (donor) NK cells offers “off-the-shelf” products that may be standardized and applied rapidly to cancer patients with highly reduced costs.

[0029] Moreover, the cellular product according to the invention is characterized by the following advantages:

[0030] Is a stand-alone treatment and does not require further gene modifications or treatments;

[0031] Uses lymphocytes derived from the innate immune system, such as NK and gamma / delta T cells, which can be used also in an allogenic setting since they are unable to induce GVHD;

[0032] Fuses together different features which make the generated cellular product more robust and effective and applicable also in settings for which there are no applications to date such as for example in the early posttransplant setting, allowing to work in a minimal residual disease condition.

[0033] Therefore, the cellular products according to the invention advantageously represent an alternative, cost-effective, and improved therapeutic option both in terms of survival and quality-of-life as compared to CAR-T cells or other approaches.

[0034] The therapeutic strategy proposed according to the present invention advantageously represents a true scientific advance based on new knowledge going far beyond the current state-of-the-art, paving the way for a totally novel generation of immunotherapeutic products, circumventing several of the challenges and obstacles that impair the present cellular and gene therapy of cancer.

[0035] A deeper analysis of the cellular components and the expression of IC in the TME to generate medicinal assets in the fight against cancer, with a potentially improved safety profile over other current cellular therapies, will be a major contribution to cancer treatment and a unique paradigm of “circular economy” in medicine. While the driving molecular alterations within each tumor histotype may differ, the TME is a common characteristic for many tumors.

[0036] The outcome of the administration of one or more boosts of the third-party NK cell product according to the invention may result in remission or complete eradication of tumors without side effects. Importantly, it will be possible to treat patients immediately after the failure of rescue therapies, avoiding long waiting times necessary instead in the case of other therapies such as CAR T cell products. The product provided by the present invention is highly innovative and has a major translational impact since it is based on an optimized immunotherapeutic approach using allogeneic donor-derived gmNK cells with innovative chimeric activating checkpoint domains able to subvert with one medication several tumor-driven immunosuppression.

[0037] In summary, the present invention could greatly improve cell-based immunotherapies, since its mechanisms of action may allow broader applications (inflammatory and tumor pathologies) and larger tumor patient populations.

[0038] Therefore, the present invention concerns a chimeric receptor for an immune checkpoint (IC) ligand, said chimeric receptor comprising or consisting of, from N-terminus to C-terminus,

[0039] - a signal peptide, fused to

[0040] - an extracellular domain of an immune checkpoint molecule (exIC), which is able to bind the respective immune checkpoint ligand, fused to

[0041] - a transmembrane domain of said immune checkpoint molecule, fused to

[0042] - one or more costimulatory domains.

[0043] In particular, according to the present invention, said one or more costimulatory domains represent an activating domain able to activate the immune cell, for example the NK cell, carrying the chimeric receptor. Said one or more costimulatory domains can be one or more intracellular domains.

[0044] More in detail, it is an object of the present invention a nucleotide sequence encoding a) a chimeric receptor for an immune checkpoint ligand, said chimeric receptor comprising or consisting of, from N-terminus to C- terminus,

[0045] - a signal peptide, fused to

[0046] - an extracellular domain of an immune checkpoint molecule, fused to

[0047] - a transmembrane domain of said immune checkpoint molecule, fused to

[0048] - one or more costimulatory domains; b) a soluble molecule able to bind a target, wherein said molecule is chosen from an antibody fragment (or antibody-derived fragment), a ligand of said target or a receptor of said target and wherein said target is chosen from an immune checkpoint molecule or an immunosuppressive molecule; c) one or more soluble factors chosen from a cytokine or a chemokine; said nucleotide sequence being a single nucleotide sequence or more than one nucleotide sequence.

[0049] According to the present invention, said nucleotide sequence encoding a), b) and c) is preferably a single nucleotide sequence encoding all three a), b) and c). Alternatively, said nucleotide sequence can be more than one sequence, for example: a first sequence encoding a) and b) and a second sequence encoding c); a first sequence encoding a) and c) and a second sequence encoding b); a first sequence encoding a) and a second sequence encoding b) and c); or a first sequence encoding a), a second sequence encoding b) and a third sequence encoding c).

[0050] According to the present invention, when said nucleotide sequence is a single nucleotide sequence or when said more than one nucleotide sequences comprise a sequence encoding at least two of a), b) or c), said nucleotide sequence can further comprise a sequence encoding a selfcleaving peptide before the sequence encoding b) and / or c) for the release of b) and / or c) after translation.

[0051] In fact, according to the present invention, chimeric receptor a) encoded by the said nucleotide sequence is expressed on an innate immune cell, whereas b) and c) can be released outside the same cell thanks to a self-cleaving peptide. In particular, a self-cleaving peptide can link the nucleotide sequence encoding b) to the nucleotide sequence encoding a) or it can link the nucleotide sequence encoding c) to the nucleotide sequence encoding b). The sequence encoding a self-cleaving peptide can be fused at the 5’ -end of the sequence encoding b) and / or at the 5’-end of the sequence encoding c).

[0052] According to the present invention, said immune checkpoint molecule can be chosen from the group consisting of PD-1 , TIGIT, B7-H3, B7-H4, Lag-3, TIM-3, CTLA-4.

[0053] Preferably, said immune checkpoint molecule is PD-1. In particular, the sequence of said extracellular domain of PD-1 can be FLDSPDRPWNPPTFSPALLWTEGDNATFTCSFSNTSESFVLNWYRMSP SNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSWRARRNDSG TYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTL V (SEQ ID NO:20), whereas the sequence of said transmembrane domain of PD-1 can be VGWGGLLGSLVLLVWVLAV (SEQ ID NO:21 ).

[0054] According to another preferred embodiment of the present invention, said immune checkpoint molecule can be TIGIT. In particular, the sequence of said extracellular domain of TIGIT can be MMTGTIETTGNISAEKGGSIILQCHLSSTTAQVTQVNWEQQDQLLAICNA DLGWHISPSFKDRVAPGPGLGLTLQSLTVNDTGEYFCIYHTYPDGTYTG RIFLEVLESSVAEHGARFQIP (SEQ ID NO:32), whereas the sequence of said transmembrane domain of TIGIT can be LLGAMAATLWICTAVIVWAM (SEQ ID NO:33).

[0055] According to the present invention, the signal peptide can be a short peptide sequence capable of translocating the target protein in a specific location in the cell or secrete it. In particular, according to the invention, the signal peptide is preferably a sequence able to translocate the immune checkpoint molecule on the membrane of the cell carrying the chimeric receptor so that the extracellular domain of said immune checkpoint molecule is exposed on the outside surface of the cell membrane. More in particular, according to the present invention, said signal peptide can be chosen from the group consisting of MQIPQAPWPWWAVLQLGWRPGW (SEQ ID NO: 19) and MRWCLLLIWAQGLRQAPLASG (SEQ ID NO:31 ).

[0056] According to the present invention, said one or more costimulatory domains can be chosen from the group consisting of NKG2D, 4-1 BB, CD3 , DAP10, DAP12, CD28, OX-40, CD27, ICOS. According to the present invention, said one or more costimulatory domains can be at least two or at least three or at least four costimulatory domains, preferably chosen from the group consisting of NKG2D, 4-1 BB, CD3 , DAP10, DAP12, CD28, OX-40, CD27, ICOS. Preferably, said one or more costimulatory domains are two costimulatory domains fused to each other. For example, said activating domain may comprise a first costimulatory domain chosen from the group consisting of NKG2D, 4-1 BB, DAP10, DAP12, CD28, OX-40, CD27, ICOS and a second costimulatory domain chosen from the group consisting of NKG2D, 4-1 BB, CD3 , DAP10, DAP12, CD28, OX-40, CD27, ICOS. Preferably, when said one or more co-stimulatory domains are more than one and they comprise CD3 , said CD3 is positioned at the N- terminus of the sequence with respect to the other co-stimulatory domain or domains.

[0057] Preferably, said costimulatory domains are chosen from NKG2D, 4- 1 BB, CD3 , DAP10 and DAP12. In particular, the sequence of NKG2D can be GWIRGRRSRHSWEMSEFHNYNLDLKKSDFSTRWQKQRCPWKSKCRE NAS (SEQ ID NO:22), the sequence of 4-1 BB can be KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRA (SEQ ID NO:23), the sequence of CD3 can be RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGG KPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLS TATKDTYDALHMQALPPR (SEQ ID NO:34), the sequence of DAP10 can be LCARPRRSPAQEDGKVYINMPGRG (SEQ ID NO:35) and the sequence of DAP 12 can be

[0058] YFLGRLVPRGRGAAEAATRKQRITETESPYQELQGQRSDVYSDLNTQR PYYK (SEQ ID NO:36).

[0059] According to a preferred embodiment of the present invention, said one or more costimulatory domains are two costimulatory domains chosen from the group consisting of NKG2D and 4-1 BB; 4-1 BB and CD3 4-1 BB and DAP10; or 4-1 BB and DAP12. In particular, said two costimulatory domains may be chosen from the group consisting of: NKG2D fused to 4- 1 BB (NKG2D.4-1 BB), wherein NKG2D is at the N-terminus of the sequence, for example SEQ ID NO:22 fused to SEQ ID NO:23; 4-1 BB fused to CD3 (4-1 BB.CD3 , wherein 4-1 BB is at the N-terminus of the sequence, therefore for example SEQ ID NO:23 fused to SEQ ID NO:34; 4-1 BB fused to DAP10 (4-1 BB.DAP10), wherein 4-1 BB is at the N- terminus of the sequence, therefore, for example, SEQ ID NO:23 fused to SEQ ID NO:35; or 4-1 BB fused to DAP12 (4-1 BB.DAP12), wherein 4-1 BB is at the N-terminus of the sequence, therefore, for example, SEQ ID NO:23 fused to SEQ ID NO:36.

[0060] Preferably, according to the present invention, said immune checkpoint molecule is PD-1 and said costimulatory domains are two costimulatory domains chosen from NKG2D and 4-1 BB; 4-1 BB and CD3 4- I BB and DAP10; or 4-1 BB and DAP12.

[0061] According to an embodiment of the invention, said immune checkpoint molecule is PD-1 and

[0062] - said signal peptide is SEQ ID NO:19,

[0063] - said extracellular domain of PD-1 is SEQ ID NQ:20,

[0064] - said transmembrane domain of PD-1 is SEQ ID NO:21 ,

[0065] - said one or more costimulatory domains are two costimulatory domains consisting of

[0066] NKG2D of sequence SEQ ID NO:22 fused to 4-1 BB of sequence SEQ ID NO:23, or consisting of

[0067] 4-1 BB of sequence SEQ ID NO:23 fused to CD3 of sequence SEQ ID NO:34, or consisting of

[0068] 4-1 BB of sequence SEQ ID NO:23 fused to DAP10 of sequence SEQ ID NO:35, or consisting of

[0069] 4-1 BB of sequence SEQ ID NO:23 fused to DAP12 of sequence SEQ ID NO:36, preferably consisting of NKG2D of sequence SEQ ID NO:22 fused to 4-1 BB of sequence SEQ ID NO:23.

[0070] According to another embodiment of the invention, said immune checkpoint molecule is TIGIT and

[0071] - said signal peptide is SEQ ID NO:31 ,

[0072] - said extracellular domain of TIGIT is SEQ ID NO:32,

[0073] - said transmembrane domain of PD-1 is SEQ ID NO:33, - said one or more costimulatory domains are two costimulatory domains consisting of NKG2D of sequence SEQ ID NO:22 fused to 4-1 BB of sequence SEQ ID NO:23.

[0074] According to the present invention: the nucleotide sequence encoding the signal peptide of SEQ ID NO: 19 can be

[0075] ATGCAGATTCCACAGGCTCCTTGGCCAGTCGTTTGGGCAGTTTTGCA GCTGGGCTGGAGACCAGGCTGG (SEQ ID NO:1 ); the nucleotide sequence encoding the extracellular domain of PD1 of sequence SEQ ID NQ:20 can be TTCCTTGATAGCCCTGACAGACCTTGGAACCCCCCAACTTTTAGTCCA GCCCTGCTTGTGGTGACTGAGGGGGACAACGCCACCTTTACCTGCA GTTTTAGCAACACGAGTGAATCTTTTGTCCTGAATTGGTATAGGATGA GTCCATCCAACCAGACGGATAAGCTGGCCGCTTTCCCTGAAGATAGG TCACAGCCCGGGCAAGACTGTCGGTTTAGAGTCACCCAGCTGCCCA ACGGTAGAGACTTTCACATGTCAGTGGTTCGAGCAAGAAGGAATGAT TCAGGAACCTACCTGTGTGGTGCTATCTCACTTGCACCTAAGGCCCA AATTAAAGAGTCACTTAGAGCCGAGCTCAGGGTCACTGAACGAAGGG CAGAAGTTCCCACTGCACACCCATCTCCATCACCTAGACCAGCCGGC CAATTTCAAACGCTTGTC (SEQ ID NO:2); the nucleotide sequence encoding the transmembrane domain of PD1 of sequence SEQ ID NO:21 can be GTTGGAGTGGTAGGGGGTTTGCTCGGATCCCTCGTTCTGCTCGTGTG GGTGTTGGCCGTC (SEQ ID NO:3); the nucleotide sequence encoding NKG2D of sequence SEQ ID NO:22 can be

[0076] GGCTGGATCAGGGGCAGGAGGAGCAGGCACAGCTGGGAGATGAGC GAGTTCCACAACTACAACCTGGACCTGAAGAAGAGCGACTTCAGCAC CAGGTGGCAGAAGCAGAGGTGCCCCGTGGTGAAGAGCAAGTGCAG GGAGAACGCCAGC (SEQ ID NO:4), the nucleotide sequence encoding 4-1 BB of sequence SEQ ID NO:23 can be AAACGCGGCCGCAAGAAACTCCTGTATATATTCAAACAACCATTTATG AGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATT TCCAGAAGAAGAAGAAGGAGGATGTGAACTG (SEQ ID NO:5), the nucleotide sequence encoding CD3 of sequence SEQ ID NO:34 can be AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGG GCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAG TACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGG GAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACT GCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAA GGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGT CTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGC CCTGCCCCCTCGC (SEQ ID NO:16), the nucleotide sequence encoding DAP10 of sequence SEQ ID NO:35 can be TTGTGTGCAAGACCAAGAAGAAGTCCAGCCCAGGAAGACGGCAAGG TCTACATTAACATGCCCGGGAGGGGA (SEQ ID NO:17) and the nucleotide sequence encoding DAP12 of sequence SEQ ID NO:36 can be TACTTTCTGGGTAGATTGGTCCCCAGGGGAAGAGGAGCAGCTGAAG CAGCCACACGCAAACAGAGAATCACCGAGACAGAGTCCCCATATCAG GAGTTGCAGGGTCAGAGGTCTGACGTGTACTCCGACCTCAACACTCA AAGGCCATATTACAAA (SEQ ID NO: 18); the nucleotide sequence encoding the signal peptide of sequence SEQ ID NO:31 can be

[0077] ATGAGATGGTGTTTGCTGCTGATTTGGGCCCAAGGGCTTAGGCAAGC ACCCCTGGCTTCTGGT (SEQ ID NO: 13); the nucleotide sequence encoding the extracellular domain of TIGIT of sequence SEQ ID NO:32 can be ATGATGACCGGAACCATAGAAACAACCGGGAACATCTCCGCGGAAAA AGGCGGATCTATCATCCTCCAGTGTCACCTTTCTTCTACGACCGCCC AAGTCACTCAGGTGAACTGGGAGCAACAGGATCAGCTCCTCGCTATC TGTAACGCTGACCTCGGATGGCATATTTCCCCCTCTTTCAAAGACCG CGTAGCACCAGGTCCAGGTTTGGGACTGACGCTTCAGTCCCTGACC GTAAATGATACCGGGGAGTACTTCTGCATCTACCACACTTATCCTGAC GGCACATACACCGGTCGGATATTTCTGGAGGTCCTTGAAAGTTCTGT CGCTGAGCATGGAGCCAGGTTTCAAATACCT (SEQ ID NO: 14); the nucleotide sequence encoding the transmembrane domain of TIGIT of sequence SEQ ID NO:33 can be TTGCTGGGCGCAATGGCTGCCACACTGGTTGTAATTTGTACTGCCGT CATTGTAGTGGTGGCTATG (SEQ ID NO: 15). The nucleotide sequence according to the invention can be a nucleotide sequence comprising each of the above listed specific sequences independently or it can comprise more of the above listed sequences.

[0078] As stated above, according to the present invention said molecule b) encoded by said nucleotide sequence can be an antibody fragment, such as a single chain variable fragment or a nanobody, having an immune checkpoint (IC) or an immunosuppressive molecule as a target. In this case, said sequence can further comprises a nucleotide sequence encoding a self-cleaving peptide before the sequence encoding the antibody fragment, wherein preferably said nucleotide sequence encoding a self-cleaving peptide links the nucleotide sequence encoding the antibody fragment to the nucleotide sequence encoding the chimeric receptor.

[0079] According to the present invention, said antibody fragment can be a single chain variable fragment (scFv) comprising a variable heavy chain (VH) and a variable light chain (VL) linked to each other by a linker, preferably wherein said single chain variable fragment is linked to the nucleotide sequence encoding said chimeric receptor by said nucleotide sequence encoding a self-cleaving peptide.

[0080] Therefore, the nucleotide sequence encoding a chimeric receptor as defined above can further comprise a nucleotide sequence encoding for an antibody fragment, such as for example a scFv comprising the VH chain followed by a linker and the VL chain. As stated above, the nucleotide sequence encoding the antibody fragment, such as the scFv, is preferably linked to the sequence encoding said chimeric receptor by a nucleotide sequence encoding a self-cleaving peptide, for example T2A, P2A, E2A, F2A, IRES. Preferably, the nucleotide sequence encoding the antibody fragment, such as the scFv, is linked to the C-terminus of the nucleotide sequence of the chimeric receptor, for example, it can be linked to the sequence encoding the intracellular activating domain.

[0081] According to an embodiment of the present invention, said nucleotide sequence can further comprise a nucleotide sequence encoding an immunoglobulin heavy chain signal peptide linked to the sequence encoding said antibody fragment. The immunoglobulin heavy chain signal peptide, when present, is preferably placed between the sequence encoding the self-cleaving peptide and the sequence encoding the antibody fragment, such as the scFV.

[0082] According to the present invention, said immunoglobulin heavy chain signal peptide can be a signal peptide derived from immunoglobulins, cytokines, chemokines or derived from other secretory molecules. The immunoglobulin heavy chain signal peptide is intended to secrete the antibody fragment outside the cell. According to the present invention, the sequence of said immunoglobulin heavy chain signal peptide can be MEFGLSWLFLVAILKGVQCSR (SEQ ID NO:25). For example, according to the present invention, the nucleotide sequence encoding for SEQ ID NO:25 can be

[0083] ATGGAGTTCGGCCTGAGCTGGCTGTTCCTGGTGGCCATCCTGAAGG GCGTGCAGTGCAGCAGG (SEQ ID NO:7).

[0084] According to the present invention, the target of said antibody fragment can be chosen from NKG2a and HLA-G, preferably NKG2a. Preferably, said antibody fragment is a scFv having a target chosen from NKG2a and HLA-G, preferably NKG2a.

[0085] According to a preferred embodiment of the present invention, said antibody fragment is a single chain variable fragment (scFv) having NKG2a as a target. In particular, the sequences of VL and VH of said single chain variable fragment having NKG2a as a target can be the following:

[0086] VL sequence:

[0087] QIVLTQSPALMSASPGEKVTMTCSASSSVSYIYWYQQKPRSSPKPWIYL TSNLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSGNPYTF GGGTKLEIKR (SEQ ID NO:26)

[0088] VH sequence:

[0089] EVQLVESGGGLVKPGGSLKLSCAASGFTFSSYAMSWVRQSPEKRLEWV AEISSGGSYTYYPDTVTGRFTISRDNAKNTLYLEISSLRSEDTAMYYCTR HGDYPRFFDVWGAGTTVTVSS (SEQ ID NO:28).

[0090] For example, the nucleotide sequences encoding SEQ ID NO:26 and SEQ ID NO:28 can be the following

[0091] VL sequence

[0092] CAGATCGTGCTGACCCAGAGCCCCGCCCTGATGAGCGCCAGCCCCG GCGAGAAGGTGACCATGACCTGCAGCGCCAGCAGCAGCGTGAGCTA CATCTACTGGTACCAGCAGAAGCCCAGGAGCAGCCCCAAGCCCTGG ATCTACCTGACCAGCAACCTGGCCAGCGGCGTGCCCGCCAGGTTCA GCGGCAGCGGCAGCGGCACCAGCTACAGCCTGACCATCAGCAGCAT GGAGGCCGAGGACGCCGCCACCTACTACTGCCAGCAGTGGAGCGG CAACCCCTACACCTTCGGCGGCGGCACCAAGCTGGAGATCAAGAGG (SEQ ID NO:8)

[0093] VH sequence GAGGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGGTGAAGCC CGGCGGCAGCCTGAAGCTGAGCTGCGCCGCCAGCGGCTTCACCTTC AGCAGCTACGCCATGAGCTGGGTGAGGCAGAGCCCCGAGAAGAGG CTGGAGTGGGTGGCCGAGATCAGCAGCGGCGGCAGCTACACCTACT ACCCCGACACCGTGACCGGCAGGTTCACCATCAGCAGGGACAACGC CAAGAACACCCTGTACCTGGAGATCAGCAGCCTGAGGAGCGAGGAC ACCGCCATGTACTACTGCACCAGGCACGGCGACTACCCCAGGTTCTT CGACGTGTGGGGCGCCGGCACCACCGTGACCGTGAGCAGC (SEQ ID NQ:10).

[0094] The sequence of the linker of said single chain variable fragment can be GGGSGGGG (SEQ ID NO:27). For example, the nucleotide sequence encoding SEQ ID NO:27 can be GGCGGCGGCAGCGGCGGCGGCGGC (SEQ ID NO:9).

[0095] According to the present invention, said self-cleaving peptide can be chosen from T2A, P2A, E2A, F2A, IRES, and for molecule b), such as the scFV mentioned above, the cleaving peptide is preferably T2A. In particular, the sequence of T2A can be EGRGSLLTCGDVEENPGP (SEQ ID NO:24). According to the present invention, the nucleotide sequence encoding SEQ ID NO:24 can be SEQ ID NO:6.

[0096] As stated above, the nucleotide sequence according to the invention further comprises a nucleotide sequence encoding for one or more cytokines, such as one or more interleukins, or chemokines, which is preferably linked to the nucleotide sequence encoding said chimeric receptor or to the nucleotide sequence encoding said antibody fragment by a nucleotide sequence encoding a self-cleaving peptide.

[0097] In particular, said one or more cytokines can be chosen from the group consisting of IL15, IL2, IL7, IL21 , IL18, IL1 |3, lper-IL15, cytokine’s fusion, preferably IL15. According to the invention, “cytokine’s fusion” means a chimeric protein where two or more cytokines are linked to each other by a linker sequence or where one or more cytokines are fused to a Fc region of an antibody (Gillies, S.D., Lan, Y., Brunkhorst, B. et al. Bifunctional cytokine fusion proteins for gene therapy and antibody-targeted treatment of cancer. Cancer Immunol Immunother 51 , 449-460 (2002). https: / / doi.org / 10.1007 / s00262-002-0302-6; Jinyu Zhang, Xuan Zhao. Administration of fusion cytokines induces tumor regression and systemic antitumor immunity. Med Comm, vol. 1 Issue 2, pp. 256-268). According to an embodiment of the present invention, said cytokine is IL15 of sequence

[0098] MRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANVWN VISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGD ASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQ MFINTS (SEQ ID NO:30). The nucleotide sequence encoding SEQ ID NO:30 can be

[0099] ATGCGGATCAGCAAGCCCCACCTGCGGAGCATCAGCATCCAGTGCT ACCTGTGCCTGCTGCTGAACAGCCACTTCCTGACCGAGGCCGGCAT CCACGTGTTCATCCTGGGCTGCTTCAGCGCCGGACTGCCCAAGACC GAGGCCAACTGGGTGAACGTGATCAGCGACCTGAAGAAGATCGAGG ACCTGATCCAGAGCATGCACATCGACGCCACCCTGTACACCGAGAG CGACGTGCACCCCAGCTGCAAGGTGACCGCCATGAAGTGCTTTCTG CTGGAACTGCAGGTGATCAGCCTGGAAAGCGGCGACGCCAGCATCC ACGACACCGTGGAGAACCTGATCATCCTGGCCAACAACAGCCTGAG CAGCAACGGCAACGTGACCGAGAGCGGCTGCAAAGAGTGCGAGGAA CTGGAAGAGAAGAACATCAAAGAGTTTCTGCAGAGCTTCGTGCACAT CGTGCAGATGTTCATCAACACCAGCTGA (SEQ ID NO:12). As stated above, according to the present invention, said selfcleaving peptide can be chosen from the group consisting of E2A, P2A, T2A, F2A, and IRES, and for molecule c) is preferably E2A. According to the invention, the sequence of E2A self-cleaving peptide can be GGPQCTNYALLKLAGDVESNPGP (SEQ ID NO:29). The nucleotide sequence encoding SEQ ID NO:29 can be CAGTGTACTAATTATGCTCTCTTGAAATTGGCTGGAGATGTTGAGAGC AATCCCGGGCCC (SEQ ID NO:11 ). Therefore, the nucleotide sequence according to the present invention can encode for the chimeric receptor, an antibody fragment, such as a scFv, a ligand or a receptor targeting an immune checkpoint molecule or an immunosuppressive molecule and a cytokine, such as an interleukin, or a chemokine. Both the antibody fragment, ligand or receptor and the cytokine or chemokine, when present, are intended to be secreted outside the cell. For example, the nucleotide sequence according to the invention can encode for a chimeric receptor having PD-1 as IC, an scFv targeting NKG2a and the interleukin IL15 and can be one of the following sequences: ATGCAGATTCCACAGGCTCCTTGGCCAGTCGTTTGGGCAGTTT

[0100] TGCAGCTGGGCTGGAGACCAGGCTGGTTCCTTGATAGCCCTGACAG

[0101] ACCTTGGAACCCCCCAACTTTTAGTCCAGCCCTGCTTGTGGTGACTG

[0102] AGGGGGACAACGCCACCTTTACCTGCAGTTTTAGCAACACGAGTGAA

[0103] TCTTTTGTCCTGAATTGGTATAGGATGAGTCCATCCAACCAGACGGAT

[0104] AAGCTGGCCGCTTTCCCTGAAGATAGGTCACAGCCCGGGCAAGACT

[0105] GTCGGTTTAGAGTCACCCAGCTGCCCAACGGTAGAGACTTTCACATG

[0106] TCAGTGGTTCGAGCAAGAAGGAATGATTCAGGAACCTACCTGTGTGG

[0107] TGCTATCTCACTTGCACCTAAGGCCCAAATTAAAGAGTCACTTAGAGC

[0108] CGAGCTCAGGGTCACTGAACGAAGGGCAGAAGTTCCCACTGCACAC

[0109] CCATCTCCATCACCTAGACCAGCCGGCCAATTTCAAACGCTTGTCGT

[0110] TGGAGTGGTAGGGGGTTTGCTCGGATCCCTCGTTCTGCTCGTGTGG

[0111] GTGTTGGCCGTCGGCTGGATCAGGGGCAGGAGGAGCAGGCACAGC

[0112] TGGGAGATGAGCGAGTTCCACAACTACAACCTGGACCTGAAGAAGAG

[0113] CGACTTCAGCACCAGGTGGCAGAAGCAGAGGTGCCCCGTGGTGAAG

[0114] AGCAAGTGCAGGGAGAACGCCAGCAAACGCGGCCGCAAGAAACTCC

[0115] TGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGA

[0116] GGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGA

[0117] TGTGAACTGGAGGGCAGGGGCAGCCTGCTGACCTGCGGCGACGTG

[0118] GAGGAGAACCCCGGCCCCATGGAGTTCGGCCTGAGCTGGCTGTTCC

[0119] TGGTGGCCATCCTGAAGGGCGTGCAGTGCAGCAGGCAGATCGTGCT

[0120] GACCCAGAGCCCCGCCCTGATGAGCGCCAGCCCCGGCGAGAAGGT

[0121] GACCATGACCTGCAGCGCCAGCAGCAGCGTGAGCTACATCTACTGG

[0122] TACCAGCAGAAGCCCAGGAGCAGCCCCAAGCCCTGGATCTACCTGA

[0123] CCAGCAACCTGGCCAGCGGCGTGCCCGCCAGGTTCAGCGGCAGCG

[0124] GCAGCGGCACCAGCTACAGCCTGACCATCAGCAGCATGGAGGCCGA

[0125] GGACGCCGCCACCTACTACTGCCAGCAGTGGAGCGGCAACCCCTAC

[0126] ACCTTCGGCGGCGGCACCAAGCTGGAGATCAAGAGGGGCGGCGGC

[0127] AGCGGCGGCGGCGGCGAGGTGCAGCTGGTGGAGAGCGGCGGCGG

[0128] CCTGGTGAAGCCCGGCGGCAGCCTGAAGCTGAGCTGCGCCGCCAG

[0129] CGGCTTCACCTTCAGCAGCTACGCCATGAGCTGGGTGAGGCAGAGC

[0130] CCCGAGAAGAGGCTGGAGTGGGTGGCCGAGATCAGCAGCGGCGGC

[0131] AGCTACACCTACTACCCCGACACCGTGACCGGCAGGTTCACCATCAG

[0132] CAGGGACAACGCCAAGAACACCCTGTACCTGGAGATCAGCAGCCTG

[0133] AGGAGCGAGGACACCGCCATGTACTACTGCACCAGGCACGGCGACT

[0134] ACCCCAGGTTCTTCGACGTGTGGGGCGCCGGCACCACCGTGACCGT

[0135] GAGCAGCCAGTGTACTAATTATGCTCTCTTGAAATTGGCTGGAGATGT TGAGAGCAATCCCGGGCCCATGCGGATCAGCAAGCCCCACCTGCGG AGCATCAGCATCCAGTGCTACCTGTGCCTGCTGCTGAACAGCCACTT CCTGACCGAGGCCGGCATCCACGTGTTCATCCTGGGCTGCTTCAGC GCCGGACTGCCCAAGACCGAGGCCAACTGGGTGAACGTGATCAGCG ACCTGAAGAAGATCGAGGACCTGATCCAGAGCATGCACATCGACGC CACCCTGTACACCGAGAGCGACGTGCACCCCAGCTGCAAGGTGACC GCCATGAAGTGCTTTCTGCTGGAACTGCAGGTGATCAGCCTGGAAAG CGGCGACGCCAGCATCCACGACACCGTGGAGAACCTGATCATCCTG GCCAACAACAGCCTGAGCAGCAACGGCAACGTGACCGAGAGCGGCT GCAAAGAGTGCGAGGAACTGGAAGAGAAGAACATCAAAGAGTTTCTG CAGAGCTTCGTGCACATCGTGCAGATGTTCATCAACACCAGCTGA (SEQ ID NO:37)(PD1 Signal peptide of SEQ ID N0:1 + PD1 extracellular domain of SEQ ID NO:2 + PD1 transmembrane domain of SEQ ID NO:3 + NKG2D of SEQ ID NO:4 + 4-1 BB of SEQ ID NO:5 + T2A of SEQ ID NO:6 + Immunoglobulin heavy chain signal peptide of SEQ ID NO:7 + VL NKG2A of SEQ ID NO:8 + Linker of SEQ ID NO:9 + VH NKG2A of SEQ ID NQ:10 + E2A of SEQ D NO:11 + IL15 of SEQ ID NO:12);

[0136] ATGCAGATTCCACAGGCTCCTTGGCCAGTCGTTTGGGCAGTTTTGCA GCTGGGCTGGAGACCAGGCTGGTTCCTTGATAGCCCTGACAGACCT TGGAACCCCCCAACTTTTAGTCCAGCCCTGCTTGTGGTGACTGAGGG GGACAACGCCACCTTTACCTGCAGTTTTAGCAACACGAGTGAATCTTT TGTCCTGAATTGGTATAGGATGAGTCCATCCAACCAGACGGATAAGC TGGCCGCTTTCCCTGAAGATAGGTCACAGCCCGGGCAAGACTGTCG GTTTAGAGTCACCCAGCTGCCCAACGGTAGAGACTTTCACATGTCAG TGGTTCGAGCAAGAAGGAATGATTCAGGAACCTACCTGTGTGGTGCT ATCTCACTTGCACCTAAGGCCCAAATTAAAGAGTCACTTAGAGCCGA GCTCAGGGTCACTGAACGAAGGGCAGAAGTTCCCACTGCACACCCA TCTCCATCACCTAGACCAGCCGGCCAATTTCAAACGCTTGTCGTTGG AGTGGTAGGGGGTTTGCTCGGATCCCTCGTTCTGCTCGTGTGGGTGT TGGCCGTCAAACGCGGCCGCAAGAAACTCCTGTATATATTCAAACAA CCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAG CTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGA AGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAA CCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATG TTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCC GAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAA GATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGC >

[0137] GCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTAC AGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCC CCTCGCGAGGGCAGGGGCAGCCTGCTGACCTGCGGCGACGTGGAG GAGAACCCCGGCCCCATGGAGTTCGGCCTGAGCTGGCTGTTCCTGG TGGCCATCCTGAAGGGCGTGCAGTGCAGCAGGCAGATCGTGCTGAC CCAGAGCCCCGCCCTGATGAGCGCCAGCCCCGGCGAGAAGGTGAC CATGACCTGCAGCGCCAGCAGCAGCGTGAGCTACATCTACTGGTAC CAGCAGAAGCCCAGGAGCAGCCCCAAGCCCTGGATCTACCTGACCA GCAACCTGGCCAGCGGCGTGCCCGCCAGGTTCAGCGGCAGCGGCA

[0138] GCGGCACCAGCTACAGCCTGACCATCAGCAGCATGGAGGCCGAGGA CGCCGCCACCTACTACTGCCAGCAGTGGAGCGGCAACCCCTACACC TTCGGCGGCGGCACCAAGCTGGAGATCAAGAGGGGCGGCGGCAGC GGCGGCGGCGGCGAGGTGCAGCTGGTGGAGAGCGGCGGCGGCCT GGTGAAGCCCGGCGGCAGCCTGAAGCTGAGCTGCGCCGCCAGCGG CTTCACCTTCAGCAGCTACGCCATGAGCTGGGTGAGGCAGAGCCCC GAGAAGAGGCTGGAGTGGGTGGCCGAGATCAGCAGCGGCGGCAGC TACACCTACTACCCCGACACCGTGACCGGCAGGTTCACCATCAGCAG GGACAACGCCAAGAACACCCTGTACCTGGAGATCAGCAGCCTGAGG AGCGAGGACACCGCCATGTACTACTGCACCAGGCACGGCGACTACC CCAGGTTCTTCGACGTGTGGGGCGCCGGCACCACCGTGACCGTGAG CAGCCAGTGTACTAATTATGCTCTCTTGAAATTGGCTGGAGATGTTGA GAGCAATCCCGGGCCCATGCGGATCAGCAAGCCCCACCTGCGGAGC ATCAGCATCCAGTGCTACCTGTGCCTGCTGCTGAACAGCCACTTCCT GACCGAGGCCGGCATCCACGTGTTCATCCTGGGCTGCTTCAGCGCC GGACTGCCCAAGACCGAGGCCAACTGGGTGAACGTGATCAGCGACC TGAAGAAGATCGAGGACCTGATCCAGAGCATGCACATCGACGCCAC CCTGTACACCGAGAGCGACGTGCACCCCAGCTGCAAGGTGACCGCC ATGAAGTGCTTTCTGCTGGAACTGCAGGTGATCAGCCTGGAAAGCGG CGACGCCAGCATCCACGACACCGTGGAGAACCTGATCATCCTGGCC AACAACAGCCTGAGCAGCAACGGCAACGTGACCGAGAGCGGCTGCA AAGAGTGCGAGGAACTGGAAGAGAAGAACATCAAAGAGTTTCTGCAG

[0139] AGCTTCGTGCACATCGTGCAGATGTTCATCAACACCAGCTGA (SEQ ID NO:38)(PD1 Signal peptide of SEQ ID N0:1 + PD1 extracellular domain of SEQ ID NO:2 + PD1 transmembrane domain of SEQ ID NO:3 + 4-1 BB of SEQ ID NO:5 + CD3 of SEQ ID NO:16 + T2A of SEQ ID NO:6 + Immunoglobulin heavy chain signal peptide of SEQ ID NO:7 + VL NKG2A of SEQ ID NO:8 + Linker of SEQ ID NO:9 + VH NKG2A of SEQ ID NO:10 + E2A of SEQ D NO:11 + IL15 of SEQ ID NO:12);

[0140] ATGCAGATTCCACAGGCTCCTTGGCCAGTCGTTTGGGCAGTTTTGCA GCTGGGCTGGAGACCAGGCTGGTTCCTTGATAGCCCTGACAGACCT TGGAACCCCCCAACTTTTAGTCCAGCCCTGCTTGTGGTGACTGAGGG GGACAACGCCACCTTTACCTGCAGTTTTAGCAACACGAGTGAATCTTT TGTCCTGAATTGGTATAGGATGAGTCCATCCAACCAGACGGATAAGC TGGCCGCTTTCCCTGAAGATAGGTCACAGCCCGGGCAAGACTGTCG GTTTAGAGTCACCCAGCTGCCCAACGGTAGAGACTTTCACATGTCAG TGGTTCGAGCAAGAAGGAATGATTCAGGAACCTACCTGTGTGGTGCT ATCTCACTTGCACCTAAGGCCCAAATTAAAGAGTCACTTAGAGCCGA GCTCAGGGTCACTGAACGAAGGGCAGAAGTTCCCACTGCACACCCA TCTCCATCACCTAGACCAGCCGGCCAATTTCAAACGCTTGTCGTTGG AGTGGTAGGGGGTTTGCTCGGATCCCTCGTTCTGCTCGTGTGGGTGT TGGCCGTCAAACGCGGCCGCAAGAAACTCCTGTATATATTCAAACAA CCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAG CTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGTTGTGTG CAAGACCAAGAAGAAGTCCAGCCCAGGAAGACGGCAAGGTCTACATT AACATGCCCGGGAGGGGAGAGGGCAGGGGCAGCCTGCTGACCTGC GGCGACGTGGAGGAGAACCCCGGCCCCATGGAGTTCGGCCTGAGC TGGCTGTTCCTGGTGGCCATCCTGAAGGGCGTGCAGTGCAGCAGGC AGATCGTGCTGACCCAGAGCCCCGCCCTGATGAGCGCCAGCCCCGG CGAGAAGGTGACCATGACCTGCAGCGCCAGCAGCAGCGTGAGCTAC ATCTACTGGTACCAGCAGAAGCCCAGGAGCAGCCCCAAGCCCTGGA TCTACCTGACCAGCAACCTGGCCAGCGGCGTGCCCGCCAGGTTCAG CGGCAGCGGCAGCGGCACCAGCTACAGCCTGACCATCAGCAGCATG GAGGCCGAGGACGCCGCCACCTACTACTGCCAGCAGTGGAGCGGC AACCCCTACACCTTCGGCGGCGGCACCAAGCTGGAGATCAAGAGGG GCGGCGGCAGCGGCGGCGGCGGCGAGGTGCAGCTGGTGGAGAGC GGCGGCGGCCTGGTGAAGCCCGGCGGCAGCCTGAAGCTGAGCTGC GCCGCCAGCGGCTTCACCTTCAGCAGCTACGCCATGAGCTGGGTGA GGCAGAGCCCCGAGAAGAGGCTGGAGTGGGTGGCCGAGATCAGCA GCGGCGGCAGCTACACCTACTACCCCGACACCGTGACCGGCAGGTT CACCATCAGCAGGGACAACGCCAAGAACACCCTGTACCTGGAGATCA GCAGCCTGAGGAGCGAGGACACCGCCATGTACTACTGCACCAGGCA CGGCGACTACCCCAGGTTCTTCGACGTGTGGGGCGCCGGCACCACC GTGACCGTGAGCAGCCAGTGTACTAATTATGCTCTCTTGAAATTGGCT GGAGATGTTGAGAGCAATCCCGGGCCCATGCGGATCAGCAAGCCCC ACCTGCGGAGCATCAGCATCCAGTGCTACCTGTGCCTGCTGCTGAAC AGCCACTTCCTGACCGAGGCCGGCATCCACGTGTTCATCCTGGGCT GCTTCAGCGCCGGACTGCCCAAGACCGAGGCCAACTGGGTGAACGT GATCAGCGACCTGAAGAAGATCGAGGACCTGATCCAGAGCATGCAC ATCGACGCCACCCTGTACACCGAGAGCGACGTGCACCCCAGCTGCA AGGTGACCGCCATGAAGTGCTTTCTGCTGGAACTGCAGGTGATCAGC CTGGAAAGCGGCGACGCCAGCATCCACGACACCGTGGAGAACCTGA TCATCCTGGCCAACAACAGCCTGAGCAGCAACGGCAACGTGACCGA GAGCGGCTGCAAAGAGTGCGAGGAACTGGAAGAGAAGAACATCAAA GAGTTTCTGCAGAGCTTCGTGCACATCGTGCAGATGTTCATCAACAC CAGCTGA (SEQ ID NO:39) (PD1 Signal peptide of SEQ ID N0:1 + PD1 extracellular domain of SEQ ID NO:2 + PD1 transmembrane domain of SEQ ID NO:3 + 4-1 BB of SEQ ID NO:5 + DAP10 of SEQ ID NO:17 + T2A of SEQ ID NO:6 + Immunoglobulin heavy chain signal peptide of SEQ ID NO:7 + VL NKG2A of SEQ ID NO:8 + Linker of SEQ ID NO:9 + VH NKG2A of SEQ ID NQ:10 + E2A of SEQ D NO:11 + IL15 of SEQ ID NO:12);

[0141] ATGCAGATTCCACAGGCTCCTTGGCCAGTCGTTTGGGCAGTTTTGCA GCTGGGCTGGAGACCAGGCTGGTTCCTTGATAGCCCTGACAGACCT TGGAACCCCCCAACTTTTAGTCCAGCCCTGCTTGTGGTGACTGAGGG GGACAACGCCACCTTTACCTGCAGTTTTAGCAACACGAGTGAATCTTT TGTCCTGAATTGGTATAGGATGAGTCCATCCAACCAGACGGATAAGC TGGCCGCTTTCCCTGAAGATAGGTCACAGCCCGGGCAAGACTGTCG GTTTAGAGTCACCCAGCTGCCCAACGGTAGAGACTTTCACATGTCAG TGGTTCGAGCAAGAAGGAATGATTCAGGAACCTACCTGTGTGGTGCT ATCTCACTTGCACCTAAGGCCCAAATTAAAGAGTCACTTAGAGCCGA GCTCAGGGTCACTGAACGAAGGGCAGAAGTTCCCACTGCACACCCA TCTCCATCACCTAGACCAGCCGGCCAATTTCAAACGCTTGTCGTTGG AGTGGTAGGGGGTTTGCTCGGATCCCTCGTTCTGCTCGTGTGGGTGT TGGCCGTCAAACGCGGCCGCAAGAAACTCCTGTATATATTCAAACAA CCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAG CTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGTACTTTC TGGGTAGATTGGTCCCCAGGGGAAGAGGAGCAGCTGAAGCAGCCAC ACGCAAACAGAGAATCACCGAGACAGAGTCCCCATATCAGGAGTTGC AGGGTCAGAGGTCTGACGTGTACTCCGACCTCAACACTCAAAGGCCA TATTACAAAGAGGGCAGGGGCAGCCTGCTGACCTGCGGCGACGTGG AGGAGAACCCCGGCCCCATGGAGTTCGGCCTGAGCTGGCTGTTCCT GGTGGCCATCCTGAAGGGCGTGCAGTGCAGCAGGCAGATCGTGCTG ACCCAGAGCCCCGCCCTGATGAGCGCCAGCCCCGGCGAGAAGGTG ACCATGACCTGCAGCGCCAGCAGCAGCGTGAGCTACATCTACTGGTA CCAGCAGAAGCCCAGGAGCAGCCCCAAGCCCTGGATCTACCTGACC AGCAACCTGGCCAGCGGCGTGCCCGCCAGGTTCAGCGGCAGCGGC AGCGGCACCAGCTACAGCCTGACCATCAGCAGCATGGAGGCCGAGG ACGCCGCCACCTACTACTGCCAGCAGTGGAGCGGCAACCCCTACAC CTTCGGCGGCGGCACCAAGCTGGAGATCAAGAGGGGCGGCGGCAG CGGCGGCGGCGGCGAGGTGCAGCTGGTGGAGAGCGGCGGCGGCC TGGTGAAGCCCGGCGGCAGCCTGAAGCTGAGCTGCGCCGCCAGCG GCTTCACCTTCAGCAGCTACGCCATGAGCTGGGTGAGGCAGAGCCC CGAGAAGAGGCTGGAGTGGGTGGCCGAGATCAGCAGCGGCGGCAG CTACACCTACTACCCCGACACCGTGACCGGCAGGTTCACCATCAGCA GGGACAACGCCAAGAACACCCTGTACCTGGAGATCAGCAGCCTGAG GAGCGAGGACACCGCCATGTACTACTGCACCAGGCACGGCGACTAC CCCAGGTTCTTCGACGTGTGGGGCGCCGGCACCACCGTGACCGTGA GCAGCCAGTGTACTAATTATGCTCTCTTGAAATTGGCTGGAGATGTTG AGAGCAATCCCGGGCCCATGCGGATCAGCAAGCCCCACCTGCGGAG CATCAGCATCCAGTGCTACCTGTGCCTGCTGCTGAACAGCCACTTCC TGACCGAGGCCGGCATCCACGTGTTCATCCTGGGCTGCTTCAGCGC CGGACTGCCCAAGACCGAGGCCAACTGGGTGAACGTGATCAGCGAC CTGAAGAAGATCGAGGACCTGATCCAGAGCATGCACATCGACGCCA CCCTGTACACCGAGAGCGACGTGCACCCCAGCTGCAAGGTGACCGC CATGAAGTGCTTTCTGCTGGAACTGCAGGTGATCAGCCTGGAAAGCG GCGACGCCAGCATCCACGACACCGTGGAGAACCTGATCATCCTGGC CAACAACAGCCTGAGCAGCAACGGCAACGTGACCGAGAGCGGCTGC AAAGAGTGCGAGGAACTGGAAGAGAAGAACATCAAAGAGTTTCTGCA GAGCTTCGTGCACATCGTGCAGATGTTCATCAACACCAGCTGA (SEQ ID NO:40)(PD1 Signal peptide of SEQ ID N0:1 + PD1 extracellular domain of SEQ ID NO:2 + PD1 transmembrane domain of SEQ ID NO:3 + 4-1 BB of SEQ ID NO:5 + DAP12 of SEQ ID NO:18 + T2A of SEQ ID NO:6 + Immunoglobulin heavy chain signal peptide of SEQ ID NO:7 + VL NKG2A of SEQ ID NO:8 + Linker of SEQ ID NO:9 + VH NKG2A of SEQ ID NQ:10 + E2A of SEQ D NO:11 + IL15 of SEQ ID NO:12). Alternatively, the nucleotide sequence according to the invention can encode for a chimeric receptor having TIGIT as IC, an scFv targeting NKG2a and the interleukin IL15 and can be the following sequence:

[0142] ATGAGATGGTGTTTGCTGCTGATTTGGGCCCAAGGGCTTAGGCAAGC ACCCCTGGCTTCTGGTATGATGACCGGAACCATAGAAACAACCGGGA ACATCTCCGCGGAAAAAGGCGGATCTATCATCCTCCAGTGTCACCTT TCTTCTACGACCGCCCAAGTCACTCAGGTGAACTGGGAGCAACAGGA TCAGCTCCTCGCTATCTGTAACGCTGACCTCGGATGGCATATTTCCC CCTCTTTCAAAGACCGCGTAGCACCAGGTCCAGGTTTGGGACTGACG CTTCAGTCCCTGACCGTAAATGATACCGGGGAGTACTTCTGCATCTA CCACACTTATCCTGACGGCACATACACCGGTCGGATATTTCTGGAGG TCCTTGAAAGTTCTGTCGCTGAGCATGGAGCCAGGTTTCAAATACCTT TGCTGGGCGCAATGGCTGCCACACTGGTTGTAATTTGTACTGCCGTC ATTGTAGTGGTGGCTATGGGCTGGATCAGGGGCAGGAGGAGCAGGC ACAGCTGGGAGATGAGCGAGTTCCACAACTACAACCTGGACCTGAAG AAGAGCGACTTCAGCACCAGGTGGCAGAAGCAGAGGTGCCCCGTGG TGAAGAGCAAGTGCAGGGAGAACGCCAGCAAACGCGGCCGCAAGAA ACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACT CAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAG GAGGATGTGAACTGGAGGGCAGGGGCAGCCTGCTGACCTGCGGCG ACGTGGAGGAGAACCCCGGCCCCATGGAGTTCGGCCTGAGCTGGCT GTTCCTGGTGGCCATCCTGAAGGGCGTGCAGTGCAGCAGGCAGATC GTGCTGACCCAGAGCCCCGCCCTGATGAGCGCCAGCCCCGGCGAG AAGGTGACCATGACCTGCAGCGCCAGCAGCAGCGTGAGCTACATCT ACTGGTACCAGCAGAAGCCCAGGAGCAGCCCCAAGCCCTGGATCTA CCTGACCAGCAACCTGGCCAGCGGCGTGCCCGCCAGGTTCAGCGG CAGCGGCAGCGGCACCAGCTACAGCCTGACCATCAGCAGCATGGAG GCCGAGGACGCCGCCACCTACTACTGCCAGCAGTGGAGCGGCAACC CCTACACCTTCGGCGGCGGCACCAAGCTGGAGATCAAGAGGGGCGG CGGCAGCGGCGGCGGCGGCGAGGTGCAGCTGGTGGAGAGCGGCG GCGGCCTGGTGAAGCCCGGCGGCAGCCTGAAGCTGAGCTGCGCCG CCAGCGGCTTCACCTTCAGCAGCTACGCCATGAGCTGGGTGAGGCA GAGCCCCGAGAAGAGGCTGGAGTGGGTGGCCGAGATCAGCAGCGG CGGCAGCTACACCTACTACCCCGACACCGTGACCGGCAGGTTCACC ATCAGCAGGGACAACGCCAAGAACACCCTGTACCTGGAGATCAGCA GCCTGAGGAGCGAGGACACCGCCATGTACTACTGCACCAGGCACGG CGACTACCCCAGGTTCTTCGACGTGTGGGGCGCCGGCACCACCGTG ACCGTGAGCAGCCAGTGTACTAATTATGCTCTCTTGAAATTGGCTGG AGATGTTGAGAGCAATCCCGGGCCCATGCGGATCAGCAAGCCCCAC CTGCGGAGCATCAGCATCCAGTGCTACCTGTGCCTGCTGCTGAACAG CCACTTCCTGACCGAGGCCGGCATCCACGTGTTCATCCTGGGCTGCT TCAGCGCCGGACTGCCCAAGACCGAGGCCAACTGGGTGAACGTGAT CAGCGACCTGAAGAAGATCGAGGACCTGATCCAGAGCATGCACATC GACGCCACCCTGTACACCGAGAGCGACGTGCACCCCAGCTGCAAGG TGACCGCCATGAAGTGCTTTCTGCTGGAACTGCAGGTGATCAGCCTG GAAAGCGGCGACGCCAGCATCCACGACACCGTGGAGAACCTGATCA TCCTGGCCAACAACAGCCTGAGCAGCAACGGCAACGTGACCGAGAG CGGCTGCAAAGAGTGCGAGGAACTGGAAGAGAAGAACATCAAAGAG TTTCTGCAGAGCTTCGTGCACATCGTGCAGATGTTCATCAACACCAG CTGA (SEQ ID N0:41)(TIGIT Signal peptide of SEQ ID N0:13 + TIGIT extracellular domain of SEQ ID NO: 14 + TIGIT transmembrane domain of SEQ ID NO:15 + NKG2D of SEQ ID NO:4 + 4-1 BB of SEQ ID N0:5 + T2A of SEQ ID N0:6 + Immunoglobulin heavy chain signal peptide of SEQ ID NO:7 + VL NKG2A of SEQ ID NO:8 + Linker of SEQ ID NO:9 + VH NKG2A of SEQ ID NQ:10 + E2A of SEQ D NO:11 + IL15 of SEQ ID NO:12).

[0143] Table 1 below shows the cytokines, the immune checkpoint molecules (ICs), the costimulatory domains and the scFvs which could be used according to the present invention.

[0144] Tab e 1 The present invention also concerns a vector comprising a nucleotide sequence as defined above.

[0145] According to the invention, said vector can be chosen from the group consisting of retroviral vectors, such as a y-retroviral vector, a DNA vector, a RNA vector, a plasmid, a lentivirus vector, adenoviral vector, or non-viral vector.

[0146] The present invention also concerns a cell comprising a chimeric receptor as defined above or a nucleotide sequence as defined above or a vector as defined above.

[0147] In particular, the cell according to the invention is preferably a cell transduced with said vector, preferably a retroviral vector, which comprises the nucleotide sequence according to the invention as defined above. Therefore, said transduced cell, once administered to a patient, is able to express and expose said chimeric receptor a) on its cellular membrane and, at the same time, is able to express and secrete outside the cell said molecule b) and said soluble factor c).

[0148] Preferably, said cell is an innate immune cell.

[0149] In particular, according to the present invention, said cell can be a NK cell, a gamma delta T cell or a myeloid cell, such as a macrophage, preferably a NK cell.

[0150] The present invention concerns also a pharmaceutical composition comprising a nucleotide sequence as defined above, a vector as defined above or a cell as defined above together with one or more pharmaceutically acceptable excipients and / or adjuvants.

[0151] A further object of the present invention is a nucleotide sequence as defined above, a vector as defined above, a cell as defined above or a pharmaceutical composition as defined above for medical use.

[0152] Moreover, the present invention concerns also a nucleotide sequence as defined above, a vector as defined above, a cell as defined above or a pharmaceutical composition as defined above for use in the treatment of tumors or of auto-immune diseases.

[0153] In particular, according to the present invention said tumor can be a tumor expressing an immune checkpoint (IC) ligand. Preferably, said tumor expresses an IC ligand chosen from a PD-1 ligand, such as PDL-1 , a TIGIT ligand, a B7-H3 ligand, a B7-H4 ligand, a Lag-3 ligand, a TIM-3 ligand, a CTLA-4 ligand. For example, the tumor can be chosen from the group consisting of neuroblastoma, metastatic or non-metastatic pancreas cancer, lung adenocarcinoma.

[0154] According to the present invention, said auto-immune disease can be systemic Lupus Erythematosus (LES).

[0155] Moreover, a further object of the present invention is a combination of a nucleotide sequence, a vector comprising said nucleotide sequence or a cell comprising said vector or nucleotide sequence, wherein said nucleotide sequence encodes: i. a) a chimeric receptor of an immune checkpoint ligand, said chimeric receptor comprising or consisting of, from N-terminus to C- terminus,

[0156] - a signal peptide, fused to

[0157] - an extracellular domain of an immune checkpoint molecule, fused to

[0158] - a transmembrane domain of said immune checkpoint molecule, fused to

[0159] - one or more costimulatory domains; or ii. the chimeric receptor a) and b) a soluble molecule able to bind a target, wherein said molecule is chosen from an antibody fragment (or antibody-derived fragment), a ligand of said target or a receptor of said target and wherein said target being chosen from an immune checkpoint molecule or an immunosuppressive molecule; or iii. the chimeric receptor a) and c) one or more soluble factors chosen from a cytokine or a chemokine; with

[0160] I. b) a soluble molecule able to bind a target, wherein said molecule is chosen from an antibody fragment, a ligand of said target or a receptor of said target and wherein said target being chosen from an immune checkpoint molecule or an immunosuppressive molecule and c) one or more soluble factors chosen from a cytokine or a chemokine; or with c) and a nucleotide sequence encoding b); or with b) and a nucleotide sequence encoding c); or

[0161] II. c) one or more soluble factors chosen from a cytokine or a chemokine; or

[0162] III. b) a soluble molecule able to bind a target, wherein said molecule is chosen from an antibody fragment, a ligand of said target or a receptor of said target and wherein said target being chosen from an immune checkpoint molecule or an immunosuppressive molecule; respectively.

[0163] Therefore, according to the present invention, said combination can consist of a nucleotide sequence, vector or cell as defined in point i with one of the alternatives defined in point I, or it can consist of a nucleotide sequence, vector, or cell as defined in point ii with the amino acid sequence of c) as defined in point II, or it can consist of a nucleotide sequence, vector or cell as defined in point iii with the amino acid sequence of b) as defined in point III.

[0164] Said combination according to the present invention can be for use in the treatment of tumors, preferably a tumor expressing an immune checkpoint ligand, or of auto-immune diseases.

[0165] According to the present invention, “separate use” is understood as meaning the administration, at the same time, of the compounds of the combination according to the invention in distinct pharmaceutical forms, whereas

[0166] “sequential use” is understood as meaning the successive administration of the two compounds of the combination according to the invention, each in a distinct pharmaceutical form.

[0167] As stated above, said tumor can be chosen from the group consisting of neuroblastoma, metastatic or non-metastatic pancreas cancer, lung adenocarcinoma; and said auto-immune disease can be systemic Lupus Erythematosus (LES).

[0168] The present invention also concerns a method of treating a tumor, preferably a tumor expressing an immune checkpoint ligand (such as for example a tumor can be chosen from the group consisting of neuroblastoma, metastatic or non-metastatic pancreas cancer, lung adenocarcinoma) or an auto-immune diseases (such as for example systemic Lupus Erythematosus), said method comprising administering to a patient in need thereof a nucleotide sequence, a vector, a cell, a pharmaceutical composition or a combination as defined above.

[0169] The present invention now will be described by an illustrative, but not limitative way, according to preferred embodiments thereof, with particular reference to the examples and the enclosed drawings, wherein:

[0170] - Figure 1 shows a schematic representation of the constructs used in the study for producing gmNK cells according to the invention. - Figure 2 shows (A-B) % of transduction of NK cells engineered with an (A)exPD1 or (B)exTIGIT vectors at day 10. (C) CD57 and CD16 expression on non-transduced (NT)-NK cells andexPD1 -NK cells at day 20 of culture. These data suggested that the release of IL-15 by transduced NK cells did not induce exhaustion / anergy. (D) pg / ml of IL-15 released byexPD1 -NK cells stimulated (black bars) or not (white bars) with coated recombinant (r) PDL1 - ligand. Notably,exPD1-NK cells released higher amounts of IL15 upon triggering with rPD-L1. (E) The absolute number of gmNK cells (black bars) and control NK cells (white bars), (F) Agarose gel of scFv purified from supernatants of HEK cell line transduced withexPD1 - vector.

[0171] - Figure 3 shows (A) Expression of NKG2A molecule on NK cells that were preincubated or not with purified scfv NKG2A. Dot plots indicated the specificity of scfv. (B) Percentages of cell lysis mediated by NK cells at different E:T ratios, in the absence (white) or in the presence (black) of purified scfv NKG2A. HLA-G transfected LCL.221 -G cells (that also express HLA-E) cells were used as target.

[0172] - Figure 4 shows (A) % of remaining PD-L1negativeand PD-L1+tumor cells after 72h of co-culture with NT-NK (black bars) orexPD1 -NK cells (white bars) at different effector: target ratio (E:T ratio). (B) % of killed tumor target cells (PDL-1negativeand PD-L1+tumor cells) by NT-NK (black bars) andexPD1 -NK cells (white bars) after 4 hours of incubation.

[0173] - Figure 5 shows (A) in vitro 3D cell culture model of patient- derived organoids (PDO) from a biopsy of tumor patient in the absence or in the presence of NT-NK or gmNK cells from 4 to 72 hours. (B) Absolute quantification of PDO size after treatment with NT-NK or gmNK cells. (C) PDL-1 expression on PDO cultured alone or in the presence of NT-NK or gmNK cells after 72 hours. (D) OCR and ECAR level in NT-NK and gmNK cells after co-culture with PDL-1 + tumor cells in basal and stressed conditions.

[0174] - Figure 6 shows (A) In vivo bioluminescence imaging of NSG mice engrafted with NALM-18 PD-L1+tumor cells and untreated (control) or treated with NT-NK orexPD1 -NK cells at day 21 after administration of effector cells. (B) The absolute number of tumor cells in the BM of mice at day 21 after administration of effector cells: control (dark grey), NT-NK (black), orexPD1 -NK cells (white). These data indicated thatexPD1 -NK efficiently controls tumour growth. (C) Absolute number of human NK cells (identified as CD45+CD3’CD56+) in the BM and spleen of mice at day 21 after treatment with NT (black bars) orexPD1-NK cells (white bars). These data indicated thatexPD1-NK cells were present in mice for long time intervals after administration in different organs and control tumor growth. (D) Counts of bioluminescence of mice engrafted with NALM-18 PD- L1+tumor cells and treated with NT-NK (black bars) orexPD1-NK cells (white bars) at different time points. Results showed a very low count of tumor cells in mice treated withexPD1 -NK cells (white bars) suggesting the efficacy of the treatment.

[0175] - Figure 7 shows percentages of cell lysis of NALM-18 target cells mediated by NK cells at different E:T ratios. NK cells were genetically modified with different vectors containing different co-stimuli (CD3 or DAPIO or DAP12).

[0176] EXAMPLE 1: Design and production of genetically modified NK cells according to the present invention and study of their anti-tumor activity.

[0177] MATERIALS AND METHODS

[0178] The geographical origin and the code of the cell lines that have been used in the experiments are shown in Table 2 below.

[0179] Table 2

[0180] The biological material of human origin used in these experiments was sampled after the donors signed a written informed consent, in accordance with rules set by the Institutional Review Board (IRB) of Bambino Gesu Children’s Hospital of Rome, as shown in Table 3.

[0181] Table 3

[0182] (OPBG; Approval of Ethical Committee N°969 / 2015 prot. N°669LB).

[0183] Regarding GMO, the following authorizations have been obtained: authorization number: Az. VI 1 / 3-740.320-01 / 04 (UKW, Germany) and AU- 740.134-01 / 21 (UKW, Germany) and RM / IC / 0p2 / 18 / 006 (OPBG, Italy).

[0184] NK cell isolation and expansion

[0185] Peripheral blood mononuclear cells (PBMC) were obtained from patients and healthy donors (HD) and isolated after density gradient centrifugation (Ficoll-Lympholyte, Cederlane). PBMC of HD were obtained from a buffy coat (IRCCS Bambino Gesu Children’s Hospital, Rome). NK cells were purified using NK isolation kit II (Miltenyi) or Rosettsep (StemCell) and shortly activated (72h) with IL15 (150U / ml, Miltenyi), IL2 (50U / ml) and IL1 b (2000U / ml) in MACSMix medium (Miltenyi) supplement with 5% human serum and 2 mM Glutamax. Cells were maintained in a humidified atmosphere containing 8% CO2 at 37°C.

[0186] Design of constructs y-retroviral vectors have been generated encoding PD-1-NKG2D.4- 1 BB.2a.NKG2A(scfv).2a.lL15 and TIGIT-NKG2D.4-

[0187] 1 BB.2a.NKG2A(scfv).2a.lL15. Briefly, these vectors will allow the expression of a transmembrane chimeric protein capable of binding the IC ligands by PD-1 or TIGIT ectodomain, transduce then the signal to the cytoplasmatic NKG2D and 4.1 BB co-stimulatory domains and activate the NK cells. The expression of this receptor is then in frame with a secretable NKG2A single chain variable fragment (scFv), allowing the blocking of NKG2A / HLA-E interaction, and with IL15 to support NK and T cell survival and proliferation (Table 4). Then, using the PD-1 construct as a backbone, several variations on the chimeric protein have been generated. In particular, the NKG2D sequence has been replaced by the 4-1 BB while the 4-1 BB was exchanged with a sequence encoding for CD3 7, or DAP10, or DAP 12. Therefore, the other constructs generated were PD-1 -4- 1 BB.CD3 <.2a.NKG2A(scfv).2a.lL15, PD-1-4-

[0188] 1 BB.DAP10.2a.NKG2A(scfv).2a.lL15 and PD-1-4-

[0189] 1 BB.DAP12.2a.NKG2A(scfv).2a.lL15 (Figure 6).

[0190] In Table 4 the nucleotide sequences used in the above-mentioned constructs are reported.

[0191] Table 4 In Table 5 the amino acid sequences resulting from the abovedescribed constructs are reported.

[0192] Table 5

[0193] Generation of retroviral vectors and NK cell transduction

[0194] Retroviral supernatant was generated in 293T-cells (PMID:32381575, PMID:20686963) and quantified by Retro-X™ qRT-PCR Titration Kit (Takara). The supernatant was used to transduce primary NK cells derived from peripheral blood mononuclear cells of healthy donors. In particular, NK lymphocytes were activated by using IL15 (150U / ml, Miltenyi), IL2 (50U / ml), and IL1 b (2000U / ml).

[0195] Activated NK cells were transduced on day 3 in 24-well plates precoated with recombinant human RetroNectin (Takara-Bio. Inc; Japan) using a specific retroviral supernatant and the specific above-described cytokines. At day 5 from transduction, the NK cells are expanded in “MACS MIX complete medium” (Miltenyi) supplemented with 5% human serum and 2 mM Glutamax, and fed twice a week with the specific abovedescribed cytokines (PMID: 29872565). The transduction efficiency was determined by anti-PD-1 mAb.

[0196] Generation of eGFP-Firefly-Luciferase cell lines.

[0197] The retroviral vector encoding eGFP-Firefly-Luciferase (eGFP- FFLuc) was used in selected experiments to label positive (PDL-1 +) or negative (PDL-1-) tumor cells. Cells were maintained in a humidified atmosphere containing 5% CO2 at 37°C. All cell lines were routinely tested for mycoplasma and for surface expression of target antigens. All cell lines have been authenticated by STR analysis in the certificated lab "BMR Genomics s.r.l."

[0198] Co-culture assays

[0199] For co-culture experiments, NT and gmNK cells were plated at 0.2x106cells / well in 24-well plates in the presence or absence of different tumor target cell lines (expressing or not the PDL-1 ) at the indicated E:T ratios. Following 4 or 72 hours of incubation at 37°C, tumor cells, and NK cells were collected, and residual tumor cells and NK cells were assessed by flow cytometry based on propidium iodine (PI) and GFP (tumor cell line) expression.

[0200] Phenotypic analysis

[0201] Expression of cell surface molecules was determined by flowcytometry. The following mAbs were used: CD3, CD14, CD15, CD16, CD19, CD25, CD33, CD45, CD56, CD57, CD66b, CD94, CD95, CD279 (PD1 ), DNAM1 , GD2, HLA-DR, NKG2A, NKG2D, TIGIT and NKp46. These mAbs were purchased by BD Bioscience, Miltenyi, BioLegend (San Diego, CA, USA) and Beckman Coulter. Samples were analyzed on a Cytoflex S and LX (Beckman Coulter). Data were analyzed using Cytexpert (Beckman Coulter) and FlowJo 10 software (BD Biosciences). For each sample, we analyzed a minimum of 50,000 events.

[0202] In vivo experiments To investigate the in vivo antitumor activity of gmNK cells on tumor model, 0.25x106Karpas-GFP-FF-Luc cells were intravenously injected (i.v.), in 6-8 week old female NSG mice (NOD.Cg-Prkdcscid Il2rgtm1 Wjl / SzJ; from Charles River). After tumor engraftment, the mice received only one i.v. injection of gmNK cells (7x106 / mouse). Tumor growth was evaluated using the IVIS imaging system (PerkinElmer, USA), Briefly, a constant region of interest was drawn over the mouse, and the intensity of the signal was measured, every week, as total photon / sec / cm2 / sr (p / s / cm2 / sr), as previously described (PMID: 20686963). Mice were maintained in the animal facility at Plaisant Castel Romano (Rome, Italy). All in vivo experiments were in compliance with the ethical international, EU, and national requirements and were approved by the Italian Health Ministry (N°88 / 2016-PR). The circulating human gmNK cells were evaluated at the end of experiments in different organs.

[0203] Statistical Analysis.

[0204] Data are summarized as average ± standard deviation (SD). Student t-test (two-sided) was used to determine statistically significant differences between samples, with p-value <0.05 indicating a significant difference. The murine survival data were analyzed using the Kaplan- Meier curves; the log-rank test was used to measure differences between groups. No valuable samples were excluded from the analyses. Animals were excluded only in the event of death after tumor implant but before NK cell infusion. Mice were matched based on the tumor signal for control and treatment groups before infusion of control or gmNK cells. To compare the growth of tumors over time, bioluminescence signal intensity was collected in a blind fashion. Bioluminescence signal intensity was log-transformed and then compared using a two-sample t-test.

[0205] RESULTS

[0206] 1. Generation of genetically modified NK cells with innovative “activating” immune checkpoint receptors capable of counteracting the immunosuppression of TME.

[0207] One of the main tumor escape mechanisms, common to different tumors, is the expression of immune checkpoints (IC) ligands on tumor cells and tumor-associated (TA)-cells. The interaction between IC-ligands and their corresponding receptors, expressed on immune effector cells, strongly impairs the anti-tumor activity of both T and NK cells. Therefore, according to the present invention, a ready-to-use cellular product suitable for the treatment of many different relapsed / refractory tumors was generated, based on the use of genetically modified (gm) NK cells armed with innovative "activating” immune checkpoint receptors. As IC-ligands are intended the molecules that are strongly upregulated in pathological conditions and which following the binding with the corresponding receptors, expressed by the immune effector cells, are capable of mediating an inhibitory signal of the function of the latter (negative regulation).

[0208] 2.exIC-NK cells expressing the innovative “activating” receptors with IC ectodomains.

[0209] Since PD-L1 and PD-L2 IC-ligands are highly expressed on both malignant cells and different TA-cells in many different tumors, in this experiment, as a proof of concept, PD-1 was first analyzed as a target module of the extracellularexIC (in particular, all PD-1 constructs shown in Figure 1 were tested). Second, a protocol was developed for NK cell expansion from healthy donor (HD) peripheral blood mononuclear cells achieving a suitable expansion rate and high transduction efficacy using PD-1 vector (Figure 2A) and TIGIT vector (Figure 2B). Since the vector is designed with an innovative “activating" ICI, the Receptor / Ligand interaction (PD-1 / PD-L1-2) results in a potent NK cell activation and high tumor killing. In addition, NKG2D and 4.1 BB costimulatory molecules present in this vector, have been chosen to guarantee a strong activation and function but not exhaustion (Figure 2C) of NK cells. Then, the release of IL-15 is able to support NK / T cell migration, survival, and proliferation (Figure 2D-E).

[0210] Notably, as explained above in the present patent application, it is important to underline that other cytokines or IC or costimulatory molecules could be used in replacement of the one suggested in this study.

[0211] It is well known that tumor cells are able to downregulate classical HLA-Class 1 molecules to evade T cell recognition, however, the non- classical HLA-Class 1 (HLA-E and HLA-G) remain partially expressed by tumors. The release of NKG2A-scfv by the gmNK according to the invention is providing a further mechanism to overcome this tumor escape mechanism by blocking the NKG2A / HLA-E binding and reducing the inhibitory pressure present within TME (Figure 2 F). In order to evaluate the capability of NKG2A scfv to affect the inhibitory signal mediated by NKG2A and HLA-E interaction on tumor cells, healthy donor NK cells (that physiologically express NKG2A) were pre-incubated or not with supernatants obtained by HEK cells stable transduced with ourexPD1 vector (See figure 2F) and the capability of preincubated or not NK cells to kill HLA-E+tumor cell line was analyzed. As shown in Figure 3, NK cells incubated with NKG2A scfv were not able to bind NKG2A mAb (Figure 3A) indicating the specificity and the capability of masking mediated by scfv. In addition, NK cells incubated with NKG2A displayed a higher capability to kill HLA-E+tumour cells as compared to untreated (ctr) NK cells (Figure 3B).

[0212] In order to better analyze the anti-tumor activity ofexPD-1 NK cells, cytotoxic assays were performed using as target cells both PDL-1+and PDL-1negativetumor cell lines. As shown in Figure 4,exPD1 -NK cells display a higher cytolytic activity as compared to non-transduced (NT)-NK cells against PD-L1+tumors at different effector: target (E:T) ratios, while PDL-1 negative cells were killed similarly byexPD-1 -NK and NT-NK cells.

[0213] These results were obtained after both long-term co-cultures (72 hours, Figure 4 A) and short-time intervals of co-culture (4 hours, Figure 4B).

[0214] Notably, the capability to kill tumor organoids after long-term coculture was more efficient using gmNK cells compared to NT-NK cells suggesting that the capability to release IL15 and NKG2a scFv maintains the anti-tumor capacity and increased the penetration efficiency of gmNK cells (Figure 5 A-B). In addition, upon incubation with gmNK cells releasing IL-15 and NKG2a scFv, tumor organoids increased their expression of PDL-1 further implementing the gmNK tumor recognition (Figure 5 C).

[0215] The seahorse assay on gmNK cells displayed high metabolic fitness compared to NT-NK cells upon incubation with target tumor cells suggesting that the release of IL-15 and NKG2a scFv supports gmNK cell fitness in terms of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) (Figure 5D).

[0216] To prove theexIC-NK cell efficacy in vivo, immunodeficient NSG (NOD.Cg-Prkdcscid Il2rgtm 1 Wjl / SzJ) mice were engrafted (intra-peritoneal infusion) with human IC-ligands+tumor cell lines stably transduced with FireFly luciferase. In particular, in this set of experiments NALM-18 stably transduced with FireFly luciferase was used, and this cell line expressed PD-L1. After tumor engraftment was monitored by live imaging (IVIS), mice were adoptively transferred intravenously with different doses (from 7x106cells / mouse) of theexIC-NK cells. Mice treated with NT-NK cells were used as a control for theexIC-NK group. Tumour growth was then monitored weekly by IVIS (Figure 6 A).

[0217] The overall survival and disease-free survival of mice were assessed (Figure 6 D). At the end of the experiments, mice were sacrificed and tumor infiltratingexIC-NK cells were analyzed phenotypically (by flow cytometry) in tumors, metastases, and different organs (blood, spleen, and bone marrow) (Figure 6 B-C). The results showed thatexPD1 -NK efficiently controls tumor growth (Fig. 5B) and thatexPD1 -NK cells were present in mice for long time intervals after administration in different organs and able to control tumor growth (Fig. 5C). Moreover, a very low count of tumor cells in mice treated withexPD1 -NK cells were detected (Figure 6 D), meaning that the treatment was effective.

[0218] In order to test other costimuli, NK cells were genetically modified with different vectors containing different co-stimuli (4.1 BB+ CD3 or DAP10 or DAP12) and their cytolytic activity was assessed against NALM- 18 (PDL-1 +) tumor target cells (Figure 7). All vectors displayed an increased ability (% of PI+ cells) to kill target cells as compared to not transduced NK cells.

Claims

CLAIMS1 ) A nucleotide sequence encoding a) a chimeric receptor for an immune checkpoint ligand, said chimeric receptor comprising or consisting of, from N-terminus to C- terminus,- a signal peptide, fused to- an extracellular domain of an immune checkpoint molecule, fused to- a transmembrane domain of said immune checkpoint molecule, fused to- one or more costimulatory domains; b) a molecule able to bind a target, wherein said molecule is chosen from an antibody fragment, a ligand of said target or a receptor of said target and wherein said target is chosen from an immune checkpoint molecule or an immunosuppressive molecule; c) one or more soluble factors chosen from a cytokine or a chemokine; said nucleotide sequence being a single nucleotide sequence or more than one nucleotide sequence.2) Nucleotide sequence according to claim 1 , wherein, when said nucleotide sequence is a single nucleotide sequence or when said more than one nucleotide sequences comprise a sequence encoding at least two of a), b) or c), said nucleotide sequence further comprises a sequence encoding a self-cleaving peptide before the sequence encoding b) and / or c).3) Nucleotide sequence according to any one of claims 1 -2, wherein said immune checkpoint molecule is chosen from the group consisting of PD-1 , TIGIT, B7-H3, B7-H4, Lag-3, TIM-3, CTLA-4.4) Nucleotide sequence according to any one of claims 1 -3, wherein said signal peptide is chosen from the group consisting of MQIPQAPWPWWAVLQLGWRPGW (SEQ ID NO: 19) and MRWCLLLIWAQGLRQAPLASG (SEQ ID NO:31 ).5) Nucleotide sequence according to any one of claims 1 -4, wherein said one or more costimulatory domains are chosen from the group consisting of NKG2D, 4-1 BB, CD3 , DAP10, DAP12, CD28, OX-40, CD27, ICOS.6) Nucleotide sequence according to claim 5, wherein said one or more costimulatory domains are two costimulatory domains chosen from the group consisting DAP10; or 4-1 BB an7) Nucleotide sequence according to any one of claims 1 -6, wherein said immune checkpoint molecule is PD-1 and wherein- said signal peptide is SEQ ID NO: 19,- said extracellular domain of PD-1 is SEQ ID NO:20,- said transmembrane domain of PD-1 is SEQ ID NO:21 ,- said one or more costimulatory domains are two costimulatory domains consisting ofNKG2D of sequence SEQ ID NO:22 fused to 4-1 BB of sequence SEQ ID NO:23, or consisting of4-1 BB of sequence SEQ ID NO:23 fused to CD3 of sequence SEQ ID NO:34, or consisting of4-1 BB of sequence SEQ ID NO:23 fused to DAP10 of sequence SEQ ID NO:35, or consisting of4-1 BB of sequence SEQ ID NO:23 fused to DAP12 of sequence SEQ ID NO:36.8) Nucleotide sequence according to any one of claims 1 -6, wherein said immune checkpoint molecule is TIGIT and wherein- said signal peptide is SEQ ID NO:31 ,- said extracellular domain of TIGIT is SEQ ID NO:32,- said transmembrane domain of PD-1 is SEQ ID NO:33,- said one or more costimulatory domains are two costimulatory domains consisting of NKG2D of sequence SEQ ID NO:22 fused to 4-1 BB of sequence SEQ ID NO:23.9) Nucleotide sequence according to any one of claims 1 -8, wherein the nucleotide sequence encoding the signal peptide of SEQ IDNO:19 isATGCAGATTCCACAGGCTCCTTGGCCAGTCGTTTGGGCAGTTTTGCA GCTGGGCTGGAGACCAGGCTGG (SEQ ID NO:1 ); the nucleotide sequence encoding the extracellular domain of PD1 of sequence SEQ ID NQ:20 is TTCCTTGATAGCCCTGACAGACCTTGGAACCCCCCAACTTTTAGTCCA GCCCTGCTTGTGGTGACTGAGGGGGACAACGCCACCTTTACCTGCA GTTTTAGCAACACGAGTGAATCTTTTGTCCTGAATTGGTATAGGATGAGTCCATCCAACCAGACGGATAAGCTGGCCGCTTTCCCTGAAGATAGG TCACAGCCCGGGCAAGACTGTCGGTTTAGAGTCACCCAGCTGCCCA ACGGTAGAGACTTTCACATGTCAGTGGTTCGAGCAAGAAGGAATGAT TCAGGAACCTACCTGTGTGGTGCTATCTCACTTGCACCTAAGGCCCA AATTAAAGAGTCACTTAGAGCCGAGCTCAGGGTCACTGAACGAAGGG CAGAAGTTCCCACTGCACACCCATCTCCATCACCTAGACCAGCCGGC CAATTTCAAACGCTTGTC (SEQ ID NO:2); the nucleotide sequence encoding the transmembrane domain of PD1 of sequence SEQ ID NO:21 is GTTGGAGTGGTAGGGGGTTTGCTCGGATCCCTCGTTCTGCTCGTGTG GGTGTTGGCCGTC (SEQ ID NO:3); the nucleotide sequence encoding NKG2D of sequence SEQ ID NO:22 isGGCTGGATCAGGGGCAGGAGGAGCAGGCACAGCTGGGAGATGAGC GAGTTCCACAACTACAACCTGGACCTGAAGAAGAGCGACTTCAGCAC CAGGTGGCAGAAGCAGAGGTGCCCCGTGGTGAAGAGCAAGTGCAG GGAGAACGCCAGC (SEQ ID NO:4), the nucleotide sequence encoding 4-1 BB of sequence SEQ ID NO:23 is AAACGCGGCCGCAAGAAACTCCTGTATATATTCAAACAACCATTTATG AGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATT TCCAGAAGAAGAAGAAGGAGGATGTGAACTG (SEQ ID NO:5), the nucleotide sequence encoding CD3 of sequence SEQ ID NO:34 is AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGG GCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAG TACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGG GAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACT GCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAA GGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGT CTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGC CCTGCCCCCTCGC (SEQ ID NO:16), the nucleotide sequence encoding DAP10 of sequence SEQ ID NO:35 is TTGTGTGCAAGACCAAGAAGAAGTCCAGCCCAGGAAGACGGCAAGG TCTACATTAACATGCCCGGGAGGGGA (SEQ ID NO:17) and the nucleotide sequence encoding DAP12 of sequence SEQ ID NO:36 is TACTTTCTGGGTAGATTGGTCCCCAGGGGAAGAGGAGCAGCTGAAG CAGCCACACGCAAACAGAGAATCACCGAGACAGAGTCCCCATATCAGGAGTTGCAGGGTCAGAGGTCTGACGTGTACTCCGACCTCAACACTCA AAGGCCATATTACAAA (SEQ ID NO: 18); the nucleotide sequence encoding the signal peptide of sequence SEQ ID NO:31 isATGAGATGGTGTTTGCTGCTGATTTGGGCCCAAGGGCTTAGGCAAGC ACCCCTGGCTTCTGGT (SEQ ID NO: 13); the nucleotide sequence encoding the extracellular domain of TIGIT of sequence SEQ ID NO:32 is ATGATGACCGGAACCATAGAAACAACCGGGAACATCTCCGCGGAAAA AGGCGGATCTATCATCCTCCAGTGTCACCTTTCTTCTACGACCGCCC AAGTCACTCAGGTGAACTGGGAGCAACAGGATCAGCTCCTCGCTATC TGTAACGCTGACCTCGGATGGCATATTTCCCCCTCTTTCAAAGACCG CGTAGCACCAGGTCCAGGTTTGGGACTGACGCTTCAGTCCCTGACC GTAAATGATACCGGGGAGTACTTCTGCATCTACCACACTTATCCTGAC GGCACATACACCGGTCGGATATTTCTGGAGGTCCTTGAAAGTTCTGT CGCTGAGCATGGAGCCAGGTTTCAAATACCT (SEQ ID NO: 14); the nucleotide sequence encoding the transmembrane domain of TIGIT of sequence SEQ ID NO:33 is TTGCTGGGCGCAATGGCTGCCACACTGGTTGTAATTTGTACTGCCGT CATTGTAGTGGTGGCTATG (SEQ ID NO: 15).10) Nucleotide sequence according to any one of claims 1 -9, wherein said antibody fragment is a single chain variable fragment comprising a variable heavy chain (VH) and a variable light chain (VL) linked to each other by a linker.11 ) Nucleotide sequence according to any one of claims 1 -10, wherein, when molecule b) is an antibody-fragment, said nucleotide sequence further comprises a nucleotide sequence encoding an immunoglobulin heavy chain signal peptide linked to the sequence encoding said antibody fragment.12) Nucleotide sequence according to claim 11 , wherein the sequence of said immunoglobulin heavy chain signal peptide is MEFGLSWLFLVAILKGVQCSR (SEQ ID NO:25).13) Nucleotide sequence according to any one of claims 1 -12, wherein, when molecule b) is an antibody-fragment, the target of said antibody fragment is chosen from NKG2a and HLA-G, preferably NKG2a.14) Nucleotide sequence according to any one of claims 1 -13, wherein said antibody fragment is a single chain variable fragment (scFv)having NKG2a as a target and wherein the sequences of VL and VH of said single chain variable fragment are the following:VL sequence:QIVLTQSPALMSASPGEKVTMTCSASSSVSYIYWYQQKPRSSPKPWIYL TSNLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSGNPYTF GGGTKLEIKR (SEQ ID NO:26)VH sequence:EVQLVESGGGLVKPGGSLKLSCAASGFTFSSYAMSWVRQSPEKRLEWV AEISSGGSYTYYPDTVTGRFTISRDNAKNTLYLEISSLRSEDTAMYYCTR HGDYPRFFDVWGAGTTVTVSS (SEQ ID NO:28).15) Nucleotide sequence according to claim 14, wherein the sequence of the linker of said single chain variable fragment is GGGSGGGG (SEQ ID NO:27).16) Nucleotide sequence according to any one of claims 2-15, wherein said self-cleaving peptide is chosen from T2A, P2A, E2A, F2A, IRES.17) Nucleotide sequence according to any one of claims 2-16, wherein the self-cleaving peptide before the sequence encoding b) is T2A and / or the self-cleaving peptide before the sequence encoding c) is E2A.18) Nucleotide sequence according to any one of claims 1 -17, wherein said one or more cytokines are chosen from the group consisting of IL15, IL2, IL7, IL21 , IL18, IL1 (3, lper-IL15, cytokine’s fusion, preferably IL15.19) Nucleotide sequence according to claim 18, wherein said cytokine is IL15 of sequenceMRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVN VISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGD ASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQ MFINTS (SEQ ID NQ:30).20) Vector comprising a nucleotide sequence as defined in any one of claims 1 -19.21 ) A vector according to claim 20, said vector being chosen from the group consisting of retroviral vectors, such as a y-retroviral vector, a DNA vector, a RNA vector, a plasmid, a lentivirus vector, adenoviral vector, or non-viral vector.22) A cell comprising a nucleotide sequence as defined in any one of claims 1 -19 or a vector as defined in any one of claims 20-21 .23) Cell according to claim 22, said cell being a NK cell, a gamma delta T cell or a myeloid cell, such as a macrophage, preferably a NK cell.24) Pharmaceutical composition comprising a nucleotide sequence as defined in any one of claims 1 -19, a vector as defined in any one of claims 20-21 or a cell as defined in any one of claims 22-23 together with one or more excipients and / or adjuvants.25) A nucleotide sequence as defined in any one of claims 1 -19, a vector as defined in any one of claims 20-21 , a cell as defined in any one of claims 22-23 or a pharmaceutical composition as defined in claim 24 for medical use.26) A nucleotide sequence as defined in any one of claims 1 -19, a vector as defined in any one of claims 20-21 , a cell as defined in any one of claims 22-23 or a pharmaceutical composition as defined in claim 24 for use in the treatment of tumors or of auto-immune diseases.27) Nucleotide sequence, vector, cell or pharmaceutical composition for use according to claim 26, wherein said tumor is a tumor expressing an immune checkpoint ligand.28) Nucleotide sequence, vector, cell or pharmaceutical composition for use according to claim 27, wherein said tumor is a tumor expressing an IC ligand chosen from a PD-1 ligand, such as PDL-1 , a TIGIT ligand, a B7-H3 ligand, a B7-H4 ligand, a Lag-3 ligand, a TIM-3 ligand, a CTLA-4 ligand.29) Nucleotide sequence, vector, cell or pharmaceutical composition for use according to any one of claims 26-28, wherein said tumor can be chosen from the group consisting of neuroblastoma, metastatic or non-metastatic pancreas cancer, lung adenocarcinoma.30) Nucleotide sequence, vector, cell or pharmaceutical composition for use according to claim 26, wherein said auto-immune disease is systemic Lupus Erythematosus (LES).31 ) A combination of a nucleotide sequence, a vector comprising said nucleotide sequence or a cell comprising said vector or nucleotide sequence, wherein said nucleotide sequence encodes: i. a) a chimeric receptor of an immune checkpoint ligand, said chimeric receptor comprising or consisting of, from N-terminus to C- terminus,- a signal peptide, fused to- an extracellular domain of an immune checkpoint molecule, fused to- a transmembrane domain of said immune checkpoint molecule, fused to- one or more costimulatory domains; or ii. the chimeric receptor a) and b) a molecule able to bind a target, wherein said molecule is chosen from an antibody fragment, a ligand of said target or a receptor of said target and wherein said target being chosen from an immune checkpoint molecule or an immunosuppressive molecule; or iii. the chimeric receptor a) and c) one or more soluble factors chosen from a cytokine or a chemokine; withI. b) and c); or with c) and a nucleotide sequence encoding b); or with b) and a nucleotide sequence encoding c); orII. c); orIII. b); respectively.32) Combination according to claim 31 , for separate and sequential use in in the treatment of tumors, preferably a tumor expressing an immune checkpoint ligand, or of auto-immune diseases.

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