Cell therapy approaches

Abstract

The present invention is in the field of cell therapy and provides compositions and methods for treating cancer and / or viral infections in patients. The present invention provides lymphocytes comprising synthetic polynucleotides encoding at least one iron regulatory protein and, optionally, a chimeric antigen receptor. The present invention also provides methods for generating these lymphocytes and administering them to patients.

Description

[0001] summary

[0002] The present invention is in the field of cell therapy and provides compositions and methods for treating cancer and / or viral infections in patients. The present invention provides lymphocytes comprising synthetic polynucleotides encoding at least one iron regulatory protein and, optionally, a chimeric antigen receptor. The present invention also provides methods for generating these lymphocytes and administering them to patients.

[0003] introduce

[0004] Over the past few decades, the potential of the immune system in the development and treatment of cancer has been a major focus of research. Although targeted therapies and immunotherapies using immune checkpoint blockade have greatly improved the survival rates of many cancer patients, a large proportion of patients still experience disease progression after receiving these treatments. Adoptive cell therapy (ACT) may provide another treatment option for these patients, and involves the intravenous transfer of tumor-resident or peripheral blood modified immune cells into cancer patients to modulate anti-tumor functions. Currently, ACT can be divided into three different types based on their respective mechanisms of action, namely ACT using tumor-infiltrating lymphocytes (TILs), ACT using T cell receptor (TCR) gene therapy, and ACT using chimeric antigen receptor (CAR) modified T cells. The use of other immune cell types such as natural killer cells as the basis for cell therapy is also an area of ​​current research.

[0005] Rosenberg and colleagues at the Department of Surgery, National Institutes of Health (SB, NIH, Bethesda, Maryland, US) conducted the first study of TILs, in which TILs were cultured from different mouse tumors and showed in vivo anti-tumor activity. Current TIL therapy consists of ex vivo expansion of TILs from resected tumor material and adoptive transfer into the patient after a lymphodepletion preparatory protocol and subsequent support with interleukin 2 (IL-2). Using this regimen, approximately 50% of significant objective tumor responses were achieved in patients with metastatic melanoma in several Phase I / II clinical trials. Following the success of TILs in melanoma patients, the generation of TILs from other solid tumor types was also studied. So far, TILs have been cultured from non-melanoma tumor types (such as cervical cancer, renal cell carcinoma, breast cancer, and non-small cell lung cancer) with different tumor response rates.

[0006] In addition to naturally occurring TILs in tumors and treatment options based on this, peripheral blood T cells can be isolated and genetically modified in vitro to express TCRs targeting specific tumor antigens for use in ACT. Using this method, a large number of tumor-specific T cells can be produced, with effective anti-tumor activity and objective clinical responses observed in up to 30% of treated patients. For recognition achieved by modifying TCR, antigen presentation through the major histocompatibility complex (MHC) is required. However, it is well known that many cancer types can escape T cell-mediated immune responses by downregulating or losing their MHC expression. In order to avoid the need for MHC to be present on tumor cells for tumor-specific T cell recognition, artificial receptors such as CAR molecules have been developed. ACT using CAR-modified T cells has the same effector function as TCR-modified T cells, but is independent of MHC expression. In addition to using protein antigens, other antigens, such as carbohydrate or glycolipid antigens, have also been explored. Impressive clinical responses have been observed in hematological malignancies using CD19-specific CAR T cells, which has also led to the exploration of using CAR therapy in solid tumors.

[0007] While hematopoietic stem cell transplantation (HSCT) offers a chance for cure for many patients with high-risk cancers or primary immunodeficiency syndromes, recipients remain susceptible to infectious complications due to prolonged and severe immunosuppression. These risks vary depending on the preparatory regimen, type of transplant, and duration of myelosuppression. Advances in conditioning regimens and improvements in post-transplant management have made an increasing number of patients eligible for HSCT from mismatched, unrelated, or haploidentical donors. While outcomes for patients with severe or otherwise untreatable illnesses have greatly improved, the immunosuppression required for transplantation and, when indicated, treatment of graft-versus-host disease (GVHD) presents a fertile window for infection. Specifically, viral infections cause significant morbidity and mortality, and the risk increases when T-cell immune reconstitution is delayed. The relationship between the effects of immunosuppression, immune reconstitution, and GVHD on infection is complex and intertwined. Medical treatment and prevention options for viral infections remain limited and often ineffective, with associated morbidity, particularly from acute kidney injury and myelosuppression. Treatments can also provoke resistance and do not provide prolonged protection, placing patients at risk for viral reactivation. In view of the correlation between T cell immune recovery delay and viral disease, adoptive cell therapy is a reasonable alternative to drug therapy. Untreated lymphocyte infusions from seropositive donors have been infused into patients with life-threatening diseases (such as EBV-associated lymphoma), demonstrating clinical efficacy and the risk mainly associated with GVHD. This strategy has been continuously developed over the past two decades, and donor lymphocyte products have been successfully used as the treatment of viral diseases (including reactivation, new exposure and lymphoma) and as prevention to rebuild viral immunity in the host. After these preliminary studies, the selection and / or amplification of virus-specific T cells (VST) have been finely improved to maximize viral cytotoxicity and minimize alloreactivity, to reduce and eliminate the risk of GVHD to a great extent. In current research, VST provides targeted therapy and has so far demonstrated very good safety profile.

[0008] Compared to T cells, the potential of natural killer cells (NK cells) in ACT has been less explored. However, several properties of NK cells make them ideal candidates for adoptive cell therapy. In addition to being highly cytotoxic effectors, NK cells are not restricted by antigen specificity, and they rapidly produce proinflammatory cytokines that enhance adaptive immune responses.

[0009] NK cells from cancer patients are often dysfunctional, manifested by reduced proliferation rate, reduced response to cytokine stimulation, and reduced effector function. Therefore, early immunotherapy strategies aimed to enhance or restore the function of endogenous NK cells. These strategies involved ex vivo IL-2-induced activation of autologous NK cells, which were then reinfused into the patient along with combined IL-2 therapy during treatment. Unfortunately, IL-2-activated NK cells did not affect tumor growth, and the treatment regimen had severe side effects. The use of allogeneic NK cells to treat cancer patients is more promising because allogeneic NK cells are fully functional compared to patient NK cells. In addition, allogeneic NK cells have graft-versus-leukemia / tumor (GvL / GvT) effects without causing graft-versus-host disease (GvHD), and therefore cause less immunopathology.

[0010] Clinically relevant responses have been achieved, particularly in the treatment of hematological malignancies such as acute myeloid lymphoma (AML) and non-Hodgkin's lymphoma (NHL). However, the activity of NK cells alone is often insufficient to completely control tumor growth; and due to the limitations of the tumor microenvironment, the treatment of solid tumors is particularly challenging. Therefore, strategies to enhance NK cell function have been widely studied. One method to enhance the anti-tumor activity of NK cells is to utilize cytokines. Various cytokines (IL-2, IL-12, IL-15, IL-18, IL-21, and type I interferon) have been used to amplify and activate NK cells in vitro before adoptive transfer.

[0011] A promising cytokine combination that maximizes NK cell function is the combined use of IL-12, IL-18, and IL-15. This combined stimulation induces a population of NK cells with "memory-like" characteristics (such as prolonged survival and enhanced effector functions). Preclinical studies have shown that cytokine-enhanced (CE) NK cells have great potential as anti-leukemia cell therapy. In in vivo tumor models of lymphoma or melanoma, CE NK cells have enhanced effector functions (IFN-γ production and cytotoxicity). In addition, after adoptive transfer into immunodeficient NOD-SCID-γc- / - mice (NSG), IL-2-enhanced CE NK cells lasted longer than control NK cells. Finally, in NSG mice with AML xenografts, CE NK cells significantly reduced AML burden and improved overall survival.

[0012] The molecular mechanisms driving the increased effector function of CE NK cells are currently unknown. For T cells, it is well established that the function of certain subsets (e.g., naive CD8+ T cells versus memory CD8+ T cells) is associated with distinct metabolic regulatory mechanisms. Therefore, altered metabolic patterns in CE NK cells could support enhanced function. Elucidating the molecular mechanisms underlying CE NK cell differentiation and their superior effector functionality is an important prerequisite for improving clinical efficacy.

[0013] In general, highly proliferative cells strictly rely on iron to support basic processes such as energy metabolism / respiration, DNA synthesis and repair, and cell cycle control. A large amount of iron required for proliferating cells including lymphocytes is provided by transferrin, which is absorbed by the cell surface receptor CD71. CD71 is commonly used as a lymphocyte activation marker and is expressed on activated NK cells. Only a few studies have investigated the importance of iron metabolism to lymphocyte function so far. It has recently been shown that mutations in the CD71 receptor (TFRCY20H / Y20H) damage T and B cell function due to impaired proliferation.

[0014] It has been proposed that reduced iron levels impair NK cell cytotoxicity and dysfunctional NK cells, and in this case, may promote cancer development in rats. Furthermore, low serum ferritin levels have been associated with reduced NK cell activity in humans. However, the specific effects of iron on NK cell-mediated immunity remain unclear.

[0015] Cellular iron homeostasis is a strictly regulated process that involves the coordination of iron uptake, utilization and storage. It is mainly regulated by the iron regulatory protein / iron response element (IRP / IRE) regulatory system at the post-transcriptional level. IRP1 and IRP2 are RNA binding proteins that recognize IREs in different mRNAs, thereby controlling their stability and protein translation. The activity of IRP1 and IRP2 is regulated in response to cellular iron levels. Classical IREs are present in the 5'UTR or 3'UTR of mRNAs encoding for iron acquisition, iron storage, iron utilization, ATP production and iron export.

[0016] Under conditions of iron deficiency, IRP activity is high and IRPs bind to IREs in either the 5'UTR or 3'UTR of the corresponding mRNA. Depending on the localization of the IRE, IRPs can differentially affect protein expression. Translation of mRNAs harboring an IRE in the 5'UTR (e.g., FTH1 mRNA, ferritin light chain 1) is inhibited by IRP binding. In contrast, IRP binding to 3'UTR IREs (e.g., TFRC mRNA, CD71) stabilizes the mRNA and leads to enhanced translation. Thus, the IRP / IRE regulatory network coordinates cellular iron homeostasis by selectively regulating the translation of certain mRNAs relative to cellular iron status.

[0017] Despite recent success in adoptive cell therapy, there is still a need to improve these treatments so that they can be used in more patients. A general requirement for successful adoptive cell therapy is to ensure that immune cells are robustly expanded in the patient's body after the cells are infused. On the one hand, this will result in a reduction in the number of infusions, and on the other hand, it will result in higher efficacy of the treatment. Therefore, there is a need in the art for improved means and methods related to cell therapy. More specifically, there is a need for immune cells that are robustly expanded in vivo after the cells are administered to a subject.

[0018] This technical problem is solved by the embodiments provided in the claims. That is, the present invention relates to the following items:

[0019] 1. A lymphocyte comprising a synthetic polynucleotide encoding at least one iron regulatory protein.

[0020] 2. The lymphocyte according to item 1, wherein the lymphocyte is a T cell or a natural killer cell.

[0021] 3. The lymphocyte according to any one of items 1 or 2, wherein the at least one iron regulatory protein is constitutively expressed.

[0022] 4. The lymphocyte according to any one of items 1 to 3, wherein the at least one iron regulatory protein is IRP1 (SEQ ID NO: 1) and / or IRP2 (SEQ ID NOs: 2-6).

[0023] 5. The lymphocyte according to any one of items 1 to 4, wherein the lymphocyte further comprises a chimeric antigen receptor.

[0024] 6. The lymphocyte according to item 5, wherein the chimeric antigen receptor comprises an antigen binding domain, a transmembrane domain, a co-stimulatory signaling region, and a signaling domain.

[0025] 7. The lymphocyte according to item 6, wherein the antigen-binding domain is an antibody or an antigen-binding fragment thereof, specifically, wherein the antigen-binding fragment is Fab or scFv.

[0026] 8. The lymphocyte according to any one of items 6 or 7, wherein the antigen binding domain specifically binds to a tumor antigen.

[0027] 9. The lymphocyte according to item 8, wherein the tumor antigen is present on the cell surface of the target cell population or tissue.

[0028] 10. A pharmaceutical composition comprising the lymphocyte according to any one of items 1 to 9 and a pharmaceutically acceptable carrier.

[0029] 11. Lymphocytes according to any one of items 1 to 9 or the pharmaceutical composition according to item 10 for use in therapy.

[0030] 12. The lymphocyte according to any one of items 1 to 9 or the pharmaceutical composition according to item 10, for use in treating cancer.

[0031] 13. The lymphocyte or pharmaceutical composition for the use according to item 12, wherein the cancer is a hematological cancer or a solid tumor, specifically, wherein the hematological cancer is acute lymphoblastic leukemia, diffuse large B-cell lymphoma, Hodgkin's lymphoma, acute myeloid leukemia or multiple myeloma, and wherein the solid tumor is colon cancer, breast cancer, pancreatic cancer, ovarian cancer, hepatocellular carcinoma, lung cancer, neuroblastoma, glioblastoma or sarcoma.

[0032] 14. The lymphocyte according to any one of items 1 to 9 or the pharmaceutical composition according to item 10, for use in preventing and / or treating viral infection.

[0033] 15. The lymphocyte or pharmaceutical composition for use according to item 14, wherein the viral infection is caused by human immunodeficiency virus (HIV), adenovirus, polyomavirus, influenza virus or human herpes virus, specifically, wherein the human herpes virus is cytomegalovirus (CMV), Epstein-Barr virus (EBV), herpes simplex virus (HSV), varicella-zoster virus (VZV) or human herpes virus 8 (HHV8).

[0034] 16. A method for treating a subject suffering from cancer or for preventing and / or treating a viral infection in a subject, the method comprising administering to the subject a therapeutically effective amount of the lymphocytes according to any one of items 1 to 7 or the pharmaceutical composition according to item 10.

[0035] 17. The method according to item 16, wherein the cancer is a hematological cancer or a solid tumor, specifically, wherein the hematological cancer is acute lymphoblastic leukemia, diffuse large B-cell lymphoma, Hodgkin's lymphoma, acute myeloid leukemia or multiple myeloma, and wherein the solid tumor is colon cancer, breast cancer, pancreatic cancer, ovarian cancer, hepatocellular carcinoma, lung cancer, neuroblastoma, glioblastoma or sarcoma.

[0036] 18. The method according to item 16, wherein the viral infection is caused by human immunodeficiency virus (HIV), adenovirus, polyomavirus, influenza virus or human herpes virus, specifically, wherein the human herpes virus is cytomegalovirus (CMV), Epstein-Barr virus (EBV), herpes simplex virus (HSV), varicella-zoster virus (VZV) or human herpes virus 8 (HHV8).

[0037] 19. A method for producing lymphocytes according to any one of items 1 to 9, comprising the following steps:

[0038] a) providing lymphocytes obtained from a subject;

[0039] b) introducing into said lymphocytes a synthetic polynucleotide encoding at least one iron regulatory protein; and

[0040] c) expressing one or more genes encoded in said synthetic polynucleotide.

[0041] 20. The method according to item 19, wherein a second synthetic polynucleotide encoding a chimeric antigen receptor is introduced in step (b).

[0042] 21. The method according to item 20, wherein the synthetic polynucleotide encoding the chimeric antigen receptor is combined with the synthetic polynucleotide encoding the at least one iron regulatory protein.

[0043] 22. The method according to any one of items 19 to 21, wherein the lymphocytes are activated before or after the introduction of the at least one synthetic polynucleotide into the lymphocytes.

[0044] 23. Method according to any one of items 19 to 22, wherein said at least one synthetic polynucleotide is introduced into said lymphocytes by viral transduction, in particular by retroviral transduction.

[0045] Thus, in one embodiment, the present invention relates to a lymphocyte comprising a synthetic polynucleotide encoding at least one iron regulatory protein.

[0046] Specifically, the present invention is based on the surprising discovery that CD71-mediated iron uptake is a key metabolic checkpoint in activated NK cells, acting as a go / no-go gatekeeper for cell proliferation. In cytokine-enhanced (CE) NK cells, excess iron regulatory proteins unexpectedly create a pseudo-iron-deficient state, selectively enhancing CD71 translation and thereby increasing cell proliferation.

[0047] To date, the molecular mechanisms that lead to enhanced effector function of CE NK cells remain unknown. Example 2 specifically shows that compared with naive (NV) NK cells, the expression of CD71 in CE NK cells is significantly higher in response to stimulation of IL-12 and IL-18 and tumor target cells (Figures 2B, 2C and 2D). In Example 5, it is further shown that the transcription of the TFRC gene encoding CD71 is induced after activation of NV and CE NK cells with cytokines, however, to a higher degree in CE NK cells (Figure 5B). Compared with NV NK cells, higher TFRC mRNA expression is directly converted into increased protein expression in CE NK cells (Figures 2B and C). Therefore, it is shown in Example 6 that compared with NV NK cells, the expression levels of iron regulatory proteins (IRPs) IRP1 and IRP2 in CE NK cells are higher (Figure 5B). Figure 6A Since IRPs are known to be involved in regulating the translation of TFRC mRNA by stabilizing the latter, it can be concluded that the higher amount of CD71 protein in CE NK cells compared to NV NK cells is caused by the higher amount of IRPs in these cells. These findings are surprising, because the expression of IRPs is known to be regulated in response to cellular iron levels. However, in CE NK cells, despite the abundant iron content in the surrounding medium, the expression of IRPs is upregulated, thus creating a pseudo-iron-deficient state. After stimulation, this pseudo-iron-deficient state allows for increased stability of CD71 mRNA, leading to increased CD71 protein expression and, therefore, proliferation.

[0048] Based on these findings, the inventors have concluded that inducing a pseudo-iron-deficient state in lymphocytes, such as NK cells or T cells, leads to increased proliferation and, consequently, a larger effector population upon administration to a subject. Instead of having to treat lymphocytes with cytokines and / or feeder cell lines, which are often difficult or even impossible to control in in vivo applications, the present invention provides an inventive solution for inducing a pseudo-iron-deficient state in lymphocytes by overexpressing at least one IRP in said lymphocytes. Thus, pseudo-iron-deficient lymphocytes overexpressing IRPs according to the present invention have enhanced function, in particular, enhanced proliferation, compared to lymphocytes that do not overexpress IRPs. Therefore, in an alternative embodiment, the present invention relates to lymphocytes that overexpress at least one iron-regulatory protein.

[0049] The inventors have demonstrated that forced expression of IRPs leads to increased proliferation of different types of lymphocytes. For example, the inventors showed that lentiviral overexpression of IRP2 (SEQ ID NO: 2; NCBI RefSeq: NM_004136.4) leads to increased expression of CD71 and, more importantly, increased proliferation of Jurkat T cells ( FIG8F ). In addition, the inventors showed that lentiviral overexpression induced CD4+ and CD8 + Expression of CD71 in T cells (Figure 8G and H). In addition, the inventors showed that lentiviral overexpression of IRP2 in CAR T cells led to increased proliferation of CAR T cells after antigen stimulation, while overexpression of IRP2 had no effect on the proliferation of unstimulated CAR T cells ( Figure 8K ).

[0050] Thus, the present inventors have convincingly demonstrated that the regulatory effects of IRPs on CD71 expression and the correlation between CD71 expression and cell proliferation are well conserved between different lymphocyte types, particularly between T cells and NK cells. In view of these findings, it is concluded that overexpression of IRPs in lymphocytes leads to increased proliferation of said lymphocytes. Therefore, it is reasonable to conclude that forced overexpression of IRPs represents an attractive strategy for achieving robust in vivo proliferation of lymphocytes infused into patients during lymphocyte-based therapies.

[0051] IRPs, also known as iron-responsive element-binding proteins, are proteins that bind to iron-responsive elements (IREs) and thereby regulate human iron metabolism. In humans, two different IRPs have been described, named IRP1 and IRP2. The activities of IRP1 and IRP2 are regulated in different ways. IRP1 contains an iron-sulfur cluster and functions as a cytoplasmic aconitase under conditions of iron repletion. When iron is scarce, the iron-sulfur cluster becomes iron-deficient, and IRP1 changes its conformation, enabling it to bind to the IRE of mRNA. In contrast, IRP2 is rapidly degraded by the ubiquitin proteasome system under conditions of relative iron excess. Under conditions of iron deficiency, the adapter protein FBXL5 is degraded, leading to increased IRP2 levels. Therefore, ubiquitin ligases act as iron sensors and regulators of iron homeostasis. Tissue-specific changes in the activity of IRP1 and IRP2 have been described, and IRP1 and IRP2 knockout mice have different phenotypes.

[0052] As used herein, the term "polynucleotide" refers to a sequence of nucleotides linked by phosphodiester bonds. The polynucleotides of the present invention can be deoxyribonucleic acid (DNA) molecules or ribonucleic acid (RNA) molecules in single-stranded or double-stranded form. Nucleotide bases are represented herein by single-letter codes: adenine (A), guanine (G), thymine (T), cytosine (C), inosine (I), and uracil (U). The polynucleotides of the present invention can be prepared using standard techniques well known to those of ordinary skill in the art.

[0053] As used herein, a "synthetic polynucleotide" is a polynucleotide that is not naturally derived and has been integrated into lymphocytes. Synthetic polynucleotides can be produced by recombinant techniques, including the polymerase chain reaction, or by chemical synthesis. The skilled artisan is aware of methods for producing synthetic polynucleotides having a specific polynucleotide sequence. Furthermore, the skilled artisan is aware of methods for integrating synthetic polynucleotides into lymphocytes. In the present invention, preferably, the synthetic polynucleotide comprises at least one gene or coding sequence encoding an IRP, wherein at least one gene or coding sequence is operably linked to at least one regulatory element, such as a promoter, that is not naturally associated with the endogenous gene encoding the at least one IRP. Such synthetic polynucleotides can be obtained by a variety of strategies. For example, a polynucleotide comprising a gene or coding sequence encoding an IRP can be integrated into a lymphocyte, wherein the gene or coding sequence is under the control of a promoter or another regulatory element that is not naturally associated with the endogenous gene encoding the IRP. Alternatively, the polynucleotide encoding the IRP can be integrated into the genome of the lymphocyte such that it is operably linked to a regulatory element that is not naturally associated with the endogenous gene encoding the IRP. In addition, the synthetic polynucleotides of the present invention can also be obtained by integrating a polynucleotide comprising a regulatory element that is not naturally associated with an endogenous gene encoding an IRP into a lymphocyte, such that the regulatory element is operably linked to the endogenous gene encoding the IRP of the lymphocyte. Alternatively, the synthetic polynucleotides of the present invention can also be obtained by modifying the endogenous regulatory elements of the gene encoding the IRP using genetic engineering or genome editing methods, such that the expression of the endogenous IRP gene of the lymphocyte is altered. For example, the regulatory elements of the endogenous gene encoding the IRP, such as the promoter, can be modified so that the gene encoding the IRP is constitutively expressed in the lymphocyte. Methods for genetic engineering are well known in the art and include CRISPR / Cas9, or the use of engineered nucleases, such as meganucleases, zinc finger nucleases, or TALENs.

[0054] The term "operably linked" refers to a functional connection between a regulatory sequence and a heterologous nucleic acid sequence, resulting in the expression of the latter. For example, a first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is arranged in a functional relationship with the second nucleic acid sequence. For example, if a promoter affects the transcription or expression of a coding sequence, the promoter is operably linked to the coding sequence. Typically, operably linked DNA sequences are continuous and, when it is necessary to link two protein coding regions, are in the same reading frame. As used herein, the term "promoter" is defined as a DNA sequence required for initiating specific transcription of a polynucleotide sequence that is recognized by the synthetic machinery of the cell or introduced synthetic machinery.

[0055] If the synthetic polynucleotide is present in the cell interior, i.e., surrounded by the cytoplasmic membrane of the cell, the cell, such as a lymphocyte, is referred to as comprising the synthetic polynucleotide. The synthetic polynucleotide can be delivered to the cell in any form and by any method known in the art. For example, the synthetic polynucleotide can be present in the cell interior in the form of linear DNA or as a part of a circular DNA vector (such as a plasmid), or in the form of mRNA or as a part of mRNA. However, preferably, the synthetic polynucleotide is integrated into the lymphocyte genome as DNA.

[0056] The term "encoding" refers to the intrinsic properties and the biological properties resulting therefrom of a specific nucleotide sequence (such as a coding sequence, gene, cDNA or RNA) in a polynucleotide that is used as a template for synthesizing other polymers and macromolecules with a limited nucleotide sequence (i.e., rRNA, tRNA and mRNA) or a limited amino acid sequence in a biological process. Therefore, in a cell or other biological system, if a coding sequence or gene is transcribed into mRNA and the translation of the mRNA corresponding to the coding sequence or gene produces a protein, the coding sequence or gene encodes the protein. Coding strands (whose nucleotide sequence is identical to the mRNA sequence and is generally provided in a sequence table) and non-coding strands (used as templates for gene or cDNA transcription) may be referred to as proteins or other products encoding the coding sequence, gene or cDNA.

[0057] As used herein, the term "coding sequence" refers to a nucleic acid sequence that is transcribed and translated into a polypeptide when placed under the control of an appropriate regulatory or expression control sequence. As used herein, the term "gene" refers to a DNA sequence, including but not limited to a DNA sequence that can be transcribed into mRNA (which can be translated into a polypeptide chain), transcribed into rRNA or tRNA, or used as a recognition site for enzymes and other proteins involved in DNA replication, transcription, and regulation. The term "gene" is generally understood to include all introns and other DNA sequences spliced ​​from mRNA transcripts, as well as variants produced by alternative splicing sites when used in its endogenous environment. However, when the term "gene" is used in the context of synthetic polynucleotides, the term is widely understood to further include a coding sequence corresponding to the cDNA of the splicing mRNA variants of the gene or the splicing mRNA derived from the gene in sequence.

[0058] As used herein, the term "lymphocyte" refers to any mononuclear non-phagocytic leukocyte derived from lymphoid stem cells found in blood, lymph and lymphoid tissues; lymphocytes include natural killer cells (NK cells; which play a role in cell-mediated cytotoxic innate immunity), T cells (for cell-mediated cytotoxic adaptive immunity) and B cells (for humoral, antibody-driven adaptive immunity). Preferably, the lymphocyte of the present invention is a NK cell or a T cell. Therefore, in a preferred embodiment, the present invention relates to a lymphocyte according to the present invention, wherein the lymphocyte is a T cell or a natural killer cell.

[0059] T cells are a type of lymphocyte that develops in the thymus and plays a key role in immune responses. T cells can be distinguished from other lymphocytes by the presence of T cell receptors on the cell surface. These immune cells originate as precursor cells from the bone marrow and once they migrate to the thymus, they develop into several different types of T cells. T cell differentiation continues even after leaving the thymus. The lymphocytes of the present invention can be any T cell, such as helper CD4 T cells. + T cells, cytotoxic CD8 + T cells, memory T cells, regulatory CD4 + T cells, natural killer T cells or gamma delta T cells. In certain embodiments, the T cells according to the present invention are CD4 + or CD8 + T cells, optionally CD4 + or CD8 + The T cells contain chimeric antigen receptors.

[0060] Natural killer cells, or NK cells, are a type of cytotoxic lymphocyte that is crucial to the innate immune system. NK cells play a role similar to cytotoxic T cells in the adaptive immune response of vertebrates. NK cells provide a rapid response to virally infected cells, become active approximately 3 days after infection, and respond to tumor formation. They were named "natural killer cells" because the original concept was their ability to lyse tumor cells without prior sensitization. The inhibitory receptors of NK cells engage most major histocompatibility class I (MHC class I) molecules that are ubiquitously expressed on the surface of nucleated cells. Healthy cells that express high levels of MHC class I maintain self-tolerance and are protected from killing by NK cells. In contrast, viral infection or malignant transformation triggers NK cell activation by removing inhibitory signals. Activating NK cell receptors recognize stress-induced ligands on virally infected or malignant cells. Expression of these stimulatory ligands on target cells can overcome the constitutive inhibition delivered by inhibitory receptors, thereby activating NK cells.

[0061] In the present invention, the lymphocyte may be any lymphocyte. Preferably, the lymphocyte of the present invention is a T cell or a NK cell. Therefore, in one embodiment, the present invention relates to a lymphocyte according to the present invention, wherein the lymphocyte is a T cell or a NK cell. In another embodiment, the present invention relates to a lymphocyte according to the present invention, wherein the lymphocyte is a cytotoxic CD8 + T cells, helper CD4 + In another embodiment, the present invention relates to a lymphocyte according to the present invention, wherein the lymphocyte is a cytotoxic CD8 + In another embodiment, the present invention relates to a lymphocyte according to the present invention, wherein the lymphocyte is a cytotoxic CD8 + In another embodiment, the present invention relates to a lymphocyte according to the present invention, wherein the lymphocyte is a helper CD4 + T cells.

[0062] In one embodiment the invention relates to a lymphocyte according to the invention, wherein the lymphocyte is a tumor infiltrating lymphocyte, a T cell comprising a modified TCR or a virus-specific T cell.

[0063] That is, the lymphocytes are lymphocytes suitable for cell therapy applications, such as TILs, T cells containing modified TCRs, or virus-specific T cells. Preferably, the TILs, T cells containing modified TCRs, or virus-specific T cells comprise a synthetic polynucleotide encoding at least one iron regulatory protein, preferably, wherein the iron regulatory protein is IRP1 and / or IRP2, more preferably, wherein the iron regulatory protein is IRP2, even more preferably, wherein the iron regulatory protein is IRP2 as shown in SEQ ID NO: 2.

[0064] In certain embodiments, the lymphocytes of the present invention can be tumor infiltrating lymphocytes (TIL). TIL is a white blood cell that has left the bloodstream and migrated to the tumor. They include T cells and B cells, and are a part of a larger class of "tumor infiltrating immune cells", which are composed of mononuclear and polymorphonuclear immune cells (e.g., T cells, B cells, natural killer cells, macrophages, neutrophils, dendritic cells, mast cells, eosinophils, basophils, etc.) of varying proportions. Their abundance varies due to tumor type and stage, and is related to disease prognosis in some cases. TIL can be used for cell therapy, in which TIL is separated and amplified in vitro from the patient's tumor. The specific tumor recognition of the amplified TIL can then be determined, and then the tumor-specific TIL can be returned to the patient, optionally after another amplification step.

[0065] In the present invention, a synthetic polynucleotide encoding at least one iron regulatory protein can be introduced ex vivo into TILs that have been obtained from a patient, particularly a patient's tumor. The IRP-overexpressing TILs can then be infused into the patient during cancer treatment, preferably after one or more additional expansion and / or selection steps.

[0066] In certain embodiments of the present invention, the lymphocytes of the present invention may be T cells comprising a modified T cell receptor (TCR). The term "T cell comprising a modified T cell receptor" or "TCR-modified T cell" refers to a T cell that has been genetically modified to express a specific TCR. TCR-modified T cells can be produced by obtaining a T cell population from a subject and introducing genetic elements encoding a T cell receptor into the T cell population. The TCR may be a naturally occurring TCR or an engineered TCR.

[0067] TCR modified T cells can be used in cell therapy to increase the patient's immune response to specific antigens (e.g., antigens that have been confirmed to be produced by the patient's tumor). Overexpression of iron regulatory proteins, preferably IPR1 and / or IPR2, more preferably IPR2, in the TCR modified T cell population can result in these T cells proliferating more robustly in vivo when infused into the patient's body, thereby eliciting a stronger immune response to the antigen recognized by the TCR in the patient. The TCR modified T cells of the present invention can be any type of T cell. Preferably, the TCR modified T cells of the present invention are CD4 + or CD8 + T cells.

[0068] In certain embodiments of the invention, the lymphocytes according to the invention are virus-specific T cells. "Virus-specific T cells" are T cells that have been stimulated with viral antigens, such as CD4 + or CD8 + T cells. When administered to a patient, virus-specific T cells can be used to treat viral infections in the patient. Overexpression of an iron regulatory protein, preferably IRP1 and / or IRP2, more preferably IRP2, in a population of virus-specific T cells can result in more robust proliferation of these T cells in vivo when infused into the patient, thereby eliciting a stronger immune response in the patient to the antigen recognized by the TCR.

[0069] In another embodiment, the invention relates to a lymphocyte according to the invention, wherein at least one iron regulatory protein is constitutively expressed.

[0070] The at least one IRP encoded in a synthetic polynucleotide according to the present invention and contained in a cell can be operably linked to any promoter or regulatory element known in the art. Thus, the at least one IRP can be constitutively expressed, i.e., expressed in most cell types most of the time, or inducibly expressed, i.e., expressed only under certain physiological conditions and / or in response to specific signals and / or inducer molecules. However, in the present invention, it is preferred that the at least one IRP is constitutively expressed. Alternatively, the at least one IRP can be inducibly expressed under conditions commonly encountered in cell therapy applications, such as by signals, molecules, and / or processes associated with lymphocyte activation.

[0071] As used herein, the term "expression" refers to the production of a desired end product molecule in a target cell. The end product molecule may include, for example, an RNA molecule, a peptide, a protein, or a combination thereof. In the present invention, the end product is preferably an iron regulatory protein.

[0072] A "constitutive" promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding a gene product or designated gene product, causes the gene product to be produced in a cell under most or all physiological conditions of the cell.

[0073] An example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence that can drive high-level expression of any polynucleotide sequence operably linked thereto. Another example of a suitable promoter is elongation growth factor-1a (EF-1a). However, other constitutive promoter sequences may also be used, including but not limited to simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukosis virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, and human gene promoters such as but not limited to actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter.

[0074] Therefore, in certain embodiments, the present invention relates to a lymphocyte according to the invention, wherein the synthetic polynucleotide encoding at least one iron regulatory protein is under the control of a constitutive promoter.

[0075] A polynucleotide encoding a protein or polypeptide is "under the control of a constitutive promoter" if the constitutive promoter is responsible for initiating transcription of the polynucleotide encoding the protein or polypeptide. The skilled person is aware of methods for placing a polynucleotide encoding a protein or polypeptide under the control of a promoter, for example by molecular cloning methods.

[0076] The constitutive promoter can be any constitutive promoter known in the art, preferably a constitutive promoter that initiates transcription in mammalian cells and more preferably in human cells. For example, the constitutive promoter can be any of the constitutive promoters listed above.

[0077] In certain embodiments of the present invention, the invention relates to a lymphocyte according to the invention, wherein the constitutive promoter is the EF-1α promoter.

[0078] Human elongation factor-1α (EF-1α) is a constitutive promoter of human origin that can be used to drive ectopic gene expression in a variety of in vitro and in vivo settings. Without being bound by theory, EF-1α is generally useful in situations where other promoters (such as CMV) have reduced activity or have been silenced (e.g., in embryonic stem cells).

[0079] An "inducible" promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding a gene product or a designated gene product, causes the gene product to be produced in a cell substantially only when an inducer corresponding to the promoter is present in the cell. Examples of inducible promoters include, but are not limited to, metallothionine promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

[0080] In another embodiment, the invention relates to a lymphocyte according to the invention, wherein the at least one iron regulatory protein is IRP1 (SEQ ID NO: 1) and / or IRP2 (SEQ ID NOs: 2-6).

[0081] That is, the at least one IRP encoded by the synthetic polynucleotide contained in the lymphocyte according to the present invention may be an IRP known in the art. However, preferably, the IRP is human IRP1 and / or human IRP2. In humans, four different isoforms of IRP2 have been described, of which two different sequences of isoform 3 have been described (SEQ ID NOs: 2-6). Therefore, in certain embodiments of the present invention, the lymphocyte according to the present invention may contain a synthetic polynucleotide encoding a protein having the amino acid sequence of SEQ ID NO: 1. In other embodiments of the present invention, the lymphocyte according to the present invention may contain one or more synthetic polynucleotides encoding proteins having the amino acid sequence of SEQ ID NOs: 2-6. In further embodiments of the present invention, the lymphocyte according to the present invention may contain a synthetic polynucleotide encoding a protein having one or more of SEQ ID NOs: 2-6 and the amino acid sequence of SEQ ID NO: 1. The lymphocyte according to the present invention may also contain two or more synthetic polynucleotides, wherein the first synthetic polynucleotide encodes a protein having the amino acid sequence of SEQ ID NO: 1, and wherein the second or any further synthetic polynucleotide encodes a protein having the amino acid sequence of SEQ ID NOs: 2-6.

[0082] In one embodiment of the present invention, the present invention relates to lymphocytes according to the present invention, wherein the at least one iron regulatory protein is IRP1 (SEQ ID NO: 1). In another embodiment of the present invention, the present invention relates to lymphocytes according to the present invention, wherein the at least one iron regulatory protein is IRP2 (SEQ ID NO: 2). In another embodiment of the present invention, the present invention relates to lymphocytes according to the present invention, wherein the at least one iron regulatory protein is IRP2 (SEQ ID NO: 3). In another embodiment of the present invention, the present invention relates to lymphocytes according to the present invention, wherein the at least one iron regulatory protein is IRP2 (SEQ ID NO: 4). In another embodiment of the present invention, the present invention relates to lymphocytes according to the present invention, wherein the at least one iron regulatory protein is IRP2 (SEQ ID NO: 5). In another embodiment of the present invention, the present invention relates to lymphocytes according to the present invention, wherein the at least one iron regulatory protein is IRP2 (SEQ ID NO: 6). In another embodiment of the present invention, the present invention relates to lymphocytes according to the present invention, wherein the at least one iron regulatory protein is IRP1 (SEQ ID NO: 1) and IRP2 (SEQ ID NO: 2). In a preferred embodiment of the present invention, the present invention relates to lymphocytes according to the present invention, wherein the at least one iron regulatory protein is IRP2 (SEQ ID NO: 2).

[0083] The inventors have demonstrated that overexpression of IRP2 in lymphocytes leads to more robust proliferation of these lymphocytes. The inventors further showed that silencing IRP1 in lymphocytes has a similar effect as silencing IRP2, although the effect is less pronounced than with IRP2 (Figures 8C and D). However, it must be noted that silencing IRP1 is not as effective as silencing IRP2 (Figure 8A). Therefore, it is plausible that overexpression of IRP1 may also lead to increased proliferation of lymphocytes. Furthermore, it is plausible that simultaneous overexpression of IRP1 and IRP2 may lead to increased proliferation of lymphocytes.

[0084] In one embodiment the invention relates to a lymphocyte according to the invention, wherein the lymphocyte further comprises a chimeric antigen receptor.

[0085] As used herein, the term "chimeric antigen receptor" or "CAR" or "CARs" refers to an engineered receptor that transplants antigen specificity onto lymphocytes such as T cells and NK cells. The CAR of the present invention can be any CAR known in the art. Preferably, the CAR of the present invention comprises at least one extracellular antigen binding domain, a transmembrane domain, one or more costimulatory signaling regions, and an intracellular signaling domain. In certain embodiments of the present invention, CAR can be a bispecific CAR that is specific for two different antigens or epitopes. After the antigen binding domain specifically binds to the target antigen, the signaling domain activates intracellular signaling. For example, the signaling domain can utilize the antigen binding properties of the antibody to redirect T cell specificity and reactivity to the selected target in a non-MHC restricted manner. Non-MHC restricted antigen recognition enables CAR-expressing T cells to recognize antigens independently of antigen processing, thereby bypassing the main mechanism of tumor escape. In addition, when expressed in T cells, CAR advantageously does not dimerize with endogenous T cell receptor (TCR) α and β chains. In the case of NK cells, the expression of CAR can help guide NK cells to the target antigen. However, compared to CAR T cells, CAR NK cells retain the expression of their activating and inhibitory receptors.Thus, unlike CAR-T cells, CAR-NK cells can still perform their “natural” functions even when the CAR-targeted antigen is downregulated.

[0086] In the present invention, if lymphocytes include coding sequences encoding CAR and express these coding sequences so that CAR is anchored on the membrane of lymphocytes, then lymphocytes are said to include chimeric antigen receptors. The coding sequences encoding the components of CAR may be located on one or more synthetic polynucleotides. In certain embodiments of the present invention, the coding sequences encoding the components of CAR and the one or more coding sequences encoding IRP may be located on a single synthetic polynucleotide. Alternatively, the coding sequences encoding the components of CAR and the one or more coding sequences encoding one or more IRPs may be located on two or more separate polynucleotides. For example, the polynucleotides encoding CAR and the polynucleotides encoding one or more IRPs may be introduced into cells by two independent viral transduction events and thus may be integrated into different parts of the genome. One or more synthetic polynucleotides comprising CAR coding sequences and optionally one or more IRP coding sequences may be included in lymphocytes in any form and may have been introduced into lymphocytes by any method known in the art. For example, synthetic polynucleotides encoding CAR and / or one or more iron regulatory proteins may be present inside cells as part of a circular DNA vector (e.g., a plasmid), in the form of linear DNA or as part thereof, or in the form of mRNA or as part thereof. However, it is preferred that one or more synthetic polynucleotides comprising a CAR coding sequence and optionally one or more IRP coding sequences are integrated into the genome of the lymphocyte as DNA. The skilled artisan is aware of methods for introducing DNA into the genome of the lymphocyte. Synthetic polynucleotides encoding IRP1 and / or IRP2 and optionally CAR can be introduced into the genome by any method known in the art. In certain embodiments, synthetic polynucleotides encoding IRP1 and / or IRP2 and optionally CAR can be introduced into the genome of the lymphocyte by viral transduction. However, other methods for introducing synthetic DNA into the genome of the lymphocyte, such as CRISPR / Cas9, are also encompassed herein.

[0087] In another embodiment, the invention relates to a lymphocyte according to the invention, wherein the chimeric antigen receptor comprises an antigen binding domain, a transmembrane domain, a co-stimulatory signaling region and a signaling domain.

[0088] Lymphocytes according to the present invention may include chimeric antigen receptors (CARs) comprising extracellular and intracellular domains. The extracellular domain may include one or more target-specific binding elements otherwise referred to as antigen binding moieties. The intracellular domain or cytoplasmic domain may include one or more costimulatory signaling regions and signaling domains. The costimulatory signaling region refers to a part of CAR that includes the intracellular domain of costimulatory molecules. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands required for the effective response of lymphocytes to antigens.

[0089] Between the extracellular domain and the transmembrane domain of CAR, or between the cytoplasmic domain and the transmembrane domain of CAR, a spacer domain can be incorporated. As used herein, the term "spacer domain" generally refers to any oligopeptide or polypeptide used to connect a transmembrane domain to an extracellular domain or a cytoplasmic domain in a polypeptide chain. The spacer domain may comprise up to 300 amino acids, preferably 10 to 100 amino acids and most preferably 25 to 50 amino acids.

[0090] The CAR of the present invention may include one or more target-specific binding elements otherwise referred to as antigen binding moieties. The selection of part depends on the type and quantity of the ligands that define the target cell surface. For example, the antigen binding domain can be selected to identify ligands that serve as cell surface markers on target cells associated with a specific disease state. Therefore, examples of cell surface markers that can serve as ligands for the antigen portion domains in the CAR of the present invention include those associated with viral, bacterial and parasitic infections, autoimmune diseases and cancer cells.

[0091] Depending on the desired antigen to be targeted, the CAR of the present invention can be engineered to include an appropriate antigen binding portion that is specific for the desired antigen target. For example, if CD19 is the desired antigen to be targeted, an antibody to CD19 can be used as an antigen binding portion and incorporated into the CAR of the present invention.

[0092] About transmembrane domain, CAR can be designed to include a transmembrane domain fused to the extracellular domain of CAR. In certain embodiments, a transmembrane domain naturally associated with the extracellular or cytoplasmic domain of CAR can be used. In some cases, the transmembrane domain can be selected or modified by amino acid substitution to avoid such domains from binding to the transmembrane domain of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex.

[0093] The membrane-spanning domain can be derived from natural or synthetic sources. When the source is natural, the domain can be derived from any membrane-bound or transmembrane protein. Particularly useful membrane-spanning regions in the present invention can be derived from (i.e., at least including one or more membrane-spanning regions) α, β or ζ chains of T cell receptors, CD28, CD8, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154. Alternatively, the membrane-spanning domain can be synthetic, in which case it will mainly include hydrophobic residues, such as leucine and valine. Preferably, a triplet of phenylalanine, tryptophan and valine will be present at each end of the synthetic membrane-spanning domain.

[0094] Optionally, a short oligopeptide or polypeptide linker, preferably between 2 and 10 amino acids in length, can form a connection between the transmembrane domain and the cytoplasmic signaling domain of the CAR. Glycine-serine doublets provide particularly suitable linkers.

[0095] The cytoplasmic domain or intracellular signaling domain of the CAR of the present invention is responsible for activating at least one normal effector function of the lymphocyte in which the CAR has been placed. The term "effector function" refers to the specialized function of the cell. The effector function of a T cell can be, for example, cytolytic activity or auxiliary activity, including the secretion of cytokines. Therefore, the term "intracellular signaling domain" refers to a part of a protein that transduces effector function signals and guides cells to perform specialized functions. Although a complete intracellular signaling domain can usually be used, in many cases, it is not necessary to use a complete chain. In the case of using a truncated portion of an intracellular signaling domain, as long as the truncated portion transduces the effector function signal, it can be used to replace the complete chain. Therefore, the term intracellular signaling domain is intended to include any truncated portion of the intracellular signaling domain that is sufficient to transduce the effector function signal. Preferred examples of the intracellular signaling domain used in the CAR of the present invention include cytoplasmic sequences that initiate signal transduction after antigen receptor engagement by the cooperation of T cell receptors (TCRs) and co-receptors, as well as any derivatives or variants of these sequences and any synthetic sequences with the same functional capabilities.

[0096] It is known that signals generated by the TCR alone are insufficient to fully activate T cells and that secondary or co-stimulatory signals are required. Therefore, it can be said that T cell activation is mediated by two different types of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation through the TCR (primary cytoplasmic signaling sequences) and those that act in an antigen-independent manner to provide secondary or co-stimulatory signals (secondary cytoplasmic signaling sequences).

[0097] The primary cytoplasmic signaling sequence regulates the primary activation of the TCR complex in a stimulatory or inhibitory manner. The primary cytoplasmic signaling sequence that acts in a stimulatory manner may contain a signaling motif known as an immunoreceptor tyrosine-based activation motif (ITAM).

[0098] Examples of ITAMs containing primary cytoplasmic signaling sequences that are particularly useful in the present invention include those derived from TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d. It is particularly preferred that the cytoplasmic signaling molecule in the CAR of the present invention comprises a cytoplasmic signaling sequence derived from CD3ζ.

[0099] The cytoplasmic domain of CAR can be designed to include a separate CD3ζ signaling domain or a combination thereof with any one or more other desired cytoplasmic domains useful in the context of the CAR of the present invention. For example, the cytoplasmic domain of CAR may include a CD3ζ chain portion and a costimulatory signaling region. The costimulatory signaling region refers to a portion of CAR that includes the intracellular domain of a costimulatory molecule. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands required for the effective response of lymphocytes to antigens. Examples of such molecules include CD27, CD28, 4-1BB (CD 137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and ligands that specifically bind to CD83.

[0100] The cytoplasmic signaling sequences within the cytoplasmic signaling portion of the CAR of the present invention can be connected to each other in a random or specified order. Optionally, a short oligopeptide or polypeptide linker preferably having a length between 2 and 10 amino acids can form a connection. Glycine-serine doublets provide particularly suitable linkers.

[0101] In certain embodiments, the cytoplasmic domain can be designed to include the signaling domain of CD3ζ and the signaling domain of CD28 and / or 4-1BB.

[0102] In another embodiment, the invention relates to a lymphocyte according to the invention, wherein the antigen binding domain is an antibody or an antigen binding fragment thereof, in particular, wherein the antigen binding fragment is a Fab or scFv.

[0103] That is, the antigen binding domain of CAR can be any domain known in the art that can specifically bind to a specific antigen. However, preferably, the antigen binding domain of CAR is an antibody or an antigen binding fragment of an antibody.

[0104] As used herein, the term "antibody" refers to an immunoglobulin molecule that specifically binds to an antigen. An antibody can be a complete immunoglobulin derived from a natural or recombinant source, or can be an immunoreactive portion of a complete immunoglobulin. An antibody is typically a tetramer of an immunoglobulin molecule. The antibodies in the present invention can exist in various forms, including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab, and F(ab)2, as well as single-chain antibodies and humanized antibodies (Harlow et al., 1999, Using Antibodies:A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Houston et al., 1989, Proc.A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc.Natl.Acad.Sci.USA; 85:5879-5883; Bird et al., 1988, Science; 242:423-426).

[0105] As used herein, the term "immunoglobulin" or "Ig" is defined as a class of proteins that have antibody function. Antibodies expressed by B cells are sometimes referred to as BCRs (B cell receptors) or antigen receptors. The five members included in this class of proteins are IgA, IgG, IgM, IgD, and IgE. IgA is a primary antibody present in body secretions such as saliva, tears, breast milk, gastrointestinal secretions, and mucous secretions of the respiratory and urogenital tracts. IgG is the most common circulating antibody. IgM is the main immunoglobulin produced by most subjects in the primary immune response. It is the most effective immunoglobulin in agglutination, complement fixation, and other antibody responses, and is important for defending against bacteria and viruses. IgD is an immunoglobulin that does not have a known antibody function but can be used as an antigen receptor. IgE is an immunoglobulin that mediates immediate hypersensitivity reactions by causing mast cells and basophils to release mediators when exposed to allergens.

[0106] The term "antibody fragment" refers to a portion of an intact antibody and refers to the antigen-determining variable region of an intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2 and Fv fragments, linear antibodies, scFv antibodies, and multispecific antibodies formed from antibody fragments.

[0107] As used herein, the term "Fab" is intended to refer to the region of an antibody (monovalent antigen-binding fragment) consisting of one constant domain and one variable domain of each of the heavy and light chains, but in which the heavy chain is truncated so that it lacks the CH2 and CH3 domains and may also lack some or all of the hinge region. Fab fragments can be produced by digesting whole antibodies with papain. Fab can refer to this region in isolated form, or in the context of a full-length antibody, immunoglobulin construct, or Fab fusion protein.

[0108] "scFv" refers to an antibody fragment comprising the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. See, for example, U.S. Patent Nos. 4,946,778, 5,260,203, 5,455,030, and 5,856,456. Typically, the Fv polypeptide also comprises a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding. For a review of sFv, see Pluckthun (1994) The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore, eds. (Springer-Verlag, New York), pp. 269-315. The VH and VL domain complexes of the Fv fragment can also be stabilized by disulfide bonds (U.S. Patent No. 5,747,654).

[0109] As used herein, "antibody heavy chain" refers to the larger of the two types of polypeptide chains found in all antibody molecules in their naturally occurring conformation. As used herein, "antibody light chain" refers to the smaller of the two types of polypeptide chains found in all antibody molecules in their naturally occurring conformation, and lambda light chains refer to the two major antibody light chain isotypes.

[0110] As used herein, the term "synthetic antibody" refers to an antibody produced using recombinant DNA technology, such as by phage expression. The term should also be interpreted as referring to an antibody produced by synthesizing a DNA molecule encoding the antibody, and said DNA molecule expresses an antibody protein or an amino acid sequence specifying the antibody, wherein the DNA or amino acid sequence is obtained using techniques for synthesizing DNA or amino acid sequences that are available and well known in the art.

[0111] Those skilled in the art know methods for producing CARs with different antigen binding domains, transmembrane domains, costimulatory signaling regions and / or signaling domains. In addition, those skilled in the art know methods for introducing such CARs into lymphocytes such as T cells or NK cells.

[0112] In one embodiment the invention relates to a lymphocyte according to the invention, wherein the antigen binding domain specifically binds to a tumor antigen.

[0113] That is, the antigen binding domain of CAR can bind to any antigen known in the art. However, it is preferred that the antigen binding domain of CAR specifically binds to tumor antigens. As used herein, the term "antigen" or "Ag" is defined as a molecule that causes an immune response. This immune response may involve the production of antibodies or the activation of specific immune competent cells, or both. A skilled person will understand that any macromolecule, including almost all proteins or peptides, can be used as an antigen. In addition, antigens can be derived from recombinant or genomic DNA. A skilled person will understand that any DNA comprising a nucleotide sequence or partial nucleotide sequence encoding a protein that elicits an immune response thus encodes an "antigen," as the term is used herein. In addition, a skilled person will understand that an antigen does not necessarily have to be encoded only by the full-length nucleotide sequence of a gene. It is clear that the present invention includes but is not limited to using partial nucleotide sequences of more than one gene and that these nucleotide sequences are arranged in various combinations to elicit a desired immune response. In addition, a skilled person will understand that an antigen does not necessarily have to be encoded by a "gene" at all. It is clear that an antigen can be produced, synthesized, or can be derived from a biological sample. Such a biological sample may include but is not limited to a tissue sample, a tumor sample, a cell, or a biological fluid.

[0114] Tumor antigens are proteins produced by tumor cells that trigger an immune response, particularly a T cell-mediated immune response. The choice of the antigen-binding portion of the CAR will depend on the specific type of cancer to be treated. Tumor antigens are well known in the art and include, for example, glioma-associated antigen, carcinoembryonic antigen (CEA), β-human chorionic gonadotropin, alpha-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxylesterase, muthsp70-2, M-CSF, prostase, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53, prostein, PSMA, Her2 / neu, survivin and telomerase, prostate cancer tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrin B2, CD22, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor, and mesothelin.

[0115] Tumor antigens can include one or more antigenic cancer epitopes associated with malignant tumors. Malignant tumors express a variety of proteins that can be used as target antigens for immune attack. These molecules include but are not limited to tissue-specific antigens, such as MART-1, tyrosinase and GP 100 in melanoma, and prostatic acid phosphatase (PAP) and prostate-specific antigen (PSA) in prostate cancer. Other target molecules belong to the transformation-related molecule group, such as oncogenes HER-2 / Neu / ErbB-2. Another group of target antigens is onco-fetal antigen (onco-fetal antigen), such as carcinoembryonic antigen (CEA). In B-cell lymphoma, tumor-specific idiotypic immunoglobulin constitutes the true tumor-specific immunoglobulin antigen unique to individual tumors. B cell differentiation antigens such as CD 19, CD20 and CD37 are other candidates for target antigens in B-cell lymphoma. Some of these antigens (CEA, HER-2, CD19, CD20, idiotypic) have been used as targets for monoclonal antibody passive immunotherapy, but with limited success.

[0116] The types of tumor antigens mentioned in the present invention may also be tumor-specific antigens (TSAs) or tumor-associated antigens (TAAs). TSAs are unique to tumor cells and are not present on other cells in the body. TAA-related antigens are not unique to tumor cells, but are expressed on normal cells under conditions where an immune tolerance state to the antigen cannot be induced. The expression of antigens on tumors can occur under conditions that allow the immune system to respond to the antigen. TAAs may be antigens that are expressed on normal cells during fetal development when the immune system is immature and unable to respond, or they may be antigens that are normally present at very low levels on normal cells but expressed at much higher levels on tumor cells.

[0117] Non-limiting examples of TSA or TAA antigens include the following: differentiation antigens, such as MART-1 / MelanA (MART-1), gp 100 (Pmel 17), tyrosinase, TRP-1, TRP-2, and tumor-specific multi-lineage antigens, such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15; overexpressed embryonic antigens, such as CEA; overexpressed oncogenes and mutated tumor suppressor genes, such as p53, Ras, HER-2 / neu; unique tumor antigens arising from chromosomal translocations, such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens, such as Epstein Barr virus antigen EBVA and human papillomavirus (HPV) antigens E6 and E7. Other large protein-based antigens include TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, p185erbB2, p180erb-B3, c-met, nm23_H1, PSA, TAG-72, CA 19-9, CA 72-4, CAM17.1, NuMa, K-ras, β-catenin, CDK4, Mum-1, p15, p16, 43-9F, 5T4 (791Tgp72), alpha-fetoprotein, β-HCG, BCA225, BTAA, CA 125, CA15-3\CA 27.29\BCAA, CA195, CA 242, CA-50, CAM43, CD68\I, CO-029, FGF-5, G250, Ga733\EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV 18, NB / 70K, NY-CO-1, RCAS-1, SDCCAG16, TA-90\Mac-2 binding protein, cyclophilin C-related protein, TAAL6, TAG72, TLP, and TPS.

[0118] The antigen binding portion of the CAR can also target antigens including but not limited to the following: CD19, CD20, CD22, CD30, CD123, CD171, CS-1, ROR1, mesothelin, CD33, 1L3Ra, c-Met, PSMA, glycolipid F77, EGFRvIII, GD-2, CD7, NY-ESO-1 TCR, MAGE-A3 TCR, CLL-1, GD3, BCMA, Tn Ag, PSMA, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, IL-1Ra, PSCA, PRSS21, VEGFR2, Lewis Y, CD24, PDGFR-β, SSEA-4, folate receptor alpha, ErbB2 (Her2 / neu), MUC1, EGFR, NCAM, prostase, PAP , ELF2M, ephrin B2, IGF-I receptor, CAIX, LMP2, gp100, bcr-abl, tyrosinase, EphA2, fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, folate receptor β, TEM1 / CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, polysialic acid, PLAC1, Globo H, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, LAGE-1a, legumain, HPV E6, E7, ETV6-AML, sperm protein 17, XAGE1, Tie2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, prostein, survivin and telomerase, PCTA-1 / galectin 8, MelanA / MART1, Ras mutant, hTERT, sarcoma translocation breakpoints, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, androgen receptor, cyclin B1, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxylesterase, mut hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, and IGLL1.

[0119] In certain embodiments, the lymphocytes of the present invention comprising CAR can be used to treat blood cancers, especially for treating acute lymphoblastic leukemia and / or diffuse large B cell lymphoma. In these embodiments, the antigen binding portion of CAR can specifically target CD19.

[0120] In certain embodiments, the lymphocytes of the present invention comprising CAR can be used to treat blood cancers, especially for treating refractory Hodgkin's lymphoma. In these embodiments, the antigen binding portion of CAR can specifically target CD30.

[0121] In certain embodiments, the lymphocytes of the present invention comprising CAR can be used to treat blood cancers, especially for treating acute myeloid leukemia. In these embodiments, the antigen binding portion of CAR can specifically target CD33, CD123 or FLT3.

[0122] In certain embodiments, the lymphocytes of the present invention comprising CAR can be used to treat blood cancers, particularly for treating multiple myeloma. In these embodiments, the antigen binding portion of CAR can specifically target BCMA.

[0123] In certain embodiments, the CAR contained in the lymphocyte of the present invention can bind to two antigens. In certain embodiments, the bispecific CAR can bind to CD19 and CD22 or bind to CD19 and CD20.

[0124] In general, CAR has the advantage of not relying on MHC molecules on the surface of target cells to present tumor antigens. On the contrary, in theory, CAR can bind to any molecule accessible to CAR on the surface of tumor cells, provided that the antigen binding domain of CAR specifically binds to the antigen. Therefore, the tumor antigen is preferably an antigen present on the surface of a tumor or malignant cell. More preferably, the tumor antigen is an antigen that is more abundant on the surface of a tumor or malignant cell than on the surface of a healthy or non-tumor cell. Even more preferably, the tumor antigen is an antigen present on the surface of a tumor cell or malignant cell but not on the surface of a healthy or non-tumor cell.

[0125] As used herein, the term "specific binding" with respect to an antigen-binding domain or an antibody refers to that the antigen-binding domain or antibody recognizes a specific antigen but does not substantially recognize or bind to other molecules in the sample. For example, an antigen-binding domain or an antibody that specifically binds to an antigen from a species may also bind to antigens from one or more other species. However, this cross-species reactivity itself does not change the specific classification of the antigen-binding domain or the antibody. In another example, an antigen-binding domain or an antibody that specifically binds to an antigen may also bind to antigens of different allelic forms. However, this cross-reactivity itself does not change the specific classification of the antibody. In some cases, the term "specific binding" or "specifically binds" can be used to refer to that the interaction between an antigen-binding domain, an antibody, a protein or a peptide and a second chemical substance depends on the presence of a specific structure (e.g., antigenic determinant or epitope) on the chemical substance when referring to the interaction between the antigen-binding domain, an antibody, a protein or a peptide and a second chemical substance; for example, an antigen-binding domain or an antibody recognizes and binds to a specific protein structure rather than a general protein. If the antigen binding domain or antibody is specific for epitope "A", then in a reaction containing labeled "A" and the antigen binding domain or antibody, the presence of molecules containing epitope A (or free unlabeled A) will reduce the amount of labeled A bound to the antigen binding domain or antibody.

[0126] In another embodiment, the invention relates to a lymphocyte according to the invention, wherein the tumor antigen is present on the cell surface of the target cell population or tissue.

[0127] Lymphocytes according to the present invention comprising a CAR can bind to a tumor antigen present on the cell surface of a target cell. The target cell can be part of a cell population or tissue. Generally speaking, a tumor antigen is said to be "present on the cell surface" if it is exposed by the target cell so that the tumor antigen is accessible to the antigen binding domain of the CAR.

[0128] The tumor antigen can be any protein produced by a tumor cell, or more preferably, any portion of a protein produced by a tumor cell and expressed on the cell surface of the tumor cell. Preferably, the tumor antigen is a portion of the extracellular domain of a membrane-anchored protein accessible to the antigen binding domain of the CAR.

[0129] However, the present invention also encompasses tumor antigens that are presented on the surface of target cells by another molecule, particularly an MHC molecule. In this case, the tumor antigen is preferably a peptide derived from a protein. The tumor antigen can, for example, be derived from a protein produced by a tumor cell. Alternatively, the tumor antigen can be derived from an extracellular protein that has previously been taken up by a tumor cell (e.g., by endocytosis). In both cases, the protein can be processed by the target cell into a peptide, which can then be presented on the surface of the target cell, for example, by an MHC molecule.

[0130] In one embodiment the invention relates to a lymphocyte according to the invention, wherein the antigen binding domain specifically binds to a viral antigen.

[0131] That is to say, lymphocytes according to the present invention can be used to treat viral infections of subjects.The CAR included in the lymphocytes of the present invention may include antigen binding domains that specifically bind to viral antigens.Viral antigens can be any component of virions accessible to CAR, such as antigens that form a part of the surface and / or protein coat of the virus.Preferably, the viral antigens recognized by CAR are antigens derived from human immunodeficiency virus (HIV), adenovirus, polyomavirus, influenza virus or human herpes virus, specifically, wherein the human herpes virus is cytomegalovirus (CMV), Epstein-Barr virus (EBV), herpes simplex virus (HSV), varicella-zoster virus (VZV) or human herpes virus 8 (HHV8).

[0132] In one embodiment, the invention relates to a lymphocyte according to the invention, wherein the CAR is encoded by a polynucleotide and wherein the polynucleotide encoding the CAR is transcriptionally linked to a synthetic polynucleotide encoding IRP1 and / or IRP2.

[0133] In the present invention, it is preferred that CAR is encoded by a polynucleotide that is integrated into the lymphocyte genome. More preferably, the polynucleotide encoding CAR and the polynucleotide encoding IRP1 and / or IRP2 are integrated into the same locus of the lymphocyte genome. More preferably, the polynucleotide encoding CAR and the polynucleotide encoding IRP1 and / or IRP2 are integrated into the same locus of the lymphocyte genome so that two or more polynucleotides are transcribed and connected. If the transcription of the coding sequence contained in two or more polynucleotides is driven by a single promoter so that a single transcript encoding two or more polypeptides is obtained, then the two or more polynucleotides are referred to as being transcribed and connected. Preferably, the promoter is located upstream (5') of the coding sequence or polynucleotide that is transcribed and connected. That is, the coding sequence encoding CAR and the coding sequence encoding one or more IRP1 and / or IRP2 can be transcribed by a single promoter.

[0134] To enable synthesis of functional proteins, the coding sequence encoding CAR and one or more coding sequences encoding IRP1 and / or IRP2 may be separated by an internal ribosome entry site (IRES) or may be linked by a polynucleotide encoding a self-cleaving peptide.

[0135] When the coding sequence encoding CAR and one or more coding sequences encoding IRP1 and / or IRP2 are separated by IRES, each coding sequence contained in the transcript is translated independently. However, if the coding sequence encoding CAR and one or more coding sequences encoding IRP1 and / or IRP2 are linked by a self-cleaving peptide, the entire transcript is translated into a polyprotein, which is then cleaved into single proteins by autocleavage during or after translation.

[0136] In one embodiment, the invention relates to a lymphocyte according to the invention, wherein the polynucleotide encoding the CAR and the synthetic polynucleotide encoding IRP1 and / or IRP2 are linked via a polynucleotide encoding a self-cleaving peptide.

[0137] As used herein, the term "self-cleaving peptide" refers to a peptide sequence associated with a cleavage activity that occurs between two amino acid residues within the peptide sequence itself. For example, in a 2A / 2B peptide or a 2A / 2B-like peptide, cleavage occurs between a glycine residue on the 2A peptide and a proline residue on the 2B peptide. This occurs through a "ribosome skipping mechanism" during translation, in which the normal peptide bond formation between the 2A glycine residue and the 2B proline residue of the 2A / 2B peptide is impaired without affecting the translation of the rest of the 2B peptide. Such ribosome skipping mechanisms are well known in the art and are known to be used by several viruses to express several proteins encoded by a single messenger RNA.

[0138] Therefore, in one embodiment the invention relates to a lymphocyte according to the invention, wherein the self-cleaving peptide is a 2A self-cleaving peptide.

[0139] In a preferred embodiment, the present invention relates to a lymphocyte according to the present invention, wherein the self-cleaving peptide is T2A. T2A is a self-cleaving peptide comprising the peptide sequence EGRGSLLTCGDVEENPGP (SEQ ID NO: 7).

[0140] When two coding sequences are linked by a polynucleotide encoding a self-cleaving peptide, it is understood that the coding sequence encoding the first polypeptide, the coding sequence encoding the self-cleaving peptide and the coding sequence encoding the second polypeptide are encoded in the same reading frame.

[0141] In the present invention, it is preferred that the polynucleotide encoding CAR and one or more polynucleotides encoding IRP1 and / or IRP2 are encoded on the same synthetic polynucleotide. In certain embodiments, the synthetic polynucleotide encoding CAR, IRP1 and / or IRP2 has been integrated into the genome of the lymphocyte by viral transduction. In certain embodiments, the polynucleotide encoding CAR and the polynucleotide encoding IRP1 contained in the synthetic polynucleotide are separated by IRES. In another embodiment, the polynucleotide encoding CAR and the polynucleotide encoding IRP2 contained in the synthetic polynucleotide are separated by IRES. In other embodiments, the polynucleotide encoding CAR and the polynucleotide encoding IRP1 contained in the synthetic polynucleotide are connected by encoding a self-cleaving peptide, particularly a 2A self-cleaving peptide, particularly a T2A polynucleotide. In other embodiments, the polynucleotide encoding CAR and the polynucleotide encoding IRP2 contained in the synthetic polynucleotide are connected by encoding a self-cleaving peptide, particularly a 2A self-cleaving peptide, particularly a T2A polynucleotide.

[0142] In certain embodiments, the synthetic polynucleotide encoding CAR, IRP1 and / or IRP2 is under the control of a constitutive promoter. In certain embodiments, the promoter is part of the synthetic polynucleotide. In certain embodiments, the constitutive promoter is the EF-1α promoter. However, it should be understood that those skilled in the art know a wide variety of promoters that can be used instead of the promoter EF-1α. In addition, it should be understood that Example 10 only represents a proof of concept and that more efficient in vivo proliferation of lymphocytes can be achieved by optimizing the expression of CAR and / or IRP1 / 2 in lymphocytes.

[0143] In certain embodiments, the synthetic polynucleotide has the following structure: 5'-CAR-self-cleaving peptide-IRP1-3'. In other embodiments, the synthetic polynucleotide has the following structure: 5'-CAR-self-cleaving peptide-IRP2-3'. In other embodiments, the synthetic polynucleotide has the following structure: 5'-constitutive promoter-CAR-self-cleaving peptide-IRP1-3'. In other embodiments, the synthetic polynucleotide has the following structure: 5'-constitutive promoter-CAR-self-cleaving peptide-IRP2-3'. In other embodiments, the synthetic polynucleotide has the following structure: 5'-constitutive promoter-CAR-self-cleaving peptide-IRP1-3'. In other embodiments, the synthetic polynucleotide has the following structure: 5'-constitutive promoter-CAR-T2A-IRP1-3'. In other embodiments, the synthetic polynucleotide has the following structure: 5'-constitutive promoter-CAR-T2A-IRP2-3'.

[0144] In certain embodiments, the synthetic polynucleotide has the following structure: 5'-IRP1-self-cleaving peptide-CAR-3'. In other embodiments, the synthetic polynucleotide has the following structure: 5'-IRP2-self-cleaving peptide-CAR-3'. In other embodiments, the synthetic polynucleotide has the following structure: 5'-constitutive promoter-IRP1-self-cleaving peptide-CAR-3'. In other embodiments, the synthetic polynucleotide has the following structure: 5'-constitutive promoter-IRP2-self-cleaving peptide-CAR-3'. In other embodiments, the synthetic polynucleotide has the following structure: 5'-constitutive promoter-IRP1-T2A-CAR-3'. In other embodiments, the synthetic polynucleotide has the following structure: 5'-constitutive promoter-IRP2-T2A-CAR-3'. In other embodiments, the synthetic polynucleotide has the following structure: 5'-constitutive promoter-IRP1-P2A-CAR-3'. In other embodiments, the synthetic polynucleotide has the following structure: 5'-constitutive promoter-IRP2-P2A-CAR-3'. In other embodiments, the synthetic polynucleotide has the following structure: 5'-Constitutive Promoter-IRP1-E2A-CAR-3'. In other embodiments, the synthetic polynucleotide has the following structure: 5'-Constitutive Promoter-IRP2-E2A-CAR-3'. In other embodiments, the synthetic polynucleotide has the following structure: 5'-Constitutive Promoter-IRP1-F2A-CAR-3'. In other embodiments, the synthetic polynucleotide has the following structure: 5'-Constitutive Promoter-IRP2-F2A-CAR-3'.

[0145] However, it should be understood that the present invention also encompasses lymphocytes in which the first polynucleotide encoding IRP1 and / or IRP2 and the second polynucleotide encoding a CAR are integrated into different locations in the lymphocyte genome and expressed independently. Preferably, the polynucleotide encoding IRP1 and / or IRP2 and the polynucleotide encoding a CAR are integrated into the genome of the lymphocyte by two independent viral transduction events. The two independent viral transduction events may occur simultaneously or in a stepwise manner.

[0146] In another embodiment, the present invention relates to a viral vector comprising at least one polynucleotide encoding IRP1 (SEQ ID NO: 1) and / or IRP2 (SEQ ID NOs: 2-6).

[0147] That is, the present invention also relates to viral vectors that can be used to integrate iron regulatory proteins into cells, preferably lymphocytes. The viral vector can be any viral vector suitable for integrating polynucleotides into cells, preferably lymphocytes. Therefore, in certain embodiments, the present invention relates to a viral vector according to the present invention, wherein the viral vector is derived from a lentivirus, an adeno-associated virus (AAV), an adenovirus, a herpes simplex virus, a retrovirus, an alphavirus, a flavivirus, a rhabdovirus, a measles virus, a Newcastle disease virus or a poxvirus. In a preferred embodiment, the present invention relates to a viral vector according to the present invention, wherein the viral vector is derived from a lentivirus or an adeno-associated virus (AAV). In a more preferred embodiment, the present invention relates to a viral vector according to the present invention, wherein the viral vector is derived from a lentivirus.

[0148] The viral vector may comprise one or more transgenes. As used herein, the term "transgene" refers to a specific nucleic acid sequence encoding a polypeptide or a portion of a polypeptide to be expressed in a cell, which is inserted into the cell. The term transgenic is intended to include (1) a nucleic acid sequence that is not naturally present in the cell (i.e., a heterologous nucleic acid sequence, such as a nucleic acid encoding a CAR); (2) a nucleic acid sequence that is a mutant form of a nucleic acid sequence that is naturally present in the cell into which it has been introduced; (3) a nucleic acid sequence that is used to add additional copies of itself (i.e., homologous) or a similar nucleic acid sequence that is naturally present in the cell into which it has been introduced (such as IRP1 and / or IRP2; or (4) a silent naturally occurring or homologous nucleic acid sequence, the expression of which is induced in the cell into which it has been introduced. A mutant form refers to a nucleic acid sequence containing one or more nucleotides that are different from a wild-type or naturally occurring sequence, i.e., a mutant nucleic acid sequence contains one or more nucleotide substitutions, deletions, and / or insertions. In some cases, the transgene may also include a sequence encoding a leader peptide or signal sequence so that the transgenic product will be secreted from the cell.

[0149] The synthetic polynucleotide encoding IRP1 and / or IRP2 and optionally a promoter and / or CAR contained in the lymphocyte according to the present invention can preferably be integrated into the lymphocyte by viral transduction. Therefore, it should be understood that the synthetic polynucleotides disclosed for use in the lymphocyte according to the present invention can also be contained in the viral vector according to the present invention.

[0150] In certain embodiments, the viral vector comprises a single transgene. For example, in certain embodiments, the viral vector comprises a polynucleotide encoding IRP1 (SEQ ID NO: 1). In other embodiments, the viral vector comprises a polynucleotide encoding IRP2 (SEQ ID NO: 2). In other embodiments, the viral vector comprises a polynucleotide encoding IRP2 (SEQ ID NO: 3). In other embodiments, the viral vector comprises a polynucleotide encoding IRP2 (SEQ ID NO: 4). In other embodiments, the viral vector comprises a polynucleotide encoding IRP2 (SEQ ID NO: 5). In other embodiments, the viral vector comprises a polynucleotide encoding IRP2 (SEQ ID NO: 6). Preferably, the viral vector comprises a polynucleotide encoding IRP2 (SEQ ID NO: 2).

[0151] In certain embodiments, a viral vector may comprise more than one transgene. For example, a viral vector may comprise two or more polynucleotides encoding IRP1 (SEQ ID NO: 1) and one or more isoforms of IRP2 (SEQ ID NOs: 2-6). In another embodiment, a viral vector may comprise two or more polynucleotides encoding two or more isoforms of IRP2 (SEQ ID NOs: 2-6).

[0152] In addition, the viral vector may comprise one or more polynucleotides encoding IRP1 and / or IRP2 and another polynucleotide encoding CAR. Therefore, in one embodiment, the present invention relates to a viral vector according to the present invention, wherein the viral vector comprises another polynucleotide encoding CAR. Therefore, the viral vector can be used to simultaneously integrate the polynucleotide encoding CAR and at least one polynucleotide encoding IRP1 and / or IRP2 into cells, preferably lymphocytes.

[0153] In certain embodiments, the present invention relates to a viral vector according to the present invention, wherein the polynucleotide encoding CAR is transcriptionally linked to one or more polynucleotides encoding IRP1 and / or IRP2. The polynucleotide encoding CAR and the one or more polynucleotides encoding IRP1 and / or IRP2 may be transcriptionally linked as described above. That is, the polynucleotide encoding CAR and the one or more polynucleotides encoding IRP1 and / or IRP2 may be under the control of a common promoter.

[0154] As described herein, the polynucleotide encoding a CAR and the one or more polynucleotides encoding IRP1 and / or IRP2 can be separated by one or more IRESs, or can be linked by one or more polynucleotides encoding a self-cleaving peptide.

[0155] In certain embodiments, the present invention relates to a viral vector according to the present invention, wherein the polynucleotide encoding CAR and one or more polynucleotides encoding IRP1 and / or IRP2 are linked via a polynucleotide encoding a self-cleaving peptide.

[0156] In certain embodiments, the present invention relates to a viral vector according to the present invention, wherein the self-cleaving peptide is a 2A self-cleaving peptide.

[0157] In certain embodiments, the present invention relates to a viral vector according to the present invention, wherein the self-cleaving peptide is T2A.

[0158] In another embodiment, the viral vector may further comprise a promoter for controlling the expression of one or more polynucleotides encoding CAR and IRP1 and / or IRP2. Therefore, in another embodiment, the present invention relates to a viral vector according to the present invention, wherein at least one polynucleotide encoding IRP1 and / or IRP2 and optionally CAR is under the control of a promoter. The promoter may be a constitutive promoter or an inducible promoter, such as one of the constitutive or inducible promoters specified elsewhere herein. Preferably, the promoter is a constitutive promoter, such as the promoter EF-1α. Therefore, in a certain embodiment, the present invention relates to a viral vector according to the present invention, wherein the constitutive promoter is an EF-1α promoter.

[0159] In certain embodiments, the viral vector may comprise a polynucleotide having the following structure: 5'-CAR-self-cleaving peptide-IRP1-3'. In other embodiments, the viral vector may comprise a polynucleotide having the following structure: 5'-CAR-self-cleaving peptide-IRP2-3'. In other embodiments, the viral vector may comprise a polynucleotide having the following structure: 5'-constitutive promoter-CAR-self-cleaving peptide-IRP1-3'. In other embodiments, the viral vector may comprise a polynucleotide having the following structure: 5'-constitutive promoter-CAR-self-cleaving peptide-IRP2-3'. In other embodiments, the viral vector may comprise a polynucleotide having the following structure: 5'-constitutive promoter-CAR-T2A-IRP1-3'. In other embodiments, the viral vector may comprise a polynucleotide having the following structure: 5'-constitutive promoter-CAR-T2A-IRP2-3'. In other embodiments, the viral vector may comprise a polynucleotide having the following structure: 5'-constitutive promoter-CAR-P2A-IRP1-3'. In other embodiments, the viral vector may comprise a polynucleotide having the following structure: 5'-Constitutive Promoter-CAR-P2A-IRP2-3'. In other embodiments, the viral vector may comprise a polynucleotide having the following structure: 5'-Constitutive Promoter-CAR-E2A-IRP1-3'. In other embodiments, the viral vector may comprise a polynucleotide having the following structure: 5'-Constitutive Promoter-CAR-E2A-IRP2-3'. In other embodiments, the viral vector may comprise a polynucleotide having the following structure: 5'-Constitutive Promoter-CAR-F2A-IRP1-3'. In other embodiments, the viral vector may comprise a polynucleotide having the following structure: 5'-Constitutive Promoter-CAR-F2A-IRP2-3'.

[0160] In certain embodiments, the viral vector may comprise a polynucleotide having the following structure: 5'-IRP1-self-cleaving peptide-CAR-3'. In other embodiments, the viral vector may comprise a polynucleotide having the following structure: 5'-IRP2-self-cleaving peptide-CAR-3'. In other embodiments, the viral vector may comprise a polynucleotide having the following structure: 5'-constitutive promoter-IRP1-self-cleaving peptide-CAR-3'. In other embodiments, the viral vector may comprise a polynucleotide having the following structure: 5'-constitutive promoter-IRP2-self-cleaving peptide-CAR-3'. In other embodiments, the viral vector may comprise a polynucleotide having the following structure: 5'-constitutive promoter-IRP1-T2A-CAR-3'. In other embodiments, the viral vector may comprise a polynucleotide having the following structure: 5'-constitutive promoter-IRP2-T2A-CAR-3'. In other embodiments, the viral vector may comprise a polynucleotide having the following structure: 5'-constitutive promoter-IRP1-P2A-CAR-3'. In other embodiments, the viral vector may comprise a polynucleotide having the following structure: 5'-Constitutive Promoter-IRP2-P2A-CAR-3'. In other embodiments, the viral vector may comprise a polynucleotide having the following structure: 5'-Constitutive Promoter-IRP1-E2A-CAR-3'. In other embodiments, the viral vector may comprise a polynucleotide having the following structure: 5'-Constitutive Promoter-IRP2-E2A-CAR-3'. In other embodiments, the viral vector may comprise a polynucleotide having the following structure: 5'-Constitutive Promoter-IRP1-F2A-CAR-3'. In other embodiments, the viral vector may comprise a polynucleotide having the following structure: 5'-Constitutive Promoter-IRP2-F2A-CAR-3'.

[0161] The skilled artisan is aware of molecular biological methods for introducing transgenes and / or regulatory elements such as promoters into viral vectors.

[0162] In one embodiment, the invention relates to a pharmaceutical composition comprising lymphocytes according to the invention and a pharmaceutically acceptable carrier.

[0163] Lymphocytes of the present invention can be administered alone or as a pharmaceutical composition comprising lymphocytes of the present invention. In simple terms, the pharmaceutical composition of the present invention may comprise lymphocytes or lymphocyte groups as described herein in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions may include buffers such as neutral buffered saline, phosphate buffered saline, etc.; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. Pharmaceutical compositions according to the present invention may be administered in combination with diluents and / or other components such as IL-2 or other cytokines or cell populations. The compositions of the present invention are preferably formulated for intravenous administration.

[0164] In another embodiment, the present invention relates to a pharmaceutical composition comprising the viral vector according to the present invention and a pharmaceutically acceptable carrier.

[0165] In certain embodiments, it is contemplated that the subject will be treated directly by direct introduction of the vector. Viral vector compositions can be formulated for delivery by any available route, including but not limited to parenteral (e.g., intravenous), intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, rectal, and vaginal. Common routes of delivery include inhalation, parenteral, and transmucosal.

[0166] In certain embodiments, the pharmaceutical composition according to the present invention may comprise a viral vector comprising a polynucleotide encoding at least one iron regulatory protein according to the present invention and a second viral vector comprising a polynucleotide encoding a CAR. That is, the one or more polynucleotides encoding one or more iron regulatory proteins and the polynucleotide encoding a CAR may be located on two separate viral vectors, but may be contained in the same pharmaceutical composition.

[0167] In various embodiments, the pharmaceutical composition may comprise a viral vector in combination with a pharmaceutically acceptable carrier. As used herein, the language "pharmaceutically acceptable carrier" includes solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc., that are compatible with drug administration. Supplementary active compounds may also be incorporated into the composition.

[0168] In some embodiments, the active agent, i.e., the viral vector described herein and / or other agents to be administered with the vector, is prepared together with a carrier that will protect the compound from rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable biocompatible polymers such as ethylene-vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. The method for preparing such compositions will be apparent to those skilled in the art. Suitable materials can also be commercially available from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomes can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811. In some embodiments, the composition targets specific cell types or cells infected by viruses. For example, monoclonal antibodies targeting compositions for cell surface markers (e.g., endogenous markers or viral antigens expressed on the surface of infected cells) can be used.

[0169] It is advantageous to formulate the composition in dosage unit form for ease of administration and uniformity of dosage. As used herein, dosage unit form refers to physically discrete units suitable as unit dosages for the subject to be treated; each unit contains a predetermined quantity of viral vector and pharmaceutical carrier calculated to produce the desired therapeutic effect.

[0170] A unit dose need not be administered as a single injection, but may comprise a continuous infusion over a set period of time. A unit dose of the viral vectors described herein may conveniently be described in terms of transducing units (TU) of the viral vector, as defined by titrating the vector on a cell line such as HeLa or 293. In certain embodiments, a unit dose may be administered in 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 TU and higher range.

[0171] The pharmaceutical composition can be administered at various intervals and over various time periods as needed, for example, once a week for between about 1 and about 10 weeks; between about 2 and about 8 weeks; between about 3 and about 7 weeks; about 4 weeks; about 5 weeks; about 6 weeks, etc. It may be necessary to administer the therapeutic composition indefinitely. The skilled artisan will recognize that certain factors may influence the dosage and duration required to effectively treat a subject, including but not limited to the severity of the disease or condition, previous treatments, the general health and / or age of the subject, and the presence of other diseases. Treatment of a subject with a viral vector may include a single treatment or, in many cases, may include a series of treatments.

[0172] Exemplary dosages for administering viral vectors and methods for determining appropriate dosages are known in the art. It should also be understood that the appropriate dosage of the viral vector may depend on the specific recipient and mode of administration. The appropriate dosage level for any particular subject may depend on a variety of factors, including the subject's age, weight, general health, sex, and diet, time of administration, route of administration, excretion rate, other administered therapeutic agents, and the like.

[0173] In certain embodiments, viral vectors can be delivered to a subject by, for example, intravenous injection, topical administration, or stereotactic injection (see, for example, Chen et al. (1994) Proc. Natl. Acad. Sci. USA, 91: 3054). In certain embodiments, the vector can be delivered orally or by inhalation, and can be encapsulated or otherwise manipulated to protect it from degradation, enhance tissue or cell uptake, etc. Pharmaceutical preparations can include viral vectors in an acceptable diluent, or can include a sustained-release matrix in which the viral vector is embedded. Alternatively or additionally, when the vector can be produced intact from recombinant cells, such as in the case of retroviral or lentiviral vectors, the pharmaceutical preparation can include one or more cells that produce the vector. A pharmaceutical composition comprising a viral vector as described herein can be included in a container, package, or dispenser, optionally together with instructions for use.

[0174] The foregoing compositions, methods and uses are intended to be illustrative and not limiting. Other variations on compositions, methods and uses will be readily apparent to those skilled in the art using the teachings provided herein.

[0175] In another embodiment, the invention relates to a lymphocyte according to the invention, a viral vector according to the invention or a pharmaceutical composition according to the invention for use in therapy.

[0176] That is, the lymphocytes according to the present invention, the viral vectors according to the present invention, or the pharmaceutical composition comprising the lymphocytes and / or viral vectors according to the present invention can be used for treatment.

[0177] In certain embodiments, the present invention relates to a lymphocyte according to the invention, a viral vector according to the invention or a pharmaceutical composition according to the invention for use in the treatment of cancer.

[0178] The present invention provides the use of CAR as defined in the present invention to redirect the specificity of lymphocytes (e.g., T cells or NK cells) to tumor antigens. Disclosed herein is a type of cell therapy in which lymphocytes are genetically modified to express at least one IRP and CAR, wherein the resulting cells are infused into a recipient in need. The infused cells are capable of killing tumor cells in the recipient. Unlike antibody therapy, lymphocytes according to the present invention are able to replicate in vivo, thereby causing long-term persistence, which can lead to sustained tumor control.

[0179] Due to the overexpression of at least one IRP, the lymphocytes described herein can undergo robust in vivo expansion and can persist for extended periods of time. Without being bound by any particular theory, the anti-tumor immune response elicited by the lymphocytes of the present invention can be an active or passive immune response. In addition, the CAR-mediated immune response can be part of an adoptive immunotherapy approach, wherein the CAR-modified lymphocytes induce an immune response specific for the antigen-binding portion of the CAR.

[0180] While lymphocytes expressing at least one IRP and a CAR are preferred for use in treating cancer, it is also contemplated that lymphocytes expressing only at least one IRP but not a CAR can be used. In this case, a synthetic polynucleotide encoding at least one IRP can be introduced into lymphocytes ex vivo, and the genetically engineered lymphocytes can then be administered to a subject suffering from cancer. Optionally, lymphocytes can be stimulated ex vivo with tumor antigens to increase specificity for a certain type of cancer or tumor. In certain embodiments, genetically engineered lymphocytes expressing at least one IRP can be injected directly into a tumor.

[0181] However, it should be understood that the present invention also encompasses the use of lymphocytes that overexpress IRP1 and / or IRP2 but do not overexpress CAR in the treatment of cancer.

[0182] For example, IRP1 and / or IRP2 can be overexpressed in TILs or TCR-modified T cells, which are then used for cell therapy. The inventors have demonstrated that overexpression of IRPs in lymphocytes leads to more robust lymphocyte proliferation. Therefore, it is reasonable to overexpress IRPs in TILs or TCR-modified T cells to produce more effective cell therapy.

[0183] Furthermore, IRP1 and / or IRP2 can be overexpressed in NK cells, which are then used in cell therapy.In certain embodiments, the NK cells are allogeneic NK cells.

[0184] "Treating" a disease, as the term is used herein, means reducing the frequency or severity of at least one sign or symptom of the disease or condition experienced by a subject.

[0185] A "disease" is a state of health in an animal (including a human) in which the animal is unable to maintain homeostasis and in which the animal's health would continue to deteriorate if the disease were not ameliorated. In contrast, a "disorder" in an animal is a state of health in which the animal is able to maintain homeostasis, but the animal's health is not as good as it would be in the absence of the disorder. A disorder does not necessarily lead to a further decline in the animal's health if left untreated.

[0186] The terms "patient," "subject," "individual," and the like are used interchangeably herein and refer to any animal or cell thereof suitable for use in the methods described herein, whether in vitro or in situ. In certain non-limiting embodiments, the patient, subject, or individual is a human.

[0187] As used herein, the term "cancer" is defined as a disease characterized by the rapid and uncontrolled growth of abnormal cells. Cancer cells can spread locally or to other parts of the body through the bloodstream and lymphatic system.

[0188] Cancers that can be treated with lymphocytes according to the present invention include tumors that are not vascularized or substantially not yet vascularized, as well as vascularized tumors. Cancers can include non-solid tumors (such as hematological tumors, for example leukemias and lymphomas) or can include solid tumors. Cancer types to be treated with lymphocytes of the present invention include, but are not limited to, carcinomas, blastomas, and sarcomas, as well as certain leukemias or lymphoid malignancies, benign and malignant tumors, and malignant tumors, such as sarcomas, carcinomas, and melanomas. Adult tumors / cancers and pediatric tumors / cancers are also included.

[0189] In one embodiment, the invention relates to a lymphocyte, a viral vector or a pharmaceutical composition for use according to the invention, wherein the cancer is a hematological cancer or a solid tumor. In a specific embodiment, the hematological cancer is acute lymphoblastic leukemia, diffuse large B-cell lymphoma, Hodgkin lymphoma, acute myeloid leukemia or multiple myeloma and the solid tumor is colon cancer, breast cancer, pancreatic cancer, ovarian cancer, hepatocellular carcinoma, lung cancer, neuroblastoma, glioblastoma or sarcoma.

[0190] Hematologic cancers are cancers of the blood or bone marrow. Examples of hematologic (or blood-borne) cancers include leukemias, including acute leukemias (such as acute lymphocytic leukemia, acute myeloid leukemia, acute myeloid leukemia, and myeloblastic, promyelocytic, granulocytic, monocytic, and erythroleukemias), chronic leukemias (such as chronic myeloid (granulocytic) leukemia, chronic myeloid leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphomas, Hodgkin's disease, non-Hodgkin's lymphomas (indolent and advanced forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia, and myelodysplasia.

[0191] Solid tumors are abnormal masses of tissue that do not typically contain cysts or fluid areas. Solid tumors can be benign or malignant. Different types of solid tumors are named after the cell types that form them (such as sarcomas, carcinomas, and lymphomas). Examples of solid tumors (such as sarcomas and carcinomas) include fibrosarcomas, myosarcomas, liposarcoma, chondrosarcomas, osteosarcomas, and other sarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, lymphoid malignancies, pancreatic cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland cancer, medullary thyroid cancer, papillary thyroid cancer, pheochromocytoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatocarcinoma, bile duct cancer, chorionic villus, choriocarcinoma, cholangiocarcinoma, choriocarcinoma, cholangiocarcinoma, choriocarcinoma, leukemia, leukemia, ulcer, ulcer, thrombus, ulcer, cystic fibrosis ... Choroidal carcinoma, Wilms' tumor, cervical cancer, testicular tumors, seminoma, bladder cancer, melanoma, and CNS tumors such as gliomas (such as brain stem gliomas and mixed gliomas), glioblastomas (also known as glioblastoma multiforme), astrocytomas, CNS lymphomas, germ cell tumors, medulloblastomas, schwannomas, craniopharyngiomas, ependymomas, pinealomas, hemangioblastomas, acoustic neuromas, oligodendrogliomas, meningiomas, neuroblastomas, retinoblastomas, and brain metastases.

[0192] The lymphocytes of the present invention can be designed to target CD19 and can be used to treat cancers and disorders, including but not limited to pre-B ALL (pediatric indications), adult ALL, mantle cell lymphoma, diffuse large B-cell lymphoma, rescue post-allogeneic bone marrow transplantation, etc.

[0193] The CAR-modified lymphocytes described herein can also be used as a vaccine for ex vivo immunization and / or in vivo treatment of a subject. Preferably, the subject is a human.

[0194] With respect to ex vivo immunization, at least one of the following occurs in vitro prior to administering the lymphocytes to a subject: i) expansion of the cells, ii) introduction of at least one synthetic nucleotide encoding at least one IRP and / or CAR into the cells, and / or iii) cryopreservation of the cells.

[0195] In vitro procedures are well known in the art. In simple terms, lymphocytes are isolated from a subject (preferably a human) and genetically modified (i.e., in vitro transduction or transfection) with at least one vector expressing at least one IRP and / or CAR disclosed herein. Cells modified by CAR expressing at least one IRP can be administered to a recipient to provide therapeutic benefits. The recipient can be a human and the modified lymphocytes can be autologous relative to the recipient. Alternatively, the lymphocytes can be allogeneic, syngeneic, or xenogeneic relative to the recipient.

[0196] The ex vivo expansion procedures for hematopoietic stem and progenitor cells described in U.S. Patent No. 5,199,942 can be applied to the cells of the present invention. Other suitable methods are known in the art, and thus the present invention is not limited to any particular method for ex vivo expansion of cells. Briefly, the ex vivo culture and expansion of lymphocytes comprises: (1) collecting CD34+ hematopoietic stem and progenitor cells from a peripheral blood harvest or bone marrow explant of a mammal; and (2) expanding such cells ex vivo. In addition to the cell growth factors described in U.S. Patent No. 5,199,942, other factors such as flt3-L, IL-1, IL-3, and c-kit ligand can be used for the culture and expansion of cells.

[0197] In addition to the use of cell-based vaccines for ex vivo immunization, the present disclosure also provides compositions and methods for in vivo immunization to elicit an immune response to an antigen in a patient.

[0198] Typically, lymphocytes activated and amplified as described herein can be used to treat and prevent diseases that occur in immunocompromised individuals. Specifically, the CAR-modified lymphocytes described herein can be used to treat chronic lymphocytic leukemia (CCL). In certain embodiments, the lymphocytes described herein can be used to treat patients at risk of developing CCL. Therefore, the present disclosure provides treatment or prevention of CCL, comprising administering a therapeutically effective amount of lymphocytes of the present invention to a subject in need thereof.

[0199] Alternatively, the lymphocytes used to treat cancer can be NK cells, TILs, or TCR-modified lymphocytes. NK cells, TILs, or TCR-modified T cells can be modified using the methods of the present invention so that they overexpress IRP1 and / or IRP2, which can result in more efficient proliferation of NK cells, TILs, or TCR-modified T cells in vivo.

[0200] NK cells used in cancer treatment can be allogeneic NK cells because allogeneic NK cells have graft-versus-leukemia / tumor (GvL / GvT) effects without causing graft-versus-host disease (GvHD), thus causing less immunopathology.

[0201] TILs for cancer treatment can be obtained as described in WO 2018 / 182817 and can be further modified by introducing at least one polynucleotide encoding at least one IRP.

[0202] In one embodiment, the present invention relates to a lymphocyte according to the invention, a viral vector according to the invention or a pharmaceutical composition according to the invention for use in the prevention and / or treatment of a viral infection.

[0203] That is to say, lymphocytes according to the present invention can also be used for preventing and treating viral infections. Known virus-specific T cells can be used for preventing or treating viral infections, such as, but not limited to, in subjects receiving hematopoietic stem cell transplantation. Virus-specific T cells can be produced by stimulating and amplifying T cells with viral antigens, such as antigen-presenting cells that display viral antigen peptides, complete viral particles, viral lysates, complete viral proteins or viral vectors. Alternatively, virus-specific T cells can be produced by expressing natural or engineered T cell receptors known to bind specific viral antigens. The virus-specific T cells obtained can then be administered to subjects suffering from viral infections or subjects who are at risk of acquiring viral infections.

[0204] Expressing at least one IRP in virus-specific T cells, thereby creating a pseudo-iron-deficient state, can result in virus-specific T cells that proliferate more robustly after administration to a subject. Thus, virus-specific T cells genetically engineered to express at least one IRP may be more effective in preventing or treating viral infections compared to non-genetically engineered virus-specific T cells. Synthetic polynucleotides encoding at least one IRP can be introduced into T cells before, during, or after stimulation of the T cells with viral antigens.

[0205] The prevention and treatment of viral infections as described above do not necessarily require the presence of a CAR. However, the use of lymphocytes further comprising a CAR according to the present invention can improve the recognition of viruses by lymphocytes in at least some cases. In this case, the CAR may preferably comprise an antigen binding domain that specifically binds to a viral antigen.

[0206] In one embodiment, the present invention relates to a lymphocyte, a viral vector or a pharmaceutical composition for use according to the present invention, wherein the viral infection is caused by human immunodeficiency virus (HIV), adenovirus, polyomavirus, influenza virus or human herpes virus, in particular, wherein the human herpes virus is cytomegalovirus (CMV), Epstein-Barr virus (EBV), herpes simplex virus (HSV), varicella-zoster virus (VZV) or human herpes virus 8 (HHV8).

[0207] Human cytomegalovirus is a ubiquitous beta herpes virus with a prevalence of 50-100% in the general population. Although it may manifest as a mild self-limiting disease in a host with normal immune function, CMV can cause serious life-threatening diseases in a host with compromised immune function. Since CMV persists in a latent form after acute infection, CMV-specific CD4+ and CD8+ T cells are necessary to maintain viral quiescence. In patients after HSCT, in the absence of donor immunity and in other immunodeficiency states, CMV can be reactivated in the form of retinitis, limited pneumonia (pneumonitis), hepatitis or enterocolitis. The adoptive transfer of CMV-specific T cells is a reasonable strategy for treating and preventing CMV reactivation in such individuals, and many clinical trials have confirmed the overall excellent efficacy of virus-specific T cells. CMV-specific VSTs produced by naive T cells in umbilical cord blood (UCB) have also been shown to be effective. These VSTs show specificity for atypical epitopes while maintaining functionality.

[0208] EBV is a ubiquitous, highly immunogenic gamma-herpes virus that can cause unique complications after transplantation. More than 90% of the general population has been infected and remains serum positive for life. The manifestations of primary EBV infection vary greatly, from asymptomatic infection to a debilitating viral disease. Thereafter, in most cases, EBV remains dormant in B cells and mucosal epithelial reservoirs for life under continuous T cell immune surveillance. In these healthy individuals, up to 2% of circulating T cells are EBV-specific. During the immunodeficiency phase after HSCT, EBV reactivation can lead to viremia and life-threatening post-transplant lymphoproliferative disease (PTLD). Although the monoclonal antibody rituximab has successfully treated severe EBV disease in many patients by eliminating B cells in which EBV virus resides, it causes a long-term reduction in antibody production and is not always successful in controlling PTLD.

[0209] Adenovirus infections can range from mild upper respiratory tract infections to a range of life-threatening pneumonia, gastrointestinal, hepatic, renal, and neurological complications. Following infection, the virus remains latent in lymphoid tissues, but can reactivate during prolonged periods of lack of T cell immunity. Adenovirus causes potentially fatal viral complications in recipients after HSCT. Antiviral drugs such as ribavirin are largely ineffective. However, adenovirus-specific T cells generated from healthy donors have been shown to be effective in treating even advanced disease. For this reason, adenovirus antigens are often incorporated into the production of multi-virus-specific T cell products.

[0210] BK and JC polyomaviruses, which are usually latent in the healthy tissues of most adult individuals, reactivate after HSCT and in immunodeficient individuals. BK virus can manifest as nephropathy and life-threatening hemorrhagic cystitis (HC). In rare cases, the closely related JC virus causes fatal brain damage caused by progressive multifocal leukoencephalopathy. Polyomavirus-specific VSTs are being developed to combat these viruses. A case report describes the successful use of a BK VST, after which the patient's HC completely resolved without bystander organ toxicity, GVHD, or graft rejection. It is clear that the platform developed for ex vivo selection and amplification of VSTs is easily adaptable to many other viruses that complicate immunodeficiency states, and future developments include developing VSTs to target a range of viruses including VZV, HHV, and even HIV or influenza.

[0211] In one embodiment, the present invention relates to a method for treating a subject suffering from cancer or for preventing and / or treating a viral infection in a subject, the method comprising administering to the subject a therapeutically effective amount of lymphocytes according to the invention, a viral vector according to the invention or a pharmaceutical composition according to the invention.

[0212] The lymphocytes or pharmaceutical compositions described herein can be administered in a manner suitable for the disease to be treated (or prevented). The amount and frequency of administration will be determined by factors such as the patient's condition and the type and severity of the patient's disease, but the appropriate dosage can be determined by clinical trials.

[0213] As used herein, an "effective amount" refers to an amount that provides a therapeutic or preventive beneficial effect. The term "therapeutically effective amount" refers to the amount of the subject compound that will elicit a biological or medical response in a tissue, system, or subject that is sought by a researcher, veterinarian, physician, or other clinician. The term "therapeutically effective amount" includes an amount of a compound that, upon administration, is sufficient to prevent the development of one or more signs or symptoms of the condition or disease being treated or to alleviate such signs or symptoms to some extent. The therapeutically effective amount will vary depending on the compound, the disease and its severity, and the age, weight, etc., of the subject to be treated.

[0214] When an "immunologically effective amount," "anti-tumor effective amount," "tumor suppressive effective amount," or "therapeutic amount" is indicated, the precise amount of the lymphocytes or composition of the present invention to be administered can be determined by a physician taking into account individual differences in the patient's (subject's) age, weight, tumor size, extent of infection or metastasis, type of viral infection, severity of viral infection, and / or condition. In general, 10 4 to 10 9 cells / kg body weight, preferably 10 5 to 10 6 The pharmaceutical compositions comprising the lymphocytes described herein are administered at a dose of 10 cells / kg body weight (including all integer values ​​within those ranges). Lymphocyte compositions can also be administered multiple times at these doses. Lymphocytes can be administered using infusion techniques commonly known in immunotherapy (Rosenberg et al., 1988, New Eng. J. of Med; 319: 1676.). A person skilled in the art of medicine can easily determine the optimal dosage and treatment regimen for a particular patient by monitoring the patient's symptoms and adjusting the treatment accordingly.

[0215] It may be desirable to administer activated lymphocytes to the experimenter, and then draw blood (or perform apheresis), activate lymphocytes from therein according to the present invention, and return these activated and amplified lymphocytes to the patient. This process can be carried out repeatedly every few weeks. Lymphocytes can be activated from the drawing of blood of 10mL to 400mL, for example, lymphocytes can be activated from the drawing of blood of 20mL, 30mL, 40mL, 50mL, 60mL, 70mL, 80mL, 90mL or 100mL. Without being bound by theory, using this multiple drawing of blood / multiple return scheme can be used for selecting certain lymphocyte populations.

[0216] The administration of lymphocytes or compositions can be carried out in any convenient manner, including by aerosol inhalation, injection, intake, infusion, implantation or transplantation. Lymphocytes as herein described or compositions can be administered subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous (iv) injection or intraperitoneally to the subject. For example, lymphocytes as herein described or compositions can be administered to the patient by intradermal or subcutaneous injection. In another example, lymphocytes as herein described or compositions can preferably be administered by intravenous injection. Lymphocytes or compositions can be directly injected into a tumor, lymph node or site of infection.

[0217] In some cases, lymphocytes activated and expanded using the methods described herein or other methods known in the art for expanding lymphocytes to therapeutic levels are administered to a patient along with (e.g., before, simultaneously with, or after) any number of related therapeutic modalities, including but not limited to treatment with agents such as antiviral therapy, cidofovir and interleukin 2, ribavirin, rituximab, cytarabine (also known as ARA-C), or natalizumab treatment for MS patients or efalizumab treatment for psoriasis patients or other treatments for PML patients. It is also disclosed herein that the lymphocytes of the present invention can be used in combination with chemotherapy, radiation, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolate mofetil and FK506, antibodies or other immunoablative agents such as CAMPATH, anti-CD3 antibodies or other antibody therapy, cytotoxins, fludaribine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines and irradiation. These drugs inhibit the calcium-dependent phosphatase calcineurin (cyclosporine and FK506) or inhibit p70S6 kinase (rapamycin), which is important for growth factor-induced signaling (Liu et al., 1991, Cell; 66: 807-815; Henderson et al., 1991, Immun; 73: 316-321; Bierer et al., 1993, Curr. Opin. Immun; 5: 763-773). It is also disclosed herein that the lymphocytes or compositions of the present invention can be administered to a patient in combination with (e.g., before, simultaneously with, or after) bone marrow transplantation, T cell ablation therapy using chemotherapeutic agents (such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide), or antibodies (such as OKT3 or CAMPATH). It is also described herein that the lymphocytes or compositions of the present invention can be administered after B cell ablation therapy such as agents that react with CD20 (e.g., Rituxan). For example, a subject can undergo standard treatment with high-dose chemotherapy followed by a peripheral blood stem cell transplant. In some cases, after the transplant, the subject can receive an infusion of the expanded immune cells of the present invention, or the expanded cells can be administered before or after surgery.

[0218] The dosage of the above treatment to be administered to a subject will vary with the exact nature of the condition being treated and the recipient of the treatment. Adjustments to human administration dosages may be made according to practices recognized in the art.

[0219] In one embodiment, the invention relates to a method according to the invention, wherein the cancer is a hematological cancer or a solid tumor, in particular, wherein the hematological cancer is acute lymphoblastic leukemia, diffuse large B-cell lymphoma, Hodgkin's lymphoma, acute myeloid leukemia and multiple myeloma, or wherein the solid tumor is colon cancer, breast cancer, pancreatic cancer, ovarian cancer, hepatocellular carcinoma, lung cancer, neuroblastoma, glioblastoma and sarcoma.

[0220] In one embodiment, the invention relates to a method according to the invention, wherein the viral infection is caused by human immunodeficiency virus (HIV), adenovirus, polyomavirus, influenza virus or human herpes virus, in particular, wherein the human herpes virus is cytomegalovirus (CMV), Epstein-Barr virus (EBV), herpes simplex virus (HSV), varicella-zoster virus (VZV) or human herpes virus 8 (HHV8).

[0221] In one embodiment, the present invention relates to a method for producing a lymphocyte according to the present invention, the method comprising the steps of: a) providing lymphocytes obtained from a subject; b) introducing a synthetic polynucleotide encoding at least one iron regulatory protein into the lymphocyte of step (a), wherein the iron regulatory protein is IRP1 (SEQ ID NO: 1) and / or IRP2 (SEQ ID NO: 2-6) and c) expressing the at least one iron regulatory protein encoded by the synthetic polynucleotide introduced into the lymphocyte in step (b). It will be understood that the lymphocyte may be any lymphocyte disclosed herein.

[0222] Optionally, the present invention also relates to a method according to the invention, wherein in step (b) a second synthetic polynucleotide encoding a chimeric antigen receptor (CAR) is introduced into the lymphocyte.

[0223] That is, the method according to the present invention can be used to produce lymphocytes that overexpress IRP1 and / or IRP2. In addition, the method according to the present invention can be used to produce lymphocytes comprising two synthetic polynucleotides (i.e., a first synthetic polynucleotide encoding IRP1 and / or IRP2 and a second synthetic polynucleotide encoding CAR). In the latter case, it should be understood that the first synthetic polynucleotide encoding IRP1 and / or IRP2 and the second synthetic polynucleotide encoding CAR can be fused to each other as described herein. Alternatively, the first synthetic polynucleotide encoding IRP1 and / or IRP2 and the second synthetic polynucleotide encoding CAR can be unrelated. That is, the first synthetic polynucleotide encoding IRP1 and / or IRP2 and the second synthetic polypeptide encoding CAR can be independently introduced into lymphocytes. Preferably, the synthetic polynucleotide is introduced into lymphocytes by viral transduction and incorporated into the genome of the lymphocytes. Therefore, the first synthetic polynucleotide encoding IRP1 and / or IRP2 and the second synthetic polypeptide encoding CAR can be contained in different viral vectors. A first viral vector comprising a synthetic polynucleotide encoding IRP1 and / or IRP2 and a second viral vector comprising a synthetic polynucleotide encoding CAR can be introduced into lymphocytes in a single transduction experiment. Alternatively, a first viral vector comprising a synthetic polynucleotide encoding IRP1 and / or IRP2 and a second viral vector comprising a synthetic polynucleotide encoding CAR can be used to transduce lymphocytes in a stepwise manner. For example, lymphocytes according to the present invention can first be transduced with a viral vector comprising a synthetic polynucleotide encoding CAR to produce CAR T cells or CAR NK cells without restriction, and the lymphocytes can be transduced with a viral vector comprising a synthetic polynucleotide encoding IRP1 and / or IRP2 in a second step. Alternatively, lymphocytes can first be transduced with a viral vector comprising a synthetic polynucleotide encoding IRP1 and / or IRP2, and the lymphocytes can be transduced with a viral vector comprising a synthetic polynucleotide encoding CAR in a second step.

[0224] Before the lymphocytes of the present invention are genetically modified, a lymphocyte source can be obtained from a subject. Lymphocytes can be obtained from a variety of sources, including peripheral blood, mononuclear cells, bone marrow, lymph node tissue, blood, thymus tissue, tissue from an infected site, ascites, pleural effusion, spleen tissue, and tumors. In the present invention, any type of lymphocyte available in the art can be used. Generally speaking, the skilled artisan is aware of methods for isolating a certain type of lymphocyte from a suitable source.

[0225] Certain types of lymphocytes, particularly peripheral blood mononuclear cells (PBMCs), can be isolated using a number of techniques known to those skilled in the art, such as Ficoll. TMSeparation) is obtained from a blood unit collected from a subject. In addition, cells from an individual's circulating blood can be obtained by apheresis. Apheresis products typically contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. Cells collected by apheresis can be washed to remove the plasma fraction and placed in an appropriate buffer or culture medium for subsequent processing steps. For example, cells can be washed with phosphate-buffered saline (PBS). Alternatively, the washing solution can lack calcium and magnesium or can lack multiple (if not all) divalent cations. As will be readily understood by those of ordinary skill in the art, the washing step can be accomplished by methods known to those skilled in the art, such as by using a semi-automated "flow-through" centrifuge (e.g., Cobe 29 cell processor, Baxter CytoMate, or Haemonetics Cell Saver 5) according to the manufacturer's instructions. After washing, the cells can be resuspended in a variety of biocompatible buffers, such as Ca+-free, Mg+-free PBS, PlasmaLyte A, or other saline solutions with or without buffers. Alternatively, undesirable components of the apheresis sample can be removed and the cells can be resuspended directly in culture medium. Alternatively, the apheresis sample can be resuspended in culture medium, for example, by PERCOLL TM Certain types of lymphocytes are isolated by centrifugation of gradients or by counterflow centrifugal elutriation to lyse red blood cells and remove mononuclear cells.

[0226] Specific subsets of lymphocytes, such as CD3+, CD28+, CD4+, CD8+, CD45RA+ and CD45RO+ T cells, CD16+ and CD56+ NK cells or CD3+, CD56+ and CD161+ NKT cells, can be further separated by positive or negative selection techniques. It is known in the art which surface antigens are present on the corresponding type of lymphocytes. Thus, the skilled person is able to select positive or negative selection conditions that allow enrichment or isolation of a particular type of lymphocyte. In addition, the skilled person is aware of commercial kits for enriching and / or isolating certain types of lymphocytes.

[0227] In certain embodiments, T cells can be expressed by beads conjugated to anti-CD3 / anti-CD28, such as M-450CD3 / CD28T) are incubated together for a period of time to separate, and this period of time is enough to positively select the desired T cells. The scope of the time period can be 30 minutes to 36 hours or longer, and can include all integer values ​​therebetween. In certain embodiments, the time period is at least 0.5, 1, 2, 3, 4, 5 or 6 hours. Alternatively, the time period is 10 to 24 hours. For separating T cells from leukemia patients, using a longer incubation time (such as 24 hours) can improve cell yield. In any case where there are fewer T cells compared to other cell types, such as when separating tumor infiltrating lymphocytes (TIL) from tumor tissue or from immunocompromised individuals, a longer incubation time can be used to separate T cells. In addition, using a longer incubation time can improve the efficiency of capturing CD8+T cells. Therefore, by simply shortening or extending the time allowing T cells to bind to the CD3 / CD28 beads and / or by increasing or reducing the ratio of beads to T cells, it is possible to preferentially select or target a subpopulation of T cells at the start of culture or at other time points during the process. In addition, by increasing or decreasing the ratio of anti-CD3 and / or anti-CD28 antibodies on beads or other surfaces, subpopulations of T cells can be preferentially selected or targeted at the start of culture or at other time points. The skilled artisan will recognize that multiple rounds of selection can also be used in the context of the present invention. In certain embodiments, it may be desirable to perform a selection procedure and use "unselected" cells during activation and amplification. "Unselected" cells may also undergo other rounds of selection.

[0228] Lymphocyte populations enriched by negative selection can be completed with a combination of antibodies for surface markers unique to negatively selected cells. One method is to perform cell sorting and / or selection by negative magnetic immunoadhesion or flow cytometry, which uses a mixture of monoclonal antibodies for cell surface markers present on negatively selected cells. For example, in order to enrich CD4+ cells by negative selection, monoclonal antibody mixtures typically include antibodies for CD14, CD20, CD11b, CD16, HLA-DR, and CD8. In certain embodiments, it is desirable to enrich or positively select regulatory T cells that typically express CD4+, CD25+, CD62L, GITR+, and FoxP3+. Alternatively, in certain embodiments, T regulatory cells can be removed by anti-C25-conjugated beads or other similar selection methods.

[0229] In certain embodiments, NK cells from healthy donors or patients can be enriched from PBMC or directly from blood using a human NK cell negative selection separation kit (Miltenyi Biotec or STEMCELL Technologies) by incubating with magnetic beads according to the manufacturer's instructions. For example, when using the separation kit from Miltenyi Biotec, unwanted cells (i.e., T cells, B cells, macrophages, and monocytes) can be removed with a mixture of monoclonal anti-human antibodies conjugated to biotin for antigens that NK cells do not express, and the PBMC concentration is 2.5 billion cells / mL. NK cell MicroBead Cocktail can then be used to magnetically label unwanted cells labeled with biotin-conjugated antibodies and removed using MACS columns. For example, when using the separation kit from STEMCELL Technologies, unwanted cells (i.e., T cells, B cells, macrophages, and monocytes) can be removed with a tetrameric anti-human antibody complex for antigens that NK cells do not express, and the PBMC concentration is 50 million cells / mL. Unwanted cells labeled with the tetrameric antibody complex can then be magnetically labeled with dextran-coated magnetic particles and removed using a magnet.

[0230] In order to separate the desired lymphocyte population by positive selection or negative selection, the concentration and surface (for example, particles, such as beads) of cells can be changed. In certain embodiments, it can be expected that the volume (i.e., increasing the concentration of cells) that beads and cells are mixed together can be significantly reduced to ensure the maximum contact of cells with beads. For example, in one embodiment, a concentration of 2,000,000,000 cells / mL can be used. In one embodiment, a concentration of 1,000,000,000 cells / mL can be used. In another embodiment, a cell concentration greater than 100,000,000 cells / mL can be used. In another embodiment, a cell concentration of 0,0.15,0.2,0.25,0.3,0.35,0.4,0.45 or 0.500,000 cells / mL can be used. In another embodiment, a cell concentration of 0.75,0.8,0.85,0.9,0.95 or 100,000,000 cells / mL can be used. In other embodiments, a concentration of 1.25 or 150,000,000 cells / mL can be used. Use high concentration to produce increased cell yield, cell activation and cell expansion. In addition, the use of high cell concentrations can make it possible to more effectively capture cells that can weakly express the target antigen of interest (such as CD28-negative T cells), or cells from samples where many tumor cells are present (i.e., leukemic blood, tumor tissue, etc.). Such cell populations may have therapeutic value and would be desirable to obtain. For example, the use of high cell concentrations can make it possible to more effectively select CD8+ T cells that typically have weak CD28 expression.

[0231] In a related embodiment, it may be desirable to use lower concentrations of cells. The interaction between the particles and cells can be minimized by significantly diluting the mixture of cells and a surface (e.g., particles, such as beads). This can select cells that express a large amount of the desired antigen to be bound to the particles. For example, at dilute concentrations, CD4+ T cells may express higher levels of CD28 than CD8+ T cells and be captured more effectively.

[0232] Lymphocytes can be incubated at 2-10°C or at room temperature on a rotator at different speeds for different lengths of time. Cells used for stimulation can also be frozen after the washing step. Without being bound by theory, the freezing and subsequent thawing steps can provide a more uniform product by removing granulocytes and, to some extent, monocytes from the cell population. Following the washing steps to remove plasma and platelets, the cells can be suspended in a freezing solution. Although many freezing solutions and parameters are known in the art and will be used in the present invention, one method involves using PBS containing 20% ​​DMSO and 8% human serum albumin, or culture medium containing 10% dextran 40 and 5% dextrose, 20% human serum albumin and 7.5% DMSO, or 1.25% Plasmalyte-A, 3.25% dextrose 5%, 0.45% NaCl, 10% dextran 40 and 5% dextrose, 20% human serum albumin and 7.5% DMSO, or other suitable cell freezing media containing, for example, Hespan and PlasmaLyte-A. The cells can then be frozen to -80°C at a rate of 1°C per minute and stored in the vapor phase of a liquid nitrogen tank. In addition to immediate uncontrolled freezing at -20°C or in liquid nitrogen, other controlled freezing methods can be used.

[0233] Cryopreserved cells can be thawed and washed as described herein and allowed to stand at room temperature for one hour prior to activation using the methods of the invention.

[0234] In the context of the present invention, it is also contemplated that a blood sample or apheresis product may be collected from a subject in the time period before the amplification cells as described herein may be needed. Therefore, the source of cells to be amplified can be collected at any necessary time point, and desired cells (such as lymphocytes) are separated and frozen for later use in the cell therapy of any number of diseases or conditions that will benefit from cell therapy (such as those described herein). A blood sample or apheresis sample can be obtained from a generally healthy subject. In certain embodiments, a blood sample or apheresis sample can be obtained from a generally healthy subject with the risk of developing a disease, but not yet developing a disease, and the target cells can be separated and frozen for later use. In certain embodiments, cells can be amplified, frozen and used at a subsequent time. In certain embodiments, a sample can be collected from a patient shortly after diagnosing a specific disease as described herein but before any treatment. In addition, cells can be isolated from a blood sample or apheresis sample of a subject prior to any number of relevant treatment modalities, including but not limited to treatment with agents such as natalizumab, efalizumab, antiviral agents, chemotherapy, radiation, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolate mofetil and FK506, antibodies or other immunoablative agents such as CAMPATH, anti-CD3 antibodies, cyclophosphamide (cytoxan), fludarabine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228 and irradiation. These drugs inhibit the calcium-dependent phosphatase calcineurin (cyclosporine and FK506) or inhibit p70S6 kinase (rapamycin) which is important for growth factor-induced signaling (99-101). In certain embodiments, cells can be isolated from a patient and frozen for later use in conjunction with (e.g., before, concurrently with, or after) bone marrow or stem cell transplantation, T cell ablative therapy using chemotherapeutic agents (such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide), or antibodies (directed against OKT3 or CAMPATH). In certain embodiments, cells can be isolated prior to treatment following B cell ablative therapy (such as an agent reactive with CD20, e.g., Rituximab) and can be frozen for later use in such treatment.

[0235] In certain embodiments of the present invention, lymphocytes can be obtained directly from the patient after treatment. In this respect, it has been observed that in certain cancer treatments, especially after treatment with drugs that damage the immune system, soon after treatment, during the time period when the patient will usually recover from treatment, the quality of the lymphocytes obtained in terms of their ex vivo expansion capacity can be optimal or improved. Similarly, after using the method described herein to manipulate in vitro, these cells can be in a preferred state for enhanced transplantation and in vivo expansion. Therefore, in the present invention, it is expected that blood cells, including lymphocytes, dendritic cells or other cells of hematopoietic lineages, will be collected during this recovery phase. In addition, in certain embodiments, mobilization (such as mobilization with GM-CSF) and conditioning regimens can be used to produce the following state in the subject, wherein the re-proliferation, recirculation, regeneration and / or amplification of specific cell types are advantageous, especially during the limited time window after treatment. Exemplary cell types include T cells, NK cells, B cells, dendritic cells and other immune system cells.

[0236] The present invention encompasses one or more synthetic polynucleotides comprising a polynucleotide sequence encoding one or more IRPs and optionally a CAR. Synthetic polynucleotides encoding the desired molecule can be obtained using recombinant methods known in the art, for example, by screening a library from cells expressing the gene, by obtaining the gene from a vector known to include the gene, or by direct isolation from cells and tissues containing the gene using standard techniques. Alternatively, the gene of interest can be produced synthetically rather than by cloning.

[0237] The present invention also provides vectors into which the synthetic polynucleotides of the present invention can be inserted. Vectors derived from retroviruses (such as lentiviruses) are suitable tools for achieving long-term gene transfer because they allow transgenes to be stably integrated over a long period of time and to propagate in daughter cells. Lentiviral vectors have additional advantages over vectors derived from onco-retroviruses (such as murine leukemia viruses) because they can transduce non-proliferating cells, such as hepatocytes. They also have the additional advantage of low immunogenicity.

[0238] A "vector" is a composition of matter that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid to the interior of a cell. Many vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides associated with ions or amphiphilic compounds, plasmids, and viruses. Therefore, the term "vector" includes autonomously replicating plasmids or viruses. The term should also be interpreted to include non-plasmid and non-viral compounds that promote the transfer of nucleic acids into cells, such as polylysine compounds, liposomes, etc. Examples of viral vectors include but are not limited to adenoviral vectors, adeno-associated viral vectors, retroviral vectors, etc.

[0239] In short, the expression of natural or synthetic polynucleotides encoding IRPs and optional CARs is generally achieved by operably linking the polynucleotide encoding the IRP or optional CAR polypeptide or portion thereof to a promoter and incorporating the construct into an expression vector. The vector may be suitable for replication and / or integration in eukaryotic organisms. Typical cloning vectors contain transcriptional and translational terminators, initiation sequences, and promoters that can be used to regulate expression of the desired polynucleotide.

[0240] "Expression vector" refers to a vector comprising a recombinant polynucleotide comprising an expression control sequence operably linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression; other elements for expression may be provided by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate a recombinant polynucleotide.

[0241] The expression constructs of the present invention can also be used for nucleic acid immunization and gene therapy using standard gene delivery protocols.Methods for gene delivery are known in the art.

[0242] The synthetic polynucleotide encoding the IRP and, optionally, the CAR, can be cloned into a variety of vector types. For example, the polynucleotide can be cloned into vectors including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0243] In addition, expression vector can be provided to cell in the form of viral vector.Viral vector technology is well known in the art, and is for example described in Sambrook et al. (2001, Molecular Cloning:A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals. The virus that can be used as a vector includes but is not limited to retrovirus, adenovirus, adeno-associated virus, herpes virus and slow virus. Generally, suitable vectors are included in at least one organism with a functional origin of replication, promoter sequence, convenient restriction endonuclease site and one or more selective markers (for example, WO 01 / 96584; WO 01 / 29058; and U.S. Patent number 6,326,193).

[0244] A variety of virus-based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. The selected gene can be inserted into a vector and packaged into retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to the subject's cells in vivo or ex vivo. A variety of retroviral systems are known in the art. In the present invention, adenoviral vectors or lentiviral vectors are preferably used.

[0245] In some embodiments, the promoter element of the present invention is the promoter of the transcriptional initiation site.Other promoter elements, such as enhancers, can be used to regulate the frequency of transcription initiation.Usually, these elements are located in the zone of 30-110bp upstream of the start site, but have recently shown that many promoters also contain functional elements downstream of the start site.The spacing between the promoter elements is normally flexible, so when the elements are inverted or moved relative to each other, the promoter function can be retained.In the thymidine kinase (tk) promoter, before the activity began to decline, the spacing between the promoter elements can be increased to 50bp apart.Depending on the promoter, it seems that each element can work in a coordinated or independent manner to activate transcription.

[0246] An example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence that can drive the high-level expression of any polynucleotide sequence operably connected thereto. Another example of a suitable promoter is elongation growth factor-1a (EF-1a). However, other constitutive promoter sequences can also be used, including but not limited to simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukosis virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, and human gene promoters, such as but not limited to actin promoter, myosin promoter, hemoglobin promoter and creatine kinase promoter. In addition, the present invention should not be limited to the use of constitutive promoters, and inducible promoters are also contemplated as a part of the present invention. The use of inducible promoters provides a molecular switch that can, when desired expression is desired, open the expression of the polynucleotide sequence operably connected thereto or, when undesired expression is desired, close expression. Examples of inducible promoters include, but are not limited to, the metallothionine promoter, the glucocorticoid promoter, the progesterone promoter, and the tetracycline promoter.

[0247] In order to evaluate the expression of IRP or optional CAR polypeptide or portion thereof, the expression vector to be introduced into the cell may also contain a selective marker gene or a reporter gene or both to facilitate identification and selection of expressing cells from a cell population that is attempted to be transfected or infected by a viral vector. In other embodiments, the selective marker can be carried on a separate DNA fragment and used in a co-transfection procedure. Both the selective marker and the reporter gene may be flanked by appropriate regulatory sequences to enable their expression in the host cell. Available selective markers include, for example, antibiotic resistance genes such as neo.

[0248] Reporter gene is used to identify potential transfected cells and evaluate the functionality of regulatory sequences. Generally, reporter gene is a gene that is not present in or is not expressed by a recipient organism or tissue, and encodes a polypeptide that is expressed by some easily detectable properties (such as enzymatic activity). The expression of reporter gene can be measured at the appropriate time after DNA is introduced into recipient cells. Suitable reporter gene can include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secretory alkaline phosphatase or green fluorescent protein gene (Ui-Tei et al., 2000, FEBS Letters; 479: 79-82). Suitable expression systems are well known and can be prepared using known techniques or commercially available.

[0249] Methods for introducing genes into cells and expressing them are known in the art. In the context of expression vectors, vectors can be readily introduced into host cells, such as mammalian, bacterial, yeast, or insect cells, by any method known in the art. For example, expression vectors can be transferred to host cells by physical, chemical, or biological means.

[0250] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for generating cells containing vectors and / or exogenous polynucleotides are well known in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). A preferred method for introducing polynucleotides into host cells is calcium phosphate transfection.

[0251] Biological methods for introducing target polynucleotides into host cells include the use of DNA and RNA vectors. Viral vectors and especially retroviral vectors have become the most widely used methods for inserting genes into mammalian (e.g., human) cells. Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses. See, for example, U.S. Patent Nos. 5,350,674 and 5,585,362.

[0252] Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles and liposomes. The exemplary colloidal system used as a delivery vehicle in vitro and in vivo is a liposome (for example, an artificial membrane vesicle). In the case of using a non-viral delivery system, an exemplary delivery vehicle can be a liposome. It is envisioned that lipid formulations are used to introduce polynucleotides into host cells (in vitro, in vitro or in vivo). In another embodiment, polynucleotides can be relevant to lipids. The polynucleotides relevant to lipids can be encapsulated in the aqueous interior of liposomes, be interspersed in the lipid bilayer of liposomes, be connected to liposomes by the connecting molecule relevant to liposomes and polynucleotides, be embedded in liposomes, be compounded with liposomes, be dispersed in the solution containing lipids, be mixed with lipids, be combined with lipids, be included in lipids as a suspension, include micelles or be compounded with them, or otherwise be relevant to lipids. Lipid, lipid / DNA or lipid / expression vector related compositions are not limited to any specific structure in solution. For example, they can exist as bilayer structures, micelles, or have a "collapsed" structure. They can also simply be dispersed in a solution, possibly forming aggregates of uneven size or shape. Lipids are fatty substances that can be naturally occurring or synthetic lipids. For example, lipids include fat droplets naturally present in the cytoplasm and a class of compounds containing long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[0253] Lipids suitable for use are available from commercial sources. For example, dimyristylphosphatidylcholine ("DMPC") can be obtained from Sigma, St. Louis, MO; dicetyl phosphate ("DCP") can be obtained from K&K Laboratories (Plainview, NY); cholesterol ("Choi") can be obtained from Calbiochem-Behring; dimyristylphosphatidylglycerol ("DMPG") and other lipids can be obtained from Avanti Polar Lipids, Inc. (Birmingham, AL). Stock solutions of lipids in chloroform or chloroform / methanol can be stored at approximately -20°C. Chloroform can be used as the sole solvent because it evaporates more easily than methanol. "Liposome" is a general term that includes a variety of unilamellar and multilamellar lipid vehicles formed by producing encapsulated lipid bilayers or aggregates. Liposomes can be characterized as having a vesicle structure having a phospholipid bilayer membrane and an internal aqueous medium. Multilamellar liposomes can have multiple lipid layers separated by an aqueous medium. They can form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components can rearrange themselves before forming a closed structure and entrain water and dissolved solutes between the lipid bilayers (Ghosh et al., 1991, Glycobiology; 5; 505-10). However, compositions that have structures in solution that differ from normal vesicular structures are also included. For example, lipids can assume micellar structures or exist only as heterogeneous aggregates of lipid molecules. Lipofectamine-nucleic acid complexes are also contemplated.

[0254] As used herein, the term "transfection" or "transformation" or "transduction" refers to the process of transferring or introducing exogenous nucleic acid into a host cell. A "transfected" or "transformed" or "transduced" cell is a cell that has been transfected, transformed, or transduced with an exogenous nucleic acid. Such cells include the primary subject cell and its progeny.

[0255] Regardless of the method used to introduce the exogenous synthetic polynucleotide into the host cell, a variety of assays can be performed to confirm the presence of the recombinant DNA sequence in the host cell. Such assays include, for example, "molecular biology" assays well known to those skilled in the art, such as Southern and Northern blotting, RT-PCR and PCR; "biochemical" assays, such as detecting the presence or absence of specific peptides, for example, by immunological means (ELISA, Western blotting, flow cytometry) or by assays described herein to identify agents falling within the scope of the present invention.

[0256] In another embodiment, the present invention relates to a method according to the present invention, wherein a synthetic polynucleotide encoding a chimeric antigen receptor (CAR) is combined with a synthetic polynucleotide encoding at least one iron regulatory protein, in particular, wherein the at least one iron regulatory protein is IRP1 and / or IRP2.

[0257] The chimeric antigen receptor and at least one IRP may be encoded on separate synthetic polynucleotides that are not contiguous or directly linked to each other. In this case, the synthetic polynucleotide encoding at least one IRP and the synthetic polynucleotide encoding the CAR may be introduced into the cell separately using the same or different methods. Alternatively, one or more genes encoding at least one IRP and the gene encoding the CAR may be combined in a single synthetic polynucleotide. If the combined synthetic polynucleotide contains all the genes encoded in two separate synthetic polypeptides, then the two synthetic polynucleotides are said to be combined.

[0258] For example, if genes encoding at least one IRP and a CAR are intended to be integrated into lymphocytes by viral transduction, the gene or genes encoding the at least one IRP and the gene encoding the CAR can be contained in separate viral vectors or can be combined in a single viral vector.

[0259] In another embodiment, the invention relates to a method according to the invention, wherein the lymphocytes are activated before or after introduction of the one or more synthetic polynucleotides into the lymphocytes.

[0260] Whether before or after the lymphocytes are genetically modified to express at least one IRP or optionally the desired CAR, they can be activated and expanded prior to administration to a subject. The skilled artisan is aware that specific conditions are required to activate different types of lymphocytes.

[0261] Technicians know the method for activating NK cells.For example, NK cells as described herein can be activated by culturing in suitable culture medium (for example, minimum essential medium or RPMI culture medium 1640 or X-vivo 15 (Lonza), CellGro culture medium (Cellgenix), IMDM (Gibco)), and the culture medium can contain the factor necessary for proliferation and survival, including serum (for example, fetal bovine, human or horse serum) supplemented with IL-15 and / or IL-12 and / or IL-18. The activation of NK cells can also be achieved by supplementing IL-2 in culture medium. The activation of NK cells can be improved by adding feeder cell lines to culture. The suitable feeder cell lines for activating NK cells are lymphoblastoid cell lines or autologous peripheral blood mononuclear cells (irradiated) transformed by cancer cell lines, genetically modified K562 cells or EBV.

[0262] Typically, the T cells described herein can be activated by contacting with a reagent that stimulates CD3 / TCR complex-related signals and a surface ligand that stimulates the co-stimulatory molecules on the T cell surface. Specifically, T cell populations can be stimulated by contacting with anti-CD3 antibodies or their antigen-binding fragments or anti-CD2 antibodies fixed on the surface, or by contacting with protein kinase C activators (such as bryostatin) and calcium ion carriers. In order to costimulate the auxiliary molecules on the T cell surface, a ligand that binds the auxiliary molecules can be used. For example, T cell populations can be contacted with anti-CD3 antibodies and anti-CD28 antibodies under conditions suitable for stimulating T cell proliferation. In order to stimulate the proliferation of CD4+T cells or CD8+T cells, anti-CD3 antibodies and anti-CD28 antibodies can be used. Anti-CD28 antibodies 9.3, B-T3, XR-CD28 (Diaclone, France), other methods known in the art may also be used (Berg et al., 1998, Transplant Proc; 30(8): 3975-3977; Haanen et al., 1999, J. Exp. Med; 190: 1319-1328; Garland et al., 1999, J. Immunol Meth. 227: 53-63).

[0263] The main stimulation signal and costimulatory signal of T cell can be provided by different schemes.For example, the reagent providing each signal can be in solution form or coupled to a surface.When coupled to a surface, the reagent can be coupled to the same surface (i.e., in " cis " form) or coupled to a separate surface (i.e., in " trans " form). Alternatively, a kind of reagent can be coupled to a surface, and another reagent can be in solution form. For example, the reagent providing a costimulatory signal can be combined with the surface, and the reagent providing the main activation signal can be in solution form or coupled to the surface, or both reagents can be in solution form. Alternatively, reagent can be a soluble form, subsequently cross-linked with the surface, such as cells or antibodies expressing Fc receptors or other binding agents in conjunction with the reagent. In this regard, referring to, for example, artificial antigen presenting cells (aAPCs) in U.S. Patent Application Publication 20040101519 and 20060034810, it is expected that the cell can be used for activation and amplification of T cells.

[0264] The two reagents are immobilized on the beads, either on the same bead, i.e., "cis", or on separate beads, i.e., "trans". By way of example, the reagent providing the primary activation signal can be an anti-CD3 antibody or an antigen-binding fragment thereof, and the reagent providing the co-stimulatory signal can be an anti-CD28 antibody or an antigen-binding fragment thereof; and the two reagents can be co-immobilized on the same bead with equal molecular weight. For example, a 1:1 ratio of each antibody bound to the beads for CD4+ T cell expansion and T cell growth can be used. In some cases, a certain anti-CD3:CD28 antibody ratio bound to the beads can be used so that an increase in T cell expansion is observed compared to the expansion observed using a 1:1 ratio. An increase of about 1 to about 3 times can be observed compared to the expansion observed using a 1:1 ratio. The ratio of CD3:CD28 antibodies bound to the beads can be in the range of 100:1 to 1:100 and all integer values ​​therebetween. In one embodiment, there may be more anti-CD28 antibodies than anti-CD3 antibodies bound to the particles, i.e., the CD3:CD28 ratio may be less than 1. In some cases, the ratio of anti-CD28 antibodies to anti-CD3 antibodies bound to the beads may be greater than 2:1. For example, a 1:100 CD3:CD28 ratio of antibodies bound to the beads may be used, a 1:75 CD3:CD28 ratio of antibodies bound to the beads may be used, a 1:50 CD3:CD28 ratio of antibodies bound to the beads may be used, a 1:30 CD3:CD28 ratio of antibodies bound to the beads may be used, a 1:10 CD3:CD28 ratio of antibodies bound to the beads may be used, or a 1:3 CD3:CD28 ratio of antibodies bound to the beads may be used. Alternatively, a 3:1 CD3:CD28 ratio of antibodies bound to the beads may be used.

[0265] The ratio of particles to cells can be in the range of 1:500 to 500:1, and any integer value therebetween can be used to stimulate T cells or other target cells. As one of ordinary skill in the art will readily appreciate, the ratio of particles to cells can depend on the size of the particles relative to the target cells. For example, small beads may only bind to a few cells, while larger beads may bind to many cells. As described above, the ratio of anti-CD3 and anti-CD28 coupled particles to T cells that result in T cell stimulation can vary, but some preferred values ​​include 1:100, 1:50, 1:40, 1:30, 1:20, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, and 15:1, with a preferred ratio being at least 1:1 particle / T cell. Alternatively, a particle-to-cell ratio of 1:1 or less can be used. A preferred particle-to-cell ratio can be 1:5. The particle-to-cell ratio can vary depending on the number of days of stimulation. For example, on the first day, the particle-to-cell ratio can be 1:1 to 10:1, and additional particles can be added to the cells every day or every other day for up to 10 days, with a final ratio of 1:1 to 1:10 (based on the cell count on the day of addition). Alternatively, the particle-to-cell ratio can be 1:1 on the first day of stimulation and adjusted to 1:5 on the third and fifth days of stimulation. In another case, particles can be added every day or every other day to reach a final ratio of 1:1 on the first day of stimulation and 1:5 on the third and fifth days of stimulation. In another case, the particle-to-cell ratio can be 2:1 on the first day of stimulation and adjusted to 1:10 on the third and fifth days of stimulation. In another case, particles can be added every day or every other day to reach a final ratio of 1:1 on the first day of stimulation and 1:10 on the third and fifth days of stimulation. Those skilled in the art will appreciate that a variety of other ratios may be suitable for use with the present invention. Specifically, the ratio will vary depending on the particle size and cell size and type.

[0266] T cells can be combined with reagent-coated beads, which can then be separated from the cells and the cells can then be cultured. Alternatively, the reagent-coated beads and cells may not be separated prior to cultivation, but may be cultured together. In another embodiment, the beads and cells may first be concentrated by applying a force (such as a magnetic force) to increase the attachment of cell surface markers, thereby inducing cell stimulation.

[0267] Cells (e.g. 10 4 to 10 9 T cells) and beads (e.g., 1:1 ratio of In one embodiment, the cells are mixed with M-450 CD3 / CD28T paramagnetic beads in a buffer solution, preferably PBS (without divalent cations such as calcium and magnesium). Again, one of ordinary skill will readily appreciate that any cell concentration can be used. It may be desirable to significantly reduce the volume in which the particles and cells are mixed together (i.e., increase the concentration of the cells) to ensure maximum contact between the cells and the particles. For example, a concentration of approximately 2 billion cells / mL may be used. In another embodiment, a concentration exceeding 100 million cells / mL may be used. In another embodiment, a cell concentration of 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, or 0.50 million cells / mL may be used. In another embodiment, a cell concentration of 0.75, 0.8, 0.85, 0.9, 0.95, or 100 million cells / mL may be used. In yet another embodiment, a concentration of 125 or 150 million cells / mL may be used. Using high concentrations can result in increased cell yield, cell activation, and cell expansion. In addition, the use of high cell concentrations can allow for more efficient capture of cells that may weakly express the target antigen of interest, such as CD28-negative T cells. Such cell populations may have therapeutic value and, in certain embodiments, would be desirable. For example, the use of high cell concentrations can allow for more efficient selection of CD8+ T cells, which typically have weak CD28 expression.

[0268] The mixture can be cultured for a number of hours (about 3 hours) to about 14 days or any integer number of hours therebetween. The mixture can be cultured for 21 days. In one embodiment, the beads and T cells can be cultured together for about eight days. In another embodiment, the beads and T cells can be cultured together for 2-3 days. It is also desirable to have several stimulation cycles so that the culture time of the T cells can be 60 days or longer.

[0269] T cells exposed to different stimulation times can show different characteristics. For example, the helper T cell group (TH, CD4+) of the peripheral blood mononuclear cell product obtained by typical blood or apheresis is more than cytotoxic or suppressive T cell group (TC, CD8). T cell group is produced by stimulating CD3 and CD28 receptors in vitro amplification of T cells, and the cell group is mainly composed of TH cells before about 8-9 days, and after about 8-9 days, the T cell group includes more and more TC cell groups. Therefore, depending on the purpose of treatment, it can be advantageous to infuse a T cell group mainly comprising TH cells to a subject. Similarly, if the antigen-specific subgroup of TC cells has been separated, it can be beneficial to amplify this subgroup to a greater extent.

[0270] Furthermore, phenotypic markers other than CD4 and CD8 also varied significantly during cell expansion, but were largely reproducible. This reproducibility therefore enables the customization of activated T cell products for specific purposes.

[0271] Suitable conditions for lymphocyte culture include an appropriate culture medium (e.g., minimum essential medium or RPMI medium 1640 or X-vivo 15 (Lonza), CellGro medium (Cellgenix), IMDM (Gibco)) that may contain factors required for proliferation and survival, including serum (e.g., fetal bovine, human, or horse serum), interleukin-2 (IL-2), insulin, IFN-γ, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, TGFβ, and TNF-α, or any other additives known to the skilled artisan for cell growth. Other additives for cell growth may include, but are not limited to, surfactants, human plasma protein powder (plasmanate), and reducing agents such as N-acetyl-cysteine ​​and 2-mercaptoethanol. Culture medium can include RPMI1640, AIM-V, DMEM, MEM, α-MEM, F-12, X-Vivo 15, X-Vivo 20, IMDM and CellGro, Optimizer, which is added with amino acids, sodium pyruvate and vitamins, does not contain serum or is supplemented with an appropriate amount of serum (or plasma) or a limited hormone group, and / or is added with cytokines sufficient to grow and amplify NK cells and T cells. Antibiotics (such as penicillin and streptomycin) can be included only in experimental cultures, rather than in cultures of cells to be infused into a subject. Lymphocytes can be maintained under conditions necessary to support growth (e.g., suitable temperature (e.g., 37°C) and atmosphere (e.g., air plus 5% CO2)).

[0272] In another embodiment, the invention relates to a method according to the invention, wherein the at least one synthetic polynucleotide is introduced into the lymphocytes by viral transduction, in particular by lentiviral transduction.

[0273] As described above, one or more synthetic polynucleotides encoding at least one IRP1 and optionally a CAR can be introduced into lymphocytes by any method known in the art. However, as described above, it is preferred that one or more synthetic polynucleotides be introduced into lymphocytes by viral transduction. More preferably, the viral vector used to introduce synthetic polynucleotides into lymphocytes is a lentiviral vector. Since viral vectors are typically integrated into the host cell genome at random locations, it is preferred that the synthetic polynucleotides comprise at least one gene encoding an IRP and regulatory elements required for expression of at least one gene encoding an IRP in the host cell.

[0274] As used herein, "lentivirus" refers to a genus of the Retroviridae family. Lentiviruses are unique among retroviruses in that they are able to infect non-dividing cells; they can deliver large amounts of genetic information into the host cell's DNA, making them one of the most effective methods of gene delivery vectors. HIV, SIV, and FIV are all examples of lentiviruses. Vectors derived from lentiviruses provide a means to achieve significant levels of gene transfer in vivo.

[0275] In one embodiment, the invention relates to a method according to the invention, wherein the synthetic polynucleotide encoding the CAR is transcriptionally linked to a synthetic polynucleotide encoding an IRP1 and / or an IRP2.

[0276] In one embodiment, the invention relates to a method according to the invention, wherein the synthetic polynucleotide encoding the CAR and the polynucleotide encoding the IRP1 and / or IRP2 are linked via a polynucleotide encoding a self-cleaving peptide.

[0277] In one embodiment the invention relates to a method according to the invention, wherein the self-cleaving peptide is a 2A self-cleaving peptide.

[0278] In one embodiment the invention relates to a method according to the invention, wherein the self-cleaving peptide is T2A.

[0279] In one embodiment, the invention relates to a method according to the invention, wherein the one or more synthetic polynucleotides are introduced into the lymphocytes by viral transduction.

[0280] In one embodiment, the invention relates to a method according to the invention, wherein the viral transduction is performed using a viral vector according to any embodiment provided herein.

[0281] Examples of synthetic polynucleotides encoding IRP1 and / or IRP2 and optionally CAR, promoters and / or other regulatory elements (such as IRES or polynucleotides encoding self-cleaving peptides) are disclosed elsewhere herein and are mutatis mutandis applicable to the claimed methods. Preferably, the viral vector according to the present invention is used in the method according to the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0282] Figure 1: Naive and cytokine-enhanced NK cells similarly rely on glycolysis for IFN-γ production

[0283] (A) Schematic diagram of the experiment used to generate CE NK cells. (B) IFN-γ production by NV and CE NK cells that were unstimulated (no stimulation) or stimulated with IL-12 / IL-18 (mean ± SEM, n = 18 donors). (C) GMFI of CD69 expression on NV and CE NK cells that were unstimulated (no stimulation) or stimulated with IL-12 / IL-18 (mean ± SEM, n = 13 donors). (D) PCA of transcriptomic data depicting group relationships in NV and CE NK cells that were unstimulated (no stimulation) or stimulated with IL-12 / IL-18. The proportion of component variance is expressed as a percentage (n = 5 donors). (E) Heat map of transcriptomic data showing the relative expression of mRNA encoding glycolytic genes from NV and CE NK cells that were unstimulated (no stimulation) or stimulated with IL-12 / IL-18 (n = 5 donors). (F) Upper panel: Representative mitochondrial perturbation assays of NV and CE NK cells that were not stimulated (no stimulation) or stimulated with IL-12 / IL-18. Glycolysis (extracellular acidification rate - ECAR) was measured in the hippocampus after injection of oligomycin, FCCP, and rotenone. Lower panel: Basal and maximum rates of ECAR in NV and CE NK cells that were not stimulated (no stimulation) or stimulated with IL-12 / IL-18 analyzed by mitochondrial perturbation assay (mean ± SEM, n = 12 donors). (G) Upper panel: Representative histograms of NBDG uptake in NV and CE NK cells that were not stimulated (no stimulation) or stimulated with IL-12 / IL-18. Lower panel: GMFI of NBDG uptake in NV and CE NK cells that were not stimulated (no stimulation) or stimulated with IL-12 / IL-18 (mean ± SEM, n = 15 donors). (H) Expression of IFNG mRNA in NV and CE NK cells that were unstimulated (no stimulation) or stimulated with IL-12 / IL-18, IL-12 / IL-18+2-DG. Transcript levels were determined relative to 18S mRNA levels and normalized to unstimulated (no stimulation) NV NK cells (mean ± SEM, n = 6 donors). (I) Upper panel: IFN-γ production by NV and CE NK cells that were unstimulated (no stimulation) or stimulated with IL-12 / IL-18, IL-12 / IL-18+2-DG (mean ± SEM, n = 6 donors). Lower panel: IFN-γ production by NV and CE NK cells that were unstimulated (no stimulation) or stimulated with IL-12 / IL-18 in 10 mM glucose and 2 mM glucose (mean ± SEM, n = 5 donors). Statistical significance was assessed by paired two-tailed Student's t-test (C, F, H, I) or linear regression analysis (B, G, H, I).*p<0.05, **p<0.01, ***p<0.001; ns, not significant.

[0284] Figure 2: Activated CE NK cells are characterized by high levels of cell surface CD71 and rapid cell proliferation.

[0285] (A) Upper panel: Representative histograms of CD98 expression on NV and CE NK cells that were unstimulated (no stimulation) or stimulated with IL-12 / IL-18. Lower panel: MFI of CD98 expression on NV and CE NK cells that were unstimulated (no stimulation) or stimulated with IL-12 / IL-18 (mean ± SEM, n = 8 donors). (B) Upper panel: Representative histograms of CD71 expression on NV and CE NK cells that were unstimulated (no stimulation) or stimulated with IL-12 / IL-18. Lower panel: GMFI and percentage of CD71 expression on NV and CE NK cells that were unstimulated (no stimulation) or stimulated with IL-12 / IL-18 (mean ± SEM, n = 14 donors). (C) Left panel: Representative Western blot of total CD71 expression in NV and CE NK cells that were unstimulated (no stimulation) or stimulated with IL-12 / IL-18. Right: Total CD71 expression normalized to actin in NV and CE NK cells that were unstimulated (no stimulation) or stimulated with IL-12 / IL-18 (mean ± SEM, n = 13 donors). (D) Left: GMFI of CD71 expression on NV and CE NK cells that were unstimulated (no stimulation) or stimulated with K562 (mean ± SEM, n = 6). Right: Percentage of CD71+ NK cells on NV and CE NK cells that were unstimulated (no stimulation) or stimulated with K562 (mean ± SEM, n = 6 donors). (E) Top: Representative histograms of Tf-488 uptake in NV and CE NK cells that were unstimulated (no stimulation) or stimulated with IL-12 / IL-18. Bottom: GMFI of Tf-488 uptake in NV and CE NK cells that were unstimulated (no stimulation) or stimulated with IL-12 / IL-18 (mean ± SEM, n = 10 donors). (F) Top: Schematic diagram of the experiment used to analyze CFSE dilution in NV and CE NK cells. Middle: Representative histograms of CFSE dilution in NV and CE NK cells that were unstimulated (no stimulation) or stimulated with IL-12 / IL-18. Bottom: Percentage of proliferating NV and CE NK cells that were unstimulated (no stimulation) or stimulated with IL-12 / IL-18 analyzed by CFSE dilution (mean ± SEM, n = 13 donors). (G) Heat map of relative expression of mRNA encoding cell cycle genes (GO: 0006098) in NV and CE NK cells that were unstimulated (no stimulation) or stimulated with IL-12 / IL-18 (n = 5 donors).(H) Percentage of proliferating NV and CE NK cells unstimulated (no stimulation) or stimulated with IL-12 / IL-18, IL-12 / IL-18 + BIP (1, 10, and 50 μM) analyzed by CFSE dilution (mean ± SEM; n = 11 donors for unstimulated, IL-12 / IL-18, and IL-12 / IL-18 + BIP 10 μM stimulation; n = 8 donors for IL-12 / IL-18 + BIP 1 μM stimulation; n = 3 donors for IL-12 / IL-18 + BIP 50 μM stimulation). (I) GMFI of CD69 expression on NV and CE NK cells unstimulated (no stimulation) or stimulated with IL-12 / IL-18, IL-12 / IL-18 + BIP 100 μM (mean ± SEM, n = 5 donors). (J) Upper panel: Heat map of the relative expression of mRNA encoding the PPP gene (GO:0006098) in NV and CE NK cells that were not stimulated (no stimulation) or stimulated with IL-12 / IL-18 (n = 5 donors). Lower panel: Percentage of proliferating cells in NV and CE NK cells that were not stimulated (no stimulation) or stimulated with IL-12 / IL-18, IL-12 / IL-18+6AN 50 μM analyzed by CFSE dilution (mean ± SEM, n = 6 donors). Statistical significance was assessed by paired two-tailed Student's t-test (F, H, I, J) or linear regression analysis (A, B, C, E). *p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant.

[0286] Figure 3: CD71-mediated iron uptake and dietary iron availability influence NK cell function

[0287] (A) upper figure: schematic diagram of the experiment for analyzing the CFSE dilution in WT and TfrcY20H / Y20H NK cells from the spleen. Lower left figure: representative histogram of CFSE dilution in WT and TfrcY20H / Y20H NK1.1+NK cells from the spleen under IL-12 / IL-18 stimulation. Lower right figure: the percentage of proliferation WT and TfrcY20H / Y20H NK1.1+NK cells from the spleen in IL-15LD analyzed by CFSE dilution or stimulated with IL-12 / IL-18 (mean ± SEM; for WT NK cells, n = 5; for TfrcY20H / Y20H NK cells, n = 6). (B) upper figure: schematic diagram of MCMV infection experiment in mice fed + / – iron diet for 6 weeks. Lower panel: Serum levels of iron, ferritin, UIBC, TIBC from mice fed a + / - iron diet for 6 weeks; and hematocrit (mean ± SEM; n = 8-18 for iron, ferritin, UIBC, and TIBC; and n = 3 for hematocrit). (C) Left panel: Percentage of NK1.1+ NK cells in the spleen of mice fed a + / - iron diet for 6 weeks (mean ± SEM, n = 5). Right panel: Percentage of CD8+, CD4+, CD19+ cells in the spleen of mice fed a + / - iron diet for 6 weeks (mean ± SEM, n = 5). (D) Left panel: Percentage of CD27+CD11b–, CD27+CD11b+, CD27–CD11b+ on NK1.1+ NK cells in the spleen of mice fed a + / - iron diet for 6 weeks (mean ± SEM, n = 5). Right: Percentage of KLRG1+ and CD62L+ on NK1.1+ NK cells in the spleen of mice fed a + / - iron diet for 6 weeks (mean ± SEM, n = 5). (E) Left: Virus titers in the liver and spleen of WT MCMV-infected mice 3 dpi fed a + / - iron diet for 6 weeks (each point represents the data of cells isolated from one mouse, the data are expressed as the fold change difference normalized to the mice fed a + iron diet, the horizontal line represents the median, n = 10). Right: Virus titers in the liver and spleen of Δm157 MCMV-infected mice 3 dpi fed a + / - iron diet for 6 weeks (each point represents the data of cells isolated from one mouse, the data are expressed as the fold change difference normalized to the mice fed a + iron diet, the horizontal line represents the median, n = 9-10). (F) Percentage of IFN-γ+ in NK1.1+ NK cells in the liver and spleen of WT MCMV-infected mice fed the + / - diet for 6 weeks at 1.5 dpi (mean ± SEM, n = 4-5). Statistical significance was assessed by unpaired two-tailed Student's t-test (A, B, C, D, E, F).*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; ns, not significant.

[0288] Figure 4: CD71 supports NK cell proliferation and optimal effector function during viral infection

[0289] (A) Left panel: Percentage and absolute number of NK1.1+ NK cells in the liver of Tfrcfl / fl and Tfrcfl / flNcr1Cre mice (mean ± SEM, n = 10). Right panel: Percentage and absolute number of NK1.1+ NK cells in the spleen of Tfrcfl / fl and Tfrcfl / flNcr1Cre mice (mean ± SEM, n = 17-22). (B) Upper left panel: Percentage and absolute number of CD8+ cells in the liver of Tfrcfl / fl and Tfrcfl / flNcr1Cre mice (mean ± SEM, n = 10). Upper right panel: Percentage and absolute number of CD4+ cells in the liver of Tfrcfl / fl and Tfrcfl / flNcr1Cre mice (mean ± SEM, n = 4). Lower panel: Percentage and absolute number of CD19+ cells in the liver of Tfrcfl / fl and Tfrcfl / flNcr1Cre mice (mean ± SEM, n = 10). (C) Upper left panel: Percentage and absolute number of CD8+ cells in the spleen of Tfrcfl / fl and Tfrcfl / flNcr1Cre mice (mean ± SEM, n = 9-19). Upper right panel: Percentage and absolute number of CD4+ cells in the spleen of Tfrcfl / fl and Tfrcfl / flNcr1Cre mice (mean ± SEM, n = 11-22). Lower panel: Percentage and absolute number of CD19+ cells in the spleen of Tfrcfl / fl and Tfrcfl / flNcr1Cre mice (mean ± SEM, n = 12-22). (D) Upper panel: Percentages of CD27+CD11b–, CD27+CD11b+, and CD27–CD11b+ on NK1.1+ NK cells in the livers of Tfrcfl / fl and Tfrcfl / flNcr1Cre mice (mean ± SEM, n = 10). Lower panel: Percentages of CD62L+ and Ly6C+ on NK1.1+ NK cells in the livers of Tfrcfl / fl and Tfrcfl / flNcr1Cre mice (mean ± SEM, n = 6). (E) Upper panel: Percentages of CD27+CD11b–, CD27+CD11b+, and CD27–CD11b+ on NK1.1+ NK cells in the spleens of Tfrcfl / fl and Tfrcfl / flNcr1Cre mice (mean ± SEM, n = 5). Bottom: Percentages of KLRG1+, CD62L+, and Ly6C+ on NK1.1+ NK cells in the spleens of Tfrcfl / fl and Tfrcfl / flNcr1Cre mice (mean ± SEM, n=5). (F) Percentages of Ly49H+ on NK1.1+ NK cells in the livers and spleens of Tfrcfl / fl and Tfrcfl / flNcr1Cre mice (mean ± SEM, n=5-6).(G) Upper left panel: Schematic diagram of adoptive transfer experiments to Klra8- / - recipients to track the expansion of WT and Tfrcfl / fl NK cells after MCMV infection. Upper right panel: Representative flow chart of gating of adoptively transferred CD45.1+ and CD45.2+ (Ly49H+NK1.1+) NK cells in the liver of WT MCMV-infected recipients at 7 dpi. Lower panel: Percentage of adoptively transferred WT (Ly49H+NK1.1+CD45.1+) and Tfrcfl / flNcr1Cre (Ly49H+NK1.1+CD45.2+) NK cells in the liver, spleen, lung, and blood of WT MCMV-infected recipients at 7 and 30 dpi (each point represents data from cells isolated from one mouse; bars represent ± SEM; two independent experiments, the first for 7 dpi, n=5 and for 30 dpi, n=3-5; the second for 7 dpi, n=4 and for 30 dpi, n=2). (H) Left panel: Schematic diagram of adoptive transfer experiments performed on Rag2- / -IL2rg- / - recipients to track the expansion of WT and Tfrcfl / fl NK cells. Right panel: Percentage of adoptively transferred WT (Ly49H+NK1.1+CD45.1+) and Tfrcfl / flNcr1Cre (Ly49H+NK1.1+CD45.2+) NK cells in liver, spleen, lung, and blood at 6 dpt (each point represents data from cells isolated from one mouse, bars represent ± SEM, n=3-4). (I) Upper left panel: Schematic representation of adoptive transfer experiments performed on Klra8- / - recipients to analyze CFSE dilution in WT and Tfrcfl / fl NK cells after WT MCMV infection. Upper right panel: Representative histogram of CFSE dilution in adoptively transferred WT and Tfrcfl / flNcr1Cre NK1.1+ NK cells in the liver of WT MCMV-infected recipients at 3.5 dpi. Lower panel: CFSE GMFI of adoptively transferred WT (Ly49H+NK1.1+CD45.1+) and Tfrcfl / flNcr1Cre (Ly49H+NK1.1+CD45.2+) NK cells in the liver and spleen of WT MCMV-infected recipients at 3.5 dpi (mean ± SEM, two independent experiments, first n = 5, second n = 4). (J) Percentage of proliferating Tfrcfl / fl and Tfrcfl / flNcr1CreNK1.1+ NK cells in IL-15LD or from the spleen stimulated with IL-12 / IL-18 analyzed by CFSE dilution (mean ± SEM; n = 3-4). (K) Upper panel: Schematic diagram of MCMV infection experiments in Tfrcfl / fl and Tfrcfl / flNcr1cre mice.Middle panel: Percentage and absolute number of NK1.1+ NK cells in the liver of WT MCMV-infected Tfrcfl / fl and Tfrcfl / flNcr1cre mice at 3.5 and 5.5 dpi (mean ± SEM, n = 4-6). Lower panel: Percentage and absolute number of NK1.1+ NK cells in the spleen of WT MCMV-infected Tfrcfl / fl and Tfrcfl / flNcr1cre mice at 3.5 and 5.5 dpi (mean ± SEM, n = 3-6). (L) Virus titer in the liver and spleen of WT MCMV-infected Tfrcfl / fl and Tfrcfl / flNcr1cre mice at 3.5 dpi (each point represents data from cells isolated from one mouse, the horizontal line represents the median, n = 5). (M) Percentage of IFN-γ+ on NK1.1+ NK cells in the liver and spleen of WT MCMV-infected Tfrcfl / fl and Tfrcfl / flNcr1Cre mice at 1.5 dpi (mean ± SEM, n = 4). (N) Left panel: Percentage of CD27+CD11b–, CD27+CD11b+, and CD27–CD11b+ on NK1.1+ NK cells in the spleen of WT MCMV-infected Tfrcfl / fl and Tfrcfl / flNcr1Cre mice at 5.5 dpi (mean ± SEM, n = 3-5). Right panel: Percentage of KLRG1+ on NK1.1+ NK cells in the spleen of WT MCMV-infected Tfrcfl / fl and Tfrcfl / flNcr1Cre mice at 5.5 dpi (mean ± SEM, n = 3-5). Statistical significance was assessed by unpaired two-tailed Student's t-test (A, B, C, D, E, F, I, J, K, L, M, N). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns, not significant.

[0290] Figure 5: Glycolysis is required for the induction of CD71 in activated NK cells (A) Top: Representative Western blot of total CD71 expression in NV and CE NK cells that were unstimulated (no stimulation) or stimulated with IL-12 / IL-18, IL-12 / IL-18+ActD (1 and 10 μM) and IL-12 / IL-18+CHX (10 and 100 μg / ml). Bottom left: Total CD71 expression normalized to actin in NV and CE NK cells that were unstimulated (no stimulation) or stimulated with IL-12 / IL-18, IL-12 / IL-18+ActD 10 μM (mean ± SEM, n = 3 donors). Lower right panel: Total CD71 expression normalized to actin in NV and CE NK cells that were unstimulated (no stimulation) or stimulated with IL-12 / IL-18, IL-12 / IL-18+CHX 100 μg / ml (mean ± SEM, n = 2 donors). (B) Expression of TFRC mRNA in NV and CE NK cells that were unstimulated (no stimulation) or stimulated with IL-12 / IL-18, IL-12 / IL-18+2-DG. Transcript levels were determined relative to 18S mRNA levels and normalized to unstimulated (no stimulation) NV NK cells (mean ± SEM, n = 6 donors). (C) Upper left panel: GMFI of CD71 expression on NV and CE NK cells that were unstimulated (no stimulation) or stimulated with IL-12 / IL-18, IL-12 / IL-18+2-DG (mean ± SEM, n = 6 donors). Upper right panel: Representative Western blot of total CD71 expression on NV and CE NK cells that were not stimulated (no stimulation) or stimulated with IL-12 / IL-18, IL-12 / IL-18+2-DG. Lower panel: Total CD71 expression normalized to actin in NV and CE NK cells that were not stimulated (no stimulation) or stimulated with IL-12 / IL-18, IL-12 / IL-18+2DG (mean ± SEM, n = 5 donors). (D) GMFI of CD71 expression on NV and CE NK cells that were not stimulated (no stimulation) or stimulated with IL-12 / IL-18 in 10 mM glucose and 2 mM glucose (mean ± SEM, n = 5 donors). (E) GMFI of Tf-488 uptake in NV and CE NK cells unstimulated (no stimulation) or stimulated with IL-12 / IL-18, IL-12 / IL-18 + 2-DG (mean ± SEM, n = 5 donors). (F) Top: Representative Western blot of total c-Myc expression in NV and CE NK cells unstimulated (no stimulation) or stimulated with IL-12 / IL-18.Bottom: Total c-Myc expression normalized to actin in NV and CE NK cells unstimulated (no stimulation) or stimulated with IL-12 / IL-18 (mean ± SEM, n = 6 donors). Statistical significance was assessed by paired two-tailed Student's t-test (A, B, C, D, E, F) or linear regression analysis (B, C, E). *p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant.

[0291] Figure 6: Cytokine priming induces the IRP / IRE regulatory system

[0292] (A) Top: Transcriptome data for ACO1 and IREB2 mRNA expression in NV and CE NK cells that were unstimulated (no stimulation) or stimulated with IL-12 / IL-18 (n = 5 donors). Middle: Representative Western blot of total IRP1 and IRP2 expression in NV and CE NK cells that were unstimulated (no stimulation) or stimulated with IL-12 / IL-18. Bottom: Total IRP1 and IRP2 expression normalized to actin in NV and CE NK cells that were unstimulated (no stimulation) or stimulated with IL-12 / IL-18 (mean ± SEM; for IRP1, n = 7 donors; for IRP2, n = 6 donors). (B) Heat map of relative expression of mRNA encoding genes carrying IREs in NV and CE NK cells that were unstimulated (no stimulation) or stimulated with IL-12 / IL-18 (n = 5 donors). (C) Upper left panel: Expression of eIF4E mRNA in NV and CE NK cells that were unstimulated (no stimulation) or stimulated with IL-12 / IL-18 from transcriptome data (n = 5 donors). Upper right panel: Representative Western blot of total eIF4E expression in NV and CE NK cells that were unstimulated (no stimulation) or stimulated with IL-12 / IL-18. Lower panel: Total eIF4E expression normalized to actin in NV and CE NK cells that were unstimulated (no stimulation) or stimulated with IL-12 / IL-18 (mean ± SEM, n = 6 donors). (D) Upper panel: Representative histogram of HPG incorporation in NV and CE NK cells that were unstimulated (no stimulation) or stimulated with IL-12 / IL-18. Lower panel: GMFI of HPG incorporation in NV and CE NK cells that were unstimulated (no stimulation) or stimulated with IL-12 / IL-18 (mean ± SEM, n = 5 donors). (E) Left: Representative Western blot of total ferritin heavy chain 1 expression in NV and CE NK cells that were unstimulated (no stimulation) or stimulated with IL-12 / IL-18. Right: Total ferritin heavy chain 1 expression normalized to actin in NV and CE NK cells that were unstimulated (no stimulation) or stimulated with IL-12 / IL-18 (mean ± SEM, n = 4 donors). Statistical significance was assessed by paired two-tailed Student's t-test (A, C, E) or linear regression analysis (D). *p < 0.05, **p < 0.01; ns, not significant.

[0293] Figure 7: The IRP / IRE regulatory system coordinates CD71 expression in NK cells

[0294] (A) TFRC mRNA expression in NV and CE NK cells without stimulation with IL-12 / IL-18, data derived from transcriptome data (n=5). (B) FTH1 mRNA expression in NV and CE NK cells without stimulation with IL-12 / IL-18 (n=5). (C) Representative Western blot of total IRP1 expression in NK92 cells transfected with control or Aco1 siRNA. Total IRP1 expression in NK92 cells transfected with control or Aco1 siRNA (n=7). (D) Representative Western blot of total IRP2 expression in NK92 cells transfected with control or IREB2 siRNA. Total IRP2 expression in NK92 cells transfected with control or IREB2 siRNA (n=6). (E) Left panel: Representative histogram of CD71 expression on NK92 cells transfected with control, Aco1, or IREB2 siRNA. Right panel: GMFI of CD71 expression on NK92 cells transfected with control, Aco1, or IREB2 siRNA (n=4-5). (F) Left panel: Representative Western blot of total FTH1 expression in NK92 cells transfected with control, Aco1, and IREB2 siRNA. Right panel: Total FTH1 expression in NK92 cells transfected with control, Aco1, and IREB2 siRNA (n=5). (G) Left panel: Representative Western blot of total IRP1 expression in NKL cells transfected with control or Aco1 siRNA. Right panel: Total IRP1 expression in NKL cells transfected with control or Aco1 siRNA (n=6). (H) Left panel: Representative Western blot of total IRP2 expression in NKL cells transfected with control or IREB2 siRNA. Right panel: Total IRP2 expression in NKL cells transfected with control or IREB2 siRNA (n=7). (I) Left panel: Representative histograms of CD71 expression on NKL cells transfected with control, ACO1, or IREB2 siRNA. Right panel: GMFI of CD71 expression on NKL cells transfected with control, ACO1, or IREB2 siRNA (n=5). (J) Left panel: Representative Western blot of total FTH1 expression in NKL cells transfected with control and Aco1 siRNA. Right panel: Total FTH1 expression in NKL cells transfected with control and Aco1 siRNA (n=6). (K) Left panel: Representative Western blot of total FTH1 expression in NKL cells transfected with control and IREB2 siRNA. Right panel: Total FTH1 expression in NKL cells transfected with control and IREB2 siRNA (n=5). All average data are expressed as mean ± sem and analyzed using unpaired two-tailed Student's t-test (a, b, d, e) or ANOWA (c).Asterisks indicate significance between groups. *p<0.05, **p<0.01; ns, not significant. (L) Left panel: Representative Western blot of total IRP2 expression in NK92 cells transfected with control or IREB2 sgRNA. Right panel: Total IRP2 expression in NK92 cells transfected with control or IREB2 sgRNA (n=3). (M) Left panel: GMFI of CD71 expression on NK92 cells transfected with control or IREB2 sgRNA (n=5). Right panel: Number of NK92 cells transfected with control or IREB2 sgRNA (n=4). All average data are expressed as mean ± sem and analyzed using two-tailed Student's t-test (AB), unpaired two-tailed Student's t-test (C, D, G, H, J, K, L, M) or ANOWA (E, F, I). Asterisks indicate significance between groups. *p<0.05, **p<0.01, ***p<0.001; ns, not significant.

[0295] FIG8 : Forced IRP expression is a molecular module that also supports T cell proliferation.

[0296] (A) Left panel: Representative Western blot of total IRP1 expression in Jurkat cells transfected with control or Aco1 siRNA. Right panel: Total IRP1 expression in Jurkat cells transfected with control or Aco1 siRNA (n=5). (B) Left panel: Representative Western blot of total IRP2 expression in Jurkat cells transfected with control or IREB2 siRNA. Right panel: Total IRP2 expression in Jurkat cells transfected with control or IREB2 siRNA (n=4). (C) Left panel: Representative histogram of CD71 expression on Jurkat cells transfected with control, ACO1, or IREB2 siRNA. Right panel: GMFI of CD71 expression on Jurkat cells transfected with control, ACO1, or IREB2 siRNA (n=7). (D) Left panel: Representative Western blot of total FTH1 expression in Jurkat cells transfected with control, Aco1, or IREB2 siRNA. Right panel: Total FTH1 expression in Jurkat cells transfected with control, Aco1, and IREB2 siRNA (n=5). (E) Representative Western blot of total IRP2 expression in IRP2 knockout (ko) Jurkat cells transduced with control vectors encoding mCherry (LV-mCherry) or IREB2 (LV-IREB2). (F) Top panel: Representative histograms of CD71 expression on IRP2 ko Jurkat cells transduced with LV-mCherry or LV-IREB2. Bottom left panel: GMFI of CD71 expression on IRP2 ko Jurkat cells transduced with LV-mCherry and LV-IREB2 (n=4). Bottom right panel: Number of IRP2 ko Jurkat cells transduced with LV-mCherry and LV-IREB2 (n=3). (G) Left panel: Primary CD4 + Representative histograms of CD71 expression on T cells. Right: Primary CD4 T cells transduced with LV-mCherry and LV-IREB2. + GMFI of CD71 expression on T cells (n=2). (H) Left: Primary CD8 + Representative histograms of CD71 expression on T cells. Right: Primary CD8 T cells transduced with LV-mCherry and IREB2 (LV-IREB2) + GMFI of CD71 expression on T cells (n=2). (I) Untransduced CD4 + T cells (UTD), PSMA-specific CAR CD4 +T cells (CAR) and PSMA-specific CARCD4 co-expressing IRP2 + Representative Western blots of total IRP2 expression in T cells (CAR-IREB2). (J) Top: Unstimulated UTD, CAR, and CAR-IREB2 transduced CD4 + Representative histograms of CD71 expression on T cells. Unstimulated UTD, CAR, and CAR-IREB2 transduced CD4 + MFI of CD71 expression on T cells (n=3). Bottom: Fab-stimulated UTD, CAR, and CAR-IREB2-transduced CD4 + Representative histograms of CD71 expression on T cells. Fab-stimulated UTD, CAR, and CAR-IREB2-transduced CD4 + MFI of CD71 expression on T cells (n=3). (K) Upper panel: Unstimulated UTD, CAR, and CAR-IREB2 transduced CD4 T cells that have entered 0, 1, and 2 cell proliferation cycles. + Percentage of T cells (n=3). Bottom panel: Fab-stimulated UTD, CAR, and CAR-IREB2-transduced CD4 T cells that have entered 0, 1, and 2 cell proliferation cycles. + Percentage of T cells (n=3). All average data are expressed as mean ± sem and analyzed using unpaired two-tailed Student's t-test (a, b, f) or ANOWA (c, d). Asterisks indicate significance between groups. *p < 0.05, **p < 0.01; ns, not significant. Example

[0297] Example 1: Naive and cytokine-boosted NK cells similarly rely on glycolysis for IFN-γ production

[0298] The enhanced recall response of cytokine-enhanced (CE) NK cells reflects a promising feature for immune cell therapy against cancer. Whether and how CE NK cell metabolism supports cytokine production, target cell clearance and proliferation remains unknown. In order to clarify these key features of CE NK cells, the inventors used an established in vitro CE NK cell model that allows comparison of naive (NV) and CE NK cells. In brief, the inventors primed freshly isolated human NK cells with IL-12 and IL-18 (IL-12 / IL-18) for 16 hours and then allowed to stand for a period of time in low-dose IL-15 (IL-15LD) to support survival. After standing for 7 days, the characteristics of NV and CE NK cells after stimulation were compared (Figure 1A). Consistent with previous data, priming NK cells with IL-12 / IL-18 enhanced their ability to produce IFN-γ after restimulation (Figure 1B). It is worth noting that NK cells are similarly activated after stimulation, as indicated by CD69 expression (Figure 1C). To explore how cellular metabolism relates to the function of NV and CE NK cells at the transcriptional level, RNA sequencing (RNA-seq) was performed using unstimulated and cytokine-stimulated cells. In principal component analysis (PCA), unstimulated and activated NV and CE NK cells clustered separately. However, activation was a stronger overall discriminator, indicating relative similarity between the transcriptomes of NV and CE NK cells (Figure 1D).

[0299] Rapid upregulation of aerobic glycolysis is a metabolic hallmark of activated lymphocytes, including NK cells. Unexpectedly, NV and CE NK cells similarly upregulated transcripts of genes encoding glycolytic enzymes upon stimulation, with the exception of HK2, which was higher in CE than in NV NK cells (Figure 1E). Consistent with the transcriptomic data, metabolic flux assays showed that basal and maximal glycolytic rates were increased in activated cells compared to unstimulated cells, but no differences were observed between NV and CE NK cells (Figure 1E). Figure 1F ).

[0300] Likewise, uptake of the glucose analog 2-NBDG did not differ between unstimulated and activated NV and CE NK cells ( Figure 1G). To assess whether the increase in glycolytic metabolism is related to the ability of NV and CE NK cells to produce IFN-γ, the inventors stimulated NV and CE NK cells with IL-12 / IL-18 in the presence of the hexokinase inhibitor 2-deoxy-d-glucose (2-DG). Inhibition of glycolysis during cytokine stimulation similarly reduced IFNG mRNA abundance and IFN-γ secretion in NV and CE NK cells (Figures 1H and 1I, left). Similarly, culturing NK cells in low glucose reduced IFN-γ production in both subpopulations (Figure 1I, top). Together, these data confirm a similar increase in basal and maximum glycolytic activity after activation of NV and CE NK cells, which is required for the efficient production of the key inflammatory cytokine IFN-γ in both subpopulations.

[0301] Example 2: Activated CE NK cells are characterized by high levels of cell surface CD71 and rapid cell proliferation

[0302] To further characterize the metabolic profiles of NV and CE NK cells, we analyzed the surface expression of nutrient transporters CD98 and CD71, which are reported to be upregulated on activated NK cells. Upon stimulation, a small but comparable increase in CD98 expression was observed on both NK cell subsets ( Figure 2A ). In contrast, the upregulation of the transferrin receptor CD71 was much greater on CE relative to NV NK cells, when both were expressed as GMFI and percentage of positive cells (Figure 2B). As assessed by immunoblot analysis of whole-cell lysates, the increase in cell surface expression of CD71 was reflected in the overall greater cell abundance of CD71 protein (Figure 2C). In order to test whether the differential cell surface expression of CD71 can also be driven by NK cell stimulation through activating receptors, both subpopulations were stimulated with HLA-deficient target cells (K562 cell line). Similar to cytokine stimulation, the upregulation of CD71 was more obvious on CE exposed to K562 than on NV NK cells (Figure 2D). In order to assess the functional capacity of increased CD71 expression, the inventors used fluorescently labeled transferrin to monitor transferrin uptake in NV and CE NK cells. These experiments revealed that transferrin uptake in activated CE increased compared to NV NK cells (Figure 2E).

[0303] CD71 expression has previously been correlated with proliferation rate in neoplastic cells. To test whether this association also applies to NK cells, a CFSE dilution assay was used to monitor the proliferation of NV and CE NK cells. Under steady-state conditions and after stimulation, CE NK cells proliferated to a greater extent than their NV counterparts ( Figure 2F). Stimulated CE NK cells were clustered according to their different proliferation rates when testing the abundance of transcripts encoding cell cycle progression genes (Figure 2G). To elucidate whether increased transferrin uptake is associated with increased cell proliferation, the inventors used the intracellular iron chelator 2,2′-bipyridyl (BIP). These experiments revealed that BIP inhibited NK cell proliferation in a dose-dependent manner in both NV and CE NK cells (Figure 2H). Notably, BIP had little effect on cell viability (data not shown) and had no effect on NK cell activation, as assessed by CD69 expression (Figure 2I).

[0304] The pentose phosphate pathway (PPP), which provides ribose 5-phosphate and NADPH for nucleotide synthesis and reducing equivalents, respectively, supports cell proliferation. Consistent with the increased proliferation observed in CE NK cells, cytokine stimulation increased the mRNA abundance of several PPP-related genes in CE more significantly than in NV NK cells (Figure 2J, upper panel). The PPP inhibitor 6-aminonicotinamide (6AN) prevented the expansion of cytokine-stimulated NK cells, further supporting the relevance of PPP in promoting the proliferation of NV and CE NK cells (Figure 2J, lower panel). In summary, these experiments determined that (i) CD71 is preferentially upregulated on activated CE relative to NV NK cells and (ii) relative to activated NV NK cells, the proliferation of activated CE NK cells is increased, which is dependent on PPP activity.

[0305] Example 3: CD71-mediated iron uptake and dietary iron availability affect NK cell function

[0306] Recently, mutations in the TFRC gene (TFRCY20H / Y20H) have been shown to impair B and T cell function, leading to primary immunodeficiency (PID). This mutation affects receptor-mediated endocytosis and impairs CD71-mediated iron uptake in human cells and when introduced into mice. The number of NK cells in patients carrying this mutation is normal, however, functional properties have not been evaluated previously. To test whether CD71 function and NK cell proliferation are related, the inventors evaluated CFSE dilution in wild-type (WT) and TfrcY20H / Y20H murine NK cells stimulated with IL-15LD and IL-12 / IL-18 in vitro. These experiments revealed a significant lack of IL-15LD and IL-12 / IL-18-induced proliferation in NK cells carrying Tfrc mutations ( Figure 3A ).

[0307] In view of this strong phenotype, the inventors wanted to know whether mild iron deficiency could be enough to cause NK cell dysfunction. In order to explore this concept, the inventors first established systemic iron deficiency in a mouse model (Fig. 3B, upper figure). As expected, compared with mice maintaining a control diet, mice maintained on an iron-deficient diet for 6 weeks showed reduced levels of iron, ferritin and hematocrit in peripheral blood, while unsaturated iron binding capacity (UIBC) and total iron binding capacity (TIBC) increased (Fig. 3B, lower figure). Although the number of spleen T and B cells in iron-deficient mice was normal, the number of NK cells was often low, which may indicate the selective sensitivity of these cells to systemic iron abundance (Fig. 3C). The effect of iron deficiency on NK cell maturation phenotype was not observed (Fig. 3D). However, after MCMV infection, in mice maintained on an iron-deficient diet, the virus control mediated by spleen NK cells and the IFN-γ production of NK cells tended to decrease, indicating that NK cell function was impaired (Fig. 3E, left figure and 8F). Notably, the replication of Δm157 MCMV, which evades NK cell-mediated control, was not affected by reduced iron levels (Figure 3E, right panel). Together, these data establish that CD71-mediated iron uptake plays an important role in regulating NK cell proliferation. Furthermore, reduced systemic iron levels significantly impair immune control of MCMV infection in vivo, likely by reducing NK cell function. Whether the impairment of NK cell-mediated immunity is caused by factors intrinsic to or extrinsic to NK cells remains to be determined.

[0308] Example 4: CD71 supports NK cell proliferation and optimal effector function during viral infection

[0309] To test the functional importance of CD71-mediated iron uptake in NK cells, the inventors generated mice that specifically lack CD71 in NK cells by crossing Ncr1Cre mice with Tfrcfl / fl mice (Tfrcfl / flNcr1Cre). Under steady-state conditions, the percentage and absolute number of NK cells in the liver and spleen of Tfrcfl / flNcr1Cre mice were slightly reduced compared to Tfrcfl / fl littermate controls (Figure 4A). The percentages and absolute numbers of CD8+ and CD4+ T cells and CD19+ B cells were not affected by the NK cell-specific deletion of CD71 (Figures 4B and 4C). In addition, the expression of terminal NK cell maturation markers was comparable between Tfrcfl / flNcr1Cre and Tfrcfl / fl mice (CD27, CD11b, KLRG1, CD62L and Ly6C) (Figures 4D and 4E). Likewise, the NK cell-activating receptor Ly49H, which is important for controlling MCMV infection, was equally expressed on Tfrcfl / flNcr1Cre and Tfrcfl / fl NK cells (Fig. 4F).

[0310] MCMV activation of NK cells drives the proliferation of Ly49H+NK cells. To examine whether the absence of CD71 affects antigen-specific NK cell expansion in vivo, the inventors co-transferred congenic Ly49H+WT and Tfrcfl / flNcr1Cre NK cells into Ly49H-deficient (Klra8- / -) recipients (Figure 4G, upper panel). The inventors then infected recipient mice with MCMV and tracked the expansion of transferred NK cells. Compared to Tfrcfl / flNcr1Cre NK cells, WT NK cells robustly expanded in the liver, spleen, lungs, and blood, constituting 80-90% of the Ly49H+NK cell pool at 7 and 30 days post-infection (dpi) (Figure 4G, lower panel). The inventors next addressed the question of whether the expansion of NK cells driven by the availability of common g-chain-dependent cytokines in lymphopenic hosts also depends on CD71. To this end, the inventors transferred equal ratios of WT and Tfrcfl / flNcr1Cre NK cells into Rag2- / -IL2rg- / - recipient mice (Figure 4H, left panel). Similar to the infection experiment, at 6dpi, the frequency of Tfrcfl / flNcr1Cre NK cells was much lower than that of WT cells (Figure 4H, right panel). The reduction in the number of Tfrcfl / flNcr1Cre NK cells in the two adoptive transfer experiments may be caused by lack of amplification, increased cell death, or a combination of the two. In order to assess how the absence of CD71 in NK cells is related to their proliferation in vivo, WT and Tfrcfl / flNcr1Cre NK cells were labeled with CFSE and transferred into recipient mice at equal ratios (Figure 4I, top panel). The recipients were then infected with MCMV and the donor cells were harvested at 3.5dpi. The CFSE dilution and thus proliferation of adoptively transferred Tfrcfl / flNcr1Cre NK cells in the liver and spleen were significantly lower than those of WT cells (Figure 4I, bottom panel). These findings were further confirmed in in vitro proliferation studies, in which IL-15LD and IL-12 / IL-18 stimulated Tfrcfl / flNcr1Cre NK cells showed reduced proliferation compared to control cells (Figure 4J).

[0311] To address the question of whether the loss of CD71 affects NK cell-mediated viral control, the inventors challenged Tfrcfl / fl and Tfrcfl / flNcr1Cre mice with MCMV ( Figure 4K Consistent with the competitive transfer assay described above, a significant decrease in both the percentage and absolute number of NK cells was observed in the liver and spleen of Tfrcfl / flNcr1Cre mice at 3.5 and 5.5 dpi ( Figure 4K, middle and lower figures). At 3.5 dpi, insufficient expansion of NK cells in Tfrcfl / flNcr1Cre mice was associated with higher spleen virus titers, and a similar trend was observed in the liver (Figure 4L). In addition, after MCMV infection, IFN-γ production by spleen and liver infiltrating Tfrcfl / flNcr1Cre NK cells was reduced (Figure 4M). It is worth noting that despite poor expansion and reduced effector capacity, CD71 deficiency did not impair the final maturation of MCMV-stimulated CD71-deficient NK cells, as indicated by CD27, CD11b and KLRG1 expression (Figure 4N). In summary, these data demonstrate the key role of CD71 in NK cell proliferation during infection and in a lymphopenic environment.

[0312] Example 5: Glycolysis is required for the induction of CD71 in activated NK cells

[0313] Our experiments identified (i) iron uptake by CD71 as a key metabolic checkpoint controlling NK cell proliferation; and (ii) CD71 is highly preferentially upregulated on activated CE relative to NV NK cells. These findings prompted the inventors to ask how CD71 itself is regulated in NV and CE NK cells. To address this question, the inventors first assessed whether the induction of CD71 is dependent on NK cell transcriptional activity. As in previous experiments, CD71 was induced to a greater extent in cytokine-stimulated CE than NV NK cells (Figure 5A). In both subsets, transcriptional inhibition (using actinomycin D) completely prevented stimulation-induced CD71 upregulation, as did blocking translation (using cycloheximide) (Figure 5A). Therefore, transcription and translation are similarly required in both cell subsets. It has been previously shown that glycolytic reprogramming drives transcription in activated NK cells (72). Since glycolysis is similarly triggered in activated NV and CE NK cells ( Figure 1F ), the inventors examined the possibility that glycolytic metabolism could affect the transcription of TFRC (which encodes CD71) differently between NV and CE NK cells. TFRC mRNA abundance was indeed higher in activated CE than in NV NK cells, but was similarly reduced when glycolysis was inhibited with 2-DG (Figure 5B). When the cells were exposed to 2-DG, the cell surface expression of CD71 and the total CD71 levels followed the same pattern (Figure 5C). The glucose dependence of inducing CD71 expression after NK cell activation in low glucose medium was summarized and translated into a reduction in transferrin uptake in 2-DG-treated NK cells (Figures 5D and 5E). Therefore, glycolysis makes the transcription and translation of CD71 possible. However, there is no evidence that glycolysis regulates the differential abundance of CD71 in activated CE relative to NV NK cells.

[0314] c-Myc has been identified as a key regulator of TFRC transcription in various immune cells. Given that the abundance of TFRC mRNA in activated CE is higher than that in NV NK cells (Figure 5B), preferential c-Myc induction in CE NK cells may explain the differential regulation of CD71 between activated CE and NV NK cells. However, c-Myc was robustly but equally induced in both NK cell subsets (Figure 5F). Together, these data establish a symmetrical requirement in activated NV and CE NK cells for (i) continuous transcription and translation to support CD71 expression and (ii) glycolytic reprogramming as a metabolic requirement for CD71 expression.

[0315] Example 6: Cytokine Sensitization Induces IRP / IRE Regulatory System

[0316] Many genes involved in cellular iron homeostasis contain iron response elements (IREs) in the 5' or 3'UTR of their mRNA. Iron regulatory proteins 1 and 2 (IRP1 and IRP2) bind to IREs, thereby controlling mRNA stability and translation. TFRC mRNA contains five IREs in the 3'UTR; binding of IRPs stabilizes the mRNA and promotes translation. As described above, this would occur under conditions of iron deficiency. Therefore, the inventors hypothesized that increasing the abundance of IRPs selectively in CE NK cells could be a possible mechanism for regulating enhanced CD71 expression in activated CE NK cells. At the mRNA level, the abundance of the IRP transcripts ACO1 and IREB2 was similar in NV and CE NK cells ( Figure 6A , upper panel). However, the protein abundance of IRP1 and IRP2 was higher in resting and activated CE NK cells ( Figure 6A , lower panel). This finding is consistent with a novel role for IRPs, namely, to generate a pseudo-iron-deficient state in a cell-subpopulation-specific manner, thereby posttranscriptionally controlling the abundance of a distinct set of proteins.

[0317] To expand on this observation, the inventors analyzed the transcript abundance of known IRE-containing mRNAs expressed in NK cells (Figure 6B). The inventors included the key eukaryotic translation initiation factor 4E (eIF4E) in the list of mRNAs containing IRE because the search for iron-responsive elements (SIRE) algorithm revealed an IRE-like motif in the 3'UTR of EIF4E mRNA (data not shown). This analysis prompted the inventors to evaluate the transcription and translation patterns of EIF4E. The pattern observed for CD71 was summarized to some extent, with activated CE NK cells expressing more EIF4E transcripts and significantly more eIF4E proteins (Figure 6C). However, compared to NV NK cells, CE NK cells that had been stationary expressed increased levels of eIF4E. It is worth noting that although the abundance of eIF4E is higher, there is no significant difference in overall protein translation between activated NV and CE NK cells, as assessed by using L-homopropargylglycine (HPG) incorporation assay (Figure 6D). In addition, the inventors noticed an increase in FTH1 mRNA abundance in activated CE NK cells (Figure 6B). FTH1 mRNA contains an IRE in its 5'UTR, and the binding of IRP to the 5'UTRs IRE inhibits translation. This series of similar situations led the inventors to test the hypothesis that pseudo-iron deficiency is driven by a selective increase in IRP abundance in CE NK cells. In fact, despite high transcription levels, the protein abundance (if any) of ferritin heavy chain 1 (the gene product of FTH1) was low in both unstimulated and activated CE NK cells (Figure 6E). This finding highly suggests that IRP is involved in regulating FTH1 mRNA translation with its CE and NV NK cell-specific abundance. Together, these data determine a regulatory axis selectively induced in CE NK cells, in which pseudo-iron deficiency achieves increased translation of CD71 in activated CE NK cells - and therefore increased proliferation.

[0318] Example 7: Expression of IRP in CAR T cells

[0319] CAR T cell production

[0320] Clone the sequences of the antigen-binding domain, transmembrane domain, CD3ζ domain, and CD28 costimulatory domain of the CAR and IRP1 and / or IRP2 into the corresponding lentiviral vectors. If necessary, clone the IRP and CAR sequences into separate lentiviral packaging vectors. Produce lentivirus in an appropriate cell line.

[0321] CD8+ and / or CD4+ T cells are isolated from peripheral blood mononuclear cells from patients or healthy donors and activated with anti-CD3 and anti-CD28 (soluble or bead-bound) in the presence of IL-2. One to two days after activation, the soluble antibodies or beads are removed and the cells are transduced with lentivirus for approximately 18 hours, and the culture medium is replaced with fresh culture medium supplemented with IL-2. CAR and IRP expression will be confirmed by flow cytometry at the specified time points.

[0322] Optional:

[0323] CD8+ and / or CD4+ T cells are isolated from peripheral blood mononuclear cells from patients or healthy donors and activated with anti-CD3 and anti-CD28 (soluble or bead-bound) in the presence of IL-2. One day after activation, cells are transduced with lentivirus for approximately 48 hours, and the culture medium is replaced with fresh culture medium supplemented with IL-2. Five days after activation, beads are removed and replaced with culture medium containing IL-15 and IL-7. CAR and IRP expression will be confirmed by flow cytometry at the specified time point.

[0324] In vitro proliferation of CAR T cells

[0325] To analyze the cell proliferation of CAR T cells, before activation, the cells were loaded with the cell proliferation dye carboxyfluorescein succinimidyl ester (CFSE, 1 μM, Molecular probes, USA) and seeded in 96-well plates. Before sample collection, dead cells were excluded using a fixable live-dead cell stain (Fixable Viability Dye, eBioscience or ZombieAqua, Biolegend). CFSE dilution was analyzed by flow cytometry at different time points after stimulation.

[0326] In vivo proliferation of CAR T cells

[0327] To analyze the in vivo cell proliferation of CAR T cells, CAR T cells overexpressing at least one IRP and CAR T cells that do not overexpress any IRP were adoptively transferred into the corresponding murine tumor model, and the frequency and number of transferred cells were analyzed at different time points. In some experiments, CAR T cells overexpressing at least one IRP and CAR T cells that do not overexpress any IRP were loaded with the cell proliferation dye CFSE before transfer to analyze in vivo proliferation.

[0328] Mouse tumor model

[0329] CAR T cells overexpressing at least one IRP and CAR T cells not overexpressing any IRP were adoptively transferred into the corresponding murine tumor models. Depending on the tumor model, tumor diameter was measured in some experiments. Depending on the tumor model, in some experiments, lungs were dissected and fixed in the corresponding buffer, and the number of nodules was counted using a microscope. Depending on the tumor model, in some experiments, survival was analyzed.

[0330] Example 8: Materials and Methods

[0331] mice

[0332] Animal experiments performed at the University of Rijeka, Faculty of Medicine were approved by the Ethical Committee of the Faculty of Medicine, University of Rijeka and the Ethical Committee at the Croatian Ministry of Agriculture, Veterinary and Food Safety Directorate (UP / I-322-01 / 18-01 / 44). Mice were strictly age- and sex-matched and maintained under SPF conditions. Animal handling complied with the guidelines contained in the International Guiding Principles for Biomedical Research Involving Animals.

[0333] Wild-type C57BL / 6J (B6, strain 000664), B6 ​​Ly5.1 (strain 002014), Tfrcfl / fl (strain 028363), and Rag2– / –γc– / – (strain 014593) mice were purchased from Jackson Laboratory. Ncr1Cre mice were kindly provided by V.Sexl (Vienna, Austria), and B6.Ly49h– / – mice were kindly provided by Silvia M. Vidal (Montreal, Canada). In some experiments, mice were placed on an iron-deficient diet and corresponding control diet for 6 weeks (C1038 and C1000, Altromin).

[0334] Animal experiments at the University of Basel were conducted in accordance with local regulations for the care and use of laboratory animals. Mice were age- and sex-matched and maintained under SPF conditions. Wild-type C57BL / 6J (B6, strain 000664) mice were purchased from Jackson Laboratories (USA), and TfrcY20H / Y20H mice were kindly provided by R. Geha (Boston, USA).

[0335] Hematological analysis

[0336] Serum iron, ferritin, unsaturated iron binding capacity (UIBC) and total iron binding capacity (TIBC) were determined using AU5800 analyzer (Beckman Coulter). Hematocrit was determined using hematology analyzer DxH500 (Beckman Coulter). Measurements were performed at the Clinical Institute of Laboratory Diagnostics (Clinical Hospital Center, Rijeka, Croatia).

[0337] Virus

[0338] The bacterial artificial chromosome-derived murine cytomegalovirus (BAC-MCMV) strain pSM3fr-MCK-2fl clone 3.3 has been previously shown to be biologically equivalent to the MCMV Smith strain (VR-1399; ATCC) and is hereinafter referred to as wild-type (WT) MCMV231. pSM3fr-MCK-2fl clone 3.3 and Δm157 were propagated on mouse embryonic fibroblast (MEF) 232 cells. 2×10 5 Animals were infected intravenously (iv) with 100 plaque forming units (PFU). Viral titers were determined on MEFs by standard plaque assay.

[0339] Adoptive transfer experiments

[0340] Adoptive co-transfer studies were performed by transferring splenocytes from WT B6 (CD45.1) and Tfrcfl / flNcr1Cre (CD45.2) mice at equal ratios into B6.Ly49h− / − and Rag2− / −γc− / − recipients, respectively, 1 day before MCMV infection. For in vivo cell proliferation assays, splenocytes were loaded with the cell proliferation dye carboxyfluorescein succinimidyl ester (5 μM CFSE, Molecular probes, USA) before transfer.

[0341] Human NK cell isolation and cell culture

[0342] Blood samples were obtained from healthy donors after written informed consent. Peripheral blood mononuclear cells were isolated by standard density gradient centrifugation protocol (Lymphoprep; Fresenius Kabi). NK cells were negatively selected using the EasySep negative NK cell isolation kit (Stemcell). Human NK cells were maintained in RPMI-1640 medium (Invitrogen) (R10AB) supplemented with 10% heat-inactivated human AB serum, 50U / ml penicillin (Invitrogen) and 50μg / ml streptomycin (Invitrogen). To generate CE NK cells, the isolated NK cells were sensitized overnight in R10AB containing IL-12 (10ng / ml, R&D systems), IL-15 (1ng / ml, PeproTech) and IL-18 (50ng / ml, R&D systems). The next day, cells were washed twice with PBS and maintained in R10AB containing IL-15 (1 ng / ml) until stimulation. 50% of the medium was replaced with fresh IL-15 (1 ng / ml) every 2-3 days. After 7 days, cells were stimulated in R10AB containing IL-12 (10 ng / ml), IL-15 (1 ng / ml), and IL-18 (50 ng / ml) or with the K562 leukemia target (effector: target ratio, 5:1) for 6 hours. When indicated, cells were preincubated with 2-deoxy-D-glucose (10 mM, Sigma-Aldrich), actinomycin D (1 and 10 μM, Sigma-Aldrich), cycloheximide (10 and 100 μg / ml, Sigma-Aldrich), 2,2′-bipyridine (1, 10, 50, and 100 μM, Sigma-Aldrich), or 6-aminonicotinamide (50 μM, Sigma-Aldrich) for 30 min and then stimulated in R10AB containing IL-12 (10 ng / ml), IL-15 (1 ng / ml), and IL-18 (50 ng / ml) for 6 h.

[0343] NK cell lines NK92 and NKL were maintained in R10AB supplemented with IL-2 (50 U / ml). Jurkat and K562 cell lines were maintained in RPMI-1640 medium (Invitrogen) (R10FBS) supplemented with 10% heat-inactivated human fetal bovine serum (FBS), 50 U / ml penicillin (Invitrogen) and 50 μg / ml streptomycin (Invitrogen). 293T human embryonic kidney (HEK-293T) cells were maintained in DMEM medium (Invitrogen) supplemented with 10% heat-inactivated human fetal bovine serum (FBS), 50 U / ml penicillin (Invitrogen) and 50 μg / ml streptomycin (Invitrogen).

[0344] Flow cytometric analysis of human cells

[0345] For surface staining, NK cells were stained with saturating concentrations of antibodies at 4°C for 30 min. The following antibodies were used: anti-human CD71 (clone CY1G4, Biolegend), anti-human CD69 (clone FN50, Immunotools), anti-human CD98 (clone MEM-108, Biolegend). Samples were collected using a BD AccuriC6 or CytoFLEX flow cytometer (Beckman Coulter). Data were analyzed using ELISA_V10.5 (Tree Star, USA).

[0346] For cell proliferation assays, before activation, NK cells were loaded with cell proliferation dye CFSE (1 μM, Molecular probes, USA) and seeded in 96-well plates. When the presence of inhibitors was indicated, the cells were washed twice and maintained in R10AB with IL-15 (1 ng / ml). Before sample collection, dead cells were excluded using a fixable live-dead cell stain (Fixable Viability Dye, eBioscience or Zombie Aqua, Biolegend). CFSE dilution was analyzed by flow cytometry 65 hours after stimulation. Samples were collected using a BD AccuriC6 or CytoFLEX flow cytometer (Beckman Coulter). (Tree Star, USA) analyzed the data.

[0347] Flow cytometric analysis of mouse cells

[0348] Lymphocytes in the spleen were isolated by meshing the organ and filtering through a 100-μm filter. To isolate lymphocytes from the liver, the tissue was meshed and filtered through a 100 μm filter and purified using a discontinuous gradient of 40% to 80% (40% over 80%) Percoll. Red blood cells in the spleen and liver were lysed using red blood cell lysis buffer. Cells were pretreated with Fc block (clone 2.4G2) and dead cells were excluded using a fixable live-dead cell stain (Fixable Viability Dye, eBioscience). Cells were stained with saturating concentrations of antibodies for 30 min at 4°C. The following antibodies were purchased from Thermo Fisher Scientific: anti-mouse CD8α (clone 53-6.7), anti-mouse CD45.2 (clone 104), anti-mouse CD4 (clone RM4-5), anti-mouse CD69 (clone H1.2F3), anti-mouse CD45.1 (clone A20), anti-mouse CD3ε (clone 145-2C11), anti-mouse CD19 (clone 1D3), anti-mouse NK1.1 (clone PK136), anti-mouse NKp46 (clone 29A1.4), anti-mouse CD62L (clone MEL-14), anti-mouse Ly6c (clone HK1.4), anti-mouse KLRG1 (clone 2F1), anti-mouse Ly49H (clone 3D10), anti-mouse CD11b (clone M1 / 70), and anti-mouse CD27 (clone O323). Samples were acquired using a BD FACSAria. Data were analyzed using ELISA_V10.5 (Tree Star, USA).

[0349] For intracellular cytokine staining after MCMV infection, lymphocytes from the spleen and liver of MCMV-infected mice were isolated as indicated above. The cells were resuspended in RPMI-1640 medium (R10FBS) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific), 50 U / ml penicillin (Invitrogen), 50 μg / ml streptomycin (Invitrogen) and 50 μM 2-mercaptoethanol (Thermo Fisher Scientific) in the presence of IL-2 (500 IU / ml). The cells were incubated at 37°C for 5 hours in the presence of Brefeldin A (eBioscience). The cells were surface stained and then fixed and permeabilized according to the manufacturer's protocol (BD Biosciences). Intracellular cytokine staining was performed using mouse anti-IFN-γ (clone XMG1.2, Thermo Fisher Scientific). Samples were collected using a BD FACSAria. Data were analyzed using ELISA_V10.5 (Tree Star, USA).

[0350] For cell proliferation assay, lymphocytes were loaded with the cell proliferation dye CFSE (1 μM, Molecular probes, USA) before activation and seeded in U-bottom 96-well plates (5 × 10 5 Cells were stimulated for 16 hours in R10FBS containing IL-12 (10 ng / ml, PeproTech), IL-15 (10 ng / ml, PeproTech) and IL-18 (50 ng / ml, R&D Systems). The cells were washed twice and maintained in R10FBS containing IL-15 (10 ng / ml). CFSE dilution was analyzed by flow cytometry 65 hours after stimulation. Dead cells were excluded using a fixable live-dead cell stain (Fixable Viability Dye, eBioscience or Zombie Aqua, Biolegend). Samples were collected using a BDFACSAria or CytoFLEX flow cytometer (Beckman Coulter). Data were analyzed using ELISA_V10.5 (Tree Star, USA).

[0351] Seahorse Metabolic Flux Analyzer

[0352] The metabolic profile of cells was determined using a Seahorse XF-96e extracellular flux analyzer (Seahorse Bioscience, Agilent). NK cells were plated (3 × 10 5 Cells were plated on Celltak (Corning, USA)-coated plates (1 μM cells / well). Mitochondrial perturbation experiments were performed by sequentially adding oligomycin (1 μM, Sigma), FCCP (2 μM, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone, Sigma), and rotenone (1 μM, Sigma). Oxygen consumption rate (OCR, pmol / min) and extracellular acidification rate (ECAR, mPH / min) were monitored in real time after injection of each compound.

[0353] 2-NBDG uptake

[0354] NK cells were seeded in a U-bottom 96-well plate (2×10 5 Cells were pre-incubated with inhibitors for 30 min and stimulated for 6 h in R10AB containing IL-12 (10 ng / ml), IL-15 (1 ng / ml), and IL-18 (50 ng / ml) when indicated. Cells were then incubated in medium containing 20 μM 2-NBDG (Invitrogen) for 15 min and analyzed by flow cytometry. Samples were collected using a BD AccuriC6 flow cytometer. Data were analyzed using TreeStar V10.5 (TreeStar, USA).

[0355] IFN-γ measurement in human NK cells

[0356] NK cells were seeded in U-bottom 96-well plates using R10AB (2 × 10 5 Cells / well). Where indicated, cells were pre-incubated with inhibitors for 30 min and stimulated for 6 h in R10AB containing IL-12 (10 ng / ml), IL-15 (1 ng / ml), and IL-18 (50 ng / ml). Cell supernatants were harvested after stimulation and IFN-γ was measured using an immunoassay based on human Th1 cytokine beads (Legendplex, Biolegend) according to the manufacturer's protocol.

[0357] Transferrin uptake assay

[0358] NK cells were seeded in a U-bottom 96-well plate (2×10 5Cells / well). When indicated, cells were pre-incubated with inhibitors for 30 min and stimulated for 4 hours in R10AB containing IL-12 (10 ng / ml), IL-15 (1 ng / ml) and IL-18 (50 ng / ml). Cells were stimulated for 2 hours in RPMI-1640 culture medium containing 5% BSA and IL-12 (10 ng / ml), IL-15 (1 ng / ml) and IL-18 (50 ng / ml). After stimulation, cells were washed with RPMI-1640 containing 0.5% BSA and then incubated for 15 min with transferrin-alexa488 conjugate (Tf-488, 10 μg / ml, Thermo Fisher Scientific). Transferrin uptake was stopped by washing cells in ice-cold acidic buffer (150 mM NaCl, 20 mM citric acid and pH: 5). Cells were resuspended in FACS buffer and analyzed by flow cytometry. Samples were collected using a BD AccuriC6 flow cytometer. (Tree Star, USA) analyzed the data.

[0359] HPG incorporation assay

[0360] NK cells were seeded in 96-well plates (2 × 10 5 Cells were stimulated in R10AB containing IL-12 (10 ng / ml), IL-15 (1 ng / ml), and IL-18 (50 ng / ml) for 4.5 h, and then incubated in methionine-free RPMI-1640 medium containing 10% dialyzed FBS and IL-12 (10 ng / ml), IL-15 (1 ng / ml), and IL-18 (50 ng / ml) for 1.5 h. HPG (50 μM, Life Technologies). HPG was incorporated into NK cells using The reaction mixture was stained (Thermo Fisher Scientific) and analyzed by flow cytometry. Samples were collected using a BD AccuriC6 flow. (Tree Star, USA) analyzed the data.

[0361] Immunoblot analysis

[0362] Protein concentration was determined by BCA protein assay kit (Thermo Fisher Scientific). Total cell lysates were separated using 4%-15% Mini Protean TGX Gel (Bio-Rad, Hercules CA, USA) and transferred to nitrocellulose membranes using Trans-Blot Turbo Transfer (Bio-Rad, Hercules CA, USA). The membranes were probed with the following antibodies: anti-human CD71 mAb (13113), anti-human IRP1 mAb (20272), anti-human IRP2 mAb (37135), anti-human FTH1 mAb (4393), anti-human eIF4E mAb (2067), anti-human c-Myc mAb (5605), and anti-human β-actin mAb (3700) (all from Cell Signaling, USA). Blots were stained with appropriate secondary antibodies and visualized using the Odyssey imaging system (LICOR, Lincoln NE, USA) and quantified using ImageJ software (1.48v).

[0363] RNA sequencing

[0364] RNA-seq was performed by Admera Health (USA). Briefly, samples were isolated using ethanol precipitation. Quality was checked using the Tapestation RNA HS assay (Agilent Technologies, USA) and quantified using the Qubit RNA HS assay (Thermo Fisher Scientific). Ribosomal RNA cleanup was performed using the Ribo-zero Magnetic Gold kit (MRZG12324, Illumina Inc., USA). Sequencing was performed based on the manufacturer's recommendations ( Ultra TM RNA Library Prep Kit for ) samples were randomly primed and fragmented. The first strand was synthesized using Protoscript II reverse transcriptase with a longer extension period (40 min, 42°C). All other steps of library construction were performed according to UltraTM RNA Library Prep Kit for An Illumina 8-nt dual index was used. Samples were pooled and sequenced on a HiSeq with a 150 paired-end read length configuration.

[0365] Using STAR(2.5.2 4 Reads were aligned to the human genome (UCSC version hg38AnalysisSet) with the multi-mapping settings "--outFilterMultimapNmax 10 --outSAMmultNmax 1". The output was sorted and indexed using samtools (version 1.7), and samples run on different sequencing lanes were collapsed using picard markDuplicates (version 2.9.2). Exon union model was assumed (RefSeq genes were generated on 2017-09-01 5 The number of reads (5' end) overlapping with exons of each gene was calculated using the qCount function of QuasR (version 1.20.0) (downloaded from UCSC). All subsequent gene expression data analyses were performed in R software (R Foundation for Statistical Computing, Vienna, Austria). Differentially expressed genes were identified using the edgeR package (version 3.22.5).

[0366] Quantitative real-time PCR

[0367] RNA was isolated from NK cells using Trizol (Thermo Fisher Scientific) and chloroform (Sigma-Aldrich) according to the manufacturer's protocol and then purified using RNeasy RNA purification mini kit (QIAGEN, Germany). RNA concentration was determined using NanoDrop 2000C (Thermo Fisher Scientific). cDNA was synthesized using reverse transcriptase kit GoScript™ Reverse Transcriptase (Promega) from the purified RNA. Quantitative PCR of IFNG, TFRC, and 18S mRNA was performed three times using commercially designed primers (Hs00989291_m1, Hs00951083_m1, Hs03003631_g1) from Life Technologies. PCR reactions were performed using Go Tag G2 DNA polymerase (Promega) according to the manufacturer's protocol.

[0368] RNA-mediated interference

[0369] NK92, NKL, or Jurkat cells (2 × 10 cells / mL) were transfected with a pool of siRNA targeting ACO1, IREB2, or control scrambled siRNA (10 pmol each) (QIAGEN) using the AMAXA Cell Line V Nucleofection Kit (Lonza).6 The cells were then rested for 72 hours and subjected to phenotypic and functional analyses. Knockdown efficiency was assessed by immunoblotting of the corresponding proteins.

[0370] CRISPR editing

[0371] A 24-well cell culture plate was prepared with 1 ml of R10AB (50 U / ml; NK92 cells) or R10FBS (Jurkat cells) containing IL-2 and preheated at 37°C. For CRISPR-Cas9-mediated IREB2 gene knockout, the following sgRNAs from IDT were used: Hs.Cas9.IREB2.1 AA (reference number 220257866) or Alt-R CRISPR-Cas9 negative control (reference number 224163224). The guide RNA complex was formed by combining equimolar amounts of crRNA and tracrRNA in IDT Duplex buffer (30 mM HEPES, pH 4.5, 100 mM potassium acetate) at a concentration of 20 μM, heating the oligos at 95°C for 5 min and slowly cooling to room temperature. An equal volume of CAS9 nuclease (QB3 MacroLab, University of California, Berkeley) was added and incubated at room temperature for 15 min. NK92 or Jurkat cells (2×10 6 ) were washed in PBS and resuspended in electroporation solution (AMAXA Cell Line V Nucleofection Kit, Lonza). RNP solution was added (final RNP concentration was 3 μM) and electroporation was performed using the recommended procedure. The cells were transferred to preheated culture medium and the cells were allowed to rest at the specified time points. The NK92 cells were allowed to rest for 5 days and then subjected to phenotypic and functional analysis. The knockdown efficiency of IRP2 was assessed by immunoblotting. Jurkat cells were allowed to rest for 2 days and then single cells were sorted into 96-well plates. The clones were expanded and the knockout efficiency of IRP2 was assessed by immunoblotting analysis, and the positive clones were expanded for phenotypic and functional analysis and lentiviral transduction of IRP2.

[0372] IRP2 CAR construction

[0373] Human IRP2 was synthesized as a gene string (GeneArt, Thermo Fischer Scientific). IRP2 (NM_004136.4) was then cloned into the third-generation self-inactivating lentiviral expression vector pELNS, where its expression was driven by the elongation factor 1α (EF-1α) promoter, in the same frame as T2A and the second-generation anti-PSMA CAR. The scFv of the anti-PSMA CAR derived from the monoclonal antibody J591 was used as the tumor targeting portion, while the intracellular domain consisted of the CD28 costimulatory domain and the CD3ζ chain. In the control vector, IRP2 was replaced by the reporter gene eGFP.

[0374] Recombinant lentivirus production

[0375] 24 hours before transfection, HEK-293 cells (5×10 6 All plasmid DNA was purified using an Endotoxin-free Plasmid Maxiprep Kit (Sigma). HEK-293T cells were transfected with 1.3 pmol psPAX2 (lentiviral packaging plasmid), 0.72 pmol pMD2G (VSV-G envelope expression plasmid), and 1.64 pmol of pLV-EF1A>mCherry(ns):P2A:EGF or PLV-EIF1A>hIREB2:P2A:EGFP (VectorBuilder) using Lipofectamine 2000 (Invitrogen) and Optimem medium (Invitrogen, Life Technologies). Viral supernatants were collected 48 and 72 hours after transduction. Viral particles were concentrated using VIVASPIN 20 (Sartorius) and stored at -80°C.

[0376] Lentiviral particles of the CAR construct were generated as described in Giordano-Attianese et al., Nat Biotechnol, 2020, 38, 426-432).

[0377] Lentiviral transduction of Jurkat cells

[0378] Jurkat cells were seeded in U-bottom 96-well plates (5 × 10 5The viral supernatant was thawed and transduced into Jurkat cells at various virus dilutions ranging from 1:16 to 1:1,160,000. The plate was centrifuged at 400 × g for 3 minutes and incubated at 37°C for 24 hours. The medium was then changed and the cells were allowed to rest for another 2 days. Transduction efficiency was assessed by analyzing GFP expression using flow cytometry. + Cells were flow-sorted (frequency 10-30% positive cells) and expanded for phenotypic analysis. Lentiviral overexpression of IRP2 was assessed by immunoblot analysis.

[0379] Lentiviral transduction of primary T cells

[0380] Blood samples were obtained from healthy donors after written informed consent. Peripheral blood mononuclear cells (PBMCs) were isolated by standard density gradient centrifugation protocol (Lymphoprep; Fresenius Kabi). Magnetic CD4 + and CD8 + Beads (Miltenyi Biotec) for CD4 + and CD8 + T cells were positively selected. Purified CD4 + and CD8 + T cells. CD4 + and CD8 + T cells were plated into 24-well cell culture plates and stimulated with anti-CD3 and anti-CD28 monoclonal antibody-coated beads (Invitrogen, Life Technologies) at a 1:1 ratio in R10AB containing IL-2 (150 U / ml). T cells were transduced with lentiviral particles in Retronectin (Takara Bio)-coated cell culture plates 18-22 hours after activation. The culture medium was replaced with fresh IL-2 (150 U / ml) every 24 hours. Five days after transduction, cells were analyzed by flow cytometry for CD71 expression. Samples were collected using a CytoFLEX flow cytometer (Beckman Coulter). Data were analyzed using CAR constructs. Primary T cells were lentivirally transduced as described. 7 .

[0381] Activation and proliferation of transduced primary CAR T cells

[0382] Transduced T cells expressing CAR or CAR_IREB2 were adjusted to perform equivalent CAR expression. To stimulate CAR, polyclonal anti-Fab antibodies (Jackson Immuno Research) were used. Briefly, 96-well plates were coated with 20 μg / ml anti-Fab in PBS for 4 hours at 37 ° C. Prior to activation, primary T cells were loaded with the cell proliferation dye Cell Trace Purple (CTV; 1 μM, Thermo Fisher Scientific). The plate was washed twice and the stained T cells were inoculated (1 × 10 5 / well) and stimulated for 5 days. CTV dilution and CD71 expression were analyzed by flow cytometry. Samples were collected using a BD FACS LSR II flow cytometer (BD Bioscience). Data were analyzed using ELISA_V10.5 (Tree Star, USA).

[0383] Statistical analysis

[0384] Data are provided as mean ± SEM. Using GraphPad Prism 8.00 (GraphPad Software), statistical significance was determined by using unpaired two-tailed Student's t test or paired two-tailed Student's t test. In order to compare the increase in paired samples (before relative to after), a simple linear regression model was used. P values ​​less than 0.05 were considered statistically significant.

[0385] Example 9: IRP / IRE regulatory system coordinates CD71 expression in NK cells

[0386] To date, the experiments described have identified (i) iron uptake by CD71 as a key metabolic checkpoint controlling NK cell proliferation; and (ii) CD71 is preferentially upregulated on activated CE compared to NV NK cells. Next, the inventors' question was how CD71 itself is regulated in NV and CE NK cells. To address this question, the inventors first assessed whether the induction of CD71 depends on NK cell transcriptional activity. As in previous experiments, CD71 was induced to a greater extent in cytokine-stimulated CE than in NV NK cells (Figure 5A, top). In both subpopulations, transcriptional inhibition - using actinomycin - completely prevented the stimulation-induced CD71 upregulation, as did blocking translation using cycloheximide (Figure 5A bottom). Therefore, transcription and translation are similarly required in the two cell subpopulations. Next, we examined the possibility that transcription of TFRC could be differentially regulated between NV and CE NK cells. To this end, we analyzed the transcript abundance of TFRC. Indeed, TFRC mRNA levels were higher in activated CE than in NV NK cells, but were further induced in both subpopulations after stimulation (Figure 5A, bottom). Figure 7A ). c-Myc is a key transcription factor that regulates TFRC in various immune cells. Given that the abundance of TFRC mRNA in activated CE is higher than that in NV NK cells, preferential c-Myc induction in CE NK cells can therefore explain the differential regulation of CD71 between activated CE and NV NK cells. However, c-Myc was induced equally in both NK cell subsets (Figure 5F). Together, these data confirm the symmetrical requirement of activated NV and CE NK cells for continuous transcription and translation to support the expression of CD71.

[0387] Many genes involved in cellular iron homeostasis contain iron response elements (IREs) in the 5' or 3'UTR of their mRNA. Iron regulatory proteins 1 and 2 (IRP1 and IRP2) bind to IREs, thereby controlling mRNA stability and translation. TFRC mRNA contains five IREs in the 3'UTR; binding of these IRPs stabilizes the mRNA and promotes translation. According to the textbook, this occurs under conditions of iron deficiency. The inventors reasoned that regardless of cellular iron abundance, increased IRP expression selectively in CE NK cells would explain the higher TFRC transcript abundance and enhanced activation-dependent CD71 expression in these cells. Supporting this notion, protein abundance of IRP1 and IRP2 was higher in both resting and activated CE NK cells ( Figure 6AMiddle and bottom panels). These data are consistent with IRPs creating a pseudo-iron-deficient state in a cell subset-specific manner (i.e., in CE NK cells), thereby selectively controlling the abundance of a distinct set of proteins at the posttranscriptional level. To further explore this concept, we analyzed transcript and protein abundance of FTH1 (encoding ferritin heavy chain), which contains an IRE in its 5'UTR—where binding of IRPs inhibits translation. FTH1 mRNA is increased in activated CE NK cells ( Figure 7B Despite these higher transcript levels, the protein abundance of ferritin heavy chain, if any, was lower in both unstimulated and activated CE NK cells (Figure 6E). This finding strongly suggests that IRPs, with their CE- versus NV-specific abundance, are involved in regulating the translation of IRE-containing mRNAs in these cells.

[0388] To genetically investigate the role of IRPs in regulating CD71 in NK cells—and thus their proliferation—the inventors went on to utilize the NK cell line NK92. In these cells, both IRP1 and IRP2 levels were selectively reduced using an siRNA approach ( Figure 7C Notably, no consistent changes in CD71 protein expression were observed after IRP1 silencing, whereas CD71 abundance was significantly decreased in IRP2-silenced cells ( Figure 7E Thus, when IRP2 was silenced, the expression of ferritin heavy chain was also increased, but IRP1 was not ( Figure 7F To determine the robustness of this finding, the inventors repeated these experiments using a second NK cell line (NKL cells). Similar to NK92 cells, silencing of IRP2 also significantly reduced CD71 and increased ferritin heavy chain expression in this cell line ( Figure 7G -K). Finally, CRISPR / Cas9 technology was used to knock out IRP2 ( Figure 7L ) also reduced CD71 expression in NK92 cells, which directly translated into a reduced proliferation rate ( Figure 7M Thus, these data recapitulate findings obtained through genetic manipulation in CE versus NV NK cells and identify the IRE / IRP regulatory axis—specifically IRE / IRP2—as an important system for regulating proliferation in NK cells / NK cell lineages by controlling CD71 expression.

[0389] Example 10: Forced IRP expression is a molecular module that also supports T cell proliferation.

[0390] Adoptive cell therapy using engineered chimeric antigen receptor (CAR) T cells is a promising approach for controlling various malignancies, particularly hematological malignancies. However, not all patients respond to CAR T cell therapy, some relapse - and the treatment of solid cancers remains uniquely challenging. Based on the results of the NK cell studies, the inventors reasoned that genetically enforced pseudo-iron deficiency might also specifically improve the activation-driven (i.e., context-dependent) proliferation of (CAR) T cells - and therefore therapeutic potential. To begin to examine the role of IRPs in regulating CD71 expression and interrelated proliferation in T cells, the inventors first inhibited the abundance of IRP1 and IRP2 in Jurkat T cells using siRNA technology ( Figure 8A-B Similar to NK cell lines, IRP2 reduction was the dominant factor in reducing CD71 expression and increasing protein abundance of ferritin heavy chain ( Figure 8C-D In contrast, lentiviral overexpression of IRP2 (LV-IREB2) in IRP2 knockout (ko) Jurkat cells increased CD71 expression compared to cells transduced with a control vector encoding mCherry (LV-mCherry). Figure 8E-F , upper and lower left panels). LV-IREB2-dependent increased CD71 cell surface expression was associated with faster Jurkat T cell proliferation (Fig. 8F, lower right panel). Importantly, CD71 expression was not observed in primary human CD4 + and CD8 + T cells were also regulated by lentiviral transduction of LV-IREB2 ( Figure 8G-H ).

[0391] Encouraged by these observations, the inventors went on to test how pseudo-iron deficiency—enforced by expression of IREB2—affected CD71 regulation and interconnected proliferation in primary human T cells expressing CAR. For these proof-of-concept experiments, a CAR T cell model targeting human prostate-specific membrane antigen (hPSMA) was used. + T cells were lentivirally transduced with vectors encoding CAR (CAR) or both CAR and IRP2 (CAR_IREB2). Overexpression of IRP2 in CAR_IREB2 T cells was confirmed by Western blot analysis (Figure 8I). When evaluated under non-activating conditions, overexpression of IRP2 did not affect the cell surface expression of CD71 ( Figure 8J , upper panel). However, after cross-linking CAR with α-Fab antibody, CD71 expression was consistently higher on CAR_IREB2 compared to CAR T cells ( Figure 8J CAR_IREB2 T cells do not proliferate spontaneously ( Figure 8K , upper panel), but increased IRP2-driven and therefore strictly activation-dependent CD71 expression was sufficient to drive superior proliferation ( Figure 8K , lower panel). Together, these data suggest that IRP2 also regulates CD71 expression and interrelated cell proliferation in T cells, particularly CAR T cells. Importantly, inducing pseudo-iron deficiency by overexpressing IRP2 in CAR T cells enhances proliferation in a strictly activation-dependent (i.e., proximity-dependent) manner. Sequence Listing <110> University of Basel <120> Cell therapy approaches <130> AC2078 PCT BS <150> EP 19 19 2299.6 <151> 2019-08-19 <160> 7 <170> BiSSAP 1.3.6 <210> 1 <211> 889 <212> PRT <213> Homo sapiens <220> <223> IRP1 <400> 1 Met Ser Asn Pro Phe Ala His Leu Ala Glu Pro Leu Asp Pro Val Gln 1 5 10 15 Pro Gly Lys Lys Phe Phe Asn Leu Asn Lys Leu Glu Asp Ser Arg Tyr 20 25 30 Gly Arg Leu Pro Phe Ser Ile Arg Val Leu Leu Glu Ala Ala Ile Arg 35 40 45 Asn Cys Asp Glu Phe Leu Val Lys Lys Gln Asp Ile Glu Asn Ile Leu 50 55 60 His Trp Asn Val Thr Gln His Lys Asn Ile Glu Val Pro Phe Lys Pro 65 70 75 80 Ala Arg Val Ile Leu Gln Asp Phe Thr Gly Val Pro Ala Val Val Asp 85 90 95 Phe Ala Ala Met Arg Asp Ala Val Lys Lys Leu Gly Gly Asp Pro Glu 100 105 110 Lys Ile Asn Pro Val Cys Pro Ala Asp Leu Val Ile Asp His Ser Ile 115 120 125 Gln Val Asp Phe Asn Arg Arg Ala Asp Ser Leu Gln Lys Asn Gln Asp 130 135 140 Leu Glu Phe Glu Arg Asn Arg Glu Arg Phe Glu Phe Leu Lys Trp Gly 145 150 155 160 Ser Gln Ala Phe His Asn Met Arg Ile Ile Pro Pro Gly Ser Gly Ile 165 170 175 Ile His Gln Val Asn Leu Glu Tyr Leu Ala Arg Val Val Phe Asp Gln 180 185 190 Asp Gly Tyr Tyr Tyr Pro Asp Ser Leu Val Gly Thr Asp Ser His Thr 195 200 205 Thr Met Ile Asp Gly Leu Gly Ile Leu Gly Trp Gly Val Gly Gly Ile 210 215 220 Glu Ala Glu Ala Val Met Leu Gly Gln Pro Ile Ser Met Val Leu Pro 225 230 235 240 Gln Val Ile Gly Tyr Arg Leu Met Gly Lys Pro His Pro Leu Val Thr 245 250 255 Ser Thr Asp Ile Val Leu Thr Ile Thr Lys His Leu Arg Gln Val Gly 260 265 270 Val Val Gly Lys Phe Val Glu Phe Phe Gly Pro Gly Val Ala Gln Leu 275 280 285 Ser Ile Ala Asp Arg Ala Thr Ile Ala Asn Met Cys Pro Glu Tyr Gly 290 295 300 Ala Thr Ala Ala Phe Phe Pro Val Asp Glu Val Ser Ile Thr Tyr Leu 305 310 315 320 Val Gln Thr Gly Arg Asp Glu Glu Lys Leu Lys Tyr Ile Lys Lys Tyr 325 330 335 Leu Gln Ala Val Gly Met Phe Arg Asp Phe Asn Asp Pro Ser Gln Asp 340 345 350 Pro Asp Phe Thr Gln Val Val Glu Leu Asp Leu Lys Thr Val Val Pro 355 360 365 Cys Cys Ser Gly Pro Lys Arg Pro Gln Asp Lys Val Ala Val Ser Asp 370 375 380 Met Lys Lys Asp Phe Glu Ser Cys Leu Gly Ala Lys Gln Gly Phe Lys 385 390 395 400 Gly Phe Gln Val Ala Pro Glu His His Asn Asp His Lys Thr Phe Ile 405 410 415 Tyr Asp Asn Thr Glu Phe Thr Leu Ala His Gly Ser Val Val Ile Ala 420 425 430 Ala Ile Thr Ser Cys Thr Asn Thr Ser Asn Pro Ser Val Met Leu Gly 435 440 445 Ala Gly Leu Leu Ala Lys Lys Ala Val Asp Ala Gly Leu Asn Val Met 450 455 460 Pro Tyr Ile Lys Thr Ser Leu Ser Pro Gly Ser Gly Val Val Thr Tyr 465 470 475 480 Tyr Leu Gln Glu Ser Gly Val Met Pro Tyr Leu Ser Gln Leu Gly Phe 485 490 495 Asp Val Val Gly Tyr Gly Cys Met Thr Cys Ile Gly Asn Ser Gly Pro 500 505 510 Leu Pro Glu Pro Val Val Glu Ala Ile Thr Gln Gly Asp Leu Val Ala 515 520 525 Val Gly Val Leu Ser Gly Asn Arg Asn Phe Glu Gly Arg Val His Pro 530 535 540 Asn Thr Arg Ala Asn Tyr Leu Ala Ser Pro Pro Leu Val Ile Ala Tyr 545 550 555 560 Ala Ile Ala Gly Thr Ile Arg Ile Asp Phe Glu Lys Glu Pro Leu Gly 565 570 575 Val Asn Ala Lys Gly Gln Gln Val Phe Leu Lys Asp Ile Trp Pro Thr 580 585 590 Arg Asp Glu Ile Gln Ala Val Glu Arg Gln Tyr Val Ile Pro Gly Met 595 600 605 Phe Lys Glu Val Tyr Gln Lys Ile Glu Thr Val Asn Glu Ser Trp Asn 610 615 620 Ala Leu Ala Thr Pro Ser Asp Lys Leu Phe Phe Trp Asn Ser Lys Ser 625 630 635 640 Thr Tyr Ile Lys Ser Pro Pro Phe Phe Glu Asn Leu Thr Leu Asp Leu 645 650 655 Gln Pro Pro Lys Ser Ile Val Asp Ala Tyr Val Leu Leu Asn Leu Gly 660 665 670 Asp Ser Val Thr Thr Asp His Ile Ser Pro Ala Gly Asn Ile Ala Arg 675 680 685 Asn Ser Pro Ala Ala Arg Tyr Leu Thr Asn Arg Gly Leu Thr Pro Arg 690 695 700 Glu Phe Asn Ser Tyr Gly Ser Arg Arg Gly Asn Asp Ala Val Met Ala 705 710 715 720 Arg Gly Thr Phe Ala Asn Ile Arg Leu Leu Asn Arg Phe Leu Asn Lys 725 730 735 Gln Ala Pro Gln Thr Ile His Leu Pro Ser Gly Glu Ile Leu Asp Val 740 745 750 Phe Asp Ala Ala Glu Arg Tyr Gln Gln Ala Gly Leu Pro Leu Ile Val 755 760 765 Leu Ala Gly Lys Glu Tyr Gly Ala Gly Ser Ser Arg Asp Trp Ala Ala 770 775 780 Lys Gly Pro Phe Leu Leu Gly Ile Lys Ala Val Leu Ala Glu Ser Tyr 785 790 795 800 Glu Arg Ile His Arg Ser Asn Leu Val Gly Met Gly Val Ile Pro Leu 805 810 815 Glu Tyr Leu Pro Gly Glu Asn Ala Asp Ala Leu Gly Leu Thr Gly Gln 820 825 830 Glu Arg Tyr Thr Ile Ile Ile Pro Glu Asn Leu Lys Pro Gln Met Lys 835 840 845 Val Gln Val Lys Leu Asp Thr Gly Lys Thr Phe Gln Ala Val Met Arg 850 855 860 Phe Asp Thr Asp Val Glu Leu Thr Tyr Phe Leu Asn Gly Gly Ile Leu 865 870 875 880 Asn Tyr Met Ile Arg Lys Met Ala Lys 885 <210> 2 <211> 963 <212> PRT <213> Homo sapiens <220> <223> IRP2 Isoform 1 <400> 2 Met Asp Ala Pro Lys Ala Gly Tyr Ala Phe Glu Tyr Leu Ile Glu Thr 1 5 10 15 Leu Asn Asp Ser Ser His Lys Lys Phe Phe Asp Val Ser Lys Leu Gly 20 25 30 Thr Lys Tyr Asp Val Leu Pro Tyr Ser Ile Arg Val Leu Leu Glu Ala 35 40 45 Ala Val Arg Asn Cys Asp Gly Phe Leu Met Lys Lys Glu Asp Val Met 50 55 60 Asn Ile Leu Asp Trp Lys Thr Lys Gln Ser Asn Val Glu Val Pro Phe 65 70 75 80 Phe Pro Ala Arg Val Leu Leu Gln Asp Phe Thr Gly Ile Pro Ala Met 85 90 95 Val Asp Phe Ala Ala Met Arg Glu Ala Val Lys Thr Leu Gly Gly Asp 100 105 110 Pro Glu Lys Val His Pro Ala Cys Pro Thr Asp Leu Thr Val Asp His 115 120 125 Ser Leu Gln Ile Asp Phe Ser Lys Cys Ala Ile Gln Asn Ala Pro Asn 130 135 140 Pro Gly Gly Gly Asp Leu Gln Lys Ala Gly Lys Leu Ser Pro Leu Lys 145 150 155 160 Val Gln Pro Lys Lys Leu Pro Cys Arg Gly Gln Thr Thr Cys Arg Gly 165 170 175 Ser Cys Asp Ser Gly Glu Leu Gly Arg Asn Ser Gly Thr Phe Ser Ser 180 185 190 Gln Ile Glu Asn Thr Pro Ile Leu Cys Pro Phe His Leu Gln Pro Val 195 200 205 Pro Glu Pro Glu Thr Val Leu Lys Asn Gln Glu Val Glu Phe Gly Arg 210 215 220 Asn Arg Glu Arg Leu Gln Phe Phe Lys Trp Ser Ser Arg Val Phe Lys 225 230 235 240 Asn Val Ala Val Ile Pro Pro Gly Thr Gly Met Ala His Gln Ile Asn 245 250 255 Leu Glu Tyr Leu Ser Arg Val Val Phe Glu Glu Lys Asp Leu Leu Phe 260 265 270 Pro Asp Ser Val Val Gly Thr Asp Ser His Ile Thr Met Val Asn Gly 275 280 285 Leu Gly Ile Leu Gly Trp Gly Val Gly Gly Ile Glu Thr Glu Ala Val 290 295 300 Met Leu Gly Leu Pro Val Ser Leu Thr Leu Pro Glu Val Val Gly Cys 305 310 315 320 Glu Leu Thr Gly Ser Ser Asn Pro Phe Val Thr Ser Ile Asp Val Val 325 330 335 Leu Gly Ile Thr Lys His Leu Arg Gln Val Gly Val Ala Gly Lys Phe 340 345 350 Val Glu Phe Phe Gly Ser Gly Val Ser Gln Leu Ser Ile Val Asp Arg 355 360 365 Thr Thr Ile Ala Asn Met Cys Pro Glu Tyr Gly Ala Ile Leu Ser Phe 370 375 380 Phe Pro Val Asp Asn Val Thr Leu Lys His Leu Glu His Thr Gly Phe 385 390 395 400 Ser Lys Ala Lys Leu Glu Ser Met Glu Thr Tyr Leu Lys Ala Val Lys 405 410 415 Leu Phe Arg Asn Asp Gln Asn Ser Ser Gly Glu Pro Glu Tyr Ser Gln 420 425 430 Val Ile Gln Ile Asn Leu Asn Ser Ile Val Pro Ser Val Ser Gly Pro 435 440 445 Lys Arg Pro Gln Asp Arg Val Ala Val Thr Asp Met Lys Ser Asp Phe 450 455 460 Gln Ala Cys Leu Asn Glu Lys Val Gly Phe Lys Gly Phe Gln Ile Ala 465 470 475 480 Ala Glu Lys Gln Lys Asp Ile Val Ser Ile His Tyr Glu Gly Ser Glu 485 490 495 Tyr Lys Leu Ser His Gly Ser Val Val Ile Ala Ala Val Ile Ser Cys 500 505 510 Thr Asn Asn Cys Asn Pro Ser Val Met Leu Ala Ala Gly Leu Leu Ala 515 520 525 Lys Lys Ala Val Glu Ala Gly Leu Arg Val Lys Pro Tyr Ile Arg Thr 530 535 540 Ser Leu Ser Pro Gly Ser Gly Met Val Thr His Tyr Leu Ser Ser Ser 545 550 555 560 Gly Val Leu Pro Tyr Leu Ser Lys Leu Gly Phe Glu Ile Val Gly Tyr 565 570 575 Gly Cys Ser Ile Cys Val Gly Asn Thr Ala Pro Leu Ser Asp Ala Val 580 585 590 Leu Asn Ala Val Lys Gln Gly Asp Leu Val Thr Cys Gly Ile Leu Ser 595 600 605 Gly Asn Lys Asn Phe Glu Gly Arg Leu Cys Asp Cys Val Arg Ala Asn 610 615 620 Tyr Leu Ala Ser Pro Pro Leu Val Val Ala Tyr Ala Ile Ala Gly Thr 625 630 635 640 Val Asn Ile Asp Phe Gln Thr Glu Pro Leu Gly Thr Asp Pro Thr Gly 645 650 655 Lys Asn Ile Tyr Leu His Asp Ile Trp Pro Ser Arg Glu Glu Val His 660 665 670 Arg Val Glu Glu Glu His Val Ile Leu Ser Met Phe Lys Ala Leu Lys 675 680 685 Asp Lys Ile Glu Met Gly Asn Lys Arg Trp Asn Ser Leu Glu Ala Pro 690 695 700 Asp Ser Val Leu Phe Pro Trp Asp Leu Lys Ser Thr Tyr Ile Arg Cys 705 710 715 720 Pro Ser Phe Phe Asp Lys Leu Thr Lys Glu Pro Ile Ala Leu Gln Ala 725 730 735 Ile Glu Asn Ala His Val Leu Leu Tyr Leu Gly Asp Ser Val Thr Thr 740 745 750 Asp His Ile Ser Pro Ala Gly Ser Ile Ala Arg Asn Ser Ala Ala Ala 755 760 765 Lys Tyr Leu Thr Asn Arg Gly Leu Thr Pro Arg Glu Phe Asn Ser Tyr 770 775 780 Gly Ala Arg Arg Gly Asn Asp Ala Val Met Thr Arg Gly Thr Phe Ala 785 790 795 800 Asn Ile Lys Leu Phe Asn Lys Phe Ile Gly Lys Pro Ala Pro Lys Thr 805 810 815 Ile His Phe Pro Ser Gly Gln Thr Leu Asp Val Phe Glu Ala Ala Glu 820 825 830 Leu Tyr Gln Lys Glu Gly Ile Pro Leu Ile Ile Leu Ala Gly Lys Lys 835 840 845 Tyr Gly Ser Gly Asn Ser Arg Asp Trp Ala Ala Lys Gly Pro Tyr Leu 850 855 860 Leu Gly Val Lys Ala Val Leu Ala Glu Ser Tyr Glu Lys Ile His Lys 865 870 875 880 Asp His Leu Ile Gly Ile Gly Ile Ala Pro Leu Gln Phe Leu Pro Gly 885 890 895 Glu Asn Ala Asp Ser Leu Gly Leu Ser Gly Arg Glu Thr Phe Ser Leu 900 905 910 Thr Phe Pro Glu Glu Leu Ser Pro Gly Ile Thr Leu Asn Ile Gln Thr 915 920 925 Ser Thr Gly Lys Val Phe Ser Val Ile Ala Ser Phe Glu Asp Asp Val 930 935 940 Glu Ile Thr Leu Tyr Lys His Gly Gly Leu Leu Asn Phe Val Ala Arg 945 950 955 960 Lys Phe Ser <210> 3 <211> 713 <212> PRT <213> Homo sapiens <220> <223> IRP2 Isoform 2 <400> 3 Met Ala His Gln Ile Asn Leu Glu Tyr Leu Ser Arg Val Val Phe Glu 1 5 10 15 Glu Lys Asp Leu Leu Phe Pro Asp Ser Val Val Gly Thr Asp Ser His 20 25 30 Ile Thr Met Val Asn Gly Leu Gly Ile Leu Gly Trp Gly Val Gly Gly 35 40 45 Ile Glu Thr Glu Ala Val Met Leu Gly Leu Pro Val Ser Leu Thr Leu 50 55 60 Pro Glu Val Val Gly Cys Glu Leu Thr Gly Ser Ser Asn Pro Phe Val 65 70 75 80 Thr Ser Ile Asp Val Val Leu Gly Ile Thr Lys His Leu Arg Gln Val 85 90 95 Gly Val Ala Gly Lys Phe Val Glu Phe Phe Gly Ser Gly Val Ser Gln 100 105 110 Leu Ser Ile Val Asp Arg Thr Thr Ile Ala Asn Met Cys Pro Glu Tyr 115 120 125 Gly Ala Ile Leu Ser Phe Phe Pro Val Asp Asn Val Thr Leu Lys His 130 135 140 Leu Glu His Thr Gly Phe Ser Lys Ala Lys Leu Glu Ser Met Glu Thr 145 150 155 160 Tyr Leu Lys Ala Val Lys Leu Phe Arg Asn Asp Gln Asn Ser Ser Gly 165 170 175 Glu Pro Glu Tyr Ser Gln Val Ile Gln Ile Asn Leu Asn Ser Ile Val 180 185 190 Pro Ser Val Ser Gly Pro Lys Arg Pro Gln Asp Arg Val Ala Val Thr 195 200 205 Asp Met Lys Ser Asp Phe Gln Ala Cys Leu Asn Glu Lys Val Gly Phe 210 215 220 Lys Gly Phe Gln Ile Ala Ala Glu Lys Gln Lys Asp Ile Val Ser Ile 225 230 235 240 His Tyr Glu Gly Ser Glu Tyr Lys Leu Ser His Gly Ser Val Val Ile 245 250 255 Ala Ala Val Ile Ser Cys Thr Asn Asn Cys Asn Pro Ser Val Met Leu 260 265 270 Ala Ala Gly Leu Leu Ala Lys Lys Ala Val Glu Ala Gly Leu Arg Val 275 280 285 Lys Pro Tyr Ile Arg Thr Ser Leu Ser Pro Gly Ser Gly Met Val Thr 290 295 300 His Tyr Leu Ser Ser Ser Gly Val Leu Pro Tyr Leu Ser Lys Leu Gly 305 310 315 320 Phe Glu Ile Val Gly Tyr Gly Cys Ser Ile Cys Val Gly Asn Thr Ala 325 330 335 Pro Leu Ser Asp Ala Val Leu Asn Ala Val Lys Gln Gly Asp Leu Val 340 345 350 Thr Cys Gly Ile Leu Ser Gly Asn Lys Asn Phe Glu Gly Arg Leu Cys 355 360 365 Asp Cys Val Arg Ala Asn Tyr Leu Ala Ser Pro Pro Leu Val Val Ala 370 375 380 Tyr Ala Ile Ala Gly Thr Val Asn Ile Asp Phe Gln Thr Glu Pro Leu 385 390 395 400 Gly Thr Asp Pro Thr Gly Lys Asn Ile Tyr Leu His Asp Ile Trp Pro 405 410 415 Ser Arg Glu Glu Val His Arg Val Glu Glu Glu His Val Ile Leu Ser 420 425 430 Met Phe Lys Ala Leu Lys Asp Lys Ile Glu Met Gly Asn Lys Arg Trp 435 440 445 Asn Ser Leu Glu Ala Pro Asp Ser Val Leu Phe Pro Trp Asp Leu Lys 450 455 460 Ser Thr Tyr Ile Arg Cys Pro Ser Phe Phe Asp Lys Leu Thr Lys Glu 465 470 475 480 Pro Ile Ala Leu Gln Ala Ile Glu Asn Ala His Val Leu Leu Tyr Leu 485 490 495 Gly Asp Ser Val Thr Thr Asp His Ile Ser Pro Ala Gly Ser Ile Ala 500 505 510 Arg Asn Ser Ala Ala Ala Lys Tyr Leu Thr Asn Arg Gly Leu Thr Pro 515 520 525 Arg Glu Phe Asn Ser Tyr Gly Ala Arg Arg Gly Asn Asp Ala Val Met 530 535 540 Thr Arg Gly Thr Phe Ala Asn Ile Lys Leu Phe Asn Lys Phe Ile Gly 545 550 555 560 Lys Pro Ala Pro Lys Thr Ile His Phe Pro Ser Gly Gln Thr Leu Asp 565 570 575 Val Phe Glu Ala Ala Glu Leu Tyr Gln Lys Glu Gly Ile Pro Leu Ile 580 585 590 Ile Leu Ala Gly Lys Lys Tyr Gly Ser Gly Asn Ser Arg Asp Trp Ala 595 600 605 Ala Lys Gly Pro Tyr Leu Leu Gly Val Lys Ala Val Leu Ala Glu Ser 610 615 620 Tyr Glu Lys Ile His Lys Asp His Leu Ile Gly Ile Gly Ile Ala Pro 625 630 635 640 Leu Gln Phe Leu Pro Gly Glu Asn Ala Asp Ser Leu Gly Leu Ser Gly 645 650 655 Arg Glu Thr Phe Ser Leu Thr Phe Pro Glu Glu Leu Ser Pro Gly Ile 660 665 670 Thr Leu Asn Ile Gln Thr Ser Thr Gly Lys Val Phe Ser Val Ile Ala 675 680 685 Ser Phe Glu Asp Asp Val Glu Ile Thr Leu Tyr Lys His Gly Gly Leu 690 695 700 Leu Asn Phe Val Ala Arg Lys Phe Ser 705 710 <210> 4 <211> 906 <212> PRT <213> Homo sapiens <220> <223> IRP2 Isoform 3 Sequence 1 <400> 4 Met Lys Lys Glu Asp Val Met Asn Ile Leu Asp Trp Lys Thr Lys Gln 1 5 10 15 Ser Asn Val Glu Val Pro Phe Phe Pro Ala Arg Val Leu Leu Gln Asp 20 25 30 Phe Thr Gly Ile Pro Ala Met Val Asp Phe Ala Ala Met Arg Glu Ala 35 40 45 Val Lys Thr Leu Gly Gly Asp Pro Glu Lys Val His Pro Ala Cys Pro 50 55 60 Thr Asp Leu Thr Val Asp His Ser Leu Gln Ile Asp Phe Ser Lys Cys 65 70 75 80 Ala Ile Gln Asn Ala Pro Asn Pro Gly Gly Gly Asp Leu Gln Lys Ala 85 90 95 Gly Lys Leu Ser Pro Leu Lys Val Gln Pro Lys Lys Leu Pro Cys Arg 100 105 110 Gly Gln Thr Thr Cys Arg Gly Ser Cys Asp Ser Gly Glu Leu Gly Arg 115 120 125 Asn Ser Gly Thr Phe Ser Ser Gln Ile Glu Asn Thr Pro Ile Leu Cys 130 135 140 Pro Phe His Leu Gln Pro Val Pro Glu Pro Glu Thr Val Leu Lys Asn 145 150 155 160 Gln Glu Val Glu Phe Gly Arg Asn Arg Glu Arg Leu Gln Phe Phe Lys 165 170 175 Trp Ser Ser Arg Val Phe Lys Asn Val Ala Val Ile Pro Pro Gly Thr 180 185 190 Gly Met Ala His Gln Ile Asn Leu Glu Tyr Leu Ser Arg Val Val Phe 195 200 205 Glu Glu Lys Asp Leu Leu Phe Pro Asp Ser Val Val Gly Thr Asp Ser 210 215 220 His Ile Thr Met Val Asn Gly Leu Gly Ile Leu Gly Trp Gly Val Gly 225 230 235 240 Gly Ile Glu Thr Glu Ala Val Met Leu Gly Leu Pro Val Ser Leu Thr 245 250 255 Leu Pro Glu Val Val Gly Cys Glu Leu Thr Gly Ser Ser Asn Pro Phe 260 265 270 Val Thr Ser Ile Asp Val Val Leu Gly Ile Thr Lys His Leu Arg Gln 275 280 285 Val Gly Val Ala Gly Lys Phe Val Glu Phe Phe Gly Ser Gly Val Ser 290 295 300 Gln Leu Ser Ile Val Asp Arg Thr Thr Ile Ala Asn Met Cys Pro Glu 305 310 315 320 Tyr Gly Ala Ile Leu Ser Phe Phe Pro Val Asp Asn Val Thr Leu Lys 325 330 335 His Leu Glu His Thr Gly Phe Ser Lys Ala Lys Leu Glu Ser Met Glu 340 345 350 Thr Tyr Leu Lys Ala Val Lys Leu Phe Arg Asn Asp Gln Asn Ser Ser 355 360 365 Gly Glu Pro Glu Tyr Ser Gln Val Ile Gln Ile Asn Leu Asn Ser Ile 370 375 380 Val Pro Ser Val Ser Gly Pro Lys Arg Pro Gln Asp Arg Val Ala Val 385 390 395 400 Thr Asp Met Lys Ser Asp Phe Gln Ala Cys Leu Asn Glu Lys Val Gly 405 410 415 Phe Lys Gly Phe Gln Ile Ala Ala Glu Lys Gln Lys Asp Ile Val Ser 420 425 430 Ile His Tyr Glu Gly Ser Glu Tyr Lys Leu Ser His Gly Ser Val Val 435 440 445 Ile Ala Ala Val Ile Ser Cys Thr Asn Asn Cys Asn Pro Ser Val Met 450 455 460 Leu Ala Ala Gly Leu Leu Ala Lys Lys Ala Val Glu Ala Gly Leu Arg 465 470 475 480 Val Lys Pro Tyr Ile Arg Thr Ser Leu Ser Pro Gly Ser Gly Met Val 485 490 495 Thr His Tyr Leu Ser Ser Ser Gly Val Leu Pro Tyr Leu Ser Lys Leu 500 505 510 Gly Phe Glu Ile Val Gly Tyr Gly Cys Ser Ile Cys Val Gly Asn Thr 515 520 525 Ala Pro Leu Ser Asp Ala Val Leu Asn Ala Val Lys Gln Gly Asp Leu 530 535 540 Val Thr Cys Gly Ile Leu Ser Gly Asn Lys Asn Phe Glu Gly Arg Leu 545 550 555 560 Cys Asp Cys Val Arg Ala Asn Tyr Leu Ala Ser Pro Pro Leu Val Val 565 570 575 Ala Tyr Ala Ile Ala Gly Thr Val Asn Ile Asp Phe Gln Thr Glu Pro 580 585 590 Leu Gly Thr Asp Pro Thr Gly Lys Asn Ile Tyr Leu His Asp Ile Trp 595 600 605 Pro Ser Arg Glu Glu Val His Arg Val Glu Glu Glu His Val Ile Leu 610 615 620 Ser Met Phe Lys Ala Leu Lys Asp Lys Ile Glu Met Gly Asn Lys Arg 625 630 635 640 Trp Asn Ser Leu Glu Ala Pro Asp Ser Val Leu Phe Pro Trp Asp Leu 645 650 655 Lys Ser Thr Tyr Ile Arg Cys Pro Ser Phe Phe Asp Lys Leu Thr Lys 660 665 670 Glu Pro Ile Ala Leu Gln Ala Ile Glu Asn Ala His Val Leu Leu Tyr 675 680 685 Leu Gly Asp Ser Val Thr Thr Asp His Ile Ser Pro Ala Gly Ser Ile 690 695 700 Ala Arg Asn Ser Ala Ala Ala Lys Tyr Leu Thr Asn Arg Gly Leu Thr 705 710 715 720 Pro Arg Glu Phe Asn Ser Tyr Gly Ala Arg Arg Gly Asn Asp Ala Val 725 730 735 Met Thr Arg Gly Thr Phe Ala Asn Ile Lys Leu Phe Asn Lys Phe Ile 740 745 750 Gly Lys Pro Ala Pro Lys Thr Ile His Phe Pro Ser Gly Gln Thr Leu 755 760 765 Asp Val Phe Glu Ala Ala Glu Leu Tyr Gln Lys Glu Gly Ile Pro Leu 770 775 780 Ile Ile Leu Ala Gly Lys Lys Tyr Gly Ser Gly Asn Ser Arg Asp Trp 785 790 795 800 Ala Ala Lys Gly Pro Tyr Leu Leu Gly Val Lys Ala Val Leu Ala Glu 805 810 815 Ser Tyr Glu Lys Ile His Lys Asp His Leu Ile Gly Ile Gly Ile Ala 820 825 830 Pro Leu Gln Phe Leu Pro Gly Glu Asn Ala Asp Ser Leu Gly Leu Ser 835 840 845 Gly Arg Glu Thr Phe Ser Leu Thr Phe Pro Glu Glu Leu Ser Pro Gly 850 855 860 Ile Thr Leu Asn Ile Gln Thr Ser Thr Gly Lys Val Phe Ser Val Ile 865 870 875 880 Ala Ser Phe Glu Asp Asp Val Glu Ile Thr Leu Tyr Lys His Gly Gly 885 890 895 Leu Leu Asn Phe Val Ala Arg Lys Phe Ser 900 905 <210> 5 <211> 906 <212> PRT <213> Homo sapiens <220> <223> IRP2 Isoform 3 Sequence 2 <400> 5 Met Lys Lys Glu Asp Val Met Asn Ile Leu Asp Trp Lys Thr Lys Gln 1 5 10 15 Ser Asn Val Glu Val Pro Phe Phe Pro Ala Arg Val Leu Leu Gln Asp 20 25 30 Phe Thr Gly Ile Pro Ala Met Val Asp Phe Ala Ala Met Arg Glu Ala 35 40 45 Val Lys Thr Leu Gly Gly Asp Pro Glu Lys Val His Pro Ala Cys Pro 50 55 60 Thr Asp Leu Thr Val Asp His Ser Leu Gln Ile Asp Phe Ser Lys Cys 65 70 75 80 Ala Ile Gln Asn Ala Pro Asn Pro Gly Gly Gly Asp Leu Gln Lys Ala 85 90 95 Gly Lys Leu Ser Pro Leu Lys Val Gln Pro Lys Lys Leu Pro Cys Arg 100 105 110 Gly Gln Thr Thr Cys Arg Gly Ser Cys Asp Ser Gly Glu Leu Gly Arg 115 120 125 Asn Ser Gly Thr Phe Ser Ser Gln Ile Glu Asn Thr Pro Ile Leu Cys 130 135 140 Pro Phe His Leu Gln Pro Val Pro Glu Pro Glu Thr Val Leu Lys Asn 145 150 155 160 Gln Glu Val Glu Phe Gly Arg Asn Arg Glu Arg Leu Gln Phe Phe Lys 165 170 175 Trp Ser Ser Arg Val Phe Lys Asn Val Ala Val Ile Pro Pro Gly Thr 180 185 190 Gly Met Ala His Gln Ile Asn Leu Glu Tyr Leu Ser Arg Val Val Phe 195 200 205 Glu Glu Lys Asp Leu Leu Phe Pro Asp Ser Val Val Gly Thr Asp Ser 210 215 220 His Ile Thr Met Val Asn Gly Leu Gly Ile Leu Gly Trp Gly Val Gly 225 230 235 240 Gly Ile Glu Thr Glu Ala Val Met Leu Gly Leu Pro Val Ser Leu Thr 245 250 255 Leu Pro Glu Val Val Gly Cys Glu Leu Thr Gly Ser Ser Asn Pro Phe 260 265 270 Val Thr Ser Ile Asp Val Val Leu Gly Ile Thr Lys His Leu Arg Gln 275 280 285 Val Gly Val Ala Gly Lys Phe Val Glu Phe Phe Gly Ser Gly Val Ser 290 295 300 Gln Leu Ser Ile Val Asp Arg Thr Thr Ile Ala Asn Met Cys Pro Glu 305 310 315 320 Tyr Gly Ala Ile Leu Ser Phe Phe Pro Val Asp Asn Val Thr Leu Lys 325 330 335 His Leu Glu His Thr Gly Phe Ser Lys Ala Lys Leu Glu Ser Met Glu 340 345 350 Thr Tyr Leu Lys Ala Val Lys Leu Phe Arg Asn Asp Gln Asn Ser Ser 355 360 365 Gly Glu Pro Glu Tyr Ser Gln Val Ile Gln Ile Asn Leu Asn Ser Ile 370 375 380 Val Pro Ser Val Ser Gly Pro Lys Arg Pro Gln Asp Arg Val Ala Val 385 390 395 400 Thr Asp Met Lys Ser Asp Phe Gln Ala Cys Leu Asn Glu Lys Val Gly 405 410 415 Phe Lys Gly Phe Gln Ile Ala Ala Glu Lys Gln Lys Asp Ile Val Ser 420 425 430 Ile His Tyr Glu Gly Ser Glu Tyr Lys Leu Ser His Gly Ser Val Val 435 440 445 Ile Ala Ala Val Ile Ser Cys Thr Asn Asn Cys Asn Pro Ser Val Met 450 455 460 Leu Ala Ala Gly Leu Leu Ala Lys Lys Ala Val Glu Ala Gly Leu Arg 465 470 475 480 Val Lys Pro Tyr Ile Arg Thr Ser Leu Ser Pro Gly Ser Gly Met Val 485 490 495 Thr His Tyr Leu Ser Ser Ser Gly Val Leu Pro Tyr Leu Ser Lys Leu 500 505 510 Gly Phe Glu Ile Val Gly Tyr Gly Cys Ser Thr Cys Val Gly Asn Thr 515 520 525 Ala Pro Leu Ser Asp Ala Val Leu Asn Ala Val Lys Gln Gly Asp Leu 530 535 540 Val Thr Cys Gly Ile Leu Ser Gly Asn Lys Asn Phe Glu Gly Arg Leu 545 550 555 560 Cys Asp Cys Val Arg Ala Asn Tyr Leu Ala Ser Pro Pro Leu Val Val 565 570 575 Ala Tyr Ala Ile Ala Gly Thr Val Asn Ile Asp Phe Gln Thr Glu Pro 580 585 590 Leu Gly Thr Asp Pro Thr Gly Lys Asn Ile Tyr Leu His Asp Ile Trp 595 600 605 Pro Ser Arg Glu Glu Val His Arg Val Glu Glu Glu His Val Ile Leu 610 615 620 Ser Met Phe Lys Ala Leu Lys Asp Lys Ile Glu Met Gly Asn Lys Arg 625 630 635 640 Trp Asn Ser Leu Glu Ala Pro Asp Ser Val Leu Phe Pro Trp Asp Leu 645 650 655 Lys Ser Thr Tyr Ile Arg Cys Pro Ser Phe Phe Asp Lys Leu Thr Lys 660 665 670 Glu Pro Ile Ala Leu Gln Ala Ile Glu Asn Ala His Val Leu Leu Tyr 675 680 685 Leu Gly Asp Ser Val Thr Thr Asp His Ile Ser Pro Ala Gly Ser Ile 690 695 700 Ala Arg Asn Ser Ala Ala Ala Lys Tyr Leu Thr Asn Arg Gly Leu Thr 705 710 715 720 Pro Arg Glu Phe Asn Ser Tyr Gly Ala Arg Arg Gly Asn Asp Ala Val 725 730 735 Met Thr Arg Gly Thr Phe Ala Asn Ile Lys Leu Phe Asn Lys Phe Ile 740 745 750 Gly Lys Pro Ala Pro Lys Thr Ile His Phe Pro Ser Gly Gln Thr Leu 755 760 765 Asp Val Phe Glu Ala Ala Glu Leu Tyr Gln Lys Glu Gly Ile Pro Leu 770 775 780 Ile Ile Leu Ala Gly Lys Lys Tyr Gly Ser Gly Asn Ser Arg Asp Trp 785 790 795 800 Ala Ala Lys Gly Pro Tyr Leu Leu Gly Val Lys Ala Val Leu Ala Glu 805 810 815 Ser Tyr Glu Lys Ile His Lys Asp His Leu Ile Gly Ile Gly Ile Ala 820 825 830 Pro Leu Gln Phe Leu Pro Gly Glu Asn Ala Asp Ser Leu Gly Leu Ser 835 840 845 Gly Arg Glu Thr Phe Ser Leu Thr Phe Pro Glu Glu Leu Ser Pro Gly 850 855 860 Ile Thr Leu Asn Ile Gln Thr Ser Thr Gly Lys Val Phe Ser Val Ile 865 870 875 880 Ala Ser Phe Glu Asp Asp Val Glu Ile Thr Leu Tyr Lys His Gly Gly 885 890 895 Leu Leu Asn Phe Val Ala Arg Lys Phe Ser 900 905 <210> 6 <211> 343 <212> PRT <213> Homo sapiens <220> <223> IRP2 Isoform 4 <400> 6 Met Asp Ala Pro Lys Ala Gly Tyr Ala Phe Glu Tyr Leu Ile Glu Thr 1 5 10 15 Leu Asn Asp Ser Ser His Lys Lys Phe Phe Asp Val Ser Lys Leu Gly 20 25 30 Thr Lys Tyr Asp Val Leu Pro Tyr Ser Ile Arg Val Leu Leu Glu Ala 35 40 45 Ala Val Arg Asn Cys Asp Gly Phe Leu Met Lys Lys Glu Asp Val Met 50 55 60 Asn Ile Leu Asp Trp Lys Thr Lys Gln Ser Asn Val Glu Val Pro Phe 65 70 75 80 Phe Pro Ala Arg Val Leu Leu Gln Asp Phe Thr Gly Ile Pro Ala Met 85 90 95 Val Asp Phe Ala Ala Met Arg Glu Ala Val Lys Thr Leu Gly Gly Asp 100 105 110 Pro Glu Lys Val His Pro Ala Cys Pro Thr Asp Leu Thr Val Asp His 115 120 125 Ser Leu Gln Ile Asp Phe Ser Lys Cys Ala Ile Gln Asn Ala Pro Asn 130 135 140 Pro Gly Gly Gly Asp Leu Gln Lys Ala Gly Lys Leu Ser Pro Leu Lys 145 150 155 160 Val Gln Pro Lys Lys Leu Pro Cys Arg Gly Gln Thr Thr Cys Arg Gly 165 170 175 Ser Cys Asp Ser Gly Glu Leu Gly Arg Asn Ser Gly Thr Phe Ser Ser 180 185 190 Gln Ile Glu Asn Thr Pro Ile Leu Cys Pro Phe His Leu Gln Pro Val 195 200 205 Pro Glu Pro Glu Thr Val Leu Lys Asn Gln Glu Val Glu Phe Gly Arg 210 215 220 Asn Arg Glu Arg Leu Gln Phe Phe Lys Trp Ser Ser Arg Val Phe Lys 225 230 235 240 Asn Val Ala Val Ile Pro Pro Gly Thr Gly Met Ala His Gln Ile Asn 245 250 255 Leu Glu Tyr Leu Ser Arg Val Val Phe Glu Glu Lys Asp Leu Leu Phe 260 265 270 Pro Asp Ser Val Val Gly Thr Asp Ser His Ile Thr Met Val Asn Gly 275 280 285 Leu Gly Ile Leu Gly Trp Gly Val Gly Gly Ile Glu Thr Glu Ala Val 290 295 300 Met Leu Gly Leu Pro Val Ser Leu Thr Leu Pro Glu Val Val Gly Cys 305 310 315 320 Glu Leu Thr Gly Ser Ser Asn Pro Phe Val Thr Ser Ile Asp Val Val 325 330 335 Leu Gly Ile Thr Lys Val Ser 340 <210> 7 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> T2A Peptide <400> 7 Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu Glu Asn Pro 1 5 10 15 Gly Pro

Claims

1. A lymphocyte comprising a synthetic polynucleotide encoding an iron regulatory protein (IRP), wherein the iron regulatory protein is IRP2 as shown in SEQ ID NO: 2; the lymphocyte is a T cell; and the lymphocyte further comprises a chimeric antigen receptor (CAR).

2. The lymphocyte according to claim 1, wherein the lymphocyte is a modified T cell or a virus-specific T cell.

3. The lymphocyte according to claim 1 or 2, wherein IRP2 is constitutively expressed.

4. The lymphocyte of claim 1 or 2, wherein the synthetic polynucleotide encoding IRP2 is under the control of a constitutive promoter. The lymphocyte according to claim 4 , wherein the constitutive promoter is the EF-1α promoter.

6. The lymphocyte of claim 1, wherein the CAR comprises an antigen binding domain, a transmembrane domain, a co-stimulatory signaling region, and a signaling domain.

7. The lymphocyte of claim 6, wherein the antigen binding domain is an antibody or an antigen binding fragment thereof.

8. The lymphocyte according to claim 7, wherein the antigen binding fragment is Fab or scFv.

9. The lymphocyte according to any one of claims 6 to 8, wherein the antigen binding domain specifically binds to a tumor antigen or a viral antigen.

10. The lymphocyte according to claim 9, wherein the tumor antigen is present on the cell surface of a target cell population or tissue.

11. The lymphocyte of claim 1, wherein the CAR is encoded by a polynucleotide, wherein the polynucleotide encoding the CAR is transcriptionally linked to a synthetic polynucleotide encoding IRP2.

12. The lymphocyte of claim 11, wherein the polynucleotide encoding CAR and the synthetic polynucleotide encoding IRP2 are linked by a polynucleotide encoding a self-cleaving peptide.

13. The lymphocyte according to claim 12, wherein the self-cleaving peptide is a 2A self-cleaving peptide.

14. The lymphocyte according to claim 12 or 13, wherein the self-cleaving peptide is T2A.

15. A viral vector comprising at least one polynucleotide encoding IRP2 as shown in SEQ ID NO: 2, wherein the viral vector comprises another polynucleotide encoding CAR.

16. The viral vector of claim 15, wherein the viral vector is derived from a lentivirus, adeno-associated virus (AAV), adenovirus, herpes simplex virus, retrovirus, alphavirus, flavivirus, rhabdovirus, measles virus, Newcastle disease virus, or poxvirus. The viral vector according to claim 16 , wherein the viral vector is derived from a lentivirus.

18. The viral vector of claim 15 or 16, wherein the at least one polynucleotide encoding IRP2 is under the control of a constitutive promoter. The viral vector according to claim 18 , wherein the constitutive promoter is the EF-1α promoter.

20. The viral vector of claim 15, wherein the polynucleotide encoding CAR is transcriptionally linked to the polynucleotide encoding IRP2.

21. The viral vector of claim 20, wherein the polynucleotide encoding CAR and the polynucleotide encoding IRP2 are linked by a polynucleotide encoding a self-cleaving peptide.

22. The viral vector according to claim 21, wherein the self-cleaving peptide is a 2A self-cleaving peptide.

23. The viral vector according to claim 21 or 22, wherein the self-cleaving peptide is T2A.

24. A pharmaceutical composition comprising the lymphocyte according to any one of claims 1 to 14 or the viral vector according to any one of claims 15 to 23 and a pharmaceutically acceptable carrier.

25. The lymphocyte according to any one of claims 1 to 14, the viral vector according to any one of claims 15 to 23 or the pharmaceutical composition according to claim 24 for use in therapy.

26. The lymphocyte according to any one of claims 1 to 14, the viral vector according to any one of claims 15 to 23 or the pharmaceutical composition according to claim 24 for use in treating cancer.

27. The lymphocyte, viral vector or pharmaceutical composition of claim 26, wherein the cancer is a hematological cancer or a solid tumor.

28. The lymphocyte, viral vector or pharmaceutical composition of claim 27, wherein the hematological cancer is acute lymphoblastic leukemia, diffuse large B-cell lymphoma, Hodgkin lymphoma, acute myeloid leukemia or multiple myeloma, and wherein the solid tumor is colon cancer, breast cancer, pancreatic cancer, ovarian cancer, hepatocellular carcinoma, lung cancer, neuroblastoma, glioblastoma or sarcoma.

29. The lymphocyte according to any one of claims 1 to 14, the viral vector according to any one of claims 15 to 23 or the pharmaceutical composition according to claim 24, for use in preventing and / or treating viral infections.

30. The lymphocyte, viral vector or pharmaceutical composition according to claim 29, wherein the viral infection is caused by human immunodeficiency virus (HIV), adenovirus, polyomavirus, influenza virus or human herpes virus, specifically, wherein the human herpes virus is cytomegalovirus (CMV), Epstein-Barr virus (EBV), herpes simplex virus (HSV), varicella-zoster virus (VZV) or human herpes virus 8 (HHV8).

31. A method for producing lymphocytes according to any one of claims 1 to 14 in vitro, the method comprising the following steps: a) providing lymphocytes obtained from a subject; b) introducing a synthetic polynucleotide encoding an iron regulatory protein into the lymphocytes of step (a), wherein the iron regulatory protein is IRP2; and c) expressing IRP2 in lymphocytes obtained from the subject, wherein in step (b) a second synthetic polynucleotide encoding a chimeric antigen receptor (CAR) is introduced into the lymphocyte.

32. The method of claim 31 , wherein the synthetic polynucleotide encoding CAR is combined with a synthetic polynucleotide encoding IRP2.

33. The method of claim 31 or 32, wherein the synthetic polynucleotide encoding CAR is transcriptionally linked to the synthetic polynucleotide encoding IRP2.

34. The method of claim 31 or 32, wherein the synthetic polynucleotide encoding CAR and the polynucleotide encoding IRP2 are linked by a polynucleotide encoding a self-cleaving peptide.

35. The method of claim 34, wherein the self-cleaving peptide is a 2A self-cleaving peptide.

36. The method of claim 34, wherein the self-cleaving peptide is T2A.

37. The method of claim 31 or 32, wherein one or more synthetic polynucleotides are introduced into the lymphocytes by viral transduction.

38. The method of claim 37, wherein viral transduction is performed using a viral vector according to any one of claims 15 to 23.

39. The method of claim 31 or 32, wherein the lymphocytes are activated before or after introduction of one or more synthetic polynucleotides into the lymphocytes.

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