Compositions of compound chimeric antigen receptors (cCARs) targeting multiple antigens and methods of using the same

Abstract

In one embodiment, the present disclosure provides an engineered cell having a first chimeric antigen receptor polypeptide comprising a first antigen recognition domain, a first signal peptide, a first hinge region, a first transmembrane domain, a first costimulatory domain, and a first signaling domain, and a second chimeric antigen receptor polypeptide comprising a second antigen recognition domain, a second signal peptide, a second hinge region, a second transmembrane domain, a second costimulatory domain, and a second signaling domain, wherein the first antigen recognition domain is different from the second antigen recognition domain.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 62 / 571,608, filed Oct. 12, 2017, and U.S. Provisional Patent Application No. 62 / 628,973, filed Feb. 10, 2018. All of these are hereby incorporated by reference in their entirety into this specification.

Background Art

[0002] Background T cells, a type of lymphocyte, play a central role in cellular immunity. They are distinguished from other lymphocytes such as B cells and natural killer (NK) cells by the presence of T - cell receptors (TCRs) on their cell surfaces. Helper T cells, called CD4+T or CD4 T cells, have CD4 glycoproteins expressed on their cell surfaces. Helper T cells are activated when exposed to peptide antigens presented by major histocompatibility complex (MHC) class II molecules. Once activated, these cells rapidly proliferate and secrete cytokines that regulate the immune response. Cytotoxic T cells, also known as CD8+T cells or CD8 T cells, have CD8 glycoproteins expressed on their cell surfaces. CD8+T cells are activated when exposed to peptide antigens presented by MHC class I molecules. Memory T cells, a subset of T cells, survive for a long time and provide the immune system with “memory” of past infections and / or tumor cells, enabling them to respond to their homologous antigens.

[0003] T cells can be genetically engineered to produce special receptors called chimeric antigen receptors (CARs) on their surfaces. CARs are proteins on T cells that can recognize specific proteins (antigens) on tumor cells. These engineered CAR T cells are grown in the laboratory until they reach billions in number. Then, the population of expanded CAR T cells is injected into patients.

[0004] In clinical trials using chimeric antigen receptor (CAR) T cells to date, it has shown great promise as a treatment for hematological malignancies resistant to standard chemotherapy. Most notably, CD19-specific CAR (CD19CAR) T cell therapy has shown significantly favorable outcomes, including long-term remission in B cell malignancies. (References: Kochenderfer, Wilson et al. 2010, Kalos, Levine et al. 2011, Porter, Levine et al. 2011, Davila, Riviere et al. 2013, Grupp, Frey et al. 2013, Grupp, Kalos et al. 2013, Kalos, Nazimuddin et al. 2013, Kochenderfer, Dudley et al. 2013, Kochenderfer, Dudley et al. 2013, Lee, Shah et al. 2013, Park, Riviere et al. 2013, Maude, Frey et al. 2014)

[0005] Despite the success of CAR therapy in B cell leukemia and lymphoma, the application of CAR therapy for soft tissue tumors has not yet been fully established. Malignant soft tissue tumors have a dramatically worse prognosis compared to B cell malignancies (Abramson, Feldman et al. 2014), and in this regard, CAR therapy holds the potential to address a greater clinical need.

[0006] There are several obstacles that prevent the adoption of a more extensive approach in CAR therapy. The most common issues are: (1) the selection of antigen targets and chimeric antigen receptors, (2) the design of CARs, and (3) tumor heterogeneity, particularly the variability in surface expression of tumor antigens. Targeting a single antigen poses a risk of immune escape, which can be overcome by targeting multiple desired antigens.

[0007] Most chimeric antigen receptors (CARs) are single-chain variable fragments (scFvs) derived from monoclonal antibodies, and some of these monoclonal antibodies are used in clinical trials or treatment of diseases. However, their effectiveness is limited, suggesting the need for alternative and more potent approaches to targets such as CARs.

[0008] Since there are no general rules to guarantee or guide effective CAR design, target discovery and selection are the first steps.

[0009] ScFv is the most commonly used chimeric antigen receptor in CARs. However, the affinity binding of CARs and the position of the recognized epitope on the antigen may potentially affect its function. Furthermore, the expression level of surface CARs on T cells or NK cells is affected by appropriate leader sequences and promoters. Additionally, overexpressed CAR proteins can also be toxic to cells.

[0010] Therefore, there remains a need for improved chimeric antigen receptor-based therapies that are more effective, safe, and efficient against T cell-related malignancies.

[0011] Furthermore, CARs targeting neuroblastoma are very difficult due to the presence of heterogeneous tumor populations and suppression in the tumor microenvironment. Antigen-specific immunotherapies for neuroblastoma have been pursued for a long time to improve patient treatment outcomes, but the success has been limited so far because many of these therapies have been ineffective in the clinic or have an uncertain impact on patient outcomes. Ideal targets have not been established for neuroblastoma or other soft tissue tumors (such as sarcomas), diseases with high antigen diversity. Identification of appropriate targets is an important step for CAR design, and CAR design is necessary to address tumor heterogeneity, CAR persistence, and suppression of the tumor microenvironment. There are no general rules for CAR design to be effective and safe.

[0012] Therefore, there remains a need for improved chimeric antigen receptor-based therapies that enable more effective, safe, and efficient targeting of soft tissue tumors.

Prior Art Documents

Patent Documents

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Patent Document 1

Non-Patent Documents

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Summary of the Invention

Problems to be Solved by the Invention

[0015] Therefore, there remains a need for improved chimeric antigen receptor-based therapies that enable more effective, safe, and efficient targeting of soft tissue tumors.

Means for Solving the Problems

[0016] In one embodiment, the present disclosure provides a first chimeric antigen receptor polypeptide comprising a first antigen recognition domain, a first signal peptide, a first hinge region, a first transmembrane domain, a first co-stimulatory domain, and a first signaling domain, and a second chimeric antigen receptor polypeptide comprising a second antigen recognition domain, a second signal peptide, a second hinge region, a second transmembrane domain, a second co-stimulatory domain, and a second signaling domain, wherein the first antigen recognition domain and the second antigen rejection domain are selected from the group consisting of interleukin 6 receptor, NY-ESO-1, alpha-fetoprotein (AFP), (GPC3), BAFF-R, BAFF, APRIL, BCMA, TACI, LeY, CD5, CD4, CD3, CD2, CD52, GD2, CD13, CD14, CD15 CD19, CD20, CD22, CD33, CD30, CD41, CD45, CD61, CD64, CD68, CD117, CD123, CD138, CD267, CD269, CD38, Flt3 receptor, CD4, CLL-1 and CS1 (SLAMF7).

[0017] In another embodiment, the present disclosure provides a modified polypeptide comprising a chimeric antigen receptor and an enhancer.

[0018] In another embodiment, the present disclosure provides a method of reducing the number of target cells, comprising: (i) contacting an effective amount of engineered cells having at least one chimeric antigen receptor polypeptide with the target cells, wherein the engineered cells have multiple chimeric antigen receptor polypeptides and each chimeric antigen receptor polypeptide is independent; and (ii) optionally, measuring a decrease in the number of the target cells. The target cells include at least one cell surface antigen selected from the group consisting of GD2, GD3, ROR1, PSMA, PSCA (prostate stem cell antigen), MAGE A3, glycolipid, glypican 3, F77, GD-2, WT1, CEA, HER-2 / neu, MAGE-3, MAGE-4, MAGE-5, MAGE-6, alpha-fetoprotein, CA 19-9, CA 72-4, NY-ESO, FAP, ErbB, c-Met, MART-1, MUC1, MUC2, MUC3, MUC4, MUC5, CD30, EGFRvIII, CD33, CD123, CLL-1, immunoglobulin kappa and lambda, CD38, CD52, CD19, CD20, CD22, CD38, BCMA, CS1, NKG2D receptor, April receptor, BAFF receptor, TACI, CD3, CD4, CD8, CD5, CD7, CD2, and CD138. The target antigens also include viral or fungal antigens such as E6 and E7 derived from human papillomavirus (HPV) or Epstein-Barr virus (EBV) antigens.

[0019] In another embodiment, the present disclosure provides a method of treating B cell lymphoma, T cell lymphoma, multiple myeloma, chronic myelogenous leukemia, acute myeloid leukemia, myelodysplastic syndrome, chronic myeloproliferative neoplasm, B cell acute lymphoblastic leukemia (B-ALL), acute plasmacytoid dendritic cell tumor, lung cancer, liver cancer, brain cancer, osteosarcoma, breast cancer, prostate cancer, and proliferative diseases, comprising administering to a patient in need of any of the engineered cells described above.

[0020] In another embodiment, the present disclosure provides a method for treating an autoimmune disease, the method comprising administering the engineered cells according to claim 1 to a patient in need thereof. Here, the autoimmune disease includes systemic lupus erythematosus (SLE), multiple sclerosis (MS), inflammatory bowel disease (IBD), rheumatoid arthritis, Sjogren's syndrome, dermatomyositis, autoimmune hemolytic anemia, neuromyelitis optica (NMO), NMO spectrum disorder (NMOSD), idiopathic thrombocytopenic purpura (ITP), systemic autoimmune small-vessel vasculitis syndrome or antineutrophil cytoplasmic autoantibody (ANCA) associated with microscopic polyangiitis (MPA), granulomatosis with polyangiitis (GPA, Wegener's granulomatosis), or eosinophilic granulomatosis with polyangiitis (EGPA, Churg-Strauss syndrome) and TTP (thrombotic thrombocytopenic purpura).

[0021] The present disclosure provides chimeric antigen receptors (CARs) targeting non-hematological malignancies, compositions thereof, and methods of use.

[0022] In one embodiment, the present disclosure provides engineered cells having a first chimeric antigen receptor polypeptide comprising a first antigen recognition domain, a first signal peptide, a first hinge region, a first transmembrane domain, a first co-stimulatory domain, and a first signaling domain, and having a second chimeric antigen receptor polypeptide comprising a second antigen recognition domain, a second signal peptide, a second hinge region, a second transmembrane domain, a second co-stimulatory domain, and a second signaling domain. Here, the first antigen recognition domain is different from the second antigen recognition domain.

[0023] In another embodiment, the present disclosure provides a modified polypeptide comprising a chimeric antigen receptor and an enhancer. In a further aspect, the enhancer can be selected from at least one of a group including, but not limited to, IL-2, IL-7, IL-12, IL-15, IL-15 / IL-15sush, IL-15 / IL-15sushi anchor, IL-15 / IL-15RA, IL-18, IL-21, IL-21 anchor, PD-1, PD-L1, CSF1R, CTAL-4, TIM-3, the cytoplasmic domain of the IL-15 receptor alpha, 4-1BBL, IL-21, IL-21 anchor, and the TGFR beta receptor.

[0024] In some embodiments, the CAR having an antigen recognition domain is part of an expression cassette. In a preferred embodiment, the expressed gene or cassette can include an accessory gene or tag or a part thereof. The accessory gene may be an inducible suicide gene or a part thereof including, but not limited to, the caspase 9 gene. The ablation approach by the "suicide gene" improves the safety of gene therapy and kills cells only when activated by a specific compound or molecule. In some embodiments, the epitope tag is a c-myc tag, CD52, streptavidin binding peptide (SBP), truncated EGFR gene (EGFRt), or a part or combination thereof.

[0025] In some embodiments, CAR cells can be removed by administering an anti-CD52 monoclonal antibody (CAMPATH) to the subject.

[0026] In another embodiment, the present disclosure provides a method for treating soft tissue tumors, carcinomas, sarcomas, leukemias, and cell proliferative diseases by administering any of the above engineered cells to a patient in need thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0027]

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Mode for Carrying Out the Invention

[0028] Detailed Description The present disclosure provides chimeric antigen receptor (CAR) compositions, methods and their production, and methods of using the CAR compositions.

[0029] Compositions Chimeric antigen receptor polypeptide In one embodiment, the present disclosure provides a chimeric antigen receptor (CAR) polypeptide having a signal peptide, an antigen recognition domain, a hinge region, a transmembrane domain, at least one co-stimulatory domain, and a signaling domain.

[0030] As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably and refer to a compound having amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that can be the sequence of the protein or peptide. A polypeptide includes any peptide or protein having two or more amino acids bonded to each other by peptide bonds. As used herein, short chains, generally referred to in the art as peptides, oligopeptides, and oligomers, and long chains, generally referred to in the art as proteins, of which there are many types, are referred to for both. “Polypeptide” includes, for example, among others, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins. Polypeptides include natural peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0031] A “signal peptide” is a peptide sequence that directs transport and localization and includes any peptide sequence that can bind to a certain cell organelle (such as the endoplasmic reticulum) and / or the cell surface.

[0032] In the present disclosure, a signal peptide is a peptide or polypeptide of any secretory protein or transmembrane protein that directs transport to the cell membrane and cell surface and provides accurate localization. In particular, the signal peptides of the present disclosure are the polypeptides of the present disclosure that direct to the cell membrane in the present disclosure, the extracellular portions of those polypeptides are presented on the cell surface, the transmembrane portions extend into the plasma membrane, and the active domains are present in the cytoplasmic portion or inside the cell.

[0033] In this example, the signal peptide is cleaved after passing through the endoplasmic reticulum (ER), i.e., it is a cleavable signal peptide. In this example, the signal peptide is a type I, type II, type III, or type IV human protein. In this example, the signal peptide includes an immunoglobulin heavy chain signal peptide.

[0034] The "antigen recognition domain" includes a polypeptide that is selective for a target antigen, receptor, peptide ligand, or protein ligand, or a polypeptide that is selective for the polypeptide targeted. The target-specific antigen recognition domain preferably comprises an antigen-binding domain that can target an antibody against the target antigen or a peptide that binds to the target antigen, or a peptide or protein that binds to an antibody that binds to the target antigen, or a peptide or protein ligand (including but not limited to growth factors, cytokines, or hormones) that binds to a receptor on the target, or a domain derived from a receptor that binds to a peptide or protein ligand that binds on the target (including but not limited to growth factor receptors, cytokine receptors, or hormone receptors). The targets include GD2 and GD3. In another embodiment, the target comprises any portion of GD2 and GD3. In another embodiment, the target is ganglioside GD2, and its structure is GD2 = bDGalpNAc(1-4)[aNeu5Ac(2-8)aNeu5Ac(2-3)] bDGalp(1-4)bDGlcp(1-1)Cer. In another embodiment, the target is ganglioside GD3, and its structure is GD3 = aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer.

[0035] In this example, the antigen recognition domain comprises the binding portion or variable region of a (selective) monoclonal or polyclonal antibody against the target.

[0036] In one embodiment, the antigen recognition domain comprises an antigen-binding fragment (Fab). In another embodiment, the antigen recognition domain comprises a single-chain variable fragment (scFv). The scFv is a fusion protein of the variable regions of the heavy chain (VH) and light chain (VL) of an immunoglobulin, connected by a short linker peptide.

[0037] In another embodiment, the antigen recognition domain comprises a single domain antibody of camelidae or a portion thereof. In this embodiment, the camelidae single domain antibody comprises a heavy chain antibody found in camelidae animals, or a VHH antibody. Camel VHH antibodies (e.g., camel, dromedary, llama, and alpaca) refer to the variable fragments of camelidae single chain antibodies (see Nguyen et al., 2001; Muyldermans, 2001), and also include isolated VHH antibodies of camel, recombinant VHH antibodies of camel, or synthetic VHH antibodies of camel, etc.

[0038] In another embodiment, the antigen recognition domain comprises a ligand involved in their cognate receptor. In another embodiment, the antigen recognition domain is humanized.

[0039] It is understood that the antigen recognition domain can contain some variability within its sequence and still be selective for the targets disclosed herein. Thus, the polypeptide of the antigen recognition domain is at least 95%, at least 90%, at least 80%, or at least 70% identical to the antigen recognition domain polypeptide disclosed herein and is still considered to be within the scope of the present disclosure as described herein.

[0040] In another embodiment, the antigen recognition domain is selective for ganglioside GD2 and ganglioside GD3.

[0041] The hinge region is located between sequences including, but not limited to, for example, chimeric antigen receptors, and at least one co-stimulatory domain and signaling domain. The hinge sequence can be obtained from any suitable sequence from any genus including, for example, humans or a part thereof. Such hinge regions are known in the art. In this example, the hinge region includes the hinge regions of human proteins including CD-8 alpha, CD28, 4-1BB, OX40, CD3-zeta, T cell receptor alpha or beta chain, CD3 zeta chain, CD28, CD3ε, CD45, CD4, CD5, CD8, CD8a, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, their functional derivatives, and combinations thereof.

[0042] In this example, the hinge region includes the CD8 hinge region.

[0043] In some examples, the hinge region includes, but is not limited to, those selected from immunoglobulins (e.g., IgG1, IgG2, IgG3, IgG4, and IgD).

[0044] The transmembrane domain includes a hydrophobic polypeptide spanning the cell membrane. In particular, the transmembrane domain spans from one side of the cell membrane (extracellular) to the other side of the cell membrane (intracellular or cytoplasmic).

[0045] The transmembrane domain can be in the shape of an alpha helix or a beta barrel, or a combination thereof. The transmembrane domain includes many transmembrane segments, polytopic proteins having each alpha - helix, beta sheet, or a combination thereof.

[0046] In this example, a transmembrane domain originally associated with one of the domains of the CAR structure is used. In another example, to avoid the transmembrane domains of the same or different surface membrane proteins and the binding of such domains, or to minimize the interaction with other members of the receptor complex, the transmembrane domain is modified by selection or amino acid substitution.

[0047] For example, the transmembrane domain includes the transmembrane domains of the T cell receptor α or β chain, CD3 zeta chain, CD28, CD3ε, CD45, CD4, CD5, CD7, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD68, CD134, CD137, ICOS, CD41, CD154, their functional derivatives, and combinations thereof.

[0048] An artificially designed transmembrane domain is a polypeptide mainly containing hydrophobic residues such as leucine and valine. In one embodiment, a triplet of phenylalanine, tryptophan, and valine is found at each end of the synthetic transmembrane domain.

[0049] In this example, the transmembrane domain is the CD8 transmembrane domain. In another example, the transmembrane domain is the CD28 transmembrane domain. Such transmembrane domains are known in the art.

[0050] The signaling domain and the costimulatory domain include polypeptides for activating immune cells or at least activating the immune cell signaling pathway in at least some aspects.

[0051] In this example, the signaling domain includes polypeptides of those functional signaling domains, including CD3 zeta, common FcRγ (FCER1G), FcγRIIa, FcRβ (Fc epsilon Rib), CD3 gamma, CD3 delta, CD3 epsilon, CD79a, CD79b, DNAX activation protein 10 (DAP10), DNAX activation protein 12 (DAP12), their active fragments, their functional derivatives, and combinations thereof. Such signaling domains are known in the art.

[0052] In this example, the CAR polypeptide comprises one or more co-stimulatory domains. In one embodiment, the co-stimulatory domain is selected from at least one protein including, but not limited to, IL-15 receptor alpha; IL-15 receptor alpha cytoplasmic domain; B7-1 / CD80, CD28, 4-1BB, 4-1BBL, B7-2 / CD86, CTLA-4, B7-H1 / PD-L1, ICOS; B7-H2, PD-1, B7-H3, PD-L2, B7-H4, PDCD6, BTLA, 4-1BB / TNFRSF9 / CD137, CD40 ligand / TNFSF5, 4-1BB ligand / TNFSF9, GITR / TNFRSF18, BAFF / BLyS / TNFSF13B, GITR ligand / TNFSF18, BAFF R / TNFRSF13C, HVEM / TNFRSF14, CD27 / TNFRSF7, LIGHT / TNFSF14, CD27 ligand / TNFSF7, OX40 / TNFRSF4, CD30 / TNFRSF8, OX40 ligand / TNFSF4, CD30 ligand / TNFSF8, TACI / TNFRSF13B, CD40 / TNFRSF5, 2B4 / CD244 / SLAMF4, CD84 / SLAMF5, BLAME / SLAMF8, CD229 / SLAMF3, CD2, CD27, CRACC / SLAMF7, CD2F-10 / SLAMF9, NTB-A / SLAMF6, CD48 / SLAMF2, SLAM / CD150, CD58 / LFA-3, Ikaros, CD53, integrin alpha4 / CD49d, CD82 / Kai-1, integrin alpha4beta1, CD90 / Thy1, integrin alpha4beta7 / LPAM-1, CD96, LAG-3, CD160, LMIR1 / CD300A, CRTAM, TCL1A, DAP12, TIM-1 / KIM-1 / HAVCR, dectin-1 / CLEC7A, TIM-4, DPPIV / CD26, TSLP, EphB6, TSLP R and HLA-DR, which is a functional signaling domain.

[0053] The present disclosure further provides a polynucleotide encoding the above chimeric antigen receptor polypeptide. The polynucleotide encoding the CAR can be readily prepared from the amino acid sequence of a particular CAR by any conventional method. The nucleotide sequence encoding the amino acid sequence can be obtained from the aforementioned NCBI RefSeq ID or the amino acid sequence of each domain from the GenBank accession number, and the nucleic acids of the present disclosure can be prepared using standard molecular biological and / or chemical procedures. For example, based on the nucleotide sequence, polynucleotides can be synthesized, and the polynucleotides of the present disclosure can be prepared by combining DNA fragments obtained from a cDNA library using the polymerase chain reaction (PCR).

[0054] In one embodiment, the polynucleotides disclosed herein are part of a gene, or an expression or cloning cassette.

[0055] As used herein, the term "polynucleotide" is defined as a chain of nucleotides. Polynucleotides include DNA and RNA. Further, nucleic acids are polymers of nucleotides. Thus, as used herein, nucleic acids and polynucleotides are also synonymous. One of ordinary skill in the art has the general knowledge that nucleic acids are polynucleotides and can be hydrolyzed to monomeric "nucleotides". Monomeric nucleotides can be hydrolyzed to nucleosides. The polynucleotides used herein include, but are not limited to, all nucleic acid sequences obtained by any means available in the art, such as cloning by recombinant libraries or conventional cloning techniques from cells and synthetic means such as the polymerase chain reaction (PCR).

[0056] Polynucleotide vector The above polynucleotide can be cloned into a vector. A "vector" is a composition of substances that contains an isolated polynucleotide and can be used to deliver the isolated polynucleotide into the interior of a cell. A number of vectors are known in the art, including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, phagemids, cosmids, and viruses. Viruses include phages and phage derivatives. Thus, the term "vector" includes autonomously replicating plasmids or viruses. This term should also be construed to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells, such as, for example, polylysine compounds and liposomes. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, and lentiviral vectors.

[0057] In this example, vectors include cloning vectors, expression vectors, replication vectors, probe generation vectors, integration vectors, and sequencing vectors.

[0058] In this example, the vector is a viral vector. In this example, the viral vector is a retroviral vector or a lentiviral vector. In one embodiment, the engineered cell is transduced with a virus to express a polynucleotide sequence.

[0059] Many virus-based systems for gene delivery into mammalian cells have been developed. For example, retroviruses provide a convenient platform for gene delivery systems. The selected gene is inserted into a vector and packaged into retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to the patient's cells by either in vivo or ex vivo means. Many retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. Many adenoviral vectors are known in the art. In this embodiment, lentiviral vectors are used.

[0060] Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals. Viruses useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses. Generally, suitable vectors include an origin of replication that functions in at least one living organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers (e.g., WO 01 / 96584; WO 01 / 29058; and U.S. Patent No. 6,326,193).

[0061] The expression of chimeric antigen receptor polynucleotides is achieved using, for example, but not limited to, the SFFV (spleen focus forming virus) or human elongation factor 11α (EF) promoter, the CAG (chicken β-actin promoter with CMV enhancer) promoter, or the human elongation factor 1α (EF) promoter. Examples of low-intensity / low-expression promoters include, but are not limited to, the simian virus 40 (SV40) early promoter, the cytomegalovirus (CMV) immediate early promoter, the ubiquitin C (UBC) promoter, and the phosphoglycerate kinase 1 (PGK) promoter, or portions thereof, including but not limited to these, are used. Inducible expression of chimeric antigen receptors may be achieved, for example, but not limited to, using tetracycline-responsive promoters including TRE3GV (Tet response element including all generations, preferably the third generation), inducible promoters (Clontech Laboratories, Mountain View, CA), or portions or combinations thereof.

[0062] An example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of inducing high-level expression of any operable polynucleotide sequence linked thereto. Another example of a suitable promoter is elongation growth factor-1a (EF-1a). However, simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein - Barr virus immediate early promoter, Rous sarcoma virus promoter, and human gene promoters such as actin promoter, myosin promoter, hemoglobin promoter, creatine kinase promoter, etc. are included, but not limited to these, and other constitutive promoter sequences are also used. Furthermore, the present disclosure should not be limited to the use of constitutive promoters, and inducible promoters are also considered as part of the present disclosure. By using an inducible promoter, a molecular switch that can turn on the expression when the expression of the linked polynucleotide sequence is desired, or a molecular switch that can turn off the expression when the expression is not desired, is provided. Examples of inducible promoters include, but are not limited to, metallothionein promoter, glucocorticoid promoter, progesterone promoter, and tetracycline promoter.

[0063] An "expression vector" refers to a vector containing a recombinant polynucleotide expression control sequence operably linked to a nucleotide sequence to be expressed. The expression vector contains sufficient cis-acting elements for expression, and other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentivirus, retrovirus, adenovirus, and adeno-associated virus) that incorporate the recombinant polynucleotide.

[0064] Additional promoter elements, such as enhancers, regulate the frequency of transcription initiation. Usually, these are located in the region 30 - 100 bp upstream of the start site, but recently many promoters have been shown to contain functional elements also downstream of the start site. Since the spacing between promoter elements is often flexible, the promoter function is maintained even when the elements are inverted or moved relative to each other. For the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased by 50 bp before a decrease in activity begins. Depending on the promoter, individual elements can apparently function either cooperatively or independently to activate transcription.

[0065] To assess the expression of the CAR polypeptide or a portion thereof, the expression vector introduced into the cells may contain a selectable marker gene and / or a reporter gene and be transfected or infected via a viral vector that can also facilitate the identification and selection of expressing cells from a cell population. In another aspect, where an attempt is made to transfect or infect via a viral vector, the selectable marker may be carried on a separate DNA and used in a cotransfection procedure. Appropriate regulatory sequences that allow expression in the host cell may flank both the selectable marker and the reporter gene. Useful selectable markers include, for example, antibiotic resistance genes such as neo.

[0066] Reporter genes are used to identify cells that may have been transfected and to evaluate the function of regulatory sequences. In general, a reporter gene is a gene that is not present or expressed in the recipient organism or tissue and encodes a polypeptide whose expression is manifested by some easily detectable property, such as enzyme activity. The expression of the reporter gene is assayed at an appropriate time after the DNA has been introduced into the recipient cells. Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein genes (e.g., Ui-Tei et al., 2000 FEBS Letters 479: 79-82). Suitable expression systems are well known and can be prepared using known techniques or obtained commercially. In general, a construct with the minimal 5' flanking region that shows the highest level of expression of the reporter gene is identified as the promoter. Such promoter regions can be linked to the reporter gene and used to evaluate, as a proxy, the ability to regulate promoter-driven transcription.

[0067] Methods for introducing and expressing genes into cells are known in the art. In the context of expression vectors, the vectors can be readily introduced into host cells, such as mammalian, bacterial, yeast, or insect cells, by any method in the art. For example, the expression vectors can be transferred into host cells by physical, chemical, or biological means.

[0068] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for generating cells containing vectors and / or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). A preferred method for introducing polynucleotides into host cells is calcium phosphate transfection.

[0069] Biological methods for introducing a polynucleotide of interest into host cells include the use of DNA and RNA vectors. Viral vectors, particularly retroviral vectors, have become the most widely used method for inserting genes into mammals such as human cells. Other viral vectors can be derived from, for example, lentivirus, poxvirus, herpes simplex virus I, adenovirus, and adeno-associated virus. See, for example, U.S. Patent Nos. 5,350,674 and 5,585,362.

[0070] Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems such as polymer complexes, nanocapsules, microspheres, beads, and lipid-based systems including water-in-oil emulsions, micelles, mixed micelles, and liposomes. Exemplary colloidal systems for use as delivery vehicles in vitro and in vivo are liposomes (e.g., artificial membrane vesicles). When non-viral delivery systems are utilized, an exemplary delivery vehicle is a liposome. The use of lipid formulations is contemplated for the introduction of nucleic acids into host cells (in vitro, ex vivo or in vivo). In another aspect, the nucleic acid may be associated with a lipid. Nucleic acids involved with lipids are dispersed within the lipid bilayer of liposomes, attached to liposomes via linking molecules associated with both liposomes and oligonucleotides, entrapped within liposomes, complexed with liposomes, dispersed in a lipid-containing solution, mixed with lipids, combined with lipids, included as a suspension in lipids, included or complexed with micelles, or encapsulated within the aqueous interior of something associated with lipids. Lipid, lipid / DNA, or lipid / expression vector-related compositions are not limited to a particular structure in solution. For example, they may exist as micelles or in a bilayer structure with a "disrupted" structure. They may also simply be dispersed in solution and form aggregates that are not uniform in size or shape. Lipids are fatty substances that may be natural or synthetic lipids. For example, lipids include fatty droplets that occur naturally in the cytoplasm and classes of compounds including long-chain aliphatic hydrocarbons and their derivatives such as fatty acids, alcohols, amines, amino alcohols, aldehydes, etc.

[0071] Lipids suitable for use are available from commercial sources. For example, dimyristoylphosphatidylcholine ("DMPC") is available from Sigma in St. Louis, Missouri, dicetyl phosphate ("DCP") is available from K&K Laboratories (Plainview, New York), cholesterol ("Choi") is available from Calbiochem-Behring, and dimyristoylphosphatidylglycerol ("DMPG") and other lipids can be obtained from Avanti Polar Lipids, Inc. (Birmingham, Alabama). Stock solutions of lipids in chloroform or chloroform / methanol can be stored at about -20°C. Since chloroform evaporates more readily than methanol, it is used as the sole solvent.

[0072] "Liposome" is a general term encompassing lipid media of various single and multi-layer lipids formed by the formation of enclosed lipid bilayers or aggregates. Liposomes can be characterized as having a vesicular structure with a phospholipid bilayer membrane and an internal aqueous medium. Multilamellar liposomes have multiple lipid layers separated by an aqueous medium. They are formed spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-arrangement before a closed structure is formed, trapping water and solutes between the lipid bilayers (Ghosh et al., 191 Glycobiology 5; 505 - 10). However, compositions with solution structures different from normal vesicular structures are also included. For example, the lipids may form micellar structures or exist only as heterogeneous aggregates of lipid molecules. Lipofectamine-nucleic acid complexes are also conceivable.

[0073] Regardless of the method used to introduce an exogenous polynucleotide into a host cell or to expose a cell to a polynucleotide of the present disclosure, various assays can be performed to confirm the presence of a recombinant DNA sequence in the host cell. Such assays include, for example, "molecular biology" assays well known to those skilled in the art, such as Southern and Northern blotting, RT-PCR, and PCR, and "biochemical" assays for detecting the presence of specific peptides, such as immunological means (ELISA and Western blot), or the assays described herein for identifying agents within the scope of the present disclosure.

[0074] engineered cell In another embodiment, the present disclosure provides an engineered cell that expresses the chimeric antigen receptor polypeptide described above or encodes the same and expresses the polynucleotide described above.

[0075] "Engineered cell" means any cell of a living organism that is modified, transformed, or engineered by the addition or modification of a gene, DNA or RNA sequence, or protein or polypeptide. The isolated cells, host cells, and genetically engineered cells of the present disclosure contain a DNA or RNA sequence encoding a chimeric antigen receptor or chimeric antigen receptor complex and include isolated immune cells, such as NK cells and T cells, that express the chimeric receptor on the cell surface. The isolated host cells and engineered cells can be used, for example, to enhance NK cell activity or T lymphocyte activity, treat cancer, and treat infectious diseases.

[0076] Any cell that can express and / or incorporate the chimeric antigen receptor polypeptide disclosed herein into its membrane can be used.

[0077] In this example, the engineered cells include immunomodulatory cells. Immunomodulatory cells include T cells such as CD4 T cells (helper T cells), CD8 T cells (cytotoxic T cells, CTLs), and memory T cells or memory T stem cells. In another example, the T cells include natural killer T cells (NK T cells).

[0078] T cells are composed of CD4 and CD8 cells. CD4 is a glycoprotein present on the surface of immune cells such as helper T cells and is important for T cell activation and the receptor for HIV. Some monocytes or macrophages also express CD4. CD4 is also called OKT4. Cytotoxic T cells are also known as CD8 + T cells or CD8 T cells that express the CD8 glycoprotein on their surface. These CD8 + T cells are activated when exposed to peptide antigens presented by MHC class I.

[0079] In one embodiment, the engineered cells include NK T cells. NK T cells are well known in the art.

[0080] In one embodiment, the engineered cells include natural killer cells. Natural killer cells are well known in the art. In one embodiment, natural killer cells include cell lines such as NK-92 cells. Further examples of NK cell lines include NKG, YT, NK-YS, HANK-1, YTS cells, and NKL cells.

[0081] NK cells mediate an anti-tumor effect without the risk of GvHD and have a shorter lifespan compared to T cells. Therefore, NK cells are used up soon after destroying cancer cells, reducing the need for an inducible suicide gene on the CAR construct to remove the engineered cells. As used herein, CDXCAR refers to a chimeric antigen receptor having a CDX antigen recognition domain. As used herein, CDX can be either GD2 or GD3.

[0082] TCR-deficient T cells used to lodge CAR In one embodiment, modified cells, particularly allogeneic T cells obtained from a donor, can be modified to inactivate components of the TCR (T cell receptor) involved in MHC recognition. As a result, TCR-deficient T cells do not cause graft-versus-host disease (GVHD).

[0083] Cell source Modified cells may be obtained from peripheral blood, umbilical cord blood, bone marrow, tumor-infiltrating lymphocytes, lymph node tissue, or thymus tissue. Host cells may include placental cells, embryonic stem cells, induced pluripotent stem cells, or hematopoietic stem cells. Cells can be obtained from humans, monkeys, chimpanzees, dogs, cats, mice, rats, and their transgenic species. Cells can be obtained from established cell lines.

[0084] The above cells can be obtained by any known means. The cells may be autologous, syngeneic, allogeneic, or xenogeneic to the recipient of the modified cells. The term "autologous" refers to any material derived from the same individual that is later reintroduced into that individual.

[0085] The term "allogeneic" refers to a material derived from a different animal of the same species as the individual into which the substance is introduced. When the genes at one or more loci are not identical, two or more individuals are said to be allogeneic to each other. In some aspects, allogeneic materials from individuals of the same species can be sufficiently different to interact antigenically genetically.

[0086] The term "xenogeneic" refers to a graft derived from an animal of a different species.

[0087] The term "syngeneic" refers to genetic similarity or identity that is extremely similar, particularly with respect to antigens or immunological reactions. Syngeneic includes, for example, models in which an organ and a cell (e.g., a cancer cell and its non-cancerous counterpart) are derived from the same individual, and / or models in which an organ and a cell are derived from different individual animals of the same inbred strain.

[0088] In certain embodiments, the T and NK cells are derived from human peripheral blood mononuclear cells (PBMCs), peripheral blood stem cell products (PBSCs), human embryonic stem cells (hESCs), induced pluripotent stem cells (iPSCs), bone marrow, or umbilical cord blood.

[0089] Potential drawbacks of using NK cells in CAR therapy include a lack of persistence that may reduce long-term efficacy.

[0090] Finding matching donor T cells for generating CAR T cells is difficult because mismatched T cells can attach to the recipient's tissues and cause graft-versus-host disease (GVHD).

[0091] Recent studies have shown that CRISPR-Cas9-mediated gene editing for generating universal CAR T cells increases cancer risk by creating unintended mutations and disrupting the function of the p53 repair protein. Given this risk, it is important to explore ways to avoid genome editing when planning CAR therapy for patients. Natural killer (NK) cells are an ideal platform for creating universal CARs that avoid risks associated with genome editing. However, the average lifespan of NK CAR cells in vivo is very short, with a lifespan of approximately one week.

[0092] In one embodiment, the disclosure includes a method of generating chimeric antigen receptor (CAR)-modified NK cells having long lifespan or long-term persistence in vivo for treating a disease. Surprisingly, it has been found that CAR NK cells co-expressing IL-15 / IL-15sushi or IL-15 / IL-15 sushi anchor extend long-term survival.

[0093] In further embodiments, extension of the survival of CAR NK cells can be achieved by co-expressing an IL-15 / IL-15 anchor.

[0094] In some embodiments, CAR NK cells co-expressing IL-15 / IL-15sushi or IL-15 / IL-15sushi anchor can be scaled up and used as an off-the-shelf product.

[0095] In one embodiment, CAR NK cells co-expressing IL-15 / IL-15sushi or IL-15 / IL-15sushi anchor can continue supportive cytokine signaling, which is important for post-injection survival in patients.

[0096] In some embodiments, CAR NK T cells co-expressing IL-15 / IL-15sushi or IL-15 / IL-15sushi anchor can be scaled up and used as an off-the-shelf product.

[0097] In one embodiment, CAR NK T cells co-expressing IL-15 / IL-15sushi or IL-15 / IL-15sushi anchor can continue supportive cytokine signaling, which is important for post-injection survival in patients.

[0098] In a further embodiment, the extension of CAR NK cell survival can be achieved by co-expressing a cytokine selected from the group consisting of IL-7, IL-15, IL-15 / IL-15 anchor, IL-15 / IL-15RA IL-12, IL-18, and IL-21.

[0099] In one embodiment, CAR NK T cells co-expressing IL-15 / IL-15sushi or IL-15 / IL-15sushi anchor can continue supportive cytokine signaling, which is important for post-injection survival in patients.

[0100] In a further embodiment, the extension of CAR NK cell survival can be achieved by co-expressing a cytokine selected from the group consisting of IL-7, IL-15, IL-15 / IL-15 anchor, IL-15 / IL-15RA, IL-12, IL-18, and IL-21.

[0101] Natural killer T (NK T) cells are a group of T cells that share the characteristics of both T cells and natural killer cells.

[0102] In one embodiment, the IL-15 product is modified to generate a disulfide bond that links the IL-15 / sushi domain complex to an Fc region such as IgG1. In this embodiment, the IL-15 / sushi complex can be linked to the Fc region, which forms a dimer having a disulfide bridge that links the two molecules. In one embodiment, leucine 52 of IL-15 is replaced with cysteine, and serine 40 of the Sushi domain is replaced with cysteine.

[0103] In one embodiment, the inducible promoter causes expression upon activation of a cellular pathway such as the T cell receptor pathway. In this embodiment, the expression of the gene of interest is induced by activation of a similar pathway that is activated by the T cell receptor or a CAR. Those skilled in the art will understand that this includes promoters such as those under the control of the nuclear factor of activated T cells (NFAT) promoter, which includes a portion of the well-described IL-2 promoter or a synthetic promoter consisting of an NFAT binding motif, or a synthetic promoter consisting of an NFAT binding motif. These are merely examples of inducible promoters and are not limiting.

[0104] uCAR NK cells Allogeneic CAR T cells can cause GVHD (graft-versus-host disease) in recipients, so most current clinical trials or treatments involve injecting autologous CAR T cells. This autologous approach has had clinically significant success, but the manufacturing process for patient-specific T cell products is time-consuming and expensive. Furthermore, it is not always possible to obtain sufficient T cells from patients who have received intensive treatment in order to successfully produce a sufficient dose of CAR T cells. There is a great need for the development of autologous allogeneic CAR products. NK cells are similar to T cells in that they are highly cytotoxic immune effectors. In contrast to T cells, NK cells have the property of killing targets in a specific way. NK cells can be used as autologous allogeneic products because they usually do not have the potential to cause GVHD. The main drawback of using NK cells is their lack of persistence in vivo, with a half-life of only about one week.

[0105] In some embodiments, the present invention discloses forms of universal CAR-expressing NK cells or NK T cells derived from healthy donors that can be stored and then injected into an individual upon request. In further embodiments, the present invention includes methods for producing a universal CAR NK product from allogeneic healthy donors that can be injected into any patient without causing GVHD.

[0106] In some embodiments, the NK cells or NK T cells are obtained from cord blood banks and peripheral blood banks. In further embodiments, the NK cells are induced pluripotent stem cells or embryonic stem cells or NK-92 cells.

[0107] In some embodiments, the present disclosure includes methods having a CAR or compound CAR (cCAR) that co-expresses IL-15 / IL-15sushi in NK cells. These engineered NK cells are called uCAR NK cells.

[0108] In some embodiments, the uCAR NK cells have a CAR or cCAR that co-expresses IL-15 / IL-15sushi. In further embodiments, the uCAR NK cells can persist in vivo for more than one week.

[0109] In some embodiments, the disclosure includes methods for uCAR NK cells having a vector that expresses a CAR or cCAR comprising IL-15 / IL-15sushi.

[0110] In some embodiments, the co-expression of IL-15 / IL-15sushi with the CAR or cCAR provides for long-term persistence of NK cells in a subject.

[0111] In some embodiments, the co-expression of the CAR or cCAR and IL-15 / IL-15sushi results in long-term remission in a patient by enhancing the sensitivity of CAR recognition of target cancer cells or by recruiting innate immune cells to the cancer cells.

[0112] In some embodiments, the present disclosure includes a method of generating NK cells having one CAR or cCAR that co-expresses IL-15 / IL-15sushi. In further embodiments, specific tumor antigens targeted by the antigen recognition domain within the CAR are GD2, GD3, interleukin 6 receptor, FSHR, ROR1, PSMA, PSCA (prostate stem cell antigen), MAGE A3, glycolipid, glypican 3, F77, WT1, CEA, HER-2 / neu, MAGE-3, MAGE-4, MAGE-5, MAGE-6, alpha-fetoprotein, CA 19-9, CA 72-4, NY-ESO, FAP, ErbB, c-Met, MART-1, MUC1, MUC2, MUC3, MUC4, MUC5, MMG49 epitope, CD30, EGFRvIII, CD33, CD123, CLL-1, NKG2D, NKG2D receptor, immunoglobulin kappa and lambda, CD38, CD52, CD47, CD200, CD70, CD56, CD19, CD20, CD22, CD38, BCMA, CS1, BAFF receptor, TACI, CD3, CD4, CD8, CD5, CD7, CD2, and CD138, and may be selected from the group consisting of, but not limited to, these.

[0113] In some embodiments, the present disclosure includes a method of treating a disorder or disease by injecting, in a therapeutically effective amount, NK cells genetically engineered to express IL-15 / IL-15sushi and / or a CAR having an antigen recognition domain for a specific tumor antigen. In further embodiments, the specific tumor antigen targeted by the antigen recognition domain can be selected from the group consisting of GD2, GD3, interleukin 6 receptor, FSHR, ROR1, PSMA, PSCA (prostate stem cell antigen), MAGE A3, glycolipids, glypican 3, F77, WT1, CEA, HER-2 / neu, MAGE-3, MAGE-4, MAGE-5, MAGE-6, alpha-fetoprotein, CA 19-9, CA 72-4, NY-ESO, FAP, ErbB, c-Met, MART-1, MUC1, MUC2, MUC3, MUC4, MUC5, MMG49 epitope, CD30, EGFRvIII, CD33, CD123, CLL-1, NKG2D, NKG2D receptor, immunoglobulin kappa and lambda, CD38, CD52, CD47, CD200, CD70, CD56, CD19, CD20, CD22, CD38, BCMA, CS1, BAFF receptor, TACI, CD3, CD4, CD8, CD5, CD7, CD2, and CD138, but is not limited thereto. In some embodiments, administration of high doses of uCAR NK cells can cause cytokine release syndrome (CRS). The present disclosure includes methods of reducing or avoiding CRS by providing low doses or split doses of uCAR NK cells to a subject.

[0114] Suicide and safety switch systems The modified cells of the present disclosure may also include a suicide system. The suicide system provides a mechanism by which the cells engineered as described above are inactivated or destroyed. Such a feature allows for precise therapeutic control of any treatment in which the engineered cells are used. As used herein, a suicide system provides a mechanism that can inactivate or destroy cells having the suicide system. Suicide systems are well known in the art.

[0115] In one embodiment, the suicide system comprises a gene that can be pharmacologically activated to remove the contained cells as needed. In certain aspects, the suicide gene is not immunogenic to the polynucleotide or the host harboring the cells. In one example, the suicide system comprises a gene that expresses CD20 on the cell surface of the genetically engineered cells. Thus, administration of rituximab can be used to destroy the engineered cells containing the gene.

[0116] In some embodiments, the suicide system comprises an epitope tag. Examples of epitope tags include the c-myc tag, the CD52 streptavidin-binding peptide (SBP), and the truncated EGFR gene (EGFRt). In this embodiment, the epitope tag is expressed in the engineered cells. Thus, administration of an antibody against the epitope tag can be used to destroy the engineered cells containing the gene.

[0117] In another embodiment, the suicide system comprises a gene that expresses a truncated epidermal growth factor receptor on the surface of the engineered cells. Thus, administration of cetuximab can be used to destroy the engineered cells containing the gene.

[0118] In another embodiment, the suicide system comprises CD52 expressed on the surface of the engineered cells. Thus, administration of an anti-52 monoclonal antibody (CAMPATH, alemtuzumab) can destroy the artificial cells containing the gene.

[0119] In another embodiment, the suicide system comprises CAMPATH (alemtuzumab). Thus, due to the high expression of CD52 by CAR T cells or T cells, administration of an anti-52 monoclonal antibody (CAMPATH) can destroy the engineered cells without expressing the tag or gene.

[0120] In another embodiment, the suicide gene may include caspase 8 gene, caspase 9 gene, thymidine kinase, cytosine deaminase (CD), or cytochrome P450.

[0121] Examples of additional suicide systems include those described by Jones et al. (Jones BS, Lamb LS, Goldman F and Di Stasi A (2014) Improving the safety of cell therapy products by suicide gene transfer. Front. Pharmacol. 5:254. doi: 10.3389 / fphar.2014.00254).

[0122] Compound CAR (cCAR) As used herein, compound CAR (cCAR) or multiple CAR refers to engineered cells having at least two complete and distinct chimeric antigen receptor polypeptides. As used herein, "different chimeric antigen receptor polypeptides" have unique antigen recognition domains, signal peptides, hinge regions, transmembrane domains, at least one co-stimulatory domain, and signaling domains. Thus, two unique chimeric antigen receptor polypeptides will have different antigen recognition domains. The signal peptide, hinge region, transmembrane domain, at least one co-stimulatory domain, and signaling domain may be the same or different between two different chimeric antigen receptor polypeptides. As used herein, a chimeric antigen receptor (CAR) unit refers to different chimeric antigen receptor polypeptides, or polynucleotides encoding the same.

[0123] As used herein, a unique antigen recognition domain is either specific for a target or one that targets a single target or a single epitope of a target.

[0124] As used herein, a compound CAR in context. A single chimeric antigen receptor polypeptide has only a single unique antigen recognition domain. By way of further explanation, this single antigen recognition domain recognizes and binds only a single antigen or a single antigen epitope.

[0125] In some embodiments, the compound CARs target the same antigen. For example, cCARs target different epitopes or portions of a single antigen. In some embodiments, each of the CAR units present in the compound CAR targets a different antigen specific to the same or different disease states or side effects caused by a disease state.

[0126] In some embodiments, the compound CAR targets two different antigens. The design of compound CARs with different CAR units is very challenging. (1) CAR-CAR interactions may have a detrimental effect, and appropriate CAR design is the key to offsetting this adverse effect. (2) Compound CARs of a single construct may increase the length of the expression cassette, which may result in a decrease in viral titer and protein expression levels. (3) An appropriate design containing various CAR body elements is required to select a strategy for expressing multiple CARs within a single vector. (4) A strong promoter is particularly important for compound CARs with additional units of CAR. (5) The hinge region of the CAR needs to be designed so that interactions between the hinge regions of each CAR unit are avoided as much as possible. (6) Two or more CAR units expressed in cells may cause toxic effects (CAR-CAR interactions). The applicant provides novel and surprising CAR compositions and methods for overcoming these hurdles.

[0127] The transduction efficiency of cCAR (the proportion of CAR T cells) is often lower than that of a single unit CAR. There are several ways to improve efficiency in both the transfection and transduction steps. To improve the viral titer for producing cCAR, it is preferable to use the LentiX™ 293 T (Clontech / Takara) packaging cell line selected for high-titer lentivirus production instead of the commonly used HEK-293FT. Also, to increase the transfection efficiency, when transfecting the packaging cells, it is preferable to increase the amount of plasmid DNA (including the cCAR construct) by 1.5 to 2.0 times. The amounts of the viral packaging plasmid and the transfection reagent remain the same during complex formation. The transduction efficiency can be further enhanced by reducing the ratio of T cells to the viral vector to 0.3 x 10 6 cells per mL and increasing the amount of the lentivirus supernatant or lentivirus.

[0128] In this example, the present disclosure provides engineered cells having multiple CAR units. This enables a single engineered cell to target multiple antigens. By targeting multiple surface markers or antigens simultaneously with multiple CAR units, the selection of resistant clones is hindered and tumor recurrence is reduced. Multiple CAR T cell immunotherapy using individual component CARs containing various domains and activation sites has not yet been developed for any malignant tumors.

[0129] In one aspect of the invention, cCAR includes multiple CAR units. In some examples, cCAR includes at least two CAR units. In another example, cCAR includes at least three CAR units. In another example, cCAR includes at least four units.

[0130] In one embodiment, the present disclosure provides engineered cells having at least two distinct chimeric antigen receptor polypeptides, each having a different antigen recognition domain.

[0131] In this example, the engineered cells having at least two different chimeric antigen receptor polypeptides are T cells or NK T cells. The T cells can be engineered not to express cell surface antigens. For example, the T cells can be engineered not to express the CD45 cell surface antigen.

[0132] In a preferred example, the engineered cells having at least two different chimeric antigen receptor polypeptides are primary NK cells or NK T cells isolated from peripheral blood or cord blood, and are also NK-92 cells, and are administered as "off-the-shelf" products to any mammal having a disease or cancer.

[0133] In this example, the engineered cells comprise (i) a first chimeric antigen receptor polypeptide comprising a first antigen recognition domain, a first signal peptide, a first hinge region, a first transmembrane domain, a first co-stimulatory domain, and a first signaling domain, and (ii) a second chimeric antigen receptor polypeptide comprising a second antigen recognition domain, a second signal peptide, a second hinge region, a second transmembrane domain, a second co-stimulatory domain, and a second signaling domain. The first antigen recognition domain is different from the second antigen recognition domain.

[0134] In a preferred example, each engineered CAR unit polynucleotide has a different nucleotide sequence to avoid homologous recombination.

[0135] In one embodiment, the targets of the first antigen recognition domain include, but are not limited to, GD2, GD3, interleukin 6 receptor, ROR1, PSMA, PSCA (prostate stem cell antigen), MAGE A3, glycolipid, glypican 3, F77, GD-2, WT1, CEA, HER-2 / neu, MAGE-3, MAGE-4, MAGE-5, MAGE-6, alpha-fetoprotein, CA 19-9, CA 72-4, NY-ESO, FAP, ErbB, c-Met, MART-1, MUC1, MUC2, MUC3, MUC4, MUC5, CD30, EGFRvIII, CD33, CD123, CLL-1, NKG2D, NKG2D receptor, immunoglobulin kappa and lambda, CD38, CD52, CD19, CD20, CD22, CD38, BCMA, CS1, BAFF receptor, TACI, CD3, CD4, CD8, CD5, CD7, CD2, and CD138. Also, the targets of the second recognition domain are selected from the group consisting of GD2, GD3, interleukin 6 receptor, ROR1, PSMA, PSCA (prostate stem cell antigen), MAGE A3, glycolipid, glypican 3, F77, GD-2, WT1, CEA, HER-2 / neu, MAGE-3, MAGE-4, MAGE-5, MAGE-6, alpha-fetoprotein, CA 19-9, CA 72-4, NY-ESO, FAP, ErbB, c-Met, MART-1, MUC1, MUC2, MUC3, MUC4, MUC5, CD30, EGFRvIII, CD33, CD123, CLL-1, NKG2D, NKG2D receptor, immunoglobulin kappa and lambda, CD38, CD52, CD19, CD20, CD22, CD38, BCMA, CS1, BAFF, BAFF receptor, April receptor, TACI, CD3, CD4, CD8, CD5, CD7, CD2, and CD138.

[0136] In one embodiment, the target of the first antigen recognition domain is selected from the group consisting of, but not limited to, GD2, GD3, CD19, CD20, CD22, CD38, CD138, BCMA, CS1, BAFF, BAFF receptor, TACI, April, April receptor, CD3, CD4, CD5, CD7, CD2, CLL-1, CD33, CD123, NKG2D receptor and CD30. The target of the second recognition domain is selected from the group consisting of GD2, GD3, CD19, CD20, CD22, CD38, CD138, BCMA, CS1, BAFF, April, April receptor, BAFF receptor, TACI, CD3, CD4, CD5, CD7, CD2, CLL-1, CD33, CD123, NKG2D receptor and CD30.

[0137] In one embodiment, each CAR unit includes the same or different hinge regions. In another embodiment, each CAR unit includes the same or different transmembrane regions. In another embodiment, each CAR unit includes the same or different intracellular domains.

[0138] In this example, each CAR unit includes a CD3 zeta chain signaling domain.

[0139] In one embodiment, each distinct CAR unit includes a different co-stimulatory domain. For example, the first chimeric antigen receptor polypeptide includes a 4-1BB co-stimulatory domain, and the second chimeric antigen receptor polypeptide includes a CD28 co-stimulatory domain.

[0140] In one embodiment, each distinct CAR unit includes the same co-stimulatory domain. For example, the first chimeric antigen receptor polypeptide includes a 4-1BB co-stimulatory domain, and the second chimeric antigen receptor polypeptide includes a 4-1BB co-stimulatory domain.

[0141] In another embodiment, the hinge region is designed to exclude amino acids that can cause unwanted intramolecular or intermolecular interactions. For example, the hinge region is designed to exclude or minimize cysteine residues to prevent the formation of disulfide bonds. In another embodiment, the hinge region is designed to exclude or minimize hydrophobic residues to prevent unwanted hydrophobic interactions.

[0142] Compound CAR can kill targets alone or in combination. Multiple or compound CARs contain the same or different hinge regions, the same or different transmembrane regions, the same or different co-stimulatory regions, and the same or different intracellular domains. Preferably, the hinge region is selected to avoid interaction sites.

[0143] The compound CARs of the present invention can target the same or different tumor populations in T cells or NK cells. For example, the first CAR targets a large tumor population, and the next, or second CAR, may eradicate cancer or leukemia stem cells, for example, to avoid cancer recurrence.

[0144] According to the present disclosure, surprisingly, it has been found that compound CARs in T or NK cells targeting different or the same tumor populations fight tumor factors that cause cancer cells resistant to CAR killing activity, thereby causing down-regulation of target antigens from the cancer cell surface. Surprisingly, this also makes it possible for cancer cells to "hide" from CAR therapy due to "antigen escape" and tumor heterogeneity in which different tumor cells exhibit different surface antigen expression profiles. As shown in the present disclosure below, surprisingly, compound CARs have been found to have significant advantages over single CAR therapy due to their multi-targeting ability. Although a single antigen may be lost under the pressure of antigen-specific selection, the likelihood of two major antigens being lost simultaneously is much lower.

[0145] In one embodiment, the antigen recognition domain comprises the binding portion or variable region of a (selective) humanized monoclonal or polyclonal antibody against the target.

[0146] In one aspect of the invention, the antigen recognition domain can be a bispecific tandem chimeric antigen receptor comprising two targeting domains. In further embodiments, there are multispecific tandem chimeric antigen receptors comprising three or more targeting domains.

[0147] In certain aspects of the invention, the antigen recognition domain can be a bispecific chimeric antigen receptor (derived from a bispecific antibody) comprising two targeting domains.

[0148] In one embodiment, the bispecific tandem chimeric antigen receptor or bispecific chimeric antigen receptor effectively counteracts tumor escape or loss of antigen and enhances the sensitivity of antigen recognition.

[0149] In another example, the antigen recognition domain comprises a single domain antibody of the camelid family or a part thereof. In this example, the camelid single domain antibody includes the heavy chain antibody found in camelid animals, or VHH antibody. Camelid VHH antibodies (e.g., camel, human cobra camel, llama, and alpaca) refer to the variable fragments of camelid single chain antibodies (see Nguyen et al., 2001; Muyldermans, 2001), and also include isolated VHH antibodies of camelids, recombinant VHH antibodies of camelids, or synthetic VHH antibodies of camelids.

[0150] Enhancer of CAR function and promotion of T cell and natural cell increase or proliferation IL-15 / IL15sushi enhancer

[0151] In one embodiment, a CAR construct having an IL-15 / IL15sushi enhancer is shown in FIG. 53. The CAR comprises a secreted IL-15 / IL-15sushi complex. The CAR with IL-15 / IL-15sushi is linked with a P2A self-cleavage sequence. The IL-15 / IL-15sushi moiety consists of an IL-2 signal peptide that is fused to IL-15 and bound to the sushi of the IL-15 alpha receptor via a 26-amino acid polyproline linker. The CAR has a scFv, a co-stimulatory domain (including but not limited to CD28 or 4-1BB), and an intracellular signaling, CD3 zeta chain. The IL-15 signal peptide in IL-15 is replaced with an IL-2 signal peptide (leader sequence), which is a potent signal peptide that provides high-efficiency IL-15 / IL-15sushi secretion. The self-cleavage peptides of the construct include, but are not limited to, P2A, T2A, F2A, and E2A. The secretion enhancers of the construct may also include, but are not limited to, IL-15 / IL-15sushi, IL-15, IL-21, IL-18, IL-7, and IL-12. Secretion enhancers such as IL-15 / IL-15sushi enhance the proliferation and persistence of CAR T or NK cells. The soluble IL-15 / IL-15sushi fusion is stable and functions as an unexpected potent immunomodulatory factor for CAR T / NK cells and their adjacent tumor immune response cells. The soluble IL-15 / IL-15sushi fusion is stable, improves the persistence of CAR T / NK cells, and stimulates the proliferation of tumor-infiltrating lymphocytes and anti-tumor activity. The soluble IL-15 / IL-15sushi fusion reprograms the body's immune system to provide effects like an anti-tumor vaccine to fight cancer.

[0152] IL-15 can be a variant of IL-15N72D as described in other writings, US8507222 B2.

[0153] IL-15 / IL15sushi anchor enhancer In one embodiment, a CAR construct having an IL-15 / IL-15sushi anchor is shown in FIG. 54. The CAR IL-15 / IL15sushi anchor construct consists of an SFFV promoter that drives the expression of a CAR and an IL-15 / IL-15sushi anchor (also referred to as an anchor) linked by a P2A peptide. When this P2A peptide cleaves, the IL-15 / IL-15 anchor CAR separates into a CAR and an IL-15 / IL15sushi anchor. The IL-15 / IL-15sushi portion of the anchor is composed of an IL-2 signal peptide fused to IL-15 and bound to the sushi domain of the IL-15 alpha receptor via a 26-amino acid polyproline linker. Both the CAR and the anchor include a hinge (H) region and a transmembrane domain (TM). The CAR has a scFv, a co-stimulatory domain (including but not limited to CD28 or 4-1BB), and an intracellular signaling, CD3 zeta chain, but the anchor does not include these components. The IL-15 / IL-15sushi anchor provides a synergistic effect on T cell activation or anti-tumor activity with CD28 or 4-1BB. With the IL-15 / IL-15sushi anchor, the CAR becomes more potent. (FIG. 54)

[0154] IL-15 can be a variant of IL-15 N72D as described in other writings, US8507222 B2.

[0155] 4-1BBL enhancer In another embodiment, a CAR construct having a 4-1BBL enhancer is shown in FIG. 55. The CAR 4-1BBL construct consists of a CAR linked by a P2A peptide and an enhancer, an SFFV promoter that promotes the expression of 4-1BBL (CD137L). When this P2A peptide cleaves, the CAR construct with 4-1BBL is separated into a CAR polypeptide and a full-length 4-1BBL protein. The CAR includes a leader sequence, scFv, a hinge (H) region, and a transmembrane domain (TM). The CAR also has a co-stimulatory domain (including but not limited to CD28 or 4-1BB) and an intracellular signaling, CD3 zeta chain, but 4-1BBL does not have these components. 4-1BBL provides a synergistic effect on T cell activation or anti-tumor activity with CD28 or 4-1BB. When equipped with 4-1BBL, the CAR becomes more potent.

[0156] IL-15 enhancer The function of the CAR can be enhanced by incorporating the secreted enhancer IL-15 shown in FIG. 56. The CAR 4-IL-15 construct consists of a CAR linked by a P2A peptide and an SFFV promoter that drives the expression of the enhancer IL-15. When this P2A peptide cleaves, the CAR construct containing IL-15 is separated into a CAR polypeptide and a full-length IL-15 protein. The CAR includes a leader sequence, scFv, a hinge (H) region, and a transmembrane domain (TM). The CAR also has a co-stimulatory domain (including but not limited to CD28 or 4-1BB) and an intracellular signaling, CD3 zeta chain, but IL-15 does not have these components. The secretion of IL-15 results in a synergistic effect on T cell activation or anti-tumor activity with CD28 or 4-1BB. When secreting IL-15, the CAR is more potent. The IL-15 signal peptide in IL-15 is replaced with the IL-2 signal peptide (leader sequence), a potent signal peptide that provides high-efficiency IL-15 secretion.

[0157] CAR with multiple enhancers Example of the production of a CAR using multiple enhancers (CAR super). FIG. 57, a schematic diagram showing a CAR enhancer construct. The construct consists of a CAR linked by P2A and T2A peptides respectively, an enhancer 4-1BBL (CD137L), and an SFFV promoter that drives the expression of IL-15 / IL-15sushi. After cleavage of the P2A and T2A peptides, the CAR construct containing 4-1BBL and IL-15 / IL-15sushi is divided into the full lengths of the CAR, 4-1BBL protein, and secreted IL-15 / IL-15sushi. The CAR is composed of a leader sequence, scFv, a hinge (H) region, and a transmembrane domain (TM). The CAR has a co-stimulatory domain (including but not limited to CD28 or 4-1BB) and an intracellular signaling CD3 zeta chain. 4-1BBL provides a synergistic effect on the activation of CD28 or 4-1BB and T or NK cells or antitumor activity (but not limited thereto). The self-cleaving peptides of the construct include, but are not limited to, P2A, T2A, F2A, and E2A. The secreted enhancers of the construct may also include, but are not limited to, IL-15 / IL-15sushi, IL-15, IL-21, IL-18, IL-7, and IL-12. Secreted enhancers such as IL-15 / IL-15sushi enhance the proliferation and persistence of CAR T or NK cells. The soluble IL-15 / IL-15sushi fusion is stable and functions as an unexpectedly potent immunomodulatory factor for CAR T / NK cells and their adjacent tumor immune response cells. The soluble IL-15 / IL-15sushi fusion is stable, improves the persistence of CAR T / NK cells, and stimulates the proliferation of tumor-infiltrating lymphocytes and antitumor activity. The soluble IL-15 / IL-15sushi fusion reprograms the body's immune system to provide effects like an antitumor vaccine to fight cancer.

[0158] BCMA-CS1 compound CAR (BCMA-CS1 cCAR) Multiple myeloma (MM) is a blood cancer caused by the rapid proliferation of abnormal plasma cells and accounts for 18% of all blood cancers in the United States. Treatment options for MM include chemotherapy, corticosteroid therapy, targeted therapy, high-dose chemotherapy with stem cell transplantation, biologic therapy, radiation therapy, monoclonal antibodies, proteasome inhibitors, and surgery. Even with these available treatment methods, the 5-year survival rate of MM remains at 49.6%. However, there is no cure for MM, and almost all patients relapse after treatment.

[0159] Current efforts in CAR technology for multiple myeloma include the use of CART cells targeting BCMA (CD269) for bulk disease, led by James Kochenderfer (NIH). Patients who achieve remission after BCMA CAR treatment eventually relapse, which may be due to the fact that some myeloma cells have dim (weak) or negative expression for BCMA. Therefore, a single target in CAR-based therapy may not be sufficient to prevent myeloma relapse. CS1 (SLAMF7) is another excellent target for myeloma because its expression in myeloma cells is generally high and uniform, and it is involved in myeloma cell adhesion and tumor formation.

[0160] This disclosure includes that a single CAR T cell expressing two separate CAR units with independent signaling domains on a vector can be used as a new approach to target multiple antigens and potentially avoid tumor recurrence. Compound CAR (cCAR) is a BCMA CAR linked to a CS1 CAR via a self-cleaving P2A peptide and includes the expression of both functional CAR molecules on the surface of T cells.

[0161] In the present disclosure, surprisingly, it has been found that these BCMA-CS1 cCAR (BC1cCAR) T cells exhibit potent and specific anti-tumor activity in vitro and control significant tumor growth in vivo. For the first time, we demonstrate that two units of individual CARs can effectively target both antigens in vitro and potentially impact more comprehensive clinical outcomes. It is unexpected that a compound CAR targeting both BCMA and CS1 in combination is a very powerful strategy for targeting multiple myeloma. This new approach avoids antigen escape (loss of a single antigen) due to selection pressure in single CAR therapy by the pressure from the combination in the compound design.

[0162] BCMA (B cell maturation antigen) and CS1 (SLAMF7) are expressed as surface antigens in most myeloma cases, either alone or both, and since these antigens are not present on hematopoietic stem cells, they are preferably selected as targets for the compound CAR. Using two different targets, BCMA and CS1, which are widely expressed in plasma cells, improves coverage and prevents antigen escape, enabling effective eradication of cancer cells.

[0163] In the present disclosure, surprisingly, the addition of CS1 to the BCMA CAR enhanced the anti-tumor response by eliminating residual BCMA - CS1 + myeloma cells and reducing the risk of recurrence. Both BCMA and CS1 (CD319) are widely expressed in MM cells, and this high expression potentially allows the BCMA-CS1 cCAR to comprehensively cover all cancer cells. This enables more complete elimination of cancer cells and reduction of antigen escape by strongly hitting multiple targets simultaneously before resistance develops.

[0164] In one embodiment, the BCMA-CS1cCAR (BC1cCAR) therapy for BCMA-CS1 is a "bridge" to bone marrow transplantation (BMT) or a combination of high-dose chemotherapy and BMT. BCMA-CS1 cCAR can offer a path to a potentially curative BMT option for many patients who previously had residual disease. Current literature supports the idea that reducing the minimal residual disease (MRD) level to undetectable levels may be associated with improved patient outcomes. This is very beneficial in terms of preventing recurrence of difficult-to-treat and highly aggressive malignancies.

[0165] In another embodiment, the BCMA-CS1 cCAR therapy can reduce the disease burden to the lowest possible level before transplantation or completely eliminate MRD, which can be expected to reduce the recurrence rate and increase the long-term disease-free survival rate, and the patient outcome will be dramatically improved.

[0166] In one embodiment, the BCMA-CS1 cCAR therapy can have further uses in BCMA+ and / or CS1+ multiple myeloma patients beyond the bridge to bone marrow transplantation. BCMA-CS1cCAR therapy as a monotherapy or as part of an individualized immunochemotherapy regimen for patients. For elderly patients or patients with co-morbidities who cannot tolerate very intensive chemotherapy or BMT, this may be a promising strategy to extend the patient's survival and ensure a better quality of life.

[0167] In some embodiments, the BCMA-CS1cCAR T cell therapy can be developed as a supplement to chemotherapy, as a "bridge to transplantation", or as a monotherapy for multiple myeloma patients.

[0168] In some embodiments, the present disclosure provides compound CAR polypeptide-modified cells that target cells expressing BCMA or CS1 antigen or both. The target cells may be cancer cells such as, but not limited to, lymphoma, leukemia, plasma cell neoplasms, etc. In a further embodiment, the plasma cell neoplasm is selected from plasma cell leukemia, multiple myeloma, plasmacytoma, heavy chain disease, amyloidosis, Waldenström macroglobulinemia, heavy chain disease, solitary bone plasmacytoma, monoclonal gammopathy of undetermined significance (MGUS), and smoldering multiple myeloma. It was surprising to find that co-expression of IL-15 / IL-15sushi and cCAR can enhance the sensitivity of CAR recognition of target cancer cells or recruit natural immune cells against cancer cells, resulting in long-term tolerance remission in patients.

[0169] Without being bound by theory, co-expression of IL-15 / IL-15sushi anchor or 4-1BBL and BCMA-CS1 cCAR is thought to result in long-term tolerance remission in patients by enhancing the sensitivity of CAR recognition of target cancer cells or by recruiting natural immune cells against cancer cells.

[0170] Without being bound by theory, co-expression of IL-21 or IL-21 anchor and BCMA-CS1 cCAR is thought to result in long-term tolerance remission in patients by enhancing the sensitivity of CAR recognition of target cancer cells or by recruiting natural immune cells against cancer cells.

[0171] In one embodiment, the engineered cells comprise a BCMA-CS1 cCAR polypeptide and IL-15 / IL-15sushi (SEQ ID NO: 42), and the corresponding nucleotide (SEQ ID NO: 43).

[0172] BCMA1-BCMA2 compound CAR (BCMA1-BCMA2 cCAR) (Figure 38) Initial remission is seen in most B-ALL with CD19 CAR T therapy, but relapse with epitope loss occurs in 10% - 20% of responders.

[0173] Current efforts with CAR technology in multiple myeloma include the use of CART cells targeting BCMA (CD269) for multiple myeloma, led by James Kochenderfer (NIH). Patients in remission after BCMA CAR therapy eventually relapse, which may be due to the fact that some myeloma cells have dim (weak) or negative expression for BCMA. Additionally, there is also the issue of the efficacy of a single CAR in eliminating a patient's multiple myeloma cells. Therefore, CAR-based therapy with a single target may not be sufficient to prevent relapse of myeloma.

[0174] In one embodiment, the antibody recognition domain includes the binding variable region of a monoclonal antibody, a single-chain fragment variable (scFv). The scFv contains one light-chain antibody and one heavy-chain antibody. In certain embodiments, the antigen recognition domain is composed of two different heavy-chain domains (VHH). Each heavy-chain domain binds to a different epitope of the same antigen or different antigens. VHH antibodies are more stable and robust than the whole antibody.

[0175] In some embodiments, the compound CAR targets the same antigen. For example, cCAR targets different epitopes or parts of a single antigen. In some embodiments, each of the CAR units present in the compound CAR targets different epitopes specific to the same antigen but at different positions.

[0176] In some embodiments, the compound CAR targets different epitopes on one antigen.

[0177] The present disclosure includes that a vector having an independent signaling domain, in which a single CAR T cell expressing two individual CAR units targets different epitopes on one antigen, can be used as a novel approach to potentially avoid tumor epitope skipping or epitope loss or epitope escape. Compound cCAR (BCMA1-BCMA2 cCAR) is composed of one BCMA CAR (BCMA1 CAR) linked via a self-cleaving P2A peptide to another BCMA CAR (BCMA2 CAR), and expresses both functional CAR molecules on the surface of T cells. Both CAR units of the cCAR target the same antigen BCMA.

[0178] In one embodiment, the engineered cell comprises a first chimeric antigen receptor polypeptide having a BCMA antigen recognition epitope and a second chimeric antigen receptor polypeptide having a different BCMA recognition epitope. In this example, the engineered cell comprises the polypeptide of SEQ ID NO: 3 and the polynucleotide corresponding to SEQ ID NO: 4.

[0179] Surprisingly, in the present disclosure, it has been found that this BCMA1-BCMA2 cCAR T cell exhibits potent and specific anti-tumor activity in vitro and controls significant tumor growth in vivo. We show for the first time that two units of individual CARs can effectively target both different epitopes of one antigen, BCMA, in vitro, which may potentially affect more comprehensive clinical outcomes. It is unexpected that a compound CAR targeting different epitopes in combination is a very powerful strategy for targeting multiple myeloma. This new approach avoids antigen escape (loss of a single antigen or antigen skipping) due to selection pressure in single CAR therapy due to the pressure by combination in compound design.

[0180] Surprisingly, in this disclosure, it has been found that for BCMA CAR, the addition of epitopes enhances the anti-tumor response and reduces the risk of relapse of multiple myeloma due to the loss of BCMA epitopes.

[0181] In one embodiment, the therapy for BCMA1 - BCMA2 is a "bridge" for bone marrow transplantation (BMT) or a combination of high-dose chemotherapy and BMT. The BCMA1 - BCMA2 cCAR can enhance the recognition sensitivity to the BCMA antigen and provide a path to a potentially curative BMT option for many patients who previously had residual disease. Current literature supports the idea that reducing the minimal residual disease (MRD) level to undetectable levels may be associated with improved patient outcomes. This is very beneficial in terms of preventing relapse of difficult-to-treat and highly aggressive malignancies.

[0182] In another embodiment, the BCMA1 - BCMA2 cCAR therapy can reduce the disease burden to the lowest possible level before transplantation or completely eliminate MRD, which is expected to reduce the relapse rate and increase the long-term disease-free survival rate, and the patient outcome is dramatically improved.

[0183] In some embodiments, the present disclosure provides compound CAR polypeptide-modified cells that target two different epitopes of the BCMA antigen. The target cells may be cancer cells such as, but not limited to, lymphoma, leukemia, plasma cell neoplasms, etc. In further embodiments, the plasma cell neoplasms are selected from plasma cell leukemia, multiple myeloma, plasmacytoma, heavy chain disease, amyloidosis, Waldenström macroglobulinemia, heavy chain disease, solitary bone plasmacytoma, monoclonal gammopathy of undetermined significance (MGUS), and smoldering multiple myeloma.

[0184] Without being bound by theory, co-expression of IL-15 / IL-15sushi or IL-15 / IL-15sushi anchor or 4-1BBL and BCMA1-BCMA2 cCAR is thought to result in long-term remission with tolerance in patients by enhancing the sensitivity of CAR-mediated recognition of target cancer cells or by recruiting innate immune cells against cancer cells.

[0185] Without being bound by theory, co-expression of IL-21 or IL-21 anchor and BCMA1-BCMA2 cCAR is thought to result in long-term remission with tolerance in patients by enhancing the sensitivity of CAR-mediated recognition of target cancer cells or by recruiting innate immune cells against cancer cells.

[0186] CD123-CD33 compound CAR (CD123-CD33 cCAR) To translate the success of CAR to AML, it is necessary to carefully understand the disease-specific characteristics. The feature of AML is the presence of blast cells, which are very aggressive and rapidly dividing cells that form most of the disease. Different from B cell malignancies, AML is very difficult to treat due to the role of leukemia stem cells (LSCs). LSCs are a cell population that expresses the markers of hematopoietic stem cells (CD34 + CD38-) capable of initiating and maintaining hematopoietic malignancies and generate a clonal cell population that overrides healthy bone marrow. Since LSCs are mostly in the quiescent phase of the cell cycle, chemotherapy against rapidly dividing tumor populations does not affect LSCs. In most cases, this elusive population contains minimal residual disease (MRD), which is the cause of inevitable relapse after AML treatment. Transplanting CAR therapy to AML to completely eliminate the disease and prevent relapse requires careful antigen selection that can eradicate not only the bulk leukemia disease but also leukemia stem cells. A CD123-CD33 cCAR is expected that can eliminate both CD33+ and CD123+ cells without causing CAR-CAR interaction. A convenient analogy in this case is to consider AML as a cancer tree with leaves and roots. The leaves account for most / more than half of the disease (these are CD33+ AML blasts), but trimming these leaves will not prevent further growth of the tree unless the roots (these are CD123+ CD34+ CD38- LSCs) are removed. In a study of 319 AML patients, 87.8% of the cases expressed CD33, so targeting CD33 may make most leukemia cells a target. However, patients treated with gemtuzumab ozogamicin, an anti-CD33 antibody therapy conjugated with calicheamicin, relapsed in CD33+ AML, probably due to acquired chemotherapy resistance to calicheamicin. Therefore, while targeting CD33 eliminates most of the disease, it is also necessary to target chemotherapy-resistant LSCs; otherwise, relapse will occur. This can be achieved by targeting CD123, which is overexpressed in CD34+ CD38- LSCs compared to healthy hematopoietic stem cells. Considering that 97.2% of AML cases express at least one of the two targets, targeting both CD123 and CD33 will eliminate all cancer cells in the majority of patients, enhance the therapeutic effect, and uproot the cancer tree.

[0187] AML is a rapidly progressing blood cancer that accounts for approximately 15 - 20% of acute childhood leukemia and 80% of acute adult leukemia. Patients are still currently treated potentially with high-dose combination chemotherapy, followed by hematopoietic stem cell transplantation. Despite such intensive treatment, in many cases, about 60 - 70% of AML patients still relapse due to acquired treatment resistance or the reappearance of LSCs, often with significant toxicity and even death. Furthermore, the 5-year survival rate from AML remains at 27%. However, there are limited clinical trials attempting to use CARs for the treatment of AM.

[0188] The present disclosure includes that a single CAR T cell expressing two separate CAR units in a vector having an independent signaling domain can be used as a novel approach to target multiple antigens and potentially avoid tumor recurrence. Compound CAR (cCAR) includes those in which a CD123 CAR is linked to a CD33 CAR via a self-cleaving P2A peptide, expressing both functional CAR molecules on the surface of T cells.

[0189] In the present disclosure, surprisingly, it has been found that this CD123-CD33 cCAR T cell exhibits potent and specific anti-tumor activity in vitro and controls significant tumor growth in vivo. We demonstrate for the first time that two units of individual CARs can effectively target both antigens in vitro and potentially impact more comprehensive clinical outcomes. Targeting AML with a compound CAR that targets both CD123 and Cd33 in combination is unexpectedly a very powerful strategy. This new approach avoids recurrence of diseases associated with LSCs and, due to the pressure from the combination in the compound design, avoids antigen escape (loss of a single antigen) resulting from the selection pressure in single CAR therapy.

[0190] In the present disclosure, surprisingly, the addition of CD123 to CD33 CAR enhanced the anti-tumor response by eliminating both leukemic blasts and the LSCs that are their root, reducing the risk of recurrence. Thereby, by eliminating both slowly growing LSCs and proliferative leukemic cells, cancer cells can be more completely eliminated and disease recurrence can be reduced.

[0191] In the present disclosure, surprisingly, it has been found that CD123-CD33 cCAR T cells can eliminate normal leukemic cells and leukemic progenitor cells, reduce the risk of recurrence, and enhance anti-tumor activity.

[0192] In this disclosure, surprisingly, CD123-CD33 cCAR T cells show more complete removal of cancer cells by strongly hitting multiple targets simultaneously before resistance occurs, reducing antigen escape.

[0193] In one embodiment, CD123-CD33 cCAR T cell therapy can be deployed as "bridging to transplantation", as an adjunct to chemotherapy, or as checkpoint blockade (including but not limited to PD-L1, CTLA-4 inhibitors), or as monotherapy for patients with diseases including but not limited to acute myeloid leukemia, myelodysplastic syndromes, chronic myeloid leukemia, and chronic myeloproliferative disorders.

[0194] In another embodiment, CD123-CD33 cCAR T cell therapy can reduce the disease burden to the lowest possible level before transplantation or completely eliminate MRD, with an expected reduction in the recurrence rate and an increase in long-term disease-free survival, and the patient outcome is dramatically improved.

[0195] In one embodiment, CD123-CD33 cCAR T cell therapy can have further uses in patients with Cd123+ and / or CD33+ multiple myeloma beyond bridging to bone marrow transplantation. CD123-CD33 cCAR T cell therapy as monotherapy or as part of an individualized immunochemotherapy regimen for the patient. For elderly patients or patients with co-existing diseases who cannot tolerate very intensive chemotherapy or BMT, this may be a promising strategy to extend the patient's survival and ensure a better quality of life.

[0196] Without being bound by theory, co-expression of IL-15 / IL-15sushi or IL-15 / IL-15sushi anchor or 4-1BBL with CD123-CD33 cCAR is thought to result in long-term tolerance remission in patients by enhancing the sensitivity of CAR recognition to target cancer cells or by recruiting natural immune cells against cancer cells.

[0197] Without being bound by theory, co-expression of IL-21 or an IL-21 anchor with CD123-CD33 cCAR is thought to result in long-term remission in patients by enhancing the sensitivity of CAR-mediated recognition of target cancer cells or by recruiting innate immune cells to cancer cells.

[0198] In one embodiment, the engineered cells comprise a CD123-CD33 cCAR polypeptide and IL-15 / IL-15sushi (SEQ ID NO: 34), and the corresponding nucleotide (SEQ ID NO: 35).

[0199] CLL-1-CD33 compound CAR (CLL-1-CD33 cCAR) The cCAR includes two units of CAR, a CLL-1CAR and a CD33 CAR, which target tumor cells expressing CLL-1 and CD33, respectively. The CD33b CAR and CLL-1 CAR were used to construct the version of the cCAR shown in FIG. 92. The construct includes an SFFV promoter that drives the expression of multiple modular units of CAR linked by a P2A peptide. When the linker cleaves, the cCAR separates and engages targets expressing CD33 and CLL-1. The activation domains of the construct include 4-1BB on the CD33b (CD33) CAR unit and CD28 on the CLL-1 CAR unit. This CD33b-CLL-1 cCAR was designed to eliminate myeloid leukemia cells, including leukemia stem cells.

[0200] Currently, the treatment methods for MDS, MPN (myeloproliferative neoplasms), and AML focus on leukemic blast cells because they are very abundant and clearly represent the most urgent problems for patients. Importantly, leukemia stem cells (LSCs) are quite different from most of the other leukemia cells ("blast" cells), and they form a rare subpopulation. Killing blast cells provides short-term relief, but if LSCs are not destroyed, they will always regrow and the patient's disease will relapse. To achieve a permanent cure for MDS diseases, it is essential to destroy LSCs. Unfortunately, standard drug regimens are not effective against LSCs in MDS or MPN or AML. Therefore, it is important to develop new treatment methods that can specifically target both the leukemia stem cell population and the large leukemia population. The compound CAR disclosed in the present invention targets both of these populations and is specifically described herein.

[0201] In one aspect of the present disclosure, the CLL-1 antigen is one of the targets in cCAR therapy. C-type lectin-like 1 (CLL-1) is also known as MICL, CLEC12A, CLEC-1, and DCAL2. CLL-1 is a glycoprotein receptor and is expressed in hematopoietic cells. CLL-1 is not present in uncommitted CD34 + / CD38- or CD34 + / CD33- stem cells, but is present in a subset of CD34 + / CD38 + or CD34 + / CD33 + progenitor cells (Bakker et al, 2004). Furthermore, CLL-1 is not expressed in other tissues.

[0202] CLL-1 expression is seen in the blasts and leukemia stem cells of acute myeloid leukemia (AML). CLL-1 is expressed in various leukemias including myelomonocytic leukemia (M4), acute monocytic leukemia (M5), acute promyelocytic leukemia (M3), chronic myeloid leukemia (CML), chronic myeloproliferative neoplasms, and myelodysplastic syndrome (MDS).

[0203] CLL-1 is expressed in a subset of leukemia cells associated with leukemia stem cells (LSCs), and its removal is essential for preventing disease refractoriness and recurrence.

[0204] CD33 (Siglec-3) is a myeloid lineage-specific antigen that is expressed in early myeloid progenitor cells, most monocyte cells, and approximately 90% of AML blasts, but is absent in normal HSCs.

[0205] In one aspect of the present disclosure, the CD33 antigen is one of the targets of cCAR therapy. CD33 is a transmembrane receptor expressed in 90% of malignant cells of acute myeloid leukemia. Thus, according to the present disclosure, the CLL-1 and CD33 target antigens are particularly attractive from a safety perspective.

[0206] According to the present disclosure, compound CLL-1-CD33 can be very effective in treating chronic myeloid leukemia (CML) populations. There is a rare subset of cells that are CD34 + CD38- in chronic myeloid leukemia (CML). This population is considered to be composed of LSCs. An increase in the number of LSCs is associated with disease progression. Small molecule Bcr-Abl tyrosine kinase inhibitors (TKIs) have been shown to significantly improve the overall survival of CP-CML patients. However, LSCs are thought to be resistant to TKI therapy. There is an urgent need for new therapies targeting CML-resistant LSCs, and the new therapy is embodied in the compound CD33CLL-1 CAR disclosed in the present disclosure. The expression of CLL-1 is elevated in the CD34 + CD38- population. According to the present disclosure, compound CD33CLL-1 CAR is very effective in the therapeutic treatment of this population.

[0207] In one embodiment of the present disclosure, leukemia cells expressing both CD33 and CLL-1 in cCAR are used as a therapeutic treatment. CD33 is expressed in cells of the myeloid lineage, myeloid leukemia blasts, and mature monocytes, but not in normal pluripotent hematopoietic stem cells. CD33 is widely expressed in leukemia cells of CML, myeloproliferative neoplasms, and MDS.

[0208] A significant number of patients with acute myeloid leukemia (AML) are resistant to standard chemotherapy regimens or experience disease recurrence after treatment (Burnett 2012), and the development of CAR T cell immunotherapy for AML holds the potential to address a huge clinical need. In most of these patients, leukemia cells express both CLL-1 and CD33, giving the compound CLL-1-CD33 cCAR disclosed herein broad clinical applicability. Accordingly, the present disclosure discloses novel multiple cCAR T / NK cell constructs comprising multiple CARs targeting multiple leukemia-associated antigens, thereby counteracting the mechanism of antigen escape targeting leukemia cells including leukemia stem cells by the synergistic effect of co-stimulatory domain activation, thereby providing a more potent, safe and effective treatment.

[0209] In a further embodiment, the present disclosure provides a method for eradicating or killing leukemia stem cells (LSCs) or bulk leukemia cells that express CLL-1 and / or CD33. In this embodiment, T or NK engineered cells having CD33 units and CLL-1 units are administered to a patient in need thereof.

[0210] In a further embodiment, compound CARs in T or NK cells can be used to eradicate or kill CD34+ CD38- leukemia stem cells or bulk leukemia cells that express CLL-1 or CD33 or both.

[0211] The present disclosure further discloses compound CAR constructs having enhanced efficacy with enhanced antitumor activity against cells co-expressing target antigens, while also retaining sensitivity to tumor cells expressing only one antigen. Furthermore, each CAR of the compound CAR contains one or two co-stimulatory domains and exhibits potent killing ability in the presence of a specific target.

[0212] In the present disclosure, surprisingly, it has been found that CLL-1-CD33 cCAR T cells can eliminate normal leukemia cells and leukemic precursor cells, reduce the risk of recurrence, and enhance antitumor activity.

[0213] In this disclosure, surprisingly, CLL-1-CD33 cCAR T cells reduce antigen escape and show more complete removal of cancer cells by strongly hitting multiple targets simultaneously before resistance occurs.

[0214] In one embodiment, CLL-1-CD33 cCAR T cell therapy can be deployed as a "bridge to transplantation", as an adjunct to chemotherapy, or as checkpoint blockade (including, but not limited to, PD-L1 and CTLA-4 inhibitors), or as monotherapy for patients with diseases including, but not limited to, acute myeloid leukemia, myelodysplastic syndromes, chronic myeloid leukemia, and chronic myeloproliferative disorders.

[0215] In another embodiment, CLL-1-CD33 cCAR T cell therapy can reduce the disease burden to the lowest possible level before transplantation or completely eliminate MRD, which can be expected to reduce the recurrence rate and increase the long-term disease-free survival rate, and the patient outcome will be dramatically improved.

[0216] In one embodiment, CLL-1-CD33 cCAR T cell therapy can have additional uses in patients with CLL-1+ and / or CD33+ multiple myeloma beyond bridging to bone marrow transplantation. CLL-1-CD33 cCAR T cell therapy as monotherapy or as part of an individualized immunochemotherapy regimen for patients. For elderly patients or patients with coexisting diseases who cannot tolerate very intensive chemotherapy or BMT, this may be a promising strategy to extend the patient's survival and ensure a better quality of life.

[0217] Without being bound by theory, co-expression of IL-15 / IL-15sushi or IL-15 / IL-15sushi anchor or 4-1BBL and CLL-1-CD33 cCAR is thought to result in long-term durable remission in patients by enhancing the sensitivity of CAR recognition of target cancer cells or by recruiting natural immune cells against cancer cells.

[0218] Without being bound by theory, co-expression of IL-21 or IL-21 anchor and CLL-1-CD33 cCAR is thought to result in long-term durable remission in patients by enhancing the sensitivity of CAR recognition of target cancer cells or by recruiting natural immune cells against cancer cells.

[0219] In one embodiment, the engineered cell comprises a CLL1-CD33 cCAR polypeptide and IL-15 / IL-15sushi (SEQ ID NO: 60), and the corresponding nucleotide (SEQ ID NO: 61).

[0220] In one embodiment, the engineered cell comprises a CLL-1 CAR polypeptide, 4-1BBL and IL-15 / IL-15sushi (SEQ ID NO: 44), and the corresponding nucleotide (SEQ ID NO: 45).

[0221] In one embodiment, the engineered cell comprises a CD33 CAR polypeptide, 4-1BBL and IL-15 / IL-15sushi (SEQ ID NO: 20), and the corresponding nucleotide (SEQ ID NO: 21).

[0222] CD123-NKG2D cCAR or CLL-1-NKG2D cCAR or CD33-NKG2D cCAR or BCMA-NKG2D cCAR NKG2D (NKG2D receptor) is considered a transmembrane protein belonging to the CD94 / NKG2 family of C-type lectin-like receptors. NKG2D can bind to at least eight different ligands that are naturally expressed in AML, multiple myeloma, or other leukemias. NKG2D ligands are induced self-proteins that are rarely present or present at very low levels on the surface of normal cells, but are overexpressed in cancer cells such as AML and multiple myeloma. Therefore, these are excellent candidates for CAR targeting.

[0223] cCAR contains two units of CAR, CD123 CAR and NKG2D CAR, which target tumor cells expressing CD123 and NKG2D ligand, respectively.

[0224] cCAR contains two units of CAR, CLL-1 CAR and NKG2D CAR, which target tumor cells expressing CLL-1 and NKG2D ligand, respectively.

[0225] CD123-NKG2D cCAR or CLL-1-NKG2D cCAR or CD33-NKG2D cCAR can eliminate leukemias such as AML, MDS, CML, and MPN.

[0226] In the present disclosure, BCMA-NKG2D cCAR can eliminate multiple myeloma. In the present disclosure, since NKG2D is widely expressed in AML, MDS, CML, and MPN, the addition of NKG2D to CD123 CAR or CLL-1 CAR or CD33 CAR enhances the anti-tumor response and reduces the risk of antigen escape associated with disease recurrence.

[0227] Both BCMA and NKG2D ligands are widely expressed in multiple myeloma cells, and due to this high expression, BCMA-NKG2D cCAR has the potential to comprehensively cover all cancer cells. This enables more complete elimination of cancer cells and reduction of antigen escape by strongly targeting multiple targets simultaneously before resistance develops.

[0228] BCMA-CD38 compound CAR (BCMA-CD38 cCAR) Current efforts with CAR technology in multiple myeloma include the use of CART cells targeting BCMA (CD269) for bulk disease, led by James Kochenderfer (NIH). Patients in remission after BCMA CAR therapy ultimately relapse, which may be due to the fact that some myeloma cells have dim (weak) or negative expression for BCMA. Therefore, CAR-based therapy with a single target may not be sufficient to prevent relapse of myeloma.

[0229] CD38, also known as cyclic ADP-ribose hydrolase, is a glycoprotein present on the surface of many immune cells including CD4 +, CD8 +, B lymphocytes, plasma cells, and natural killer cells.

[0230] CD38 is another excellent target for myeloma because its expression is generally high and uniform in myeloma cells and lymphoma cells.

[0231] This disclosure includes that a single CAR T cell expressing two separate CAR units with independent signaling domains in a vector can be utilized as a new approach to target multiple antigens and potentially avoid tumor relapse. Compound CAR (cCAR) is a BCMA CAR linked to a CD38 CAR via a self-cleaving P2A peptide, expressing both functional CAR molecules on the surface of T cells. The expression of this compound cCAR is controlled by a strong promoter SFFV to ensure proper CAR expression.

[0232] In the present disclosure, BCMA-CD38 cCAR T cells can provide potent and specific anti-tumor activity in the control of multiple myeloma (Figure 37). Targeting multiple myeloma with a compound CAR that targets both BCMA and CD38 in combination is a very powerful strategy. This new approach avoids antigen escape (loss of a single antigen) due to selection pressure in single CAR therapy by the pressure from the combination in compound design.

[0233] In this disclosure, the addition of CD38 to BCMA CAR enhanced the anti-tumor response by eliminating surviving BCMA-CD38 + multiple myeloma cells and reducing the risk of recurrence.

[0234] Both BCMA and CD38 are widely expressed in multiple myeloma cells, and this high expression enables BCMA-CD38 cCAR to potentially cover all cancer cells comprehensively. This allows for more complete elimination of cancer cells and reduction of antigen escape by strongly hitting multiple targets simultaneously before resistance develops.

[0235] In one embodiment, BCMA-CD38 cCAR therapy for BCMA-CD38 is a "bridge" to bone marrow transplantation (BMT) or a combination of high-dose chemotherapy and BMT. BCMA-CD38 cCAR can provide a path to a potentially curative BMT option for many patients who previously had residual disease. Current literature supports the idea that reducing the level of minimal residual disease (MRD) to undetectable levels may be associated with improved patient outcomes. This is very beneficial in preventing recurrence of difficult-to-treat and very aggressive malignancies.

[0236] In another embodiment, the BCMA-CD38 cCAR therapy can reduce the disease burden to the lowest possible level before transplantation or completely eliminate MRD, which is expected to reduce the recurrence rate and increase the long-term disease-free survival rate, and the patient outcome will be dramatically improved.

[0237] In one embodiment, the BCMA-CD38 cCAR therapy can have further uses in patients with BCMA+ and / or CD38+ multiple myeloma beyond bridging to bone marrow transplantation. The BCMA-CD38 cCAR therapy as a monotherapy or as part of an individualized immunochemotherapy regimen for patients. For elderly patients or patients with co-existing diseases who cannot tolerate very intensive chemotherapy or BMT, this may be a promising strategy to extend the patient's survival and ensure a better quality of life.

[0238] In some embodiments, the present disclosure provides compound CAR polypeptide-modified cells that target cells expressing BCMA or CD38 antigen or both. The target cells may be cancer cells such as, but not limited to, lymphoma, leukemia, plasma cell neoplasms, etc. In a further embodiment, the plasma cell neoplasm is selected from plasma cell leukemia, multiple myeloma, plasmacytoma, heavy chain disease, amyloidosis, Waldenström macroglobulinemia, heavy chain disease, solitary bone plasmacytoma, monoclonal gammopathy of undetermined significance (MGUS), and smoldering multiple myeloma.

[0239] It was surprisingly found that co-expression of IL-15 / IL-15sushi or IL-15 / IL-15sushi anchor or 4-1BBL with BCMA-CD38 cCAR can result in long-term tolerance remission in patients by enhancing the sensitivity of target cancer cell recognition by CAR or by recruiting natural immune cells against cancer cells.

[0240] Without being bound by theory, co-expression of IL-21 or an IL-21 anchor with BCMA-CD38 cCAR is thought to result in long-term remission in patients by enhancing the sensitivity of CAR recognition to target cancer cells or by recruiting natural immune cells against cancer cells.

[0241] In one embodiment, the engineered cells comprise a BCMA-CD38 cCAR polypeptide, 4-1BBL and IL-15 / IL-15sushi (SEQ ID NO: 40), and the corresponding nucleotide (SEQ ID NO: 41).

[0242] Without being bound by theory, BCMA-CD38 compound CAR engineered cells are thought to provide better treatment outcomes for patients suffering from organ rejection due to autoimmune disorders or depletion of B cells and plasma cells associated with autoimmune disorders.

[0243] In some embodiments, the compound CAR (BCMA-CD38 cCAR) targets cells that express BCMA or CD38 antigen or both. The target cells may be cancer cells such as, but not limited to, lymphoma, leukemia, plasma cell neoplasms, etc. In further embodiments, the plasma cell neoplasms are selected from plasma cell leukemia, multiple myeloma, plasmacytoma, heavy chain disease, amyloidosis, Waldenström macroglobulinemia, heavy chain disease, solitary bone plasmacytoma, monoclonal gammopathy of undetermined significance (MGUS), and smoldering multiple myeloma.

[0244] The target cells of BCMA-CD38 cCAR are B cells, immature B cells, memory B cells, plasmablasts, long-lived plasma cells, or plasma cells of patients with autoimmune diseases. Autoimmune diseases include systemic sclerosis, multiple sclerosis, psoriasis, dermatitis, inflammatory bowel disease (such as Crohn's disease and ulcerative colitis), systemic lupus erythematosus, vasculitis, rheumatoid arthritis, Sjögren's syndrome, polymyositis, alveolar proteinosis, granulomatosis and vasculitis, Addison's disease, antigen-antibody complex-mediated diseases, and anti-glomerular basement membrane disease.

[0245] In another embodiment, the present disclosure provides a method of treating an autoimmune disease. The autoimmune disease is selected from the group of diseases including systemic lupus erythematosus (SLE), multiple sclerosis (MS), inflammatory bowel disease (IBD), rheumatoid arthritis, Sjogren's syndrome, dermatomyositis, autoimmune hemolytic anemia, neuromyelitis optica (NMO), NMO spectrum disorder (NMOSD), idiopathic thrombocytopenic purpura (ITP), systemic autoimmune small-vessel vasculitis syndrome or microscopic polyangiitis (MPA)-associated antineutrophil cytoplasmic autoantibodies (ANCAs), granulomatosis with polyangiitis (GPA, Wegener's granulomatosis), pemphigus vulgaris (PV) and pemphigus foliaceus (PF). Pemphigus vulgaris (PV) and pemphigus foliaceus (PF) are chronic and life-threatening blistering diseases caused by autoantibodies.

[0246] Compound CD123-CLL-1 Unlike B-cell and plasma cell malignancies, AML is very difficult to treat due to the role of leukemia stem cells (LSCs). LSCs are a population of cells that express the markers of hematopoietic stem cells (CD34 + CD38-) capable of initiating and maintaining hematopoietic malignancies and generate a clonal population of cells that outcompete healthy bone marrow. Since LSCs are mostly in the quiescent phase of the cell cycle, chemotherapy against rapidly dividing tumor populations does not affect LSCs. In most cases, it is this elusive population, including minimal residual disease (MRD), that causes inevitable relapses after AML treatment. Transplanting CAR therapy into AML to completely eliminate the disease and prevent relapse requires careful antigen selection that can eradicate not only bulk leukemia disease but also leukemia stem cells.

[0247] Single CAR therapy has recently achieved a breakthrough by achieving high remission rates in the treatment of previously refractory and relapsed B-cell malignancies. Conversely, new treatment approaches for AML are lacking, and CAR therapy offers a glimmer of hope. In particular, the application of compound CAR therapy to AML has the potential to completely transform the treatment.

[0248] CD123 and C-type lectin-like molecule-1 (CLL-1) are present on most AML CD34+CD38− cells of AML patients. Without being bound by theory, it is believed that a compound CAR can enable a single T cell encoding two separate CAR units to simultaneously target and eradicate LSCs more extensively and prevent disease recurrence.

[0249] The present disclosure includes that a single CAR T cell expressing two separate CAR units in a vector having independent signaling domains can be utilized as a novel approach to target multiple antigens and potentially avoid tumor recurrence. Compound CAR (cCAR) includes those in which a CD123 CAR is linked to a CLL-1 CAR via a self-cleaving P2A peptide and both functional CAR molecules are expressed on the surface of T cells.

[0250] In one embodiment, the CD123-CLL-1 cCAR T cell therapy can be developed as a monotherapy for patients with diseases including, but not limited to, "bridging to transplantation", supplementing chemotherapy, or checkpoint blockade (including, but not limited to, PD-L1 and CTLA-4 inhibitors), or acute myeloid leukemia, myelodysplastic syndromes, chronic myeloid leukemia, and chronic myeloproliferative disorders.

[0251] In another embodiment, the CD123-CLL-1 cCAR T cell therapy can be used to completely eliminate MRD. It is expected that the recurrence rate will decrease, the long-term disease-free survival rate will increase, and the patient outcome will be dramatically improved.

[0252] In one embodiment, the CD123-CLL1 cCAR T cell therapy can have further uses in patients with CD123+ and / or CLL-1+ multiple myeloma that crosses over to bone marrow transplantation. CD123-CLL1 cCAR T cell therapy as a monotherapy or as part of a patient-specific immunochemotherapy regimen. For elderly patients or patients with co-existing diseases who cannot tolerate very intensive chemotherapy or BMT, this may be a promising strategy to extend the patient's survival and ensure a better quality of life.

[0253] Without being bound by theory, co-expression of IL-15 / IL-15sushi or IL-15 / IL-15sushi anchor or 4-1BBL with CD123-CDLL-1 cCAR is thought to result in long-term tolerance remission in patients by enhancing the sensitivity of CAR recognition to target cancer cells or by recruiting natural immune cells against cancer cells.

[0254] Without being bound by theory, co-expression of IL-21 or IL-21 anchor with CD123-CLL-1 cCAR is thought to result in long-term tolerance remission in patients by enhancing the sensitivity of CAR recognition to target cancer cells or by recruiting natural immune cells against cancer cells.

[0255] Compound CD38 CARs for T cell malignancies The present disclosure includes that a single CAR T cell expressing two separate CAR units in a vector having independent signaling domains can be used as a novel approach to target multiple antigens and potentially avoid tumor recurrence. The CD38-based compound CAR (cCAR) includes a CD4 CAR or CD5 CAR or CD3 CAR or CD7 CAR linked to the CD38 CAR via a self-cleaving P2A peptide, and expresses both functional CAR molecules on the surface of T cells.

[0256] The present disclosure includes that a single NK cell expressing two distinct CAR units in a vector having an independent signaling domain can be utilized as a novel approach for targeting multiple antigens and potentially avoiding tumor recurrence. The CD38-based compound CAR (cCAR) includes a CD4 CAR or a CD5 CAR or a CD3 CAR or a CD7 CAR linked to the CD38 CAR via a self-cleaving P2A peptide, and expresses both functional CAR molecules on the surface of T cells.

[0257] Without being bound by theory, it is believed that CD38-based compound cCAR T or NK cells remove T cell lymphoma / leukemia cells, reduce the risk of recurrence due to antigen escape, and enhance anti-tumor activity.

[0258] The CD4-CD38 compound CAR (cCAR) containing a CD4 CAR is linked to the CD38 CAR via a self-cleaving P2A peptide and expresses both functional CAR molecules on the surface of T cells.

[0259] The CD5-CD38 compound CAR (cCAR) containing a CD5 CAR is linked to the CD38 CAR via a self-cleaving P2A peptide and expresses both functional CAR molecules on the surface of T cells.

[0260] In one embodiment, the engineered cell includes a CD5-CD38 chimeric antigen receptor polypeptide (SEQ ID NO: 18) and the corresponding nucleotide (SEQ ID NO: 19).

[0261] The CD7-CD38 compound CAR (cCAR) containing a CD4 CAR is linked to the CD38 CAR via a self-cleaving P2A peptide and expresses both functional CAR molecules on the surface of T cells.

[0262] CD56-CD38 CAR for lymphoma / leukemia CD56 is a glycoprotein that functions as a neural cell adhesion molecule. The antigen is expressed on NK cells. CD56 or CD38 is usually present in most cases of 1) aggressive NK cell leukemia / lymphoma, 2) extranodal NK / T lymphoma (nasal type), hepatosplenic T cell lymphoma, and 4) chronic NK cell lymphocytosis.

[0263] Similar to CD38, CD56 is also expressed in non-blood cells such as brain cells. The off-target effect is serious for patients who receive only CD56 or CD38 CAR T cells.

[0264] Without being bound by theory, it is believed that compound cCAR T cells with two CARs targeting different antigens have a higher binding affinity for cells with two antigens targeted by the cCAR than for cells with the antigen targeted by a single cCAR. As a result, compound cCAR T cells are believed to have a higher trafficking ability to tumors than single CAR T cells. Thus, surprisingly, the applicant has discovered that concerns about off-target effects are significantly reduced when compound cCAR cell-based therapies are used.

[0265] CD56 is a glycoprotein that functions as a neural cell adhesion molecule. The antigen is expressed on NK cells. Similar to CD38, CD56 is also expressed in non-blood cells such as brain cells. The off-target effect is serious for patients who receive only CD56 or CD38 CAR T cells. Therefore, the disclosure of the present invention provides a method for producing CD56-CD38 cCAR to reduce concerns about off-target effects associated with the use of CD56 CAR or CD38 CAR alone.

[0266] The present invention consists of a single T cell expressing two separate CAR units in a vector having independent signaling domains that can be used as a novel approach for simultaneously targeting CD56 and CD38 and potentially avoiding tumor recurrence. The CD56-CD38 compound CAR (cCAR) holds the CD56 CAR linked to the CD38 CAR via a self-cleaving P2A peptide and expresses both functional CAR molecules on the surface of T cells.

[0267] The present invention consists of a single T cell expressing two separate CAR units in a vector having independent signaling domains that can be used as a novel approach for simultaneously targeting CD56 and CD38 and potentially avoiding tumor recurrence. The CD56-CD38 compound CAR (cCAR) holds the CD56 CAR linked to the CD38 CAR via a self-cleaving P2A peptide and expresses both functional CAR molecules on the surface of NK cells.

[0268] CD19-CD38 compound CAR (CD19-CD38 cCAR) Treatment with CD19CAR in B-ALL has an initial remission rate of approximately 90%, but most of these relapse within 1 year. The relapse is due, at least in part, to antigen escape. Therefore, a more effective CAR T cell therapy for preventing relapse is urgently needed.

[0269] CD38 is another excellent target for lymphoma because its expression is generally high and uniform in lymphoma cells. CD38 is expressed in various lymphomas, including chronic lymphocytic leukemia / small lymphocytic lymphoma, follicular lymphoma, primary effusion lymphoma, diffuse large B-cell lymphoma, and lymphoplasmacytic lymphoma.

[0270] The present disclosure includes that a single CAR T cell expressing two separate CAR units in a vector having an independent signaling domain can be used as a novel approach to target multiple antigens and potentially avoid tumor recurrence. Compound CAR (cCAR) includes a CD19 CAR linked to a CD38 CAR via a self-cleaving P2A peptide, expressing both functional CAR molecules on the surface of T cells. The expression of this compound cCAR is controlled by a strong promoter SFFV to ensure proper CAR expression.

[0271] In the present disclosure, CD19-CD38 cCAR T cells can provide potent and specific anti-tumor activity in the control of lymphoma. Targeting multiple myeloma with a compound CAR that targets both BCMA and CD19 in combination is a very powerful strategy. This new approach avoids antigen escape (loss of a single antigen) resulting from selection pressure in single CAR therapy due to the pressure from the combination in the compound design.

[0272] In the present disclosure, the addition of CD38 to the BCMA CAR enhanced the anti-tumor response by eliminating surviving BCMA-CD38 + lymphoma and reducing the risk of recurrence.

[0273] Both CD19 and CD38 are widely expressed in multiple myeloma cells, and this high expression enables CD19-CD38 cCAR to potentially cover all lymphoma cells comprehensively. This allows for more complete elimination of cancer cells and reduction of antigen escape by strongly hitting multiple targets simultaneously before resistance develops.

[0274] In one embodiment, the BCMA-CD38 cCAR therapy for CD19-CD38 is a "bridge" to bone marrow transplantation (BMT) or a combination of high-dose chemotherapy and BMT. CD19-CD38 cCAR can provide a path to a potentially curative BMT option for many patients who previously had residual disease. Current literature supports the idea that reducing the minimal residual disease (MRD) level to undetectable levels may be associated with improved patient outcomes. This is very beneficial in terms of preventing recurrence of difficult-to-treat and highly aggressive malignancies.

[0275] In another embodiment, the CD19-CD38 cCAR therapy can reduce the disease burden to the lowest possible level before transplantation or completely eliminate MRD, resulting in a reduced recurrence rate and an increased long-term disease-free survival rate, and it is expected that the patient outcome will be dramatically improved.

[0276] In one embodiment, the CD19-CD38 cCAR therapy can have further uses in patients with CD19+ and / or CD38+ multiple myeloma beyond the bridge to bone marrow transplantation. CD19-CD38 cCAR therapy as a monotherapy or as part of an individualized immunochemotherapy regimen for a patient. For elderly patients or patients with co-existing diseases who cannot tolerate very intensive chemotherapy or BMT, this may be a promising strategy to extend the patient's survival and ensure a better quality of life.

[0277] In some embodiments, the present disclosure provides compound CAR polypeptide-modified cells that target cells expressing CD19 or CD38 antigen or both. The target cells may be cancer cells such as lymphoma, but are not limited thereto. In further embodiments, the lymphoma is selected without limitation from B-ALL, high-grade B-cell lymphoma, low-grade B-cell lymphoma, diffuse large B-cell lymphoma, Burkitt lymphoma, mantle cell lymphoma, CLL, marginal zone B-cell lymphoma, and follicular lymphoma.

[0278] Without being bound by theory, co-expression of IL-15 / IL-15sushi or IL-15 / IL-15sushi anchor or 4-1BBL and CD19-CD38 cCAR is thought to result in long-term durable remission in patients by enhancing the sensitivity of CAR recognition to target cancer cells or by recruiting natural immune cells against cancer cells.

[0279] Without being bound by theory, co-expression of IL-21 or IL-21 anchor and CD19-CD38 cCAR is thought to result in long-term durable remission in patients by enhancing the sensitivity of CAR recognition to target cancer cells or by recruiting natural immune cells against cancer cells.

[0280] Without being bound by theory, CD19-CD38 compound CAR engineered cells are thought to provide better treatment outcomes for patients suffering from organ rejection due to autoimmune disorders or depletion of B cells and plasma cells associated with autoimmune disorders.

[0281] In some embodiments, the compound CAR (BCMA-CD38 cCAR) targets cells that express BCMA or CD38 antigen or both. The target cells may be cancer cells such as, but not limited to, lymphoma, leukemia, plasma cell neoplasms, etc. In further embodiments, the plasma cell neoplasm is selected from plasma cell leukemia, multiple myeloma, plasmacytoma, heavy chain disease, amyloidosis, Waldenström macroglobulinemia, heavy chain disease, solitary bone plasmacytoma, monoclonal gammopathy of undetermined significance (MGUS), and smoldering multiple myeloma.

[0282] The target cells of the BCMA-CD38 cCAR are B cells, immature B cells, memory B cells, plasmablasts, long-lived plasma cells, or plasma cells of patients with autoimmune diseases. Autoimmune diseases include systemic sclerosis, multiple sclerosis, psoriasis, dermatitis, inflammatory bowel diseases (such as Crohn's disease and ulcerative colitis), systemic lupus erythematosus, vasculitis, rheumatoid arthritis, Sjögren's syndrome, polymyositis, alveolar proteinosis, granulomatosis with polyangiitis, Addison's disease, antigen-antibody complex-mediated diseases, and anti-glomerular basement membrane diseases.

[0283] In another embodiment, the present disclosure provides a method for treating an autoimmune disease. The autoimmune disease is selected from the group of diseases including systemic lupus erythematosus (SLE) including systemic lupus, multiple sclerosis (MS), inflammatory bowel disease (IBD), rheumatoid arthritis, Sjögren's syndrome, dermatomyositis, autoimmune hemolytic anemia, neuromyelitis optica (NMO), NMO spectrum disorder (NMOSD), idiopathic thrombocytopenic purpura (ITP), antineutrophil cytoplasmic autoantibodies (ANCAs) associated with systemic autoimmune small-vessel vasculitis or microscopic polyangiitis (MPA), granulomatosis with polyangiitis (GPA, Wegener's granulomatosis), pemphigus vulgaris (PV) and pemphigus foliaceus (PF). Pemphigus vulgaris (PV) and pemphigus foliaceus (PF) are chronic and life-threatening blistering diseases caused by autoantibodies.

[0284] BCMA-CD19 compound CAR (BCMA-CD19 cCAR ) Killing multiple myeloma cells results in short-term remission, but LSCs (multiple myeloma leukemia stem cells) always regrow if not destroyed, causing relapse in patients. To achieve a permanent cure for multiple myeloma, it is essential to destroy LSCs. Although not wishing to be bound by theory, a small subset of multiple myeloma cells is CD19-positive and is the stem cell associated with disease progression and relapse, and the large multiple myeloma cell population is thought to be BCMA-positive. Therefore, it is important to develop new therapies that can specifically target both the myeloma stem cell population and the large myeloma population. CompoundCAR in the present disclosure targets both the BCMA+ and / or CD19+9 positive populations of multiple myeloma cells and is embodied herein.

[0285] In some embodiments, the present disclosure provides a method of eradicating or killing myeloma stem cells (LSCs) or bulk myeloma cells that express CD19 and / or BCMA. In this embodiment, T or NK engineered cells having BCMA and CD19 units are administered to a patient in need thereof.

[0286] In some embodiments, the disclosed disclosure includes methods and compositions for eliminating both BCMA and CD19 populations to prevent relapse of multiple myeloma using BCMA-CD19 cCAR. The CAR is more potent at removing myeloma cells when the two units of BCMA and CD19 (BCMA-CD19) are combined either intracellularly or in a vector.

[0287] In a further embodiment, BCMA-CD19 cCAR, a compound CAR in T or NK cells, can be used to eradicate or kill BCMA+CD19+ or BCMA+CD19- or BCMA-CD19+ populations.

[0288] In some embodiments, the disclosed disclosure includes methods and compositions for eliminating both BCMA and CD19 populations using BCMA-CD19 cCAR to prevent recurrence of multiple myeloma. The CAR is more potent at removing myeloma cells when the two units of BCMA and CD19 (BCMA-CD19) are combined intracellularly or in a vector.

[0289] In some embodiments, the CD19+ population can be an early precursor of multiple myeloma cells, and the CD19-BCMA+ cells can be more differentiated malignant multiple myeloma cells. In some embodiments, the disclosed invention includes methods and compositions for eliminating both early precursors of multiple myeloma cells and more differentiated malignant multiple myeloma cells using BCMA-CD19b cCAR (a version of BCMA-CD19 cCAR) T or NK cells. In further embodiments, the disclosed disclosure includes methods and compositions for targeting both early precursor cells and more differentiated malignant cells and completely eliminating the malignant clones of multiple myeloma using BCMA-CD19b cCAR T or NK cells.

[0290] The present disclosure further discloses a compound CAR construct having enhanced efficacy of anti-myeloma cell activity against cells co-expressing target antigens, while also retaining sensitivity to tumor cells expressing only one antigen. Further, each CAR of the compound CAR includes one or two co-stimulatory domains and exhibits potent killing ability in the presence of a specific target.

[0291] Without being bound by theory, co-expression of IL-15 / IL-15sushi or IL-15 / IL-15sushi anchor or 4-1BBL with BCMA-CD19 cCAR is thought to result in long-term tolerance remission in patients by enhancing the sensitivity of CAR recognition to target myeloma cells or by recruiting natural immune cells against myeloma cells.

[0292] In some embodiments, the compound CAR (BCMA-CD19 cCAR) targets cells expressing BCMA or CD19 antigen or both. The target cells may be cancer cells such as, but not limited to, lymphoma, leukemia, plasma cell neoplasms, etc. In further embodiments, the plasma cell neoplasms are selected from plasma cell leukemia, multiple myeloma, plasmacytoma, heavy chain disease, amyloidosis, Waldenström macroglobulinemia, heavy chain disease, solitary bone plasmacytoma, monoclonal gammopathy of undetermined significance (MGUS), and smoldering multiple myeloma.

[0293] Without being bound by theory, co-expression of IL-21 or an IL-21 anchor with BCMA-CD19 cCAR is thought to result in long-term durable remission in patients by enhancing the sensitivity of CAR recognition to target myeloma cells or by recruiting natural immune cells against myeloma cells.

[0294] Without being bound by theory, co-expression of IL-18 or an IL-18 anchor with BCMA-CD19 cCAR is thought to result in long-term durable remission in patients by enhancing the sensitivity of CAR recognition to target myeloma cells or by recruiting natural immune cells against myeloma cells.

[0295] In some embodiments, the present disclosure provides a method of depleting B cells, immature B cells, memory B cells, plasmablasts, long-lived plasma cells, or plasma cells in a patient having an autoimmune disease by administering CAR or compound CAR (BCMA-CD19 cCAR) T cells or NK cells to the patient.

[0296] The target cells of BCMA-CD19 cCAR are B cells, immature B cells, memory B cells, plasmablasts, long-lived plasma cells, or plasma cells of patients with autoimmune diseases. Autoimmune diseases include systemic sclerosis, multiple sclerosis, psoriasis, dermatitis, inflammatory bowel diseases (such as Crohn's disease and ulcerative colitis), systemic lupus erythematosus, vasculitis, rheumatoid arthritis, Sjögren's syndrome, polymyositis, alveolar proteinosis, granulomatosis with polyangiitis, Addison's disease, antigen-antibody complex-mediated diseases, and anti-glomerular basement membrane diseases.

[0297] In some embodiments, the present disclosure provides a method of depleting B cells, immature B cells, memory B cells, plasmablasts, long-lived plasma cells, or plasma cells of a patient having an autoimmune disease by administering BCMA and CD19 bispecific CAR T cells or bispecific antibodies to the patient.

[0298] In another embodiment, the present disclosure provides a method of treating an autoimmune disease. The autoimmune disease is selected from the group of diseases including systemic lupus erythematosus (SLE), multiple sclerosis (MS), inflammatory bowel disease (IBD), rheumatoid arthritis, Sjogren's syndrome, dermatomyositis, autoimmune hemolytic anemia, neuromyelitis optica (NMO), NMO spectrum disorder (NMOSD), idiopathic thrombocytopenic purpura (ITP), antineutrophil cytoplasmic autoantibodies (ANCAs) associated with systemic autoimmune small-vessel vasculitis syndromes or microscopic polyangiitis (MPA), granulomatosis with polyangiitis (GPA, Wegener's granulomatosis), pemphigus vulgaris (PV) and pemphigus foliaceus (PF). Organ transplantation symbolizes a new life for a person, and transplantable organs include kidneys, hearts, lungs, pancreases, and intestines. However, many patients are unable to receive life-saving organs due to existing or developing donor-specific antibodies against donor antigens such as human leukocyte antigen (HLA). Thus, patients may lose the provided organs. Currently, there are few treatment options available for antibody-mediated rejection, and there is a large unmet need in the field for effective treatment of antibody-mediated rejection reactions. Deletion of B cells or plasma cells or both using CAR T / NK cells provides a treatment for antibody-mediated rejection.

[0299] The target cells of BCMA-CD19 cCAR or CD19-CD38 cCAR or BCMA-CD38 cCAR are B cells, immature B cells, memory B cells, plasmablasts, long-lived plasma cells, or plasma cells of a patient with an antibody-mediated rejection reaction associated with organ rejection.

[0300] Engineered cells having a CAR polypeptide and an enhancer In another example, the present disclosure provides engineered cells having at least one chimeric antigen receptor polypeptide and an enhancer.

[0301] In another example, the present disclosure provides engineered cells having at least one chimeric antigen receptor polypeptide and at least one enhancer.

[0302] In this example, the present disclosure provides engineered cells having at least two different chimeric antigen receptor polypeptides and an enhancer.

[0303] In this example, the present disclosure provides engineered cells having at least two different chimeric antigen receptor polypeptides and at least one enhancer.

[0304] As used herein, an enhancer includes a biomolecule that promotes or enhances the activity of an engineered cell having a chimeric antigen receptor polypeptide. Cytokines are also included in the enhancer. In another example, the enhancer includes IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, IL-21 anchor, PD-1, PD-L1, CSF1R, CTAL-4, TIM-3, and TGFR-beta, the receptors, and functional fragments thereof.

[0305] The enhancer can be expressed by the engineered cells described herein and presented on the surface of the engineered cells, or the enhancer can be secreted into the surrounding extracellular space by the engineered cells. Methods of surface display and secretion are well known in the art. For example, the enhancer may be a fusion protein with a peptide that provides surface display or secretion into the extracellular space.

[0306] The effect of the enhancer may be complemented by additional factors such as enhancer receptors and functional fragments thereof. The additional factors may be co-expressed with the enhancer as a fusion protein, or expressed as a separate peptide and secreted into the extracellular space.

[0307] The enhancer is a cytokine secreted from engineered CAR cells and is designed to co-express with the CAR polypeptide. Massive release occurs upon the involvement of cognate antigen and CAR. Inflammatory cells surrounding tumor cells have a significant correlation with the progression and metastasis of cancer cells. Inflammatory cells include innate immune response cells such as T cells and NK cells, macrophages, dendritic cells, etc., and their proliferation and anti-tumor activity are regulated by cytokines. CAR cells such as CAR T cells or NK cells bind to target cancer cells and induce massive secretion of the enhancer from the proliferation of CAR T / NK cells. The secreted enhancer efficiently promotes the survival, differentiation, and activation of immune response cells against cancer cells. Co-expression of the enhancer and CAR can compensate for the drawback that CAR T or NK cells cannot eliminate non-target cancer cells.

[0308] CAR cells are carriers of cytokines and can deliver cytokines to target cancer sites by CAR cells to reduce the systemic toxicity caused by high-dose exogenous cytokines.

[0309] To improve the persistent survival or long-term persistence of CAR cells, a membrane-bound enhancer can be co-expressed with CAR to improve CAR persistence.

[0310] In this example, the enhancer is IL-15. In this case, the above additional factors are the IL-15 receptor and its functional fragments. Functional fragments include the IL-15 receptor, IL-15RA, and the sushi domain of IL-15RA (IL-15sushi). Soluble IL-15RA or IL15sushi significantly enhances IL-15 functional activity by preventing the degradation of IL-15. The soluble IL-15 / IL-15RA or IL-15 / IL-15sushi complex is much more stable and stimulatory in vivo than IL-15 alone.

[0311] In this example, IL-15 is co-expressed as a fusion protein with at least one of the sushi domains of the IL-15 receptor, IL-15RA, and IL-15RA (IL-15sushi). In this example, the IL-15 receptor, IL-15RA, or the sushi domain of IL-15RA (IL-15sushi) is at the N-terminus of IL-15. In another example, the IL-15 receptor, IL-15RA, or the sushi domain of IL-15RA (IL-15sushi) is at the C-terminus of IL-15. As used herein, IL-15 / IL-15sushi indicates that IL-15sushi is at the C-terminus of IL-15 in the fusion protein, and IL-15sushi / il-15 indicates that IL-15sushi is at the N-terminus of IL-15 in the fusion protein.

[0312] In some examples, IL-15 and the IL-15 receptor or a functional fragment polypeptide thereof are on a single polypeptide molecule, separated by a peptide linker, and the peptide linker is 1 to 25 amino acid residues in length, 25 to 100 amino acid residues in length, or 50 to 200 amino acid residues in length. This linker may contain the highly efficient cleavage sites described herein.

[0313] Interleukin (IL)-15 and its specific receptor chain, IL-15Rα (IL-15-RA), play important functional roles in various effector cells, including NK and CD8 T cells. CD8 + T cells are modified to express autocrine growth factors, including, but not limited to, IL-2, IL-7, IL21, or IL-15, to maintain their survival after transfer in vivo. Without wishing to be bound by theory, IL-15 is thought to overcome CD4 deficiency, induce primary memory, and recall memory CD8 T cells. Overexpression of IL-15-RA or an IL-15 IL-RA fusion on CD8 T cells significantly enhances their survival and proliferation in vitro and in vivo. In some embodiments, a CD4 CAR or a CD3 CAR or a CD5 CAR or a CD20 CAR, CD33 CAR, CLL-1 or CD123 CAR, CD19 CAR or CCD45 CAR or GD2 CAR, BCMA CAR, or any CAR co-expresses at least one, or a portion, or a combination of IL-15, IL15RA, and IL-15 / IL-15RA or IL15-RA / IL-15 or IL-15 / IL-15 sushi to enhance the survival or proliferation of CAR T or NK and improve the proliferation of memory CAR CD8 + T cells.

[0314] A CD4 CAR or a CD7 CAR, a CD3 CAR or a CD5 CAR or a CD20 CAR, CD33 CAR, CLL-1 or CD123 CAR, CD19 CAR or GD2 CAR or CD45 CAR or BCMA CAR, or any CAR co-expresses at least one, or a portion, or a combination of IL-15 / IL-15 sushi to enhance the survival or proliferation of CAR NK and improve the proliferation of memory CAR CD8 + T cells.

[0315] Surprisingly, it has been revealed that co-expression of IL-15 / IL-15sushi and CAR is important for the longer persistence and enhanced activity of T cells and NK cells targeting tumor cells. Surprisingly, co-expression of IL-15 / IL-15sushi and CAR has been found to be important for T cells, NK T cells, and NK cells to target tumor cells and prevent cancer recurrence. Surprisingly, it has been found that co-expressing IL-15 / IL-15sushi with CAR in NK cells and NK cells can extend the survival period.

[0316] The present disclosure provides engineered cells having at least one of IL-15, IL-15RA, IL-15sushi, IL-15 / IL-15RA, IL15-RA / IL-15, IL-15 / IL-15sushi, IL15sushi / IL-15, fragments thereof, combinations thereof, to enhance the survival or persistence or proliferation of CAR T or NK for treating a patient's cancer with the CAR polypeptides described herein.

[0317] In one embodiment, the engineered cells comprise a CD5 chimeric antigen receptor polypeptide and IL-15 / IL-15sushi (SEQ ID NO: 48), and the corresponding nucleotide (SEQ ID NO: 49).

[0318] In one embodiment, the engineered cells comprise a CD4 chimeric antigen receptor polypeptide and IL-15 / IL-15sushi (SEQ ID NO: 22), and the corresponding nucleotide (SEQ ID NO: 23).

[0319] In one embodiment, the engineered cells comprise a C4 chimeric antigen receptor polypeptide, 4-1BBL and IL-15 / IL-15sushi (SEQ ID NO: 20), and the corresponding nucleotide (SEQ ID NO: 21).

[0320] In one embodiment, the engineered cells comprise a CD3 chimeric antigen receptor polypeptide, 4-1BBL and IL-15 / IL-15sushi (SEQ ID NO: 18), and the corresponding nucleotide (SEQ ID NO: 19).

[0321] In one embodiment, the engineered cells comprise a CD19 chimeric antigen receptor polypeptide and IL-15 / IL-15sushi (SEQ ID NO: 24), and the corresponding nucleotide (SEQ ID NO: 25).

[0322] In one embodiment, the engineered cells comprise a CD19 chimeric antigen receptor polypeptide, 4-1BBL and IL-15 / IL-15sushi (SEQ ID NO: 26), and the corresponding nucleotide (SEQ ID NO: 27).

[0323] In one embodiment, the engineered cells comprise a CD33 chimeric antigen receptor polypeptide, 4-1BBL and IL-15 / IL-15sushi (SEQ ID NO: 30), and the corresponding nucleotide (SEQ ID NO: 31).

[0324] In one embodiment, the engineered cells comprise a CD123 chimeric antigen receptor polypeptide, 4-1BBL and IL-15 / IL-15sushi (SEQ ID NO: 32), and the corresponding nucleotide (SEQ ID NO: 33).

[0325] In one embodiment, the engineered cells comprise a BCMA chimeric antigen receptor polypeptide, 4-1BBL and IL-15 / IL-15sushi (SEQ ID NO: 38), and the corresponding nucleotide (SEQ ID NO: 39).

[0326] In one embodiment, the engineered cells comprise a GD2 chimeric antigen receptor polypeptide, 4-1BBL and IL-15 / IL-15sushi (SEQ ID NO: 46), and the corresponding nucleotide (SEQ ID NO: 47).

[0327] In one embodiment, the engineered cell comprises a GD2 chimeric antigen receptor polypeptide (SEQ ID NO: 56) and the corresponding nucleotide (SEQ ID NO: 57).

[0328] In one embodiment, the engineered cell comprises a GD2 chimeric antigen receptor polypeptide and 4-1BBL (SEQ ID NO: 56) and the corresponding nucleotide (SEQ ID NO: 57).

[0329] In one embodiment, the engineered cell comprises a CD45 chimeric antigen receptor polypeptide and IL-15 / IL-15sushi (SEQ ID NO: 54) and the corresponding nucleotide (SEQ ID NO: 55).

[0330] In another example, the present disclosure provides engineered cells comprising recombinant IL-15, IL-15RA, IL-15sushi, IL-15 / IL-15RA, IL15-RA / IL-15, IL-15 / IL-15sushi, IL15sushi / IL-15, functional fragments thereof, and combinations thereof, and at least one distinct CAR polypeptide, wherein the antigen recognition domain comprises at least one selected from GD2, GD3, interleukin 6 receptor, ROR1, PSMA, PSCA (prostate stem cell antigen), MAGE A3, glycolipid, glypican 3, F77, GD-2, WT1, CEA, HER-2 / neu, MAGE-3, MAGE-4, MAGE-5, MAGE-6, alpha-fetoprotein, CA 19-9, CA 72-4, NY-ESO, FAP, ErbB, c-Met, MART-1, MUC1, MUC2, MUC3, MUC4, MUC5, CD30, EGFRvIII, CD33, CD123, CLL-1, immunoglobulin kappa and lambda, CD38, CD52, CD19, CD20, CD22, CD38, BCMA, CS1, BAFF receptor, TACI, CD3, CD4, CD8, CD5, CD7, CD2, and CD138.

[0331] Without being bound by theory, IL-15 / IL-15sushi and other types of IL-15 or IL-15RA proteins or protein fragments thereof are thought to provide a synergistic effect of the CAR polypeptide when combined with a checkpoint inhibitor or modulator (e.g., anti-PD-1).

[0332] In one embodiment, the present disclosure provides a method of providing long-term sustained remission to a patient suffering from cancer by administering to the patient in need thereof CAR-engineered cells that co-express an IL-21 or IL-12 anchor (FIGS. 24 and 25). Without being bound by theory, co-expression of IL-21 or an IL-21 anchor and a CAR is thought to result in long-term remission in the patient by enhancing the persistence of the CAR-engineered cells.

[0333] Co-expression of secreted IL-21 and a CAR polypeptide is thought to result in long-term remission in the patient by affecting the tumor microenvironment and resulting in a decrease in immunosuppression and promotion of natural cell proliferation or function.

[0334] Without being bound by theory, co-expression of secreted IL-21 or an IL-21 anchor and a CAR is thought to be important for the long-term persistence and activation of T cells, NK T cells, and NK cells that target tumor cells. CAR NK cells or NK cells or NK T cells can have their survival period extended by co-expressing with IL-21 or an IL-21 anchor.

[0335] In one embodiment, the present disclosure provides methods related to the ability of CAR T or NK cells targeting tumor cells to be carriers for delivering an enhancer, IL-21, to the tumor microenvironment. The CAR T or NK cells are designed to co-express secreted IL-21. The CAR T or NK T cells or NK cells engineered in the tumor microenvironment target tumor cells, bind to the CAR targeting antigen, and cause lysis of the tumor cells and induction of massive secretion of soluble IL-21 by the proliferation of the CAR T or NK T cells or NK cells.

[0336] In certain embodiments, tumor elimination can be achieved by at least one or a combination of the following steps: (1) Binding of some of the tumor cells by the CAR engineered T cells or NK cells or NK T cells disclosed herein by targeting the CAR antigen. (2) Triggering massive secretion of IL-21 by the proliferation of CAR T / NK cells co-expressing this molecule. (3) Recruiting and stimulating various innate and adaptive immune cells against the tumor. (4) Reducing tumor suppression present in the tumor by administration of checkpoint blockade such as PD-L1 and CTLA-4 inhibitors.

[0337] Without being bound by theory, the combination of the above steps is thought to provide a potent anti-tumor effect through coordinated innate and adaptive immune responses.

[0338] In another embodiment, the present disclosure includes IL-21, or an IL-21 anchor, a functional fragment thereof, and combinations thereof, and at least one distinct CAR polypeptide, wherein the antigen recognition domain is GD2, GD3, interleukin-6 receptor, ROR1, PSMA, PSCA (prostate stem cell antigen), MAGE A3, glycolipid, glypican 3, F77, GD-2, WT1, CEA, HER-2 / neu, MAGE-3, MAGE-4, MAGE-5, MAGE-6, alpha-fetoprotein, CA 19-9, CA 72-4, NY-ESO, FAP, ErbB, c-Met, MART-1, MUC1, MUC2, MUC3, MUC4, MUC5, CD30, EGFRvIII, CD33, CD123, CLL-1, immunoglobulin kappa and lambda, CD38, CD52, CD19, CD20, CD22, CD38, BCMA, CS1, BAFF receptor, TACI, CD3, CD4, CD8, CD5, CD7, CD2 and CD138, and provides engineered cells comprising at least one of them.

[0339] In one embodiment, the present disclosure provides a method of providing long-term durable remission to a patient suffering from cancer by administering to the patient in need thereof CAR-engineered cells that co-express IL-18 or an IL-18 anchor (Figures 26 and 27). Without being bound by theory, co-expression of IL-18 or an IL-18 anchor and a CAR is thought to result in long-term remission in the patient by enhancing the persistence of the cells engineered with the CAR.

[0340] Co-expression of secreted IL-18 and a CAR polypeptide is thought to result in long-term remission in the patient by affecting the tumor microenvironment, resulting in reduced immunosuppression and promotion of natural cell proliferation or function.

[0341] Without being bound by theory, co-expression of secreted IL-18 or an IL-18 anchor and a CAR is thought to be important for the long-term persistence and enhanced activity of T cells and NK cells targeting tumor cells. CAR NK cells or NK cells can have their survival extended by co-expression with IL-18 or an IL-18 anchor.

[0342] In one embodiment, the present disclosure provides methods related to the possibility that CAR T or NK cells targeting tumor cells can be carriers for delivering an enhancer, IL-18, to the tumor microenvironment. CAR T or NK cells are designed to co-express secreted IL-18. CAR T or NK cells engineered in the tumor microenvironment target tumor cells, bind to the CAR targeting antigen, and cause lysis of the tumor cells and induction of massive secretion of soluble IL-18 by proliferation of the CAR T or NK cells.

[0343] In certain embodiments, tumor removal can be achieved by at least one or a combination of the following steps: (1) Binding of the CAR-engineered T cells or NK cells disclosed herein to a portion of the tumor cells by targeting the CAR antigen. (2) Triggering massive secretion of IL-18 by proliferation of the CAR T / NK cells co-expressing this molecule. (3) Recruiting and stimulating various innate and adaptive immune cells against the tumor. (4) Reducing tumor suppression present in the tumor by administration of checkpoint blockade such as PD-L1 and CTLA-4 inhibitors.

[0344] Without being bound by theory, the combination of the above steps is thought to provide a potent anti-tumor effect through coordinated innate and adaptive immune responses.

[0345] In another embodiment, the present disclosure includes IL-18, or an IL-18 anchor, a functional fragment thereof, and combinations thereof, and at least one distinct CAR polypeptide, wherein the antigen recognition domain is GD2, GD3, interleukin-6 receptor, ROR1, PSMA, PSCA (prostate stem cell antigen), MAGE A3, glycolipid, glypican 3, F77, GD-2, WT1, CEA, HER-2 / neu, MAGE-3, MAGE-4, MAGE-5, MAGE-6, alpha-fetoprotein, CA 19-9, CA 72-4, NY-ESO, FAP, ErbB, c-Met, MART-1, MUC1, MUC2, MUC3, MUC4, MUC5, CD30, EGFRvIII, CD33, CD123, CLL-1, immunoglobulin kappa and lambda, CD38, CD52, CD19, CD20, CD22, CD38, BCMA, CS1, BAFF receptor, TACI, CD3, CD4, CD8, CD5, CD7, CD2, and CD138, and provides engineered cells comprising at least one of them.

[0346] In some embodiments, two or more different antigens can be targeted by pooled CAR-engineered cells produced by at least two distinct CART or NK cells.

[0347] As used herein, pooled CAR-engineered cells include a population of engineered cells having one or more different CAR polypeptide units. As an example, the pooled engineered cells include a population of engineered cells having different CAR polypeptides and a population of engineered cells having different and distinct CAR polypeptides. Further, pooled CAR-engineered cells include engineered cells having a cCAR polypeptide.

[0348] Methods for producing engineered cells Any of the polynucleotides disclosed herein can be introduced into engineered cells by any method known in the art.

[0349] In this example, the CAR polynucleotide is delivered to cells engineered by any of the viral vectors disclosed herein.

[0350] In this example, to achieve an improved safety profile or therapeutic index, any engineered cells disclosed herein are constructed as transient RNA-modified "biodegradable" versions or derivatives, or combinations thereof. The RNA-modified CARs of the invention may be electroporated into T cells or NK cells. Expression of the compound CAR may gradually decrease over several days.

[0351] In some examples of the invention, any engineered cells disclosed herein can be constructed with a transposon system (also called "Sleeping Beauty") that integrates CAR DNA into the host genome without a viral vector.

[0352] In some embodiments of the present disclosure, any engineered cells disclosed herein can be introduced by two vectors, each vector carrying a unit of CAR or enhancer. A method for producing an artificial cell having multiple CAR units

[0353] In another example, the present disclosure provides a method for producing engineered cells having at least two CAR units.

[0354] In some embodiments, multiple units of the CAR are expressed in T cells or NK cells using a bicistronic or multicistronic expression vector. There are several strategies that can be used to construct a bicistronic or multicistronic vector, including but not limited to: (1) multiple promoters fused to the open reading frame of the CAR, (2) insertion of splicing signals between units of the CAR, fusion of the CARs such that expression is driven by a single promoter, (3) insertion of a proteolytic cleavage site (self-cleaving peptide) between units of the CAR, and (iv) insertion of internal ribosome entry sites (IRESs), (5) separating the different units of the CAR into two vectors, etc.

[0355] In a preferred embodiment, multiple CAR units are expressed in a single open reading frame (ORF), thereby producing a single polypeptide having multiple CAR units. In this embodiment, an amino acid sequence or linker containing a highly efficient cleavage site is placed between each CAR unit.

[0356] As used herein, high cleavage efficiency is defined as 50% or more, 70% or more, 80% or more, or 90% or more of the translated protein being cleaved. Cleavage efficiency may be evaluated by Western blot analysis as described by Kim 2011.

[0357] Furthermore, in a preferred embodiment, as shown by Western blot analysis, equal amounts of cleavage products are present.

[0358] Examples of highly efficient cleavage sites include, but are not limited to, Porcine teschovirus-1 2A (P2A), FMDV 2A (abbreviated as F2A herein), Equine rhinitis A virus (ERAV) 2A (E2A), Thosea asigna virus (T2A), Bombyx cytoplasmic polyhedrosis virus 2A (BmCPV2A) and Bombyx mori infectious flacherie virus 2A (BmIFV2A), or combinations thereof. In a preferred embodiment, the highly efficient cleavage site is P2A. The highly efficient cleavage site is used herein with reference to the description in Kim JH, Lee S-R, Li L-H, Park H-J, Park J-H, Lee KY, et al. (2011) High Cleavage Efficiency of a 2A Peptide Derived from Porcine Teschovirus-1 in Human Cell Lines, Zebrafish and Mice. PLoS ONE 6(4): e18556.

[0359] In the examples herein, multiple CAR units are expressed under a single open reading frame (ORF), and the expression is under the control of a strong promoter. Examples of strong promoters include the SFFV promoter and its derivatives.

[0360] When designing longer gene constructs, the level of protein expression decreases significantly with each additional 1 kb in length. Therefore, it is preferred to initially screen several antigen recognition sequences to find combinations that result in both the highest transduction efficiency and the highest target cell lysis. Additionally, it is preferred to avoid very high CAR expression that leads to tonic effects and poor lysis caused by single-chain aggregation on the cell surface.

[0361] In embodiments where multiple CAR units are expressed intracellularly, it is preferred to avoid CAR-CAR interactions between the hinge regions of each individual CAR. It is preferred to exclude the hinge interaction sites or for each unit of the CAR to use a different hinge region to avoid interactions.

[0362] In some embodiments where multiple CAR units are expressed in cells, it is preferred that the different nucleotide sequences common to each domain, such as the leader sequence, hinge and transmembrane region, and CD3 zeta region, avoid homologous recombination while maintaining the same amino acid sequence.

[0363] In some embodiments where multiple CAR units are created, based on medical knowledge and background, and based on obtaining the best therapeutic effect, the selection of the target antigen is preferred.

[0364] In some embodiments, two or more different antigens can be targeted by pooled CAR-engineered cells produced by at least two separate CART or NK cells.

[0365] To test specificity, it is preferred to perform co-culture lysis experiments on both on-target and off-target cell lines using CAR T or NK cells. Further, to show the ability of each component CAR to lyse, it is preferred to use cell lines that express only one targeted antigen each. To do this, it is preferred to create off-target cell lines and synthetically express the desired antigen.

[0366] In some embodiments, two or more different antigens can be targeted by pooled CAR-engineered cells produced by at least two separate CART or NK cells.

[0367] As used herein, pooled CAR-engineered cells include a population of engineered cells having one or more different CAR polypeptide units. As an example, pooled engineered cells include a population of engineered cells having different CAR polypeptides and a population of engineered cells having different and distinct CAR polypeptides.

[0368] Engineered cells with a CAR polypeptide and an enhancer. In another embodiment, the present disclosure provides a method of generating engineered cells that express at least one CAR unit and an enhancer.

[0369] In some embodiments, at least one CAR unit and an enhancer are expressed in T cells or NK cells using a bicistronic or multicistronic expression vector. There are several strategies that can be used to construct a bicistronic or multicistronic vector, including but not limited to: (1) multiple promoters fused to the open reading frame of the CAR, (2) insertion of a splicing signal between units of the CAR, and fusion of the CAR whose expression is driven by a single promoter, (3) insertion of a proteolytic cleavage site (self-cleaving peptide) between units of the CAR, and (4) insertion of an internal ribosome entry site (IRES).

[0370] In some embodiments, expression of at least one CAR and an enhancer in T cells or NK cells can be achieved by two separate vectors or viruses.

[0371] In a preferred embodiment, at least one CAR unit and an enhancer are expressed in a single open reading frame (ORF), thereby producing a single polypeptide having at least one CAR unit and an enhancer. In this embodiment, an amino acid sequence or linker containing a highly efficient cleavage site is placed between each CAR unit and between the CAR unit and the enhancer. In this embodiment, the ORF is under the control of a strong promoter. Examples of strong promoters include the SFFV promoter and its derivatives.

[0372] Furthermore, in a preferred embodiment, equal amounts of cleavage products are present as shown by Western blot analysis.

[0373] Combination therapy Using the compositions and methods of the present disclosure, a population of CAR T lymphocytes or NK cells can be generated that delivers both primary and costimulatory signals for use in immunotherapy in the treatment of cancer. In further embodiments, the invention with respect to clinical aspects is combined with other agents effective in the treatment of proliferative diseases such as anti-cancer agents. The anti-cancer agent can reduce the tumor burden in a subject. Anti-cancer agents include chemotherapy, radiotherapy, and immunotherapy.

[0374] More than 50% of cancer patients undergo some type of surgery. Curative surgery includes resection in which all or part of the cancerous tissue is physically removed, excised, and / or destroyed.

[0375] The compositions and methods described in the present disclosure may be utilized in conjunction with other types of cancer therapies such as chemotherapy, surgery, radiotherapy, gene therapy, and the like.

[0376] According to the present disclosure, natural killer (NK) cells exhibit alternative cytotoxic effectors for CAR-mediated killing. Unlike T cells, NK cells do not require pre-activation and constitutively exert a cytolytic function. Further expression of cCAR in NK cells enables NK cells to effectively kill cancer, particularly cancer cells resistant to NK cell therapy.

[0377] Furthermore, NK cells are known to mediate anti-cancer effects without the risk of inducing graft-versus-host disease (GvHD).

[0378] The present disclosure may be better understood with reference to the examples described below. The following examples are presented to provide those skilled in the art with a complete disclosure and description of how the compounds, compositions, articles, devices, and / or methods claimed herein are made and evaluated and are purely illustrative in intent and are not intended to limit the disclosure.

[0379] In the administration of any of the engineered cells described herein, co - administration of a CAR enhancer may be added. Examples of CAR enhancers include, but are not limited to, immunomodulatory agents that enhance CAR activity to enhance the therapeutic effect, such as agents that target the immune checkpoint pathway, inhibitors of colony - stimulating factor - 1 receptor (CSF1R), etc. Agents that target the immune checkpoint pathway include small molecules, proteins or antibodies that bind to the inhibitory immune receptors CTLA - 4, PD - 1, and PD - L1 and result in CTLA - 4 and PD - 1 / PD - L1 blockade. As used herein, enhancer also includes the enhancers described above.

[0380] As used herein, "patient" includes mammals. The mammals referred to herein can be any mammal. The term "mammal" as used herein means any mammal including, but not limited to, rodent mammals such as mice and hamsters, and lagomorph mammals such as rabbits. Mammals can also be from the order Carnivora including the families Felidae (cats) and Canidae (dogs). Mammals can also be from the order Artiodactyla including the genus Bos (cows) and the genus Sus (pigs) or from the order Perissodactyla including the genus Equus (horses). Mammals can be from the order Primates, the suborder Prosimii, or the suborder Simiiformes (monkeys), or the parvorder Catarrhini (humans and apes). Preferably, the mammal is a human. A patient includes a subject.

[0381] In certain embodiments, the patient is a human of 0 - 6 months old, 6 - 12 months old, 1 - 5 years old, 5 - 10 years old, 5 - 12 years old, 10 - 15 years old, 15 - 20 years old, 13 - 19 years old, 20 - 25 years old, 25 - 30 years old, 20 - 65 years old, 30 - 35 years old, 35 - 40 years old, 40 - 45 years old, 45 - 50 years old, 50 - 55 years old, 55 - 60 years old, 60 - 65 years old, 65 - 70 years old, 70 - 75 years old, 75 - 80 years old, 80 - 85 years old, 85 - 90 years old, 90 - 95 years old, or 95 - 100 years old.

[0382] As described herein, the terms "effective amount" and "therapeutically effective amount" of the engineered cells mean an amount of the engineered cells sufficient to provide the desired treatment or physiological or effect or therapeutic effect. Such effects or therapeutic effects include the alleviation or amelioration of the symptoms of a cellular disease. Undesirable effects, such as side effects, may sometimes appear along with the desired therapeutic effect. Therefore, the practitioner balances the potential effects and potential risks to determine what the appropriate "effective amount" is. The exact amount required will vary from patient to patient depending on the species, age and general condition of the subject, the mode of administration, and others. Therefore, it may not be possible to specify the exact "effective amount". However, for any individual, the appropriate "effective amount" can be determined by one of ordinary skill in the art using only routine experimentation. Generally, a particular engineered cell or group of engineered cells is administered in an amount and under conditions sufficient to reduce the growth of the target cells.

[0383] After administration in a delivery system for the treatment, inhibition or prevention of cancer, the effectiveness of the engineered cells for treatment can be evaluated using various techniques well known to skilled experts. For example, one of ordinary skill in the art will understand that an engineered cell engineered for treatment delivered with a chemotherapy adjuvant is effective for treating or inhibiting the cancer of a subject by observing whether it reduces the cancer cell load or prevents further growth. The cancer cell load can be measured by methods known in the art, for example, using a polymerase chain reaction assay to detect the presence of nucleic acids of specific cancer cells, or by measuring a sample (but not limited to blood) from a subject or patient for the identification of specific cancer cell markers in the blood, such as using an antibody, or by measuring the amount of circulating cancer cell antibody levels in the patient. Throughout this specification, amounts are defined by ranges and the lower and upper limits of the ranges. Each lower limit can be combined with each upper limit to define a range. The lower and upper limits should be considered as separate elements.

[0384] As used throughout this specification, the terms "one embodiment", "an embodiment", "one example", or "an example" refer to a particular feature, structure, or characteristic described in connection with an embodiment or example that is included in at least one embodiment of the present application. Thus, the appearances of the phrases "in one embodiment" or "an example" in various places throughout this specification are not necessarily all referring to the same embodiment or example. Further, the particular features, structures, or characteristics may be combined in any suitable combination and / or sub-combination in one or more embodiments or examples. Additionally, the figures provided in this specification are intended to be illustrative to those skilled in the art and are not necessarily drawn to scale.

[0385] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having", or any other variation thereof are intended to cover a non-exclusive inclusion. For example, a process, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements; it may include other elements not expressly listed or inherent to such process, article, or apparatus.

[0386] Further, unless expressly stated to the contrary, "or" as used herein is intended to be inclusive and not exclusive. For example, the condition A or B is satisfied by any of the following: A is true (or present) and B is false (or absent), A is false (or absent) and B is true (or present), and both A and B are true (or present).

[0387] Furthermore, any example or figure disclosed in the present specification is not to be considered as limiting, restricting, or representing the definition of any term used. Instead, these examples or figures are described with respect to one particular embodiment and are considered illustrative only. Those skilled in the art will understand that any term used in these examples or figures encompasses other embodiments, whether provided together with it or not elsewhere in the present specification, and that all such embodiments are intended to be included within the scope of that term. Such language designations of non-limiting examples and figures include, but are not limited to, "for example", "for instance", "e.g.", and "in one embodiment".

[0388] In the present specification, various groups of parameters including multiple elements are described. Among a group of parameters, each element can be combined with any one or more other elements to additionally form sub-groups. For example, if a group of elements is a, b, c, d, and e, specifically conceivable additional sub-groups include those containing any 1, 2, 3, or 4 elements (e.g., a and c; a, d, and e; and b, c, d, and e, etc.).

[0389] As used herein, the XXXX antigen recognition domain is a polypeptide selective for XXXX. "XXXX" refers to the target described herein and above. For example, the CD38 antigen recognition domain is a polypeptide specific for CD38.

[0390] As used herein, CDXCAR refers to a chimeric antigen receptor having a CDX antigen recognition domain.

[0391] Usage examples BCMA-CS1 cCAR targeting plasma cell diseases such as multiple myeloma Preparation of BCMA-CS1 cCAR (BC1cCAR) T cells The BC1cCAR construct is a two-unit CAR composed of a complete BCMA-CAR fused to a complete CS1-CAR by a self-cleaving P2A peptide, allowing for the individual expression of both CAR receptors on the T cell surface (Figure 1A). Expression assayed by FACS revealed distinct transduced cells (Figure 1B). A leader, scFv, hinge domain (H), transmembrane domain (TM), co-stimulatory domain (CD28 or 4-1BB), and intracellular signaling domain CD3 zeta (CD3) are included in each CAR unit. A strong spleen focus-forming virus promoter (SFFV) and CD8 leader sequence were used for efficient expression of the BCMA-CS1 cCAR molecule on the T cell surface.

[0392] BC1cCAR T cells specifically lyse BCMA+ and CS1+ myeloma cell lines To evaluate the cytotoxic activity of BC1cCAR T-cells, co-culture assays were performed with myeloma cell lines such as MM1S (BCMA+ CS1+), RPMI-8226 (BCMA + CS1 dim ) and U266 (BCMA + CS1 dim ). FACS analysis of BC1cCAR cytotoxicity in 24-hour co-cultures showed substantial complete lysis (>90%) of MM1S cells at all E:T ratios (Figure 2A). Similar trends were observed for RPMI-8226 and U266 cells in culture (Figures 2A, 2B), demonstrating effective bulk cytotoxicity against target populations with varying levels of antigen expression (Figure 2C).

[0393] BC1cCAR T cells specifically target BCMA+ and CS1+ populations in primary patient myeloma samples To further evaluate the ability of BC1cCAR to kill diverse primary myeloma cell types, primary samples were selected and the spectrum of target antigen expression was shown (Figure 3). Flow cytometry analysis of the MM10-G sample revealed a mixed tumor of a subset of the BCMA + CS1 + double-positive and CS1 + only parent populations. The MM7-G sample showed a complete BCMA + CS1 + phenotype, and the bone marrow aspirate MM11-G showed a noisy BCMA dim CS1 dim phenotype. BC1cCAR T cells showed strong (> 80%) dose-dependent ablation in the MM7-G primary patient sample (Figure 4A).

[0394] BC1cCAR also showed a specific lysis ability by significantly ablating both subsets of the parent populations of BCMA + CS1 + and BCMA - CS1 + in co-culture with MM10-G. At an E:T ratio of 2:1, BC1cCAR T cells ablated 60% of the BCMA + CS1 + population and 70% of the CS1 + only population (Figure 4B). BC1cCAR T cells were also able to demonstrate dose-dependent cytotoxic activity against MM11-G cells (Figure 4C). Through cytotoxic screening, BC1cCAR T cells showed potent antitumor activity against both myeloma cell lines and primary tumor cells expressing different combinations of BCMA and CS1 (Figure 4D).

[0395] Functional evaluation of BC1cCAR antigen-specific activity We established a model in which the BC1cCAR scFv function could be tested individually. The CML cell line K562, which is negative for myeloma markers, was overexpressed with either CS1 (CS1-K562) or BCMA (BCMA-K562). After confirming independent antigen expression in each cell line (Figure 5A), the targeting function of BC1cCAR T cells was determined through co-culture experiments.

[0396] In short-term culture (overnight), BC1cCAR T cells showed cytotoxic activity against BCMA-K562 cells. There was no off-target effect on wild-type K562 cells that were negative for both antigens (Figure 5B). Similar responses were also seen against CS1-expressing target cells in short-term culture with CS1-K562 cells. Furthermore, BC1cCAR T cells appeared to have a stronger cytotoxic effect against CS1-K562 cells than the CS1-specific CAR (Figure 5B).

[0397] Residual tumor populations bearing non-target antigens may lead to recurrence in patients treated with single antigen CARs. Therefore, to model more clinically relevant mixed antigen-expressing cell populations, we performed co-culture experiments in combination. BCMA-K562 cells and CS1-K562 cells were mixed at a 1:1 ratio in continuous culture (48 hours), and the remaining antigen-positive populations were assayed. Next, histograms representing the populations of T cells and target tumor cells were created, and the remaining gated target tumor populations were marked (Figure 5C). Compared to control T cells, the BCMA-specific CAR and the CS1-specific CAR showed significant cytotoxic effects against their respective target parent populations. However, the BCMA-CAR achieved significant cytotoxicity but left a small population of CS1 + whereas the CS1-CAR left a significant residual BCMA + population. In contrast, BC1cCAR T cells effectively depleted both target populations (Figure 5C).

[0398] Tumor re-challenge demonstrated the time-dependent killing ability of BC1cCAR T cells Next, the ability of BC1cCAR T cells to continuously kill tumor cells in an unfavorable microenvironment caused by cell lysis, debris, and tumor re-challenge was investigated. Using the scheme in Figure 6A, MM1S cells were co-cultured for a long time as a myeloma model tumor, and tumor growth or recurrence was simulated by periodically re-adding fresh MM1S cells to BC1cCAR T cells and single BCMA-CAR and CS1-CAR T cells. Even without exogenous cytokines, target antigens were depleted after 48 hours in all CAR treatments, and significant clustering and T cell proliferation were observed (Figure 6B). In contrast, control T cells did not show a response or proliferation and doubled the tumor cell population to its initial size. When fresh MM1S cells were re-loaded into all treatment wells, all CARs were found to still retain high cytotoxicity. By 108 hours, new MM1S cells were substantially depleted by both BCMA-CAR and BC1cCAR, while CS1-CAR showed incomplete killing of new MM1S cells (Figure 6C). Tumor lysis and cytotoxicity via all CARs ceased after 168 hours, but BCMA-CAR and BC1cCAR still showed a detectable minority T cell population, while control T cells and CS1-CAR T cells were virtually undetectable (data not shown).

[0399] BC1cCAR T cells show significant tumor control and reduction in vivo To evaluate the in vivo activity of BC1cCAR T cells, a myeloma mouse model was developed that induces fluorescence-visible tumor formation using luciferase-expressing MM1S cells. BC1cCAR T cells significantly reduced the tumor burden and extended the survival of MM1S-injected mice compared to control T cells. Mice were administered a single dose of BC1cCAR or control T cells, and the tumor burden was measured by IVIS imaging (Figure 7A). After day 6, there was a highly significant difference (P < 0.0003) in the IVIS measurement of the tumor burden between the control group and the BC1cCAR treatment group (Figure 7B). Mice injected with CAR also showed a significant and better survival outcome (Figure 7C).

[0400] The mixed antigen population mouse model shows excellent tumor burden control by cells expressing cCAR and cells expressing a single CAR. To model heterogeneous cell populations and potential antigen escape, a 4:1 mixture of K562 cells expressing BCMA:CS1 was injected into mice and treated on day 3 with either 7.5 x 10 6 of control, BCMA-CAR, or BC1cCAR T cells. On day 3, as a result of the injection treatment, two control mice died and were excluded from the analysis. Tumor burden was visualized by fluorescence (Figure 8A). By day 10, both CARs showed more than 50% tumor reduction compared to the GFP control, and the reduction increased to more than 60% by day 12 (Figure 8A - right). By day 10, BC1cCAR exceeded BCMA-CAR by 6% in tumor suppression, and this spread increased to 17% by day 12, potentially modeling the inability of BCMA-CAR to lyse remaining CS1-K562 cells (20% of the tumor was injected). The survival outcome of all CAR T cell-treated mice was significantly improved compared to the control group. The survival rates of both the BC1cCAR group and the BCMA-CAR group were also significantly improved (p < 0.05) (Figure 8B). Both CARs are effective in controlling tumor growth, but BC1cCAR shows stronger control compared to the single target option.

[0401] Enhanced T cell persistence and maintenance of tumor depletion by compound CAR T cells in an independent antigen mouse model To assay the depletion of cells expressing specific BCMA and CS1 antigens and verify the efficacy of the scFv of the compound, a third mouse model was constructed in which tumor groups consisting of four groups of five mice each were injected with either BCMA-K562 or CS1-K562 cells and infused with control or BC1cCAR T cells (n = 19 as a result of early natural death of mice). At the time of sacrifice (various: 30 days to over 80 days), T cells and tumor populations were screened in the whole blood and liver tissues of the mice. Both blood tissue types showed consistent tumor presence in the control group compared to the cCAR group (Figures 9A, 9B, 10A, 10B). Pooled tissue analysis of the averaged tumor cell populations in both tissues showed a consistent trend such as depletion of tumor burden in the cCAR-treated mouse group (Figure 9B). In both blood and liver, control T cells could not survive beyond 30 days and showed significant tumor amounts in both tissue types (Figures 9B, 9C). In contrast, cCAR-treated mice showed significant T cell proliferation and persistence compared to control T cells in all mice even after day 30 (Figure 9C), correlated with an increase in anti-tumor activity and supported an overall improvement in survival.

[0402] Structure of BCMA-CS1-IL-15 / IL-15sushi (CD269-A7D-CS1-hu63-IL15 / IL15sushi) BCMA-CS1-IL-15 / IL-15sushi (Figure 11A) contains two independent CAR units, CD269-A7D (also called BCMA CAR or anti-CD269 CAR), and CS1 CAR (also called CS1-hu63 CAR or anti-CS1 CAR). The BCMA-CS1-IL-15 / IL-15sushi CAR can secrete IL-15 / IL-15sushi. The soluble IL-15 / IL-15sushi fusion is stable and functions as an unexpected potent immunomodulatory factor for CAR T / NK cells and their adjacent tumor immune response cells. The soluble IL-15 / IL-15sushi fusion is stable, improves the persistence of CAR T / NK cells, stimulates the proliferation of tumor-infiltrating lymphocytes, and anti-tumor activity. The soluble IL-15 / IL-15sushi fusion reprograms the body's immune system to provide effects like an anti-tumor vaccine to fight cancer.

[0403] Expression of CAR Activated human peripheral blood T cells were transduced with a lentiviral vector of CD269-A7D-CS1-hu63-IL15 / IL15sushi CAR. Figure 11B shows the transduction efficiency among activated T cells transduced with either a control vector or a CD269-A7D-hu63-IL15 / IL15sushi CAR vector, determined by labeling with goat anti-mouse F(Ab’)2 antibody. Activated T cells transduced with the CAR vector yielded 23.7% F(Ab’)2-positive cells against CD269-A7D-hu63-IL15 / IL15sushi (Figure 11B). These CAR T cells were used in the following in vitro killing assays.

[0404] CD269-A7D-CS1-hu63-IL15 / IL15sushi CAR T cells can lyse tumor cell lines expressing the CD269 or CS1 antigen in in vitro assays The ability of CD269-A7D-CS1-hu63-IL15 / IL15sushi CAR T cells of Figure 11B to specifically lyse both K562 cells synthetically expressing either the CD269 (BCMA) or CS1 (CD319) antigen was measured. Wild-type K562 cells were transduced with a lentiviral vector for CD269 antigen or CS1 antigen expression and positively selected by FACS (FACS-Aria, BD). Co-culture with K562-BCMAxp or K562-CS1xp synthetic expression cells was set up at effector cell:target cell ratios of 2:1 and 5:1 for 48 hours. After this incubation, cells were stained using a mouse anti-human CD3 antibody (in all cases), and either a mouse anti-human CD269 or CS1, and analyzed by flow cytometry. At a 2:1 E:T ratio, 58% of K562-BCMAxp tumor cells were lysed, while at a 5:1 ratio, 91% of tumor cells were lysed (Figure 11C). In co-culture with K562-CS1-xp tumor cells, the lysis rates were 33% and 72% respectively (Figure 11C). These results indicate that each CAR component of CD269-A7D-CS1-hu63-IL15 / IL15sushi CAR T cells is capable of lysing the target cells of interest.

[0405] CD269-A7D-CS1-hu63-IL15 / IL15sushi CAR T cells exhibit significant antitumor activity in a xenogeneic mouse model To evaluate the in vivo antitumor activity of CD269-A7D-CS1-hu63-IL15 / IL15sushi CAR T cells, we developed a xenogeneic mouse model by intravenously injecting 4 x 10 6 luciferase-expressing MM.1S wild-type multiple myeloma cells at a sublethal dose into irradiated NSG mice. Eight days after injection of the tumor cells, all mice were injected with 15 x 10 6They were intravenously injected in individual courses. On the 8th day (the day before T cell treatment) and the 12th day (72 hours after treatment), IVIS imaging of the mice was performed to measure the tumor volume.

[0406] The lysis rate of target cells was determined by comparing the average fluorescence intensity measured in MM.1S mice injected with CD269-A7D-CS1-hu63-IL15 / IL15sushi CAR T cells with that in mice injected with control T cells. The results showed that on the 3rd day after treatment with T cells (the 12th day), the mice treated with CD269-A7D-CS1-hu63-IL15 / IL15sushi CAR T cells had a 90% lower tumor burden than the mice given control T cells (Figure 11D). These results indicate the effectiveness of CD269-A7D-CS1-hu63-IL15 / IL15sushi CAR T cells against multiple myeloma cell lines in vivo. Furthermore, the blood collected at the time of sacrifice showed significantly higher levels of human IL-15 / IL-15sushi in MM.1S mice injected with CD269-A7D-CS1-hu63-IL15 / IL15sushi CAR T cells than in control mice where it was undetectable.

[0407] Function of IL15 in CD269-A7D-CS1-hu63-IL15 / IL15sushi CAR NK cells. To further determine whether IL-15 / IL15sushi was secreted, the NK-92 cell line was transduced with a lentiviral vector containing CD269-A7D-CS1-hu63-IL15 / IL15sushi CAR. To sort NK cells with a positive F(Ab’)2 phenotype, the cells were sorted using a BD FACS Aria (Figure 11E). The sorted cells were expanded, and after 72 hours, the supernatant was collected and ELISA was performed in a 96-well plate pre-coated with an IL-15 antibody. According to the instructions of the manufacturer (Boster), the results of the colorimetric analysis obtained with a plate reader were compared with a standard curve prepared with human IL-15 to determine the IL-15 concentration in the supernatant. It was confirmed that IL-15 was detected at 285.9 pg / mL in the supernatant (Figure 11F). In comparison, the concentration in the supernatant containing wild-type control NK-92 cells of the same cell number was only 0.33 pg / mL.

[0408] IL15 / IL15sushi secreted from CD269-A7D-CS1-hu63-IL15 / IL15sushi CAR NK cells can replace the function of IL-2 in vitro for the proliferation of T cells The culture of NK-92 cells requires the presence of IL-2. IL-15 can replace the absence of IL-2 for the proliferation or amplification of NK-92 cells in vitro. This system is used to test the function of the fusion IL-15 / IL-15sushi secreted from NK cells transduced with CD269-A7D-CS1-hu63-IL15 / IL15sushi CAR. The selected CD269-A7D-CS1-hu63-IL15 / IL15sushi CAR NK cells, and wild-type NK-92 cells were cultured in 24-well plates at a density of 0.5 x 10e6 cells per mL and a total volume of 1 mL. Cells were added to wells in duplicate, and one well of each pair contained 300 IU / mL of IL-2, while the other well did not. After 48 hours (day 2), the cells were counted and the volume was increased to a concentration of approximately 0.5 x 10e6 cells / mL. This process was repeated on days 4, 6, and 8. As shown in the graph of Figure 11E, CD269-A7D-CS1-hu63-IL15 / IL15sushi NK CAR cells cultured for 8 days without IL-2 proliferated at the same rate as wild-type NK-92 cells cultured with added IL-2, while all wild-type NK-92 cells cultured without added IL-2 died by day 6. This indicates that IL-15 secreted from NK CAR cells can substitute for the proliferative activity of IL-2.

[0409] In one embodiment, the engineered cells comprise a BCMA-CS1 cCAR polypeptide and IL-15 / IL-15sushi (SEQ ID NO: 42), and the corresponding nucleotide (SEQ ID NO: 43). Example of targeting CD123+ and / or CD33+ leukemia / lymphoma by CD123b-CD33b cCAR (version of CD123-CD33 cCAR) T cells

[0410] Generation of CD123b-CD33b cCAR T cells Cytotoxic effector T cells transfected with lentivirus were designed to express two complete units of CAR linked by a self-cleaving P2A peptide (Figure 12A). The resulting compound CAR (CD123b-CD33b cCAR) can target CD123+ and / or CD33+ leukemia cells (Figure 12B). A leader, scFv, hinge domain (H), transmembrane domain (TM), co-stimulatory domain (CD28 or 4-1BB), and intracellular signaling domain CD3 zeta (CD3) are included in each CAR unit. A strong spleen focus-forming virus promoter (SFFV) and a CD8 leader sequence were used for efficient expression of the CD123b-CD33b cCAR molecule on the T cell surface.

[0411] Transduction efficiency of CD123b-CD33b cCAR T cells To evaluate the CD123b-CD33b cCAR expression level on the T cell surface after transduction, flow cytometry analysis was used (Figure 13). The transduction efficiency was determined to be 25%.

[0412] CD123b-CD33b cCAR T cells effectively lyse acute myeloid leukemia cell lines To evaluate the anti-tumor activity of CD123b-CD33b chimeric antigen receptor (cCAR) T cells, co-cultures were performed using the AML cell line MOLM13 (CD33 + CD123 +) and the monoblast U937 cell line (CD33 + CD123-). Cells were stained with CD3 to distinguish target leukemia (MOLM13 and U927; both CD3-) from effector T cells (CD3 +) during flow cytometry. Co-culture assays with effector-to-target (E:T) ratios of 2:1 and 5:1 were performed for 24 hours, and flow cytometry analysis was used to determine the cytotoxicity rates by CD123b-CD33b cCAR T cells or control T cells (Figures 14A, 14B). At an E:T ratio of 2:1, CD123b-CD33b cCAR T cells were able to lyse approximately 98% of CD123 + CD33 + MOLM13 cells and 99.9% of CD33 + U937 cells compared to control T cells. Furthermore, at a ratio of 5:1, 100% lysis of both cell lines was observed (Figure 14C). Surface markers expressed on both the MOLM13 and U937 cell lines were also verified (Figure 14C). Overall, these results suggest that CD123b-CD33b cCAR T cells specifically and efficiently eliminate tumor cells expressing either or both antigens. Additionally, the finding that CD123b-CD33b cCAR T cells effectively ablated U937 cells expressing only CD33 but not CD123 supports the fact that each individual unit of the compound CAR targets its antigen independently and can eliminate targets expressing only one antigen or both antigens.

[0413] We further evaluated the dose-dependent tumor-lysing ability of CD123b-CD33b cCAR T cells by varying and decreasing the E:T ratios against two other cell lines, KG1a (CD123dimCD33+) and HL60 (CD123dimCD33+). CD123b-CD33b cCAR T cells were cultured against KG1a and HL60 cell lines at E:T ratios of 0.25:1, 0.5:1, 1:1, 2:1, 5:1, and 10:1, and showed tumor-lysing ability of over 75% even at a ratio of 0.25:1. Overall, there was a strong correlation between dose and tumor lysis up to saturation at a ratio of 5:1 (Figure 14D).

[0414] CD123b-CD33b cCAR T-cells effectively lyse primary myeloid leukemia tumor cells Next, the anti-tumor properties of CD123b-CD33b cCAR T cells against primary tumor cells were established. To distinguish CAR T cells from CD3 - leukemia samples, the cells were stained with CD3. Primary leukemia samples from various patients, including two CD123 + CD33 + AML and two CD123 + B-ALL samples (PT1: AML, PT2: B-ALL, PT3: AML, and PT4: B-ALL), were assayed with this panel, and flow cytometry analysis was performed to verify tumor lysis surrounding the depleted target population (Figure 15). Compared with previous anti-tumor cytotoxicity results regarding the AML cell line (Figure 14), CD123b-CD33b cCAR T cells showed positive results with tumor lysis rates of over 80% at a ratio of 2:1 and over 98% at a ratio of 5:1 for all patient samples (Figure 15). Furthermore, the finding that CD123b-CD33b cCAR T cells effectively ablated PT2 cells that express only CD123 but not CD33, similar to our cell line, supports the fact that each individual unit of the compound CAR targets its antigen independently and can eliminate cells expressing only one of its target antigens (seen for CD33 + U937 and CD123 + PT2 cells) or both target antigens (seen for CD123 + CD33 + MOLM13 and PT1 cells). Overall, these results suggest that CD123b-CD33b cCAR T cells exhibit high killing effects against tumor cells of patients expressing one or both antigens.

[0415] We also specifically investigated the ability of CD123b-CD33b cCAR to eliminate specific cell populations, including leukemic stem cells (CD123 + CD34 + CD38-) of the PT3 sample and myeloid leukemia bulk disease (CD34variableCD33 +) of the PT4 sample (Figure 15C, 15D). We found that CD123b-CD33b cCAR T cells normally ablated both LSCs and bulk disease cells.

[0416] The distinct receptor units of CD123b-CD33b cCART cells lyse target cells independently in an antigen-specific manner. To further confirm the independent antigen targeting ability of cCAR, Jurkat artificial cell lines expressing either CD123 or CD33 were generated, and CD123b-CD33b cCAR T cells were tested against these cells, in addition to wild-type Jurkat cells that do not express either antigen (Figure 16). We found that CD123b-CD33b cCAR T cells specifically and potently eliminated cells expressing either the CD123 or CD33 antigen, compared to wild-type Jurkat cells that do not express either antigen (Figures 16A, 16B, 16C). Overall, we conclude that our CD123b-CD33b cCAR T cells act via stimulation of either CAR receptor, can target cells expressing only one or both target antigens equally, and can eliminate targets with high efficiency.

[0417] CD123b-CD33b cCAR T cells exhibit potent antitumor activity in two xenograft mouse models of AML using MOLM13 and U937 cells. To evaluate the in vivo antitumor activity of CD123b-CD33b cCAR T cells as a predictor of patient treatment efficacy, two xenograft mouse models were developed (Figure 17). NSG mice irradiated below the lethal dose (2.0 Gy) were intravenously injected with either 1.0 x 10 6 individual firefly luciferase-expressing MOLM13 cells or 1.0 x 10 6 individual firefly luciferase-expressing U937 cells. Four days after engraftment of MOLM13 or U937, mice were injected with 10×10 6Individual cells were injected intravenously. To evaluate the tumor burden in mice, RediJect D-luciferin (Perkin-Elmer) was injected intraperitoneally on days 6, 9, and 13, and the mice were subjected to IVIS imaging to quantify luciferase activity (Caliper LifeSciences) (Figures 17A, 17B). As observed by IVIS imaging, the total flux level continuously increased in control mice with a substantial increase in tumor mass. In contrast, mice treated with CD123b-CD33b cCAR significantly suppressed the tumor burden by day 3. By day 6, the tumor burden decreased by more than 80% in both models in mice treated with cCAR (Figures 17A, 17B). The total flux in mice treated with CD123b-CD33b cCAR remained near the background null value, showing a statistically significant difference from mice treated with control T cells. This tumor suppression was maintained and the efficacy increased until day 13.

[0418] We also evaluated the persistence of tumor cells and CAR T cells at the time of sacrifice.

[0419] Peripheral blood was collected from each experimental mouse together with control mice at the time of sacrifice, and the presence or absence of transplanted tumors (MOLM13 or U937 cells) and T cells (cCAR or control) was analyzed via flow cytometry. MOLM13 and U937 cells are CD3- cells and can be distinguished from CD3 + CAR or control T cells. Mouse peripheral blood cells were gated by side scatter light and human CD45 antibody and divided into CD3 and CD33. Treatment mice in the control showed a significant residual tumor population (about 75 - 87%) in peripheral blood, while treatment mice with CD123b - CD33b cCAR showed substantial depletion of all tumors in control mice (Figure 17C). Furthermore, treatment mice with CD123b - CD33b cCAR showed significant T cell proliferation in human cells that are substantially CAR T cells in peripheral blood. This confirms the ability and persistence of cCAR T cells to maintain a long-term response. Additionally, treatment mice with CD123b - CD33b cCAR showed a significantly increased survival outcome compared to control-treated mice (Figures 17A, 17B).

[0420] In vivo depletion of injected cCAR T cells after treatment with CAMPATH In clinical treatment with CAR T cells for acute myeloid leukemia, due to unexpected side effects of CAR therapy, it may be necessary to establish a safe method to eliminate CAR T cells from patients after tumor depletion or in emergency situations. T cells and B cells express CD52 on the cell surface, and CAMPATH (alemtuzumab), a CD52-specific antibody, can eliminate CD52+ cells from the circulatory system. To evaluate the effect of CAR removal by treatment with CAMPATH, as described, we performed an in vivo procedure (Figure 18A). Irradiated mice were injected with 10 x 10 6Individual cCAR T cells were injected intravenously. The next day, 0.1 mg / kg of CAMPATH or PBS was administered to three mice in each group via IP injection. Peripheral blood was collected 6 hours and 24 hours after CAMPATH treatment, and the presence of cCAR T cells was confirmed by FACS analysis. cCAR T cells were gated by side scatter light (SSC) and expression of CD3 and CD3 and CD45 expression, and were distinguished from mouse cells. CAMPATH injection depleted cCAR T cells in the blood at both 6 hours and 24 hours (Figures 18B, 18C). These findings support the use of CAMPATH as a safety switch to rapidly deplete CAR-T cells from the circulation.

[0421] In one embodiment, the engineered cell comprises a CD123-CD33 cCAR polypeptide, and IL-15 / IL-15sushi (SEQ ID NO: 34), and the corresponding nucleotide (SEQ ID NO: 35).

[0422] In one embodiment, the engineered cell comprises a CD123-CLL1 cCAR polypeptide, and IL-15 / IL-15sushi (SEQ ID NO: 36), and the corresponding nucleotide (SEQ ID NO: 37).

[0423] Examples of targets for B-ALL and other leukemias by CD19b-CD123 cCAR (a version of CD19-CD123 cCAR)

[0424] Generation of CD19b-CD123 cCAR T cells Cytotoxic effector cells, i.e., T cells transfected with a lentivirus, are designed to express an anti-CD19 single-chain variable fragment (scFv1, CD19b) region fused to an anti-CD123 fragment (scFv2, CD123) by a self-cleaving P2A peptide. These antibody domains are linked to 4-1BB and CD28 co-activation domains and the CD3ζ signaling domain by a hinge (H) and transmembrane (TM) region derived from CD8 (Figure 19). A strong spleen focus-forming virus promoter (SFFV) and a CD8 leader sequence were used for efficient expression of the CD19b-CD123 cCAR molecule on the T cell surface.

[0425] Transduction efficiency of CD19b-CD123 cCAR T cells T cells isolated from umbilical cord blood (UCB) buffy coats were transduced with a CD19b-CD123 cCAR lentivirus 2 days after activation. The transduction efficiency of CD19b-CD123 cCAR was measured by flow cytometry to be 26% (Figure 20).

[0426] CD19b-CD123 cCAR-2G T cells effectively lyse CD19-positive and CD123-positive leukemia cell lines To evaluate the cytotoxicity of CD19b-CD123 cCAR T cells, a co-culture assay was performed at an effector:target (E:T) ratio of 5:1 against leukemia / lymphoma cell lines artificially expressing CD19 and CD123. K562 cells (a myeloid leukemia cell line) were used to express the CD19 antigen by lentiviral infection (named K19), and the wild-type K562 cell line was used as a control. Similarly, Jurkat cells were used to express the CD123 antigen (named J123), and the wild-type Jurkat cell line was used as a control.

[0427] Lysis of target cells by CD19b-CD123 cCAR T cells was quantified by flow cytometry. In a 16-hour co-culture, CD19b-CD123 cCAR T cells lysed more than 66% of K19 cells at 16 hours and more than 99% at 48 hours (Figure 21A). More than 88% of J123 cells were lysed at 16 hours and reached saturation (Figures 21B and 21D). Control K562 and control Jurkat cells were not significantly lysed, with less than 20% lysis. The finding that CD19b-CD123 cCAR T cells effectively ablate both artificially induced single-positive CD19 cells and CD123 cells supports the idea that each individual unit of the compound CAR targets its antigen independently and can eliminate targets expressing only one antigen or both antigens.

[0428] Furthermore, the lack of cell lysis of control K562 and Jurkat cells indicates that CD19b-CD123 cCAR T cells exhibit antigen-specific cytotoxicity.

[0429] We next evaluated the ability of CD19b-CD123 cCAR T cells to target leukemia / lymphoma cell lines such as human mantle cell lymphoma SP53 (CD19+ CD123-) and human acute myeloid leukemia KG1a (CD19- CD123+), which express the naturally occurring CD19 / CD123 antigens. In a 16-hour co-culture, CD19b-CD123 cCAR showed substantial complete lysis of SP53 cells, with 86% of the target cells disappearing and reaching saturation (Figure 21C). In KG1a, CD19b-CD123 cCAR lysed more than 69% of CD123+ target cells at 16 hours and more than 94% at 48 hours (Figures 21C and 21D). Overall, CD19b-CD123 cCAR T cells specifically and effectively lyse target populations expressing either antigen and exhibit potent bulk cytotoxicity.

[0430] CD19b-CD123 cCAR-2G T cells effectively lyse primary B-cell acute lymphoblastic leukemia (B-ALL) and acute myeloid leukemia (AML) tumor cells. To evaluate the ability of CD19b-CD123 cCAR T cells to kill various types of primary leukemia cells, co-cultures were performed using CD19b-CD123 cCAR T cells against primary tumor cells. Primary tumor cells and CAR T cells were distinguished by staining patient samples with CMTMR Cytotracker Dye. Co-cultures were performed with two samples, PT1:B-ALL and PT2:AML, and flow cytometry was performed to confirm tumor lysis. In the flow cytometry analysis of the PT1 sample, an almost complete CD19+ phenotype was seen, including a distinct CD19 + CD123+ population. The PT2 sample showed a phenotype of a mixed tumor, including a partial CD123 + CD19- phenotype (Figure 22A). CD19b-CD123 cCAR T cells showed strong ablation of the PT1 primary B-ALL sample and were almost completely lysed at an E:T ratio of 5:1 at 24 hours (Figures 22B and 22D). CD19b-CD123cCAR T cells also ablated the PT2 primary AML sample, with 31% lysis at 24 hours and 67% lysis at 48 hours (Figures 22C and 22D). In summary, CD19b-CD123 cCAR T cells showed strong antitumor activity against both leukemia cell lines and primary tumor cells expressing different combinations of CD19 and CD123 (Figure 22D).

[0431] CD19b-CD123 cCAR-3G T cells show strong antitumor activity in two xenograft mouse models of AML and B-ALL using MOLM-13 and REH cells. To evaluate the in vivo antitumor activity of CD19b-CD123 cCAR T cells, we developed two models that could induce measurable tumor formation, one with MOLM13 cells (CD123 + CD19-) expressing luciferase and the other with REH cells (CD19 + CD123-) expressing luciferase. Mice were administered a single dose of CD19b-CD123 cCAR T cells or control GFP cells, and tumor burden was measured on days 3, 6, 8, and 11 (Figure 23A). In the MOLM13 model, there was a significant difference (P < 0.01) between the cCAR treatment group and the control group by day 6, with lower light intensity and thus less tumor burden in the group injected with CD19b-CD123 cCAR T cells compared to the control group (Figure 23B). Mice injected with CD19b-CD123 CAR T cells had a 99% reduction in tumor burden compared to control mice by day 11. Next, the survival rates of mice were compared between the two groups. Following the aforementioned IVIS imaging experiment, mice were observed daily for severe symptoms and sacrificed when their movement was severely impaired. All control mice died by day 18, while mice treated with CD19b-CD123 CAR T survived up to 15 days longer than control mice (p = 0.0031) (Figure 23C).

[0432] Similar results were seen in the REH mouse model (Figure 23D). REH leukemia mice injected with CD19b-CD123 cCAR T cells showed a 99% lower tumor burden than control mice on day 16 (Figure 23E). Comparing the survival rates of mice between the groups treated with cCAR and control, mice injected with CD19b-CD123 cCAR T survived much longer than control mice (Figure 23F) (p = 0.0031). In summary, these in vivo data indicate that CD19b-CD123 cCAR T cells significantly reduce tumor mass and extend the survival of MOLM13-injected and REH-injected NSG mice compared to control T cells.

[0433] Screening and evaluation of several versions of cCAR targeting BCMA+ and / or CS1+ leukemia cells, particularly multiple myeloma cells using co-culture killing assays. Generation of different versions of BCMA(CD269)-CS1 cCAR. As described above, the creation of compound CARs with different CAR units can be very difficult. Different CAR body elements were selected to express multiple units of CAR in a single vector using a strong promoter and a P2A self-cleaving site. The hinge region of the CAR was selected so as to avoid the interaction of the hinge regions between each CAR unit. Cytotoxic effector cells, i.e., T cells transfected with lentivirus, were designed to express an anti-BCMA(CD269) single-chain variable fragment (scFv1) region fused to an anti-CS1 fragment (scFv2) by a self-cleaving P2A peptide. These scFv domains are linked to 4-1BB and CD28 co-activation domains and CD3ζ (CD3) signaling domains by a hinge (H) and transmembrane (TM) region derived from CD8 (Figure 30). A strong spleen focus-forming virus promoter (SFFV) and CD8 leader sequence were used for efficient expression of the compound CAR molecule on the T cell surface. Finally, the constructed constructs were screened and their expression and function were evaluated. scFv1 represents different scFv versions (A7D or C11D) against the BCMA antigen. scFv 2 represents different scFv versions (hu63 or mu34 or mu90) against the CS1 antigen.

[0434] Different levels of CAR expression in T cells transduced with different versions of BCMA-CS1 cCAR lentivirus. Peripheral blood mononuclear buffy coat cells were activated for 3 days and transduced with lentiviral vectors of 6 different sequence variations of cCARs containing CD269 (A7D or C11D) and CS1 (hu63, mu34 or mu90) CARs, or control vectors. Expression of CAR on the T cell surface was demonstrated 3 days after transduction by staining the transduced T cells with goat anti-mouse Fab antibody and mouse anti-human CD3. Figure 30A shows the surface expression for each of the CD269-CS1 CARs: A7D-mu34 was 11.2%, A7D-mu90 was 23.1%, A7D-hu63 was 28.5%, C11D-mu34 was 28.0%, C11Dmu90 was 13.6% and C11Dhu63 was 42%. This indicates the need to find pairs of CAR units that result in the highest levels of CAR expression. A high-efficiency lentiviral packaging cell line is important for the generation of high titers of these constructs (Figure 30B). To achieve high viral titers for the compound CAR constructs, we used the lenti-X 293 T cell line as the packaging system. The Lenti-X 293T packaging cell line was clearly superior to other cell lines and produced 2- to 6-fold more virus than 293 FT cells.

[0435] The transduction efficiency of cCAR (the proportion of CAR T cells) is often lower than that of a single unit of CAR. There are several ways to improve the efficiency in both the transfection and transduction steps. To improve the viral titer for producing cCAR, it is preferable to use the LentiX™ 293 T (Clontech / Takara) packaging cell line selected for high-titer lentivirus production instead of the commonly used HEK-293FT. To enhance the transfection efficiency, when transfecting the packaging cells, it is also recommended to increase the amount of plasmid DNA (including the cCAR construct) by 1.5 - 2.0 times. The amounts of the viral packaging plasmid and the transfection reagent remain the same for complex formation. To further increase the transduction efficiency, in the transduction step, lower the ratio of T cells to the viral vector to 0.3 x 10 6 cells / mL and increase the amount of the lentivirus supernatant or lentivirus.

[0436] Testing of CAR expression in T cells transduced with various anti-BCMA lentiviral vectors. Based on the above studies, CD269-A7D (alias A7D) and CS1-hu63 (alias hu63) were selected as suitable candidates for the production of enhanced CAR or compound CAR (cCAR). We also produced a cCAR (CD269-A7D-C11D-2G) that targets two epitopes on the same antigen BCMA. In this cCAR, each unit of the CAR has a different scFv that targets a different epitope of BCMA. Enhanced CARs are CD269-A7D-IL15 / IL15sushi and CD269-A7D-41BBL-2G that target the BCMA antigen. Compound CARs are CD269-A7D-CD19b-2G that targets the BCMA and CD19 antigens, and CD269-A7D-CS1-hu63 or CD269-C11D-CS1-hu63-BB that target the BCMA and CS1 antigens.

[0437] Peripheral blood mononuclear buffy coat cells were activated for 3 days and transduced with anti-BCMA lentiviral vectors or control vectors of single CAR (CD269-A7D-2G, CD269-A7D-IL15 / IL15sushi, CD269-A7D-41BBL-2G) and cCAR (CD269-A-A7D-C11D-2G, CD269-A7D-CD19b-2G, CD269-A7D-CS1-hu63, CD269-C11D-CS1-hu63-BB) (Figure 30B). Expression of CAR on the T cell surface was demonstrated 3 days after transduction by staining the transduced T cells with goat anti-mouse Fab antibody and mouse anti-human CD3. Figure 30B shows the surface expression for each of the lentiviral CARs: CD269-A7D-2G was 48.4%, CD269-A7D-IL15 / IL15sushi was 32.2%, CD269-A7D-41BBL-2G was 36%, CD269-A7D-C11D-2G was 27.4%, CD269-A7D-CD19b-2G was 30.6%, CD269-A7D-CS1-hu63 was 28.5%; and CD269-C11D-CS1-hu63-BB was 42.0%.

[0438] CD269-A7D-CD19b cCAR T cells efficiently lyse both BCMA and / or CD19-expressing tumor cell lines CD269-A7D-CD19b cCAR T cells were tested for their ability to lyse individual target cell lines in an in vitro co-culture assay (Figures 30C and 30D). K562 cells were engineered to synthetically express either BCMA (CD269) (referred to as K-BCMA) or CD19 (referred to as K-19) on the cell surface. After 18 hours of co-culture, the cells were labeled with anti-human CD3 and anti-human CD269 or CD19 and analyzed by flow cytometry (Figures 30C and CD30E). CD269-A7D-CD19b cCAR T cells were able to lyse 31% of the target K-BCMA cells at a 2:1 E:T ratio and 65% at a 5:1 ratio. CD269-A7D-CD19b cCAR T cells were also able to lyse 60% of the target K-CD19 cells at a 2:1 E:T ratio and nearly all at a 5:1 ratio (Figures 30D and CD30E). These results confirm that each CAR unit (CD269 and CD19b CAR) effectively lyses its specific target cell.

[0439] CD269-A7D-41BBL, CD269-A7D-CS1-hu63, and CD269-A7D-C11D cCAR T cells efficiently lyse the MM1S tumor cell line The various versions of BCMA-CS1 cCAR T cells prepared as described above were tested for their ability to lyse specific target cell lines in an in vitro co-culture assay. The human multiple myeloma cell line MM1S was co-cultured with CD269-A7D-41BBL CAR, CD269-A7D-CS1-hu63 cCAR, CD269-A7D-C11D cCAR T cells, or control T cells at 2:1 and 5:1 E:T ratios (Figure 30F). After 18 hours of co-culture, the cells were labeled with CMTMR (Cell Tracker) and anti-human CD269 and analyzed by flow cytometry. While the CD269-A7D-CS1-hu63 cCAR T cells were lysing, the CD269-A7D-41BBL CAR T cells were able to lyse 74% of the target MM1S cells at a 2:1 E:T ratio and 90% at a 5:1 ratio, while the CD269-A7D-CS1-hu63 cCAR T cells lysed 59% and 90% of the MM1S cells at 2:1 and 5:1 ratios, respectively, and the CD269-A7D-C11D CAR T cells lysed 62% and 86% of the MM1S cells at 2:1 and 5:1 ratios, respectively (Figure 30F). These compound CARs did not appear to show evidence of CAR-CAR interaction. Using the methods described in PCT / US2016 / 01995 and PCT / US2016 / 039306, in vivo antitumor activity and cell killing in a xenogeneic mouse model were performed, and target cells expressing BCMA or CS1 or both were eliminated or suppressed by cCAR T or NK cells.

[0440] CD269-A7D-41BBL, CD269-A7D-CS1-hu63, and CD269-A7D-C11D CAR T cells efficiently lyse the cell line K562 that synthetically expresses BCMA or CS1 The various versions of BCMA-CS1 cCAR T cells prepared as described above were tested for their ability to lyse specific target cell lines in an in vitro co-culture assay. K562 cells were modified to synthetically express either BCMA (CD269) or CS1 on the cell surface and were then co-cultured with CD269-A7D-41BBL, CD269-A7D-CS1-hu63, CD269-A7D-C11D cCAR T cells or control T cells at E:T ratios of 2:1 and 5:1. After 18 hours of co-culture, the cells were labeled with anti-human CD3 and anti-human CD269 (or CS1) and analyzed by flow cytometry. CD269-A7D-41BBL CAR T cells lysed 56% of the target K-BCMA cells at a 2:1 E:T ratio and completely eliminated all target cells at a 5:1 ratio, while CD269-A7D-CS1-hu63 cCAR T cells lysed 38% and 79% of K-BCMA cells at 2:1 and 5:1 ratios, respectively, and CD269-A7D-C11D CAR T cells lysed 16% and 74% of K-BCMA cells at 2:1 and 5:1 ratios, respectively (Figure 30G). Only CD269-A7D-CS1-hu63 and CD269-A7D-C11D cCAR T cells were tested in co-culture against K-CS1 cells (Figure 30H). CD269-A7D-CS1-hu63 cCAR T cells lysed 18% and 54% of K-CS1 cells at 2:1 and 5:1 ratios, respectively, while the compound CAR targeting two different epitopes on the BCMA antigen, CD269-A7D-C11D cCAR T cells, showed no ability to lyse K-CS1 cells at either ratio...

Claims

**Claim 1** Ex vivo engineered T or NK cells that co-express two different chimeric antigen receptor (CAR) units on the cell surface, wherein the engineered T or NK cells comprise, from 5' to 3', a first chimeric antigen receptor polypeptide (first CAR), a viral self-cleaving peptide, a second chimeric antigen receptor polypeptide (second CAR), and an enhancer that is IL-15 / IL-15sushi, under the transcriptional control of a single promoter, and encode a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 42, SEQ ID NO: 28, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 40, or SEQ ID NO: 60, and contain a polynucleotide, (i.) the first CAR polypeptide comprises a first antigen recognition domain, a first signal peptide, a first hinge region, a first transmembrane domain, a first co-stimulatory domain, and a first signaling domain, (ii.) the second CAR polypeptide comprises a second antigen recognition domain, a second signal peptide, a second hinge region, a second transmembrane domain, a second co-stimulatory domain, and a second signaling domain, the first antigen recognition domain and the second antigen recognition domain are different, the engineered T or NK cells. **Claim 2** The engineered T or NK cells according to claim 1, comprising a polynucleotide encoding a polypeptide consisting of the amino acid sequence represented by SEQ ID NO:

60. **Claim 3** The engineered T or NK cells according to claim 1, wherein the enhancer is secreted by the engineered T or NK cells. **Claim 4** The engineered T or NK cells according to claim 1, which are NK T cells, T cells, or NK cells. **Claim 5** The engineered T or NK cells according to claim 1, comprising at least two enhancers.

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