PDE-enhanced CAR-T cells

Abstract

The present invention relates generally to the field of CAR-T cells. Specifically, the present invention relates to improved methods for obtaining CAR-T cells, and improved CAR-T cells and uses thereof. The method further relates to activating or expressing phosphodiesterase (PDE) in T cells during CAR-T cell generation.

Description

[Technical Field]

[0001] The present invention relates generally to the field of CAR-T cells. Specifically, the present invention relates to improved methods for obtaining CAR-T cells, and improved CAR-T cells and uses thereof. The method further relates to activating or expressing phosphodiesterase (PDE) in T cells during CAR-T cell generation. [Background technology]

[0002] The background discussion includes information that may be useful in understanding the present invention. No admission is made that any of the information provided herein is prior art or related to the invention claimed herein, or that any of the articles specifically or implicitly referenced are prior art.

[0003] For decades, the cornerstones of cancer treatment have been surgery, chemotherapy, and radiation therapy. While these remain important treatment mainstays, novel categories of treatment have helped transform the care of people with cancer in recent years. The role of the immune system in cancer prevention and treatment has become more evident, leading, for example, to the development of immune checkpoint inhibitor-based therapies.

[0004] More recently, chimeric antigen receptor (CAR)-expressing T cells (CAR-T cells) have become popular as a method for inducing the host immune system to respond to blood or solid tumors. Using this method, chimeric antigen receptors targeting tumor-specific antigens are expressed on T cells. This allows the host T cells to be used, thereby avoiding an immune response directed at the T cells themselves. The CAR expressed on the T cells targets the cells to the tumor, and the T cells can hopefully kill the malignant cells and induce further immune responses from the host directed at the tumor cells. Summary of the Invention [Problem to be solved by the invention]

[0005] A challenge with existing CAR-T cell-based therapies is the need to generate and expand large numbers of CAR-T cells, which is costly and time-consuming. Therefore, improving the efficacy of CAR-T cells is highly desirable. Furthermore, the efficacy of CAR-T cells in killing their intended target cells is generally less than 100%, and therefore there is a need to improve cytotoxicity against their intended targets.

[0006] The present invention aims to address the above-mentioned problems, inter alia, by the methods, products and uses defined in the appended claims. [Means for solving the problem]

[0007] In a first aspect, the present invention provides an ex vivo or in vitro method for generating improved CAR-T cells, the method comprising: providing T cells; introducing a chimeric antigen receptor (CAR) gene into T cells to obtain CAR-T cells; Including, The method further includes upregulating the enzymatic activity of a phosphodiesterase (PDE) protein in the T cell before, during, or after introducing the CAR gene into the cell.

[0008] In a second aspect, the present invention relates to a CAR-T cell having an upregulated enzymatic activity of a PDE protein, or a CAR-T cell obtained or obtainable by the method defined in the first aspect of the invention.

[0009] In a third aspect, the method relates to a CAR-T cell according to the second aspect of the invention for use as a medicament.

[0010] definition Portions of this disclosure contain material that is subject to copyright protection (such as, but not limited to, diagrams, device photographs, or any other aspect of this application that may be copyrighted or available under any authority). The copyright owner has no objection to the complete facsimile reproduction by any one of the patent documents or patent publications as noted in the patent office patent file or records, but otherwise reserves all copyright whatsoever.

[0011] Various terms relating to the methods, compositions, uses, and other aspects of the present invention are used throughout the specification and claims. Such terms are to be given their ordinary meaning in the art to which the invention pertains, unless otherwise specified. Other specifically defined terms are to be interpreted in a manner consistent with the definitions provided herein. Although any methods and materials similar or equivalent to those described herein can be used in the practice of testing of the present invention, the preferred materials and methods are described herein.

[0012] For purposes of the present invention, the following terms are defined below.

[0013] As used herein, the singular includes plural references unless the context clearly dictates otherwise. For example, a method for administering an agent includes administering a plurality of molecules (e.g., tens, hundreds, thousands, tens of thousands, hundreds of thousands, millions, or more molecules).

[0014] As used herein, "about" and "approximately," when referring to measurable values ​​such as amounts, durations, etc., are meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and even more preferably ±0.1% from the particular value, as variations are appropriate to practice the disclosed invention. Unless otherwise clear from the context, all numerical values ​​provided herein include the numerical value modified by the term "about."

[0015] As used herein, "and / or" refers to a situation in which one or more of the stated instances may occur, either alone or in combination with at least one of the stated instances, up to all of the stated instances.

[0016] As used herein, the term "at least" plural value means a particular value or more. For example, "at least two" is understood to be the same as "two or more," i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, etc. As used herein, the term "up to" plural value means a particular value or less. For example, "up to five" is understood to be the same as "five or less," i.e., 5, 4, 3, -10, -11, etc.

[0017] As used herein, "comprising" is to be interpreted as inclusive and open-ended, not exclusive. Specifically, the term and variations thereof mean that the specified features, steps, or components are included. These terms are not to be interpreted as excluding the presence of other features, steps, or components. It also encompasses the more restrictive "consisting of."

[0018] As used herein, "prior art" or "methods known to those skilled in the art" refers to situations in which it would be clear to one skilled in the art how to carry out the prior art used in the methods of the present invention. The practice of prior art techniques in molecular biology, biochemistry, cell culture, genomics, sequencing, medical treatment, pharmacology, immunology and related fields is well known to those skilled in the art and is discussed in various handbooks and literature.

[0019] As used herein, "exemplary" or "for example" means "serving as an example, instance, or illustration" and should not be construed as excluding other configurations, including those disclosed herein.

[0020] Throughout this disclosure, various aspects of the invention may be presented in a variety of formats. It should be understood that the descriptions of ranges in these formats are merely for convenience and should not be construed as limitations on the scope of the invention. The description of a range is considered to include all possible subranges specifically disclosed, as well as individual numerical values ​​within that range, including both integers and non-integer values. For example, the description of a range such as 1 to 6 is considered to include specifically disclosed subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numerical values ​​within that range, e.g., 1, 2, 2.7, 3, 4, 5, 5.3, 6, etc. This applies regardless of the breadth of the range.

[0021] As used herein, "cancer" refers to a physiological condition in mammals that is typically characterized by uncontrolled cell growth. The terms "cancer," "neoplasm," and "tumor" are often used interchangeably to describe cells that have undergone malignant transformation that makes them pathological to the host organism. Primary cancer cells can be distinguished from non-cancerous cells by techniques known to those skilled in the art. Cancer cells, as used herein, include not only primary cancer cells, but also cancer cells derived from such primary cancer cells, including metastatic (secondary) cancer cells and cell lines derived from cancer cells. Examples include solid tumors and non-solid tumors, or hematologic tumors. Treating cancer in a subject includes treating a tumor in the subject.

[0022] The drugs, therapeutic agents, medicaments, and pharmaceutical compositions described herein can be formulated for administration by many routes, including, but not limited to, parenteral, intravenous, intraarterial, intramuscular, intratumoral, and oral. The drugs, therapeutic agents, medicaments, and compositions can be formulated in liquid or solid form. Liquid formulations can be formulated for administration by injection into selected regions of the human or animal body. Preferably, the cells described herein, when used as pharmaceuticals, are formulated for administration in a liquid formulation suitable for injection, for example, intravenous, intraarterial, intramuscular, or intratumoral delivery or injection.

[0023] As used herein, the term "pharmaceutical composition" refers to a composition formulated in a pharmaceutically or physiologically acceptable composition for administration to a cell or subject. The compositions of the present invention may also be administered in combination with other agents, so long as the additional agents do not adversely affect the ability of the composition to deliver the intended therapy. Pharmaceutical compositions often contain one or more pharmaceutically acceptable carriers (or excipients) in addition to the pharmaceutically active agent.

[0024] As used herein, "subject" refers to an organism to be treated, e.g., to which administration is being considered. The subject can be any subject described in the present invention, including, but not limited to, a human, male, female, infant, child, adolescent, adult, young adult, middle-aged adult, or elderly person, and / or other primate or mammal. Preferably, the subject is a human patient. In some embodiments, the subject may be diagnosed with cancer, an immune-related disorder, a bleeding disorder, a disorder associated with protein overexpression, or a disorder associated with protein underexpression. In some embodiments, the subject may be at risk of developing a disease or disorder that can be prevented or alleviated by vaccination.

[0025] As used herein, "T cells" may be selected from the group consisting of, for example, inflammatory T lymphocytes, cytotoxic T lymphocytes, regulatory T lymphocytes, or helper T lymphocytes. In another embodiment, the cells may be derived from the group consisting of CD4+ T lymphocytes and CD8+ T lymphocytes. They may be extracted from blood or derived from stem cells. T cells can be obtained from many sources, including, but not limited to, peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, and tumors. In certain embodiments of the present invention, any T cell line available and known to those of skill in the art may be used. In another embodiment, the cells may be derived from a healthy donor, a patient diagnosed with cancer, or a patient diagnosed with an infectious disease. In another embodiment, the cells are part of a mixed population of cells exhibiting different phenotypic characteristics.

[0026] As used herein, in the context of a subject being treated, "treatment," "treating," "alleviating," "relieving," and "alleviating" all refer to an approach to obtaining a beneficial or desired result, including, but not limited to, a therapeutic benefit. Therapeutic benefit refers to the eradication or alleviation of the underlying disease being treated. A therapeutic benefit may also be achieved by the eradication or alleviation of one or more physiological symptoms associated with the underlying disease, so long as an improvement is observed in the patient, even though the patient still suffers from the underlying disease. As used herein, "prevention" and "preventing" refer to an approach to partially or completely reducing the chance of developing adverse effects, for example, those typically associated with the use of a particular drug or medication. Within the context of the present invention, for example, the terms may refer to preventing, treating, or reducing the effects of cancer, immune-related disorders, bleeding disorders, disorders associated with protein overexpression, or disorders associated with protein underexpression. In some embodiments, preventing may also refer to the effect of preventing disease through vaccination.

[0027] As used herein, the term "nucleic acid" or "polynucleotide" refers to any polymer or oligomer of (contiguous) nucleotides. Nucleic acids may be DNA or RNA, or mixtures thereof, and may exist permanently or transiently in single- or double-stranded form, including homodimers, heterodimers, and hybrid states. The present invention contemplates any deoxyribonucleotide, ribonucleotide, or peptide nucleic acid component, and any chemical variants thereof, such as methylated, hydroxymethylated, or glycosylated forms of these bases. The polymer or oligomer may be heterogeneous or homogeneous in composition and may be isolated from a naturally occurring source or produced artificially or synthetically. The term "isolated," therefore, means isolated from a naturally occurring source or produced artificially or synthetically.

[0028] As used herein, "percent (%) amino acid sequence identity" is defined as the percentage of amino acid residues in a candidate sequence that are identical to those in a reference polypeptide sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and without considering any conservative substitutions as part of the sequence identity. Alignment to determine percent amino acid sequence identity can be achieved in a variety of ways within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximum alignment over the entire length of the sequences being compared. However, for purposes herein, percent amino acid sequence identity values ​​are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was created by Genentech, Inc., and the source code is on file with user documentation in the U.S. Copyright Office, Washington, DC 20559, and is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, Calif., or may be compiled from the source code. The ALIGN-2 program should be compiled for use on UNIX operating systems, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary. In situations where ALIGN-2 is used for amino acid sequence comparison, the % amino acid sequence identity of a given amino acid sequence A to, with, or relative to a given amino acid sequence B (alternatively, it may be expressed as a given amino acid sequence A having or comprising a certain % amino acid sequence identity to, with, or relative to a given amino acid sequence B) is calculated as follows: Fraction X / Y×100 where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in a program alignment of A and B, and Y is the total number of amino acid residues in B. It will be understood that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not be equal to the % amino acid sequence identity of B to A. Unless specifically stated otherwise, all % amino acid sequence identities used herein are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program.

[0029] As used herein, the terms "protein" and "polypeptide" refer to molecules consisting of chains of amino acids, regardless of a particular mode of action, size, three-dimensional structure, or origin. A "fragment" or "portion" or "part" of a polypeptide may therefore still be referred to as a "polypeptide." An "isolated protein" or "isolated polypeptide" is used to refer to a protein or polypeptide that is no longer in its natural environment, for example, in vitro or in a recombinant host cell.

[0030] As used herein, the terms "construct" or "nucleic acid construct" or "vector" refer to an artificial nucleic acid molecule resulting from the use of recombinant DNA technology and used to deliver exogenous DNA into a host cell, often for the expression in the host cell of the DNA region contained on the construct. The vector backbone of the construct may be, for example, a plasmid into which a (chimeric) gene is integrated; if suitable transcription control sequences are already present, only the desired nucleic acid sequence (e.g., coding sequence) is integrated downstream of the transcription control sequence. Vectors may contain additional genetic elements that facilitate their use in molecular cloning, such as, for example, selectable markers, multiple cloning sites, etc.

[0031] Embodiments of the present invention are further described below with reference to the accompanying drawings. [Brief explanation of the drawings]

[0032] [Figure 1]

[0023] Figure 1 provides an exemplary vector map of a CART expression vector driven by the EF-1 alpha promoter, expressing a CAR. The exemplary CAR comprises an antigen recognition domain (scFv), a CD28 domain, a 4-1BB domain, and a CD3 zeta domain (third generation CAR). [Figure 2] FIG. 1 provides an exemplary vector map of a CART expression vector that expresses CAR driven by the EF-1 alpha promoter and further contains a PDE4D2-encoding gene driven by the PGK promoter. [Figure 3] Figure 1 shows flow cytometry results for different T cells. Plots show side scatter on the Y-axis and anti-APC on the X-axis. Illustrated are control T cells without and with secondary anti-mouse Fab647 antibody (first and second panels), control CAR-T cells (third panel), and PDE4D2-expressing CAR-T cells (fourth panel). The percentage of positively identified cells is shown in each panel and is 0% (control without secondary antibody), 4% (control with secondary antibody), 25% (control CAR-T cells), and 80% (PDE4D2 CAR-T cells), respectively. [Figure 4] Figure 1 shows a graph plotting the percentage of target cell killing at different E / T (effector / target) ratios. PSMA-targeting CAR-T cells were used together with PSMA-overexpressing CHO cells. On the left, control CAR-T cells are plotted (two replicates of the same experiment), and on the right, PDE4D2-expressing CAR-T cells are plotted. At each ratio, PDE4D2-expressing CAR-T cells show a significant increase in the percentage of target cell killing. [Figure 5]1 shows a graph plotting IL-2 release at different E / T (effector / target) ratios. PSMA-targeting CAR-T cells were used with PSMA-overexpressing CHO cells. On the left, control CAR-T cells are plotted (two replicates of the same experiment), and on the right, PDE4D2-expressing CAR-T cells are plotted. At each ratio, PDE4D2-expressing CAR-T cells show a significant increase in IL-2 release. [Figure 6] Figure 1 shows a graph plotting interferon gamma (IFNG) release at different E / T (effector / target) ratios. PSMA-targeting CAR-T cells were used with PSMA-overexpressing CHO cells. On the left, control CAR-T cells are plotted (two replicates of the same experiment), and on the right, PDE4D2-expressing CAR-T cells are plotted. Only at E / T (effector / target) ratios of 1:1 and 2.5:1 was an effect observed on IFNG release between the different test conditions. [Figure 7] Figure 1 shows the expression levels of PDE4A, PDE4B, and PDE4D before and 6, 24, and 72 hours after T cell activation. T cells were activated with anti-CD3 and anti-CD28 antibodies. Significant differences are indicated in each panel for gene expression levels that deviate significantly (P<0.05) between the indicated time points. [Figure 8] Western blot analysis of (A) Pan4D and (B) VSV-tag expression. Whole cell lysates from HEK293 cells transfected with the CAR-PDE4D2 plasmid were run on a 4-12% gradient gel. After transfer to a nitrocellulose membrane, the blotted bands were immunodetected. Lane 1: molecular weight marker; lane 2: untransfected control (UT); lanes 3-6: various amounts (1-7 μg) of transfected plasmid DNA. [Figure 9]

[0023] Figure 1 shows the results of a PDE activity assay. Whole HEK293 cell lysates were used, either untransfected or transfected with a CAR-T plasmid containing PDE4D2. Transfected cells without or with a PDE inhibitor (IBMX or Rolipram) were compared. DETAILED DESCRIPTION OF THE INVENTION

[0033] The present invention is defined herein with particularity in the appended claims. Objects not encompassed by the scope of the claims do not form a part of the claimed invention.

[0034] It is contemplated that any method, use, or composition described herein can be implemented with respect to any other method, use, or composition described herein. Embodiments or preferences discussed in the context of a method, use, and / or composition of the invention can likewise be used with respect to any other method, use, or composition described herein. Thus, embodiments or preferences pertaining to one method, use, or composition can likewise be applied to other methods, uses, and compositions of the invention.

[0035] Any reference in the description of a method of treatment refers to the compounds, pharmaceutical compositions and medicaments of the invention for use in that method for treatment of the human (or animal) body by therapy.

[0036] As embodied and broadly described herein, the present invention is directed to the surprising discovery that generating CAR-T cells by introducing a CAR-expressing vector activates phosphodiesterase activity in T cells, resulting in (1) a significant increase in the efficacy of the CAR-expressing T cells, and (2) an increase in the efficacy of the resulting CAR-T cells to kill their respective target cells. As described in the Examples below and the accompanying figures, expressing a vector encoding a chimeric antigen receptor (CAR) typically results in approximately 25% of cells (CAR-T cells) that positively express the CAR on their surface. Figure 3 shows that coexpression of a gene encoding PDE4D isoform 2 (PDE4D2; encoded by PDE4D transcript variant 7) surprisingly increases this percentage to approximately 80%. When comparing control CAR-T cells with PDE4D2-expressing CAR-T cells, the percentage of killed cells was found to be clearly increased as shown in Figures 4, 5, and 6, indicating that PDE4D2-expressing CAR-T cells increased IL-2 expression but showed limited significant differences in interferon gamma secretion. Without wishing to be bound by theory, the inventors therefore theorize that the increased efficacy of PDE4D2-expressing CAR-T cells in killing their respective target cells is largely mediated by an IL-2-dependent mechanism.

[0037] In the experiments presented herein, the inventors used a CAR targeting prostate-specific membrane antigen (PSMA) and tested these CAR-T cells with CHO cells overexpressing PSMA. Those skilled in the art have no reason to expect that the results presented herein relate to the specific CAR or antigen used in these experiments, and therefore, the present invention can be applied to any CAR and any antigen targeted by the CAR. For the experiments described herein, a PDE-encoding gene is included in a CAR-T expression vector. Those skilled in the art will know that this is simply a means of introducing and overexpressing a PDE protein, with the end result of increased PDE enzymatic activity; therefore, those skilled in the art will readily understand that the same results can be achieved by any means of activating or overexpressing PDE in T cells. The results described below use PDE4D2 as an exemplary phosphodiesterase, but it is well known that there are many phosphodiesterases with very similar structures and functions. Those skilled in the art will readily understand that other phosphodiesterases can be used to achieve the same effect, and therefore, the experimental results should not be interpreted as limiting the present invention to PDE4D2 alone.

[0038] Thus, in a first embodiment, the present invention provides an ex vivo or in vitro method for generating improved CAR-T cells, comprising: providing T cells; and introducing a chimeric antigen receptor (CAR) gene into T cells to obtain CAR-T cells. Further comprising the step of upregulating the enzymatic activity of a phosphodiesterase (PDE) protein in the T cell before, during, or after introducing the CAR gene into the T cell. Regarding the method.

[0039] Thus, the invention described herein broadly defines an improved method for generating CAR-T cells that upregulates the enzymatic activity of phosphodiesterase (PDE) proteins in T cells. As shown in the experimental results, the effect of activating PDE is two-fold: (1) it significantly increases the number of T cells expressing chimeric antigens (CARs), thus surprisingly increasing the efficacy of T cell transformation or T cell expansion in vitro; and (2) the expression of PDE surprisingly leads to increased efficacy of CAR-T cells, as PDE-activated CAR-T cells result in a higher percentage of killed target cells (cells expressing the antigen to which the CAR is directed).

[0040] As used herein, CAR (chimeric antigen receptor) refers to an artificial T cell receptor typically used in immunotherapy. CARs, also known as chimeric immune receptors, chimeric T cell receptors, or artificial T cell receptors, are engineered receptor proteins to confer new capabilities to T cells for targeting specific antigens. The receptors are chimeric because they combine both antigen binding and T cell activation functions in a single receptor. Typically, chimeric receptors include a single-chain variable fragment (scFv) domain that targets the antigen; a hinge region (also called a spacer) that can be derived from, for example, IgG or CD8 protein; a transmembrane domain typically consisting of a hydrophobic alpha helix, for example, the transmembrane domain derived from CD28; and an intracellular T cell signaling domain, for example, the CD3-zeta cytoplasmic domain. It is understood that not all of the domains and regions listed above are required to be present in a CAR; they may be replaced with different domains or regions or omitted entirely. Thus, the term CAR is understood to include any engineered receptor that, when expressed on a T cell, allows for targeting of the T cell to a specific antigen and initiation of an immune response.

[0041] As used herein, a CAR-T cell refers to a T cell that expresses a CAR as defined herein.

[0042] The T cells provided herein may be obtained from a subject intended to be treated with the CAR-T cells obtained by the present method, although it is understood that the present method does not limit the origin of the T cells. Thus, the T cells may be obtained, for example, from a donor, or may be obtained by differentiating progenitor cells in vitro and obtained from a subject intended to be treated with the resulting CAR-T cells. While an obvious application of the CAR-T cells obtained by the present method is their use in treating subjects, it is further understood that the present method is not intended to be limited to such applications and may also be used, for example, in research, product development, or screening methods.

[0043] The term phosphodiesterase (PDE), as used herein, refers to an enzyme that cleaves a phosphodiester bond. More specifically, as used herein, phosphodiesterase refers to a cyclic nucleotide phosphodiesterase that can convert cyclic nucleotides into nucleotide monophosphates. Typically, phosphodiesterases convert cAMP or cGMP into AMP or GMP, respectively. As used herein, the term PDE can refer to a protein having phosphodiesterase enzyme activity or a nucleic acid (e.g., a gene) encoding such a protein. The term should not be construed as limiting and should therefore be understood to refer to both PDEs from humans or animals (e.g., rodents, primates, mammals, fish, amphibians, reptiles, birds, vertebrates, insects, etc.), although in a preferred embodiment, PDE refers to a human PDE. Therefore, the term PDE comprises at least PDE1A, PDE1B, PDE1C, PDE2A, PDE3A, PDE4A, PDE4B, PDE4C, PDE4D, PDE5A, PDE6A, PDE6B, PDE6C, PDE6D, PDE6G, PDE6H, PDE7A, PDE7B, PDE8A, PDE8B, PDE9A, PDE10A or PDE11A, or any one of these specific isoforms.In one embodiment, the above-mentioned PDE is human PDE.In one embodiment, PDE is the protein with phosphodiesterase activity, preferably the enzyme classified by EC number 3.1.4.17. In one embodiment, the PDE is a protein or enzyme defined according to Table 1 below, or that has sequence identity (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity) with a protein defined in Table 1, or that is encoded by a nucleotide sequence that has sequence identity (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity) with a sequence defined in Table 1.

[0044] [Table 1] TIFF2025537884000003.tif255151

[0045] The above table refers to nucleotide and protein identifiers available in GenBank, accessible at https: / / www.ncbi.nlm.nih.gov / nuccore. It is understood that isoforms are available for each protein. The table lists specific isoforms of the PDE2 and PDE4 genes / proteins, but for the remaining PDEs (PDE1A, PDE1B, ..., PDE3A, etc.), specific isoforms are not listed, and specific gene and protein sequences are representative isoforms. The specific nomenclature used for different isoforms is based on protein consensus nomenclature; therefore, it is possible for an mRNA to have a sequence variant number that does not correspond to an isoform number. For example, PDE4D isoform 2 protein is encoded by a sequence annotated as PDE4D sequence variant 7. When a specific isoform (e.g., PDE4D2) is referred to herein, reference to the protein nomenclature consensus is intended. It is understood that specific isoforms have been identified for each of these PDEs, and the present invention should not be construed as being limited to the specific listed isoforms.

[0046] As used herein, an isoform refers to a variant protein encoded by the same gene locus that has a different mRNA and / or protein sequence. A non-limiting example of an isoform is a splice variant. It is understood that the nucleotide sequences referenced in Table 1 above correspond to mRNA sequences and therefore may include 5'UTR and / or 3'UTR sequences. Those skilled in the art can determine the coding sequence based on information available, for example, in genome reference databases. It is understood that UTRs can be altered without changing the coding sequence; therefore, variations in the nucleotide sequences presented above are preferably based on sequence homology based solely on the coding sequence, meaning that a sequence with 99% sequence homology to SEQ ID NO:1 is intended to cover those sequences that have at least 99% sequence homology with the coding sequence defined in SEQ ID NO:1, ignoring the UTRs. The present invention also intends to cover isoforms of the above-referenced genes that are not explicitly listed.

[0047] The term "PDE1A" refers to the phosphodiesterase 1A gene (Ensembl: ENSG00000115252), e.g., the sequence defined in NCBI Reference Sequence NM_005019.7, in particular the nucleotide sequence set forth in SEQ ID NO: 1, which corresponds to the sequence of the above-mentioned NCBI Reference Sequence for the PDE1A transcript, and also to the corresponding amino acid sequence set forth in SEQ ID NO: 2, which corresponds to the protein sequence defined in NCBI Protein Accession Reference Sequence NP_005010.2, which encodes the PDE1A polypeptide.

[0048] The term "PDE1A" refers to a nucleotide sequence that exhibits high homology to PDE1A, for example, a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 1, or an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 2. Also included are sequences, or nucleic acid sequences that encode an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO:2, or amino acid sequences encoded by nucleic acid sequences that are at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO:1.

[0049] The term "PDE1B" refers to the phosphodiesterase 1B gene (Ensembl: ENSG00000123360), e.g., the sequence defined in NCBI Reference Sequence NM_000924.4, in particular the nucleotide sequence set forth in SEQ ID NO: 3, which corresponds to the sequence of the above-mentioned NCBI Reference Sequence for the PDE1B transcript, and also to the corresponding amino acid sequence set forth in SEQ ID NO: 4, which corresponds to the protein sequence defined in NCBI Protein Accession Reference Sequence NP_000915.1, which encodes the PDE1B polypeptide.

[0050] The term "PDE1B" refers to a nucleotide sequence that exhibits high homology to PDE1B, for example, a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO:3, or an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO:4. Also included are sequences, or nucleic acid sequences that encode an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO:4, or amino acid sequences encoded by nucleic acid sequences that are at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO:3.

[0051] The term "PDE1C" refers to the phosphodiesterase 1C gene (Ensembl: ENSG00000154678), for example the sequence defined in NCBI Reference Sequence NM_001191056.3, in particular the nucleotide sequence set forth in SEQ ID NO: 5, which corresponds to the sequence of the above-mentioned NCBI Reference Sequence for the PDE1C transcript, and also to the corresponding amino acid sequence set forth in SEQ ID NO: 6, which corresponds to the protein sequence defined in NCBI Protein Accession Reference Sequence NP_001177985.1, which encodes the PDE1C polypeptide.

[0052] The term "PDE1C" refers to a nucleotide sequence that exhibits high homology to PDE1C, for example, a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO:5, or an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO:6. Also included are sequences, or nucleic acid sequences that encode an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO:6, or amino acid sequences encoded by nucleic acid sequences that are at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO:5.

[0053] The term "PDE2A" refers to the phosphodiesterase 2A gene (Ensembl: ENSG00000186642), e.g., the sequence defined in NCBI Reference Sequence NM_002599.5, NM_001143839.4, NM_001243784.2, or NM_001146209.3, in particular the nucleotide sequence set forth in SEQ ID NO: 7, 9, 11, or 13, which corresponds to the sequence of the above-mentioned NCBI Reference Sequence for the PDE2A transcript, and also to the corresponding amino acid sequence set forth in SEQ ID NO: 8, 10, 12, or 14, which corresponds to the protein sequence defined in NCBI Protein Accession Reference Sequence NP_002590.1, NP_001137311.1, NP_001230713.1, or NP_001139681.1, which encodes a PDE2A polypeptide.

[0054] The term "PDE2A" refers to a nucleotide sequence that exhibits high homology to PDE2A, for example, a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 7, 9, 11, or 13, or an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 8, 10, 12, or 14. Also included are nucleic acid sequences that encode an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 8, 10, 12, or 14, or an amino acid sequence encoded by a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 7, 9, 11, or 13.

[0055] The term "PDE3A" refers to the phosphodiesterase 3A gene (Ensembl: ENSG00000172572), e.g., the sequence defined in NCBI Reference Sequence NM_000921.5, in particular the nucleotide sequence set forth in SEQ ID NO: 15, which corresponds to the sequence of the above-mentioned NCBI Reference Sequence for the PDE3A transcript, and also to the corresponding amino acid sequence set forth in SEQ ID NO: 16, which corresponds to the protein sequence defined in NCBI Protein Accession Reference Sequence NP_000912.3, which encodes the PDE3A polypeptide.

[0056] The term "PDE3A" refers to a nucleotide sequence that exhibits high homology to PDE3A, for example, a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 15, or an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 16. Also included are sequences, or nucleic acid sequences that encode an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO:16, or amino acid sequences encoded by nucleic acid sequences that are at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO:15.

[0057] The term "PDE3B" refers to the phosphodiesterase 3B gene (Ensembl: ENSG00000152270), e.g. the sequence defined in NCBI Reference Sequence NM_000922.4, in particular the nucleotide sequence set forth in SEQ ID NO: 17, which corresponds to the sequence of the above-mentioned NCBI Reference Sequence for the PDE3B transcript, and also to the corresponding amino acid sequence set forth in SEQ ID NO: 18, which corresponds to the protein sequence defined in NCBI Protein Accession Reference Sequence NP_000913.2, which encodes a PDE3B polypeptide.

[0058] The term "PDE3B" refers to a nucleotide sequence that exhibits high homology to PDE3B, for example, a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 17, or an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 18. Also included are nucleic acid sequences that encode an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO:18, or an amino acid sequence encoded by a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO:17.

[0059] The term "PDE4A" refers to the phosphodiesterase 4A gene (Ensembl: ENSG00000065989), e.g., the sequences defined in the NCBI reference sequences NM_001111307.2, NM_001111308.1, NM_001111309.1, NM_006202.3, or NM_001243121.2, in particular SEQ ID NOs: 19, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109 23, 25, or 27, and also relates to the corresponding amino acid sequence set forth in SEQ ID NO: 20, 22, 24, 26, or 28, which correspond to the protein sequence defined in, for example, NCBI Protein Accession Reference Sequence NP_001104777.1, NP_001104778.1, NP_001104779.1, NP_006193.1, or NP_001230050.1 encoding a PDE4A polypeptide.

[0060] The term "PDE4A" refers to a nucleotide sequence that exhibits high homology to PDE4A, for example, a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 19, 21, 23, 25, or 27, or an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 20, 22, 24, 26, or 28. Also included are nucleic acid sequences that encode an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 20, 22, 24, 26, or 28, or an amino acid sequence encoded by a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 19, 21, 23, 25, or 27.

[0061] The term "PDE4B" refers to the phosphodiesterase 4B gene (Ensembl: ENSG00000184588), e.g., the sequences defined in the NCBI reference sequences NM_002600.4, NM_001037339.2, NM_001037340.3, NM_001037341.2, NM_001297440.2, NM_001297441.1, or NM_001297442.2, in particular SEQ ID NOs: 29, 31, 33, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 110, 111, 120, 1 37, 39, or 41, and also relates to the corresponding amino acid sequence set forth in SEQ ID NO: 30, 32, 34, 36, 38, 40, or 42, which correspond to the protein sequence defined in, for example, NCBI Protein Accession Reference Sequence NP_002591.2, NP_001032416.1, NP_001032417.1, NP_001032418.1, NP_001284369.1, NP_001284370.1, or NP_001284371.1, which encodes a PDE4B polypeptide.

[0062] The term "PDE4B" refers to a nucleotide sequence that exhibits high homology to PDE4B, for example, a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 29, 31, 33, 35, 37, 39, or 41, or an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 30, 32, 34, 36, 38, 40, or 42. Also included are nucleic acid sequences that encode an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 30, 32, 34, 36, 38, 40, or 42, or an amino acid sequence encoded by a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 29, 31, 33, 35, 37, 39, or 41.

[0063] The term "PDE4C" refers to the phosphodiesterase 4C gene (Ensembl: ENSG00000105650), e.g., the sequences defined in the NCBI reference sequences NM_000923.6, NM_001098819.4, NM_001098818.4, NM_001369701.2, NM_001330172.2, or NM_001395274.1, in particular SEQ ID NOs: 43, 45, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135 49, 51, or 53, and also relates to the corresponding amino acid sequence set forth in SEQ ID NO: 44, 46, 48, 50, 52, or 54, which correspond to the protein sequence defined in, for example, NCBI Protein Accession Reference Sequence NP_000914.2, NP_001092289.1, NP_001092288.1, NP_001356630.1, NP_001317101.1, or NP_001382203.1, which encodes a PDE4C polypeptide.

[0064] The term "PDE4C" refers to a nucleotide sequence that exhibits high homology to PDE4C, for example, a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 43, 45, 47, 49, 51, or 53, or an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 44, 46, 48, 50, 52, or 54. Also included are nucleic acid sequences that encode an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 44, 46, 48, 50, 52, or 54, or an amino acid sequence encoded by a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 43, 45, 47, 49, 51, or 53.

[0065] The term "PDE4D" refers to the phosphodiesterase 4D gene (Ensembl: ENSG00000113448), e.g., NCBI reference sequences NM_001104631.2, NM_006203.5, NM_001165899.2, NM_001197218.2, NM_001197219.2, NM_001197220.2, NM_001197221.2, NM_001197222.2, NM_001197223.2, NM_001349241 .2, NM_001349242.2, NM_001349243.2, NM_001364599.1, NM_001364600.2, NM_001364601.1, NM_001364602.2, NM_001364603.1, or NM_001364604.1, specifically SEQ ID NOs: 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 1383, 1394, 149, 150, 151, 9, 81, 83, 85, 87, or 89, including, for example, the NCBI protein accession reference sequences NP_001098101.1, NP_006194.2, NP_001159371.1, NP_001184147.1, NP_001184148.1, NP_001184149.1, NP_001184150.1, NP_001184151.1, NP_001184152.1, NP_0013 The present invention also relates to the corresponding amino acid sequences set forth in SEQ ID NOs: 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, or 90, which correspond to the protein sequences defined in SEQ ID NOs: 36170.1, NP_001336171.1, NP_001336172.1, NP_001351528.1, NP_001351529.1, NP_001351530.1, NP_001351531.1, NP_001351532.1, or NP_001351533.1.

[0066] The term "PDE4D" refers to a nucleotide sequence that exhibits high homology to PDE4D, for example, a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, or 89, or an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, or 90. or a nucleic acid sequence that encodes an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, or 90, or an amino acid sequence encoded by a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, or 89.

[0067] The term "PDE5A" refers to the phosphodiesterase 5A gene (Ensembl: ENSG00000138735), e.g. the sequence defined in NCBI Reference Sequence NM_001083.4, in particular the nucleotide sequence set forth in SEQ ID NO: 91, which corresponds to the sequence of the above-mentioned NCBI Reference Sequence for the PDE5A transcript, and also relates to the corresponding amino acid sequence set forth in SEQ ID NO: 92, which corresponds to the protein sequence defined in NCBI Protein Accession Reference Sequence NP_001074.2, which encodes a PDE5A polypeptide.

[0068] The term "PDE5A" refers to a nucleotide sequence that exhibits high homology to PDE5A, for example, a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 91, or an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 92. Also included are nucleic acid sequences that encode an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO:92, or an amino acid sequence encoded by a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO:92.

[0069] The term "PDE6A" refers to the phosphodiesterase 6A gene (Ensembl: ENSG00000132915), for example the sequence defined in NCBI Reference Sequence NM_000440.3, in particular the nucleotide sequence set forth in SEQ ID NO: 93, which corresponds to the sequence of the NCBI Reference Sequence shown above for the PDE6A transcript, and also to the corresponding amino acid sequence set forth in SEQ ID NO: 94, which corresponds to the protein sequence defined in NCBI Protein Accession Reference Sequence NP_000431.2, which encodes the PDE6A polypeptide.

[0070] The term "PDE6A" refers to a nucleotide sequence that exhibits high homology to PDE6A, for example, a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 93, or an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 94. Also included are nucleic acid sequences that encode an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO:94, or an amino acid sequence encoded by a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO:93.

[0071] The term "PDE6B" refers to the phosphodiesterase 6B gene (Ensembl: ENSG00000133256), e.g. the sequence defined in NCBI Reference Sequence NM_000283.4, in particular the nucleotide sequence set forth in SEQ ID NO: 95, which corresponds to the sequence of the above-mentioned NCBI Reference Sequence for the PDE6B transcript, and also to the corresponding amino acid sequence set forth in SEQ ID NO: 96, which corresponds to the protein sequence defined in NCBI Protein Accession Reference Sequence NP_000274.3, which encodes the PDE6B polypeptide.

[0072] The term "PDE6B" refers to a nucleotide sequence that exhibits high homology to PDE6B, for example, a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 95, or an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 96. Also included are nucleic acid sequences that encode an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO:96, or an amino acid sequence encoded by a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO:95.

[0073] The term "PDE6C" refers to the phosphodiesterase 6C gene (Ensembl: ENSG00000095464), for example the sequence defined in NCBI Reference Sequence NM_006204.4, in particular the nucleotide sequence set forth in SEQ ID NO: 97, which corresponds to the sequence of the NCBI Reference Sequence shown above for the PDE6C transcript, and also to the corresponding amino acid sequence set forth in SEQ ID NO: 98, which corresponds to the protein sequence defined in NCBI Protein Accession Reference Sequence NP_006195.3, which encodes the PDE6C polypeptide.

[0074] The term "PDE6C" refers to a nucleotide sequence that exhibits high homology to PDE6C, for example, a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 97, or an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 98. Also included are nucleic acid sequences that encode an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO:98, or an amino acid sequence encoded by a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO:97.

[0075] The term "PDE6D" refers to the phosphodiesterase 6D gene (Ensembl: ENSG00000156973), for example the sequence defined in NCBI reference sequence NM_002601.4, in particular the nucleotide sequence set forth in SEQ ID NO: 99, which corresponds to the sequence of the NCBI reference sequence shown above for the PDE6D transcript, and also to the corresponding amino acid sequence set forth in SEQ ID NO: 100, which corresponds to the protein sequence defined in NCBI protein accession reference sequence NP_002592.1, which encodes the PDE6D polypeptide.

[0076] The term "PDE6D" refers to a nucleotide sequence that is highly homologous to PDE6D, for example, a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 99, or an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 100. Also included are nucleic acid sequences that encode an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 100, or an amino acid sequence encoded by a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 99.

[0077] The term "PDE6G" refers to the phosphodiesterase 6G gene (Ensembl: ENSG00000185527), for example the sequence defined in NCBI Reference Sequence NM_0026002.4, in particular the nucleotide sequence set forth in SEQ ID NO: 101, which corresponds to the sequence of the NCBI Reference Sequence shown above for the PDE6G transcript, and also to the corresponding amino acid sequence set forth in SEQ ID NO: 102, which corresponds to the protein sequence defined in NCBI Protein Accession Reference Sequence NP_002593.1, which encodes the PDE6G polypeptide.

[0078] The term "PDE6G" refers to a nucleotide sequence that exhibits high homology to PDE6G, for example, a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 101, or an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 102. Also included are nucleic acid sequences that encode an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO:102, or an amino acid sequence encoded by a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO:101.

[0079] The term "PDE6H" refers to the phosphodiesterase 6H gene (Ensembl: ENSG00000139053), e.g. the sequence defined in NCBI Reference Sequence NM_006205.3, in particular the nucleotide sequence set forth in SEQ ID NO: 103, which corresponds to the sequence of the above-mentioned NCBI Reference Sequence for the PDE6H transcript, and also relates to the corresponding amino acid sequence set forth in SEQ ID NO: 104, which corresponds to the protein sequence defined in NCBI Protein Accession Reference Sequence NP_006196.1, which encodes the PDE6H polypeptide.

[0080] The term "PDE6H" refers to a nucleotide sequence that exhibits high homology to PDE6H, for example, a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 103, or an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 104. Also included are nucleic acid sequences that encode an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 104, or an amino acid sequence encoded by a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 103.

[0081] The term "PDE7A" refers to the phosphodiesterase 7A gene (Ensembl: ENSG00000205268), e.g. the sequence defined in NCBI Reference Sequence NM_0026003.4, in particular the nucleotide sequence set forth in SEQ ID NO: 105, which corresponds to the sequence of the above-shown NCBI Reference Sequence for the PDE7A transcript, and also to the corresponding amino acid sequence set forth in SEQ ID NO: 106, which corresponds to the protein sequence defined in NCBI Protein Accession Reference Sequence NP_002594.1, which encodes the PDE7A polypeptide.

[0082] The term "PDE7A" refers to a nucleotide sequence that exhibits high homology to PDE7A, for example, a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 105, or an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 106. Also included are nucleic acid sequences that encode an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 106, or an amino acid sequence encoded by a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 105.

[0083] The term "PDE7B" refers to the phosphodiesterase 7B gene (Ensembl: ENSG00000171408), e.g. the sequence defined in NCBI Reference Sequence NM_018945.4, in particular the nucleotide sequence set forth in SEQ ID NO: 107, which corresponds to the sequence of the above-shown NCBI Reference Sequence for the PDE7B transcript, and also relates to the corresponding amino acid sequence set forth in SEQ ID NO: 108, which corresponds to the protein sequence defined in NCBI Protein Accession Reference Sequence NP_061818.1, which encodes a PDE7B polypeptide.

[0084] The term "PDE7B" refers to a nucleotide sequence that exhibits high homology to PDE7B, for example, a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 107, or an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 108. Also included are nucleic acid sequences that encode an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 108, or an amino acid sequence encoded by a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 107.

[0085] The term "PDE8A" refers to the phosphodiesterase 8A gene (Ensembl: ENSG00000073417), e.g. the sequence defined in NCBI Reference Sequence NM_002605.3, in particular the nucleotide sequence set forth in SEQ ID NO: 109, which corresponds to the sequence of the above-mentioned NCBI Reference Sequence for the PDE8A transcript, and also relates to the corresponding amino acid sequence set forth in SEQ ID NO: 110, which corresponds to the protein sequence defined in NCBI Protein Accession Reference Sequence NP_002596.1, which encodes the PDE8A polypeptide.

[0086] The term "PDE8A" refers to a nucleotide sequence that exhibits high homology to PDE8A, for example, a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 109, or an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 110. Also included are nucleic acid sequences that encode an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO:110, or an amino acid sequence encoded by a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO:109.

[0087] The term "PDE8B" refers to the phosphodiesterase 8B gene (Ensembl: ENSG00000113231), e.g. the sequence defined in NCBI Reference Sequence NM_003719.5, in particular the nucleotide sequence set forth in SEQ ID NO: 111, which corresponds to the sequence of the above-mentioned NCBI Reference Sequence for the PDE8B transcript, and also relates to the corresponding amino acid sequence set forth in SEQ ID NO: 112, which corresponds to the protein sequence defined in NCBI Protein Accession Reference Sequence NP_003710.1, which encodes the PDE8B polypeptide.

[0088] The term "PDE8B" refers to a nucleotide sequence that exhibits high homology to PDE8B, for example, a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 111, or an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 112. Also included are nucleic acid sequences that encode an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO:112, or an amino acid sequence encoded by a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO:111.

[0089] The term "PDE9A" refers to the phosphodiesterase 9A gene (Ensembl: ENSG00000160191), e.g., the sequence defined in NCBI Reference Sequence NM_002606.3, in particular the nucleotide sequence set forth in SEQ ID NO: 113, which corresponds to the sequence of the above-mentioned NCBI Reference Sequence for the PDE9A transcript, and also to the corresponding amino acid sequence set forth in SEQ ID NO: 114, which corresponds to the protein sequence defined in NCBI Protein Accession Reference Sequence NP_002597.1, which encodes the PDE9A polypeptide.

[0090] The term "PDE9A" refers to a nucleotide sequence that exhibits high homology to PDE9A, for example, a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 113, or an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 114. Also included are nucleic acid sequences that encode an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO:114, or an amino acid sequence encoded by a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO:113.

[0091] The term "PDE10A" refers to the phosphodiesterase 10A gene (Ensembl: ENSG00000112541), e.g., the sequence defined in NCBI Reference Sequence NM_006661.4, in particular the nucleotide sequence set forth in SEQ ID NO: 115, which corresponds to the sequence of the above-mentioned NCBI Reference Sequence for the PDE10A transcript, and also to the corresponding amino acid sequence set forth in SEQ ID NO: 116, which corresponds to the protein sequence defined in NCBI Protein Accession Reference Sequence NP_006652.1, which encodes the PDE10A polypeptide.

[0092] The term "PDE10A" refers to a nucleotide sequence that exhibits high homology to PDE10A, for example, a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 115, or an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 116. Also included are nucleic acid sequences encoding an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO:116, or an amino acid sequence encoded by a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO:115.

[0093] The term "PDE11A" refers to the phosphodiesterase 11A gene (Ensembl: ENSG00000128655), e.g., the sequence defined in NCBI Reference Sequence NM_005019.7, in particular the nucleotide sequence set forth in SEQ ID NO: 117, which corresponds to the sequence of the above-mentioned NCBI Reference Sequence for the PDE11A transcript, and also to the corresponding amino acid sequence set forth in SEQ ID NO: 118, which corresponds to the protein sequence defined in NCBI Protein Accession Reference Sequence NP_005010.2, which encodes the PDE11A polypeptide.

[0094] The term "PDE11A" refers to a nucleotide sequence that exhibits high homology to PDE11A, for example, a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 117, or an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 118. Also included are nucleic acid sequences encoding an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO:118, or an amino acid sequence encoded by a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO:117.

[0095] The experimental results described herein are obtained by overexpression of PDE, such as PDE4D2. It is understood that overexpression of a gene encoding a PDE protein leads to increased translation and high protein levels, which in turn leads to increased enzymatic activity. Therefore, the effects described herein of increasing the efficiency of CAR transfection of T cells and increasing the ability of T cells to kill their target cells can be achieved by any method that increases PDE enzymatic activity in cells. Therefore, as used herein, the term "upregulating enzymatic activity" is intended to cover any suitable method for increasing the enzymatic activity of PDE in T cells, for example, but not limited to, using a PDE agonist or overexpressing PDE.

[0096] Thus, in an embodiment of the method of the present invention, PDE enzyme activity is upregulated by increasing expression of a PDE protein and / or by upregulating PDE enzyme activity with a PDE agonist, preferably the PDE agonist is MR-L2 (or a derivative thereof) or Abeta peptide.

[0097] Thus, in one embodiment, the method comprises: - administering gene therapy to the T cells to induce PDE expression. - administering mRNA encoding a PDE (mRNA therapy) to T cells - administering to the T cells a compound that induces PDE expression. - administering to the T cells a compound that directly stimulates or promotes phosphodiesterase activity. - administering to the T cells a compound that indirectly stimulates or promotes phosphodiesterase activity. - administering to the T cells an inhibitor of a transcription inhibitor of PDE. - administering to the T cells an inhibitor of phosphodiesterase enzyme activity. - administering to the T cells mRNA encoding a phosphodiesterase protein. - administering a phosphodiesterase protein to the T cells - administering to T cells phosphodiesterase activity promoting amyloid beta peptides. The method includes upregulating the activity of a PDE enzyme selected from the group consisting of:

[0098] As used herein, gene therapy to induce PDE expression refers to a method of inducing a nucleotide encoding a phosphodiesterase in a cell, preferably a T cell. The nucleotide may be, for example, a viral or non-viral vector, although it is understood that any nucleotide that can be introduced into a patient's cells and result in expression of a PDE may be used.

[0099] Viral vectors are a known strategy for cell transformation, and viral vector-based cell transformation typically uses modified viruses as vehicles to introduce specific DNA or RNA sequences into cells. The vector is packaged using viral proteins that allow for infection of cells and expression of the viral genome. Typically, viral vectors are modified so that new viral particles are not produced upon infection of target cells. Non-limiting examples include retroviruses (RV), adenoviruses (AV), adeno-associated viruses (AAV), lentiviruses (LV), and herpes simplex viruses (HSV). Other methods using viral particles or viral vectors to introduce nucleotides encoding PDEs into cells, such as T cells, are known to those skilled in the art and are also contemplated as encompassed by the present invention.

[0100] Alternatively, non-viral vectors can be used for viral vector-based cell transformation. These vectors typically contain a promoter driving the expression of the relevant construct (e.g., PDE) and typically require some means of introducing the vector into target cells. Means of delivering non-viral vectors to target cells are known to those skilled in the art and are reviewed, for example, in Ramamoorth M, Narvekar A. Non-viral vectors in gene therapy—an overview. J Clin Diagn Res. 2015 Jan;9(1):GE01-6 (incorporated herein by reference in its entirety). For example, nanoparticles can be used to encapsulate the vector. Non-limiting examples include lipid-based nanoparticles, peptide-based nanoparticles, cationic lipid-based nanoparticles, apolipoprotein-based nanoparticles, and (synthetic) polymer-based nanoparticles, such as polyethyleneimine (PEI), chitosan, polylactate, polylacride, polyglucoside, dendrimer, or polymethracylate-based nanoparticles. As used herein, nanoparticles are small particles that can be used as a carrier to deliver a payload to a patient.Preferably, the payload is a nucleotide encoding a PDE, as broadly defined herein.Therefore, nanoparticles can be used to deliver a payload that induces the expression of a PDE or promotes the activity of phosphodiesterase to cells, such as T cells or CAR-T cells.Other methods that use non-viral vectors to introduce a PDE-encoding nucleotide are known to those skilled in the art and are also considered to be encompassed by the present invention.

[0101] Alternatively, mRNA-based approaches can be used for viral or non-viral vector-based cell transformation. This approach typically involves the delivery of a PDE encoding an mRNA molecule. mRNA can be modified by methods known in the art to stabilize the RNA in vitro and increase its half-life (e.g., using chemically modified nucleotides, codon optimization, optimized 5'-capping, and 3'-tailing). Furthermore, mRNA can contain a structure that optimizes the 5'-UTR to increase mRNA translation or an element that enables mRNA self-amplification. Means for delivering mRNA to target cells are known to those skilled in the art and are reviewed, for example, in Rohner E et al. Unlocking the promise of mRNA therapeutics. Nature Biotechnology 2022, 40, 1586-1600 (incorporated herein by reference in its entirety). For example, lipid nanoparticles (LNPs) can be used to encapsulate mRNA. Alternatively, extracellular vesicles (EVs), cells, or biomimetics can be used to deliver cargo that induces PDE expression or promotes phosphodiesterase activity to cells, such as T cells or CAR-T cells.

[0102] Thus, in one embodiment, the cell transformation method is selected from: a viral vector capable of expressing a PDE in the cell, or a non-viral vector capable of expressing a PDE in the cell, hi one embodiment, the PDE or a nucleotide encoding a PDE is delivered to the T cell using a nanoparticle.

[0103] As used herein, the term vector is used to denote any particle (e.g., plasmid, cosmid, lambda phage) used as a vehicle to artificially transport a foreign nucleic acid molecule, usually DNA, into another cell where it can be replicated and / or expressed.

[0104] As used herein, a compound that induces PDE expression is intended to refer to any biological or chemical compound that can increase the expression of PDE. This can be achieved, for example, by promoting the transcription of PDE genes or inhibiting the degradation of phosphodiesterase proteins. For example, a compound can be involved in a transduction pathway and indirectly promote the transcription of PDE, or directly interact with the promoter region of genomic DNA that promotes the transcription of PDE.

[0105] As used herein, promoting transcription of a PDE refers to increasing transcription of a PDE to produce a large amount of PDE mRNA transcripts and / or preferably a large amount of phosphodiesterase protein in cells. The promoting step can be specific to a particular PDE (e.g., PDE4D2), specific to all isoforms of a PDE gene (e.g., PDE4D), specific to all PDE subclasses (e.g., PDE4), or specific to all PDE family members. In other words, increasing expression of a PDE does not exclude other PDE genes, and isoforms are also increased in expression.

[0106] As used herein, the compound that directly stimulates or promotes phosphodiesterase activity is intended to refer to the compound that is directly related to the enzymatic activity of phosphodiesterase protein.Without wishing to be bound by theory, it is theorized that PDE protein (such as PDE4D2 protein) exists in active structure and inactive structure, and activity can be induced by other compounds through interaction with PDE protein, for example, by promoting the active structure of protein, or by blocking or preventing the binding of inhibitor, or by modifying protein to promote activity (for example, by phosphorylation or other known protein modification).As used herein, the enzymatic activity when referring to phosphodiesterase refers to the catalysis of the hydrolysis of cAMP and / or cGMP.

[0107] The compound MR-L2, for example, has been found to specifically increase PDE4 activity. As used herein, MR-L2 has the molecular formula C 19 H 16 C 13 FN4O, refers to the compound having the formula shown below and CAS number 2374703-19-0, also known as HY-128358. One skilled in the art will appreciate that compounds may be modified to obtain compounds with improved PDE activation activity or different selectivity, and thus the present invention further extends to esters, substitutions, prodrugs or other modifications of MR-L2 that have PDE agonist activity.

[0108] [ka]

[0109] As used herein, a compound that indirectly stimulates or promotes phosphodiesterase activity is intended to refer to a compound that is indirectly associated with the enzymatic activity of a phosphodiesterase protein. Similarly, as described above, a compound can induce activators of PDE or block inhibitors of PDE activity, thus indirectly affecting the enzymatic activity of the protein. Those skilled in the art know how to determine PDE activity. For example, commercially available products for assaying PDE activity are available, and methods are described, for example, in Blair et al. Measuring cAMP Specific Phosphodiesterase Activity: A Two-step Radioassay. Bio Protoc. 2020 Apr 5;10(7):e3581, the entire contents of which are incorporated herein by reference.

[0110] As used herein, an inhibitor of a PDE transcription inhibitor refers to a compound that can block an inhibitor of PDE gene transcription. An inhibitor of PDE gene transcription can be a direct inhibitor that can bind to genomic DNA and prevent or reduce gene transcription, or an indirect inhibitor that reduces or inhibits PDE gene transcription through downstream effects.

[0111] As used herein, an inhibitor of phosphodiesterase enzyme activity refers to a compound that can block the inhibitor of phosphodiesterase protein activity. For example, the compound can function by degrading the inhibitor, preventing the inhibitor from associating with a PDE, or inhibiting the activity of the inhibitor.

[0112] As used herein, mRNA encoding a phosphodiesterase protein refers to an RNA molecule from which PDE can be translated, as broadly defined herein above. mRNA molecules typically include untranslated elements such as 5' and 3' UTRs. It is expected that direct introduction of PDE mRNA into T cells can upregulate PDE. Methods for delivering mRNA to target cells are known to those skilled in the art, for example, using nanobodies as described above.

[0113] As used herein, phosphodiesterase protein may refer to compounds containing PDE proteins or biosimilars, or to constitutively active variants thereof with phosphodiesterase activity. It is expected that direct introduction of PDE proteins into T cells can increase PDE activity. Methods for delivering proteins to target cells are known to those skilled in the art, for example, using nanobodies as described above.

[0114] As used herein, a peptide that promotes phosphodiesterase activity refers to a peptide that can increase the activity of PDE enzymes. A non-limiting example is amyloid beta (Abeta) peptide, which has been demonstrated to specifically increase the activity of so-called long PDE4D isoforms, such as PDE4D5 or PDE4D7. As used herein, amyloid beta, also known as Aβ or Abeta, refers to a 37-49 amino acid peptide that is the main component of amyloid plaques found in the brains of people with Alzheimer's disease (Chen et al. Acta Pharmacol Sin 38, 1205-1235 (2017)). Amyloid beta peptide (Aβ) is produced by proteolytic processing of the transmembrane protein, amyloid precursor protein (APP), by β- and γ-secretase enzymes.

[0115] Therefore, the peptide that promotes phosphodiesterase activity is preferably Abeta peptide or a derivative thereof. As used herein, Abeta peptide or a derivative thereof is characterized as a peptide having a length of 16 to 50 amino acids and comprising an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence defined by SEQ ID NO: 119 (Abeta core peptide 1), and more preferably, an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence defined by SEQ ID NO: 120 (Abeta core peptide 2). Non-limiting examples are provided by Abeta42 (SEQ ID NO: 121) and Abeta40 (SEQ ID NO: 122). Thus, in one embodiment, the Abeta peptide or derivative thereof is characterized as a peptide having a length of 42 to 50 amino acids and comprising an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence defined by SEQ ID NO: 121, or a peptide having a length of 40 to 50 amino acids and comprising an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence defined by SEQ ID NO: 122. SEQ ID NO: 119 Abeta core peptide 2 HDSGYEVHHQKLVFFAEDVGSNKGAIIG SEQ ID NO: 120 Abeta core peptide 2 FRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMV SEQ ID NO: 121 Abeta 42 DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA SEQ ID NO: 122 Abeta 40 DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV

[0116] Thus, in one embodiment, the invention relates to an ex vivo or in vitro method for generating improved CAR-T cells, comprising the steps of providing T cells, introducing a chimeric antigen receptor (CAR) gene into the T cells, and obtaining the CAR-T cells, and further comprising the step of contacting the T cells with a PDE agonist, as broadly defined herein above, before, during, or after introducing the CAR gene into the T cells. Alternatively, the invention relates to an ex vivo or in vitro method for generating improved CAR-T cells, comprising the steps of providing T cells, introducing a chimeric antigen receptor (CAR) gene into the T cells, and obtaining the CAR-T cells, and further comprising the step of overexpressing a PDE gene in the T cells before, during, or after introducing the CAR gene into the T cells.

[0117] Thus, in one embodiment, the method of the present invention provides a method in which PDE protein expression is upregulated by increasing expression of a PDE gene, hi one embodiment, the expression of a PDE gene is increased by exogenously expressing a PDE gene in T cells.

[0118] As used herein, the terms "exogenous expression" or "exogenously expressing" are intended to refer to the introduction of a nucleic acid encoding a PDE enzyme into a T cell. The exogenously expressed gene may be identical to or different from the gene present in the genome of the T cell. It is hypothesized that exogenous expression of a gene results in overexpression and subsequent increased translation into protein.

[0119] Thus, the present invention provides a method for increasing the expression of a gene encoding a PDE present in a T cell, or for introducing a genetic construct into a T cell (e.g., a viral vector, an expression vector, etc.) to allow expression of an exogenous gene encoding a PDE. As used herein, a gene encoding a PDE is intended to refer to a gene encoding a PDE protein as broadly defined herein (e.g., as listed in Table 1 above for proteins having sequence homology, e.g., 80%, 85%, 90% or more, to a protein listed in Table 1), or a gene encoding a PDE is intended to refer to a gene capable of expressing an mRNA as defined in Table 1 (or an mRNA having 70%, 75%, 80%, 85%, 90% or more sequence homology to an mRNA listed in Table 1).

[0120] In one embodiment, the PDE gene is introduced together with the CAR gene in the T cell, and preferably the CAR gene and the PDE gene are contained in the same expression vector.

[0121] In one embodiment, the PDE protein is a PDE2 or PDE4 protein, preferably PDE2A, PDE4A, PDE4B, PDE4C or PDE4D, or PDE2A1, PDE2A2, PDE2A3, PDE2A4, PDE4A1, PDE4A2, PDE4A3, PDE4A4, PDE4A5, PDE4Ba, PDE4Bb, PDE4Bc, PDE4Bd, PDE4Be, PDE4Bf, PDE4Bg, PDE4C1, PDE4C2, PDE4C3, PDE4C4, PDE4 C5, PDE4C6, PDE4D1, PDE4D2, PDE4D3, PDE4D4, PDE4D5, PDE4D6, PDE4D7, PDE4D8, PDE4D9, PDE4D10, PDE4D11, PDE4D12, PDE4D14, PDE4D15, or PDE4D16, and preferably the PDE4 protein is selected from the isoforms PDE4A4, PDE4B2, PDE4D1, PDE4D2, PDE4D5, or PDE4D7.

[0122] In one embodiment, the PDE protein is selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, or 121. or the PDE is selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 4, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, or 122.

[0123] In a preferred embodiment, the PDE is a PDE4 gene and is identified by a nucleotide sequence having 80%, preferably 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a sequence selected from SEQ ID NOs: 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87 or 89. Alternatively, the PDE is a PDE4 protein having an amino acid sequence having 80%, preferably 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NOs: 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, or 90.

[0124] In one embodiment, the CAR antigen is prostate-specific membrane antigen (PSMA). It is understood that the present invention is not limited to any specific antigen targeted by CAR-T.

[0125] In a second aspect, the present invention relates to CAR-T cells having upregulated enzymatic activity of a PDE protein. As used herein, the term "CAR-T cells having upregulated enzymatic activity of a PDE protein" is intended to refer to CAR-T cells that have been specifically engineered to increase the activity of a PDE (e.g., by overexpression or any other method exemplified above), and is therefore not intended to cover upregulation or increased activity of a PDE enzyme due to a naturally occurring process in T cells. Thus, in one embodiment, the present invention relates to CAR-T cells having exogenously upregulated enzymatic activity of a PDE protein, where exogenous upregulation refers to any artificial method of increasing enzymatic activity in T cells (e.g., the methods listed above, including, but not limited to, overexpressing a PDE-encoding gene). CAR-T cells may, but are not necessarily, obtained by the method of the first aspect of the present invention.

[0126] In one embodiment, the CAR-T cells exogenously express a PDE gene, such as a PDE gene listed in Table 1, preferably a PDE4 or PDE2 gene.

[0127] Alternatively, according to a second aspect of the invention, it relates to a CAR-T cell obtained or obtainable by a method as defined in the first aspect of the invention.

[0128] In one embodiment, the present invention relates to the nucleic acid sequences of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 1 , 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, or 121. CAR-T cells exogenously expressing E protein; the PDE is selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 1 8, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, or 122.

[0129] In a preferred embodiment, the PDE is a PDE4 gene and is identified by a nucleotide sequence having 80%, preferably 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a sequence selected from SEQ ID NOs: 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87 or 89. Alternatively, the PDE is a PDE4 protein having an amino acid sequence having 80%, preferably 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NOs: 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, or 90.

[0130] In a third aspect, the present invention relates to a CAR-T cell according to the second aspect of the invention for use as a medicament. It will be appreciated that the CAR-T cells described herein may be used for therapy, for example, by administering the CAR-T cell to a subject in need thereof.

[0131] In one embodiment, the present invention relates to a CAR-T cell according to the second aspect of the present invention for use in the treatment, prevention, or amelioration of a disease. In a preferred embodiment, the disease is cancer or an immune-related disorder, and more preferably, the immune-related disorder is an autoimmune disease or a viral infection. It is understood that CAR-T cells may find applications outside of oncology, as outlined in Zmievskaya E, et al. Biomedicines. 2021 Jan 9;9(1):59.

[0132] In a particularly preferred application, the present invention relates to CAR-T cells for use in the treatment, prevention, or alleviation of prostate cancer, although it will be understood that this particular use does not limit the invention in any way.

[0133] In an alternative embodiment, a third aspect of the invention relates to a method of treating, preventing, or alleviating disease in a subject in need thereof, the method comprising administering to the subject CAR-T cells, as broadly described herein. In one embodiment, the CAR-T cells are CAR-T cells as described in the second aspect of the invention. In one embodiment, the method is for treating, preventing, or alleviating cancer or an immune-related disorder. In one embodiment, the immune-related disorder is an autoimmune disease or a viral infection. In one embodiment, the invention relates to the treatment, prevention, or alleviation of prostate cancer.

[0134] It will be understood that all details, embodiments and preferences discussed with respect to one aspect of an embodiment of the present invention may likewise be applied to any other aspect or embodiment of the present invention, and therefore it is not necessary to separately recite all such details, embodiments and preferences of all aspects.

[0135] Having now generally described the invention, it will be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended to limit the invention. Further aspects and embodiments will be apparent to those skilled in the art. [Example]

[0136] The foregoing description of specific embodiments will make fully apparent the general nature of the invention such that others, by applying knowledge within the art (including the content of the references cited herein), may, without undue experimentation, readily modify and / or adapt various applications of such specific embodiments without departing from the general concepts of the invention. Accordingly, such adaptations and modifications are intended to be within the meaning and range of equivalents of the disclosed embodiments, based on the teaching and guidance presented herein.

[0137] All references cited herein, including articles or abstracts, publications or corresponding patent specifications, patents, or any other references, are incorporated by reference herein in their entirety, including all data, tables, figures, and text presented in the cited references. Additionally, the entire contents of references cited within the references cited herein are also incorporated by reference in their entirety.

[0138] It is understood that the phrases or terminology herein are for purposes of description and not limitation, and that the terms or phrases herein would be interpreted by one of ordinary skill in the art in light of the teaching or guidance presented herein, in combination with the knowledge of such persons skilled in the art. [Example]

[0139] Testing PDE4D2-expressing CAR-T cells An anti-PSMA chimeric antigen receptor (CAR-T) vector was constructed to engineer T cells to target human PSMA. T cells were genetically modified by transduction with a lentiviral vector expressing an anti-PSMA antibody scFv linked to the CD28, CD137, and CD3ζ signaling domains (clone J591).

[0140] CD28 (cluster of differentiation 28) is a protein expressed on T cells that provides constitutive signals necessary for T cell activation and survival. CD28 is a receptor for the CD80 (B7.1) and CD86 (B7.2) proteins expressed on antigen-presenting cells (APCs). CD28 regulates primary TCR / CD3ζ signals in a manner distinct from the late costimulatory elements OX40 and 4-1BB. CD28 enhances the expression of downstream regulators that affect T cell proliferation, death, differentiation, and effector function. CAR+ T cells containing a CD28 endodomain demonstrated significantly enhanced T cell activation, proliferation, and sustained survival. CD28 generates a brightly expressed, stable receptor as a transmembrane domain. Inclusion of the CD28 costimulatory domain in CARs resulted in enhanced anti-tumor efficacy.

[0141] CD137 (also known as 4-1BB) is a surface costimulatory glycoprotein originally described on activated T lymphocytes that belongs to the tumor necrosis factor (TNF) receptor superfamily. It is expressed primarily on activated CD4+ and CD8+ T cells and binds to a high-affinity ligand (4-1BBL) expressed on several antigen-presenting cells, such as macrophages and activated B cells. Based on preclinical observations, this molecule can promote the persistence of antigen-specific and antigen-nonspecific chimeric antigen receptor T cells and significantly increase their antitumor activity.

[0142] CD3ζ, also known as T cell receptor zeta, forms the TCR-CD3 complex with the T cell receptor and the CD3γ, δ, and ε chains. ζ is expressed independently of the complex. The zeta chain plays an important role in coupling antigen recognition to several intracellular signaling pathways. CD3 zeta, which contains three ITAMs, is the most commonly used endodomain component of CARs. It transmits activation signals to T cells after antigen binding. CD3 zeta does not provide a fully competitive activation signal; additional costimulatory signals are required. For example, chimeric 4-1BB and OX40 can be used with CD3 zeta to transmit proliferation / survival signals, or all three can be used together.

[0143] The coding sequence (CDS) of the PDE4D isoform PDE4D2 was cloned into the multiple cloning site (MCS) of the CAR-T vector, generating the vector PDE4D2 CAR-T. Expression of the PDE4D2 protein was driven by the PGK promoter. To simplify detection of PDE4D2 protein expression, an 11-amino acid VSV tag was added to the 3' end of the PDE4D2 CDS following the translation termination codon. Examples of anti-PSMA CAR vectors without and with the PDE4D2 gene are shown in Figures 1 and 2. After transfection of the CAR vector into HEK293 cells, the expressed PDE4D2 protein could be detected by Western (immuno)blotting using an antibody against the PDE4D protein and an anti-VSV antibody (Figure 8).

[0144] Next, we tested the PDE activity of transfected cells using a radioactive assay that quantifies the rate of cAMP hydrolysis by PDE in the sample. We used whole cell lysates from HEK293 cells transfected with 3 μg of CAR-T plasmid containing PDE4D2. We used the nonselective PDE inhibitor IBMX dissolved in DMSO, the PDE4-selective inhibitor rolipram, or DMSO alone. Non-transfected cells showed low PDE activity (28.37 ± 22.32 pM / min / mg, Figure 9), as expected for HEK293 cells with endogenous PDE expression. Transfected cells without inhibitors showed a significant 4-fold increase in PDE activity compared to non-transfected cells (115.8 ± 25.91 pM / min / mg, P < 0.0001, Figure 9). This indicates that transfected PDE4D2 in the plasmid confers increased PDE activity for cellular cAMP degradation. Addition of both the PDE inhibitors IBMX and rolipram to transfected cells reduced PDE activity, returning it to the basal level of non-transfected cells. Transfected cells treated with IBMX had PDE activity that was not significantly different from that of non-transfected cells (40.16 ± 28.08 pM / min / mg, P = 0.6425, Figure below). Similarly, transfected cells treated with rolipram had PDE activity that was not significantly different from that of non-transfected cells (26.04 ± 25.29 pM / min / mg, P = 0.9948, Figure 9).

[0145] The percentage of T cells expressing anti-PSMA scFv antibody was examined by flow cytometry before and after CART transfection. An anti-human Fab antibody (produced in mice) was used to detect CART-expressing anti-PSMA scFv antibody. The "Control" experiment represents human T cells in flow cytometry without the anti-human Fab antibody. The "Control Mouse Fab" experiment represents human T cells in flow cytometry with the anti-human Fab antibody. The marked area in the flow cytometry shows nonspecific background signal in approximately 4% of T cells. The "CAR-T Mouse Fab" experiment represents human T cells in flow cytometry after transfection of CAR-T with the anti-human Fab antibody. The marked area in the flow cytometry shows anti-PSMA scFv antibody expression in approximately 25% of T cells. The "PDE4D2 CAR-T Mouse Fab" experiment represents human T cells in flow cytometry after transfection of CAR-T containing the PDE4D2 gene with the anti-human Fab antibody. The marked area in the flow cytometry shows anti-PSMA scFv antibody expression in approximately 80% of T cells (Figure 3).

[0146] Cytotoxicity experiments were performed using two vectors: CAR-T (Z011520 CAR-T) and PDE4D2 CAR-T (Z060719 PDE4D2 CAR-T), based on the assumption that PDE4D2 protein would increase T cell cytotoxicity over CAR-T lacking PDE4D2. This experiment was performed once with two replicates (rep1, rep2). A PSMA-overexpressing CHO cell line (FOLH1) was used as the target cells. Four different effector-to-target (E / T) cell ratios of CAR-T transfected cells to target CHO cells were used, ranging from 1:1 to 10:1. For the two different CAR-T vectors, the same number of positively transfected T cells was used in the cytotoxicity experiments. Increasing the E / T ratio increased the percentage of target cells killed by CAR-T transfected T cells. Across two replicate experiments and across all E / T ratios, addition of the PDE4D2 gene consistently resulted in an increased percentage of killed CHO target cells, with the maximum additional percentage of killed target cells being in the 25% range (Figure 4).

[0147] Cytokine release experiments measuring IL2 (interleukin 2) and IFNG (interferon gamma) were performed using two vectors: CAR-T (Z011520 CAR-T) and PDE4D2 CAR-T (Z060719 PDE4D2 CAR-T), based on the assumption that the PDE4D2 protein increases cytokine release upon T cell activation over CAR-T lacking PDE4D2. This experiment was performed once with two replicates (rep1, rep2). A PSMA-overexpressing CHO cell line (FOLH1) was used as the target cells. Four different effector-to-target (E / T) cell ratios of CAR-T transfected cells to target CHO cells were used, ranging from 1:1 to 10:1. For the two different CAR-T vectors, the same number of positively transfected T cells was used in the cytokine release experiments. Increasing the E / T ratio increased the cytokines released by CAR-T transfected T cells. Across two replicate experiments and across all E / T ratios, the addition of the PDE4D2 gene consistently demonstrated an increase in IL2 release. The maximum additional percentage of IL2 release was in the range of 25%. IFNG only increased with an E / T ratio of 1:1 and 2.5:1. At high E / T ratios, assay saturation may affect the measurement. This may be why no additional IFNG release was observed at high E / T ratios under the conditions of PDE4D2 CAR-T use in the experiment. The maximum additional percentage of IFNG release was in the range of 20% at E / T ratios of 1:1 and 2.5:1 (Figures 5 and 6). [Example]

[0148] Expression and activation of some PDE genes after T cell activation with anti-CD3 and CD28 antibodies RNA sequencing data (GSE160311; https: / / www.ncbi.nlm.nih.gov / geo) were analyzed for gene expression of PDE4 gene family members A, B, C, and D over a time course (0 h, 6 h, 24 h, 72 h) of T cell stimulation with anti-CD3 and anti-CD28 antibodies. TPM expression values ​​from three replicate experiments were analyzed by one-way ANOVA for differences in expression between time points. PDE4D gene family members A, B, and D were significantly upregulated upon T cell stimulation over a 24-hour time course. PDE4B appeared to return to baseline expression at 72 hours, while PDE4A and PDE4D expression continued to increase 72 hours after stimulation. PDE4C expression was not detected in T cells. Box plots represent data measured over three replicates per time point and per PDE4D gene family member. p-values ​​for differential expression are shown (Figure 7).

Claims

1. 1. An ex vivo or in vitro method for generating improved CAR-T cells, said method comprising: providing T cells; introducing a chimeric antigen receptor (CAR) gene into T cells to obtain CAR-T cells; Including, The method further comprises upregulating enzymatic activity of a phosphodiesterase (PDE) protein in the T cell before, during, or after introducing the CAR gene into the T cell; the PDE protein is a PDE2 or PDE4 protein, The expression of the PDE protein is upregulated by increasing the expression of the PDE gene by exogenously expressing the PDE gene in T cells. method.

2. 2. The method of claim 1, wherein the PDE gene is co-transfected with the CAR gene in a T cell, preferably wherein the CAR gene and the PDE gene are contained in the same expression vector.

3. The PDE protein is PDE2A, PDE4A, PDE4B, PDE4C, PDE4D, or PDE2A1, PDE2A2, PDE2A3, PDE2A4, PDE4A1, PDE4A2, PDE4A3, PDE4A4, PDE4A5, PDE4Ba, PDE4Bb, PDE4Bc, PDE4Bd, PDE4Be, PDE4Bf, PDE4Bg, PDE4C1, PDE a specific isoform selected from PDE4C2, PDE4C3, PDE4C4, PDE4C5, PDE4C6, PDE4D1, PDE4D2, PDE4D3, PDE4D4, PDE4D5, PDE4D6, PDE4D7, PDE4D8, PDE4D9, PDE4D10, PDE4D11, PDE4D12, PDE4D14, PDE4D15, or PDE4D16; Preferably, the PDE4 protein is selected from the isoforms PDE4A4, PDE4B2, PDE4D1, PDE4D2, PDE4D5 or PDE4D7.

3. The method according to claim 1 or 2.

4. The PDE protein is encoded by a nucleotide sequence having 80%, preferably 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with a sequence selected from SEQ ID NOs: 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87 or 89, or 4. The method of claim 1, wherein the protein is a PDE4 protein having an amino acid sequence that has 80%, preferably 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of SEQ ID NOs: 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, or 90.

5. 5. The method of any one of claims 1 to 4, wherein the CAR antigen is prostate-specific membrane antigen (PSMA).

6. A CAR-T cell with upregulated enzymatic activity of a PDE protein, wherein the CAR-T cell exogenously expresses a PDE gene selected from a PDE2 or PDE4 gene.

7. CAR-T cells obtained or obtainable by the method of any one of claims 1 to 5.

8. exogenously expressing a PDE protein encoded by a nucleotide sequence having 80%, preferably 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a sequence selected from SEQ ID NOs: 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, or 89; or The CAR-T cell of claim 6 or 7, wherein the protein is a PDE4 protein having an amino acid sequence having 80%, preferably 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of SEQ ID NOs: 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, or 90.

9. The CAR-T cell of any one of claims 6 to 8 for use as a medicament.

10. The CAR-T cell of any one of claims 6 to 8 for use in treating, preventing, or alleviating a disease, preferably wherein the disease is cancer or an immune-related disorder, more preferably wherein the immune-related disorder is an autoimmune disease or a viral infection.

11. The CAR-T cell for use according to claim 9 or 10, wherein the disease is prostate cancer.

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