PDE-enhanced CAR-T cells

Abstract

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

Description

Field of the Invention

[0001] The present invention generally relates to the field of CAR-T cells. In particular, the present invention relates to improved methods for obtaining CAR-T cells, improved CAR-T cells and their uses. The method also relates to activating or expressing phosphodiesterase (PDE) in T cells during the production of CAR-T cells. Background of the Invention

[0003] The background description includes information that may be helpful in understanding the present invention. However, it does not represent an admission that any of the information provided herein is prior art or relevant to the currently claimed invention, nor does it represent an admission that any of the publications explicitly or implicitly cited are prior art.

[0004] For decades, the basis of cancer treatment has been surgery, chemotherapy, and radiotherapy. These remain important treatment modalities to this day, but in recent years new treatment categories have helped to change the treatment landscape for cancer patients. The role of the immune system in cancer prevention and treatment has become increasingly prominent and has led to the development of, for example, immunotherapy based on immune checkpoint inhibitors.

[0005] In recent years, T cells expressing chimeric antigen receptors (CAR-T cells) have become increasingly popular as a method of triggering the host immune system to respond to hematological or solid tumors. Using this method, chimeric antigen receptors targeting tumor-specific antigens are expressed in T cells. For this purpose, host T cells can be used, thus avoiding an immune response against the T cells themselves. The CAR expressed in the T cells can target the cells to the tumor, where the T cells are expected to kill malignant cells and induce a further immune response of the host against the tumor cells.

[0006] A challenge faced by existing CAR-T cell-based therapies is the need to produce and expand large numbers of CAR-T cells, which is both expensive and time-consuming. Therefore, it is crucial to improve the potency of CAR-T cells. In addition, the potency of CAR-T cells to kill their designated target cells is usually less than 100%, so there is always a need to improve their cytotoxicity against the designated target.

[0007] The present invention aims to address the above challenges and other challenges through the methods, products and uses defined in the appended claims. Summary of the Invention

[0009] In a first aspect, the present invention relates to an ex vivo or in vitro method for producing improved CAR-T cells, the method comprising:

[0010] providing T cells;

[0011] introducing a chimeric antigen receptor (CAR) gene into the T cells to obtain CAR-T cells;

[0012] The method further includes the step of upregulating the enzymatic activity of phosphodiesterase (PDE) protein in the cells before, during, or after introducing the CAR gene into the T cells.

[0013] In a second aspect, the present invention relates to a CAR-T cell having upregulated PDE protease activity, or a CAR-T cell obtained or obtainable by the method defined in the first aspect of the present invention.

[0014] In a third aspect, the method relates to the use of the CAR-T cell according to the second aspect of the present invention as a medicament.

[0015] Definitions

[0016] Part of the present disclosure contains copyrighted material (such as but not limited to diagrams, device photographs, or any other aspect of this application that has been or may be copyrighted in any jurisdiction). The copyright owner does not object to anyone copying this patent document or patent disclosure, provided that it appears in the patent office's patent document or records, but otherwise, all copyrights are reserved.

[0017] Various terms related to the methods, compositions, uses, and other aspects of the present invention are used in this specification and claims. Unless otherwise specified, these terms shall have their ordinary meanings in the field to which the present invention pertains. Other terms with clearly defined meanings shall 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 to test the present invention, the preferred materials and methods are described herein.

[0018] For the purposes of the present invention, the following terms are defined as follows.

[0019] As used herein, the singular forms "a", "an", and "the" include their plural referents unless the context clearly dictates otherwise. For example, a method of administering an agent includes administering a plurality of molecules (such as dozens, hundreds, thousands, tens of thousands, hundreds of thousands, millions, or more molecules).

[0020] As used herein, "about" and "approximately", when referring to measurable values (such as amounts, durations, etc.), are intended to cover deviations of ±20% or ±10% from the specified value, more preferably ±5%, even more preferably ±1%, and even more preferably ±0.1%, provided that these deviations are suitable for practicing the disclosed invention. Unless the context clearly dictates otherwise, all numerical values provided herein include values modified by "about".

[0021] As used herein, "and / or" means that one or more of the stated circumstances can occur alone or in combination with at least one of the stated circumstances until all of the stated circumstances occur in combination.

[0022] As used herein, "at least" a particular value means that particular value or greater. For example, "at least 2" should be understood to mean "2 or more", i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15... etc. As used herein, the term "at most" a particular value means that particular value or less. For example, "at most 5" should be understood to mean "5 or less", i.e., 5, 4, 3... -10, -11 etc.

[0023] As used herein, "comprising" should be understood to be inclusive and open-ended, and not exclusive. Specifically, the term and its variants mean including the specific features, steps or components. These terms should not be construed as excluding the presence of other features, steps or components. It also encompasses the more restrictive "consisting of".

[0024] As used herein, "conventional techniques" or "methods known to those skilled in the art" means that the methods of using conventional techniques in the methods of the present invention are obvious to those skilled in the art. The practice of conventional 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 manuals and references.

[0025] As used herein, "exemplary" or "for example" means "by way of example, instance or illustration", and should not be understood to exclude other configurations, including those disclosed herein.

[0026] Throughout this disclosure, various aspects of the present invention may be presented in a range format. It should be understood that the description in range format is for convenience only and should not be construed as a limitation on the scope of the present invention. The description of a range should be considered to have specifically disclosed all possible sub-ranges as well as individual values within that range, including integers and non-integers. For example, the description of a range such as 1 to 6 should be considered to have specifically disclosed sub-ranges 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 numbers within that range, such as 1, 2, 2.7, 3, 4, 5, 5.3, 6 etc. This rule applies regardless of how broad the range is.

[0027] As used herein, "cancer" refers to a physiological condition in a mammal that is typically characterized by unregulated cell growth. The terms "cancer," "neoplasm," and "tumor" are generally used interchangeably to describe cells that have undergone malignant transformation and have become pathological to the host organism. Primary cancer cells can be distinguished from non-cancerous cells by techniques known to those of skill in the art. As used herein, cancer cells include not only primary cancer cells but also cancer cells derived from such primary cancer cells, including metastatic (secondary) cancer cells, as well as cell lines derived from cancer cells. Examples include solid tumors and non-solid tumors or hematological tumors. Treatment of cancer in a subject includes treatment of the tumor in the subject.

[0028] The drugs, therapeutic agents, medicaments, and pharmaceutical compositions of the present invention can be formulated for administration by a variety of routes, including but not limited to parenteral, intravenous, intraarterial, intramuscular, intratumoral, and oral administration. The drugs, therapeutic agents, medicaments, and compositions can be formulated in liquid or solid form. Liquid formulations can be formulated for administration by injection to a selected area of the human or animal body. Preferably, the cells of the present invention, when used as a drug, are formulated for administration in a liquid formulation, preferably suitable for injection administration, such as for intravenous, intraarterial, intramuscular, or intratumoral delivery or injection.

[0029] As used herein, the term "pharmaceutical composition" refers to a composition formulated to be pharmaceutically acceptable or physiologically acceptable for administration to a cell or a subject. The compositions of the present invention can also be administered in combination with other agents, provided that these other agents do not adversely affect the ability of the composition to deliver the intended therapy. The pharmaceutical composition typically further comprises one or more pharmaceutically acceptable carriers (or excipients) in addition to the pharmaceutically active agent.

[0030] As used herein, "subject" refers to an organism to be treated, such as an organism to which the composition is intended to be administered. The subject can be any subject of the present invention, including but not limited to humans, males, females, infants, children, adolescents, adults, young adults, middle-aged or elderly individuals, and / or other primates or mammals. Preferably, the subject is a human patient. In some embodiments, the subject may have been diagnosed with cancer, an immune-related disease, a bleeding disorder, a disease associated with protein overexpression, or a disease associated with protein underexpression. In some embodiments, the subject may be at risk of developing a disease or disorder that can be prevented or ameliorated by vaccination.

[0031] As used herein, "T cell" can be selected from, for example, inflammatory T lymphocytes, cytotoxic T lymphocytes, regulatory T lymphocytes, or helper T lymphocytes. In another embodiment, the cell can be derived from CD4 + T lymphocytes and CD8 +T lymphocytes. They can be extracted from blood or derived from stem cells. T cells can be obtained from many non-limiting sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, and tumors. In certain embodiments of the present invention, any available T cell line known to those skilled in the art can be used. In another embodiment, the cells can be derived from a healthy donor, a patient diagnosed with cancer, or a patient diagnosed with an infection. In another embodiment, the cells are part of a mixed cell population presenting different phenotypic characteristics.

[0032] As used herein, "treating", "treatment", "alleviating", "mitigating", and "ameliorating" in the context of a subject to be treated all refer to methods of obtaining a beneficial or desired result (including but not limited to a therapeutic benefit). A therapeutic benefit refers to eradicating or improving the underlying disease being treated. Additionally, a therapeutic benefit is obtained by eradicating or improving one or more physiological symptoms associated with the underlying disease, such that an improvement in the patient is observed, even if the patient may still have the underlying disease. As used herein, "preventing" refers to a method of partially or completely reducing the occurrence of an adverse reaction (e.g., an adverse reaction typically associated with the use of a particular drug or agent). For example, in the context of the present invention, these terms can refer to preventing, treating, or alleviating the effects of cancer, immune-related diseases, bleeding disorders, diseases associated with protein overexpression, and diseases associated with protein underexpression. In some embodiments, preventing can also refer to the effect of preventing a disease by vaccination.

[0033] As used herein, the term "nucleic acid" or "polynucleotide" refers to any (continuous) polymer or oligomer of nucleotides. The nucleic acid can be DNA or RNA, or a mixture thereof, and can exist permanently or temporarily in single-stranded or double-stranded form, including homoduplex, heteroduplex, and hybrid states. The present invention encompasses any deoxyribonucleotide, ribonucleotide, or peptide nucleic acid component, as well as any chemical variants thereof, such as methylated, hydroxymethylated, or glycosylated forms of these bases, etc. The composition of the polymer or oligomer can be heterogeneous or homogeneous, and can be isolated from natural sources or be produced artificially or synthetically. Thus, the term "isolated" refers to being isolated from natural sources or being produced artificially or synthetically.

[0034] As used herein, "percent amino acid sequence identity" with respect to a reference polypeptide sequence means the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence, the percentage being obtained by aligning the sequences and introducing gaps as needed to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. The alignment for determining percent amino acid sequence identity can be conducted in a variety of ways known to those skilled in the art, such as using publicly available computer software, such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for aligning the sequences, including any algorithms required to achieve the maximum alignment over the full length of the sequences being compared. However, for the purposes of this application, the percent amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was developed by Genentech, Inc., and its source code has been submitted to the U.S. Copyright Office (Washington, D.C., 20559) together with user documentation, and the U.S. copyright registration number is TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc. (South San Francisco, Calif.), and can also be compiled from the source code. The ALIGN-2 program should be compiled to be suitable for the UNIX operating system, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary. In the case of using ALIGN-2 for amino acid sequence comparison, the percent amino acid sequence identity of a given amino acid sequence A with a given amino acid sequence B (which can also be stated as a given amino acid sequence A has or contains a certain percent amino acid sequence identity with a given amino acid sequence B) is calculated as follows:

[0035] 100 × X / Y

[0036] where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in the alignment of A and B, and Y is the total number of amino acid residues in B. It should be understood that when the length of amino acid sequence A is not equal to the length of amino acid sequence B, the percent amino acid sequence identity of A with B is not equal to the percent amino acid sequence identity of B with A. Unless otherwise expressly stated, all percent amino acid sequence identity values used herein are obtained using the ALIGN-2 computer program as described in the previous paragraph.

[0037] As used herein, the terms "protein" and "polypeptide" refer to molecules composed of chains of amino acids, regardless of their specific mode of action, size, three-dimensional structure, or origin. Thus, a "fragment" or "portion" of a polypeptide may still be referred to as a "polypeptide". A "purified protein" or "purified polypeptide" refers to a protein or polypeptide that is no longer in its natural environment, such as in vitro or in a recombinant host cell.

[0038] As used herein, the term "construct" or "nucleic acid construct" or "vector" refers to an artificial nucleic acid molecule obtained using recombinant DNA technology for delivering foreign DNA into a host cell, typically with the aim of expressing the DNA region contained in the construct in the host cell. The vector backbone of the construct can be, for example, a plasmid incorporating (chimeric) genes, or, if appropriate transcriptional regulatory sequences are already present, only the desired nucleic acid sequence (such as a coding sequence) is integrated downstream of the transcriptional regulatory sequences. The vector can contain other genetic elements to facilitate its use in molecular cloning, such as selection markers, multiple cloning sites, and the like. Brief Description of the Drawings

[0040] Embodiments of the present invention will be further described in conjunction with the accompanying drawings, wherein:

[0041] Figure 1 An exemplary vector map of a CART expression vector expressing a CAR driven by an EF-1alpha promoter is provided. The exemplary CAR includes an antigen recognition domain (scFv), a CD28 domain, a 4-1BB domain, and a CD3zeta domain (third-generation CAR).

[0042] Figure 2 An exemplary vector map of a CART expression vector expressing a CAR driven by an EF-1alpha promoter is provided, which further includes a gene encoding PDE4D2 driven by a PGK promoter.

[0043] Figure 3 Flow cytometry results of different T cells are depicted, with the Y-axis being side scatter and the X-axis being anti-APC antibody. The figure shows control T cells without and with anti-mouse Fab 647 secondary antibody (the first and second figures), control CAR-T cells (the third figure), and CAR-T cells expressing PDE4D2 (the fourth figure). The percentage of positively identified cells is indicated in each figure, which are 0% (control group, without secondary antibody), 4% (control group, with secondary antibody), 25% (control CAR-T cells), and 80% (PDE4D2 CAR-T cells), respectively.

[0044] Figure 4A graph depicting the percentage of target cell killing at different E / T (effector / target) ratios. CAR-T cells targeting PSMA were used together with CHO cells overexpressing PSMA. Control CAR-T cells (the same experiment repeated twice) were plotted on the left, and CAR-T cells expressing PDE4D2 were plotted on the right. For each ratio, CAR-T cells expressing PDE4D2 showed a significant increase in the percentage of target cell killing.

[0045] Figure 5 A graph depicting the release of IL-2 at different E / T (effector / target) ratios. CAR-T cells targeting PSMA were used together with CHO cells overexpressing PSMA. Control CAR-T cells (the same experiment repeated twice) were plotted on the left, and CAR-T cells expressing PDE4D2 were plotted on the right. For each ratio, CAR-T cells expressing PDE4D2 showed a significant increase in the release of IL-2.

[0046] Figure 6 A graph depicting the release of interferon-γ (IFNG) at different E / T (effector / target) ratios. CAR-T cells targeting PSMA were used together with CHO cells overexpressing PSMA. Control CAR-T cells (the same experiment repeated twice) were plotted on the left, and CAR-T cells expressing PDE4D2 were plotted on the right. The effect of IFNG release under different test conditions was observed only at E / T (effector / target) ratios of 1:1 and 2.5:1.

[0047] Figure 7 A graph depicting 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. Significance was indicated for each panel as there were significant differences in gene expression levels at the designated time points (P < 0.05).

[0048] Figure 8 : (A) Western blot analysis of Pan4D and (B) VSV tag expression. Whole cell lysates of HEK293 cells transfected with the CAR-PDE4D2 plasmid were run on a 4-12% gradient gel. After transfer to a nitrocellulose membrane, the blot bands were immunodetected. Lane 1: molecular weight marker, Lane 2: untransfected control (UT), Lanes 3-6: different amounts (1-7 μg) of transfected plasmid DNA.

[0049] Figure 9Depicts the results of the PDE activity assay. Whole HEK293 cell lysates that were untransfected and transfected with a CAR-T plasmid containing PDE4D2 were used. Transfected cells with or without the PDE inhibitor (IBMX or Rolipram) were compared.

[0050] Detailed description of the embodiments

[0051] The present invention is defined herein, particularly in the appended claims. Subject matter not falling within the scope of the claims does not form part of the claimed invention.

[0052] It is contemplated that any method, use, or composition described herein can be implemented in combination with any other method, use, or composition described herein. Embodiments or preferences discussed in the context of the methods, uses, and / or compositions of the present invention are equally applicable to any other method, use, or composition described herein. Thus, embodiments or preferences associated with one method, use, or composition are also applicable to other methods, uses, and compositions of the present invention.

[0053] Any reference to a method of treatment in this description refers to the compounds, pharmaceutical compositions, and medicaments of the present invention for use in a method of treating a human (or animal) by therapy.

[0054] As embodied and broadly described herein, the present invention relates to a surprising discovery that activation of phosphodiesterase activity in T cells upon generation of CAR-T cells by introduction of a CAR expression vector results in (1) a significant increase in the potency of T cells expressing CAR, and (2) an increase in the potency of the resulting CAR-T cells to kill their respective target cells. As described in the following examples and figures, expression of a vector encoding a chimeric antigen receptor (CAR) typically results in approximately 25% of the cells being positive for surface expression of CAR (CAR-T cells). Figure 3 Show that co-expression of the 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 CAR-T cells expressing PDE4D2, a significant increase in the percentage of killer cells was found, as Figure 4 shown. Figure 5 and Figure 6 Show that CAR-T cells expressing PDE4D2 have increased IL-2 expression, but there is a limited significant difference in interferon-γ secretion. Thus, without wishing to be bound by theory, the inventors speculate that the increased potency of CAR-T cells expressing PDE4D2 to kill their respective target cells is mainly mediated by an IL-2-dependent mechanism.

[0055] For the experiments presented herein, the inventors used chimeric antigen receptors (CARs) targeting prostate-specific membrane antigen (PSMA) and tested these CAR-T cells on CHO cells overexpressing PSMA. There is no reason for a person skilled in the art to expect that the results presented herein are related to the specific CAR or antigen used in these experiments, and thus the present invention can be applied to any CAR and any antigen targeted by the CAR. For the experiments described herein, the PDE-encoding gene was included on the CAR-T expression vector. As is known to a person skilled in the art, this is merely one way to introduce and overexpress the PDE protein, and the end result is an increase in PDE enzyme activity. Thus, a person skilled in the art can immediately understand that the same result can be obtained by any means of activating or overexpressing PDE in T cells. The results described below use PDE4D2 as an exemplary phosphodiesterase; however, it is well known that there are many phosphodiesterases with very similar structures and functions. A person skilled in the art can immediately realize that other phosphodiesterases can be used to achieve the same effect, and thus the experimental results should not be construed as limiting the present invention to PDE4D2.

[0056] Accordingly, in a first embodiment, the present invention relates to an ex vivo or in vitro method of generating improved CAR-T cells, the method comprising:

[0057] providing T cells;

[0058] introducing a chimeric antigen receptor (CAR) gene into the T cells to obtain CAR-T cells;

[0059] wherein the method further comprises the step of upregulating the enzymatic activity of phosphodiesterase (PDE) protein in the T cells before, during, or after introducing the CAR gene into the T cells.

[0060] Accordingly, the present invention as described herein broadly defines an improved method of generating CAR-T cells, the method upregulating the enzymatic activity of phosphodiesterase (PDE) protein in T cells. As the experimental results show, the effect of activating PDE is twofold, namely: (1) it greatly increases the number of T cells expressing the chimeric antigen receptor (CAR), thereby surprisingly enhancing the efficacy of T cell transformation or T cell expansion in vitro; and (2) expressing PDE surprisingly leads to an increase in the efficacy of CAR-T cells, since CAR-T cells activated by PDE result in a higher percentage of target cells (cells expressing the antigen targeted by the CAR) being killed.

[0061] The CAR (chimeric antigen receptor) used in this text refers to an artificial T cell receptor, which is commonly used in immunotherapy. A CAR, also known as a chimeric immune receptor, chimeric T cell receptor, or artificial T cell receptor, is a receptor protein that has been engineered to confer upon T cells the new ability to target specific antigens. The receptors are chimeric because they combine antigen-binding and T cell activation functions into one receptor. Typically, a chimeric receptor comprises a single-chain variable fragment (scFv) domain that targets the antigen, a hinge region (also known as a spacer) that can be derived from, for example, IgG or CD8 protein, a transmembrane domain that usually consists of hydrophobic α-helices such as the transmembrane domain derived from CD28, and an intracellular T cell signaling domain such as the CD3-zeta cytoplasmic domain. It should be understood that not all of the above domains and regions are mandatory in a CAR, and these domains and regions can be replaced by other domains or regions, or completely omitted. Thus, the term "CAR" should be understood to encompass any modified receptor that, when expressed in T cells, enables the T cells to target specific antigens and initiate an immune response.

[0062] The CAR-T cells used in this text refer to T cells that express a CAR as defined herein.

[0063] The T cells provided herein can be obtained from a subject expected to receive CAR-T cell therapy obtained by the method, but it should be understood that the method is not limited to the source of the T cells. Thus, the T cells can be obtained, for example, from a donor, or can be obtained by in vitro differentiation of progenitor cells, or can be obtained from a subject expected to receive the resulting CAR-T cell therapy. It should be further understood that an obvious application of the CAR-T cells obtained by the method is their use in treating a subject, but the method is not limited to such applications and can also be used, for example, in research, product development, or screening methods.

[0064] The term phosphodiesterase (PDE) as used herein refers to an enzyme that breaks phosphodiester bonds. More specifically, the phosphodiesterase as used herein refers to a cyclic nucleotide phosphodiesterase that is capable of converting cyclic nucleotides into nucleotide monophosphates. Generally, phosphodiesterase converts cAMP or cGMP into AMP or GMP, respectively. The term PDE as used herein can refer to a protein having phosphodiesterase enzyme activity, or a nucleic acid (such as a gene) encoding such a protein. This term should not be construed restrictively and should thus be understood to refer to PDEs derived from humans or animals (such as rodents, primates, mammals, fish, amphibians, reptiles, birds, chordates, insects, etc.), although in a preferred embodiment, the PDE refers to a human PDE. Thus, the term PDE encompasses at least any one of the following: PDE1A, PDE1B, PDE1C, PDE2A, PDE3A, PDE3B, PDE4A, PDE4B, PDE4C, PDE4D, PDE5A, PDE6A, PDE6B, PDE6C, PDE6D, PDE6G, PDE6H, PDE7A, PDE7B, PDE8A, PDE8B, PDE9A, PDE10A or PDE11A, or specific isomers thereof. In one embodiment, the above-mentioned PDE is a human PDE. In one embodiment, the PDE is a protein having phosphodiesterase activity, preferably an enzyme with an EC classification number of 3.1.4.17. In one embodiment, the PDE is defined according to Table 1 below, or a protein or enzyme having sequence homology (such as 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homology) with the protein defined in Table 1, or encoded by a nucleotide sequence having sequence homology (such as 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homology) with the sequence defined in Table 1.

[0065] Table 1

[0066]

[0067]

[0068] The above table cites nucleotide and protein identifiers available on GenBank, which can be accessed via https: / / www.ncbi.nlm.nih.gov / nuccoreAccess. It should be understood that each protein has corresponding isomers. Although this table lists the specific isomers of PDE2 and PDE4 genes / proteins, for the remaining PDEs (PDE1A, PDE1B, …, PDE3A, etc.), specific isomers are not listed, and the designated gene and protein sequences are representative isomers. The specific nomenclature of different isomers is based on the consensus nomenclature of proteins, so the sequence variant numbers of mRNAs may not match the isomer numbers. For example, the PDE4D isomer 2 protein is encoded by a sequence annotated as sequence variant 7 of the PDE4D sequence. When a specific isomer (such as PDE4D2) is mentioned in this article, it refers to the protein consensus nomenclature. It should be understood that for each of these PDEs, specific isomers have been identified, and the present invention should not be construed as being limited to the specific isomers listed.

[0069] As used herein, an isomer refers to a variant protein encoded by the same gene locus with different mRNA and / or protein sequences. A non-limiting example of an isomer is a splicing variant. It should be understood that the nucleotide sequences mentioned in Table 1 above correspond to mRNA sequences and thus may include 5’UTR and / or 3’UTR sequences. A person skilled in the art can determine the coding sequence based on information available, for example, in genomic reference databases. It should be understood that the UTRs can be changed without changing the coding sequence. Therefore, changes in the above nucleotide sequences are preferably based only on sequence homology of the coding sequence, which means that a sequence having 99% sequence homology with SEQ ID NO:1 is intended to cover those sequences having at least 99% sequence homology with the coding sequence defined by SEQ ID NO:1, regardless of the UTRs. The present invention also aims to cover isomers not explicitly listed in the above genes.

[0070] The term “PDE1A” refers to the phosphodiesterase 1A gene (Ensembl: ENSG00000115252), for example, to the sequence defined by the NCBI reference sequence NM_005019.7, particularly to the nucleotide sequence shown in SEQ ID NO:1, which corresponds to the sequence of the NCBI reference sequence of the above PDE1A transcript, and also relates to, for example, the corresponding amino acid sequence shown in SEQ ID NO:2, which corresponds to the protein sequence encoding the PDE1A polypeptide defined by the NCBI protein accession reference sequence NP_005010.2.

[0071] The term "PDE1A" also includes nucleotide sequences that exhibit a high degree of homology with PDE1A, such as 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 shown in SEQ ID NO:1, or amino acid sequences that are at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence shown in SEQ ID NO:2, or nucleic acid sequences that encode amino acid sequences that are at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence shown 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 shown in SEQ ID NO:1.

[0072] The term "PDE1B" refers to the phosphodiesterase 1B gene (Ensembl: ENSG00000123360), for example, it refers to the sequence defined by the NCBI reference sequence NM_000924.4, particularly to the nucleotide sequence shown in SEQ ID NO:3, which corresponds to the sequence of the above-mentioned PDE1B transcript's NCBI reference sequence, and also relates to, for example, the corresponding amino acid sequence shown in SEQ ID NO:4, which corresponds to the protein sequence encoding the PDE1B polypeptide defined in the NCBI protein accession reference sequence NP_000915.1.

[0073] The term "PDE1B" also includes nucleotide sequences that exhibit a high degree of homology with PDE1B, such as 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 shown in SEQ ID NO:3, or amino acid sequences that are at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence shown in SEQ ID NO:4, or nucleic acid sequences that encode amino acid sequences that are at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence shown 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 shown in SEQ ID NO:3.

[0074] The term "PDE1C" refers to the phosphodiesterase 1C gene (Ensembl: ENSG00000154678), for example, the sequence defined by NCBI reference sequence NM_001191056.3, particularly the nucleotide sequence shown in SEQ ID NO:5, which corresponds to the sequence of the NCBI reference sequence of the above PDE1C transcript, and also refers to, for example, the corresponding amino acid sequence shown in SEQ ID NO:6, which corresponds to the protein sequence encoding the PDE1C polypeptide defined by NCBI protein accession reference sequence NP_001177985.1.

[0075] The term "PDE1C" also includes nucleotide sequences that exhibit a high degree of homology with PDE1C, such as 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 shown in SEQ ID NO:5, or amino acid sequences that are at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence shown in SEQ ID NO:6, or nucleic acid sequences encoding amino acid sequences that are at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence shown 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 shown in SEQ ID NO:5.

[0076] The term "PDE2A" refers to the phosphodiesterase 2A gene (Ensembl: ENSG00000186642), for example, the sequences defined by NCBI reference sequences NM_002599.5, NM_001143839.4, NM_001243784.2 or NM_001146209.3, particularly the nucleotide sequences shown in SEQ ID NO:7, 9, 11 or 13, which correspond to the sequences of the NCBI reference sequences of the above PDE2A transcripts, and also refers to, for example, the corresponding amino acid sequences shown in SEQ ID NO:8, 10, 12 or 14, which correspond to the protein sequences encoding the PDE2A polypeptides defined by NCBI protein accession reference sequences NP_002590.1, NP_001137311.1, NP_001230713.1 or NP_001139681.1.

[0077] The term "PDE2A" also includes nucleotide sequences that exhibit a high degree of homology with PDE2A, such as nucleic acid sequences that are at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequences shown in SEQ ID NO:7, 9, 11 or 13, or amino acid sequences that are at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequences shown in SEQ ID NO:8, 10, 12 or 14, or nucleic acid sequences that encode amino acid sequences that are at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequences shown in SEQ ID NO:8, 10, 12 or 14, 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 sequences shown in SEQ ID NO:7, 9, 11 or 13.

[0078] The term "PDE3A" refers to the phosphodiesterase 3A gene (Ensembl: ENSG00000172572), for example, it refers to the sequence defined by the NCBI reference sequence NM_000921.5, particularly the nucleotide sequence shown in SEQ ID NO:15, which corresponds to the sequence of the NCBI reference sequence of the above PDE3A transcript, and also relates to, for example, the corresponding amino acid sequence shown in SEQ ID NO:16, which corresponds to the protein sequence encoding the PDE3A polypeptide defined by the NCBI protein accession reference sequence NP_000912.3.

[0079] The term "PDE3A" also includes nucleotide sequences that exhibit a high degree of homology with PDE3A, such as 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 shown in SEQ ID NO:15, or amino acid sequences that are at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence shown in SEQ ID NO:16, or nucleic acid sequences that encode amino acid sequences that are at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence shown 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 shown in SEQ ID NO:15.

[0080] The term "PDE3B" refers to the phosphodiesterase 3B gene (Ensembl: ENSG00000152270), for example, it refers to the sequence defined by the NCBI reference sequence NM_000922.4, particularly to the nucleotide sequence shown in SEQ ID NO:17, which corresponds to the sequence of the NCBI reference sequence of the above PDE3B transcript, and also relates to, for example, the corresponding amino acid sequence shown in SEQ ID NO:18, which corresponds to the protein sequence encoding the PDE3B polypeptide defined by the NCBI protein accession reference sequence NP_000913.2.

[0081] The term "PDE3B" also includes nucleotide sequences that exhibit a high degree of homology with PDE3B, such as 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 shown in SEQ ID NO:17, or amino acid sequences that are at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence shown in SEQ ID NO:18, or nucleic acid sequences that encode amino acid sequences that are at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence shown in SEQ ID NO:18, 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 shown in SEQ ID NO:17.

[0082] The term "PDE4A" refers to the phosphodiesterase 4A gene (Ensembl: ENSG00000065989), for example, the sequences defined by NCBI reference sequences NM_001111307.2, NM_001111308.1, NM_001111309.1, NM_006202.3 or NM_001243121.2, particularly the nucleotide sequences shown in SEQ ID NO: 19, 21, 23, 25 or 27, which correspond to the sequences of the above-mentioned NCBI reference sequences of PDE4A transcripts, and also relates to, for example, the corresponding amino acid sequences shown in SEQ ID NO: 20, 22, 24, 26 or 28, which correspond to the protein sequences encoding PDE4A polypeptides defined by NCBI protein accession reference sequences NP_001104777.1, NP_001104778.1, NP_001104779.1, NP_006193.1 or NP_001230050.1.

[0083] The term "PDE4A" also includes nucleotide sequences that exhibit a high degree of homology with PDE4A, for example, nucleic acid sequences that are at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequences shown in SEQ ID NO: 19, 21, 23, 25 or 27, or amino acid sequences that are at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequences shown in SEQ ID NO: 20, 22, 24, 26 or 28, or nucleic acid sequences encoding amino acid sequences that are at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequences shown in SEQ ID NO: 20, 22, 24, 26 or 28, 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 sequences shown in SEQ ID NO: 19, 21, 23, 25 or 27.

[0084] The term "PDE4B" refers to the phosphodiesterase 4B gene (Ensembl: ENSG00000184588), for example, it refers to the sequences defined by 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, particularly to the nucleotide sequences shown in SEQ ID NO: 29, 31, 33, 35, 37, 39 or 41, which correspond to the sequences of the NCBI reference sequences of the above PDE4B transcripts, and also relates to, for example, the corresponding amino acid sequences shown in SEQ ID NO: 30, 32, 34, 36, 38, 40 or 42, which correspond to the protein sequences encoding the PDE4B polypeptide defined by NCBI protein accession reference sequences NP_002591.2, NP_001032416.1, NP_001032417.1, NP_001032418.1, NP_001284369.1, NP_001284370.1 or NP_001284371.1.

[0085] The term "PDE4B" also includes nucleotide sequences that show a high degree of homology with PDE4B, such as nucleic acid sequences that are at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequences shown in SEQ ID NO: 29, 31, 33, 35, 37, 39 or 41, or amino acid sequences that are at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequences shown in SEQ ID NO: 30, 32, 34, 36, 38, 40 or 42, or nucleic acid sequences encoding amino acid sequences that are at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequences shown in SEQ ID NO: 30, 32, 34, 36, 38, 40 or 42, 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 sequences shown in SEQ ID NO: 29, 31, 33, 35, 37, 39 or 41.

[0086] The term "PDE4C" refers to the phosphodiesterase 4C gene (Ensembl: ENSG00000105650), for example, the sequences defined by NCBI reference sequences NM_000923.6, NM_001098819.4, NM_001098818.4, NM_001369701.2, NM_001330172.2 or NM_001395274.1, particularly the nucleotide sequences shown in SEQ ID NO:43, 45, 47, 49, 51 or 53, which correspond to the sequences of the above-mentioned NCBI reference sequences of the PDE4C transcript, and also relates to, for example, the corresponding amino acid sequences shown in SEQ ID NO:44, 46, 48, 50, 52 or 54, which correspond to the protein sequences encoding the PDE4C polypeptide defined by NCBI protein accession reference sequences NP_000914.2, NP_001092289.1, NP_001092288.1, NP_001356630.1, NP_001317101.1 or NP_001382203.1.

[0087] The term "PDE4C" also includes nucleotide sequences that exhibit a high degree of homology with PDE4C, such as nucleic acid sequences that are at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequences shown in SEQ ID NO:43, 45, 47, 49, 51 or 53, or amino acid sequences that are at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequences shown in SEQ ID NO:44, 46, 48, 50, 52 or 54, or nucleic acid sequences encoding amino acid sequences that are at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequences shown in SEQ ID NO:44, 46, 48, 50, 52 or 54, 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 sequences shown in SEQ ID NO:43, 45, 47, 49, 51 or 53.

[0088] The term "PDE4D" refers to the phosphodiesterase 4D gene (Ensembl: ENSG00000113448), for example, the sequences defined by 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, particularly the nucleotide sequences shown in SEQ ID NO:55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87 or 89, which correspond to the sequences of the NCBI reference sequences of the above PDE4D transcripts, and also relates to, for example, the corresponding amino acid sequences shown in SEQ ID NO:66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88 or 90, which correspond to the protein sequences encoding the PDE4D polypeptide defined by 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_001336170.1, 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.

[0089] The term "PDE4D" also includes nucleotide sequences that exhibit a high degree of homology with PDE4D, such as nucleic acid sequences that are at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequences shown in SEQ ID NO: 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87 or 89, or amino acid sequences that are at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequences shown in SEQ ID NO: 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88 or 90, or nucleic acid sequences that encode amino acid sequences that are at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequences shown in SEQ ID NO: 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88 or 90, 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 sequences shown in SEQ ID NO: 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87 or 89.

[0090] The term "PDE5A" refers to the phosphodiesterase 5A gene (Ensembl: ENSG00000138735), for example, it refers to the sequence defined by NCBI reference sequence NM_001083.4, particularly to the nucleotide sequence shown in SEQ ID NO: 91, which corresponds to the sequence of the NCBI reference sequence of the above PDE5A transcript, and also relates to, for example, the corresponding amino acid sequence shown in SEQ ID NO: 92, which corresponds to the protein sequence encoding the PDE5A polypeptide defined by NCBI protein accession reference sequence NP_001074.2.

[0091] The term "PDE5A" also includes nucleotide sequences that exhibit a high degree of homology with PDE5A, such as 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 shown in SEQ ID NO:91, or amino acid sequences that are at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence shown in SEQ ID NO:92, or nucleic acid sequences that encode amino acid sequences that are at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence shown in SEQ ID NO:92, 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 shown in SEQ ID NO:92.

[0092] The term "PDE6A" refers to the phosphodiesterase 6A gene (Ensembl: ENSG00000132915), for example, it refers to the sequence defined by the NCBI reference sequence NM_000440.3, particularly to the nucleotide sequence shown in SEQ ID NO:93, which corresponds to the sequence of the NCBI reference sequence of the above-mentioned PDE6A transcript, and also relates to, for example, the corresponding amino acid sequence shown in SEQ ID NO:94, which corresponds to the protein sequence encoding the PDE6A polypeptide defined by the NCBI protein accession reference sequence NP_000431.2.

[0093] The term "PDE6A" also includes nucleotide sequences that exhibit a high degree of homology with PDE6A, such as 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 shown in SEQ ID NO:93, or amino acid sequences that are at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence shown in SEQ ID NO:94, or nucleic acid sequences that encode amino acid sequences that are at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence shown in SEQ ID NO:94, 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 shown in SEQ ID NO:93.

[0094] The term "PDE6B" refers to the phosphodiesterase 6B gene (Ensembl: ENSG00000133256), for example, it refers to the sequence defined by NCBI reference sequence NM_000283.4, particularly the nucleotide sequence shown in SEQ ID NO:95, which corresponds to the sequence of the NCBI reference sequence of the above PDE6B transcript, and also relates to, for example, the corresponding amino acid sequence shown in SEQ ID NO:96, which corresponds to the protein sequence encoding the PDE6B polypeptide defined by NCBI protein accession reference sequence NP_000274.3.

[0095] The term "PDE6B" also includes nucleotide sequences that show a high degree of homology with PDE6B, such as 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 shown in SEQ ID NO:95, or amino acid sequences that are at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence shown in SEQ ID NO:96, or nucleic acid sequences encoding amino acid sequences that are at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence shown in SEQ ID NO:96, 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 shown in SEQ ID NO:95.

[0096] The term "PDE6C" refers to the phosphodiesterase 6C gene (Ensembl: ENSG00000095464), for example, it refers to the sequence defined by NCBI reference sequence NM_006204.4, particularly the nucleotide sequence shown in SEQ ID NO:97, which corresponds to the sequence of the NCBI reference sequence of the above PDE6C transcript, and also relates to, for example, the corresponding amino acid sequence shown in SEQ ID NO:98, which corresponds to the protein sequence encoding the PDE6C polypeptide defined by NCBI protein accession reference sequence NP_006195.3.

[0097] The term "PDE6C" also includes nucleotide sequences that exhibit a high degree of homology with PDE6C, such as 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 shown in SEQ ID NO:97, or amino acid sequences that are at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence shown in SEQ ID NO:98, or nucleic acid sequences that encode amino acid sequences that are at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence shown in SEQ ID NO:98, 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 shown in SEQ ID NO:97.

[0098] The term "PDE6D" refers to the phosphodiesterase 6D gene (Ensembl: ENSG00000156973), for example, it refers to the sequence defined as in NCBI reference sequence NM_002601.4, particularly to the nucleotide sequence shown in SEQ ID NO:99, which corresponds to the sequence of the above-mentioned NCBI reference sequence of the PDE6D transcript, and also relates to, for example, the corresponding amino acid sequence shown in SEQ ID NO:100, which corresponds to the protein sequence defined by the NCBI protein accession reference sequence NP_002592.1 encoding the PDE6D polypeptide.

[0099] The term "PDE6D" also includes nucleotide sequences that exhibit a high degree of homology with PDE6D, such as 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 shown in SEQ ID NO:99, or amino acid sequences that are at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence shown in SEQ ID NO:100, or nucleic acid sequences that encode amino acid sequences that are at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence shown in SEQ ID NO:100, 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 shown in SEQ ID NO:99.

[0100] The term "PDE6G" refers to the phosphodiesterase 6G gene (Ensembl: ENSG00000185527), for example, it refers to the sequence defined by NCBI reference sequence NM_002602.4, particularly to the nucleotide sequence shown in SEQ ID NO: 101, which corresponds to the sequence of the NCBI reference sequence of the above-mentioned PDE6G transcript, and also relates to, for example, the corresponding amino acid sequence shown in SEQ ID NO: 102, which corresponds to the protein sequence encoding the PDE6G polypeptide defined by NCBI protein accession reference sequence NP_002593.1.

[0101] The term "PDE6G" also includes nucleotide sequences that show a high degree of homology with PDE6G, such as 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 shown in SEQ ID NO: 101, or amino acid sequences that are at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence shown in SEQ ID NO: 102, or nucleic acid sequences encoding amino acid sequences that are at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence shown in SEQ ID NO: 102, 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 shown in SEQ ID NO: 101.

[0102] The term "PDE6H" refers to the phosphodiesterase 6H gene (Ensembl: ENSG00000139053), for example, it refers to the sequence defined by NCBI reference sequence NM_006205.3, particularly to the nucleotide sequence shown in SEQ ID NO: 103, which corresponds to the sequence of the NCBI reference sequence of the above-mentioned PDE6H transcript, and also relates to, for example, the corresponding amino acid sequence shown in SEQ ID NO: 104, which corresponds to the protein sequence encoding the PDE6H polypeptide defined by NCBI protein accession reference sequence NP_006196.1.

[0103] The term "PDE6H" also includes nucleotide sequences that exhibit a high degree of homology with PDE6H, such as 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 shown in SEQ ID NO: 103, or amino acid sequences that are at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence shown in SEQ ID NO: 104, or nucleic acid sequences that encode amino acid sequences that are at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence shown in SEQ ID NO: 104, 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 shown in SEQ ID NO: 103.

[0104] The term "PDE7A" refers to the phosphodiesterase 7A gene (Ensembl: ENSG00000205268), for example, it refers to the sequence defined by NCBI reference sequence NM_002603.4, particularly to the nucleotide sequence shown in SEQ ID NO: 105, which corresponds to the sequence of the NCBI reference sequence of the above PDE7A transcript, and also relates to, for example, the corresponding amino acid sequence shown in SEQ ID NO: 106, which corresponds to the protein sequence encoding the PDE7A polypeptide defined by NCBI protein accession reference sequence NP_002594.1.

[0105] The term "PDE7A" also includes nucleotide sequences that exhibit a high degree of homology with PDE7A, such as 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 shown in SEQ ID NO:105, or amino acid sequences that are at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence shown in SEQ ID NO:106, or nucleic acid sequences that encode amino acid sequences that are at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence shown in SEQ ID NO:106, 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 shown in SEQ ID NO:105.

[0106] The term "PDE7B" refers to the phosphodiesterase 7B gene (Ensembl: ENSG00000171408), for example, it refers to the sequence defined by the NCBI reference sequence NM_018945.4, particularly to the nucleotide sequence shown in SEQ ID NO:107, which corresponds to the sequence of the NCBI reference sequence of the above PDE7B transcript, and also relates to, for example, the corresponding amino acid sequence shown in SEQ ID NO:108, which corresponds to the protein sequence encoding the PDE7B polypeptide defined by the NCBI protein accession reference sequence NP_061818.1.

[0107] The term "PDE7B" also includes nucleotide sequences that exhibit a high degree of homology with PDE7B, such as 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 shown in SEQ ID NO:107, or amino acid sequences that are at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence shown in SEQ ID NO:108, or nucleic acid sequences that encode amino acid sequences that are at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence shown in SEQ ID NO:108, 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 shown in SEQ ID NO:107.

[0108] The term "PDE8A" refers to the phosphodiesterase 8A gene (Ensembl: ENSG00000073417), for example, it refers to the sequence defined by the NCBI reference sequence NM_002605.3, particularly to the nucleotide sequence shown in SEQ ID NO:109, which corresponds to the sequence of the above-mentioned NCBI reference sequence of the PDE8A transcript, and also relates to, for example, the corresponding amino acid sequence shown in SEQ ID NO:110, which corresponds to the protein sequence encoding the PDE8A polypeptide defined by the NCBI protein accession reference sequence NP_002596.1.

[0109] The term "PDE8A" also includes nucleotide sequences that exhibit a high degree of homology with PDE8A, such as 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 shown in SEQ ID NO:109, or amino acid sequences that are at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence shown in SEQ ID NO:110, or nucleic acid sequences that encode amino acid sequences that are at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence shown in SEQ ID NO:110, 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 shown in SEQ ID NO:109.

[0110] The term "PDE8B" refers to the phosphodiesterase 8B gene (Ensembl: ENSG00000113231), for example, it refers to the sequence defined by the NCBI reference sequence NM_003719.5, particularly the nucleotide sequence shown in SEQ ID NO:111, which corresponds to the sequence of the NCBI reference sequence of the above PDE8B transcript, and also relates to, for example, the corresponding amino acid sequence shown in SEQ ID NO:112, which corresponds to the protein sequence encoding the PDE8B polypeptide defined by the NCBI protein accession reference sequence NP_003710.1.

[0111] The term "PDE8B" also includes nucleotide sequences that exhibit a high degree of homology with PDE8B, such as 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 shown in SEQ ID NO:111, or amino acid sequences that are at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence shown in SEQ ID NO:112, or nucleic acid sequences that encode amino acid sequences that are at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence shown in SEQ ID NO:112, 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 shown in SEQ ID NO:111.

[0112] The term "PDE9A" refers to the phosphodiesterase 9A gene (Ensembl: ENSG00000160191), for example, the sequence defined by NCBI reference sequence NM_002606.3, particularly the nucleotide sequence shown in SEQ ID NO: 113, which corresponds to the sequence of the NCBI reference sequence of the above PDE9A transcript, and also relates to, for example, the corresponding amino acid sequence shown in SEQ ID NO: 114, which corresponds to the protein sequence encoding the PDE9A polypeptide defined by NCBI protein accession reference sequence NP_002597.1.

[0113] The term "PDE9A" also includes nucleotide sequences that exhibit a high degree of homology with PDE9A, such as 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 shown in SEQ ID NO: 113, or amino acid sequences that are at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence shown in SEQ ID NO: 114, or nucleic acid sequences encoding amino acid sequences that are at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence shown in SEQ ID NO: 114, 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 shown in SEQ ID NO: 113.

[0114] The term "PDE10A" refers to the phosphodiesterase 10A gene (Ensembl: ENSG00000112541), for example, the sequence defined by NCBI reference sequence NM_006661.4, particularly the nucleotide sequence shown in SEQ ID NO: 115, which corresponds to the sequence of the NCBI reference sequence of the above PDE10A transcript, and also relates to, for example, the corresponding amino acid sequence shown in SEQ ID NO: 116, which corresponds to the protein sequence encoding the PDE10A polypeptide defined by NCBI protein accession reference sequence NP_006652.1.

[0115] The term "PDE10A" also includes nucleotide sequences that exhibit a high degree of homology with PDE10A, such as 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 shown in SEQ ID NO: 115, or amino acid sequences that are at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence shown in SEQ ID NO: 116, or nucleic acid sequences that encode amino acid sequences that are at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence shown in SEQ ID NO: 116, 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 shown in SEQ ID NO: 115.

[0116] The term "PDE11A" refers to the phosphodiesterase 11A gene (Ensembl: ENSG00000128655), such as the sequence defined by NCBI reference sequence NM_005019.7, particularly the nucleotide sequence shown in SEQ ID NO: 117, which corresponds to the sequence of the NCBI reference sequence of the above PDE11A transcript, and also relates to, for example, the corresponding amino acid sequence shown in SEQ ID NO: 118, which corresponds to the protein sequence encoding the PDE11A polypeptide defined by NCBI protein accession reference sequence NP_005010.2.

[0117] The term "PDE11A" also includes nucleotide sequences that exhibit a high degree of homology with PDE11A, such as 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 shown in SEQ ID NO: 117, or amino acid sequences that are at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence shown in SEQ ID NO: 118, or nucleic acid sequences that encode amino acid sequences that are at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence shown in SEQ ID NO: 118, 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 shown in SEQ ID NO: 117.

[0118] The experimental results described herein were obtained by overexpressing a PDE such as PDE4D2. It should be understood that overexpression of the gene encoding the PDE protein results in increased translation, thereby leading to higher protein levels and, in turn, increased enzyme activity. Thus, the potency described herein, namely, enhancing the potency of CAR-transfected T cells and enhancing the ability of T cells to kill their target cells, can be achieved by any method capable of increasing the PDE enzyme activity in cells. Thus, when used herein, the term "upregulating enzyme activity" is intended to encompass any suitable method for increasing the PDE enzyme activity in T cells, such as, but not limited to, using a PDE agonist or overexpressing a PDE.

[0119] Thus, in one embodiment of the method of the invention, the PDE enzyme activity is upregulated by increasing the expression of the PDE protein and / or by using a PDE agonist to upregulate the PDE enzyme activity, preferably wherein the PDE agonist is MR-L2 (or a derivative thereof) or an Aβ peptide.

[0120] Thus, in one embodiment, the method comprises the step of upregulating the PDE enzyme activity, the step being selected from:

[0121] - administering gene therapy for inducing PDE expression to T cells;

[0122] - administering PDE-encoding mRNA (mRNA therapy) to T cells;

[0123] - administering a compound that induces PDE expression to T cells;

[0124] - administering a compound that directly stimulates or promotes phosphodiesterase activity to T cells;

[0125] - Administering to T cells a compound that indirectly stimulates or promotes phosphodiesterase activity;

[0126] - Administering to T cells an inhibitor of a PDE transcription inhibitor;

[0127] - Administering to T cells an inhibitor of a phosphodiesterase activity inhibitor

[0128] - Administering to T cells an mRNA encoding a phosphodiesterase protein;

[0129] - Administering to T cells a phosphodiesterase protein;

[0130] - Administering to T cells an amyloid-β peptide that promotes phosphodiesterase activity.

[0131] As used herein, "gene therapy that induces PDE expression" refers to a method of introducing a nucleotide encoding a phosphodiesterase into a cell, preferably a T cell. The nucleotide can be, for example, a viral vector or a non-viral vector, but it should be understood that any nucleotide that can be introduced into a patient's cell and express PDE can be used.

[0132] Viral vectors are a known cell transformation strategy. Cell transformation using viral vectors typically uses a modified virus as a vehicle to introduce a specific DNA or RNA sequence into a cell. The vector is packaged with viral proteins so that it can infect the cell and express its viral genome. Typically, the viral vector is modified so that no new viral particles are produced after infecting the target cell. Non-limiting examples include retroviruses (RVs), adenoviruses (AVs), adeno-associated viruses (AAVs), lentiviruses (LVs), and herpes simplex viruses (HSVs). Other ways of introducing a PDE-encoding nucleotide into a cell, such as a T cell, using viral particles or viral vectors are known to those skilled in the art and are also contemplated to be included in the present invention.

[0133] Alternatively, in addition to virus vector-based cell transformation, non-viral vectors can also be used. These vectors typically contain promoters to drive the expression of relevant constructs (such as PDE), and generally require some means to introduce the vectors into target cells. The ways to deliver non-viral vectors to target cells are known to those skilled in the art. For example, Ramamoorth M, Narvekar A. Non-viral vectors in gene therapy - an overview. J Clin DiagnRes. 2015 Jan;9(1):GE01-6 reviewed the relevant content (the entire content of which is incorporated herein by reference). For example, nanoparticles can be used to encapsulate the vectors. Non-limiting examples include lipid-based nanoparticles, peptide-based nanoparticles, cationic lipid-based nanoparticles, apolipoprotein-based nanoparticles, (synthetic) polymer-based nanoparticles such as nanoparticles based on polyethyleneimine (PEI), chitosan, polylactic acid, poly(lactide), polyglucoside, dendrimers or polymethacrylates. As used herein, a "nanoparticle" is a small particle that can be used as a vector to deliver a payload to a patient. Preferably, the payload is a nucleotide encoding PDE as defined herein in a broad sense. Thus, nanoparticles can be used to deliver a payload that induces PDE expression or promotes phosphodiesterase activity to cells, such as to T cells or CAR-T cells. Other ways to introduce PDE-encoding nucleotides using non-viral vectors are known to those skilled in the art, and these ways are also contemplated to be included in the present invention.

[0134] Alternatively, in addition to virus- or non-virus vector-based cell transformation, mRNA-based methods can also be used. This method generally includes delivering mRNA molecules encoding PDE. The mRNA can be modified by methods known in the art to stabilize the RNA in vivo and extend its half-life (for example, using chemically modified nucleotides, codon optimization, optimized 5'-capping and 3'-tailing). In addition, the mRNA can contain a structure that optimizes the 5'-UTR to increase mRNA translation, or contain elements that allow the mRNA to self-amplify. The ways to deliver the mRNA to target cells are known to those skilled in the art. For example, Rohner E et al. Unlocking the promise of mRNA therapeutics. Nature Biotechnology 2022,40,1586-1600 reviewed the relevant content (the full text of which is incorporated herein by reference). For example, lipid nanoparticles (LNPs) can be used to encapsulate the mRNA. Alternatively, extracellular vesicles (EVs), cells or biomimetic materials can be used to deliver a payload that induces PDE expression or promotes phosphodiesterase activity to cells, such as to T cells or CAR-T cells.

[0135] Thus, in one embodiment, the cell transformation method is selected from: a viral vector capable of expressing PDE in cells or a non-viral vector capable of expressing PDE in cells. In one embodiment, nanoparticles are used to deliver PDE or a nucleotide encoding PDE to T cells.

[0136] As used herein, the term "vector" refers to any particle used as a vehicle (e.g., plasmid, cosmid, λ phage) that artificially carries a foreign nucleic acid molecule (usually DNA) into another cell and replicates and / or expresses therein.

[0137] As used herein, a "compound that induces PDE expression" refers to any biological or chemical compound capable of increasing PDE expression. For example, this can be achieved by promoting the transcription of the PDE gene or inhibiting the degradation of the phosphodiesterase protein. For example, the compound can participate in a signal transduction pathway to indirectly promote PDE transcription or directly interact with the promoter region of genomic DNA to promote PDE transcription.

[0138] As used herein, "promote PDE transcription" means increasing the transcription of PDE, thereby obtaining a higher amount of PDE mRNA transcript in the cell and / or preferably a higher amount of phosphodiesterase protein. This promotion can be specific to a particular PDE (e.g., PDE4D2), to all isomers of a particular PDE gene (e.g., PDE4D), to all PDEs of a particular subclass (e.g., PDE4), or even to all PDE family members. In other words, increasing the expression of one PDE does not exclude the possibility that the expression of other PDE genes or isomers may also increase.

[0139] As used herein, a "compound that directly stimulates or promotes phosphodiesterase activity" refers to a compound that directly participates in the phosphodiesterase protease activity. Without being bound by theory, theoretically, a PDE protein (e.g., the PDE4D2 protein) can exist in an active and an inactive conformation, where the activity can be induced by the interaction of other compounds with the PDE protein, e.g., by promoting the active conformation of the protein or by blocking or preventing the binding of an inhibitor, or by modifying the protein to promote activity (e.g., by phosphorylation or other known protein modifications). As used herein, the "enzymatic activity of phosphodiesterase" refers to the catalysis of the hydrolysis of cAMP and / or cGMP.

[0140] It has been found that, for example, the compound MR-L2 specifically increases PDE4 activity. As used herein, MR-L2 refers to a compound with the molecular formula C 19 H 16 C l3FN4O, a compound with the chemical formula shown below, CAS number 2374703-19-0, also known as HY-128358. Those skilled in the art know that the compound can be modified to obtain compounds with improved PDE activation activity or different selectivities. Therefore, the present invention further extends to esters, substituents, prodrugs or other modifiers of MR-L2 with PDE agonist activity.

[0141]

[0142] Compounds that indirectly stimulate or promote phosphodiesterase activity as used herein refer to compounds that indirectly participate in the protease activity of phosphodiesterase. Similarly, as described above, it is envisioned that compounds can induce PDE activators or block PDE activity inhibitors, thereby indirectly affecting the enzymatic activity of proteins. Methods for measuring PDE activity are known to those skilled in the art. For example, commercially available products can be used to measure PDE activity, and the 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 full text of which is incorporated herein by reference.

[0143] The "inhibitor of the inhibitor of PDE transcription" as used herein refers to a compound that can block the inhibitor of PDE gene transcription. The 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.

[0144] The "inhibitor of the inhibitor of phosphodiesterase enzymatic activity" as used herein refers to a compound that can block the inhibitor of phosphodiesterase protein activity. For example, the compound can act by degrading the inhibitor, preventing the inhibitor from binding to PDE, or inhibiting the activity of the inhibitor.

[0145] The mRNA encoding the phosphodiesterase protein as used herein refers to an RNA molecule from which the PDE as broadly defined above can be translated. The mRNA molecule usually contains untranslated elements, such as 5' and 3' UTRs. It is expected that PDE can be upregulated by directly introducing PDE mRNA into T cells. Methods for delivering mRNA to target cells are known to those skilled in the art, such as using the nanobodies as described above.

[0146] The phosphodiesterase proteins used herein can refer to compounds comprising a PDE protein or a biosimilar thereof, or can refer to constitutively active variants thereof having phosphodiesterase activity. It is expected that PDE activity can be increased by directly introducing the PDE protein into T cells. Methods for delivering proteins to target cells are known to those skilled in the art, such as using nanobodies as described above.

[0147] The peptides that promote phosphodiesterase activity used herein refer to peptides capable of increasing the activity of the PDE enzyme. Non-limiting examples are amyloid-beta peptides (Aβ), which have been shown to specifically increase the activity of so-called long PDE4D isoforms such as PDE4D5 or PDE4D7. The amyloid-beta peptide used herein, also referred to as Aβ or Abeta, refers to a peptide of 37 to 49 amino acids (Chen et al. Acta Pharmacol Sin 38, 1205–1235 (2017)), which is the main component of amyloid plaques found in the brains of Alzheimer's disease patients. The amyloid-beta peptide (Aβ) is generated by proteolytic cleavage of the transmembrane protein, namely amyloid precursor protein (APP), by the action of β- and γ-secretases.

[0148] Therefore, the peptide that promotes phosphodiesterase activity is preferably an Aβ peptide or a derivative thereof. The Aβ peptide or a derivative thereof used herein is characterized in that it has a length of 16 to 50 amino acids and comprises 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 (Aβ core peptide 1) below, more preferably comprises 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 (Aβ core peptide 2) below. Non-limiting examples are Aβ42 (SEQ ID NO: 121) and Aβ40 (SEQ ID NO: 122). Therefore, in one embodiment, the Aβ peptide or a derivative thereof is characterized in that: it has a length of 42 to 50 amino acids and comprises a peptide having 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 it has a length of 40 to 50 amino acids and comprises a peptide having 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.

[0149] SEQ ID NO:119: Aβ core peptide 2

[0150] HDSGYEVHHQKLVFFAEDVGSNKGAIIG

[0151] SEQ ID NO:120: Aβ core peptide 2

[0152] FRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMV

[0153] SEQ ID NO:121: Aβ42

[0154] DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA

[0155] SEQ ID NO:122: Aβ40

[0156] DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV

[0157] Accordingly, in one embodiment, the present invention relates to an ex vivo or in vitro method of generating improved CAR-T cells, the method comprising providing T cells, introducing a chimeric antigen receptor (CAR) gene into the T cells to obtain CAR-T cells, wherein the method further comprises the step of contacting the T cells with a PDE agonist as broadly defined above before, during or after introducing the CAR gene into the T cells. Alternatively, the present invention relates to an ex vivo or in vitro method of generating improved CAR-T cells, the method comprising providing T cells, introducing a chimeric antigen receptor (CAR) gene into the T cells to obtain CAR-T cells, wherein the method further comprises the step of overexpressing a PDE gene in the T cells before, during or after introducing the CAR gene into the T cells.

[0158] Accordingly, in one embodiment of the method of the present invention, PDE protein expression is upregulated by increasing the expression of the PDE gene. In one embodiment, the expression of the PDE gene is increased by exogenous expression of the PDE gene in T cells.

[0159] As used herein, the term "exogenous expression" or "exogenous gene expression" refers to introducing a nucleic acid encoding a PDE enzyme into T cells. The gene of exogenous expression can be the same as or different from the genes present in the T cell genome. It is hypothesized that exogenous expression of the gene will result in overexpression and subsequently increased translation of the protein.

[0160] Accordingly, the present invention contemplates either increasing the expression of the PDE-encoding gene in T cells or introducing a genetic construct (such as a viral vector, an expression vector, etc.) that allows the expression of an exogenous gene encoding PDE into T cells. The "gene encoding PDE" as used herein refers to a gene encoding a PDE protein as broadly defined herein (such as the proteins having sequence homology listed in Table 1 above, for example, having 80%, 85%, 90% or higher sequence homology with the proteins listed in Table 1), or the "gene encoding PDE" refers to a gene capable of expressing the mRNA defined in Table 1 (or an mRNA having 70%, 75%, 80%, 85%, 90% or higher sequence homology with the mRNA listed in Table 1).

[0161] In one embodiment, the PDE gene is introduced into T cells together with the CAR gene, preferably where the CAR gene and the PDE gene are contained on the same expression vector.

[0162] In one embodiment, the PDE protein is a PDE2 or PDE4 protein, preferably PDE2A, PDE4A, PDE4B, PDE4C or PDE4D, or a specific isoform selected from: PDE2A1, PDE2A2, PDE2A3, PDE2A4, PDE4A1, PDE4A2, PDE4A3, PDE4A4, PDE4A5, PDE4Ba, PDE4Bb, PDE4Bc, PDE4Bd, PDE4Be, PDE4Bf, PDE4Bg, PDE4C1, PDE4C2, PDE4C3, PDE4C4, PDE4C5, PDE4C6, PDE4D1, PDE4D2, PDE4D3, PDE4D4, PDE4D5, PDE4D6, PDE4D7, PDE4D8, PDE4D9, PDE4D10, PDE4D11, PDE4D12, PDE4D14, PDE4D15 or PDE4D16, preferably where the PDE4 protein is selected from the isoforms PDE4A4, PDE4B2, PDE4D1, PDE4D2, PDE4D5 or PDE4D7.

[0163] In one embodiment, the PDE protein is encoded by a nucleotide sequence having 80%, preferably 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or even 100% sequence identity to any of the sequences of SEQ ID NO: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 a protein having an amino acid sequence having 80%, preferably 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or even 100% sequence identity to any of the sequences of SEQ ID NO: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, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120 or 122.

[0164] In a preferred embodiment, the PDE is the PDE4 gene and is encoded by a nucleotide sequence having 80%, preferably 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or even 100% sequence identity with a sequence selected from SEQ ID No: 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 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 even 100% sequence identity with any one of the sequences of SEQ ID No: 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.

[0165] In one embodiment, the CAR antigen is prostate specific membrane antigen (PSMA). It should be understood, however, that the present invention is not limited to any particular antigen targeted by the CAR-T.

[0166] A second aspect of the present invention relates to a CAR-T cell with upregulated PDE protease activity. As used herein, the term "CAR-T cell with upregulated PDE protease activity" refers to a CAR-T cell that has been specifically engineered to increase PDE activity (e.g., by overexpression or any other method as exemplified above), and thus is not intended to cover an upregulation or increase in PDE enzyme activity through a naturally occurring process in T cells. Thus, in one embodiment, the present invention relates to a CAR-T cell with exogenously upregulated PDE protease activity, where exogenously upregulated refers to any artificial method of increasing T cell enzyme activity (e.g., the methods described above including but not limited to overexpressing a PDE encoding gene). The CAR-T cell may but need not be obtained by the method of the first aspect of the present invention.

[0167] In one embodiment, the CAR-T cell exogenously expresses a PDE gene, such as the PDE genes listed in Table 1, preferably the PDE4 or PDE2 gene.

[0168] Alternatively, according to the second aspect, the present invention relates to a CAR-T cell obtainable or obtained by the method defined in the first aspect of the present invention.

[0169] In one embodiment, the present invention relates to CAR-T cells as broadly described herein, which ectopically express a PDE protein encoded by a nucleotide sequence having 80%, preferably 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or even 100% sequence identity to any of the sequences of SEQ ID NO: 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 a protein having an amino acid sequence having 80%, preferably 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or even 100% sequence identity to any of the sequences of SEQ ID NO: 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, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120 or 122.

[0170] In a preferred embodiment, the PDE is the PDE4 gene and is encoded by a nucleotide sequence having 80%, preferably 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or even 100% sequence identity with a sequence selected from SEQ ID No: 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 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 even 100% sequence identity with any one of the sequences of SEQ ID No: 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.

[0171] The third aspect of the present invention relates to a CAR-T cell according to the second aspect of the present invention, which is used as a drug. It should be understood that the CAR-T cells described herein can be used for treatment, for example, by administering the CAR-T cells to a subject in need.

[0172] In one embodiment, the present invention relates to a CAR-T cell according to the second aspect of the present invention, which is used for treating, preventing or improving a disease. In a preferred embodiment, the disease is cancer or an immune-related disease, and more preferably, the immune-related disease is an autoimmune disease or a viral infection. It should be understood that CAR-T cells can also be applied outside of oncology applications, such as those reviewed in Zmievskaya E, et al. Biomedicines. 2021 Jan 9;9(1):59.

[0173] In a particularly preferred application, the present invention relates to a CAR-T cell for treating, preventing or improving prostate cancer, however, it should be understood that this specific use does not limit the present invention in any way.

[0174] In an alternative embodiment, a third aspect of the present invention relates to a method of treating, preventing or ameliorating a disease in a subject in need thereof, the method comprising administering to the subject the CAR-T cells described extensively herein. In one embodiment, the CAR-T cells are the CAR-T cells as described in the second aspect of the present invention. In one embodiment, the method is used for treating, preventing or ameliorating cancer or immune-related diseases. In one embodiment, the immune-related disease is an autoimmune disease or a viral infection. In one embodiment, the present invention relates to the treatment, prevention or amelioration of prostate cancer.

[0175] It should be understood that all details, embodiments and preferred options discussed for one aspect of the embodiments of the present invention are equally applicable to any other aspect or embodiment of the present invention, and thus there is no need to detail all these details, embodiments and preferred options for all aspects separately.

[0176] The present invention has been generally described above, and the present invention will be more easily understood by reference to the following examples, which are for illustrative purposes only and are not intended to limit the present invention. Other aspects and embodiments of the present invention will be apparent to those skilled in the art. Examples

[0177] The foregoing description of the specific embodiments will fully disclose the general nature of the present invention, so that others can, using the knowledge within the scope of the art (including the content of the references cited herein), easily modify and / or adapt these specific embodiments for various applications without undue experimentation, without departing from the general concept of the present invention. Therefore, such adaptations and modifications are intended to fall within the meaning and scope of the equivalent of the disclosed embodiments based on the teachings and guidance provided herein.

[0178] All references cited herein, including journal articles or abstracts, published or corresponding patent applications, patents or any other references, are hereby incorporated by reference in their entirety, including all data, tables, charts and text provided in the cited references. In addition, the entire content of the references cited in the references cited herein is also incorporated by reference in its entirety.

[0179] It should be understood that the language or terms herein are for descriptive purposes and not for limiting purposes, whereby those skilled in the art will interpret the terms or language in this specification based on the teachings and guidance presented herein and in combination with the knowledge of those of ordinary skill in the art.

[0180] Example 1: Detection of CAR-T Cells Expressing PDE4D2

[0181] An anti-PSMA chimeric antigen receptor (CAR-T) vector was constructed to engineer T cells to target human PSMA. The T cells were genetically modified by lentiviral vector transduction, and the vector expressed the scFv of an anti-PSMA antibody (clone J591) linked to CD28, CD137, and CD3ζ signaling domains.

[0182] CD28 (cluster of differentiation 28) is one of the proteins expressed on T cells, providing co-stimulatory signals required for T cell activation and survival. CD28 is a receptor for CD80 (B7.1) and CD86 (B7.2) proteins, which are expressed on antigen-presenting cells (APCs). CD28 regulates naive TCR / CD3ζ signaling in a manner different from the late co-stimulatory elements OX40 and 4-1BB. CD28 enhances the expression of downstream regulators, affecting T cell proliferation, death, differentiation, and effector functions. CAR+ T cells containing the intracellular domain of CD28 exhibit significantly enhanced sustained T cell activation, growth, and survival. And CD28 results in a receptor with clear and stable expression as a transmembrane domain. Inclusion of the CD28 co-stimulatory domain in CAR leads to enhanced anti-tumor efficacy.

[0183] CD137 (also known as 4-1BB) is a surface co-stimulatory glycoprotein initially described as present on activated T lymphocytes and belongs to the tumor necrosis factor (TNF) receptor superfamily. It is mainly expressed on activated CD4+ and CD8+ T cells and binds to a high-affinity ligand (4-1BBL) expressed on a variety of 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, thus significantly enhancing anti-tumor activity.

[0184] CD3ζ, also known as the T cell receptor ζ, forms the TCR-CD3 complex together with the T cell receptor and CD3γ, δ, ε chains. ζ is expressed independently of the complex. The ζ chain plays an important role in coupling antigen recognition to multiple intracellular signal transduction pathways. CD3-ζ contains 3 ITAMs and is the most commonly used intracellular domain component in CAR. It transmits activation signals to T cells after antigen binding. CD3-ζ may not provide a fully effective activation signal and requires additional co-stimulatory signals. For example, chimeric 4-1BB and OX40 can be used together with CD3-ζ to transmit proliferation / survival signals, or all three can be used together.

[0185] The coding sequence (CDS) of the PDE4D isoform PDE4D2 was cloned into the MCS (multiple cloning site) of CAR-T to obtain the vector PDE4D2 CAR-T. The expression of the PDE4D2 protein was driven by the PGK promoter. To simplify the detection of PDE4D2 protein expression, an 11-amino acid VSV tag was added to the 3'-end of the PDE4D2 CDS, followed by a translation termination codon. Figure 1 and Figure 2 Examples of anti-PSMA CAR vectors with and without the PDE4D2 gene are shown. After transfection of the CART vector into HEK293 cells, the expressed PDE4D2 protein can be detected by anti-PDE4D protein antibody and anti-VSV antibody in Western (immunoblotting) assay ( Figure 8 ).

[0186] Next, the PDE activity of the transfected cells was detected using a radiometric assay, which quantifies the hydrolysis rate of cAMP by PDE in the sample. Whole cell lysates of HEK293 cells transfected with 3 μg of the CAR-T plasmid containing PDE4D2 were used. The non-selective PDE inhibitor IBMX, the PDE4-selective inhibitor rolipram, or DMSO alone dissolved in DMSO were used. The PDE activity of untransfected cells was low (28.37 ± 22.32 pM / min / mg, Figure 9 ), which is expected because HEK293 cells have endogenous PDE expression. Compared with untransfected cells, the PDE activity of transfected cells without inhibitor increased significantly by 4-fold (115.8 ± 25.91 pM / min / mg, P < 0.0001, Figure 9 ). This indicates that the transfected PDE4D2 in the plasmid indeed confers increased cAMP-degrading PDE activity to the cells. Adding both the PDE inhibitors IBMX and rolipram to the transfected cells reduced the PDE activity to the basal level of untransfected cells. There was no significant difference in the PDE activity of transfected cells treated with IBMX and untransfected cells (40.16 ± 28.08 pM / min / mg, P = 0.6425, figure below). Similarly, there was no significant difference in the PDE activity of transfected cells treated with rolipram and untransfected cells (26.04 ± 25.29 pM / min / mg, P = 0.9948, Figure 9 ).

[0187] The percentage of T cells expressing anti-PSMA scFv antibody before and after CART transfection was studied by flow cytometry. To detect the anti-PSMA scFv antibody expressed by CART, an anti-human Fab antibody (produced in mice) was used. The "control" experiment represents human T cells without the anti-human Fab antibody in flow cytometry. The "control a-mouse Fab" experiment represents human T cells with the anti-human Fab antibody in flow cytometry. The labeled area in flow cytometry showed that about 4% of T cells had non-specific background signals. The "CAR-T a-mouse Fab" experiment represents human T cells with the anti-human Fab antibody in flow cytometry after CART transfection. The labeled area in flow cytometry showed that the anti-PSMA scFv antibody was expressed in about 25% of T cells. The "PDE4D2 CAR-T a-mouse Fab" experiment represents human T cells with the anti-human Fab antibody in flow cytometry after transfection with CAR-T cells containing the PDE4D2 gene. The labeled area in flow cytometry showed that the anti-PSMA scFv antibody was expressed in about 80% of T cells ( Figure 3 ).

[0188] Cytotoxicity experiments were performed using two vectors, CAR-T (Z011520 CAR-T) and PDE4D2 CAR-T (Z060719 PDE4D2 CAR-T), assuming that the PDE4D2 protein would increase T cell cytotoxicity compared to CAR-T without PDE4D2. This experiment was performed once in duplicate (rep1, rep2). A CHO cell line overexpressing PSMA (FOLH1) was used as the target cell. Four different effector cell to target cell (E / T) ratios were used, i.e., the ratio of CAR-T transfected cells to target CHO cells, 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 experiment. Increasing the E / T ratio increased the percentage of target cells killed by CAR-T transfected T cells. The two duplicate experiments and all E / T ratios were consistent, indicating that the addition of the PDE4D2 gene led to an increase in the percentage of CHO target cells killed. The maximum additional increase in the percentage of killed target cells was about 25% ( Figure 4 ).

[0189] Cytokine release experiments were performed using two vectors, CAR-T (Z011520CAR-T) and PDE4D2 CAR-T (Z060719PDE4D2 CAR-T), to measure IL2 (Interleukin 2) and IFNG (Interferon γ), assuming that the PDE4D2 protein increases cytokine release upon T cell activation compared to CAR-T without PDE4D2. This experiment was performed once in duplicate (rep1, rep2). A CHO cell line overexpressing PSMA (FOLH1) was used as the target cell. Four different effector-to-target cell (E / T) ratios, i.e., the ratio of CAR-T transfected cells to target CHO cells, ranging from 1:1 to 10:1, were used. For the two different CAR-T vectors, the same number of positively transfected T cells was used in the cytokine release experiment. Increasing the E / T ratio increased the cytokine release of CAR-T transfected T cells. The two replicate experiments and all E / T ratios were consistent in showing that addition of the PDE4D2 gene resulted in increased IL-2 release. The maximum additional percentage of IL-2 release was in the range of 25%. An increase in IFNG was only observed when the E / T ratios were 1:1 and 2.5:1. At higher E / T ratios, detection saturation may affect the measurement. This may be the reason why no additional IFNG release was observed for higher E / T ratios in the experiment using PDE4D2CAR-T. When the E / T ratios were 1:1 and 2.5:1, the maximum additional percentage of IFNG release was in the range of 20%( Figure 5 and Figure 6 ).

[0190] Example 2: Expression and activation of certain PDE genes after activation of T cells with anti-CD3 and anti-CD28 antibodies

[0191] RNA sequencing data (GSE160311; https: / / www.ncbi.nlm.nih.gov / geo ) were used to analyze the gene expression of members A, B, C, and D of the PDE4 gene family during stimulation of T cells with anti-CD3 and anti-CD28 antibodies for a period of time (0 hours, 6 hours, 24 hours, 72 hours). One-way ANOVA was used to analyze the expression differences at different time points for the TPM expression values of three replicate experiments. The expression of members A, B, and D of the PDE4D gene family was significantly upregulated at 24 hours of T cell stimulation. PDE4B seemed to return to baseline expression at 72 hours, while the expression of PDE4A and PDE4D continued to increase at 72 hours after stimulation. The expression of PDE4C was not detected in T cells. Box plots represent the data measured for each member of the PDE4D gene family at each time point in three replicate experiments. The p values of differential expression are shown( Figure 7 ).

Claims

1. 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 the T cells to obtain CAR-T cells; wherein the method further comprises the step of upregulating the enzymatic activity of phosphodiesterase (PDE) protein in the T cells before, during, or after introducing the CAR gene, wherein the PDE protein is a PDE2 or PDE4 protein, and wherein the expression of the PDE gene is increased by exogenous expression of the PDE gene in the T cells, thereby upregulating the expression of the PDE protein.

2. The method according to claim 1, wherein the PDE gene is introduced into the T cells together with the CAR gene, preferably wherein the CAR gene and the PDE gene are contained on the same expression vector.

3. The method according to claim 1 or 2, wherein the PDE protein is PDE2A, PDE4A, PDE4B, PDE4C, or PDE4D, or specific isomers selected from the following: PDE2A1, PDE2A2, PDE2A3, PDE2A4, PDE4A1, PDE4A2, PDE4A3, PDE4A4, PDE4A5, PDE4Ba, PDE4Bb, PDE4Bc, PDE4Bd, PDE4Be, PDE4Bf, PDE4Bg, PDE4C1, PDE4C2, PDE4C3, PDE4C4, PDE4C5, PDE4C6, PDE4D1, PDE4D1, PDE4D2, PDE4D3, PDE4D4, PDE4D5, PDE4D6, PDE4D7, PDE4D8, PDE4D9, PDE4D10, PDE4D11, PDE4D12, PDE4D14, PDE4D15, or PDE4D16, preferably wherein the PDE4 protein is selected from the isomers PDE4A4, PDE4B2, PDE4D1, PDE4D2, PDE4D5, or PDE4D7.

4. The method according to any one of the preceding claims, wherein the PDE protein is encoded by a nucleotide sequence having 80%, preferably 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or even 100% sequence identity with a sequence selected from: SEQ ID No: 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 PDE is a PDE4 protein having an amino acid sequence with 80%, preferably 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or even 100% sequence identity with any one of the following sequences: SEQ ID No: 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. The method according to any one of the preceding claims, wherein the CAR antigen is prostate-specific membrane antigen (PSMA).

6. A CAR-T cell having an up-regulated enzymatic activity of a PDE protein, wherein the CAR-T cell heterologously expresses a PDE gene selected from the PDe2 or PDE4 gene.

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

8. The CAR-T cell according to claim 6 or 7, wherein the exogenous expression is of a PDE protein encoded by a nucleotide sequence having 80%, preferably 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or even 100% sequence identity to a sequence selected from: SEQ ID No: 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 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 even 100% sequence identity to any one of the following sequences: SEQ ID No: 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 according to any one of claims 6 to 8, which is used as a drug.

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

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

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