Method for enhancing the therapeutic effect of CAR-T cells

Abstract

Provided herein is the use of LSD1 inhibitors, particularly bomedemstat, along with the use and production of immune effector cells, such as NK cells and T cells, engineered to express chimeric antigen receptors (CARs), to treat subjects having diseases associated with expression of tumor antigens.

Description

[Technical field]

[0001] The present invention relates to a method for enhancing the therapeutic effect of CAR-T cells.

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 365,075, filed May 20, 2022, the disclosure of which is incorporated by reference herein as if fully set forth herein.

[0003] Incorporation of sequence listing The sequence listing contained in the file entitled "IBIS0021-401-PC", created on May 15, 2023, which is 49.7 kilobytes as measured on a Microsoft Windows operating system, has been submitted electronically with this application and is incorporated herein by reference. [Background technology]

[0004] Adoptive transfer of T lymphocytes (T cells) engineered to recognize antigens on the surface of tumor cells can be a curative treatment. This is best demonstrated by the effectiveness of CD-19-targeted chimeric antigen receptor (CAR) T cells in treating B cell tumors. Many patients treated in this way and who achieve complete remission later relapse. Many hypotheses have been proposed to explain the eventual relapse, one of which is that the infused T cells differentiate and become exhausted as they repeatedly encounter tumor antigens in patients characterized as a loss of memory T cell phenotype.

[0005] Fully differentiated T cells are characterized by distinct epigenetic and gene expression profiles compared to naive or memory T cells. The protein PRDM1 (PR domain zinc finger protein 1; B lymphocyte-derived maturation protein 1; Blimp-1) is an epigenetic and transcriptional regulator that has been shown to mediate and be essential for the phenotypic transition from memory to differentiated T cells. Genetic knockout of PRDM1 in antitumor T cells enhances persistence of antitumor activity in in vitro and in vivo models of adoptive immunotherapy.

[0006] However, implementing PRDM1 knockout in CAR-T cells requires the approval of an entirely new drug and is not a solution to effective CAR-T therapies currently on the market or in development. Therefore, there remains a need for alternatives to PRDM1-deficient CAR-T cells that could avoid exhaustion by maintaining T cells with a memory phenotype.

[0007] Expression of the transcription factor (TF) Blimp-1 (PRDM1) is found primarily in activated T cells, and this TF is required for the production of interleukin (IL)-10 by a subset of forkhead box (Fox) p3+ regulatory T cells with an effector phenotype. Blimp-1 cooperates with other TFs to control the expression of IL-2, IL-21 and IL-10 in effector T lymphocytes. Blimp-1 is also required to induce T cell exclusion in the thymus and regulates T cell activation and proliferation in the periphery. Blimp-1 promotes T helper (Th)2 lineage commitment and limits the differentiation of Th1, Th17 and follicular helper T cells. In CD8+ T cells, Blimp-1 expression differs in heterogeneous populations at the stages of clonal expansion, differentiation, contraction and memory formation upon antigen encounter. Blimp-1 controls the formation and function of effector and memory CD8+ T cells as well as their exhaustion.

[0008] Blimp1 (PRDM1) protein is recruited to chromatin lysine-specific demethylase 1 (LSD1), which induces epigenetic changes in histone and DNA methylation status, altering the transcriptional profile of lymphocytes. Genetic knockdown of Blimp1 promotes CAR-T target cell killing and inflammatory cytokine secretion, thereby enhancing the clinical efficacy of CAR-T therapy. Thus, inhibition of LSD1 by small molecule inhibitors represents an alternative to genetic knockdown of PRDM1.

[0009] Furthermore, GFI1 and GFI1b (growth factor independent 1 and 1b), zinc finger-containing proto-oncogene transcriptional regulatory proteins required for erythroid and megakaryocytic development and differentiation, form complexes with LSD1 and other proteins to repress multiple genes involved in multilineage blood cell development and control hematopoietic differentiation through the recruitment of histone deacetylases (HDACs). Thus, LSD1 inhibitors that inhibit the demethylase activity and also the ability of GFI1 and GFI1b to bind to LSD1 are useful for immune control of malignancies. LSD1 inhibitors that affect the ability of LSD1 and its protein complexes to bind to both the GFI1 and Blimp1 transcription factor families are expected to be superior to LSD1 inhibitors that bind to and inhibit only LSD1. Summary of the Invention

[0010] Detailed Description In this specification, a therapeutic composition comprising a population of immune effector cells prepared for adoptive transfer that bind to an antigen on the surface of a target cell; The present invention provides a method for treating a disease comprising administering a lysine-specific demethylase 1 (LSD1) inhibitor to a patient.

[0011] Also provided is a method of enhancing the memory phenotype of a population of immune effector cells prepared for adoptive transfer, comprising treating the immune effector cells with a lysine-specific demethylase 1 (LSD1) inhibitor.

[0012] Also provided is a method of inhibiting the transition of T cells prepared for adoptive transfer from a memory to a differentiated phenotype, comprising treating immune effector cells with a lysine-specific demethylase 1 (LSD1) inhibitor.

[0013] Also provided is a method of inhibiting or reversing exhaustion in a population of immune effector cells prepared for adoptive transfer, comprising treating the immune effector cells with a lysine-specific demethylase 1 (LSD1) inhibitor.

[0014] In some embodiments, the LSD1 inhibitor inhibits both the demethylase activity and the ability of Growth Factor Independent 1 and / or Growth Factor Independent 1b (GFI1 and / or GFI1b and Blimp1) to bind to LSD1. In more particular embodiments, the LSD1 inhibitor is bomedemstat.

[0015] In some embodiments, the immune effector cells comprise a chimeric antigen receptor (CAR), e.g., a CAR-T cell.

[0016] The CARs disclosed herein can have the following additional embodiments, which can be present in various combinations to form additional embodiments.

[0017] In some embodiments, the CAR has a signal / leader peptide that translocates the extracellular domain of the CAR through the immune effector cell membrane to the cell surface. In some embodiments, the signal / leader peptide is a CD8α signal / leader peptide. In some embodiments, the CD8α signal / leader peptide has the amino acid sequence of any of SEQ ID NOs: 1-5.

[0018] The CARs provided herein have an antigen-binding domain. In some embodiments, the antigen-binding domain is H Chains, peptide linkers and V L In some embodiments, the V H Chains, peptide linkers and V L Together, the chains constitute an antigen-binding domain specific for CD2. In some embodiments, the V H Chains, peptide linkers and V L Together the chains make up an antigen-binding domain specific for either CD3, CD7, FLT3, CS1 and CD33.

[0019] In some embodiments, V H Chain and V L The peptide linker between the chains has the amino acid sequence GGGGS (1-4) has.

[0020] In some embodiments, V H Chain and V L The strand pairs are SEQ ID NO: 22 and SEQ ID NO: 23, SEQ ID NO:24 and SEQ ID NO:25, SEQ ID NO:26 and SEQ ID NO:27, SEQ ID NO:28 and SEQ ID NO:29, SEQ ID NO: 30 and SEQ ID NO: 31, SEQ ID NO: 32 and SEQ ID NO: 33, SEQ ID NO: 34 and SEQ ID NO: 35, SEQ ID NO:36 and SEQ ID NO:37, SEQ ID NO: 38 and SEQ ID NO: 39, SEQ ID NO: 40 and SEQ ID NO: 41, SEQ ID NO: 42 and SEQ ID NO: 43, and SEQ ID NO: 44 and SEQ ID NO: 45, SEQ ID NO: 46 and SEQ ID NO: 47, and SEQ ID NO: 48 and SEQ ID NO: 49 or wherein the scFv comprises an amino acid sequence selected from SEQ ID NO:50 and SEQ ID NO:51.

[0021] In some embodiments, the CAR is a ligand that specifically binds to CD27, CD28, 4-1BB (CD137), OX40, OX40L, CD30, CD40, ICOS, lymphocyte function associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and the following: CD83, CD8, CD4, b2c, CD80, CD86, DAP10, DAP12, MyD88, BTNL3 and NKG2D, PD-1, ICOS, CDS, ICAM-1, GITR, BAFFR, HVEM(LIGHTR), SLAMF7, NKp80(KLRF1), CD160, CD19, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, ITGB7, TNFR2, TRANCE / R ANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55) , PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, NKp44, NKp30, NKp46, and NKG2D. In some embodiments, the CAR has at least one costimulatory domain selected from CD2, CD8a, CD8b, CD28, ICOS, CD27, OX40, 4-1BB, NKG2D, and CD4. In some embodiments, the CAR has at least one costimulatory domain selected from CD28 and 4-1BB. In some embodiments, the at least one costimulatory domain is 4-1BB. In some embodiments, the 4-1BB domain has the amino acid sequence of SEQ ID NO:9. In some embodiments, at least one costimulatory domain is CD28.In some embodiments, the CD28 domain has the amino acid sequence of SEQ ID NO: 10. In some embodiments, the CAR has one costimulatory domain. In some embodiments, the CAR has two costimulatory domains. In some embodiments, the CAR has multiple costimulatory domains.

[0022] In some embodiments, the signaling domain is a CD3 zeta (CD3ζ) domain. In some embodiments, the CD3ζ domain has the amino acid sequence of SEQ ID NO:11.

[0023] Also provided herein are chimeric antigen receptors (CARs; and transgenic T cell receptors, TCRs) and immune effector cells expressing them, comprising the polypeptides disclosed herein, e.g., in Tables 2, 3, 12, and 13. CARs are recombinant fusion proteins that include 1) an extracellular ligand binding domain, i.e., an antigen recognition domain, 2) a hinge domain, 3) a transmembrane domain, and 4) a cytoplasmic signaling domain, 5) and an optional co-stimulatory domain.

[0024] Methods for the design, delivery and expression of CARs and the production of clinical grade CAR-T cell populations are known in the art. CAR design is generally tailored for each cell type.

[0025] The extracellular ligand-binding domain of a chimeric antigen receptor recognizes and specifically binds to an antigen, typically a surface-expressed antigen on a malignant cell. An extracellular ligand-binding domain is said to specifically bind to an antigen if, for example, it binds to an antigen with an affinity constant or interaction affinity (K) of about 0.1 pM to about 10 μM, or about 0.1 pM to about 1 μM, or about 0.1 pM to about 100 nM. D) to the antigen. Methods for determining the affinity of an interaction are known in the art. An extracellular ligand-binding domain may also be said to specifically bind a first polymorphic variant of an antigen if it binds to it preferentially over a second polymorphic variant of the same antigen.

[0026] The extracellular ligand binding domain suitable for use in a CAR can be any antigen-binding polypeptide, a wide variety of which are known in the art. In some cases, the extracellular ligand binding domain is a single chain Fv (scFv). Other antibody-based recognition domains [cAb V H H (Camelidae antibody variable domain) and its humanized form, lgNAR V H (shark antibody variable domain) and its humanized form, sdAb V H (single domain antibody variable domains) and "camelized" antibody variable domains] are also suitable for use. In some cases, T cell receptor (TCR) based recognition domains, such as single chain TCRs (scTv, single chain two domain TCRs including VαVβ), are also suitable for use. In some embodiments, the extracellular ligand binding domain is constructed from a natural binding partner for the target antigen or a functional fragment thereof. For example, a CAR can be constructed using a portion of the APRIL protein that generally targets the ligand for B cell maturation antigen (BCMA) and transmembrane activator and interactor of CAML (TACI), which can effectively co-target both BCMA and TACI for the treatment of multiple myeloma.

[0027] The target antigen that CAR binds to through its extracellular ligand-binding domain (or antigen-binding domain) can be an antigen expressed on malignant myeloid (AML) cells, T cells, or other cells. Antigens expressed on malignant myeloid (AML) cells include CD33, FLT3, CD123, and CLL-1. Antigens expressed on T cells include CD2, CD3, CD4, CD5, CD7, TCRα (TRAC), TCRβ, CD70, and CD1a. Antigens expressed on malignant plasma cells include BCMA, CS1, CD38, CD79A, CD79B, CD138, and CD19. Antigens expressed on malignant B cells include CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD27, CD38, and CD45.

[0028] Typically, the extracellular ligand-binding domain is linked to the intracellular domain of the chimeric antigen receptor by a transmembrane (TM) domain. A peptide hinge links the extracellular ligand-binding domain to the transmembrane domain. The hinge is generally an optional oligo- or polypeptide that functions to link the transmembrane domain to the extracellular ligand-binding domain. In particular, hinges are used to provide greater flexibility and accessibility to the extracellular antigen-binding domain. The hinge may comprise up to 300 amino acids, or 10-100 amino acids, or 25-50 amino acids. The hinge may be derived from all or a portion of a naturally occurring molecule, such as CD28, 4-1BB (CD137), OX-40 (CD134), CD3zeta, T cell receptor alpha or beta chain, CD45, CD4, CD5, CD8, CD8alpha, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, ICOS, CD154, or all or a portion of an antibody constant region. Alternatively, the hinge may be a synthetic sequence that corresponds to a naturally occurring hinge sequence, or the hinge region may be a completely synthetic hinge sequence. In some embodiments, the hinge domain comprises a portion of human CD8alpha, FcγRIIIalpha receptor, or IgG1, to which it has at least 80%, 90%, 95%, 97% or 99% sequence identity. In some embodiments, the CD8alpha hinge comprises the amino acid sequence of SEQ ID NO:6.

[0029] The transmembrane domain spans the cell membrane and anchors the CAR to the T cell surface, connecting the extracellular ligand binding to the intracellular (cytoplasmic) signaling domain, influencing the expression of the CAR on the T cell surface.

[0030] The transmembrane domain may be of either natural or synthetic origin. If of natural origin, the domain may be derived from an optionally included membrane-bound or transmembrane protein. For example, the transmembrane region may be derived from (i.e., may include at least the transmembrane region of) the alpha, beta, or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8 (e.g., CD8 alpha, CD8 beta), CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, IL2R beta, IL2R gamma, IL7R alpha, ITGA1, VLA1, CD49 a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR and PAG / Cbp. In some embodiments, the transmembrane domain is a CD28 transmembrane domain comprising the amino acid sequence of SEQ ID NO: 7. Alternatively, the transmembrane domain can be synthetic and comprise primarily hydrophobic amino acid residues (e.g., leucine and valine). In some cases, triplets of phenylalanine, tryptophan and valine are found at each end of the synthetic transmembrane domain.In some embodiments, the transmembrane domain is derived from T cell surface glycoprotein CD8 alpha chain isoform 1 precursor (NP_001139345.1) or CD28. A short (e.g., 2-10 amino acids long) oligo- or polypeptide linker may form the link between the endoplasmic reticulum domain and the transmembrane domain of the CAR. In some embodiments, the CAR has multiple transmembrane domains, which can be repeats of the same transmembrane domain or can be different transmembrane domains.

[0031] After antigen recognition, the cytoplasmic signaling domain transmits a signal to immune effector cells to activate at least one of the normal effector functions of immune effector cells. For example, the effector function of T cells can be cytolytic activity or helper activity (including secretion of cytokines). Usually, the entire cytoplasmic signaling domain can be used, but in many cases, it is not necessary to use the entire chain. To the extent that a truncated portion of the cytoplasmic signaling domain is used, such a truncated portion can be used instead of the complete chain, as long as it transmits effector function.

[0032] Cytoplasmic signaling sequences that regulate the primary activation of the stimulatory TCR complex may contain signaling motifs known as immunoreceptor tyrosine-based activation motifs (ITAMs). Examples of cytoplasmic signaling sequences that contain ITAMs include those derived from CD8, CD3zeta, CD3delta, CD3gamma, CD3epsilon, CD32 (FcgammaRIIA), DAP10, DAP12, CD79a, CD79b, FcgammaRIgamma, FcgammaRIIIgamma, FcεRIβ (FCERIB) and FcεRIgamma (FCERIG).

[0033] First generation CARs typically have a cytoplasmic signaling domain from the CD3 chain, which is the primary transmitter of signals from the endogenous TCR. Second generation CARs add cytoplasmic signaling domains from various co-stimulatory protein receptors (e.g., CD28, 4-1BB, ICOS) to the CAR cytoplasmic signaling domain to confer additional signals to the T cell.

[0034] Costimulatory domains are derived from intracellular signaling domains of costimulatory proteins that enhance cytokine production, proliferation, cytotoxicity and / or persistence in vivo. Preclinical studies have shown that second generation CAR designs improve the antitumor activity of T cells. More recent third and later generation CARs combine multiple costimulatory domains to further enhance efficacy. T cells implanted with these CARs have shown improved proliferation, activation, persistence and tumor eradication efficiency, independent of costimulatory receptor / ligand interactions.

[0035] For example, the cytoplasmic signaling domain of the CAR can be designed to include a signaling domain (e.g., CD3ζ), alone or with other desired cytoplasmic domains that may be included as desired and useful in the context of the CAR of the present invention. For example, the cytoplasmic domain of the CAR can include a signaling domain (e.g., CD3ζ) chain portion and a costimulatory signaling region. A costimulatory signaling region refers to a portion of the CAR that includes the intracellular domain of a costimulatory molecule. Examples of such molecules include: CD27, CD28, 4-1BB (CD137), OX40, OX40L, CD30, CD40, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and ligands that specifically bind to the following: CD83, CD8, CD4, b2c, CD80, CD86, DAP10, DA P12, MyD88, BTNL3 and NKG2D, PD-1, ICOS, CDS, ICAM-1, GITR, BAFFR, HVEM(LIGHTR), SLAMF7, NKp80(KLRF1) , CD160, CD19, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD4 9f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, IT GB7, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, L y9(CD229), CD160(BY55), PSGL1, CD100(SEMA4D), CD69, SLAMF6(NTB-A, Ly108), SLAM(SLAMF1, CD150, IPO -3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, NKp44, NKp30, NKp46, and NKG2D.

[0036] In some embodiments, the cytoplasmic signaling domain is a CD3 zeta (CD3ζ) signaling domain. In some embodiments, the costimulatory domain comprises the cytoplasmic domain of CD28, 4-1BB, or a combination thereof. In some cases, the costimulatory signaling region contains one, two, three, or four cytoplasmic domains of one or more intracellular signaling and / or costimulatory molecules.

[0037] In some embodiments, the CAR expression vector also contains a selection marker, such as CD34 or truncated CD34 and a P2A sequence. CD34 or TrCD34 is translated as a separate protein from the CAR, allowing for the selection of cells containing the CAR by anti-CD34 antibodies or other binding substances attached to magnetic beads or solid surfaces. In some embodiments, the nucleic acid encoding the CAR may optionally encode a peptide or polypeptide linker and a CD34 domain or a truncated CD34 (TrCD34) domain. This peptide or polypeptide linker and the CD34 domain or the truncated CD34 (TrCD34) domain are translated as separate proteins and therefore are not part of the CAR polypeptide. The separately translated CD34 domain or TrCD34 domain can be utilized for the selection of CAR-containing cells, for example, by using anti-CD34 antibodies or other CD34 binding substances attached to a surface or magnetic beads. In some embodiments, the CD34 domain has the amino acid sequence of SEQ ID NO: 17. In some embodiments, the TrCD34 domain has the amino acid sequence of SEQ ID NO: 18. In some embodiments, the polypeptide linker is P2A. In some embodiments, the P2A linker has the amino acid sequence of SEQ ID NO: 12. In some embodiments, the P2A linker and the CD34 domain are adjacent and have the amino acid sequence of SEQ ID NO: 19. In some embodiments, the P2A linker and the TrCD34 domain are adjacent and have the amino acid sequence of SEQ ID NO: 20.

[0038] Enumeration of embodiments Thus, although other embodiments may be found throughout this disclosure, the following embodiments are presented herein.

[0039] Embodiment 1. A method of treating a disease comprising administering a therapeutic composition comprising a population of immune effector cells prepared for adoptive transfer that bind to an antigen on the surface of a target cell, and a lysine-specific demethylase 1 (LSD1) inhibitor.

[0040] Embodiment 2. The method of any of embodiments 1, wherein the LSD1 inhibitor is administered simultaneously with a therapeutic composition comprising a population of immune effector cells prepared for adoptive transfer.

[0041] Embodiment 3. The method of embodiment 1, wherein the LSD1 inhibitor is administered after a therapeutic composition comprising a population of immune effector cells prepared for adoptive transfer.

[0042] Embodiment 4. The method of embodiment 1, wherein the LSD1 inhibitor is administered prior to the therapeutic composition comprising the population of immune effector cells prepared for adoptive transfer.

[0043] Embodiment 5. The method of embodiment 1, wherein the LSD1 inhibitor is administered after exhaustion of the adoptively transferred immune cells is observed.

[0044] Embodiment 6. The method of any one of embodiments 1 to 5, wherein the disease is cancer and the target cells are cancer cells.

[0045] Embodiment 7. A method of enhancing the memory phenotype of a population of immune effector cells prepared for adoptive transfer, comprising treating the immune effector cells with a lysine-specific demethylase 1 (LSD1) inhibitor.

[0046] Embodiment 8. A method of inhibiting the transition of T cells prepared for adoptive transfer from a memory to a differentiated phenotype comprising treating immune effector cells with a lysine-specific demethylase 1 (LSD1) inhibitor.

[0047] Embodiment 9. A method of suppressing or reversing exhaustion in a population of immune effector cells prepared for adoptive transfer comprising treating the immune effector cells with a lysine-specific demethylase 1 (LSD1) inhibitor.

[0048] Embodiment 10. The method of any one of embodiments 7 to 9, wherein the LSD1 inhibitor is administered after exhaustion of the adoptively transferred immune cells is observed.

[0049] Embodiment 11. The method of any one of embodiments 1 to 10, wherein the LSD1 inhibitor inhibits both the demethylase activity and the ability of Growth Factor Independent 1 and / or Growth Factor Independent 1b (GFI1 and / or GFI1b and Blimp!) to bind to LSD1.

[0050] Embodiment 12. The method of any one of embodiments 1-1, wherein the LSD1 inhibitor is a compound of any of formulas I-VI disclosed herein.

[0051] Embodiment 13 The method of embodiment 12, wherein the LSD1 inhibitor is bomedemstat.

[0052] Embodiment 14. The method of embodiment 13, wherein vomedemstat is administered once daily in an amount titrated to correspond to a platelet count of 50-150 k / μL.

[0053] Embodiment 15. The method of embodiment 12, wherein the LSD1 inhibitor is selected from bomedemstat (IMG-7289), iadademstat (ORY-1001), vafidemstat (ORY-2001), pulrodemstat (CC-90011), seclidemstat (SP-2577), INCB059872, TAS1440, SYHA1807, RO7051790, GSK2879552, MC2580 and DDP-38003.

[0054] Embodiment 16. The method of embodiment 15, wherein the LSD1 inhibitor is selected from vomedemstat (IMG-7289), iademstat (ORY-1001) and plerodemstat (CC-90011).

[0055] Embodiment 17. The method of any one of embodiments 1 to 16, wherein the immune effector cells are selected from T cells and natural killer cells.

[0056] Embodiment 18. The method of embodiment 17, wherein the immune effector cells express at least one chimeric antigen receptor (CAR).

[0057] Embodiment 19 The method of embodiment 18, wherein the immune effector cells are NK cells.

[0058] Embodiment 20 The method of embodiment 18, wherein the immune effector cell is a T cell.

[0059] Embodiment 21 The method of embodiment 19, wherein the immune effector cells are CAR-T cells.

[0060] Embodiment 22. The CAR comprises: an optional CD8a leader polypeptide, An antigen-binding domain, Hinge, Transmembrane domain, at least one costimulatory domain, and Signaling domains 21. The method of embodiment 18 or 20, comprising:

[0061] Embodiment 23. The CAR comprises: an extracellular domain including an optional CD8a leader polypeptide, an antigen binding domain and a hinge; A transmembrane domain, and An intracellular domain comprising at least one costimulatory domain and a signaling domain 21. The method of embodiment 18 or 20, comprising:

[0062] Embodiment 24 The method of any one of embodiments 22 or 23, wherein the transmembrane domain is selected from the CD28 transmembrane domain and the CD8 transmembrane domain.

[0063] Embodiment 25. The method of any one of embodiments 22 or 23, wherein the intracellular domain is selected from a CD28 intracellular signaling domain, an OX40L intracellular signaling domain, and a CD3-zeta (CD3-ζ) signaling domain.

[0064] Embodiment 26 The method of any one of embodiments 18 to 23, wherein the CAR comprises elements from the proximal N-terminus to the distal C-terminus.

[0065] Embodiment 27. The method of any one of embodiments 18 to 23, wherein the CAR specifically binds to at least one antigen expressed on malignant T cells.

[0066] Embodiment 28. The method of embodiment 27, wherein the antigen expressed on the malignant T cell is selected from one or more of CD2, CD3, CD4, CD5, CD7, TRAC, CD70, CD1a and TCRβ.

[0067] Embodiment 29. The method of any one of embodiments 18 to 28, wherein the CAR specifically binds to at least one antigen expressed on malignant plasma cells.

[0068] Embodiment 30. The method of embodiment 29, wherein the antigen expressed on malignant plasma cells is selected from one or more of BCMA, CS1, CD38, CD79A, CD79B, CD138 and CD19.

[0069] Embodiment 31 The method of any one of embodiments 18 to 28, wherein the CAR specifically binds to at least one antigen expressed on malignant B cells.

[0070] Embodiment 32. The method of embodiment 31, wherein the antigen expressed on malignant B cells is selected from one or more of CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD27, CD38 and CD45.

[0071] Embodiment 33 The method of embodiment 32, wherein the antigen expressed on malignant B cells is selected from one or more of CD19 and CD20.

[0072] Embodiment 34 The method of embodiment 33, wherein the antigen expressed on malignant B cells is CD19.

[0073] Embodiment 35. The antigen-binding domain is V H Chains, peptide linkers and V L The method of any one of embodiments 1 to 34, comprising a fusion protein chain (scFv).

[0074] Embodiment 36. The scFv comprises: SEQ ID NO: 22 and SEQ ID NO: 23, SEQ ID NO:24 and SEQ ID NO:25, SEQ ID NO:26 and SEQ ID NO:27, SEQ ID NO:28 and SEQ ID NO:29, SEQ ID NO: 30 and SEQ ID NO: 31, SEQ ID NO: 32 and SEQ ID NO: 33, SEQ ID NO: 34 and SEQ ID NO: 35, SEQ ID NO:36 and SEQ ID NO:37, SEQ ID NO: 38 and SEQ ID NO: 39, SEQ ID NO: 40 and SEQ ID NO: 41, SEQ ID NO: 42 and SEQ ID NO: 43, and SEQ ID NO: 44 and SEQ ID NO: 45, SEQ ID NO: 46 and SEQ ID NO: 47, and SEQ ID NO: 48 and SEQ ID NO: 49 V each containing one of the amino acid sequences H Chain and V L The method of embodiment 35, wherein the pair of chains or the scFv comprises an amino acid sequence selected from SEQ ID NO:50 and SEQ ID NO:51.

[0075] Embodiment 37.V H Chain and V L The peptide linker between the chains has the amino acid sequence GGGGS (1-4) The method of embodiment 35 or 36, comprising:

[0076] Embodiment 38 The method of any one of embodiments 1 to 37, wherein the CAR has a costimulatory domain.

[0077] Embodiment 39 The method of embodiment 38, wherein the CAR has multiple costimulatory domains.

[0078] Embodiment 40 The method of embodiment 39, wherein the CAR has two costimulatory domains.

[0079] Embodiment 41 The method of embodiment 38, wherein the costimulatory domain is selected from 4-1BB, CD28, OX-40 and NKG2D.

[0080] Embodiment 42. The method of any one of embodiments 1 to 41, wherein the CAR-T cell is defective in a subunit of the T cell receptor complex and / or is defective in at least one antigen to which one or more CARs specifically bind.

[0081] Embodiment 43 The method of embodiment 42, wherein the subunits of the T cell receptor complex are selected from one or more of the extracellular domains of TCR alpha, TCR beta, TCR delta, TCR gamma, CD3 epsilon, CD3 gamma and CD3 delta, and CD3 zeta.

[0082] Embodiment 44. The method of any one of embodiments 1 to 43, wherein the method is carried out in a human subject having cancer.

[0083] Embodiment 45. The method of any one of embodiments 1 to 44, wherein the cancer is a hematological malignancy.

[0084] Embodiment 46 The method of embodiment 45, wherein the hematological malignancy is a myeloproliferative neoplasm.

[0085] Embodiment 47. The method of embodiment 46, wherein the myeloproliferative neoplasm is a myelofibrosis selected from primary myelofibrosis (PMF) and post-PV / ET myelofibrosis (MF).

[0086] Embodiment 48. The method of embodiment 47, wherein the myeloproliferative neoplasm is post-PV / ET myelofibrosis (MF).

[0087] Embodiment 49 The method of embodiment 46, wherein the myeloproliferative neoplasm is polycythemia vera.

[0088] Embodiment 50 The method of embodiment 46, wherein the myeloproliferative neoplasm is essential thrombocythemia.

[0089] Embodiment 51. The method of embodiment 45, wherein the hematological malignancy is acute myeloid leukemia (AML).

[0090] Embodiment 52. The method of embodiment 45, wherein the hematological malignancy is chronic myeloid leukemia (CML).

[0091] Embodiment 53 The method of embodiment 45, wherein the hematological malignancy is multiple myeloma.

[0092] Embodiment 54 The method of embodiment 45, wherein the hematological malignancy is a T-cell malignancy.

[0093] In embodiment 55, the T-cell malignancy is T-cell acute lymphoblastic leukemia / lymphoma (T-ALL), human T-cell leukemia virus type 1 positive (HTLV-1+) adult T-cell leukemia / lymphoma (ATL), T-cell prolymphocytic leukemia (T-PLL), adult T-cell lymphoma / leukemia (HTLV-1 associated), aggressive NK-cell leukemia, anaplastic large cell lymphoma (ALCL), ALK positive, anaplastic large cell lymphoma (ALCL), ALK negative, angioimmunoblastic T-cell lymphoma (AITL), breast implant associated anaplastic large cell lymphoma, chronic lymphoproliferative disorder of NK cells, extranodal NK / T-cell lymphoma, nasal type, enteropathic type T-cell lymphoma, follicular T-cell lymphoma, hepatosplenic T-cell lymphoma, 55. The method of embodiment 54, wherein the tumor is selected from: primary cutaneous gamma delta T-cell lymphoma, indolent T-cell lymphoproliferative disorder of the gastrointestinal tract, monomorphic epitheliotropic intestinal T-cell lymphoma, mycosis fungoides, nodal peripheral T-cell lymphoma with TFH phenotype, peripheral T-cell lymphoma (PTCL), NOS, primary cutaneous gamma delta T-cell lymphoma, primary cutaneous CD8+ aggressive epidermotropic cytotoxic T-cell lymphoma, primary cutaneous acroal CD8+ T-cell lymphoma, primary cutaneous CD4+ small / medium T-cell lymphoproliferative disorder [primary cutaneous anaplastic large cell lymphoma (C-ALCL), lymphoid papulosis], Sezary syndrome, subcutaneous panniculitis-like T-cell lymphoma, systemic EBV+ T-cell lymphoma of childhood, and T-cell large granular lymphocytic leukemia (LGL).

[0094] Embodiment 56 The method of embodiment 55, wherein the T-cell malignancy is T-cell acute lymphoblastic leukemia (T-ALL).

[0095] Embodiment 57 The method of embodiment 54, wherein the T-cell malignancy is non-Hodgkin's lymphoma.

[0096] Embodiment 58. The method of embodiment 54, wherein the T-cell malignancy is T-cell chronic lymphocytic leukemia (T-CLL).

[0097] Embodiment 59 The method of embodiment 45, wherein the hematological malignancy is a B-cell malignancy.

[0098] Embodiment 60. The method of embodiment 59, wherein the B-cell malignancy is selected from diffuse large B-cell lymphoma (DLBCL), chronic lymphocytic leukemia (CLL) / small lymphocytic lymphoma (SLL) and B-cell precursor acute lymphoblastic leukemia (ALL).

[0099] Embodiment 61 The method of embodiment 45, wherein the hematological malignancy is a plasma cell malignancy.

[0100] Embodiment 62. The method of embodiment 61, wherein the plasma cell malignancy is selected from lymphoplasmacytic lymphoma, plasmacytoma, and multiple myeloma.

[0101] Also provided are corresponding uses of the recited elements (e.g., immune effector cells and lysine-specific demethylase 1 (LSD1) inhibitors) in each of the above embodiments in the treatment of cancer, in the manufacture of a medicament for treating cancer, and in kits containing instructions for treating cancer.

[0102] CAR design and construction Chimeric antigen receptor (CAR) constructs encoding chimeric receptors can be produced in the usual way. For the most part, native sequences are used, so the native genes are isolated and manipulated as necessary to allow for the appropriate binding of the various components (e.g., when using type II receptors, the immune signaling receptor component may need to be inverted). Thus, nucleic acid sequences encoding the N-terminal and C-terminal proteins of the chimeric receptor can be isolated by using polymerase chain reaction (PCR) using appropriate primers that result in the deletion of undesired parts of the gene. Alternatively, restriction digests of cloned genes can be used to generate chimeric constructs. In either case, sequences can be selected to obtain restriction sites that are blunt-ended or have complementary overlaps.

[0103] The various manipulations to produce the chimeric constructs can be performed in vitro, and in certain embodiments, standard transformation or transfection methods are used to introduce the chimeric constructs into vectors for cloning and expression in a suitable host. Thus, after each manipulation, the construct resulting from the ligation of DNA sequences is cloned, the vector is isolated, and the sequence is screened to confirm that it encodes the desired chimeric receptor. The sequence can be screened by restriction analysis, sequencing, etc.

[0104] It is envisioned that the chimeric construct can be introduced into immune effector cells as naked DNA or in a suitable vector. Methods for stably transfecting immune effector cells by electroporation using naked DNA are known in the art. See, for example, U.S. Patent No. 6,410,319. Naked DNA generally refers to the DNA encoding the chimeric receptor of the present invention contained in a plasmid expression vector in the proper orientation for expression. Advantageously, the use of naked DNA shortens the time required to generate immune effector cells expressing the chimeric receptor of the present invention.

[0105] Alternatively, a viral vector (e.g., a retroviral vector, an adenoviral vector, an adeno-associated viral vector, or a lentiviral vector) can be used to introduce the chimeric construct into immune cells, such as T cells. A suitable vector for use according to the method of the present invention is one that does not replicate in the immune effector cells of a subject. A number of viral-based vectors are known that maintain the copy number of the virus in the cell low enough to maintain the viability of the cell. Exemplary vectors include the pFB-neo vector (STRATAGENE™), as well as vectors based on HIV, SV40, EBV, HSV, or BPV. Once it is established that the transfected or transduced immune effector cells can express the chimeric receptor as a surface membrane protein with the desired regulation and at the desired level, it can be determined whether the chimeric receptor is functional in the host cell to provide the desired signal induction (e.g., production of Rantes, Mip1-alpha, GM-CSF upon stimulation with the appropriate ligand).

[0106] The engineered CAR can be introduced into the CAR-containing immune effector cells using a retrovirus that efficiently and stably integrates the nucleic acid sequence encoding the chimeric antigen receptor into the target cell genome. Other methods known in the art include lentiviral transduction, transposon-based systems, direct RNA transfection, and CRISPR / Cas systems [e.g., Cas3, Cas4, Cas5, Cas5e (or CasD), Cash, Cas6e, Cas6f, Cas7, Cas8a1, Cas8a2, Cas8b, Cas8c, Cas9, Cas10, Cas10d, CasF, CasG, CasH, Csy1, Csy2, Csy3, Cse1 (or CasA), Cse2 (or CasB), Cse3 (or C). Cas proteins such as, but not limited to, Type I, II or III systems using appropriate Cas proteins such as, for example, asE), Cse4 (or CasC), Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csz1, Csx15, Csf1, Csf2, Csf3, Csf4 and Cu1966. Zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) may also be used. See, e.g., Shearer RF and Saunders DN, “Experimental design for stable genetic manipulation in mammalian cell lines: lentivirus and alternatives,” Genes Cells 2015 January;20(1):1-10.

[0107] Editing strategies such as CRISPR-induced base editors (CBEs) allow efficient genome editing with minimal DNA breaks. Applications of CBEs in the case of engineered T cells include silencing TCR and HLA expression, as well as target antigen expression to prevent fratricide. Editing is achieved by coupling of inactivated Cas9 with nucleotide deaminase, which is guided by a target-specific RNA sequence to convert C to T. Conversion efficiency can be enhanced by including a uracil DNA glycosylase inhibitor. CBEs can result in gene silencing by introducing premature stop codons or splice site disruptions that result in non-sense mediated decay (NMD) of mRNA transcripts. Multiple iterations of CBEs are known in the art. General base editors include, but are not limited to, BE3, VQR-BE3, EQR-BE3, VRER-BE3, SaBE3, SaKKH-BE3, SaBE3-Gam, TAM, CRISPR X, ABE, and YEE-BE3.

[0108] The amino acid sequences of selected components that may be used to construct a CAR are disclosed below in Tables 1 and 2. As will be appreciated by one of skill in the art, in some embodiments, the following sequences may be modified by amino acid extension, truncation, and substitution. Also, in some embodiments, alternative variants are not listed here (e.g., additional P2A sequences and their function in inducing polypeptide "cleavage" by ribosome skipping are known in the art).

[0109] [Table 1] TIFF2025517410000002.tif138161

[0110] Cell-specific mutations The CAR components and construction methods disclosed above are suitable for use in T cells and other immune effector cells, but are not exhaustive. Certain modifications are useful in subsets of cells and are known in the art.

[0111] For example, in NK cells, the TM domain may be selected or modified from: NKG2D, FcγRIIIa, NKp44, NKp30, NKp46, actKIR, NKG2C or CD8α, the α, β or ζ chains of the T cell receptor, CD28, CD27, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64. , CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA1(CD11a, CD18), ICOS(CD278), 4-1BB(C D137), GITR, CD40, BAFFR, HVEM(LIGHTR), SLAMF7, NKp80(KLRF1), CD160, CD19, IL2Rβ, IL2Rγ, IL7Rα, ITG A1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA -1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1(CD226), SLAMF4( CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTBA, Ly108), SLAM (SLAMF1, CD150, IPO3), BLAME (SLAMF8), SELPLG (CD162), LTBR or PAG / Cbp. NK cells also express a number of transmembrane adaptors that are triggered by binding to activating receptors, resulting in NK cell-specific signal enhancement. For example, the TM adaptors can be selected or modified from FceR1γ (ITAMx1), CD3ζ (ITAMx3), DAP12 (ITAMx1) or DAP10 (YxxM / YINM).In some embodiments, the TM domain and adaptor may be paired, for example, as follows: NKG2D and DAP10, FcγRIIIa and CD3ζ or FceR1γ, NKp44 and DAP12, NKp30 and CD3ζ or FceR1γ, NKp46 and CD3ζ or FceR1γ, actKIR and DAP12, and NKG2C and DAP12.

[0112] In some embodiments, in NK cells, the hinge domain may be selected or modified from, for example, NKG2, TMα, or CD8.

[0113] In some embodiments, in NK cells, the intracellular signaling and / or costimulatory domain may include one or more of the following: CD137 / 41BB (TRAF, NFκB), DNAM-1 (Y motif), NKp80 (Y motif), 2B4 (SLAMF)::ITSM, CRACC (CS1 / SLAMF7)::ITSM, CD2 (Y motif, MAPK / Erk), CD27 (TRAF, NFκB), PD1, PD-L1, CTLA-4, TIM-3, CEACAM (e.g., CEACAM-1, CEACAM-3 and / or CEACAM-5), LAG-3, VISTA, BTLA, T IGIT, LAIR1, CD160 or TGFbeta; one or more integrins (e.g., multiple integrins); cytokine receptors associated with survival, survival or metabolism, such as IL-2 / 15R::Jak1 / 3, STAT3 / 5, PI3K / mTOR and MAPK / ERK; cytokine receptors associated with activation, such as IL-18R::NFkB; cytokine receptors associated with IFN-gamma production, such as IL-12R::STAT4; cytokine receptors associated with cytotoxicity or survival, such as IL-21R::Jak3 / Tyk2 or STAT3; and TM adaptors (as disclosed above). In some embodiments, the NK cell CAR comprises three signaling domains, a TM domain, and an optional TM adaptor.

[0114] Table 2 below lists VAGs targeting the listed antigens. H and V L Domain, or V H and V L Exemplary sequences of the scFvs themselves containing the domains are disclosed. These sequences can be incorporated into CARs as disclosed herein or with elements from Table 1. Additional VFv sequences against various targets can be used. H and V L Domains as well as scFvs are known in the art.

[0115] [Table 2] TIFF2025517410000004.tif195159

[0116] Additional cancer antigens and scFvs that bind thereto are known in the art and can be implemented in the CARs, CAR-expressing immune effector cells and methods comprising same disclosed herein.

[0117] CARs constructed according to methods known in the art and disclosed herein and expressed in immune effector cells may be in the following form: |―[(optional leader)(V L )(Linker)(V H )(hinge)(TMD)(costimulatory domain) n (Effector)]―| where the leader sequence, V L, linker, hinge, transmembrane domain, costimulatory domain, optional second and third costimulatory domains (i.e., n is 1, 2 or 3), effector domain and optional P2a sequence and tag are disclosed in Tables 1 and 2 above or known in the art. When an example includes P2A-(Tr)CD34, this reflects that the DNA sequence encoding them is present in the nucleic acid sequence encoding CAR. However, when the mRNA encoding CAR is translated, the CAR portion distal to P2A-TrCD34 and P2A-TrCD34 (or P2A-CD34) are translated as separate polypeptides. Therefore, the amino acid sequence of CAR does not include P2A-(Tr)CD34.

[0118] Similar CAR variations are also possible. For example, the CAR may be in the following form: |―[(optional leader)(V H )(Linker)(V L )(hinge)(TMD)(costimulatory domain) n (Effector)]―| wherein the sequences are disclosed in Tables 1 and 2 above. Thus, V H and V L The present invention also provides a CAR that is identical to the above, except that the positions of the sequences are reversed.

[0119] Also provided herein is an immune effector cell containing one or more CARs according to the first, second and / or third aspect. In some embodiments, the CAR-containing immune effector cell is a chimeric antigen receptor T cell (CAR-T cell) or a chimeric antigen receptor natural killer cell (CAR-NK cell).

[0120] In some embodiments, the CAR-containing immune effector cells have the additional characteristic of reducing or eliminating fratricide (fratricide), alloreactivity and / or graft-versus-host reaction. In some embodiments, the CAR-containing immune effector cells further comprise a suicide gene. In some embodiments, the CAR-containing immune effector cells lack at least one or more antigens to which one or more CARs specifically bind. In some embodiments, where the CAR-containing immune effector cells are CAR-T cells, T cell receptor-mediated signaling is blocked in the CAR-T cells. In some embodiments, the CAR-T cells lack a subunit of the T cell receptor complex. In some embodiments, the subunit of the T cell receptor complex is selected from one or more of the extracellular domains of TCRα, TCRβ, TCRδ, TCRγ, CD3ε, CD3γ and CD3δ, and CD3ζ.

[0121] Some of these characteristics of CAR-containing immune effector cells are introduced by CRISPR / Cas9-mediated editing to delete genes that code for proteins that are desired to be deleted. For example, if the CAR-containing immune effector cells target CD2, it may be desirable to delete endogenous CD2 and TRAC from the CAR-containing immune effector cells to reduce or prevent fratricide. In this case, the deletion of CD2 and TRAC genes can be confirmed by PCR (sometimes referred to as deep sequencing) on ​​gene-edited cells. Examples of guide RNAs used for gene editing and primers for confirming such editing are known in the art.

[0122] Immune effector cells In some embodiments, the CAR-containing immune effector cells specifically bind to at least one cancer-associated antigen expressed on a cancer cell. In some embodiments, the cancer cell is a malignant T cell. In some embodiments, the antigen expressed on the malignant T cell is selected from one or more of CD2, CD3, CD4, CD5, CD7, TCRα (TRAC) and TCRβ. In some embodiments, the cancer cell is a malignant plasma cell. In some embodiments, the antigen expressed on the malignant plasma cell is selected from one or more of BCMA, CS1, CD38, CD79A, CD79B, CD138 and CD19. In some embodiments, the cancer cell is a malignant B cell. In some embodiments, the antigen expressed on malignant B cells is selected from one or more of CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD27, CD38 and CD45, and the antigen expressed on malignant B cells is selected from one or more of CD19 and CD20.

[0123] Immune effector cells as disclosed herein can include T cells, NK cells and other cells, such as macrophages and their subtypes.

[0124] Any of these immune effector cells can be transduced with CAR using techniques known in the art. The resulting CAR-containing immune effector cells can be used in immunotherapy of diseases such as cancer by adoptive cell transfer (ACT) to subjects in need. CAR-containing immune effector cells include CAR-T cells, CAR-NK cells and CAR macrophages.

[0125] Immune effector cells for use in ACT can be autologous or allogeneic cells. In some embodiments, the use of allogeneic cells allows for deliberate polymorphic mismatching between the donor and recipient, which provides certain advantages, as described below.

[0126] T cells T cells are immune cells that express the T cell receptor (TCR) on their surface. Effector T cells include cytotoxic (CD8+) T cells, helper (CD4+) T cells, virus-specific cytotoxic T cells, memory T cells, and gamma delta (γδ) T cells.

[0127] T cells can be primary T cells or can be derived from progenitor cells. T cells can be derived from various sources, including peripheral or umbilical cord blood cells, stem cells or induced pluripotent stem cells (iPSCs). Methods for enriching / separating, differentiating and otherwise generating T cells are known in the art.

[0128] NK cells Natural killer (NK) cells are traditionally considered to be innate immune effector lymphocytes that mediate host defense against pathogens and antitumor immune responses by targeting and eliminating abnormal or stressed cells through various receptors on their surface and by antibody-dependent cellular cytotoxicity (ADCC) through the integration of signals from activating and inhibitory receptors.

[0129] Natural killer (NK) cells are an alternative to T cells for allogeneic cellular immunotherapy because they can be safely administered without significant toxicity, do not cause graft-versus-host disease (GvHD), do not require HLA matching or gene editing, naturally recognize and eliminate malignant cells, and are amenable to cell engineering. This means that allogeneic CAR-NK cells can be manufactured in large quantities, cryopreserved, and transported to patients on demand without the need for patient-specific engineering processes that can be lengthy and expensive. NK cells also have the potential to activate other components of the immune system and induce long-term anti-cancer immune memory.

[0130] On the other hand, autologous CAR-T cell therapy requires the manipulation of the patient's own cells, which can take a long time and may be impossible for some patients who do not have enough suitable T cells.Even if autologous CAR-T cells are successfully produced and administered, patients typically receive only one dose due to the risk of acute side effects such as ICANS and CRS.This limits the possibility of individualized administration and repeated administration based on individual patient response.The cost of autologous CAR-T therapy may also be prohibitive for many patients.

[0131] In contrast, allogeneic NK cell therapy offers several advantages, including the possibility of repeated dosing, response-based dosing, and individualized dosing based on individual patient responses. NK cells are also well tolerated without the risk of graft-versus-host disease, neurotoxicity, or cytokine release syndrome associated with other cell-based therapies. Cord blood units, which have favorable properties for enhanced clinical activity, can be selected as a source of NK cells. Furthermore, NK cells have the ability to induce multiclonal adaptive immune responses that can overcome innate immunity and lead to long-term anti-cancer immune memory.

[0132] The NK cells can be primary NK cells or can be derived from precursor cells. The NK cells can be derived from a variety of sources, including peripheral or umbilical cord blood cells, stem cells or induced pluripotent stem cells (iPSCs). Methods for enriching / separating, differentiating and otherwise generating T cells are known in the art.

[0133] NK cells express a number of transmembrane (TM) adaptors that signal activation, triggered through binding to activating receptors. This results in NK cell-specific signal enhancement by engineering the TM domain from the activating receptor to utilize endogenous adaptors. The TM adaptor can be any endogenous TM adaptor that can signal activation. In some embodiments, the TM adaptor can be selected from FceR1γ (ITAMx1), CD3ζ (ITAMx3), DAP12 (ITAMx1) or DAP10 (YxxM / YINM), NKG2D, FcγRIIIa, NKp44, NKp30, NKp46, actKIR, NKG2C, CD8α, and IL15Rb.

[0134] LSD1 inhibitors Numerous LSD1 inhibitors are known in the art. Examples of LSD1 inhibitor compounds that can be used in the methods disclosed herein include those shown and mentioned below. The LSD1 inhibitors of the following formulas I-VI, including vomedemstat, inhibit both the demethylase activity and the ability of GFI1 and GFI1b and Blimp1 to bind to LSD1.

[0135] In some embodiments, the LSD1 inhibitor has Formula I: [ka] [In the formula, Y is a bond, NR 4a ,O,C(O)NH,NHC(O),S,SO 2 and C.H. 2 Selected from; Z is a bond, NR 4b ,O,C(O)NH,NHC(O),S,SO 2 and C.H. 2 Selected from; m is an integer from 0 to 5; n is an integer from 0 to 3; R 1 and R 2are each independently selected from alkyl, aminoalkyl, alkylsulfonylalkyl, alkoxyalkyl, aryl, arylalkyl, cycloalkyl, cycloalkylalkyl, phenyl, biphenyl, heteroaryl, heteroarylalkyl, heterocycloalkyl, and heterocycloalkylalkyl; R 1 and R 2 together with the nitrogen to which they are attached form a nitrogen-containing heterocycloalkyl or heteroaryl ring which is composed of 0 to 3 R 6 optionally substituted with a group; R 3 is selected from alkylamino, cycloalkylamino, arylamino, heteroarylamino, heterocycloalkylamino, cycloalkyl, cycloalkylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, heterocycloalkyl, and heterocycloalkylalkyl, each of which may be selected from 0 to 3 R 6 optionally substituted with a group; R 4 , R 4a and R 4b is independently selected from hydrogen, alkyl, alkenyl, alkynyl, and cycloalkyl; R 5 is selected from aryl and heteroaryl, each of which is selected from 0 to 3 R 6 optionally substituted with a group; Each R 6 are independently hydrogen, halogen, alkyl, alkenyl, alkynyl, cycloalkyl, haloalkyl, haloalkoxy, aryl, aralkyl, heterocycloalkyl, heteroaryl, heteroarylalkyl, cyano, alkoxy, amino, alkylamino, dialkylamino, COR 7 , S.O. 2 R 7 , N.H.S.O. 2 R 7 , N.H.S.O. 2 NHR 7 , N.H.C.O.R. 7 ,NHCONHR 7 ,CONHR7 and CONR 7 R 8 selected from; and R 7 and R 8 is independently selected from hydrogen and lower alkyl; or R 7 and R 8 together form a nitrogen-containing heterocycloalkyl or heteroaryl ring, which is optionally substituted with lower alkyl; However, if desired, Y=CH 2 , R 4 =H, Z=R 4b In the case of m+n ≠ 3, or a salt thereof.

[0136] In some embodiments, the LSD1 inhibitor has Formula II: [ka] [In the formula, Y is a bond, NR 4a ,O,C(O)NH,NHC(O),S,SO 2 , CHOH and CH 2 Selected from; Z is a bond, NR 4b ,O,C(O)NH,NHC(O),S,SO 2 and C.H. 2 Selected from; m is selected from 0, 1, 2, 3, 4, 5; n is selected from 0, 1, 2, 3; R 1 and R 2 are each independently selected from alkyl, aminoalkyl, alkylsulfonylalkyl, alkoxyalkyl, aryl, arylalkyl, cycloalkyl, cycloalkylalkyl, phenyl, biphenyl, heteroaryl, heteroarylalkyl, heterocycloalkyl, and heterocycloalkylalkyl; R 1 and R 2together with the nitrogen to which they are attached form a nitrogen-containing heterocycloalkyl or heteroaryl ring which is composed of 0 to 3 R 6 optionally substituted with a group; R 4a and R 4b is independently selected from hydrogen, alkyl, alkenyl, alkynyl, and cycloalkyl; R 5 is selected from aryl and heteroaryl, each of which is selected from 0 to 3 R 6 optionally substituted with a group; R 6a is heteroaryl, cyano and S(O) 2 N(CH 3 ) 2 Selected from; Each R 6 are independently hydrogen, halogen, alkyl, alkylsulfonylaryl, alkenyl, alkynyl, cycloalkyl, haloalkyl, haloalkoxy, haloaryl, alkoxyaryl, aryl, aryloxy, aralkyl, heterocycloalkyl, heteroaryl, alkylheteroaryl, heteroarylalkyl, cyano, alkoxy, alkoxyaryl, amino, alkylamino, dialkylamino, oxo, COR 7 , S.O. 2 R 7 , N.H.S.O. 2 R 7 , N.H.S.O. 2 NHR 7 , N.H.C.O.R. 7 ,NHCONHR 7 ,CONHR 7 and CONR 7 R 8 selected from; and R 7 and R 8 is independently selected from hydrogen, aryl and lower alkyl; or R 7 and R 8 together form a nitrogen-containing heterocycloalkyl or heteroaryl ring, which is optionally substituted with lower alkyl; However, if desired, Y=CH 2 And Z=R 4b In the case of m+n ≠ 3, or a salt, polymorph or solvate thereof.

[0137] In some embodiments, the LSD1 inhibitor has formula IIIa or IIIb: [ka] [In the formula, Y is a bond, NR 4a ,O,C(O)NH,NHC(O),S,SO 2 and C.H. 2 Selected from; Z is a bond, NR 4b ,O,C(O)NH,NHC(O),S,SO 2 and C.H. 2 Selected from; m is an integer from 0 to 5; n is an integer from 0 to 3; R 1 and R 2 are each independently selected from alkyl, aminoalkyl, alkylsulfonylalkyl, alkoxyalkyl, aryl, arylalkyl, cycloalkyl, cycloalkylalkyl, phenyl, biphenyl, heteroaryl, heteroarylalkyl, heterocycloalkyl, and heterocycloalkylalkyl; R 1 and R 2 together with the nitrogen to which they are attached form a nitrogen-containing heterocycloalkyl or heteroaryl ring which is composed of 0 to 3 R 6 optionally substituted with a group; R 3 is selected from alkylamino, cycloalkylamino, arylamino, heteroarylamino, heterocycloalkylamino, cycloalkyl, cycloalkylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, heterocycloalkyl, and heterocycloalkylalkyl, each of which may be selected from 0 to 3 R 6optionally substituted with a group; R 4 , R 4a and R 4b is independently selected from hydrogen, alkyl, alkenyl, alkynyl, and cycloalkyl; R 5 is selected from aryl and heteroaryl, each of which is selected from 0 to 3 R 6 optionally substituted with a group; Each R 6 are independently hydrogen, halogen, alkyl, alkenyl, alkynyl, cycloalkyl, haloalkyl, haloalkoxy, aryl, aralkyl, heterocycloalkyl, heteroaryl, heteroarylalkyl, cyano, alkoxy, amino, alkylamino, dialkylamino, COR 7 , S.O. 2 R 7 , N.H.S.O. 2 R 7 , N.H.S.O. 2 NHR 7 , N.H.C.O.R. 7 ,NHCONHR 7 ,CONHR 7 and CONR 7 R 8 are independently selected from; R 7 and R 8 is independently selected from hydrogen and lower alkyl; or R 7 and R 8 together form a nitrogen-containing heterocycloalkyl or heteroaryl ring, which is optionally substituted with lower alkyl; where Y=CH 2 And Z=R 4b , m+n≠3] or a salt thereof.

[0138] In some embodiments, Z is NR 4b It is.

[0139] In some embodiments, R 4bis selected from methyl and hydrogen.

[0140] In some embodiments, alkyl, whether by itself or as a designated part of another acyclic substituent, is C 1 -C 8 It is an alkyl.

[0141] In some embodiments, R 3 is selected from aryl, arylalkyl, heteroaryl, and heteroarylalkyl, each of which is selected from 0 to 3 R 6 The group may be optionally substituted with a group.

[0142] In some embodiments, R 3 is selected from aryl and heteroaryl, each of which is selected from 0 to 3 R 6 The group may be optionally substituted with a group.

[0143] In some embodiments, m is an integer from 0 to 1; Y is NR 4a , O, S, SO 2 and C.H. 2 n is an integer from 1 to 3; and R 4a is selected from hydrogen and alkyl.

[0144] In some embodiments, m is 0; Y is CH 2 and n is an integer from 1 to 2.

[0145] In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3.

[0146] In some embodiments, R 3 is a 5-6 membered monocyclic or 8-12 membered bicyclic heteroaryl, where 1-5 ring members are heteroatoms selected from N, O and S, and is selected from 0-3 R 6 The group may be optionally substituted with a group.

[0147] In some embodiments, R 3 is a 5-6 membered monocyclic heteroaryl, where 1-4 ring members are heteroatoms selected from N, O and S, and is selected from 0-3 R 6 The group may be optionally substituted with a group.

[0148] In some embodiments, each R 6 is lower alkyl, halogen, lower alkoxy, OCF 3 and CF 3 is selected from.

[0149] In some embodiments, R 3 teeth, [ka] is selected from. In some embodiments, R 4 is hydrogen.

[0150] In some embodiments, R 4 is methyl.

[0151] In some embodiments, R 1 and R 2 However, the nitrogen-containing heterocycloalkyl or heteroaryl ring they form together with the nitrogen to which they are attached contains 3 to 8 atoms.

[0152] In some embodiments, R 1 and R 2 and together form a nitrogen-containing heterocycloalkyl, which is selected from 0 to 3 R 6 The group may be optionally substituted with a group.

[0153] In some embodiments, R 1 and R 2 However, the nitrogen-containing heterocycloalkyl they form together with the nitrogen to which they are attached is [ka] is selected from.

[0154] In some embodiments, R 1 and R 2 However, the nitrogen-containing heterocycloalkyl they form together with the nitrogen to which they are attached is [ka] is selected from.

[0155] In some embodiments, R 1 and R 2 However, the nitrogen-containing heterocycloalkyl they form together with the nitrogen to which they are attached is [ka] In some embodiments, R 1 and R 2 However, the nitrogen-containing heterocycloalkyl they form together with the nitrogen to which they are attached is [ka] In some embodiments, R 1 and R 2 However, the nitrogen-containing heterocycloalkyl they form together with the nitrogen to which they are attached is [ka] In some embodiments, R 1 and R 2 However, the nitrogen-containing heterocycloalkyl they form together with the nitrogen to which they are attached is [ka] In some embodiments, R 1 and R 2However, the nitrogen-containing heterocycloalkyl they form together with the nitrogen to which they are attached is [ka] It is.

[0156] In some embodiments, n is 2 or 3.

[0157] In some embodiments, R 1 and R 2 taken together with the nitrogen to which they are attached form a nitrogen-containing heteroaryl, which is 6 The group may be optionally substituted with a group.

[0158] In some embodiments, the nitrogen-containing heteroaryl is selected from pyrrole, imidazole, and pyrazole.

[0159] In some embodiments, R 5 is aryl, which is selected from 0 to 3 R 6 The group may be optionally substituted with a group.

[0160] In some embodiments, R 5 is phenyl, which is a group consisting of 0 to 3 R 6 The group may be optionally substituted with a group.

[0161] In some embodiments, n is 2 or 3.

[0162] In some embodiments, R 5 is heteroaryl, which is selected from 0 to 3 R 6 The group may be optionally substituted with a group.

[0163] In some embodiments, R 5 is a 5-6 membered monocyclic or 8-12 membered bicyclic heteroaryl, where 1-5 ring members are heteroatoms selected from N, O and S, and is selected from 0-3 R6 The group may be optionally substituted with a group.

[0164] In some embodiments, R 5 is a 5-6 membered monocyclic heteroaryl, where 1-5 ring members are heteroatoms selected from N, O and S, and is selected from 1 or 2 R 6 The group may be optionally substituted with a group.

[0165] In some embodiments, R 5 teeth, [ka] is selected from.

[0166] In some embodiments, n is 2 or 3.

[0167] In some embodiments, R 3 is 0 to 3 R 6 The aryl may be optionally substituted with a group.

[0168] In some embodiments, R 3 is selected from phenyl and biphenyl, each of which is selected from 0 to 3 R 6 The group may be optionally substituted with a group.

[0169] In some embodiments, m is an integer from 0 to 1; Y is NR 4a , O, S, SO 2 and C.H. 2 Selected from; n is an integer from 1 to 3; and R 4a is selected from hydrogen and alkyl.

[0170] In some embodiments, m is 0; Y is CH 2 and n is an integer from 1 to 3.

[0171] In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3.

[0172] In some embodiments, R 6 is lower alkyl, halogen, lower alkoxy, OCF 3 and CF 3 is selected from.

[0173] In some embodiments, R 4 is hydrogen.

[0174] In some embodiments, R 4 is methyl.

[0175] In some embodiments, n is 2 or 3.

[0176] In some embodiments, R 1 and R 2 However, the nitrogen-containing heterocycloalkyl or heteroaryl ring they form together with the nitrogen to which they are attached contains from 3 to 8 atoms.

[0177] In some embodiments, R 1 and R 2 and together form a nitrogen-containing heterocycloalkyl, which is selected from 0 to 3 R 6 The group may be optionally substituted with a group.

[0178] In some embodiments, R 1 and R 2 However, the nitrogen-containing heterocycloalkyl they form together with the nitrogen to which they are attached is [ka] is selected from.

[0179] In some embodiments, R 1 and R 2 However, the nitrogen-containing heterocycloalkyl they form together with the nitrogen to which they are attached is [ka] is selected from.

[0180] In some embodiments, R 1 and R 2 However, the nitrogen-containing heterocycloalkyl they form together with the nitrogen to which they are attached is [ka] In some embodiments, R 1 and R 2 However, the nitrogen-containing heterocycloalkyl they form together with the nitrogen to which they are attached is [ka] In some embodiments, R 1 and R 2 However, the nitrogen-containing heterocycloalkyl they form together with the nitrogen to which they are attached is [ka] In some embodiments, R 1 and R 2 However, the nitrogen-containing heterocycloalkyl they form together with the nitrogen to which they are attached is [ka] In some embodiments, R 1 and R 2 However, the nitrogen-containing heterocycloalkyl they form together with the nitrogen to which they are attached is [ka] It is.

[0181] In some embodiments, n is 2 or 3.

[0182] In some embodiments, R 1 and R 2 and together form a nitrogen-containing heteroaryl, which is selected from 0 to 3 R 6 The group may be optionally substituted with a group.

[0183] In some embodiments, the nitrogen-containing heteroaryl is selected from pyrrole, imidazole, and pyrazole.

[0184] In some embodiments, R 5 is aryl, which is selected from 0 to 3 R 6 groups, each of which is independently lower alkyl, halogen, lower alkoxy, OCF 3 and CF 3 is selected from.

[0185] In some embodiments, R 5 is phenyl, which is a group consisting of 0 to 3 R 6 groups, each of which is independently lower alkyl, halogen, lower alkoxy, OCF 3 and CF 3 is selected from.

[0186] In some embodiments, n is 2 or 3.

[0187] In some embodiments, R 5 is heteroaryl, which is selected from 0 to 3 R 6 The group may be optionally substituted with a group.

[0188] In some embodiments, R 5is a 5-6 membered monocyclic or 8-12 membered bicyclic heteroaryl, where 1-5 ring members are heteroatoms selected from N, O and S, and is selected from 0-3 R 6 groups, each of which is independently lower alkyl, halogen, lower alkoxy, OCF 3 and CF 3 is selected from.

[0189] In some embodiments, R 5 is a 5-6 membered monocyclic heteroaryl, where 1-5 ring members are heteroatoms selected from N, O and S, and is selected from 1 or 2 R 6 groups, each of which, if present, is independently a lower alkyl group.

[0190] In some embodiments, R 5 teeth, [ka] is selected from.

[0191] In some embodiments, n is 2 or 3.

[0192] Some embodiments of the present invention relate to a compound represented by formula (IV): [ka] [where: Y is a bond, NR 4a ,O,C(O)NH,NHC(O),S,SO 2 , CHOH and CH 2 Selected from; Z is a bond, NR 4b ,O,C(O)NH,NHC(O),S,SO 2 and C.H. 2 Selected from; m is selected from 0, 1, 2, 3, 4 and 5; n is selected from 0, 1, 2 and 3; R1 and R 2 are each independently selected from alkyl, aminoalkyl, alkylsulfonylalkyl, alkoxyalkyl, aryl, arylalkyl, cycloalkyl, cycloalkylalkyl, phenyl, biphenyl, heteroaryl, heteroarylalkyl, heterocycloalkyl, and heterocycloalkylalkyl; R 1 and R 2 together with the nitrogen to which they are attached form a nitrogen-containing heterocycloalkyl or heteroaryl ring which is composed of 0 to 3 R 6 optionally substituted with a group; R 4a and R 4b is independently selected from hydrogen, alkyl, alkenyl, alkynyl, and cycloalkyl; R 5 is selected from aryl and heteroaryl, each of which is selected from 0 to 3 R 6 optionally substituted with a group; R 6a is heteroaryl, cyano and S(O) 2 N(CH 3 ) 2 Selected from; Each R 6 are independently hydrogen, halogen, alkyl, alkylsulfonylaryl, alkenyl, alkynyl, cycloalkyl, haloalkyl, haloalkoxy, haloaryl, alkoxyaryl, aryl, aryloxy, aralkyl, heterocycloalkyl, heteroaryl, alkylheteroaryl, heteroarylalkyl, cyano, alkoxy, alkoxyaryl, amino, alkylamino, dialkylamino, oxo, COR 7 , S.O. 2 R 7 , N.H.S.O. 2 R 7 , N.H.S.O. 2 NHR 7 , N.H.C.O.R. 7 ,NHCONHR 7 ,CONHR 7 and CONR 7 R8 Selected from; R 7 and R 8 is independently selected from hydrogen, aryl and lower alkyl; or R 7 and R 8 together form a nitrogen-containing heterocycloalkyl or heteroaryl ring, which is optionally substituted with lower alkyl; where Y=CH 2 And Z=R 4b , m+n≠3] or a salt, polymorph or solvate thereof.

[0193] In some embodiments, Z in formula IV is NR 4b It is.

[0194] In some embodiments of Formula IV, R 4b is selected from methyl and hydrogen.

[0195] In some embodiments of Formula IV, R 4b is hydrogen.

[0196] In some embodiments of formula IV, alkyl, whether by itself or as a designated part of another acyclic substituent, is C 1 -C 8 It is an alkyl.

[0197] In some embodiments of Formula IV, m is 0; Y is CH 2 and n is selected from 0, 1 and 2. In some embodiments, n is 2.

[0198] In some embodiments of Formula IV, R 1 and R 2 are each independently selected from alkyl, aminoalkyl, alkylsulfonylalkyl, alkoxyalkyl, and heteroaryl; R 1 and R 2together with the nitrogen to which they are attached form a nitrogen-containing heterocycloalkyl or heteroaryl ring which is composed of 0 to 3 R 6 The group may be optionally substituted with a group.

[0199] In some embodiments of Formula IV, R 1 and R 2 However, the nitrogen-containing heterocycloalkyl or heteroaryl ring they form together with the nitrogen to which they are attached contains from 3 to 8 atoms.

[0200] In some embodiments of Formula IV, R 1 and R 2 and together form a nitrogen-containing heterocycloalkyl, which is selected from 0 to 3 R 6 The group may be optionally substituted with a group.

[0201] In some embodiments of formula IV, the nitrogen-containing heterocycloalkyl is selected from the group consisting of alkyl, halogen, CONH 2 , S.O. 2 CH 3 0 to 3 R selected from cyano, spiroheterocycloalkyl, and oxo; 6 The group may be optionally substituted with a group.

[0202] In some embodiments of formula IV, the nitrogen-containing heterocycloalkyl is [ka] Selected from TIFF2025517410000027.tif46165.

[0203] In some embodiments of formula IV, the nitrogen-containing heterocycloalkyl is [ka] is selected from.

[0204] In some embodiments of formula IV, the nitrogen-containing heterocycloalkyl is [ka] It is.

[0205] In some embodiments of Formula IV, each R 6a is cyano, S(O) 2 N(CH 3 ) 2 , [ka] is selected from.

[0206] In some embodiments of Formula IV, R 5 is phenyl, which is a group consisting of 0 to 3 R 6 The group may be optionally substituted with a group.

[0207] In some embodiments of Formula IV, R 5 teeth, [ka] where R 6 b is selected from halogen, hydroxy and methoxy.

[0208] In some embodiments of Formula IV, R 6b is selected from fluoro, methoxy and hydroxy.

[0209] In some embodiments of Formula IV, R 6b is fluoro.

[0210] In some embodiments of formula IV, the LSD1 inhibitor of formula IV is represented by formula V: [ka] [In the formula, R 1 and R 2are each independently selected from alkyl, aminoalkyl, alkylsulfonylalkyl, alkoxyalkyl, aryl, arylalkyl, cycloalkyl, cycloalkylalkyl, phenyl, biphenyl, heteroaryl, heteroarylalkyl, heterocycloalkyl, and heterocycloalkylalkyl; R 1 and R 2 together with the nitrogen to which they are attached form a nitrogen-containing heterocycloalkyl or heteroaryl ring which is composed of 0 to 3 R 6 optionally substituted with a group; R 4a is selected from hydrogen, alkyl, alkenyl, alkynyl, and cycloalkyl; R 6a is heteroaryl, cyano and S(O) 2 N(CH 3 ) 2 Selected from; Each R 6 and R 6b are independently hydrogen, halogen, alkyl, alkylsulfonylaryl, alkenyl, alkynyl, cycloalkyl, haloalkyl, haloalkoxy, haloaryl, alkoxyaryl, aryl, aryloxy, aralkyl, heterocycloalkyl, heteroaryl, alkylheteroaryl, heteroarylalkyl, cyano, alkoxy, alkoxyaryl, amino, alkylamino, dialkylamino, oxo, COR 7 , S.O. 2 R 7 , N.H.S.O. 2 R 7 , N.H.S.O. 2 NHR 7 , N.H.C.O.R. 7 ,NHCONHR 7 ,CONHR 7 and CONR 7 R 8 selected from; and R 7 and R 8 is independently selected from hydrogen, aryl and lower alkyl; or R 7 and R 8and together form a nitrogen-containing heterocycloalkyl or heteroaryl ring, which is optionally substituted with lower alkyl. or a salt, polymorph or solvate thereof.

[0211] In some embodiments of Formula V, R 4b is selected from methyl and hydrogen.

[0212] In some embodiments of Formula V, R 4b is hydrogen.

[0213] In some embodiments of Formula V, each R 6a Cyano, [ka] is selected from.

[0214] In some embodiments of Formula V, R 1 and R 2 are each independently selected from alkyl, aminoalkyl, alkylsulfonylalkyl, alkoxyalkyl, and heteroaryl; R 1 and R 2 together with the nitrogen to which they are attached form a nitrogen-containing heterocycloalkyl or heteroaryl ring which is composed of 0 to 3 R 6 The group may be optionally substituted with a group.

[0215] In some embodiments of Formula V, R 1 and R 2 However, the nitrogen-containing heterocycloalkyl or heteroaryl ring they form together with the nitrogen to which they are attached contains from 3 to 8 atoms.

[0216] In some embodiments of Formula V, R 1 and R 2 and together form a nitrogen-containing heterocycloalkyl, which is selected from 0 to 3 R 6 The group may be optionally substituted with a group.

[0217] In some embodiments of formula V, the nitrogen-containing heterocycloalkyl is [ka] Selected from TIFF2025517410000035.tif40170.

[0218] In some embodiments of formula V, the nitrogen-containing heterocycloalkyl is [ka] is selected from.

[0219] In some embodiments of formula V, the nitrogen-containing heterocycloalkyl is [ka] It is.

[0220] In some embodiments of Formula V, R 6b is selected from fluoro, methoxy and hydroxy.

[0221] In some embodiments of Formula V, R 6b is fluoro.

[0222] In some embodiments, the LSD1 inhibitor of formula V is [ka] or a salt, polymorph, or solvate thereof.

[0223] In some embodiments, the LSD1 inhibitor of formula V has formula VI: [ka] [In the formula, X is selected from tosylate, sulfate, tartrate, oxalate, besylate, fumarate, citrate, esylate, and malate; and q is an integer selected from 1 and 2. or a polymorph or solvate thereof.

[0224] In some embodiments, X is tosylate.

[0225] In some embodiments, q is 2.

[0226] In some embodiments, the LSD1 inhibitor is a compound described in WO2016130952 or WO2017079753.

[0227] In some embodiments, the LSD1 inhibitor is vomedemstat (IMG-7289), N-[(2S)-5-{[(1R,2S)-2-(4-fluorophenyl)cyclopropyl]amino}-1-(4-methylpiperazin-1-yl)-1-oxopentan-2-yl]-4-(1H-1,2,3-triazol-1-yl)benzamide, bis-tosylate salt, [ka] It is.

[0228] In some embodiments, the LSD1 inhibitor is selected from vomedemstat (IMG-7289), iademstat (ORY-1001), bafidemstat (ORY-2001), pleurodemstat (CC-90011), seclidemstat (SP-2577), INCB059872, TAS1440, SYHA1807, RO7051790, GSK2879552, MC2580, and DDP-38003.

[0229] The compounds can be synthesized as disclosed in US20160237043, WO2016130952, WO2017079753, WO2018035259 and WO2018035249.

[0230] Additional LSD1 inhibitors useful in the methods disclosed herein include those disclosed in the following references:

[0231] [Table 3]

[0232] (the contents of which are incorporated by reference in their entireties as if set forth herein) Therapeutic Compositions and Methods Also provided herein is a therapeutic composition comprising a population of CAR-containing immune effector cells according to the fourth aspect of the invention and at least one therapeutically acceptable diluent, carrier and / or adjuvant.

[0233] Also provided herein is a method of treating cancer in a patient comprising administering to the cancer patient a population of CAR-containing immune effector cells according to the third aspect of the invention or a therapeutic composition according to the fourth aspect of the invention, in some embodiments, the immune effector cells or population of CAR-containing immune effector cells are CAR-T cells or a population of CAR-T cells according to the third aspect of the invention.

[0234] In some embodiments, the cancer is a hematological malignancy. In some embodiments, the hematological malignancy is a T-cell malignancy. In some embodiments, the T-cell malignancy is a T-cell acute lymphoblastic leukemia (T-ALL). In some embodiments, the T-cell malignancy is a non-Hodgkin's lymphoma. In some embodiments, the T-cell malignancy is a T-cell chronic lymphocytic leukemia (T-CLL). In some embodiments, the T cell malignancy is selected from the group consisting of T cell acute lymphoblastic leukemia / lymphoma (T-ALL), human T cell leukemia virus type 1 positive (HTLV-1+) adult T cell leukemia / lymphoma (ATL), T cell prolymphocytic leukemia (T-PLL), adult T cell lymphoma / leukemia (HTLV-1 associated), aggressive NK cell leukemia, anaplastic large cell lymphoma (ALCL), ALK positive, anaplastic large cell lymphoma (ALCL), ALK negative, angioimmunoblastic T cell lymphoma (AITL), breast implant associated anaplastic large cell lymphoma, chronic lymphoproliferative disorder of NK cells, extranodal NK / T cell lymphoma, nasal type, enteropathic type T cell lymphoma, filtrate, and the like. Cystic T-cell lymphoma, hepatosplenic T-cell lymphoma, slowly progressive T-cell lymphoproliferative disorder of the gastrointestinal tract, monomorphic epitheliotropic intestinal T-cell lymphoma, mycosis fungoides, nodal peripheral T-cell lymphoma with TFH phenotype, peripheral T-cell lymphoma (PTCL), NOS, primary cutaneous γδ T-cell lymphoma, primary cutaneous CD8+ aggressive epidermotropic cytotoxic T-cell lymphoma primary cutaneous CD8+ T-cell lymphoma, primary cutaneous CD4+ small / medium T-cell lymphoproliferative disorder [primary cutaneous anaplastic large-cell lymphoma (C-ALCL), lymphoid papulopathy], Sézary syndrome, subcutaneous panniculitis-like T-cell lymphoma, pediatric systemic EBV+ T-cell lymphoma, and T-cell large granular lymphocytic leukemia (LGL).

[0235] In some embodiments, the hematological malignancy is a plasma cell malignancy, hi some embodiments, the plasma cell malignancy is selected from lymphoplasmacytic lymphoma, plasmacytoma, and multiple myeloma.

[0236] In some embodiments, the hematological malignancy is a B cell malignancy, hi some embodiments, the B cell malignancy is selected from diffuse large B cell lymphoma (DLBCL), chronic lymphocytic leukemia (CLL) / small lymphocytic lymphoma (SLL) and B cell precursor acute lymphoblastic leukemia (ALL).

[0237] definition Unless otherwise defined herein, all technical and scientific terms used have the same meaning as commonly understood by one of ordinary skill in the art of gene therapy, biochemistry, genetics, immunology and molecular biology. All disclosed compositions and methods similar or equivalent to those described herein can be used in the practice or testing of the present disclosure.

[0238] The term "antigen" refers to a soluble or cell membrane-bound molecule, in particular, but not limited to, a molecule that can be recognized by the adaptive immune system, such as, but not limited to, an antibody or TCR, or an engineered molecule, such as, but not limited to, a transgenic TCR, a chimeric antigen receptor (CAR), an scFv or multimer thereof, a Fab fragment or multimer thereof, an antibody or multimer thereof, a single chain antibody or multimer thereof, or any other molecule that can achieve binding to a structure with high affinity.

[0239] "Antigen binding domain" refers to an oligo- or polypeptide capable of binding to a ligand. In general, an antigen binding domain can interact with a cell surface molecule, which can be an antigen, a receptor, a peptide ligand, a target protein ligand, or a target polypeptide. An extracellular ligand binding domain has an affinity constant or interaction affinity (K) of about 0.1 pM to about 100 nM, e.g., about 0.1 pM to about 10 pM, or about 0.1 pM to about 1 pM. D ) and can specifically bind to the antigen. The affinity constant or interaction affinity (K DMethods for determining the binding site of an extracellular ligand are well known in the art. In some cases, the extracellular ligand binding domain is selected to recognize a ligand that functions as a cell surface marker on target cells associated with a particular pathology. In some embodiments, the antigen binding domain is selected to recognize a ligand that functions as a cell surface marker on target cells associated with a particular pathology. H Chains, peptide linkers and V L In some embodiments, the antibody is a single chain antibody fragment (scFv) comprising a V H Chain and V L The peptide linker between the chains has the amino acid sequence GGGGS (1-6) , e.g. GGGGS (1-4) has.

[0240] A "biologically active fragment" generally refers to a fragment of a polypeptide that substantially retains the biological activity of the intact polypeptide in the context of CAR function.

[0241] The term "cancer" is medically known as malignant neoplasm. Cancer is a broad group of diseases involving the upregulation of cell proliferation. In cancer, cells (cancer cells) divide and grow uncontrollably, forming malignant tumors that invade nearby parts of the body. Cancer can also spread to more distant parts of the body via the lymphatic system or bloodstream. There are over 200 different known cancers that affect humans.

[0242] A "CAR-containing immune effector cell" is an immune effector cell transduced with at least one CAR. A "CAR-T cell" is a T cell transduced with at least one CAR. A CAR-T cell can be a mono-, dual-, or tandem CAR-T cell. A CAR-T cell can be an autologous cell (meaning it is engineered from the subject's own cells) or an allogeneic cell (meaning it is derived from a healthy donor and is often engineered so as not to cause a host-versus-graft or graft-versus-host reaction). Donor cells can also be derived from umbilical cord blood or made from induced pluripotent stem cells.

[0243] "CAR-T cell" refers to a T cell that expresses a chimeric antigen receptor. A T cell that expresses a CAR molecule can be a helper T cell, a cytotoxic T cell, a virus-specific cytotoxic T cell, a memory T cell, or a gamma delta (γδ) T cell.

[0244] "CD8a leader", "CD8α leader", "CD8a leader polypeptide" or "CD8α leader polypeptide" refers to a CD8a protein or biological fragment thereof that functions to initiate the translocation of the extracellular domain of a CAR through an immune effector cell membrane to its membrane surface. A biologically active fragment in this context is defined as a fragment of at least 10 amino acids of the CD8α signal peptide that directs an associated polypeptide to the cell membrane and / or cell surface. Examples of functional fragments of the human CD8α signal peptide are provided in Table 1.

[0245] "Chimeric Antigen Receptor" ("CAR") means a recombinant fusion protein that comprises 1) an extracellular ligand-binding domain (also referred to herein as an antigen-binding domain or antigen domain), 2) a transmembrane domain, and 3) a signaling domain.

[0246] By "costimulatory domain" is meant an oligopeptide or polypeptide that enhances the signaling capability of a signaling domain.

[0247] "Deficient in at least one or more antigens to which one or more CARs specifically bind" means that the associated antigen of the immune effector cell to which the antigen binding domain binds is modified such that the chimeric antigen receptor no longer specifically binds to the modified antigen. In other words, a cell "defective in" an antigen is one that does not express the antigen, or expresses a reduced amount of the antigen, or expresses a modified version of the antigen, such that the chimeric antigen receptor or other protein that typically binds to the antigen exhibits a reduced or lost ability to do so.

[0248] A "dual" CAR-containing immune effector cell, e.g., a "dual CAR-T cell" or a "dual CAR-NK cell," refers to an engineered immune effector cell (e.g., a T cell or an NK cell) that has two different chimeric antigen receptor polypeptides with affinities for different target antigens expressed within the same effector cell, where each CAR functions independently. The CARs can be expressed from a single or multiple polynucleotide sequences.

[0249] "Fratricide" refers to the process that occurs when a CAR-containing immune effector cell is targeted and killed by another CAR-containing immune effector cell that contains the same chimeric antigen receptor as the target of the CAR-containing immune effector cell, because the targeted cell expresses an antigen that is specifically recognized by the chimeric antigen receptor on both cells.

[0250] The term "from the proximal N-terminus to the distal C-terminus" as used herein to describe elements contained in a chimeric antigen receptor does not exclude variations and linking sequences between elements known to those of skill in the art.

[0251] "Hinge" refers to any oligo- or polypeptide that functions to link a transmembrane domain to an antigen-binding domain. In particular, hinges are used to provide greater flexibility and accessibility to the antigen-binding domain.

[0252] "Immediately distal" means near the C-terminal end of an element, such as a linker or biological function domain. It can mean close enough or adjacent that it does not interfere with its own function, the function of the element, or their combined function.

[0253] "Immediately proximal" means near the N-terminal side of an element, such as a linker or biological function domain. It can mean close enough or adjacent that it does not interfere with its own function, the function of the element, or their combined function.

[0254] The term "immune cell" or "immune effector cell" refers to a cell that may be part of the immune system and performs a specific effector function, such as alpha-beta T cells, NK cells, NKT cells (including iNKT cells), LAK gamma-delta T cells, B cells, innate lymphoid cells (ILCs), mesenchymal stem cells or mesenchymal stromal cells (MSCs), monocytes and macrophages. Preferred immune cells are cells with cytotoxic effector function, such as alpha-beta T cells, NK cells, NKT cells (including iNKT cells), ILCs, CIK cells, LAK cells and gamma-delta T cells. "Effector function" refers to a specific function of a cell, for example, in T cells, effector function may be cytolytic activity or helper activity, including secretion of cytokines.

[0255] The term "malignant" or "malignant tumor" refers to a cell, group of cells, or tissue that constitutes a tumor, or that originates from a tumor, or that can be the source of new tumor cells. This term is used to describe tumor cells in contrast to normal or healthy cells of the tissue. Malignant tumors contrast with non-cancerous benign tumors in that they are not self-limited in their growth, but can invade adjacent tissues and may have the potential to spread to distant tissues. Benign tumors do not have any of these characteristics. Malignant tumors are characterized by undifferentiation, invasiveness and metastasis, and genomic instability. The term "pre-malignant cell" refers to a cell or tissue that is not yet malignant but is poised to become malignant.

[0256] As used herein, "sequence identity" refers to the percentage of identical nucleotides or amino acid residues at corresponding positions in two or more sequences when the sequences are aligned for maximum sequence matching, i.e., taking into account gaps and insertions. Identity can be readily calculated by known methods. Methods for determining identity are designed to give the maximum match between the sequences tested. Moreover, methods for determining identity are codified in publicly available computer programs. Optimal alignment of sequences for comparison can be performed, for example, by Smith & Waterman's local homology algorithm, homology alignment algorithm, similarity search method or computerized implementations of these algorithms (GAP, BESTFIT, PASTA and TFASTA in the GCG Wisconsin Package available from Accelrys, Inc.; see generally Altschul, SF et al., J. Mol. Biol. 215: 403-410 (1990) and Altschul et al., Nucl. Acids Res. 25: 3389-3402 (1997)). One example of an algorithm suitable for determining the percentage of sequence identity and sequence similarity is the BLAST algorithm.

[0257] "Signaling domain" or "signal transduction domain" refers to the signal transduction domain or intracellular signal transduction domain of the CAR that results in intracellular signal transduction after the antigen binding domain binds to a target, resulting in activation of the immune cell and immune response. In other words, the signal transduction domain results in activation of at least one of the normal effector functions of the immune cell in which the CAR is expressed.

[0258] The terms "specifically bind" or "specific" or "specifically recognize" with respect to an antigen recognizing receptor refer to an antigen-binding domain of the antigen recognizing receptor that recognizes and binds to a particular polymorphic variant of an antigen, but does not substantially recognize or bind other variants.

[0259] A "suicide gene" is a gene that causes immune effector cells to undergo cell death when the cells exceed a certain level of immune effector activity. Suitable suicide gene systems known in the art include, but are not limited to, some herpes simplex virus thymidine kinase (HSVtk) / ganciclovir (GCV) or inducible caspase 9 protein. In one embodiment, the suicide gene is a chimeric CD34 / thymidine kinase.

[0260] A "tandem" CAR-containing immune effector cell, e.g., a "tandem CAR-T cell" or a "tandem CAR-NK cell," refers to an engineered immune effector cell (e.g., a T cell or an NK cell) having a single chimeric antigen polypeptide that contains two or more different extracellular ligand binding domains that can interact with two or more different cell surface molecules, where the extracellular ligand binding domains are linked to each other by a flexible linker and share one or more effector / co-stimulatory domains, where binding of a first or second extracellular ligand binding domain signals via one or more co-stimulatory domains and a signaling domain. Tandem CARs can be bispecific or trispecific.

[0261] "Therapeutic composition" means a material that is suitable for use in contact with the tissues of a patient without undue toxicity, irritation, and allergic response, and / or is commensurate with a reasonable benefit / risk ratio, and / or is effective for its intended use.

[0262] "Transmembrane domain" refers to a generally hydrophobic peptide or polypeptide that spans the cell membrane and anchors the CAR to the T cell surface, and connects the antigen binding domain to the signaling domain to affect the expression of the CAR on the T cell surface. A distinctive feature of the transmembrane domain in this disclosure is that it can be expressed on the surface of an immune cell to direct an immune cell response against a predetermined target cell.

[0263] "Truncated CD34 domain" or "TrCD34 domain" refers to a fragment of the CD34 polypeptide.

[0264] Working Example The following examples are intended to further illustrate certain non-limiting embodiments of the present invention, and are not intended to limit the scope of the invention in any way.

[0265] Example 1 – Method for producing CAR-NK cells Briefly, NK cells are purified from normal donor PBMCs and incubated in IL-12 / 15 / 18. Cytokines are washed off, and then cells are incubated in high dose IL-15 and transduced with CAR lentivirus (2x). Cells are quiescent (in vivo or in vitro) and evaluated for enhanced effector function. A more detailed description of an exemplary procedure is provided below.

[0266] Primary NK cells can be purified from peripheral blood mononuclear cells (PBMCs). PBMCs are any peripheral blood cells with a round nucleus. These cells consist of lymphocytes (T cells, B cells, NK cells) and monocytes, where red blood cells and platelets do not have a nucleus, and granulocytes (neutrophils, basophils and eosinophils) have a multilobed nucleus. Other products are obtained from stem cells or cell lines.

[0267] The addition of cytokines may be important for the generation of NK cells. IL-12 / 15 / 18, or fusion proteins containing functional fragments of such cytokines, can be added. The cytokines can then be washed away and the cells cultured in IL-15 for the remainder of the assay.

[0268] Also, NK cells are sensitive to polybrene and therefore polybrene was not used in this transduction, although it is normally used.

[0269] Construction of lentiviral vectors and transduction into NK cells Lentiviruses can be used to introduce CARs. A cassette encoding a chimeric antigen receptor (CAR) can be incorporated, for example, into the MND lentiviral backbone to create a lentiviral vector. To generate lentiviral supernatants, 293T cells can be co-transfected with lentiviral vectors pMND-G, pMND-Lg and pMDN-REV using calcium chloride transfection reagent, and after 24-48 hours, the lentivirus-containing supernatants can be collected and concentrated using ultracentrifugation. For transduction, purified cytokine-activated (IL-12 / 15 / 18) NK cells can be plated in complete culture medium supplemented with 50 ng / mL IL-15. Viral supernatants can be added to the cells, which can be spinfected at room temperature for 90 minutes at 2000 rpm. Cells can be incubated at 37°C and 5% CO 2 Cells can be incubated at 4°C for 1 h. To maximize viral transduction efficiency, cells can be spinfected on days 1 and 2. Cells are then washed and used immediately or cultured in complete medium supplemented with 1 ng / mL IL-15. Maximal vector expression is expected to be achieved by day 7.

[0270] Example 2 – Method for producing CAR-T cells CAR-containing immune effector cells can be constructed, optionally using a genome editing process to delete or suppress one or more surface proteins. Such surface proteins can include, for example, those that form part of the TCR complex, which can induce GvHD if the cells are administered to a patient in an allogeneic manner, or those that are the target antigens of the CAR, which can induce fratricide if expression of the antigen on the CAR-T is not suppressed.

[0271] For example, in one protocol, on day 0, CD4+CD8+ T cells are thawed in cell culture medium. The required number of cells is centrifuged at room temperature at 200×g for 10 minutes. The supernatant is completely removed and 1×10 6Cells are resuspended in cell culture medium (TexMacs) supplemented with IL-7 (10 ng / ml) and IL-15 (10 ng / ml) at a concentration of 1 / ml. T cells are stimulated with Miltenyi research grade TransAct™ (10 μl / ml).

[0272] On day 1, the required amount of CAR-containing viral vector is added to the activated cells at the required MOI (multiplicity of infection). The cells and virus are mixed and placed back into the 37° C. incubator.

[0273] On day 3, the activated cells are washed to remove the stimuli.

[0274] If genome editing is desired, harvest and count the cells. Centrifuge the required number of cells at 100 x g for 10 minutes at room temperature. Remove the supernatant completely and resuspend the cells in electroporation buffer (1 ml) (e.g., Maxcyte EP buffer), transfer to a microcentrifuge tube, and centrifuge at 100 x g for 10 minutes at room temperature. Remove the supernatant completely and then resuspend the cells at the desired concentration (e.g., 5 x 10 7 1. Resuspend in electroporation buffer (e.g., MaxCyte EP buffer) at 1000 ng / ml.

[0275] Commercially available Cas9 protein (10 μg) and commercially synthesized gRNA (20 μg) are complexed at room temperature for 10 min.

[0276] Cells (100 μl) are transferred to the tube containing the complexed Cas9 / gRNA, mixed gently, and the whole is transferred into a MaxCyte OC100 cuvette. Electroporation is then initiated using the Maxcyte program Expanded T Cells 2. After this procedure, the activated cells are transferred to 10 ml of pre-warmed medium and returned to the incubator to grow for an additional 7-12 days.

[0277] FACS analysis can be used to demonstrate purity (CAR expression and targeted gene deletion) of CAR-transduced cells.

[0278] Example 3 – Production of viral vectors Lentivirus (e.g., containing expression cassettes for CAR and any tags) can be produced using the Lenti-X 293T cell line (Takara Bio, Mountain View, Calif.). Lenti-X 293T cells were transfected with the CAR lentiviral vector and packaging plasmids, pMD.Lg / rRRE, pMD.G, and pRSV.REV, using Invitrogen's Lipofectamine 2000 Transfection Reagent according to the manufacturer's instructions. 14 Thirty-six hours after transfection, the viral supernatant can be collected, filtered to remove cellular debris, and concentrated by ultracentrifugation (Optima LE-80K Ultracentrifuge, Beckman Coulter, Indianapolis, Ind.) at 25,000 rpm for 90 minutes at 4° C. After concentration, the virus is resuspended in phosphate-buffered saline.

[0279] Example 4 – CRISPR / Cas9 editing to remove genes Optionally, T cells may be cultured in Xcyte medium supplemented with 50 U / mL IL-2, 10 ng / ml IL-2, 10 ng / ml IL-15 and 10 ng / ml IL7 in the presence of anti-CD3 / CD28 beads (3:1 bead to cell ratio). On day +2 after activation, the stimuli are removed and 1×10 7 T cells are electroporated in 100μl of MaxCyte buffer containing 15μg of spCas9 (Trilink CA.) and 20μg of each gRNA (one or more for multiple editing; the appropriate gRNA sequence for the target gene is available in the art), for example using MaxCyte GT. Then, on day +3, lentiviral particles can be introduced into cells in the presence of polybrene (Sigma Aldrich) (final concentration 6μg / ml). Cells are then grown, for example, for 6 days after introduction, before being used in subsequent experiments.

[0280] To confirm editing, targeted deep sequencing can be used.The locus targeted by CRISPR editing can be amplified with primers.The PCR product is sequenced, for example, using the Illumina MiSeq platform (San Diego CA).Editing efficiency can be determined as the proportion of sequencing reads that contain indels aligned with the reads obtained from WT cells.

[0281] Example 5 – Suppression / reversal of terminal differentiation in CAR-T cells To mimic the tumor microenvironment (TMA) that continuously exposes antitumor T cells to tumor antigens, CAR-T cells are injected into target cells (e.g., CD19 CAR-T cells express CD19). + These expanded CAR-T cells are typically stimulated with CD45RA + CD62L + CCR7 + CD27 + CD28 + A population of T cells (stem cell-like memory T cells:T SCM ) and a central memory phenotype (T CM ) corresponds to CD45RA-CD62L + CCR7 + CD27 + CD28 + CAR-T cells stimulated by cells expressing the target antigen (e.g., K562-CD19) express higher levels of T cell exhaustion markers (e.g., PD1, LAG3, and TIM3) than those stimulated by NALM6 (presumably due to the high antigen load) and lose the ability to produce multifunctional cytokines.

[0282] CD19CAR-T cells genetically modified to delete PRDM1 using CRISPR / Cas9 and subjected to this protocol lost Blimp1 expression and displayed higher frequencies of CD45RA + / 2CD62L + CCR7 + CD27 + CD28 + IL7R + cells and elevated TNFα and cytokine production (e.g., TNFα and IL-2). SCM and T CM Shows improved phenotypic maintenance. See Yoshikawa T et al., “Genetic ablation of PRDM1 in antitumor T cells enhances therapeutic efficacy of adoptive immunotherapy,” Blood 139(14):2156-72 (2022).

[0283] This protocol can be modified to include treatment with an LSD1 inhibitor instead of PRDM1 deletion. The inhibitor can be administered at different concentration ranges and different schedules depending on the inhibitor. For example, bomedemstat can be used. Treatment with an LSD1 inhibitor can improve T SCM and T CM It is expected that treatment with vomedemstat or another LSD1 inhibitor that inhibits both the demethylase activity and the ability of Growth Factor Independent 1 and / or Growth Factor Independent 1b (GFI1 and / or GFI1b and Blimp1) to bind LSD1 will maintain the phenotype similarly and be superior to LSD1 inhibitors that do not have this dual activity.

[0284] Example 6 –Cell lines and preparation of cell lines for cancer cell killing assays Suitable cell lines, such as K562-CD19 or NALM6 cells (e.g., for assaying CD19-targeted CARs comprising scFvs from clone FMC63 linked to CD28 costimulatory domains and CD3z signaling domains), can be obtained from commercial or public sources, such as DSMZ-German collection of Microorganisms and Cell Cultures (Leibniz, Germany) and / or ATCC Global Bioresource Center (Manassas VA). Upon receipt, cell lines are subjected to mycoplasma testing. Cell lines can be transduced with GFP lentivirus, for example, at an MOI of 5:1. After transduction, GFP-positive cells are selected and expanded to establish pure GFP-expressing cell lines.

[0285] Example 7 –Cell killing by CAR-T cells: Chromium release CAR-T cells were administered at effector:target [E:T] ratios ranging from 25:1 to 0.25:1, for example. 51 Cr] labeled cell lines (e.g., 1 × 10 4 The cells can be incubated with 1000 ng / well of 10 ...

[0286] Example 8 –Cell killing by CAR-T cells: FACS To evaluate the cytotoxic activity of CAR-T cells, 1 × 10 5 CAR-T cells can be co-cultured with EGFP1 target cells at the indicated ratios. The absolute number of viable target cells is determined by flow cytometry. The frequency of viable tumor cells is calculated as a ratio of the number of cells incubated in the absence of CAR-T cells. Furthermore, dead cells are identified using the LIVE / DEAD Fixable Near-IR Dead Cell Stain Kit (Thermo Fisher Scientific). It is expected that treatment of tumor cells with the combination of CAR-T and LDS1i will result in improved cell killing, albeit with a somewhat delayed effect, compared to CAR-T alone, and that treatment with bomedemstat or another LDS1 inhibitor that inhibits both the demethylase activity and the ability of growth factor independent 1 and / or growth factor independent 1b (GFI1 and / or GFI1b and Blimp1) to bind to LSD1 will be superior to LSD1 inhibitors that do not have this dual activity.

[0287] Example 9 - T cell phenotypic analysis Cultured T cells were washed in PBS / 0.1% BSA and then solubilized at 1 × 10 in 50 μL of Brilliant Buffer (BD Biosciences) supplemented with 4% rat serum for 15 min at 4°C. 6The cells were then incubated for 30 minutes at 4° C. in 100 μL of Brilliant Buffer with the following antibody fluorophore conjugates (all from BD Biosciences unless otherwise indicated): CD2 BUV395, CD8 BUV496, CD3 BUV661, CD38 BV421, Tim-3 BV450 (BioLegend), PDL1 BV510 (BioLegend), PD1 BV605 (BioLegend), CD4 BV650, CD45 BV711, CD196 / CCR6 (BioLegend), CD45RA, Cy5.5, CD183 / CXCR3 PE, CD34 PE-Cy7 (BioLegend), Tigit APC (BioLegend), CD127 APC-eF780 (eBioscience), CD62L AF700 (BioLegend). Cells are then washed twice in PBS / 0.1% BSA and data acquired on a ZE5 (Yeti) cytometer (BioRad / PropelLabs). Compensation and analysis are performed in FlowJo V10 (TreeStar) using Fluorescence Minus One (FMO) controls. Statistical analysis can be performed in GraphPad Prism7.

[0288] Example 10 –Xenogeneic Mouse Model The anti-leukemia effect of the combination of CAR-T cells and LSD1 inhibitors can be tested in vivo using NSG mice inoculated with cancer cells. On day -5, approximately 4-10 week old male mice were inoculated with, for example, 5×10 5 On day 0, inject CAR-T (2 × 10 6 ) + LSD1 inhibitor, CAR-T (2 × 10 6 ) alone and controls are injected into the lateral tail vein.

[0289] To track tumor growth in vivo, mice are injected intraperitoneally with 50 μg / g D-luciferin (Biosynth, Itasca, IL, USA) and imaged by methods known in the art at various time points (e.g., weekly, 7th, 14th, 21st, 28th, 35th, etc.) for, for example, 80 days. It is also possible to track the persistence of CAR-T cells in vivo, as well as the percentage of sampled CAR-T cells at each time point expressing TSCM and TCM phenotypes compared to the injected pool.

[0290] The log-rank Mantel-Cox test may be used to determine significant differences in survival rates. Statistical analysis of tumor burden determined by BLI imaging may be performed using two-way ANOVA for repeated measures data, followed by step-down Bonferroni correction for multiple comparisons. BLI may be performed in a blinded fashion.

[0291] The combination of CAR-T cells and an LSD1 inhibitor demonstrated better tumor growth control, better CAR-T cell persistence, and improved T cell survival than administration of CAR-T cells alone. SCM and T CM Treatment with vomedemstat or another LSD1 inhibitor that results in maintenance of the phenotype and inhibits both the demethylase activity and the ability of Growth Factor Independent 1 and / or Growth Factor Independent 1b (GFI1 and / or GFI1b and Blimp1) to bind LSD1 is expected to be superior to LSD1 inhibitors that do not have this dual activity.

[0292] As a variation of the above procedure, mice treated with CAR-T alone can be administered with an LSD1 inhibitor at one or more times after CAR-T injection. For example, an LSD1 inhibitor can be administered when T cells start to show signs of exhaustion or when antitumor effects start to wane (i.e., accelerated progression). It is expected that such subsequent treatment with LDS1i will restore the phenotype and function of early memory T cells, and that treatment with bomedemstat or another LDS1 inhibitor that inhibits both demethylase activity and the ability of growth factor independent 1 and / or growth factor independent 1b (GFI1 and / or GFI1b and Blimp1) to bind to LSD1 is superior to an LSD1 inhibitor that does not have this dual activity.

[0293] The above detailed description is provided to aid those skilled in the art in practicing the present disclosure. However, the disclosure described and claimed herein is not limited in scope by the specific embodiments disclosed herein, since these embodiments are intended as illustrations of some aspects of the present disclosure. Any equivalent embodiments are intended to be within the scope of the present disclosure. Indeed, various modifications of the present disclosure, in addition to those shown and described herein, that do not depart from the spirit or scope of the teachings of the present invention will be apparent to those skilled in the art from the above description. Such modifications are also intended to be included within the scope of the appended claims.

Claims

1. A pharmaceutical composition for use in a method of treating a disease, comprising a therapeutic composition comprising a population of immunoeffector cells prepared for adoptive transfer that bind to an antigen on the surface of a target cell, and / or a lysine-specific demethylase 1 (LSD1) inhibitor, wherein the method comprises administering the therapeutic composition and the LSD1 inhibitor in combination.

2. The pharmaceutical composition according to claim 1, wherein the method involves administering an LSD1 inhibitor simultaneously with a therapeutic composition comprising a population of immune effector cells prepared for adoptive transfer.

3. The pharmaceutical composition according to claim 1, wherein the method involves administering an LSD1 inhibitor after a therapeutic composition comprising a population of immune effector cells prepared for adoptive transfer.

4. The pharmaceutical composition according to claim 1, wherein the method involves administering an LSD1 inhibitor before a therapeutic composition comprising a population of immune effector cells prepared for adoptive transfer.

5. The pharmaceutical composition according to claim 1, wherein, in the method described above, the LSD1 inhibitor is administered after the exhaustion of the adopted immune cells is observed.

6. The pharmaceutical composition according to claim 1, wherein the disease is cancer and the target cell is a cancer cell.

7. A pharmaceutical composition comprising a lysine-specific demethylase 1 (LSD1) inhibitor for use in a method to enhance the memory phenotype of a population of immunoeffector cells prepared for adoptive transfer, the method comprising treating the immunoeffector cells with the LSD1 inhibitor.

8. A pharmaceutical composition comprising a lysine-specific demethylase 1 (LSD1) inhibitor for use in a method for inhibiting the transition from a memory phenotype to a differentiated phenotype of T cells prepared for adoptive transfer, the method comprising treating immunoeffector cells with the LSD1 inhibitor.

9. A pharmaceutical composition comprising a lysine-specific demethylase 1 (LSD1) inhibitor for use in a method for suppressing or reversing exhaustion in a population of immunoeffector cells prepared for adoptive transfer, the method comprising treating the immunoeffector cells with the LSD1 inhibitor.

10. The pharmaceutical composition according to any one of claims 7 to 9, wherein, in the method described above, the LSD1 inhibitor is administered after the exhaustion of the adopted immune cells is observed.

11. The pharmaceutical composition according to any one of claims 1, 7 to 9, wherein the LSD1 inhibitor inhibits both demethylase activity and the ability of growth factor-independent 1 and / or growth factor-independent 1b (GFI1 and / or GFI1b and Blimp1) to bind to LSD1.

12. The pharmaceutical composition according to any one of claims 1, 7 to 9, wherein the LSD1 inhibitor is a compound of any of the formulas I to VI disclosed herein.

13. The pharmaceutical composition according to claim 12, wherein the LSD1 inhibitor is bomedemstat.

14. The pharmaceutical composition according to claim 13, wherein, in the method described above, vomedemstat is administered once daily in an amount determined to correspond to a platelet count of 50 to 150 k / μL.

15. The pharmaceutical composition according to claim 12, wherein the LSD1 inhibitor is selected from bomedemstat (IMG-7289), idademstat (ORY-1001), vafidemstat (ORY-2001), plurodemstat (CC-90011), seclidemstat (SP-2577), INCB059872, TAS1440, SYHA1807, RO7051790, GSK2879552, MC2580, and DDP-38003.

16. The pharmaceutical composition according to claim 15, wherein the LSD1 inhibitor is selected from vomedemstat (IMG-7289), iadademstat (ORY-1001), and plurodemstat (CC-90011).

17. The pharmaceutical composition according to any one of claims 1, 7 to 9, wherein the immune effector cells are selected from T cells and natural killer cells.

18. The pharmaceutical composition according to claim 17, wherein immune effector cells express at least one chimeric antigen receptor (CAR).

19. The pharmaceutical composition according to claim 18, wherein the immune effector cells are NK cells.

20. The pharmaceutical composition according to claim 18, wherein the immune effector cell is a T cell.

21. The pharmaceutical composition according to claim 19, wherein the immune effector cell is a CAR-T cell.

22. CAR, CD8a reader polypeptide, which may be included as desired. antigen-binding domain, hinge, Transmembrane domain, At least one co-stimulatory domain, and Signal transduction domains A pharmaceutical composition according to claim 18, comprising:

23. CAR, A CD8a reader polypeptide, an antigen-binding domain, and an extracellular domain including a hinge, which may be optionally included. Transmembrane domain, and Intracellular domains containing at least one co-stimulatory domain and a signal transduction domain A pharmaceutical composition according to claim 18, comprising:

24. The pharmaceutical composition according to claim 22, wherein the transmembrane domain is selected from a CD28 transmembrane domain and a CD8 transmembrane domain.

25. The pharmaceutical composition according to claim 22, wherein the intracellular domain is selected from a CD28 intracellular signaling domain, an OX40L intracellular signaling domain, and a CD3-zeta (CD3-ζ) signaling domain.

26. The pharmaceutical composition according to claim 18, wherein the CAR comprises elements from the proximal N-terminus to the distal C-terminus.

27. The pharmaceutical composition according to claim 18, wherein the CAR specifically binds to at least one antigen expressed on malignant T cells.

28. The pharmaceutical composition according to claim 27, wherein the antigen expressed on malignant T cells is selected from one or more of CD2, CD3, CD4, CD5, CD7, TRAC, CD70, CD1a, and TCRβ.

29. The pharmaceutical composition according to claim 18, wherein the CAR specifically binds to at least one antigen expressed on malignant plasma cells.

30. The pharmaceutical composition according to claim 29, wherein the antigen expressed on malignant plasma cells is selected from one or more of BCMA, CS1, CD38, CD79A, CD79B, CD138, and CD19.

31. The pharmaceutical composition according to claim 18, wherein CAR specifically binds to at least one antigen expressed on malignant B cells.

32. The pharmaceutical composition according to claim 31, wherein the antigen expressed on malignant B cells is selected from one or more of CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD27, CD38, and CD45.

33. The pharmaceutical composition according to claim 32, wherein the antigen expressed on malignant B cells is selected from one or more CD19 and CD20.

34. The pharmaceutical composition according to claim 33, wherein the antigen expressed on malignant B cells is CD19.

35. The antigen-binding domain is V Ｈ Chain, peptide linker and V Ｌ A pharmaceutical composition according to any one of claims 1, 7 to 9, comprising a chain (scFv).

36. scFv is, Sequence IDs 22 and 23, Sequence IDs 24 and 25, Sequence IDs 26 and 27, Sequence IDs 28 and 29, Sequence IDs 30 and 31, Sequence IDs 32 and 33, Sequence ID 34 and Sequence ID 35, Sequence ID 36 and Sequence ID 37, Sequence IDs 38 and 39, Sequence IDs 40 and 41, Sequence ID 42 and Sequence ID 43, Sequence ID 44 and Sequence ID 45, Sequence IDs 46 and 47, and Sequence IDs 48 and 49 V containing any of the following amino acid sequences Ｈ Chain and V Ｌ The pharmaceutical composition according to claim 35, comprising a chain pair, or wherein scFv comprises an amino acid sequence selected from SEQ ID NO: 50 and SEQ ID NO:

51.

37. V Ｈ Chain and V Ｌ The peptide linker between the chain and the amino acid sequence GGGGS （１－４） A pharmaceutical composition according to claim 35, having the following characteristics.

38. A pharmaceutical composition according to any one of claims 1, 7 to 9, wherein the CAR has a co-stimulatory domain.

39. The pharmaceutical composition according to claim 38, wherein the CAR has multiple co-stimulatory domains.

40. The pharmaceutical composition according to claim 39, wherein the CAR has two co-stimulatory domains.

41. The pharmaceutical composition according to claim 38, wherein the co-stimulatory domain is selected from 4-1BB, CD28, OX-40, and NKG2D.

42. The pharmaceutical composition according to any one of claims 1, 7 to 9, wherein the CAR-T cells lack a subunit of the T cell receptor complex and / or lack at least one antigen to which one or more CARs specifically bind.

43. The pharmaceutical composition according to claim 42, wherein the subunit of the T cell receptor complex is selected from one or more of the extracellular domains of TCRα, TCRβ, TCRδ, TCRγ, CD3ε, CD3γ and CD3δ, and CD3ζ.

44. The pharmaceutical composition according to any one of claims 1, 7 to 9, wherein the method is carried out in a human subject having cancer.

45. The pharmaceutical composition according to claim 44, wherein the cancer is a hematological malignancy.

46. The pharmaceutical composition according to claim 45, wherein the hematological malignancy is a myeloproliferative neoplasm.

47. The pharmaceutical composition according to claim 46, wherein the myeloproliferative neoplasm is a myelofibrosis selected from primary myelofibrosis (PMF) and post-PV / ET myelofibrosis (MF).

48. The pharmaceutical composition according to claim 47, wherein the myeloproliferative neoplasm is post-PV / ET myelofibrosis (MF).

49. The pharmaceutical composition according to claim 46, wherein the myeloproliferative neoplasm is polycythemia vera.

50. The pharmaceutical composition according to claim 46, wherein the myeloproliferative neoplasm is essential thrombocythemia.

51. The pharmaceutical composition according to claim 45, wherein the hematological malignancy is acute myeloid leukemia (AML).

52. The pharmaceutical composition according to claim 45, wherein the hematological malignancy is chronic myeloid leukemia (CML).

53. The pharmaceutical composition according to claim 45, wherein the hematological malignancy is multiple myeloma.

54. The pharmaceutical composition according to claim 45, wherein the hematological malignancy is a T-cell malignancy.

55. T-cell malignancies include T-cell acute lymphoblastic leukemia / lymphoma (T-ALL), human T-cell leukemia virus type 1 positive (HTLV-1+) adult T-cell leukemia / lymphoma (ATL), T-cell prelymphoblastic leukemia (T-PLL), adult T-cell lymphoma / leukemia (HTLV-1 related), aggressive NK-cell leukemia, anaplastic large cell lymphoma (ALCL), ALK-positive anaplastic large cell lymphoma (ALCL), ALK-negative angioimmunoblastic T-cell lymphoma (AITL), breast implant-associated anaplastic large cell lymphoma, chronic lymphoproliferative disorder of NK cells, extranodal NK / T-cell lymphoma, nasal type, enteropathy-type T-cell lymphoma, follicular T-cell lymphoma, and hepatosplenic T-cell lymphoma. The pharmaceutical composition according to claim 54, selected from follicular lymphoma, slowly progressive T-cell lymphoproliferative disorder of the gastrointestinal tract, monomorphic epitheliotropic enteric T-cell lymphoma, mycosis fungoides, nodal peripheral T-cell lymphoma with TFH phenotype, peripheral T-cell lymphoma (PTCL), NOS, primary cutaneous γδ T-cell lymphoma, primary cutaneous CD8+ aggressive epidermotropic cytotoxic T-cell lymphoma, primary cutaneous apical CD8+ T-cell lymphoma, primary cutaneous CD4+ small / medium T-cell lymphoproliferative disorder, primary cutaneous anaplastic large cell lymphoma (C-ALCL), lymphoid papulosis, Sézary syndrome, subcutaneous panniculitis-like T-cell lymphoma, systemic EBV+ T-cell lymphoma in children, and T-cell macrogranular lymphocytic leukemia (LGL).

56. The pharmaceutical composition according to claim 55, wherein the T-cell malignant tumor is T-cell acute lymphoblastic leukemia (T-ALL).

57. The pharmaceutical composition according to claim 54, wherein the T-cell malignant tumor is non-Hodgkin lymphoma.

58. The pharmaceutical composition according to claim 54, wherein the T-cell malignant tumor is T-cell chronic lymphocytic leukemia (T-CLL).

59. The pharmaceutical composition according to claim 45, wherein the hematological malignancy is a B-cell malignancy.

60. The pharmaceutical composition according to claim 59, wherein the B-cell malignancy is selected from diffuse large B-cell lymphoma (DLBCL), chronic lymphocytic leukemia (CLL) / small lymphocytic lymphoma (SLL), and B-cell progenitor acute lymphoblastic leukemia (ALL).

61. The pharmaceutical composition according to claim 45, wherein the hematological malignancy is a plasma cell malignancy.

62. The pharmaceutical composition according to claim 61, wherein the plasma cell malignancy is selected from lymphoplasmacytic lymphoma, plasmacytoma, and multiple myeloma.