Engineered lymphocytes expressing interleukin-15 and interleukin-21 and uses thereof

Abstract

The present invention relates to a lymphocyte comprising a recombinant nucleic acid encoding a fusion protein comprising interleukin-15 (IL-15) fused to interleukin-21 (IL-21), and optionally further comprising a recombinant antigen receptor and further optionally rendered independent of CD28 co-stimulation and resistant to exhaustion as caused by checkpoint protein expression and activation. The invention further encompasses the use of such lymphocytes, particularly in therapeutic applications such as adoptive cell therapy for cancer, autoimmune diseases, or infectious diseases. Also included within the scope of the invention are fusion proteins comprising IL-15 and IL-21 domains, nucleic acids encoding such fusion proteins, and cells—such as lymphocytes or other suitable host cells—comprising these nucleic acids.

Description

[0001] ENGINEERED LYMPHOCYTES EXPRESSING INTERLEUKIN-15 AND INTERLEUKIN-

[0002] 21 AND USES THEREOF

[0003] FIELD OF THE INVENTION

[0004] The present invention relates to the field of adoptive cell therapy for the treatment of diseases. More specifically, the invention concerns the molecular engineering of immune cells, particularly CD8+ T cells, and the therapeutic applications of such engineered cells. The invention is applicable to the treatment of cancer, autoimmune diseases, infections caused by pathogens, and cardiovascular diseases.

[0005] BACKGROUND TO THE INVENTION

[0006] Allogeneic cell therapy is a rapidly evolving field that focuses on the use of immune cells, tissues, or organs from a donor to treat diseases. Allogeneic cell therapy can be used to boost the immune response, repair damaged tissues, or replace diseased organs. The most common types of allogeneic cell therapies include allogeneic hematopoietic stem cell transplantation (HSCT), which is used to treat blood and immune system disorders, and solid organ transplantation, which is used to treat end-stage organ failure. In recent years, there has been a growing interest in developing new allogeneic immunotherapies, such as adoptive cell therapy and engineered T-cell therapy, for the treatment of cancer and other diseases. The approach relies on the ability of the immune system to recognize and eliminate abnormal or foreign cells, such as cancer cells or pathogens. Allogeneic immunotherapy offers a promising avenue for the development of novel and effective treatment that harness the power of the immune system to fight diseases.

[0007] Allogeneic adoptive cell therapy is a type of immunotherapy that involves the transfer of immune cells from a healthy donor to a patient with a disease. These cells can originate from different sources within a healthy donor such as bone marrow, cord blood, peripheral blood stem cell or mature immune cell. They can also be derived from induced pluripotent stem cells (iPSC). This approach can be used to treat various types of cancer, including blood cancers such as leukemia and lymphoma and solid tumors. It can also be used for the treatment of autoimmune diseases and viral infections. The T cell receptor (TCR) complex is a protein complex found on the surface of T cells. The TCR complex contains two chains, the alpha chain and beta chain (or gamma chain and delta chain in some T cells), that are linked by disulfide bonds. The alpha chain and the beta chain each contain a constant and a variable region, the latter being able to recognize specific antigens. The antigen is presented to the TCR complex by a specialized cell called an antigen-presenting cell (APC), which displays the antigen on its surface bound to a protein called major histocompatibility complex (MHC) class I or II.

[0008] When the TCR complex binds to the antigen-MHC complex on an APC, it triggers a series of signaling events that activate the T cell and initiate an immune response. This includes the activation of intracellular signaling pathways and the production of cytokines.

[0009] In addition to the TCR complex, T cells also express a co-receptor called CD3, which is required for the normal expression and function of the TCR complex. The CD3 complex is made up of several different protein chains, including the gamma, delta, epsilon, and zeta chains, and has a role in transmitting signals from the TCR complex into the T cell. Together, the TCR complex and CD3 co-receptor allow T cells to recognize and respond to specific antigens, which creates an immune response.

[0010] If either of the components of TCR / CD3 is absent, the whole complex cannot be expressed on the surface membrane and there is no immune response.

[0011] To produce allogeneic T cells which are useful for the treatment of disease (both in CAR (Chimeric Antigen Receptor) or TCR engineered T cells), it is necessary to remove one of the endogenous parts of the TCR / CD3 complex in order to prevent the expression of the TCR / CD3 complex on the cell surface. This maneuver prevents GvHD (graft versus host disease), and cells can be further engineered to express a CAR or a modified TCR. Such removal methods are well known to those of skill in the art (CRISPR, TALEN, zinc finger nuclease, silencing RNA, ER-induced degradation). However, recent studies show that removing the TCR severely impairs allogeneic T-cell therapy by shortening the in vivo half-life of the cells.

[0012] An anti-CD3 bispecific antibody is an engineered protein that targets two different types of cells: T cells expressing CD3 and tumor cells or autoreactive immune cells expressing a tumor associated antigen (TAA) on a cancer cell or a lineage surface marker on autoreactive B-cells. Alternatively, one arm of the bi-specific antibody can target an MHC-peptide complex presenting peptides from a tumor antigen, for instance a mutated neoantigen. It is used in immunotherapy to treat certain types of cancer and autoimmune diseases. The CD3 molecule is present on the surface of T cells, while the othertarget is a specific marker found on the surface of tumor cells or autoreactive immune cells. The anti-CD3 bispecific antibody binds to both of these targets, which helps to bring T cells into contact with the cancer or autoreactive cells. The formation of this ternary complex leads to activation of the T-cell, the release of enzymes such as granzyme B and cytokines, which cause destruction of the target cell.

[0013] By activating and directing the T cells to attack the cancer or autoreactive B cells, anti-CD3 bispecific antibodies enhance the body's own immune response, and they have been used in the treatment of some cancers, such as leukemias and lymphomas, solid tumors, as well as autoimmune diseases like lupus erythematosus and rheumatoid arthritis.

[0014] Cancer immunotherapy, including cell-based therapy, antibody therapy and cytokine therapy, is a promising strategy for treating various types of cancer owing to its potential to evade genetic and cellular mechanisms of drug resistance and to target tumor cells while sparing healthy tissues.

[0015] Cell-based therapy using e.g. T cells having a T cell receptor (TCR) specific for an antigen differentially expressed in association with an MHC class I molecule on cancer cells or having a chimeric antigen receptors (CAR) comprising an antigen recognition moiety [e.g., a single chain variable fragment (scFv)] and a T-cell activation moiety were shown to exert anti-tumor effects in several types of cancers, e.g. hematologic malignancies. However, TCRs are limited in their recognition spectrum and the MHC class. In addition, when introducing an exogenous TCR to a T cell there is a risk of hybridization between exogenous and endogenous alpha- and beta-chains, which may induce recognition of autoantigens. CAR T cells, on the other hand, despite of their advantages, have critical flaws that need to be solved to allow for full utilization of the technology in clinical treatments, including e.g. severe side effects, vigorous expansion in the presence of heavy tumor burden leading to tumor lysis syndrome and cytokine release syndrome, or development of tumor escape variants which have lost the target antigen during treatment. Further, using allogeneic T cells for therapy imposes the risk of harmful recognition of autoantigens leading to graft versus host disease (GvHD).

[0016] Antibody-based cancer immunotherapies, such as monoclonal antibodies, antibody-fusion proteins, and antibody drug conjugates (ADCs) depend on recognition of cell surface molecules that are differentially expressed on cancer cells relative to non-cancerous cells and / or immune-checkpoint blockade. Binding of an antibody-based immunotherapy to a cancer cell can lead to cancer cell death via various mechanisms, e.g., antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), direct cytotoxic activity of the payload from an antibody-drug conjugate (ADC) or suppressive checkpoint blockade. Many of these mechanisms are initiated through the binding of the Fc domain of cell-bound antibodies to specialized cell surface receptors (Fc receptors) on hematopoietic cells. In recent years another antibody-based therapy has emerged: anti-CD3 bi-specific antibodies, such as Mosunetuzumab, Odronextamab,Blinatumomab and Tebentafusp. These bi-specific antibodies engage CD3 positive T cells with tumor-associated antigen or tumor specific antigen complexed with MHC and thus promote T cells to attack tumor cells. Immunotherapies combining principles of antibody-based therapy, CAR T cells and / or TCR based immunotherapy have also been developed.

[0017] One of the primary advantages of allogeneic adoptive cell therapy in comparison to autologous adaptive cell therapy is the easy availability of cells from healthy donors, which can be used to treat a large number of patients. This is especially important for patients who do not have sufficiently healthy T-cells available for use in autologous cell therapy. In addition, allogeneic cell therapy can be designed to include more potent immune cells from healthy donors, which may provide a more effective treatment compared to using a patient's own cells in autologous cell therapy.

[0018] Advantageously, the manufacturing time for allogeneic cell therapy is generally shorter than for autologous cell therapy because donor cells can be obtained in advance and stored until needed, and allogeneic cell therapy may be less expensive than autologous cell therapy because it does not require individual cell collection and processing for each patient.

[0019] However, there are also several disadvantages of this type of therapy. Allogeneic adoptive cell therapy carries a risk of graft-versus-host disease (GvHD), a condition where the donor immune cells attack the recipient's tissues and organs. This can be a serious complication that requires careful management and monitoring. Finding a suitable donor for allogeneic cell therapy can also be challenging, and a lack of matching can increase the risk of GvHD.

[0020] In order to prevent GvHD, patients may need to be treated with immune-suppressing drugs, which can increase the risk of infections and other complications. Furthermore, allogeneic cells may not persist in the recipient's body as long as autologous cells, which can limit the effectiveness of the therapy.

[0021] In summary, allogeneic adoptive cell therapy has the potential to be a powerful tool for treating cancer and other diseases, but it also comes with some challenges and risks that need to be carefully managed.

[0022] T cell immunotherapy has emerged as a promising frontier in cancer treatment, harnessing the body's own immune system to combat malignancies. This innovative approach involves leveraging the potent capabilities of T cells, a type of white blood cell crucial for orchestrating immune responses, to target and destroy cancer cells. Unlike traditional treatments like chemotherapy or radiation, which can have significant side effects and often lack specificity, T cell immunotherapy offers the potential for highly targeted and durable anti-cancer responses.

[0023] However, despite its remarkable potential, T cell immunotherapy faces formidable challenges, particularly when it comes to treating solid tumors. Solid tumors present a complex microenvironment characterized by a myriad of immunosuppressive mechanisms, which pose significant barriers to the efficacy of immunotherapy. These obstacles include the presence of inhibitory molecules and cell types within the tumor microenvironment, as well as the heterogeneous nature of solid tumors, which can lead to immune evasion and treatment resistance.

[0024] Moreover, achieving effective ? cell infiltration into solid tumors remains a major hurdle. Even when T cells are successfully engineered or activated outside the body and reintroduced into the patient (as in the case of adoptive cell therapy), their ability to penetrate deep into the tumor and exert their anti-cancer effects is often limited. Additionally, once T cells reach the tumor site, they may encounter a hostile environment that dampens their function and promotes tumor survival.

[0025] Another critical challenge is preserving T cell activity over the long term. While T cell immunotherapy can initially induce potent anti-tumor responses, sustaining these responses over time is essential for durable clinical benefit. However, the immunosuppressive nature of the tumor microenvironment, coupled with mechanisms of T cell exhaustion and dysfunction, can lead to a decline in T cell activity and the eventual resurgence of tumor growth. Thus, strategies aimed at enhancing T cell persistence and overcoming immune exhaustion are paramount for the success of T cell-based immunotherapies against solid tumors.

[0026] Numerous factors contributing to the limited efficacy of immune cellular therapies in solid tumors have been recently reviewed \

[0027] Despite these recent advances, there remains a critical need for improved cell therapies— especially enhanced T cell therapies— that can overcome current limitations and deliver more consistent and robust clinical outcomes.

[0028] Accordingly, the objective technical problem addressed by the present invention is the provision of improved cells for use in cell therapies. SUMMARY OF THE INVENTION

[0029] The present invention is characterized in the herein provided embodiments and claims. In particular, the present invention relates, inter alia, to the following embodiments:

[0030] 1. A lymphocyte comprising a recombinant nucleic acid encoding a fusion protein comprising an interleukin-15 (IL-15) fused to an interleukin-21 (IL-21).

[0031] 2. The lymphocyte of embodiment 1, wherein the IL-15 and the IL-21 are fused with a peptide linker.

[0032] 3. The lymphocyte of embodiment 2, wherein the peptide linker is a non-cleavable peptide linker.

[0033] 4. The lymphocyte of embodiment 2 or 3, wherein the peptide linker comprises a glycineserine repeat sequence.

[0034] 5. The lymphocyte of any one of embodiments 1 to 4, wherein the fusion protein is expressed in soluble or in membrane-bound form.

[0035] 6. The lymphocyte of any one of embodiments 1 to 5, wherein the lymphocyte is selected from the group consisting of a T cell (including CD4+ T cells and CD8+ T cells), a natural killer (NK) cell, and a natural killer T (NKT) cell.

[0036] 7. The lymphocyte of any one of embodiments 1 to 6, wherein the lymphocyte further comprises a recombinant nucleic acid molecule encoding an antigen receptor.

[0037] 8. The lymphocyte of embodiment 7, wherein the antigen receptor is a T cell receptor (TCR) or an engineered TCR.

[0038] 9. The lymphocyte of embodiment 8, wherein the engineered TCR is a TCR that is lacking one or more variable domain.

[0039] 10. The lymphocyte of embodiment 7, wherein the antigen receptor is a chimeric antigen receptor (CAR).

[0040] 11. The lymphocyte of any one of embodiments 1 to 10, wherein activation of the lymphocyte is independent of CD28-mediated co-stimulation. 12. The lymphocyte of embodiment 11, wherein said independence from CD28-mediated co-stimulation is conferred by:

[0041] (i) deletion, inactivation, knockdown, or functional inhibition of the endogenous CBL-B gene; or

[0042] (ii) deletion, inactivation, knockdown, or functional inhibition of the endogenous STS1 gene; or

[0043] (iii) introduction of a gain-of-function mutation into the CARMIL2 gene, preferably a Q575E mutation or a functional equivalent thereof.

[0044] 13. A pharmaceutical composition comprising the lymphocyte of any one of embodiments 1 to 12 and at least one pharmaceutically acceptable excipient.

[0045] 14. The lymphocyte of any one of embodiments 1 to 12 or the pharmaceutical composition of claim 13 for use in cell therapy, preferably in adoptive cell therapy.

[0046] 15. The lymphocyte or pharmaceutical composition for use of embodiment 14, wherein the cell therapy is allogeneic cell therapy or autologous cell therapy.

[0047] 16. The lymphocyte or pharmaceutical composition for use of embodiment 14 or 15, wherein the cell therapy is for the treatment of a disease selected from the group consisting of cancer, an infectious disease, a cardiovascular disease and an autoimmune disease.

[0048] 17. The lymphocyte or pharmaceutical composition for use of any one of embodiments 14 to 16, wherein the lymphocyte or pharmaceutical composition is administered simultaneously or sequentially with a CD3-engaging agent, preferably a CD3-engaging bispecific molecule.

[0049] 18. A method of treating a subject in need thereof, comprising administering to the subject a lymphocyte according to any one of embodiments 1 to 12, or a pharmaceutical composition according to embodiment 13, in a cell therapy, preferably adoptive cell therapy.

[0050] 19. The method of embodiment 18, wherein the cell therapy is allogeneic cell therapy.

[0051] 20. The method of embodiment 18 or 19, wherein the cell therapy is for the treatment of a disease selected from the group consisting of cancer, an infectious disease, a cardiovascular disease and an autoimmune disease. 21. The method of any one of embodiments 18 to 20, further comprising administering to the subject a CD3-engaging agent, wherein the lymphocyte or pharmaceutical composition and the CD3-engaging agent are administered simultaneously or sequentially, and preferably wherein the CD3-engaging agent is a CD3-engaging bispecific molecule.

[0052] 22. A method of manufacturing a lymphocyte for cell therapy, the method comprising the steps of:

[0053] (i) providing a lymphocyte;

[0054] (ii) introducing into the lymphocyte a recombinant nucleic acid molecule encoding an interleukin-15 (IL-15) fused to an interleukin-21 (IL-21).

[0055] 23. The method of embodiment 22, wherein the IL-15 and the IL-21 are fused with a peptide linker.

[0056] 24. The method of embodiment 23, wherein the peptide linker is a non-cleavable peptide linker.

[0057] 25. The method of embodiment 23 or 24, wherein the peptide linker comprises a glycineserine repeat sequence.

[0058] 26. The method of any one of embodiments 22 to 25, further comprising introducing into the lymphocyte a recombinant nucleic acid molecule encoding a recombinant antigen receptor.

[0059] 27. The method of embodiment 26, wherein the recombinant antigen receptor is selected from the group consisting of a T cell receptor (TCR), an engineered TCR, and a chimeric antigen receptor (CAR).

[0060] 28. The method of any one of embodiments 22 to 27, wherein the recombinant nucleic acid molecule(s) are introduced into the lymphocyte by viral transduction, electroporation, or lipid-mediated transfection.

[0061] 29. The method according to any one of embodiments 22 to 28, further comprising modifying the lymphocyte to render it independent of CD28-mediated co-stimulation.

[0062] 30. The method of embodiment 29, wherein said independence from CD28-mediated costimulation is conferred by: (i) deletion, inactivation, knockdown, or functional inhibition of the endogenous CBL-B gene; or

[0063] (ii) deletion, inactivation, knockdown, or functional inhibition of the endogenous STS1 gene; or

[0064] (iii) introduction of a gain-of-function mutation into the CARMIL2 gene, preferably a Q575E mutation or a functional equivalent thereof.

[0065] 31. The method of any one of embodiments 22 to 30, wherein the lymphocyte is selected from the group consisting of a T cell (including CD4+ T cells and CD8+ T cells), a natural killer (NK) cell, and a natural killer T (NKT) cell.

[0066] 32. The method of any one of embodiments 22 to 31, wherein the lymphocyte is obtained from a healthy donor or a subject in need.

[0067] 33. The method of any one of embodiments 22 to 32, further comprising a step of formulating the lymphocyte in a pharmaceutical composition together with at least one pharmaceutically acceptable excipient.

[0068] 34. A method for increasing the viability of a lymphocyte and / or inducing a naive-like phenotype in a lymphocyte, the method comprising co-expressing in the lymphocyte a first recombinant nucleic acid molecule encoding interleukin-15 (IL-15) and a second recombinant nucleic acid molecule encoding interleukin-21 (IL-21).

[0069] 35. The method of embodiment 34, wherein the method is an ex vivo method.

[0070] 36. The method of embodiment 34 or 35, wherein the naive-like phenotype is characterized by expression of CD62L and / or CCR7 on the surface of the lymphocyte.

[0071] 37. The method of any one of embodiments 34 to 36, wherein the IL-15 and IL-21 are encoded by a single recombinant nucleic acid molecule.

[0072] 38. The method of any one of embodiments 34 to 37, wherein the IL-15 and IL-21 are expressed as a fusion protein.

[0073] 39. The method of embodiment 38, wherein the fusion protein comprises IL-15 and IL-21 fused by a peptide linker, preferably wherein the peptide linker is a non-cleavable peptide linker, more preferably wherein the peptide linker comprises a glycine-serine repeat sequence. 40. The method of any one of embodiments 34 to 39, wherein the lymphocyte is selected from the group consisting of a T cell (including CD4+ T cells and CD8+ T cells), a natural killer (NK) cell, and a natural killer T (NKT) cell.

[0074] 41. A recombinant nucleic acid molecule comprising:

[0075] (i) a nucleotide sequence encoding interleukin-15 (IL-15);

[0076] (ii) a nucleotide sequence encoding interleukin-21 (IL-21); and

[0077] (iii) a nucleotide sequence encoding a recombinant antigen receptor.

[0078] 42. The recombinant nucleic acid molecule of embodiment 41, wherein the recombinant antigen receptor is selected from the group consisting of a T cell receptor (TCR), an engineered TCR, and a chimeric antigen receptor (CAR).

[0079] 43. The recombinant nucleic acid molecule of embodiment 41 or 42, wherein the nucleotide sequences encoding IL-15 and IL-21 are arranged to be expressed as a fusion protein.

[0080] 44. The recombinant nucleic acid molecule of embodiment 43, wherein the fusion protein comprises IL-15 and IL-21 fused by a peptide linker, preferably a glycine-serine repeat sequence.

[0081] 45. The recombinant nucleic acid molecule of any one of embodiments 41 to 44, wherein the nucleotide sequence encoding the I L-15 / IL-21 fusion protein is operably linked to a first promoter, and the nucleotide sequence encoding the recombinant antigen receptor is operably linked to a second promoter.

[0082] 46. The recombinant nucleic acid molecule of any one of embodiments 41 to 45, wherein the recombinant nucleic acid molecule is comprised in a viral vector, preferably wherein the viral vector is selected from the group consisting of a lentiviral vector, a retroviral vector, an adenoviral vector, and an adeno-associated viral (AAV) vector.

[0083] 47. An I L-15 / 1 L-21 fusion protein comprising interleukin-15 (IL-15) and interleukin-21 (IL-21) fused by a peptide linker.

[0084] 48. The fusion protein of embodiment 47, wherein the peptide linker is a non-cleavable peptide linker.

[0085] 49. The fusion protein of embodiment 47 or 48, wherein the peptide linker comprises a glycine-serine repeat sequence. 50. The fusion protein of any one of embodiments 47 to 49, wherein the peptide linker comprises the amino acid sequence (Gly4Ser)n, wherein n is 1 to 5.

[0086] 51. The fusion protein of any one of embodiments 47 to 50, wherein the IL-15 is positioned N-terminal to the IL-21, or vice versa.

[0087] 52. The fusion protein of any one of embodiments 47 to 51, wherein the fusion protein comprises an amino acid sequence as set forth in SEQ ID NO:4.

[0088] 53. A nucleic acid molecule encoding the fusion protein of any one of embodiments 47 to 52.

[0089] 54. A cell comprising a nucleic acid molecule of any one of embodiments 41 to 46 or 53.

[0090] The present invention provides for molecular engineered immune cell which provide a potent solution to the challenges and for avoidance of the risks associated with cell therapies, in particular allogeneic cell therapies. Addressing these multifaceted challenges requires a comprehensive understanding of tumor immunology, coupled with innovative approaches to enhance T cell function, promote infiltration into solid tumors, and sustain long-term antitumor responses. By overcoming these hurdles, T cell immunotherapy holds the promise of revolutionizing cancer treatment and offering new hope to patients with solid tumors.

[0091] In one aspect, the present invention relates to a lymphocyte comprising a recombinant nucleic acid encoding a fusion protein comprising an interleukin-15 (IL-15) fused to an interleukin-21 (IL-21).

[0092] It was surprisingly demonstrated by the inventors that co-expressing interleukin-15 (IL-15) and interleukin-21 (IL-21), preferably in the form of a fusion protein, in a lymphocyte, promotes a naive or stem-like phenotype in lymphocytes.

[0093] As shown in Figure 2, lymphocytes co-expressing IL-15 and IL-21 cultured in cytokine-free media exhibited significantly higher viability compared to lymphocytes expressing IL-15 alone. The increased viability of these cells suggests that the IL-15 / IL-21 fusion protein supports lymphocyte persistence, self-renewal and resistance to apoptosis, all of which are critical attributes for effective cell-based therapies.

[0094] Further characterization of the cellular phenotype was performed by flow cytometry analysis, as depicted in Figure 5. After repeated antigenic rechallenge, lymphocytes co-expressing IL- 15 and IL-21 displayed markedly increased expression of CD62L and CCR7, two surface markers that are characteristic of naive and central memory T cells. The upregulation of these markers demonstrates that the co-expression of IL-15 and IL-21 not only enhances cell survival but also promotes the maintenance or induction of a less differentiated, stem-like state. This phenotype is associated with improved self-renewal, proliferative potential, and long-term persistence— features that are highly desirable for therapeutic lymphocytes intended for adoptive cell transfer.

[0095] Taken together, the data presented in Figures 2 and 5 provide compelling evidence that engineering lymphocytes to co-express IL-15 and IL-21, particularly as a fusion protein, results in a cell population with superior viability and a favorable, naive or stem-like phenotype. These properties are expected to enhance the efficacy and durability of lymphocyte-based immunotherapies in clinical settings.

[0096] The aspects of the present invention will be discussed in more detail in the following sections.

[0097] 1. The lymphocyte of the invention

[0098] The lymphocyte according to the invention comprises a recombinant nucleic acid encoding a fusion protein comprising an interleukin-15 (IL-15) fused to an interleukin-21 (IL-21).

[0099] The term "fusion protein" as used herein refers to a single polypeptide comprising two or more distinct protein domains or functional regions that are covalently linked together, typically by means of a peptide bond. Fusion proteins are generally produced by recombinant DNA technology, wherein the nucleotide sequences encoding the individual protein domains are joined in-frame to create a continuous open reading frame. The resulting fusion protein may combine the functional properties of the constituent domains, such as enzymatic activity, binding specificity, or signaling capability, and may include a linker sequence to facilitate proper folding or function.

[0100] A first protein domain comprised in the fusion protein is an IL-15 domain. As used herein, the term "IL-15 domain" refers to a polypeptide sequence that includes all or a portion of the amino acid sequence of interleukin-15 (IL-15), a cytokine involved in the activation and proliferation of lymphocytes. In certain embodiments, the IL-15 domain comprises the full- length IL-15 protein. In alternative embodiments, the IL-15 domain may consist of a biologically active fragment, variant, or analog of IL-15, provided that it retains IL-15 activity.

[0101] In certain embodiments, the IL-15 domain comprised in the fusion protein is derived from human IL-15 having an amino acid sequence as set forth in SEQ ID NO:1: NWVNVISDL KKIEDLIQSM HIDATLYTES DVHPSCKVTA MKCFLLELQV ISLESGDASI HDTVENLII L

[0102] ANNSLSSNGN VTESGCKECE ELEEKNIKEF LQSFVHIVQM FINTS

[0103] In some embodiments, the fusion protein comprises at least 70, 80, 90, 100, 105, 110, 115, 120, 121, 122, 123, 124, or all 125 consecutive amino acid residues of human IL-15 (SEQ ID NO:1), provided that the fusion protein retains IL-15 biological activity.

[0104] In further embodiments, the fusion protein comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to human IL-15 (SEQ ID NO:1), provided that the fusion protein retains IL-15 biological activity.

[0105] A second protein domain comprised in the fusion protein is an IL-21 domain. As used herein, the term "IL-21 domain" refers to a polypeptide sequence that includes all or a portion of the amino acid sequence of interleukin-21 (IL-21), a cytokine involved in the regulation and function of lymphocytes, including T cells, B cells, and natural killer (NK) cells. In certain embodiments, the IL-21 domain comprises the full-length IL-21 protein. In alternative embodiments, the IL-21 domain may consist of a biologically active fragment, variant, or analog of IL-21, provided that it retains IL-21 activity.

[0106] In certain embodiments, the IL-21 domain comprised in the fusion protein is derived from human IL-21 having an amino acid sequence as set forth in SEQ ID NO:2:

[0107] QDRHMIRMR QLIDIVDQLK NYVNDLVPEF LPAPEDVETN CEWSAFSCFQ KAQLKSANTG NNERIINVSI KKLKRKPPST NAGRRQKHRL TCPSCDSYEK KPPKEFLERF KSLLQKMIHQ HLSSRTHGSE DS

[0108] In some embodiments, the fusion protein comprises at least 70, 80, 90, 100, 110, 115, 120, 125, 126, 127, 128, 129, 130, 131 or all 132 consecutive amino acid residues of human IL-21 (SEQ ID NO:2), provided that the fusion protein retains IL-21 biological activity.

[0109] In further embodiments, the fusion protein comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to human IL-21 (SEQ ID NO:2), provided that the fusion protein retains IL-21 biological activity. Sequence variants may include one or more amino acid substitutions, deletions, insertions, or additions, so long as the variant maintains the functional properties of IL-21 as described herein.

[0110] In certain embodiments, the fusion protein encoded by the recombinant nucleic acid comprised in the lymphocyte includes an IL-15 domain having at least 80%, preferably at least 90%, and more preferably at least 95% sequence identity to SEQ ID NO:1, and an IL-21 domain having at least 80%, preferably at least 90%, and more preferably at least 95% sequence identity to SEQ ID NO:2, wherein the fusion protein retains the biological activity of both IL-15 and IL-21.

[0111] As used herein, the term “% sequence identity" refers to the degree of similarity between two amino acid or nucleic acid sequences, expressed as a percentage, and is determined by comparing the sequences over a specified region and calculating the proportion of identical residues at corresponding positions. Sequence identity may be determined using any suitable algorithm or computer program known in the art, such as BLAST, GAP, or the Needleman- Wunsch algorithm, with default parameters unless otherwise specified. In some embodiments, % sequence identity is calculated by aligning the two sequences, determining the number of identical residues in the alignment, dividing by the total number of residues in the reference sequence, and multiplying by 100. Unless otherwise indicated, % sequence identity is calculated over the full length of the reference sequence.

[0112] As used herein, a fusion protein is said to "retain the biological activity of IL-15 and IL-21" if it exhibits one or more functional properties characteristic of wild-type IL-15 and IL-21, respectively. For IL-15, such biological activity includes, but is not limited to, the ability to stimulate the proliferation, survival, or activation of lymphocytes, such as T cells or natural killer (NK) cells, as measured by standard in vitro or in vivo assays (for example, induction of STAT5 phosphorylation, promotion of T cell or NK cell proliferation, or enhancement of cytotoxic activity). For IL-21, such biological activity includes, but is not limited to, the ability to modulate the function, proliferation, or differentiation of lymphocytes, such as T cells, B cells, or NK cells, as measured by standard assays (for example, induction of STAT3 phosphorylation, promotion of B cell differentiation, or enhancement of effector cytokine production). The fusion protein may be considered to retain the biological activity of IL-15 and IL-21 if it demonstrates comparable activity to the corresponding wild-type cytokines in one or more relevant functional assays. In certain embodiments, the fusion protein retains at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the biological activity of the combination of wild-type IL-15 and IL-21, as determined by comparison in such functional assays.

[0113] In a preferred embodiment, the fusion protein encoded by the recombinant nucleic acid comprised in the lymphocyte includes an IL-15 domain having an amino acid as set forth in SEQ ID NO:1, and an IL-21 domain having an amino acid sequence as set forth in SEQ ID NO:2.

[0114] The IL-15 domain and the IL-21 domain are fused to each other to form a fusion protein. As used herein, the term "fused" refers to the covalent linkage of two or more protein domains or polypeptide sequences within a single polypeptide chain, typically achieved by recombinant DNA techniques. The domains may be joined directly or via a peptide linker, such that the resulting fusion protein comprises both the IL-15 domain and the IL-21 domain in a contiguous amino acid sequence. This fusion enables the combined or coordinated biological activities of the constituent domains within a single molecular entity.

[0115] That is, in certain embodiments, the IL-15 domain is directly fused to the IL-21 domain. For example, in certain embodiments, the C-terminal end of the IL-15 domain may be fused directly to the N-terminal end of the IL-21 domain. In other embodiments, the C-terminal end of the IL-21 domain may be fused directly to the N-terminal end of the IL-15 domain.

[0116] However, it is preferred herein that the IL-15 domain is fused to the IL-21 domain via a peptide linker. Thus, in a particular embodiment, the invention relates to the lymphocyte of the invention, wherein the IL-15 and the IL-21 are fused with a peptide linker.

[0117] As used herein, the term "peptide linker" refers to a short sequence of amino acids that connects two or more protein domains or polypeptide segments within a fusion protein. The peptide linker serves to provide spatial separation and flexibility between the joined domains, thereby facilitating proper folding, stability, and independent biological activity of each domain. Peptide linkers may vary in length, composition, and structure, and may be designed to be flexible, rigid, or cleavable, depending on the desired properties of the fusion protein. Common examples include glycine-serine (Gly-Ser) repeats, such as (GGGGS)n (SEQ ID NO:3), although any suitable amino acid sequence may be used as a linker as described herein.

[0118] In a particular embodiment, the peptide linker is a non-cleavable linker. As used herein, the term "non-cleavable linker" refers to a peptide linkerthat is resistant to enzymatic or chemical cleavage under physiological conditions, thereby maintaining the covalent connection between the fused protein domains throughout the lifetime of the fusion protein. Non- cleavable linkers are designed to provide stable and persistent linkage, ensuring that the functional domains of the fusion protein remain associated and capable of exerting their combined biological activities as intended. Thus, in a particular embodiment, the invention relates to the lymphocyte of the invention, wherein the peptide linker is a non-cleavable peptide linker.

[0119] In a particular embodiment, the IL-15 domain and the IL-21 domain are fused with a flexible linker, preferably comprising a glycine-serine repeat sequence. As used herein, a "flexible linker" refers to a peptide sequence that imparts conformational flexibility between the fused protein domains, thereby facilitating proper folding and independent biological activity of each domain. In preferred embodiments, the flexible linker comprises one or more repeats of glycine and serine residues, such as a (GGGGS)n motif (SEQ ID NO:3), which is known to provide both flexibility and solubility while minimizing potential interference between the functional domains of the fusion protein. Thus, in a particular embodiment, the invention relates to the lymphocyte of the invention, wherein the peptide linker comprises a glycineserine repeat sequence.

[0120] In a preferred embodiment, the linker comprises or consists of the sequence (GGGGS)n, wherein n is an integer ranging from 1 to 5, more preferably from 2 to 4, most preferably wherein n is 3.

[0121] In a particular embodiment, the fusion protein comprises or consists of the amino acid set forth in SEQ ID NO:4 (peptide linker is underlined):

[0122] NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIH DTVEN LIILANNSLSSNGN VTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSGGGGSGGGGSGGGGSQDRHMIRMRQUDIVDQLKNYVNDLV PEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKE FLERFKSLLQKMIHQHLSSRTHGSEDS*

[0123] In a particularly preferred embodiment, the fusion protein further comprises an N-terminal signal peptide to facilitate secretion of the fusion protein from the lymphocyte. The choice of signal peptide is not limiting, and any suitable signal peptide capable of directing the fusion protein to the secretory pathway may be used. For example, the signal peptide may be derived from immunoglobulin heavy chain (IgG), such as the IgG heavy chain signal peptide, or from IL-15, IL-21, or any other protein known to be efficiently secreted. The inclusion of an N- terminal signal peptide ensures that the fusion protein is effectively translocated into the endoplasmic reticulum and subsequently secreted from the cell, thereby enabling its biological activity in the extracellular environment.

[0124] The lymphocyte of the present invention is defined to comprise a recombinant nucleic acid. As used herein, the term "comprise" is intended to be open and inclusive, indicating that the lymphocyte must include at least the specified recombinant nucleic acid, but is not limited to this component alone. Accordingly, the lymphocyte may contain additional nucleic acids, proteins, or other cellular components, whether or not they are explicitly described herein, without departing from the scope of the invention. This definition ensures that the presence of the recombinant nucleic acid does not exclude the inclusion of other elements or features within the lymphocyte.

[0125] As used herein, the phrase "comprised in the lymphocyte" with respect to a recombinant nucleic acid is intended to encompass any manner by which the nucleic acid is present within the lymphocyte. The recombinant nucleic acid may be integrated into the genome of the lymphocyte, for example, through viral transduction, transposon-mediated integration, or homologous recombination. Alternatively, the recombinant nucleic acid may be present as an episomal element, plasmid, or other extrachromosomal construct, or may be introduced and maintained as an RNA molecule, such as messenger RNA (mRNA), or any other mobile genetic element. Thus, the invention is not limited by the form, structure, or mechanism by which the recombinant nucleic acid is introduced or maintained within the lymphocyte, provided that the nucleic acid is present and capable of being expressed as intended.

[0126] In a particular embodiment, the recombinant nucleic acid is integrated into the genome of the lymphocyte by viral transduction. As used herein, "viral transduction" refers to the process by which a viral vector— such as a lentivirus, retrovirus, or adenovirus— is used to deliver and stably insert the recombinant nucleic acid into the chromosomal DNA of the lymphocyte. This approach enables long-term and heritable expression of the encoded fusion protein within the lymphocyte. The use of viral vectors for genomic integration is well established in the art and allows for efficient and stable genetic modification of lymphocytes for therapeutic or research applications.

[0127] It is to be understood that the recombinant nucleic acid further comprises regulatory elements necessary for efficient expression of the fusion protein. Such regulatory elements may include, but are not limited to, promoters, enhancers, untranslated regions (UTRs), polyadenylation signals, splice sites, and other sequences that facilitate transcription, RNA processing, stability, and translation of the encoded fusion protein. The inclusion of these regulatory elements ensures that the fusion protein is expressed at levels sufficient to achieve the desired biological activity within the lymphocyte. The skilled person will be well aware of the selection and use of appropriate regulatory elements for achieving efficient expression in lymphocytes.

[0128] Accordingly, in a preferred embodiment, the invention relates to a lymphocyte that expresses a recombinant nucleic acid encoding the fusion protein of the invention. Methods for detecting protein expression in cells are well known in the art and may include, for example, flow cytometry, immunoblotting (Western blot), enzyme-linked immunosorbent assay (ELISA), or immunofluorescence microscopy. The skilled person can readily select and implement an appropriate assay to confirm expression of the fusion protein in the engineered lymphocyte.

[0129] The recombinant nucleic acid encodes the fusion protein. As used herein, the term "encode" means that the recombinant nucleic acid comprises a nucleotide sequence that contains the genetic information necessary for the biosynthesis of the fusion protein. This sequence is capable of being transcribed and translated by the cellular machinery of the lymphocyte, resulting in the production of the specified fusion protein. Accordingly, when it is stated that the recombinant nucleic acid encodes the fusion protein, it is intended that the nucleic acid includes a sequence of nucleotides which, when expressed, directs the synthesis of the fusion protein as described herein.

[0130] As used herein, the term "recombinant nucleic acid" refers to a nucleic acid molecule that has been artificially created, modified, or manipulated using molecular biology techniques, such as cloning, ligation, or amplification. Recombinant nucleic acids may comprise DNA or RNA sequences that are not naturally contiguous and may include sequences derived from different sources or organisms joined together in a mannerthat does not occur in nature. Such nucleic acids may encode one or more polypeptides of interest, regulatory elements, or other functional sequences, and may be introduced into host cells for expression, replication, or further manipulation as described herein.

[0131] In a particular embodiment, the invention relates to the lymphocyte of the invention, wherein the fusion protein is expressed in soluble or in membrane-bound form. As used herein, "soluble form" refers to the fusion protein being produced in a manner that allows it to be secreted from the cell or remain unassociated with cellular membranes, thereby enabling interaction with extracellular targets. "Membrane-bound form" refers to the fusion protein being anchored to or associated with the cell membrane, for example, by inclusion of a transmembrane domain or membrane-targeting sequence, such that the fusion protein is presented on the cell surface or localized to a cellular membrane. This embodiment encompasses lymphocytes expressing the fusion protein in either configuration, thereby providing flexibility in the design and application of the invention for therapeutic or research purposes.

[0132] For soluble expression of the fusion protein, a signal peptide is preferably utilized. As used herein, a "signal peptide" refers to a short amino acid sequence, typically located at the N- terminus of the fusion protein, that directs the nascent polypeptide to the secretory pathway of the cell. The presence of a signal peptide facilitates the translocation of the fusion protein into the endoplasmic reticulum, from where it is subsequently processed and secreted into the extracellular space. The skilled person will recognize that a variety of suitable signal peptides may be employed to achieve efficient secretion of the fusion protein, and the choice of signal peptide may be optimized based on the host cell and desired level of expression.

[0133] For membrane-bound expression of the fusion protein, the fusion protein is engineered to include one or more membrane-targeting elements that facilitate its association with the cellular membrane. Such elements may include a transmembrane domain, which anchors the protein within the lipid bilayer, or a membrane-targeting sequence, such as a glycosylphosphatidylinositol (GPI) anchor signal, lipidation motif, or other suitable sequence that directs localization to the cell surface or intracellular membranes. The inclusion of these membrane-targeting elements ensures that the fusion protein is stably presented on the exterior or interior surface of the lymphocyte membrane, thereby enabling interaction with cell-surface receptors, ligands, or other membrane-associated molecules. The choice and design of the membrane-targeting element may be optimized based on the intended application and the host cell type.

[0134] The term "lymphocyte" as used herein refers to a type of white blood cell that plays a central role in the adaptive immune response. Lymphocytes include, but are not limited to, T cells, natural killer (NK) cells or natural killer T (NKT) cell. These cells are characterized by their ability to recognize and respond to specific antigens, thereby contributing to immune surveillance and the elimination of pathogens or abnormal cells, such as cancer cells. Lymphocytes may be isolated from various sources, including peripheral blood, bone marrow, or lymphoid tissues, or may be generated ex vivo, meaning that the cells are generated from suitable precursors, modified, and / or expanded outside of a living organism in a controlled laboratory environment.

[0135] In a particular embodiment, the lymphocyte is selected from the group consisting of a T cell (including CD4+ T cells and CD8+ T cells), a natural killer (NK) cell, and a natural killer T (NKT) cell.

[0136] That is, in certain embodiments, the lymphocyte according to the invention is a T cell. As used herein, the term "T cell" refers to a lymphocyte that expresses a T cell receptor (TCR) complex on its surface and is capable of recognizing antigenic peptides presented by major histocompatibility complex (MHC) molecules. T cells include, but are not limited to, CD4+ helper T cells, which assist in the activation and regulation of other immune cells, and CD8+ cytotoxic T cells, which are primarily responsible for the direct killing of infected or abnormal cells. T cells may be isolated from a subject or donor or may be generated ex vivo from suitable precursor cells, and may be further expanded, activated, genetically modified, or otherwise manipulated for therapeutic applications as described herein.

[0137] In a particular preferred embodiment, the lymphocyte is a CD8+ T cell. As used herein, the term "CD8+ T cell" refers to a subset of T lymphocytes that express the CD8 co-receptor on their cell surface in addition to the T cell receptor (TCR) complex. CD8+ T cells are also known as cytotoxic T lymphocytes (CTLs) and are primarily responsible for recognizing and eliminating cells displaying abnormal or foreign antigens in the context of major histocompatibility complex class I (MHC I) molecules. Upon activation, CD8+ T cells are capable of directly lysing infected, malignant, or otherwise abnormal cells through the release of cytotoxic granules containing perforin and granzymes, as well as through the secretion of pro-inflammatory cytokines such as interferon-gamma (IFN-y). In a particular embodiment, the lymphocyte is a CD4+ T cell. As used herein, the term "CD4+ T cell" refers to a subset of T lymphocytes that express the CD4 co-receptor on their cell surface in addition to the T cell receptor (TCR) complex. CD4+ T cells are primarily responsible for recognizing antigens presented in the context of major histocompatibility complex class II (MHC II) molecules. Upon activation, CD4+ T cells function as helper T cells, orchestrating and regulating the immune response by providing essential signals and cytokines that promote the activation, proliferation, and differentiation of other immune cells, including B cells, CD8+ T cells, macrophages, and dendritic cells. CD4+ T cells may differentiate into various functional subsets, such as T helper 1 (Thl), T helper 2 (Th2), T helper 17 (Thl7), and regulatory T cells (Treg), each characterized by distinct cytokine profiles and immunological roles.

[0138] In certain embodiments, the T cell may be a wild-type T cell, a tumor-infiltrating lymphocyte (TIL), or an engineered T cell. Engineered T cells include, without limitation, T cells expressing a chimeric antigen receptor (CAR), T cells with an exogenously engineered T cell receptor (TCR) recognizing a tumor (neo)antigen or tumor driver mutation, T cells carrying an HLA- independent TCR (HIT) receptor, T cells carrying a synthetic TCR and antigen receptor (STAR) or Co-STAR, T cells carrying a TCR-like CAR (TCAR), T cells carrying an antibody-TCR (AbTCR) receptor, T cells carrying a TCR fusion construct (True), or T cells carrying a T cell antigen coupler (TAC). These various forms of T cells may be utilized according to the invention to enhance antigen specificity, functional activity, or therapeutic efficacy in the context of adoptive cell therapy.

[0139] Where the lymphocyte is an engineered T cell, it is preferred that expression of the endogenous T cell receptor (TCR) has been disrupted. Disruption of the endogenous TCR may be achieved using gene editing technologies such as CRISPR / Cas9, TALENs, or zinc finger nucleases, and serves to reduce or eliminate the potential for mispairing between endogenous and introduced TCR chains, thereby enhancing the specificity and safety of the engineered T cell.

[0140] In certain embodiments, the lymphocyte may be a natural killer (NK) cell. As used herein, the term "natural killer (NK) cell" refers to a subset of lymphocytes that lack a T cell receptor (TCR) complex and CD3 expression, but are characterized by the expression of surface markers such as CD56 and / or CD16 in humans. NK cells are a critical component of the innate immune system and are capable of recognizing and eliminating virally infected cells, tumor cells, and other abnormal cells without prior sensitization. NK cells mediate cytotoxicity through the release of perforin and granzymes, as well as through antibody-dependent cellular cytotoxicity (ADCC) via the CD16 (FcyRIII) receptor. In addition, NK cells secrete cytokines such as interferon-gamma (IFN-y) that modulate the immune response. In certain embodiments, the lymphocyte is a natural killer T (NKT) cell. As used herein, the term "natural killerT (NKT) cell" refers to a unique subset of lymphocytes that share properties of both conventional T cells and natural killer (NK) cells. NKT cells express a T cell receptor (TCR), typically of limited diversity, as well as surface markers commonly associated with NK cells, such as CD56 and / or CD161 in humans. Unlike conventional T cells, NKT cells recognize lipid antigens presented by the non-classical major histocompatibility complex molecule CDld, rather than peptide antigens presented by classical MHC molecules. Upon activation, NKT cells rapidly produce a wide range of cytokines and chemokines, thereby influencing both innate and adaptive immune responses. NKT cells are capable of direct cytotoxic activity against infected or malignant cells and play important roles in tumor surveillance, infection, and immune regulation.

[0141] The lymphocyte according to the invention may be a naturally occurring lymphocyte or an engineered lymphocyte. In certain embodiments, the lymphocyte may be a naturally occurring T cell, natural killer (NK) cell, or natural killer T (NKT) cell, as found in peripheral blood, bone marrow, or lymphoid tissues. These naturally occurring lymphocytes may be genetically modified to express the fusion protein of the present invention, thereby conferring a more naive-like phenotype or enhancing other desirable functional properties.

[0142] 1.1 Antigen receptors

[0143] The lymphocyte may be an engineered cell that has been further genetically modified, for example, to comprise a recombinant nucleic acid encoding an antigen receptor, such as a chimeric antigen receptor (CAR) or a recombinant T cell receptor (TCR). Such modifications may be introduced in addition to, or in combination with, the expression of the fusion protein, thereby providing a lymphocyte with enhanced specificity, functionality, and therapeutic potential. Thus, in a particular embodiment, the invention relates to the lymphocyte of the invention, wherein the lymphocyte further comprises a recombinant nucleic acid molecule encoding an antigen receptor.

[0144] As used herein, the term "antigen receptor" refers to a protein complex expressed on the surface of lymphocytes that is capable of specifically recognizing and binding to antigens. In the context of the present invention, antigen receptors include naturally occurring T cell receptors (TCRs), which recognize peptide antigens presented by major histocompatibility complex (MHC) molecules, as well as engineered chimeric antigen receptors (CARs), which are synthetic receptors designed to recognize specific target molecules independently of MHC presentation. Both TCRs and CARs enable lymphocytes to detect and respond to particular antigens, thereby directing the immune response against cells expressing those antigens. In certain embodiments, the lymphocyte of the invention comprises a recombinant nucleic acid encoding a T cell receptor (TCR). As used herein, a T cell receptor refers to a heterodimeric protein complex expressed on the surface of T cells that is capable of specifically recognizing peptide antigens presented by major histocompatibility complex (MHC) molecules. The recombinant nucleic acid may encode either a naturally occurring or an engineered TCR, and may be introduced into the lymphocyte to confer novel antigen specificity or enhance existing immune responses. The expression of a recombinant TCR enables the lymphocyte to selectively recognize and respond to target cells presenting the corresponding antigen, thereby broadening the therapeutic potential of the modified lymphocyte. Thus, in a particular embodiment, the invention relates to the lymphocyte of the invention, wherein the antigen receptor is a T cell receptor (TCR) or an engineered TCR.

[0145] In certain embodiments, the TCR is a natural, i.e., non-engineered, T cell receptor. As used herein, the term "natural T cell receptor" or "natural TCR" refers to a TCR that is derived from a naturally occurring T cell population. Natural TCRs are heterodimeric protein complexes, typically composed of an alpha (a) and a beta ( ) chain, each containing variable and constant regions. These receptors are generated through the physiological processes of V(D)J recombination and thymic selection, resulting in a diverse repertoire capable of recognizing a wide range of peptide antigens presented by major histocompatibility complex (MHC) molecules. Natural TCRs confer antigen specificity to T cells and play a central role in the adaptive immune response. Recombinant nucleic acids encoding natural TCRs may be introduced and expressed in lymphocytes to confer or redirect target specificity, thereby enabling the lymphocyte to recognize and respond to cells presenting the corresponding antigen.

[0146] In certain embodiments, the TCR is an engineered T cell receptor. As used herein, the term "engineered T cell receptor" or "engineered TCR" refers to a TCR that has been modified or generated using molecular biology techniques to alter its antigen specificity, affinity, or other functional properties. Engineered TCRs may be produced by introducing specific mutations, exchanging variable regions, or otherwise modifying the amino acid sequence of one or both TCR chains. Such modifications are designed to enhance the ability of the TCR to recognize particular peptide-MHC complexes, increase binding affinity, reduce cross-reactivity, or improve expression and stability.

[0147] The engineered TCR may be any type of engineered TCR known in the art. In certain embodiments, the engineered TCR may be a high-affinity TCR (HIT), which is generated by introducing specific mutations or modifications to increase the binding affinity of the TCR for its cognate peptide-MHC complex, thereby enhancing antigen recognition and sensitivity. In other embodiments, the engineered TCR may be a signaling and antigen recognition (STAR) receptor, which is a synthetic receptor that combines antigen recognition domains with intracellular signaling modules to augment T cell activation and function. Additionally, the engineered TCR may be a T cell receptor fusion construct (TRuC), in which the antigen-binding domain of a TCR or antibody is fused directly to a component of the endogenous TCR / CD3 complex, enabling MHC-independent antigen recognition while leveraging native TCR signaling pathways.

[0148] In further embodiments, the engineered TCR may be a costimulatory synthetic TCR and antigen receptor (Co-STAR) as described by Mog et al., Sci. Transl. Med. 16, eadg7123 (2024). As used herein, a "Co-STAR" refers to a synthetic receptor that combines the antigenrecognition components of a TCR— replaced with high-affinity antibody fragments— with integrated costimulatory modules, such as MyD88 and CD40, that drive NF-KB signaling. This design enables T cells equipped with Co-STARs to recognize target antigens presented at low densities on cells, while providing potent costimulatory signals to enhance T cell activation, expansion, and anti-tumor efficacy. Co-STARs thus integrate the sensitivity of TCRs for low- density antigens with the robust activation and costimulation features of chimeric antigen receptors (CARs), making them particularly advantageous for targeting peptide-HLA antigens in cancer and other settings where antigen density may be limiting.

[0149] These and other engineered TCR formats are designed to optimize T cell specificity, potency, and therapeutic efficacy for use in adoptive cell therapies and other immunotherapeutic applications.

[0150] In a particularly preferred embodiment, the engineered TCR is a T cell receptor that lacks one or more variable domains. As used herein, this means that the variable region of the alpha chain, the beta chain, or both, is deleted, omitted, or replaced, resulting in a receptor that does not possess the conventional antigen-binding specificity conferred by the variable domains. Such engineered TCRs may be designed to enable activation of the lymphocyte through alternative mechanisms, such as engagement with agonistic antibodies or synthetic ligands, ratherthan through recognition of peptide-MHC complexes. This configuration allows for precise control of T cell activation and function and may be particularly advantageous in therapeutic applications where targeted, antigen-independent activation is desired.

[0151] Importantly, removal of the variable domains typically results in destabilization of the TCR- CD3 complex. However, it has been demonstrated in the art that specific mutations introduced into the remaining TCR chains can restore or enhance the stability of the residual TCR-CD3 complex. Such stabilizing mutations are well described in the art, for example in WO 2023 / 012584, the entire disclosure of which is hereby incorporated by reference. These modifications enable the engineered TCR to maintain proper assembly and surface expression, thereby preserving functional signaling capacity despite the absence of one or more variable domains.

[0152] In a particular embodiment, the engineered TCR expressed by the lymphocyte of the invention is any one of the engineered TCRs disclosed in WO 2023 / 012584. In particular, the engineered TCR is preferably a TCR comprising a human TCR alpha polypeptide and a human TCR beta polypeptide, wherein said TCR is devoid of an antigen-binding domain and a heterologous extracellular binding domain, and wherein said TCR alpha and beta polypeptides comprise amino acid modifications enabling presentation of said TCR as a TCR complex on a surface of a T cell expressing same.

[0153] In a particular embodiment, the engineered TCR lacking one or more variable domains comprises a human TCR alpha chain as set forth in SEQ ID NO:5, which includes the T48C mutation and one or more additional mutations selected from S116L, G119V, and F120L. The engineered TCR further comprises a human TCR beta chain as set forth in SEQ ID NO:6, which includes the S57C mutation. These specific mutations are introduced to enhance the stability and functionality of the TCR-CD3 complex in the absence of one or more variable domains, thereby enabling effective surface expression and signaling of the engineered receptor.

[0154] In another particular embodiment, the engineered TCR lacking one or more variable domains comprises a human TCR alpha chain as set forth in SEQ ID NO:5, incorporating the mutations T48C, S116L, G119V, and F120L, and a human TCR beta chain as set forth in SEQ ID NO:6, incorporating the mutation S57C. An engineered TCR comprising all five mutations is referred to as "ENABLR," as described in WO 2023 / 012584, the entire disclosure of which is hereby incorporated by reference.

[0155] In a further embodiment, the engineered TCR may comprise additional or alternative stabilizing mutations. For example, the TCR alpha chain may include one or more of the following amino acid substitutions: P91S, E92D, S93V, and S94P, corresponding to the human TCR alpha polypeptide as set forth in SEQ ID NO:5. Similarly, the TCR beta chain may include one or more of the following amino acid substitutions: E18K, S22A, F133I, E / V136A, and Q139H, corresponding to the human TCR beta polypeptide as set forth in SEQ ID NO:6.

[0156] In yet another embodiment, the stabilizing ENABLR mutations described herein and in WO 2023 / 012584 may be combined with the Co-STAR approach disclosed by Mog et al. Specifically, the engineered TCR chains incorporating ENABLR mutations— which serve to restore or enhance the stability and surface expression of the TCR-CD3 complex in the absence of one or more variable domains— can be utilized as the structural framework for a Co-STAR receptor. In this configuration, high-affinity antibody fragments or other antigen-recognition domains may be fused to the engineered TCR, while costimulatory modules such as MyD88 and CD40 may be incorporated to provide potent NF-KB-driven activation signals. This combined strategy leverages the enhanced stability and signaling capacity conferred by the ENABLR mutations together with the robust antigen recognition and costimulatory features of the Co-STAR platform. Without being bound by theory, lymphocytes engineered with both ENABLR mutations and Co-STAR elements may exhibit improved surface expression, functional persistence, and anti-tumor efficacy, particularly in settings where antigen density is low or the tumor microenvironment is immunosuppressive.

[0157] Alternatively, instead of removing the variable domains of the TCR, the engineered TCR may be a TCR that has been engineered to decouple TCR-antigen binding from CD3 signaling. This may be achieved by introducing specific point mutation into the TCR alpha and / or beta chain, as described in US 2025 / 090583. Accordingly, the engineered TCR may be any one of the engineered TCR described in US 2025 / 090583, which is incorporated herein by reference in its entirety.

[0158] Alternatively, the lymphocyte according to the invention may comprise a recombinant nucleic acid encoding a chimeric antigen receptor (CAR). As used herein, a CAR refers to a synthetic receptor that typically comprises an extracellular antigen-binding domain, such as a singlechain variable fragment (scFv), linked to one or more intracellular signaling domains capable of activating the lymphocyte upon antigen recognition. The expression of a CAR enables the lymphocyte to recognize and respond to target antigens in an MHC-independent manner, thereby conferring novel specificity and potent effector functions for therapeutic applications, including cancer immunotherapy and other adoptive cell therapies.

[0159] The chimeric antigen receptor (CAR) may be any type of CAR known in the art and is capable of binding to a wide variety of antigens, including but not limited to protein, peptide, carbohydrate, or glycolipid antigens expressed on the surface of target cells. The CAR may comprise any suitable combination of extracellular antigen-binding domains, transmembrane regions, and intracellular signaling domains. In particular, the intracellular region may include one or more co-stimulatory domains, such as CD28, 4-1BB (CD137), 0X40 (CD134), or ICOS, in addition to the CD3 signaling domain. The design and configuration of the CAR can be selected and optimized according to the intended therapeutic application and the specific target antigen. Exemplary target antigens for CARs include, but are not limited to, CD19, CD20, CD137, BCMA, DLL3, GPCR5D, CLDN18.2, or CLDN2. Various CAR formats, structures, and technologies suitable for targeting these and other antigens are well described in the art. 1.2 By-passing CD28 co-stimulation and / or resistance to checkpoint overexpression and activation

[0160] The appended Examples demonstrate that expression of an IL-15 / IL-21 fusion protein in T cells promotes the development of a more naive or stem-like phenotype. However, it was further observed that T cells expressing the IL-15 / IL-21 fusion protein exhibited reduced cytotoxic activity compared to wild-type T cells in rechallenge killing assays (see Figure 3). These findings suggest that while the IL-15 / IL-21 fusion protein supports the maintenance of less differentiated T cell states, it may also limit the immediate effector function of the modified T cells under certain conditions.

[0161] Surprisingly, the inventors have found that the reduced cytotoxic activity observed in lymphocytes expressing the IL-15 / IL-21 fusion protein and exhibiting a naive or stem-like phenotype can be effectively overcome by knocking out the CBL-B gene (see Figure 7).

[0162] CBL-B is an E3 ubiquitin ligase that functions as a key negative regulator of T cell activation by targeting signaling proteins for ubiquitination and subsequent degradation. In particular, CBL- B plays a critical role in setting the activation threshold of T cells and enforcing the requirement for co-stimulatory signals, such as those provided by CD28, during T cell activation. Knocking out or inhibiting CBL-B removes this negative regulatory checkpoint, thereby lowering the activation threshold and enabling T cells to become fully activated in response to TCR / CD3 signaling alone, even in the absence of CD28-mediated co-stimulation. Knocking out CBL-B in addition disrupts signaling of checkpoint proteins, exemplified but not limited to PD-1, which counteract CD28 activation. This renders the lymphocyte resistant to excessive expression and activation of these checkpoint proteins, which are known to be critically involved in exhaustion and loss of cytotoxicity of T-cells. As a result, CBL-B knockout lymphocytes exhibit enhanced effector function and cytotoxicity, making this genetic modification particularly advantageous for adoptive cell therapies where co-stimulatory signals may be limited or absent in the tumor microenvironment.

[0163] Accordingly, in a preferred embodiment, the lymphocyte comprises one or more modifications that allows activation of the lymphocyte independent of CD28-mediated costimulation and / or resistant to exhaustion caused by expression and activation of checkpoint proteins, such as CTLA-4 or PD-1. Thus, in a particular embodiment, the invention relates to the lymphocyte of the invention, wherein activation of the lymphocyte is independent of CD28-mediated co-stimulation and / or resistant to exhaustion caused by expression and activation of checkpoint proteins, such as CTLA-4 or PD-1. Activation of lymphocytes independent of CD28-mediated co-stimulation and / or resistance to exhaustion by expression and activation of checkpoint proteins, such asCTLA-4 or PD-1, may be achieved by inactivating the endogenous CBL-B gene. Thus, in a particular embodiment, the invention relates to the lymphocyte of the invention, wherein said independence from CD28-mediated co-stimulation and / or resistance to exhaustion by expression and activation of checkpoint proteins, such as CTLA-4 or PD-1, is conferred by deletion, inactivation, knockdown, or functional inhibition of the endogenous CBL-B gene.

[0164] The CBL-B gene encodes an E3 ubiquitin-protein ligase that plays a crucial role in regulating immune cell activation, particularly in T cells. In humans, CBL-B is located on chromosome 3 (Chr 3: 105.66 - 105.87 Mb) and is a member of the CBL family of ubiquitin ligases.

[0165] An endogenous gene, such as CBL-B, may be rendered non-functional in a lymphocyte through a variety of genetic and molecular approaches, including deletion, inactivation, knockdown, or functional inhibition. Gene deletion or inactivation can be achieved using genome editing technologies such as CRISPR / Cas9, TALENs, or zinc finger nucleases, which introduce targeted double-strand breaks and result in frameshift mutations, insertions, or deletions that disrupt the gene's coding sequence. Knockdown of gene expression may be accomplished using RNA interference (RNAi) technologies, such as small interfering RNA (siRNA) or short hairpin RNA (shRNA), which reduce the levels of the target mRNA and thereby decrease protein production. Functional inhibition can also be achieved by expressing dominant-negative mutants, inhibitory peptides, or small molecules that block the activity of the gene product. These strategies can be used individually or in combination to effectively abrogate the function of endogenous genes like CBL-B, thereby modulating lymphocyte activity for therapeutic purposes. In a preferred embodiment, the lymphocyte according to the invention is a lymphocyte in which the endogenous CBL-B gene has been inactivated, preferably by CRISPR / Cas9.

[0166] Although inactivation of CBL-B is a preferred way of rendering the lymphocytes independent from CD28-mediated co-stimulation, alternative approaches for achieving CD28-independent activation have been described in the art.

[0167] For example, it has been recently demonstrated that a gain-of-function mutation in CARMI L2 is sufficient to trigger CD28 co-stimulatory functions in vivo (Zhang et al., J Exp Med (2025) 222 (8): e20250339). CARMIL2 is a cytosolic scaffolding protein that links the co-stimulatory receptor CD28 to the CARD11 adaptor protein, thereby facilitating CD28-dependent activation of the NF-KB signaling pathway. A gain-of-function mutation in CARMIL2, such as the CARMI L2Q575E mutation identified in humans, leads to the constitutive formation of CARMI L2-CARD11 complexes even in the absence of CD28 engagement. As a result, T cells harboring this mutation are able to execute most functions that are normally dependent on CD28 co-stimulation— including robust activation, proliferation, and anti-tumor responses— in an antigen-dependent manner. Accordingly, in certain embodiments, the lymphocyte of the invention is genetically modified to introduce a gain-of-f unction mutation in CARMIL2, in particular the Q575E mutation or its functional equivalent.

[0168] Another strategy to circumvent CD28-mediated co-stimulation of lymphocytes involves the inactivation of STS1. STS1 (Suppressor of T cell receptor Signaling 1) is an unconventional, pH- sensitive phosphatase that negatively regulates T cell activation by dephosphorylating key signaling proteins downstream of the T cell receptor (TCR), particularly in concert with the E3 ubiquitin ligase CBL-B. Upon TCR stimulation, STS1 forms a complex with CBL-B, and this interaction is critical for suppressing TCR signaling, especially under acidic conditions commonly found in the tumor microenvironment. Genetic inactivation or deletion of STS1 disrupts this negative regulatory axis, resulting in enhanced T cell activation, proliferation, and effector function, even in the absence of CD28-mediated co-stimulation. Thus, inactivation of STS1 represents an effective approach to render lymphocytes less dependent on CD28 costimulation and more resilient to immunosuppressive conditions, thereby enhancing their therapeutic potential in adoptive cell therapies.

[0169] Accordingly, the lymphocyte of the invention may comprise one or more modifications that render the lymphocyte independent of CD28-mediated co-stimulation, preferably wherein the one or more modification is selected from: (i) inactivation, deletion, knockdown, or functional inhibition of the endogenous CBL-B gene; (ii) inactivation, deletion, knockdown, or functional inhibition of the endogenous STS1 gene; and (iii) introduction of a gain-of-function mutation in the CARMIL2 gene, such as the Q575E mutation or a functional equivalent thereof. These modifications may be employed individually or in combination to enhance lymphocyte activation and effector function in the absence of CD28 co-stimulatory signals.

[0170] In one embodiment, the lymphocyte of the invention comprises a modification in which the CBL-B gene is inactivated, deleted, knocked down, or functionally inhibited.

[0171] In another embodiment, the lymphocyte of the invention comprises a modification in which the STS1 gene is inactivated, deleted, knocked down, or functionally inhibited.

[0172] In a further embodiment, the lymphocyte of the invention comprises a modification in which a gain-of-function mutation is introduced into the CARMIL2 gene, such as the Q575E mutation or a functional equivalent thereof.

[0173] In the previous paragraphs, various strategies have been discussed to render the lymphocyte independent of CD28-mediated co-stimulation and / or resistant to exhaustion caused by expression of checkpoint proteins, such as CTLA-4 or PD-1. However, it will be appreciated that alternative strategies could also be envisioned to achieve this objective.

[0174] The skilled person would have no difficulty in determining whether a particular modification renders a lymphocyte independent of CD28-mediated co-stimulation. A straightforward and well-established approach is to assess the ability of the genetically or chemically modified lymphocyte to produce interleukin-2 (IL-2) in response to TCR / CD3 stimulation alone. In unmodified CD8+ T cells, engagement of the TCR / CD3 complex without concurrent CD28 costimulation does not result in significant IL-2 production. However, modifications such as deletion of CBL-B or STS1 enable CD8+ T cells to produce IL-2 at levels comparable to those observed with both TCR and CD28 engagement. Accordingly, a modified lymphocyte may be considered "CD28-bypassing" if, upon TCR / CD3 stimulation alone, it produces at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the amount of IL-2 produced by a wild-type cell stimulated with both TCR / CD3 and CD28 co-stimulation. Such determinations can be readily made using standard in vitro assays for IL-2 expression.

[0175] As used herein, a lymphocyte is considered "resistant to exhaustion caused by expression of checkpoint proteins" if it maintains its effector functions, such as cytokine production, proliferation, and cytotoxic activity, despite the upregulation or engagement of immune checkpoint proteins. Checkpoint proteins are inhibitory receptors expressed on the surface of lymphocytes that serve to regulate immune responses and prevent overactivation, but their sustained expression in the tumor microenvironment can lead to a dysfunctional or "exhausted" state characterized by reduced immune activity. Preferred examples of checkpoint proteins include cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed cell death protein 1 (PD-1), both of which are well-established mediators of T cell exhaustion. Additional checkpoint proteins that may contribute to lymphocyte exhaustion include, but are not limited to, LAG-3 (lymphocyte-activation gene 3), TIM-3 (T cell immunoglobulin and mucin-domain containing-3), TIGIT (T cell immunoreceptor with Ig and ITIM domains), and VISTA (V-domain Ig suppressor of T cell activation). A lymphocyte may be considered resistant to exhaustion if, under conditions that would normally induce exhaustion— such as chronic antigen stimulation or exposure to the tumor microenvironment— it retains at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of its effector function relative to a non-exhausted control, as measured by standard assays for cytokine secretion, proliferation, or cytotoxicity.

[0176] 2. Pharmaceutical compositions

[0177] In another aspect, the invention relates to a pharmaceutical composition comprising the lymphocyte of the invention and at least one pharmaceutically acceptable excipient. As used herein, the term "pharmaceutical composition" refers to a preparation that includes one or more lymphocytes, such as those described in the present invention, formulated together with one or more additional components suitable for administration to a subject for therapeutic purposes. The composition is designed to be safe, stable, and effective for use in medical treatment. The term "pharmaceutically acceptable excipient" refers to any substance, other than the active lymphocyte component, that is included in the composition to facilitate the manufacture, storage, stability, administration, or efficacy of the product. In the context of the present invention, pharmaceutically acceptable excipients may include, but are not limited to, isotonic solutions, buffers, cryoprotectants, stabilizers, preservatives, and agents that support cell viability and function during storage, handling, or administration. All such excipients are selected to be compatible with the lymphocytes and suitable for use in humans or other intended subjects.

[0178] 3. Therapeutic uses

[0179] The lymphocyte disclosed herein may be used for a variety of therapeutic applications. In particular, the preferred field of application is cell therapy, including but not limited to adoptive cell transfer, immunotherapy, and other treatments involving the administration of modified or unmodified lymphocytes to a subject in need thereof. The lymphocyte of the invention may thus be employed to treat a wide range of diseases or conditions, with cell therapy representing the most advantageous and promising use.

[0180] Thus, in a particular embodiment, the invention relates to the lymphocyte of the invention, a lymphocyte co-expressing IL-15 and IL-21, orthe pharmaceutical composition of the invention for use in cell therapy, preferably in adoptive cell therapy.

[0181] That is, the lymphocyte used in cell therapy is preferably the lymphocyte disclosed herein above of a pharmaceutical composition comprising said lymphocyte.

[0182] However, the lymphocyte used in cell therapy may also be a lymphocyte that expresses IL-15 and IL-21 as individual proteins rather than as a fusion protein. In such embodiments, the lymphocyte may be genetically modified to express separate recombinant nucleic acids encoding IL-15 and IL-21, or a single recombinant nucleic acid comprising distinct expression cassettes for each cytokine, thereby enabling independent production and secretion of IL-15 and IL-21. Alternatively, IL-15 and IL-21 may be expressed as a single polypeptide chain in which the two proteins are separated by a cleavable linker, such as a 2A self-cleaving peptide sequence, allowing for co-expression and subsequent intracellular cleavage to yield the individual cytokines. As used herein, the term "cell therapy" refers to a therapeutic approach in which living cells are administered to a subject with the aim of treating, preventing, or ameliorating a disease or medical condition. Cell therapy encompasses a wide range of clinical applications, including the infusion, transplantation, or transfer of autologous, allogeneic, or genetically modified cells. A preferred form of cell therapy is "adoptive cell therapy," which involves collecting immune cells— such as lymphocytes— from a subject or donor, manipulating or expanding them ex vivo, and then administering them to the subject to achieve a therapeutic effect. Adoptive cell therapy includes, but is not limited to, the transfer of tumor-infiltrating lymphocytes (TILs), genetically modified T cells expressing chimeric antigen receptors (CARs) or engineered T cell receptors (TCRs), and the infusion of natural killer (NK) cells or other immune cell subsets. The goal of cell therapy, and particularly adoptive cell therapy, is to harness or enhance the functional properties of the administered cells to achieve therapeutic benefits such as immune modulation, tumor eradication, tissue regeneration, or correction of genetic defects.

[0183] Both autologous and allogeneic approaches to adoptive cell therapy are envisioned within the scope of the present invention. In autologous cell therapy, lymphocytes are collected from the patient, manipulated and / or expanded ex vivo, and then re-administered to the same individual. In allogeneic cell therapy, lymphocytes are obtained from a donor and administered to a different recipient. For allogeneic applications, the lymphocytes— particularly T cells— preferably comprise engineered T cell receptors (TCRs), such as the ENABLR TCRs disclosed in WO 2023 / 012584. These ENABLR TCRs lack variable domains, thereby reducing the risk of graft-versus-host disease by minimizing recognition of host antigens. This design enhances the safety and applicability of allogeneic adoptive cell therapy, enabling broader use of donor-derived immune cells for therapeutic purposes.

[0184] Accordingly, in a particular embodiment, the invention relates to the lymphocyte or pharmaceutical composition for use of the invention, wherein the cell therapy is allogeneic cell therapy or autologous cell therapy.

[0185] The lymphocyte of the invention, or a pharmaceutical composition comprising such a lymphocyte, may be used for cell therapy and is not limited to a particular disease or indication. While the invention is broadly applicable, preferred indications include cancer, infectious diseases, cardiovascular diseases, and autoimmune diseases, with cancer being the most preferred indication. This versatility allows the disclosed lymphocyte to provide therapeutic benefit across a range of clinical applications.

[0186] Thus, in certain embodiments, the lymphocyte or pharmaceutical composition of the invention may be used in the treatment of cancer. The lymphocytes disclosed herein are well- suited for cancer therapy due to their capacity to recognize, target, and eliminate malignant cells through antigen-specific immune responses. Engineered lymphocytes, such as those expressing chimeric antigen receptors (CARs) or modified T cell receptors (TCRs), can be tailored to specifically identify tumor-associated antigens, thereby enhancing the precision and efficacy of anti-tumor activity. CAR T cells are particularly relevant for cancer treatment, as they integrate the antigen-recognition properties of antibodies with the potent cytotoxic functions of T cells, enabling robust and targeted destruction of cancer cells. This strategy has already demonstrated remarkable clinical success in hematological malignancies and is being actively investigated for application in solid tumors. Alternatively, lymphocytes expressing an ENABLR TCR, as described in WO 2023 / 012584, may be employed in combination with bispecific antibodies that target both CD3 and a tumor antigen, providing an additional versatile approach for the treatment of cancer.

[0187] In certain embodiments, the lymphocyte or pharmaceutical composition of the invention may be employed for the treatment of infectious diseases, with particular preference for (chronic) viral infections. The lymphocytes described herein are especially advantageous for combating infectious diseases due to their ability to recognize and eliminate pathogen-infected cells through highly specific and potent immune responses. Engineered lymphocytes, such as those expressing chimeric antigen receptors (CARs) or modified T cell receptors (TCRs), can be designed to target antigens derived from viruses, bacteria, or other pathogens, thereby significantly enhancing both the specificity and efficacy of the immune response. For instance, CAR T cells may be engineered to recognize viral antigens or other pathogen-associated antigens presented on the surface of infected cells, enabling precise cytotoxic activity and effective clearance of infections, including persistent or chronic viral infections. Furthermore, lymphocytes expressing an ENABLR TCR, as described in WO 2023 / 012584, may be used in combination with bispecific antibodies that simultaneously target CD3 and a pathogenspecific antigen, offering a flexible and innovative approach for the treatment of infectious diseases. This strategy enables the targeting of a broad range of viral antigens and provides new opportunities for addressing infections that are refractory to conventional therapies.

[0188] In certain embodiments, the lymphocyte or pharmaceutical composition of the invention may be utilized for the treatment of autoimmune diseases. Autoimmune disorders are characterized by inappropriate or excessive immune responses directed against self-antigens, leading to chronic inflammation and progressive tissue damage. Engineered lymphocytes, such as regulatory T cells orT cells modified to express antigen-specific T cell receptors (TCRs), can be employed to restore immune tolerance and suppress pathogenic immune activity. These engineered cells may be designed to specifically target disease-associated antigens or to modulate key immune pathways, thereby reducing inflammation and preventing further tissue destruction. In addition, autoreactive B cells, which play a central role in the pathogenesis of certain autoimmune diseases, may also be targeted by engineered lymphocytes, such as CAR T cells directed against B cell markers. The application of such cellbased therapies holds significant promise forthe management of a wide range of autoimmune diseases, including, but not limited to, type 1 diabetes, multiple sclerosis, systemic lupus erythematosus, and rheumatoid arthritis.

[0189] Recent advances have demonstrated the potential of CAR T cell therapy for the treatment of myocardial infarction and cardiacfibrosis. Specifically, it has been shown that chimeric antigen receptor (CAR) T cells targeting activated cardiac fibroblasts can reduce fibrosis and improve cardiac function after injury (see Rurik JG et al., Science. 2022 Jan 6;375(6576):91-96. doi: 10.1126 / science.abm0594; PMCID: PMC9983611). Thus, in certain embodiments, the lymphocyte or pharmaceutical composition of the invention may be used in the treatment of cardiovascular diseases, in particular myocardial infarction and cardiac fibrosis.

[0190] The lymphocyte of the invention may be administered as the sole active ingredient or in combination with one or more additional therapeutic agents. Such combination therapies may include, but are not limited to, other cell-based products, small molecules, biologies, chemotherapeutic agents, immune checkpoint inhibitors, cytokines, or conventional treatments relevant to the disease or condition being addressed. The co-administration of the lymphocyte with other therapeutics may be simultaneous, sequential, or according to any regimen that optimizes clinical efficacy and safety. This flexibility allows for tailored treatment strategies to enhance therapeutic outcomes for individual patients.

[0191] In a particular embodiment, the lymphocyte of the invention is co-administered with a CD3- engaging agent. As used herein, a "CD3-engaging agent" refers to any molecule, such as a bispecific antibody or antibody fragment, that is capable of binding to the CD3 complex on the surface of T cells and facilitating their activation or redirecting their cytotoxic activity toward target cells. In a preferred embodiment, the CD3-engaging agent is a CD3-engaging bispecific molecule, which simultaneously binds to the CD3 complex on T cells and to a target antigen on diseased or abnormal cells. This dual specificity enables the recruitment and activation of T cells at the site of disease, thereby directing their cytotoxic activity specifically toward target cells. Co-administration of the lymphocyte with a CD3-engaging agent, particularly a bispecific molecule, can enhance the activation, proliferation, and effector function of the lymphocyte, significantly improving the precision, potency, and overall therapeutic efficacy of the treatment.

[0192] The CD3-engaging bispecific molecule is further characterized by its ability to bind not only to CD3 on T cells, but also to a second antigen, the selection of which is determined by the intended therapeutic application. This second antigen may include, for example, a tumor- associated antigen for cancer therapy, a pathogen-associated antigen for the treatment of infectious diseases, or any other disease-relevant antigen expressed on abnormal or target cells. In the context of cancer therapy, the tumor-associated antigen may be a cell surface protein, a lineage antigen, or a peptide / MHC complex derived from a tumor (neo)antigen or a tumor driver mutation. Lineage antigens are markers that are preferentially or exclusively expressed on cells of a particular tissue or cell lineage, while peptide / MHC complexes derived from tumor (neo)antigens or driver mutations represent highly specific targets resulting from genetic alterations unique to cancer cells. By simultaneously engaging CD3 on T cells and a specific antigen on target cells, the bispecific molecule enables the precise recruitment and activation of T cells at the site of disease, thereby enhancing both the specificity and efficacy of the immune response.

[0193] The CD3-engaging bispecific molecule may be a bispecific antibody or an ImmTAC, with the choice of format tailored to the therapeutic context. Both formats are designed to facilitate the targeted recruitment and activation of T cells by binding CD3 and a second antigen on diseased or abnormal cells. Exemplary bispecific antibodies include, without limitation, blinatumomab, epcoritamab, odronextamab, glofitamab, mosunetuzamab, teclistamab, cevostamab, talquetamab, and flotetuzumab, each of which targets specific antigens associated with various malignancies or disease states. ImmTACs (immune-mobilizing monoclonal TCRs against cancer), such as tebentafusp, represent a class of bispecific molecules that utilize a high-affinity, soluble TCR specific for a peptide / MHC complex on target cells, fused to an anti-CD3 effector domain. Additional formats, such as MANAbodies, may also be employed to achieve targeted T cell engagement. These diverse platforms provide flexible and potent strategies for redirecting T cell activity to sites of disease, thereby enhancing the precision and efficacy of immunotherapeutic interventions.

[0194] In certain embodiments, the invention provides a combination therapy comprising cells in which the CBL-B gene is knocked out or knocked down, or in which the CBL-B protein is inactivated, wherein the cells further express an ENABLR receptor and simultaneously express IL-15 and IL-21 in either soluble or membrane-tethered form, individually or as a fusion protein, in combination with a bispecific antibody. In one embodiment, the bispecific antibody is directed towards CD3 and a peptide / MHC complex derived from a tumor (neo)antigen and / or a tumor driver mutation. Examples of such bispecific antibodies include, without limitation, ImmTACs (e.g., tebentafusp) and MANAbodies.

[0195] In another preferred embodiment, the composition comprises cells as described above in combination with bispecific antibodies where one specificity is directed to CD3 and the other to a tumor-associated antigen (TAA) or a lineage-specific antigen, including, without limitation, blinatumomab, epcoritamab, odronextamab, glofitamab, mosunetuzamab, teclistamab, cevostamab, talquetamab, and flotetuzumab.

[0196] As used herein, the term "bispecific antibody" refers to an antibody-based molecule engineered to possess two distinct antigen-binding sites— one specific for the CD3 complex on T cells and the other for a second antigen, such as a tumor-associated or pathogen- associated antigen. This dual specificity enables the bispecific antibody to physically linkT cells to target cells, thereby promoting targeted cytotoxicity and enhancing immune-mediated clearance of diseased cells.

[0197] The term "ImmTAC" (immune-mobilizing monoclonal TCRs against cancer) refers to a class of bispecific molecules comprising a soluble, high-affinity T cell receptor (TCR) specific for a peptide-major histocompatibility complex (pMHC) on target cells, fused to an anti-CD3 effector domain. ImmTACs are designed to redirect T cells to recognize and eliminate cells presenting specific intracellular antigens in the context of MHC, thereby expanding the range of targetable antigens beyond those accessible to conventional antibodies.

[0198] The term "MANAbody" refers to a class of bispecific molecules engineered to harness the specificity of monoclonal antibodies (mAbs) for mutant antigens (MANAs), such as those arising from tumor-specific mutations or neoantigens. MANAbodies are designed to bind with high affinity to peptide-MHC complexes that display these mutant antigens on the surface of diseased cells, such as cancer cells. In addition to their MANA-specific binding domain, MANAbodies are typically fused to an effector domain, such as an anti-CD3 single-chain variable fragment (scFv), which enables the recruitment and activation of T cells upon engagement with the target cell. By bridging T cells to cells presenting mutant peptide-MHC complexes, MANAbodies facilitate highly specific immune responses against cells harboring disease-driving mutations, while sparing normal, healthy tissues. This dual specificity allows for the precise targeting of intracellular antigens that are otherwise inaccessible to conventional antibody therapies, thereby expanding the therapeutic potential of bispecific molecules in oncology and other disease contexts characterized by unique mutant antigens.

[0199] In a particular embodiment, the lymphocyte administered in combination with a CD3- engaging agent is a T cell comprising a non-functional T cell receptor (TCR), such as an ENABLR TCR as described in WO 2023 / 012584. These T cells are engineered so that their TCR cannot be activated by cognate antigen recognition, rendering them dependent on the presence of a CD3-engaging agent for activation. This feature is particularly advantageous for allogeneic cell therapy, as the lack of functional TCR signaling significantly reduces the risk of graft-versus- host disease (GvHD) by preventing unintended recognition of host antigens. Consequently, such non-functional T cells provide a safer and more controlled platform for adoptive cell therapies, especially in the allogeneic setting.

[0200] It is to be understood that all definitions, embodiments, and preferred applications described above with respect to the uses of the lymphocyte apply mutatis mutandis to corresponding methods of treatment. Accordingly, the explanations and interpretations provided for the therapeutic uses of the lymphocyte, pharmaceutical compositions, and related embodiments are equally applicable to methods of treatment involving the administration of such lymphocytes.

[0201] Thus, in a particular embodiment, the invention relates to a method of treating a subject in need thereof, comprising administering to the subject a lymphocyte of the invention, a lymphocyte co-expressing IL-15 and IL-21 , individually or as a fusion protein, or a pharmaceutical composition of the invention, in a cell therapy, preferably adoptive cell therapy.

[0202] In a particular embodiment, the invention relates to the method of the invention, wherein the cell therapy is allogeneic cell therapy.

[0203] In a particular embodiment, the invention relates to the method of the invention, wherein the cell therapy is for the treatment of a disease selected from the group consisting of cancer, an infectious disease, a cardiovascular disease and an autoimmune disease.

[0204] In a particular embodiment, the invention relates to the method of the invention, further comprising administering to the subject a CD3-engaging agent, wherein the lymphocyte or pharmaceutical composition and the CD3-engaging agent are administered simultaneously or sequentially, and preferably wherein the CD3-engaging agent is a CD3-engaging bispecific molecule.

[0205] As used herein, the term "subject" refers to any animal, including a human, that is the recipient of the therapeutic intervention described in the present invention. In preferred embodiments, the subject is a human, but the term may also encompass other mammals or vertebrates as appropriate for the intended application.

[0206] As used herein, the phrase "subject in need of treatment" refers to any subject who would benefit from the administration of the lymphocyte, pharmaceutical composition, or method described herein. This includes subjects who have been diagnosed with, are suspected of having, or are at risk of developing a disease or condition that can be prevented, alleviated, or treated by the invention. The terms "treat," "treatment," and "treating" refer to any intervention that results in the prevention, reduction, amelioration, or elimination of symptoms, disease progression, or pathological conditions in the subject.

[0207] As used herein, the term "administer" or "administering" refers to the act of providing or delivering a lymphocyte, pharmaceutical composition, or other therapeutic agent to a subject by any appropriate route, so as to achieve a therapeutic effect. For cell therapy products, the most preferred routes of administration are intravenous infusion and intratumoral injection. Other routes, including but not limited to intraperitoneal, subcutaneous, or oral administration, may also be used as appropriate for the specific product and clinical context.

[0208] The lymphocyte of the invention or the pharmaceutical composition comprising said lymphocyte is to be administered in an effective amount. As used herein, the term "effective amount" refers to an amount of a lymphocyte, pharmaceutical composition, or other therapeutic agent that is sufficient to achieve a desired therapeutic outcome, such as the prevention, reduction, or amelioration of symptoms, disease progression, or pathological conditions in the subject. The effective amount will vary depending on factors such as the nature of the disease or condition, the route of administration, the subject's age, weight, and overall health, and other relevant clinical parameters, and can be determined by those skilled in the art using routine methods.

[0209] 4. Methods of using the lymphocyte of the invention

[0210] The inventors have unexpectedly discovered that co-expression of interleukin-15 (IL-15) and interleukin-21 (IL-21) within a lymphocyte significantly enhances the viability of the lymphocyte and promotes the acquisition or maintenance of a more naive or stem-like phenotype. Accordingly, in another aspect, the invention provides a method for increasing the viability of a lymphocyte and / or inducing a naive-like phenotype in a lymphocyte, the method comprising co-expressing in the lymphocyte a first recombinant nucleic acid molecule encoding IL-15 and a second recombinant nucleic acid molecule encoding IL-21.

[0211] This method may be practiced either in vivo or ex vivo. As used herein, the term "in vivo" refers to procedures performed within a living organism, such as the direct delivery of recombinant nucleic acids encoding IL-15 and IL-21 to lymphocytes within a subject's body, for example by gene therapy vectors or mRNA delivery systems. In contrast, "ex vivo" refers to procedures carried out outside of the living organism, wherein lymphocytes that have already been obtained from a subject are genetically modified in a laboratory setting to coexpress IL-15 and IL-21. The modified lymphocytes may then be reintroduced into the same subject or administered to another subject. Both in vivo and ex vivo approaches may be employed to enhance lymphocyte viability and promote a naive-like or stem-like phenotype, thereby improving the therapeutic potential of lymphocyte-based treatments.

[0212] Thus, in a particular embodiment, the invention related to the method of the invention, wherein the method is an ex vivo method.

[0213] As used herein, the term "higher viability" refers to an increased proportion of living, metabolically active lymphocytes, as determined by standard cell viability assays (such as trypan blue exclusion, Annexin V / PI staining, or metabolic activity assays), compared to a comparable population of lymphocytes that does not recombinantly express IL-15 and IL-21.

[0214] The term "naive-like phenotype" refers to the expression of cell surface markers characteristic of naive or stem-like lymphocytes, in particular the lymphocyte adhesion molecule CD62L (L- selectin) and / or the chemokine receptor CCR7, and optionally the isoform CD45RA. A lymphocyte is defined as having a more naive-like phenotype if it exhibits at least an X% higher expression of CD62L, CCR7, and / or CD45RA, as measured by flow cytometry or equivalent quantitative methods, compared to a comparable lymphocyte that does not recombinantly express IL-15 and IL-21. The value of X may be, for example, at least 10%, 20%, 30%, or more, depending on the experimental context.

[0215] Engineered T cells co-expressing IL-15 and IL-21 have been shown to possess these desired biological properties, including prolonged survival and enhanced stem-like characteristics. Notably, their phenotype is predominantly CD62L+CCR7+CD45RA+, indicative of a naive or stem-like state.

[0216] Thus, in a particular embodiment, the invention relates to the method of the invention, wherein the naive-like phenotype is characterized by expression of CD62L and / or CCR7 on the lymphocyte.

[0217] Preferably, the first recombinant nucleic acid molecule encoding IL-15 and the second recombinant nucleic acid molecule encoding IL-21 are provided within a single recombinant nucleic acid construct. This single construct may be configured in various ways, including but not limited to: (i) two separate expression cassettes, each comprising its own promoter and regulatory elements for independent expression of IL-15 and IL-21; (ii) a single expression cassette encoding both IL-15 and IL-21, separated by a cleavable linker sequence, such as a self-cleaving 2A peptide, to allow for co-translational separation of the two proteins; or (iii) a single open reading frame encoding an IL-15 / IL-21 fusion protein, as defined elsewhere herein, wherein the two cytokines are joined directly or via a peptide linker. Most preferably, the IL-15 and IL-21 are expressed as a fusion protein, as discussed in more detail elsewhere herein.

[0218] Thus, in a particular embodiment, the invention relates to the method of the invention, wherein the IL-15 and IL-21 are encoded by a single recombinant nucleic acid molecule.

[0219] In a particular embodiment, the invention relates to the method of the invention, wherein the IL-15 and IL-21 are expressed as a fusion protein.

[0220] In a particular embodiment, the invention relates to the method of the invention, wherein the fusion protein comprises IL-15 and IL-21 fused by a peptide linker, preferably wherein the peptide linker is a non-cleavable peptide linker, more preferably wherein the peptide linker comprises a glycine-serine repeat sequence.

[0221] The lymphocyte used in the method of the invention may be any of the lymphocytes disclosed herein. In a particular embodiment, the invention relates to the method of the invention, wherein the lymphocyte is selected from the group consisting of a T cell (including CD4+ T cells and CD8+ T cells), a natural killer (NK) cell, and a natural killer T (NKT) cell.

[0222] In a preferred embodiment, the lymphocyte is a CD8+ T cell.

[0223] 5. Methods of manufacturing the lymphocyte of the invention

[0224] In another aspect, the present invention further provides methods for manufacturing the lymphocyte of the invention. Thus, in a particular embodiment, the invention relates to a method of manufacturing a lymphocyte for cell therapy, the method comprising the steps of:

[0225] (i) providing a lymphocyte; and

[0226] (ii) introducing into the lymphocyte a recombinant nucleic acid molecule encoding an interleukin-15 (IL-15) fused to an interleukin-21 (IL-21).

[0227] The lymphocyte and the recombinant nucleic acid utilized in the manufacturing method described herein may be as defined elsewhere in this application. All definitions, embodiments, and preferred features relating to the lymphocyte and recombinant nucleic acid set forth above are equally applicable to the present manufacturing methods.

[0228] That is, in a particular embodiment, the invention relates to the method of the invention, wherein the IL-15 and the IL-21 are fused with a peptide linker.

[0229] In a particular embodiment, the invention relates to the method of the invention, wherein the peptide linker is a non-cleavable peptide linker.

[0230] In a particular embodiment, the invention relates to the method of the invention, wherein the peptide linker comprises a glycine-serine repeat sequence. In a particular embodiment, the invention relates to the method of the invention, wherein the lymphocyte is selected from the group consisting of a T cell (including CD4+ T cells and CD8+ T cells), a natural killer (NK) cell, and a natural killer T (NKT) cell, as defined elsewhere herein.

[0231] Moreover, the lymphocyte may be obtained from any type of donor, including a healthy donor for allogeneic applications or a subject in need of treatment for autologous applications. Thus, in a particular embodiment, the invention relates to the method of the invention, wherein the lymphocyte is obtained from a healthy donor or a subject in need. This flexibility allows the manufacturing methods of the invention to be adapted for both autologous and allogeneic cell therapy approaches, depending on the clinical context and therapeutic requirements.

[0232] Lymphocytes may be obtained from a subject using standard clinical procedures. Most commonly, peripheral blood is collected by venipuncture or apheresis, the latter involving the separation of blood components to isolate peripheral blood mononuclear cells (PBMCs), which include lymphocytes. Alternatively, lymphocytes can be isolated from other sources such as bone marrow, lymph nodes, umbilical cord blood, or peripheral blood stem cell collections, depending on the intended application. In addition, lymphocytes may be derived from induced pluripotent stem cells (iPSCs) through directed differentiation protocols. The collected cells are typically processed using density gradient centrifugation or immunomagnetic separation techniques to enrich for the desired lymphocyte subset, such as T cells, natural killer (NK) cells, or natural killerT (NKT) cells. Once isolated, these lymphocytes can be further manipulated, expanded, or genetically modified as required for therapeutic use.

[0233] The method of the invention preferably includes an additional step of introducing a further nucleic acid molecule encoding a recombinant antigen receptor into the lymphocyte. Accordingly, in a particular embodiment, the invention relates to a method comprising the coexpression of IL-15 and IL-21 in a lymphocyte, further comprising the introduction of a recombinant nucleic acid encoding a recombinant antigen receptor.

[0234] The recombinant antigen receptor may be any of the antigen receptors described herein for use in the lymphocyte of the invention. In a preferred embodiment, the recombinant antigen receptor is selected from the group consisting of a T cell receptor (TCR), an engineered TCR, or a chimeric antigen receptor (CAR).

[0235] When the lymphocyte is a T cell and the method includes the introduction of a recombinant nucleic acid encoding a recombinant TCR (including engineered TCRs) or CAR, it is preferred that the T cell is one in which the expression of the endogenous TCR has been disrupted. Disruption of endogenous TCR expression may be achieved by any method known in the art, such as gene editing using CRISPR / Cas9, TALENs, or zinc finger nucleases, in order to minimize the risk of mispairing between endogenous and introduced TCR chains and to enhance the specificity and safety of the engineered T cell.

[0236] Recombinant nucleic acids, such as those encoding IL-15, IL-21, or an antigen receptor, can be introduced into a cell, particularly a lymphocyte, using a variety of established gene transfer techniques. These methods include, but are not limited to, viral vector-mediated delivery (such as lentiviral, retroviral, or adenoviral vectors), non-viral methods (such as electroporation, nucleofection, or lipofection), and transposon-based systems (such as Sleeping Beauty or PiggyBac transposons). Viral vectors are commonly used due to their high efficiency in stably integrating the recombinant nucleic acid into the host cell genome, thereby enabling long-term expression of the transgene. Non-viral methods, such as electroporation or nucleofection, involve the application of an electrical field to transiently permeabilize the cell membrane, allowing direct uptake of plasmid DNA or RNA. Lipofection utilizes lipid-based reagents to facilitate the delivery of nucleic acids into cells. The choice of method may be determined by factors such as the type of lymphocyte, the desired duration of transgene expression, safety considerations, and the intended clinical application. These techniques enable efficient genetic modification of lymphocytes to express therapeutic proteins, such as cytokines or antigen receptors, thereby enhancing their functionality for use in adoptive cell therapy and other immunotherapeutic strategies.

[0237] In a particular embodiment, the invention relates to the method of the invention, wherein the recombinant nucleic acid molecule(s) are introduced into the lymphocyte by viral transduction, electroporation, or lipid-mediated transfection.

[0238] Manufacturing the lymphocyte according to the invention may further comprise one or more additional genetic or non-genetic modifications to enhance the (therapeutic) properties of the lymphocyte. In particular embodiments, these modifications are designed to render the lymphocyte independent of CD28-mediated co-stimulation and / or resistant to exhaustion caused by the expression of immune checkpoint proteins, as described elsewhere herein.

[0239] For example, in a particular embodiment, the method of the invention further comprises modifying the lymphocyte to render it independent of CD28-mediated co-stimulation. Such independence may be achieved by genetic alteration of key intracellular regulators of T cell activation. In specific embodiments, this includes: (i) deletion, inactivation, knockdown, or functional inhibition of the endogenous CBL-B gene; (ii) deletion, inactivation, knockdown, or functional inhibition of the endogenous STS1 gene; or (iii) introduction of a gain-of-f unction mutation into the CARMIL2 gene, preferably a Q575E mutation or a functional equivalent thereof. These modifications may be accomplished using genome editing technologies such as CRISPR / Cas9, TALENs, or zinc finger nucleases, or by RNA interference or small molecule inhibitors, as appropriate.

[0240] Additional modifications may include, but are not limited to, the disruption of endogenous human leukocyte antigen (HLA) genes to reduce alloreactivity in allogeneic applications.

[0241] All such modifications may be performed sequentially or in combination, using standard gene editing or gene transfer techniques as described above. The selection and combination of specific modifications may be tailored to the intended clinical application, disease indication, and patient population, thereby providing a flexible and robust platform for the manufacture of next-generation lymphocyte-based cell therapies. The order and sequence of the various genetic modifications are not limiting and may be adapted as needed for manufacturing efficiency or product characteristics. For example, the introduction of the I L-15 / 1 L-21 fusion construct, knockout (KO) of the endogenous TCR, introduction of an engineered antigen receptor (such as ENABLR), and knockout of CBL-B may be performed in any order or sequence. In a preferred embodiment, the knockout of the endogenous TCR and CBL-B is performed simultaneously in a single step using two separate guide RNAs in conjunction with Cas9, thereby streamlining the editing process and reducing the number of cell manipulations required. Following this, the engineered TCR (e.g., ENABLR) and the I L-15 / 1 L-21 fusion protein may be introduced using a lentiviral vector, preferably a bidirectional lentiviral vector comprising two different promoters oriented in the forward and reverse directions to enable coordinated expression of multiple transgenes. This approach allows for efficient and precise engineering of lymphocytes with multiple desired attributes, supporting the development of advanced cell therapy products with enhanced safety, specificity, and therapeutic efficacy.

[0242] 6. Nucleic acids, proteins and cells of the invention

[0243] Another aspect of the present invention relates to a fusion protein comprising an interleukin- 15 (IL-15) domain and an interleukin-21 (IL-21) domain. The inventors have surprisingly demonstrated that such IL-15 / IL-21 fusion proteins promote a more naive or stem-like phenotype in lymphocytes, enhancing their therapeutic potential.

[0244] Thus, in another aspect, the invention relates to an IL-15 / IL-21 fusion protein comprising interleukin-15 (IL-15) and interleukin-21 (IL-21) fused by a peptide linker.

[0245] The IL-15 and IL-21 domains, including sequence variants thereof, as well as the linker peptide, are as defined elsewhere herein. In preferred embodiments, the peptide linker is a non- cleavable linker, such as a glycine-serine repeat sequence, for example, a (Gly4Ser)nmotif where n is 1 to 5. The orientation of the fusion protein may be IL-15 at the N-terminus and IL- 21 at the C-terminus, or vice versa. In a particularly preferred embodiment, the fusion protein comprises an amino acid sequence as set forth in SEQ ID NO:4.

[0246] The present invention further relates to a nucleic acid encoding the fusion protein of the invention. Thus, in another aspect, the invention relates to a nucleic acid molecule encoding the fusion protein of the invention.

[0247] Furtherencompassed herein are recombinant nucleic acids encoding an interleukin-15 (IL-15), an interleukin-21 (IL-21) and a recombinant antigen receptor.

[0248] Accordingly, in another aspect, the invention relates to a recombinant nucleic acid molecule comprising:

[0249] (i) a nucleotide sequence encoding interleukin-15 (IL-15);

[0250] (ii) a nucleotide sequence encoding interleukin-21 (IL-21); and

[0251] (iii) a nucleotide sequence encoding a recombinant antigen receptor.

[0252] The recombinant nucleic acid may be configured such that each component is expressed from a separate expression cassette, or two or more components may be expressed from the same expression cassette. Preferably, the nucleic acid is arranged so that the sequences encoding IL-15 and IL-21 are expressed as a fusion protein, meaning the nucleotide sequences are positioned in-frame and separated by a sequence encoding a peptide linker, thereby resulting in the translation of a single polypeptide comprising both cytokine domains. As used herein, the term "arranged to be expressed" refers to the organization of nucleotide sequences within the recombinant nucleic acid such that, upon transcription and translation in a host cell, the desired gene products are produced. In the context of a fusion protein, this means that the coding sequences are oriented in a manner that allows for continuous translation from a single mRNA, resulting in a single polypeptide chain that includes both IL-15 and IL-21 domains connected by the linker.

[0253] In a particularly preferred embodiment, the nucleotide sequence encoding the IL-15 / IL-21 fusion protein is operably linked to a first promoter, and the nucleotide sequence encoding the recombinant antigen receptor is operably linked to a second promoter. As used herein, "operably linked" means that the promoter is positioned in such a manner relative to the coding sequence that it directs transcription of the sequence in a host cell. This arrangement allows for independent and robust expression of both the fusion protein and the antigen receptor.

[0254] The recombinant nucleic acid may be any type of nucleic acid, including linear or circular DNA, plasmids, or cloning vectors. However, it is preferred that the recombinant nucleic acid is, or is comprised in, a viral vector. Suitable viral vectors include, but are not limited to, lentiviral vectors, retroviral vectors, adenoviral vectors, and adeno-associated viral (AAV) vectors. Lentiviral and retroviral vectors are commonly used for stable integration into the host genome, while adenoviral and AAV vectors are often used for transient or episomal expression. The specific configuration of the recombinant nucleic acid and its delivery vehicle may be as illustrated in Figure 1.

[0255] The recombinant antigen receptor encoded by the nucleic acid may be any of the antigen receptors described herein, including but not limited to a T cell receptor (TCR), an engineered TCR, or a chimeric antigen receptor (CAR).

[0256] In another aspect, the invention relates to a cell comprising any of the nucleic acids or recombinant nucleic acids described herein. As used herein, "comprised in a cell" means that the nucleic acid is present within the cell, either integrated into the genome or maintained episomally. Suitable cells include any of the lymphocytes described herein, such as T cells, NK cells, or NKT cells, as well as cells used for cloning or propagation purposes, including bacterial cells such as Escherichia coli.

[0257] Thus, in another aspect, the invention provides a cell comprising a nucleic acid molecule of the invention.

[0258] BRIEF DESCRIPTION OF THE DRAWINGS

[0259] FIG.l: Schematic drawing of lentiviruses

[0260] FIG.2: Survival of cells in cytokine free media. 5xl0A5 cells per group were cultured in-vitro in 2 ml of cytokine free media supplemented with FBS in a 24 well plate . ENABLR IL15 are ENABLR cells expressing soluble IL15, ENABLR IL15 IL21 are ENABLR cells expressing the soluble I L15 IL21 fusion protein. (A) Flow Cytometer reading for 3 donors at day 7 using DAPI staining showing ENABLR cells expressing soluble cytokines have better survival with the highest rate for ENABLR I L15 IL21. (B) Manual counting by Trypan blue demonstrates ENABLR cells expressing soluble cytokines to have higher cell expansion with ENABLR IL15 the strongest.

[0261] FIG.3: Rechallenge killing test in-vitro using non-edited T cells (WT), ENABLR I L15 and ENABLR I L15 IL21 cells. Cell lines (250K) were incubated with each group at 1:1 E:T for H82 and 1:3 for OPM2, ratio in cytokine free media with the addition of the proper T cell engager. Each Rechallenge is 48 hours incubations, after 48h half of the wells were used for luciferase measurement and flow cytometry and for the next rechallenge reading, 250k cells of tumor in 500ul with adequate TCE, keeping the same TCE concentration were added for each group. 0PM2- multiple myeloma, H82- small cell lung carcinoma, WT - non-edited T cell, ENABLR I L15 IL21 - TRAC k / o T cells expressing ENABLR cells expressing the soluble I L15 IL21 fusion protein.

[0262] FIG:4: Flow cytometry reading of non-edited T cells and ENABLR IL15 IL21 cells after 4threchallenge. Upon light microscope inspection on 4threchallenge, the majority of T cells changed their phenotype and became small rounded cells, unlike WT with large elongated shape. Upon flow cytometry reading, the inventors noticed that ENABLR 15 21 cells were smaller (By FSC and SCC). This observation is in line with the hypothesis that addition of IL15 and IL21 make T cells more naive / stem-like.

[0263] FIG.5: Flow cytometry reading for naive / stemness markers of non-edited T cells and ENABLR I L15GSI L21 cells after 4threchallenge. CCR7 - C-C chemokine receptor, CD62L- L-selectin: Both are markers only expressed on naive / stem-like reside in lymph nodes and spleen and not by effector cells. Upon light microscope inspection on 4threchallenge the majority of T cells changed their phenotype and became small, rounded cells, unlike WT with large, elongated shape. This observation is in line with our hypothesis that addition of IL15 and IL21 make T cells more naive / stem-like. In order to confirm this, cells were stained for CD62L and CCR7, which are only expressed on naive / stem-like reside in lymph nodes and spleen and not by effector cells. Indeed, a fraction with elevated expression of CD62L+ / CCR7+ was detected. The same phenomenon with cells from different donors was found with H82 and OPM2 cell line.

[0264] FIG.6: CBL-B k / o efficiency. Cells were stained with anti-MYC as marker for the ENABLR receptor on the cell surface, followed by anti-CBL-B intracellular staining 3 days after electroporation.

[0265] FIG.7: Rechallenge killing test in-vitro using non-edited T cells (WT), ENABLR IL15, ENABLR IL15+IL21, ENABLR IL15+IL21 CBL-B KO cells. Cell lines (250K) were incubated with each group at 1:1 E:T for H82 and 1:3 for OPM2, ratio in cytokine free media with the addition of the proper T cell engager. Each Rechallenge is 48 hours incubations, after 48h half of the wells were used for luciferase measurement and flow cytometry and for the next rechallenge reading, 250k cells of tumor in 500ul with adequate TCE, keeping the same TCE concentration were added for each group.

[0266] FIG.8: Rechallenge killing test in-vitro using non-edited T cells (WT), ENABLR IL15 CBL-B KO and IL15+IL21 CBL-B KO cells. Cells of the specific cell line (750K) were incubated with each group at 1:3 E:T for OPM2, ratio in cytokine free media with the addition of the proper T cell engager using n=3 donors. Rechallenge time is as indicated, on the same day of rechallenge wells were used for luciferase measurement (2 technical readings for each condition and donor) and for the next rechallenge reading, fresh new 0PM2 cells were added as indicated (750K x indicated cell number) in 500ul with adequate TCE, keeping the same TCE concentration were added for each group.

[0267] FIG.9: Granzyme B staining in donor 1 and 5 after 204 hours of rechallenge. Co-culture were stained with anti-CD8 then cells were permeabilized and stained with anti-GZMB according to manufacturer.

[0268] FIG.10: Scheme of pleiotropic effect of IL 21.

[0269] EXAMPLES

[0270] Background

[0271] Researchers around the world are working tirelessly to advance immunotherapy for solid tumors. In this study, the inventors opted for a more rational strategy, focusing on approaches with demonstrated effectiveness in this challenging field. At present, immune checkpoint inhibitors-particularly those targeting the PD-1 pathway-represent the most consistently effective immunotherapy for solid tumors. Even though only a minority of patients (approximately 10-15%) achieve significant responses, these individuals are often considered exceptional responders, experiencing remarkably prolonged survival2.

[0272] Numerous biomarkers have been proposed as predictors of anti-PD-1 therapy response, but none have achieved universal success, with the exception of tumor mutation burden (TMB). HigherTMB is associated with an increased likelihood of generating and expanding anti-tumor T cell clones, a key component of effective immune activation (Signal 1 in T cell activation)3.

[0273] Building on these insights, the inventors sought to identify additional unique features of the tumor microenvironment that are determinative in responders. The goal was to study these factors in depth and explore their potential integration into future T cell-based immunotherapies.

[0274] In recent years, increasing attention has been given to mature tertiary lymphoid structures (TLS) within the tumor microenvironment3'4. These structures are not only associated with improved responses to immunotherapy but are also linked to exceptional outcomes across various tumor types2'5-7. Notably, in melanoma patients who initially respond to anti-PD-1 or CTLA-4 therapies, the disappearance or regression of mature TLS into their immature forms has been observed during disease progression8.

[0275] TLS are complex assemblies composed of diverse immune cell populations, including B cells, plasma cells, stem-like progenitor CD8+ T cells (Tpex), dendritic cells, T follicular helper (TFH) CD4+ cells, macrophages, and other myeloid cells6'9-13. Within these structures, CD8+ Tpex cells serve as a reservoir for the continuous generation of effective effector T cells that can target and destroy tumor cells. These Tpex cells exist in a naive-like, slow-cycling state and have been identified as critical drivers of robust and durable anti-tumor responses following anti-PD-1 therapy in solid tumors14-17.

[0276] The inventors concentrated their efforts on T follicular helper (TFH) CD4+ cells, as they are uniquely positioned to drive the maturation of tertiary lymphoid structures (TLS)18. TFH cells are not only pivotal for TLS development, but they also undergo clonal expansion in parallel with CD8+ T cell responders. Both cell types migrate from the draining lymph nodes (DLN) into the tumor microenvironment, where they can become exhausted together during the immune response19.

[0277] A distinctive feature of TFH CD4+ cells is their almost exclusive ability to produce interleukin- 21 (IL-21), a cytokine essential for sustaining long-term immune responses, particularly in chronic infections20-25. However, it is important to note that IL-21, when acting alone, can induce apoptosis in T cells, thereby limiting their expansion by promoting excessive cell death26'27. For this reason, the inventors chose to express IL-15 in order to counteract T cell apoptosis and promote their survival. Notably, IL-15 is naturally produced by dendritic cells within the TLS, as well as in the vicinity of high endothelial venules, further supporting its role in sustaining effective immune responses 6,10,11,28-30.

[0278] Example 1: Expression of IL15-IL21 fusion protein in recombinant T cells

[0279] The effect of co-expressing IL-15 and IL-21 on T cells was tested in recombinant T cells expressing an engineered T cell receptor (TCR). The engineered TCR lacks the variable domains and the recombinant cells expressing the engineered TCR are referred to as ENABLR cells (WO 2023 / 012584). Due to the lack of the variable domain, ENABLR cells are activated using agonistic anti-CD3 antibodies.

[0280] To address technical challenges and ensure robust expression of ENABLR, the inventors engineered IL-15 and IL-21 to be produced as a single fusion protein. This was achieved by linking the N-terminus of IL-21 to the C-terminus of IL-15 using a flexible 3x(GGGGS) linker (or an alternative flexible or rigid linker if needed). Alternatively, IL-15 and IL-21 can be expressed individually or as membrane-bound forms. For gene delivery, the inventors utilized a third- generation lentiviral vector with a bidirectional promoter: the SFFV promoter drives ENABLR expression in the forward direction, while the mPGK promoter controls expression of the fused I L-15 / IL-21 protein or IL-15 alone, as illustrated in Figure 1.

[0281] To assess the impact of this engineering, the inventors cultured ENABLR I L15 and ENABLR I L15 IL21 cells in cytokine-free media for seven days. This approach allowed to evaluate cell survival without the confounding effects of externally added cytokines, which can obscure the true influence of the modifications. The results showed that ENABLR I L15 IL21 cells exhibited higher viability compared to ENABLR I L15 cells, although their overall cell numbers were lower (see Figure. 2).

[0282] To assess the functionality of the cells, each non-edited T (WT) cells and equipped ENABLR cells were tested in rechallenge killing assays, wherein every 48 hours cells were tested. Surprisingly upon rechallenge test, T cells expressing ENABLR IL15 IL21 fusion were the least efficient in killing among the non-edited cells, ENABLR I L15 (Figure 3).

[0283] To rule out the possibility that reduced killing rates were due to apoptosis, the inventors stained the cells from the 4threchallenge with trypan blue and examined them under a light microscope. Most T cells exhibited a distinct change in morphology, appearing as small, rounded cells. This contrasted with the wild-type (WT) cells, which retained a larger, elongated shape.

[0284] Flow cytometry analysis revealed that ENABLR 15 21 cells were smaller in size, as indicated by decreased forward and side scatter (FSC and SSC) values (Figure 4). This finding supports the hypothesis that the combined expression of IL-15 and IL-21 promotes a more naive or stemlike phenotype in T cells.

[0285] To further verify the presence of functional naive or stem-like characteristics, the inventors stained the cells for CD62L and CCR7 expression after the fourth rechallenge (Figure 5).

[0286] In conclusion, the findings demonstrate that ENABLR cells expressing both IL15 and IL21 exhibit enhanced viability under cytokine-free conditions, although they proliferate at a slower rate. This observation aligns with recent research indicating that Tpex cells cycle more slowly than effectorT cells31. The addition of the I L15 IL21 combination produces a particularly intriguing phenotype: T cells that display more naive-like surface markers and a reduced capacity to differentiate into effector cells. This is consistent with emerging evidence from anti-PD-1 and Tpex studies, which suggest that Tpex cells only begin to differentiate as they migrate from the lymph node to the tumor site32. Example 2: Inactivation of CBL-B increases killing activity of T cells expressing I L-15 / IL-21

[0287] For Tpex cells to successfully differentiate, they require continuous CD28 stimulation in addition to TCR / CD3 signaling, unlike naive CD8+ T cells, which can become effector cells with TCR / CD3 engagement alone36.

[0288] To overcome the need for CD28 engagement during TCR / CD3 stimulation, the inventors chose to knock out the CBL-B gene37'38.

[0289] ENABLR cells expressing the IL-15 / IL-21 fusion protein were electroporated with SpCas9 ribonucleoprotein complexes targeting the CBL-B gene, along with a constant alpha RNP. Intracellular staining confirmed a reduction in CBL-B protein levels following electroporation (Figure 6).

[0290] Now upon rechallenge tests, cells start to differentiate, produce effectors and kill tumor (Figure 7).

[0291] To determine whether the enhanced tumor-killing effect was solely attributable to CBL-B knockout, regardless of cytokine expression, we conducted rechallenge experiments. We compared non-edited T cells, ENABLR CBL-B knockout cells expressing IL-15, and ENABLR CBL- B knockout cells expressing the IL-15 IL-21 fusion protein for their ability to sustain tumor cell killing over time (Figure 8).

[0292] Results from the cytokine-free in vitro rechallenge assays clearly demonstrate that the addition of IL-21 leads to more effective tumor cell killing, indicating that the observed cytotoxicity cannot be attributed solely to IL-15 expression or CBL-B knockout. After 204 hours of repeated rechallenges, there was no significant difference in the proportion of CCR7+ / - CD62L+ cells between ENABLR IL-15 and ENABLR IL-15 IL-21 groups in donors 1 and 5, with less than 5% of these cells remaining (insufficient events were collected from donor 3). To further investigate the observed differences in cytotoxicity, the inventors stained cells from donors 1 and 5 for Granzyme B, a marker associated with potent, long-lived effector function. Previous studies have suggested that IL-21 plays a key role in generating durable, highly effective effector cells, with Granzyme B serving as a reliable indicator of this phenotype23'25(Figure 9). ENABLR cells that express I L15 IL21 expressed higher levels of Granzyme B than I L15 in corresponding with new findings that IL21 is important keeping the production of Granzyme B+.

[0293] In conclusion, ENABLR cells expressing the IL-15 IL-21 fusion exhibited higher levels of Granzyme B compared to those expressing IL-15 alone, supporting recent findings that IL-21 is crucial for sustaining Granzyme B production. Interestingly, murine IL-21 has been shown to exert pleiotropic effects on CD8+ T cells, promoting either a naive-like phenotype or a highly effective effector state-two seemingly contradictory outcomes that appear to be concentration-dependent39-42. The present system, which combines IL-15 IL-21 fusion expression with CBL-B inhibition, seems to recapitulate these dual cellular states. In the absence of strong TCR signaling (signal 1), the IL-15 IL-21 fusion maintains T cells in a naive- like state. However, when robust TCR signaling is present and CBL-B is inhibited, these cells transition from a naive-like state to a potent effector phenotype (Figure 10).

[0294] Previous studies have reported the expression of IL-21 and IL-15, but none have described the induction of a naive-like phenotype or a block in differentiation43-45. These findings are the first to reveal this unique phenotype, which the inventors attribute to the properties of the soluble IL-15 IL-21 fusion protein-promoting sternness under baseline conditions and driving robust effector differentiation upon tumor recognition.

[0295] Methods

[0296] Using a bidirectional lentivirus, the ENABLR receptor is expressed downstream of the SFFV promoter, downstream of the mPGK proper cytokines were expressed: IL15 linked to IL21 with (GSSS)x3 flexible linker (IL15-IL21) or I L15 cDNA alone.

[0297] PBMCs from healthy donors were produced by extracting PBMCs buffy coats. PBMCs were activated for 36 hours with the anti-CD3 antibody OKT3, then incubated with proper lentivirus supplemented with BX795(8pM). After 24 hours cell culture the cells were electroporated with SpCas9 RNP to knockout the constant alpha chain (TRAC locus) or TRAC and CBL-B using specific gRNAs in the same KO step. 4-5 days later, cells were positively selected using biotinylated anti-MYC (9E10) and Ultrapure anti-Biotin magnetic beads (Miltenyi 130-105- 637). Positively selected cells at this stage and used for subsequent experiments.

[0298] Cell surface staining was done for 30 min with anti-MYC (Biolegend), anti-CD3, anti-CD62L and anti CCR7 (BD). Intracellular staining using Cyto-Fast™ Fix / Perm Buffer Set (Biolegend, USA) for CBL-B and Granzyme B (SCBT,USA).#

[0299] For rechallenge tests, 250kT cells were cultured in serum-free RPMI-10% FCS without addition of cytokines. OPM2(Multiple Myeloma) and H82(Small cell lung carcinoma) cell lines expressing firefly luciferase selected after puromycin were used with indicated E:T ratio of 1:1 used for both cell lines. Teclistamab-CD3, BCMA (lOnM) and Tarlatamab-CD3,DLL3 (InM) biosimilars were added (Proteogenix, France) in 24 well plates. Each Rechallenge is 48 hours incubations, after 48h half of the wells were used for luciferase measurement and flow cytometry and for the next rechallenge reading, 250k cells of tumor in 500ul with adequate TCE, keeping the same TCE concentration were added for each group. Following microscope inspection after the 3rdchallenge, cytotoxicity was measured, and cells analyzed by flow cytometry.

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Claims

1. CLAIMS1. A lymphocyte comprising a recombinant nucleic acid encoding a fusion protein comprising an interleukin-15 (IL-15) fused to an interleukin-21 (IL-21).

2. The lymphocyte of claim 1, wherein the IL-15 and the IL-21 are fused with a peptide linker.

3. The lymphocyte of claim 2, wherein the peptide linker is a non-cleavable peptide linker.

4. The lymphocyte of claim 2 or 3, wherein the peptide linker comprises a glycine-serine repeat sequence.

5. The lymphocyte of any one of claims 1 to 4, wherein the fusion protein is expressed in soluble or in membrane-bound form.

6. The lymphocyte of any one of claims 1 to 5, wherein the lymphocyte is selected from the group consisting of a T cell (including CD4+ T cells and CD8+ T cells), a natural killer (NK) cell, and a natural killer T (NKT) cell.

7. The lymphocyte of any one of claims 1 to 6, wherein the lymphocyte further comprises a recombinant nucleic acid molecule encoding an antigen receptor.

8. The lymphocyte of claim 7, wherein the antigen receptor is a T cell receptor (TCR) or an engineered TCR.

9. The lymphocyte of claim 8, wherein the engineered TCR is a TCR that is lacking one or more variable domain.

10. The lymphocyte of claim 7, wherein the antigen receptor is a chimeric antigen receptor (CAR).

11. The lymphocyte of any one of claims 1 to 10, wherein activation of the lymphocyte is independent of CD28-mediated co-stimulation.

12. The lymphocyte of claim 11, wherein said independence from CD28-mediated costimulation is conferred by:(i) deletion, inactivation, knockdown, or functional inhibition of the endogenous CBL-B gene; or(ii) deletion, inactivation, knockdown, or functional inhibition of the endogenous STS1 gene; or(iii) introduction of a gain-of-function mutation into the CARMIL2 gene, preferably a Q575E mutation or a functional equivalent thereof.

13. A pharmaceutical composition comprising the lymphocyte of any one of claims 1 to 12 and at least one pharmaceutically acceptable excipient.

14. The lymphocyte of any one of claims 1 to 12 or the pharmaceutical composition of claim 13 for use in cell therapy, preferably in adoptive cell therapy.

15. The lymphocyte or pharmaceutical composition for use of claim 14, wherein the cell therapy is allogenic cell therapy or autologous cell therapy.

16. The lymphocyte or pharmaceutical composition for use of claim 14 or 15, wherein the cell therapy is for the treatment of a disease selected from the group consisting of cancer, an infectious disease, a cardiovascular disease and an autoimmune disease.

17. The lymphocyte or pharmaceutical composition for use of any one of claims 14 to 16, wherein the lymphocyte or pharmaceutical composition is administered simultaneously or sequentially with a CD3-engaging agent, preferably a CD3-engaging bispecific molecule.

18. A method of treating a subject in need thereof, comprising administering to the subject a lymphocyte according to any one of claims 1 to 12, or a pharmaceutical composition according to claim 13, in a cell therapy, preferably adoptive cell therapy.

19. The method of claim 18, wherein the cell therapy is allogeneic cell therapy.

20. The method of claim 18 or 19, wherein the cell therapy is for the treatment of a disease selected from the group consisting of cancer, an infectious disease, and an autoimmune disease.

21. The method of any one of claims 18 to 20, further comprising administering to the subject a CD3-engaging agent, wherein the lymphocyte or pharmaceutical composition and the CD3-engaging agent are administered simultaneously or sequentially, and preferably wherein the CD3-engaging agent is a CD3-engaging bispecific molecule.

22. A method of manufacturing a lymphocyte for cell therapy, the method comprising the steps of:(i) providing a lymphocyte;(ii) introducing into the lymphocyte a recombinant nucleic acid molecule encoding an interleukin-15 (IL-15) fused to an interleukin-21 (IL-21).

23. The method of claim 22, wherein the L-15 and the IL-21 are fused with a peptide linker.

24. The method of claim 23, wherein the peptide linker is a non-cleavable peptide linker.

25. The method of claims 23 or 24, wherein the peptide linker comprises a glycine-serine repeat sequence.

26. The method of any one of claims 22 to 25, further comprising introducing into the lymphocyte a recombinant nucleic acid molecule encoding a recombinant antigen receptor.

27. The method of claim 26, wherein the recombinant antigen receptor is selected from the group consisting of a T cell receptor (TCR), an engineered TCR, and a chimeric antigen receptor (CAR).

28. The method of any one of claims 22 to 27, wherein the recombinant nucleic acid molecule(s) are introduced into the lymphocyte by viral transduction, electroporation, or lipid-mediated transfection.

29. The method according to any one of claims 22 to 28, further comprising modifying the lymphocyte to render it independent of CD28-mediated co-stimulation.

30. The method of claim 29, wherein said independence from CD28-mediated costimulation is conferred by:(i) deletion, inactivation, knockdown, or functional inhibition of the endogenous CBL-B gene; or(ii) deletion, inactivation, knockdown, or functional inhibition of the endogenous STS1 gene; or(iii) introduction of a gain-of-function mutation into the CARMIL2 gene, preferably a Q575E mutation or a functional equivalent thereof.

31. The method of any one of claims 22 to 30, wherein the lymphocyte is selected from the group consisting of a T cell (including CD4+ T cells and CD8+ T cells), a natural killer (NK) cell, and a natural killer T (NKT) cell.

32. The method of any one of claims 22 to 31, wherein the lymphocyte is obtained from a healthy donor or a subject in need.

33. The method of any one of claims 22 to 32, further comprising a step of formulating the lymphocyte in a pharmaceutical composition together with at least one pharmaceutically acceptable excipient.

34. A method for increasing the viability of a lymphocyte and / or inducing a naive-like phenotype in a lymphocyte, the method comprising co-expressing in the lymphocyte a first recombinant nucleic acid molecule encoding interleukin-15 (IL-15) and a second recombinant nucleic acid molecule encoding interleukin-21 (IL-21).

35. The method of claim 34, wherein the method is an ex vivo method.

36. The method of claim 34 or 35, wherein the naive-like phenotype is characterized by expression of CD62L and / or CCR7 on the lymphocyte.

37. The method of any one of claims 34 to 36, wherein the IL-15 and IL-21 are encoded by a single recombinant nucleic acid molecule.

38. The method of any one of claims 34 to 37, wherein the IL-15 and IL-21 are expressed as a fusion protein.

39. The method of claim 38, wherein the fusion protein comprises IL-15 and IL-21 fused by a peptide linker, preferably wherein the peptide linker is a non-cleavable peptide linker, more preferably wherein the peptide linker comprises a glycine-serine repeat sequence.

40. The method of any one of claims 34 to 39, wherein the lymphocyte is selected from the group consisting of a T cell (including CD4+ T cells and CD8+ T cells), a natural killer (NK) cell, and a natural killer T (NKT) cell.

41. A recombinant nucleic acid molecule comprising:(i) a nucleotide sequence encoding interleukin-15 (IL-15);(ii) a nucleotide sequence encoding interleukin-21 (IL-21); and(iii) a nucleotide sequence encoding a recombinant antigen receptor.

42. The recombinant nucleic acid molecule of claim 41, wherein the recombinant antigen receptor is selected from the group consisting of a T cell receptor (TCR), an engineered TCR, and a chimeric antigen receptor (CAR).

43. The recombinant nucleic acid molecule of claim 41 or 42, wherein the nucleotide sequences encoding IL-15 and IL-21 are arranged to be expressed as a fusion protein.

44. The recombinant nucleic acid molecule of claim 43, wherein the fusion protein comprises IL-15 and IL-21 fused by a peptide linker, preferably a glycine-serine repeat sequence.

45. The recombinant nucleic acid molecule of any one of claims 41 to 44, wherein the nucleotide sequence encoding the I L-15 / 1 L-21 fusion protein is operably linked to a first promoter, and the nucleotide sequence encoding the recombinant antigen receptor is operably linked to a second promoter.

46. The recombinant nucleic acid molecule of any one of claims 41 to 45, wherein the recombinant nucleic acid molecule is comprised in a viral vector, preferably wherein the viral vector is selected from the group consisting of a lentiviral vector, a retroviral vector, an adenoviral vector, and an adeno-associated viral (AAV) vector.

47. An I L-15 / 1 L-21 fusion protein comprising interleukin-15 (IL-15) and interleukin-21 (IL-21) fused by a peptide linker.

48. The fusion protein of claim 47, wherein the peptide linker is a non-cleavable peptide linker.

49. The fusion protein of claim 47 or 48, wherein the peptide linker comprises a glycineserine repeat sequence.

50. The fusion protein of any one of claims 47 to 49, wherein the peptide linker comprises the amino acid sequence (Gly4Ser)n, wherein n is 1 to 5.

51. The fusion protein of any one of claims 47 to 50, wherein the IL-15 is positioned N- terminal to the IL-21, or vice versa.

52. The fusion protein of any one of claims 47 to 51, wherein the fusion protein comprises an amino acid sequence as set forth in SEQ ID NO:4.

53. A nucleic acid molecule encoding the fusion protein of any one of claims 47 to 52.

54. A cell comprising a nucleic acid molecule of any one of claims 39 to 44 or 51.

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