WO2022060806A1 - Compositions et procédés pour l'expression de récepteurs antigéniques chimériques anti-bcma ayant une il15 régulée par petites molécules dans des cellules t - Google Patents

Compositions et procédés pour l'expression de récepteurs antigéniques chimériques anti-bcma ayant une il15 régulée par petites molécules dans des cellules t Download PDF

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WO2022060806A1
WO2022060806A1 PCT/US2021/050420 US2021050420W WO2022060806A1 WO 2022060806 A1 WO2022060806 A1 WO 2022060806A1 US 2021050420 W US2021050420 W US 2021050420W WO 2022060806 A1 WO2022060806 A1 WO 2022060806A1
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cell
cells
bcma
nucleic acid
protein
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PCT/US2021/050420
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Dexue Sun
Michelle Lynn OLS
Geetha Hanna MYLVAGANAM
Abhishek KULKARNI
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Obsidian Therapeutics, Inc.
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Publication of WO2022060806A1 publication Critical patent/WO2022060806A1/fr

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    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/5443—IL-15
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    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
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    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
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    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A61K38/2086—IL-13 to IL-16
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    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/51—Lyases (4)
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
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    • A61K39/4631—Chimeric Antigen Receptors [CAR]
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    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464402—Receptors, cell surface antigens or cell surface determinants
    • A61K39/464416—Receptors for cytokines
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
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    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2878—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
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    • C12Y402/00—Carbon-oxygen lyases (4.2)
    • C12Y402/01—Hydro-lyases (4.2.1)
    • C12Y402/01001—Carbonate dehydratase (4.2.1.1), i.e. carbonic anhydrase
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61K2039/80—Vaccine for a specifically defined cancer
    • A61K2039/804—Blood cells [leukemia, lymphoma]
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by the route of administration
    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the dose, timing or administration schedule
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    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70578—NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • C—CHEMISTRY; METALLURGY
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    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
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    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07K2319/00—Fusion polypeptide
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    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
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    • C12N2510/00—Genetically modified cells

Definitions

  • the present disclosure relates to drug responsive domains (DRDs) derived from human carbonic anhydrase 2 (CA2), which can modulate the protein stability of at least one payload comprising human interleukin 15 (IL15) in response to a small molecule, and compositions and methods of use thereof.
  • DRDs drug responsive domains
  • CA2 human carbonic anhydrase 2
  • IL15 human interleukin 15
  • the present disclosure provides polypeptides comprising DRD-regulated IL15, and polynucleotides encoding the same.
  • the present disclosure provides cells comprising the polypeptides and/or polynucleotides in conjunction with anti-B cell maturation antigen (BCMA) chimeric antigen receptors (CAR) for use in cancer immunotherapy.
  • BCMA anti-B cell maturation antigen
  • CAR chimeric antigen receptors
  • BCMA B-cell maturation antigen
  • BAFF B cell activator of the TNF family
  • APRIL proliferation inducing ligand
  • BCMA is expressed in a majority of patients having B cell malignancies, including non-Hodgkin's lymphoma (NHL), Waldenstrom's macroglobulinemia, Burkitt lymphoma, Diffuse Large B-Cell Lymphoma and multiple myeloma (MM).
  • NHL non-Hodgkin's lymphoma
  • MM multiple myeloma
  • BCMA is widely expressed on malignant plasma cells at elevated levels in MM, and BCMA expression increases with progression from normal cells to active MM.
  • MM is the second most common hematologic malignancy with an incidence rate of 5/100,000.
  • MM is incurable and has an average 5-year survival rate of 48%.
  • the present disclosure provides, in part, novel anti-BCMA CAR-expressing T cells that are engineered with a regulated interleukin 15 (IL15), the expression or function of which can be modulated by a small molecule.
  • IL15 regulated interleukin 15
  • the presence of regulated IL15 improves anti-BCMA CAR-T therapy, resulting in clinically meaningful increased durations of response in patients.
  • the present discosure also provides nucleic acid molecules encoding the anti-BCMA CAR and regulated IL15, vectors comprising the nucleic acid molecules, and methods of using these nucleic acid molecules, vectors and cells in adoptive cell therapy.
  • one resistance mechanism to current anti- BCMA CAR-T therapy may be caused by T cell exhaustion and the need for persistence of enhanced memory and/or stem T cells.
  • the presence of regulated IL 15 decreases anti-BCMA CAR-T cell exhaustion and promotes more activated and persistent anti-tumor responses, thereby improving patient response to the anti-BCMA CAR-T therapy.
  • the regulated IL15 is a regulated membrane-bound IL15 (mbIL15) that is expressed by the anti- BCMA CAR-T cell.
  • the resulting mbIL15-expressing anti-BCMA CAR-T cells are able to deliver a more activated and persistent anti-tumor response to the cancer cells.
  • Administration of the small molecule to which the DRD binds will increase expression of mbIL15, thereby increasing the anti-BCMA CAR-T anti -tumor response.
  • Discontinuing administration of the small molecule, or lowering its dose, will decrease expression of mbIL15, which can be used, for example, if a patient is experiencing cytokine release syndrome.
  • the disclosure provides a cell comprising a first and a second protein.
  • the first protein is a chimeric antigen receptor (CAR) comprising (a) an antibody or antigen recognition moiety that binds to one or more epitopes of a human B-cell Maturation Antigen (BCMA) polypeptide, (b) a transmembrane domain, which may optionally further comprise a hinge domain, and (c) one or more intracellular T cell signaling domains.
  • the second protein comprises a drug responsive domain (DRD) operably linked to an IL 15 payload, wherein the DRD is a polypeptide derived from human carbonic anhydrase II (CA2) and comprising one, two, three or four amino acid insertions, deletions or substitutions compared to SEQ ID NO: 1.
  • DRD drug responsive domain
  • the regulated EL 15 of the present disclosure is an IL15 payload operably linked to a drug responsive domain (DRD) derived from human carbonic anhydrase 2 (CA2, having the amino acid sequence of SEQ ID NO: 1).
  • a DRD derived from human CA2 may be referred to as a CA2-DRD.
  • a DRD is a polypeptide that can modulate the stability of a polypeptide payload that is operably linked to it in response to a small molecule that binds to the DRD. In the absence of the small molecule ligand, the DRD is destabilized and causes degradation of the payload operably linked to the DRD. If the ligand is present, the DRD binds to it and its operably linked payload is stabilized. The stability of the DRD and its operably linked payload is dependent on the dose of the binding ligand.
  • the DRD is derived from the full-length CA2 polypeptide (SEQ ID NO: 1). In some embodiments, the DRD may be derived from a portion or region of the human carbonic anhydrase. In some embodiments, the DRD may comprise additional amino acids at either or both of its N- or C-termini. In some embodiments, the DRD may have one, two, three, four or more amino acid insertions, deletions or substitutions in its sequence relative to SEQ ID NO: 1. In some embodiments, the DRD may have one, two or three amino acid deletions or substitutions in its sequence relative to SEQ ID NO: 1.
  • the DRD is a polypeptide comprising an amino acid sequence having at least 90% sequence identity to amino acids 1-260 of SEQ ID NO: 1. In some embodiments, the DRD is a polypeptide comprising an amino acid sequence having at least 95% sequence identity to amino acids 1-260 of SEQ ID NO: 1. In some embodiments, the DRD is a polypeptide comprising an amino acid sequence having at least 96%, 97%, 98% or 99% sequence identity to amino acids 1-260 of SEQ ID NO: 1.
  • the DRD is a polypeptide comprising the amino acid sequence of SEQ ID NO:4, wherein the first methionine (Ml) is deleted and leucine at position 156 is substituted by histidine (L156H) relative to SEQ ID NO: 1.
  • the DRD is a polypeptide consisting of the amino acid sequence of SEQ ID NON.
  • the regulated IL15 payload comprises an amino acid sequence of human IL 15 (SEQ ID NO: 8).
  • the payload is a membrane-bound form of IL15.
  • the payload is a membrane-bound form of IL15 comprising a functional IL 15 component or domain, a transmembrane domain and an intracellular tail.
  • the membrane-bound form of IL15 further comprises a leader sequence.
  • the membrane-bound form of IL15 further comprises a linker sequence, which may be a peptide linker, between the functional IL 15 component or domain and the transmembrane domain.
  • the present disclosure provides a CA2 DRD-regulated mbIL15 polypeptide comprising, from N-terminal to C-terminal, a leader sequence, an IL 15 polypeptide comprising the amino acid sequence of SEQ ID NO: 8, a peptide linker, a transmembrane domain, and an intracellular tail.
  • the anti-BCMA CAR of the present disclosure provides a chimeric antigen receptor (CAR) comprising (a) an antibody or antigen recognition moiety that binds to one or more epitopes of a human B-cell Maturation Antigen (BCMA) polypeptide, (b) a transmembrane domain, which may optionally further comprise a hinge domain, and (c) one or more intracellular T cell signaling domains.
  • CAR chimeric antigen receptor
  • the antibody or the antigen recognition moiety of the CAR may be a camel immunoglobulin (Ig), an immunoglobulin new antigen receptor (IgNAR), an Fab fragment, an Fab' fragment, an F(ab)'2 fragment, an F(ab)'3 fragment, an Fv, a single-chain Fv antibody (scFv), a bis-scFv, an (scFv)2, a minibody, a diabody, a triabody, a tetrabody, a disulfide- stabilized Fv (dsFv), a single-domain antibody (sdAb) or a nanobody.
  • the anti-BCMA antibody or antigen recognition moiety is an scFv.
  • the scFv comprises an amino acid sequence described in Table 1.
  • the anti-BCMA scFv comprises the amino acid sequence of SEQ ID NO: 14. In some embodiments, the anti-BCMA scFv consists of the amino acid sequence ofNO:14. [0018] In some embodiments, the anti-BCMA CAR comprises a transmembrane domain that is derived from the transmembrane domain of a polypeptide selected from CD28, 4-1BB, CD8 alpha, CD4, CD19, CD3 epsilon, CD45, CD5, CD9, CD16, CD22, CD33, CD37, CD45, CD64, CD80, CD86, CD 134, CD 154, PD1, CTLA-4, an alpha chain of a T cell receptor, a beta chain of a T cell receptor, a zeta chain of a T cell receptor.
  • the anti-BCMA CAR comprises a transmembrane domain that is derived from the transmembrane domain of a polypeptide selected from CD28, CD8 alpha, CD4, CD45, PD1 or CTLA-4. In some embodiments, the anti-BCMA CAR comprises a transmembrane domain that is derived from the transmembrane domain of CD8 alpha polypeptide. In some embodiments, the transmembrane domain of the anti-BCMA CAR has the same amino acid sequence as the transmembrane domain of the polypeptide from which it is derived.
  • the anti-BCMA CAR comprises an optional hinge domain.
  • the optional hinge domain may be derived from the hinge domain of a polypeptide selected from CD28, 4-1BB, CD8 alpha, CD4, CD19, CD3 epsilon, CD45, CD5, CD9, CD16, CD22, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD154, PD1, CTLA-4, an alpha chain of a T cell receptor, a beta chain of a T cell receptor, a zeta chain of a T cell receptor.
  • the anti-BCMA CAR comprises a hinge domain that is derived from the hinge domain of a polypeptide selected from CD28, CD8 alpha, CD4, CD45, PD1 or CTLA-4. In some embodiments, the anti-BCMA CAR comprises a hinge domain that is derived from the hinge domain of CD8 alpha polypeptide. In some embodiments, the hinge domain of the anti- BCMA CAR has the same amino acid sequence as the hinge domain of the polypeptide from which it is derived.
  • the transmembrane domain and optional hinge domain are derived from different polypeptides. In some embodiments, the transmembrane domain and optional hinge domain are both derived from the same polypeptide. In some embodiments, the transmembrane domain and hinge domain are both derived from the transmembrane domain and hinge domain of a polypeptide selected from CD28, CD8 alpha, CD4, CD45, PD1 or CTLA-4. In another embodiment, the transmembrane domain and optional hinge domain of the anti-BCMA CAR are both derived from the transmembrane domain and hinge domain of CD8 alpha.
  • the transmembrane domain and hinge domain of the anti-BCMA CAR comprises the amino acid sequence of SEQ ID NO: 16. In some embodiments, the transmembrane domain and hinge domain of the anti-BCMA CAR consists of the amino acid sequence of SEQ ID NO: 16.
  • the CAR comprises one, two or three intracellular T cell signaling domains. In some embodiments, the CAR comprises two intracellular T cell signaling domains. In some embodiments, the intracellular T cell signaling domains are independently selected from an intracellular signaling domain of 4- IBB, CARD11, CD2, CD3( ⁇ , CD7, CD27, CD28, CD30, CD40, CD54, CD83, CD134, CD150, CTLA-4, CD223, CD270, CD273, CD274, CD278, DAP10, FcRy, LAT, NKD2C, SLP76, TRIM or ZAP70.
  • the intracellular T cell signaling domains are independently selected from an intracellular signaling domain of CD28, CD134, CD3( ⁇ or 4-1BB. In some embodiments, the intracellular T cell signaling domains are the intracellular signaling domains of 4-1BB and CD3( ⁇ . In some embodiments, the intracellular T cell signaling domains of the CAR comprise or consist of the intracellular signaling domain of 4-1BB, having amino acid sequence SEQ ID NO: 18, and the intracellular signaling domain of CD3( ⁇ , having amino acid sequence SEQ ID NO:20.
  • the CAR comprises, from N- to C-terminus, the antibody or antigen recognition moiety, the optional hinge domain, the transmembrane domain, and the one or more intracellular T cell signaling domains.
  • CAR further comprises a signal sequence domain N-terminal to the antibody or the antigen recognition moiety.
  • the signal sequence domain is the signal sequence of granulocyte macrophagecolony stimulating factor (GM-CSF), CD8a or IgGl heavy chain.
  • GM-CSF granulocyte macrophagecolony stimulating factor
  • CD8a IgGl heavy chain.
  • the signal sequence domain is CD8a.
  • the signal sequence domain comprises the amino acid sequence of SEQ ID NO: 12 or consists of the amino acid sequence of SEQ ID NO: 12.
  • the CAR comprises, from N-terminal to C-terminal: a CD8a leader sequence; an scFv directed against BCMA; a CD8a hinge domain or a CD28 hinge domain; a CD8a transmembrane domain or a CD28 hinge domain; an intracellular T cell signaling domain selected from a 4-1BB intracellular T cell signaling domain, an 0X40 intracellular T cell signaling domain, or a CD28 intracellular T cell signaling domain; and a CD3 ⁇ intracellular T cell signaling domain.
  • the CAR comprises, from N-terminal to C-terminal: a CD8a leader sequence; an scFv directed against BCMA; a CD8a hinge domain or a CD28 hinge domain; a CD8a transmembrane domain or a CD28 hinge domain; an intracellular T cell signaling domain selected from a 4-1BB intracellular T cell signaling domain, an 0X40 intracellular T cell signaling domain, or a CD28 intracellular T cell signaling domain; and a CD3 ⁇ intracellular T cell signaling domain.
  • the CAR comprises, from N-terminal to C-terminal: a CD8a leader sequence; an scFv directed against BCMA; a CD8a hinge domain; a CD8a transmembrane domain; a 4-1BB intracellular T cell signaling domain; and a CD3( ⁇ intracellular T cell signaling domain.
  • the CAR comprises the amino acid sequence of SEQ ID NO:34. In some embodiments, the CAR consists of the amino acid sequence of SEQ ID NO:34. [0027] In another aspect, the present disclosure provides nucleic acid molecules encoding the anti -BCMA CAR and the CA2 DRD-regulated IL 15 polypeptide. In some embodiments, one nucleic acid molecule encodes the anti-BCMA CAR and a second nucleic acid molecule encodes the CA2 DRD-regulated IL 15 polypeptide. In some embodiments, a single nucleic acid molecule comprises a nucleic acid sequence encoding the anti-BCMA CAR and a nucleic acid sequence encoding the CA2 DRD-regulated IL15.
  • nucleic acid sequence encoding the anti- BCMA CAR is 5’ to the nucleic acid sequence encoding the CA2 DRD-regulated IL15. In some embodiments in which a single nucleic acid molecule encodes both the anti-BCMA CAR and the CA2 DRD-regulated IL15, the nucleic acid sequence encoding the CA2 DRD-regulated IL15 is 5’ to the nucleic acid sequence encoding the anti-BCMA CAR.
  • nucleic acid sequence encoding the anti- BCMA CAR and the nucleic acid sequence encoding the CA2 DRD-regulated IL15 are separated by a co-expression element that promotes production of separate proteins.
  • a “co-expression element” is a nucleic acid element that promotes production of separate polypeptides from the nucleic acid sequences flanking the co-expression element.
  • the co-expression element is a nucleic acid sequence encoding a cleavable linker sequence, a peptide that causes ribosome skipping, or an internal ribosome entry site (IRES).
  • the peptide that causes ribosome skipping is a 2A peptide selected from foot-and-mouth disease virus (FMDV) 2A (F2A), equine rhinitis A virus (ERAV) 2 A (E2A), porcine teschovirus-1 2 A (P2A), and Thosea asigna virus 2 A (T2A).
  • the 2A peptide is P2A, which comprises the amino acid sequence of SEQ ID NO:22.
  • nucleic acid sequence encoding the anti-BCMA CAR is 5’ to the nucleic acid sequence encoding the CA2 DRD-regulated ELI 5
  • nucleic acid sequence encoding the P2A is 3’ to the nucleic acid sequence encoding the anti-BCMA CAR and 5’ to the nucleic acid sequence encoding the CA2 DRD-regulated IL15.
  • nucleic acid sequence encoding the CA2 DRD-regulated ELI 5 is 5’ to the nucleic acid sequence encoding the anti-BCMA CAR
  • nucleic acid sequence encoding the P2A is 3’ to the nucleic acid sequence encoding the CA2 DRD-regulated ELI 5 and 5’ to the nucleic acid sequence encoding the anti-BCMA CAR.
  • the nucleic acid molecule may further comprise a promoter.
  • a promoter is operably linked to each nucleic acid sequence encoding each protein.
  • a promoter is located 5’ to the nucleic acid sequence encoding the protein which is at the 5’ end of the nucleic acid molecule.
  • the promoter is located within 50 nucleotides, 100 nucleotides, 150 nucleotides, 200 nucleotides, 250 nucleotides, 300 nucleotides, 400 nucleotides, 500 nucleotides, 750 nucleotides, 1000 nucleotides, or 1500 nucleotides of the nucleic acid sequence encoding the protein which is at the 5’ end of the nucleic acid molecule. In some embodiments, the promoter is located within 50 nucleotides or within 100 nucleotides of the nucleic acid sequence encoding the protein which is at the 5’ end of the nucleic acid molecule.
  • the promoter is located 5’ to the nucleic acid sequence encoding the anti-BCMA CAR. In some embodiments, in which the nucleic acid sequence encoding the CA2 DRD-regulated ELI 5 is 5’ to the nucleic acid sequence encoding the anti- BCMA CAR, the promoter is located 5’ to the nucleic acid sequence encoding the CA2 DRD- regulated IL15.
  • a single promoter at the 5’ end of a tandem construct may promote transcription of both the first and the second nucleic acid sequences even though it may be located 5’ to the nucleic acid sequence encoding the first protein.
  • the promoter may be a CMV promoter, an EFla promoter or a PGK promoter. In some embodiments, the promoter may be an EFla promoter.
  • the present disclosure provides vectors comprising the nucleic acid molecules encoding anti-BCMA CAR and CA2 DRD-regulated IL15.
  • the vector may be a plasmid.
  • the vector may be an expression vector.
  • the vector may be a viral vector.
  • the viral vector may be derived from an adenovirus, adeno-associated virus (AAV), alphavirus, flavivirus, herpes virus, measles virus, rhabdovirus, retrovirus, lentivirus, Newcastle disease virus (NDV), poxvirus or picornavirus.
  • the viral vector may be a lentiviral vector, a retroviral vector, adeno-associated viral (AAV) vector, adenoviral vector, or a herpes viral vector.
  • the viral vector is a lentiviral vector or a gamma retroviral vector.
  • the present disclosure provides a cell comprising one or more nucleic acid molecules encoding anti-BCMA CAR and CA2 DRD-regulated ELI 5.
  • the present disclosure provides a cell expressing anti-BCMA CAR and CA2 DRD-regulated ELI 5.
  • the cell may be a E cell.
  • the T cell may be a human T cell.
  • the human T cell may be a CD4+ T cell, CD8+ T cell, or memory T cell.
  • the present disclosure provides a population of human T cells comprising one or more nucleic acid molecules encoding anti- BCMA CAR and CA2 DRD-regulated ELI 5.
  • the human T cells may comprise a regulated mbIL15. In some embodiments, the human T cells may comprise a mbIL15 that is N-terminal to the DRD. In some embodiments, the CA2 DRD in the human T cells is hCA2(Mldel, L156H) comprising the amino acid sequence of SEQ ID NO:4. In some embodiments, the mbIL15 in the T cells comprises the amino acid sequence of SEQ ID NO:6. In some embodiments, the human E cells may comprise a CA2 DRD-regulated ELI 5 comprising the amino acid sequence of SEQ ID NO: 10.
  • the human T cells may comprise, in addition to the CA2 DRD- regulated IL15, an anti-BCMA CAR, which is used interchangeably with the term “BCMA CAR”.
  • the human T cells comprise an anti-BCMA CAR comprising, from N-terminal to C-terminal, a CD8a leader sequence; an scFv directed against BCMA; a CD8a hinge domain or a CD28 hinge domain; a CD8a transmembrane domain or a CD28 hinge domain; an intracellular T cell signaling domain selected from a 4-1BB intracellular T cell signaling domain, an 0X40 intracellular T cell signaling domain, or a CD28 intracellular T cell signaling domain; and a CD3i ⁇ intracellular T cell signaling domain.
  • the human T cells comprise an anti-BCMA CAR comprising, from N-terminal to C-terminal, a CD8a leader sequence; an scFv directed against BCMA; a CD8a hinge domain; a CD8a transmembrane domain; a 4-1BB intracellular T cell signaling domain; and a CD3( ⁇ intracellular T cell signaling domain.
  • the human T cells comprise an anti-BCMA CAR comprising the amino acid sequence of SEQ ID NO:34.
  • the present disclosure provides pharmaceutical compositions.
  • the pharmaceutical compositions comprise the nucleic acid molecules encoding the CA2 DRD-regulated ELI 5 and the anti-BCMA CAR and a pharmaceutically acceptable excipient.
  • the pharmaceutical compositions comprise the vectors comprising the nucleic acid molecules encoding the CA2 DRD-regulated IL 15 and the anti-BCMA CAR and a pharmaceutically acceptable excipient.
  • the pharmaceutical compositions comprise the cells comprising the CA2 DRD-regulated ELI 5 and the anti-BCMA CAR and a pharmaceutically acceptable excipient.
  • the pharmaceutical compositions comprise a T cell, in some embodiments a human T cell, comprising the CA2 DRD-regulated ELI 5 and the anti-BCMA CAR and a pharmaceutically acceptable excipient.
  • the pharmaceutical compositions comprise a population of F cells, in some embodiments a population of human T cells, comprising the CA2 DRD-regulated IL 15 and the anti-BCMA CAR and a pharmaceutically acceptable excipient.
  • the pharmaceutically acceptable excipient may be a cryoprotectant.
  • the pharmaceutical composition comprises a pharmaceutically acceptable excipient and a cryoprotectant.
  • the present disclosure provides methods of making a modified cell by introducing one or more nucleic acid molecules encoding the CA2 DRD-regulated IL15 and the anti-BCMA CAR into the cell.
  • the method comprises introducing a first nucleic acid molecule encoding the anti-BCMA CAR and a second nucleic acid molecule encoding the CA2 DRD-regulated IL15 into the cell.
  • the method comprises introducing a single nucleic acid molecule encoding both the CA2 DRD-regulated IL 15 and the anti-BCMA CAR into the cell.
  • the cell is a T cell.
  • the nucleic acid molecule is introduced into the cell by a non-viral vector delivery method. In some embodiments, the nucleic acid molecule is introduced into the cell by viral transduction. In some embodiments, the nucleic acid molecule in introduced into the cell by lentiviral transduction. In some embodiments, the nucleic acid molecule is introduced into the cell by lentiviral transduction into a T cell.
  • the present disclosure provides a method of modulating the expression, function, and/or level of IL15 in a cell comprising CA2 DRD-regulated IL15 and anti-BCMA CAR, comprising administering to the cell a stimulus to which the CA2 DRD is responsive in an amount sufficient to modulate the expression, function and/or level of IL 15.
  • the stimulus is acetazolamide, celecoxib, valdecoxib, rofecoxib, methazolamide, dorzolamide, brinzolamide, diclofenamide, ethoxzolamide, zonisamide, dansylamide or dichlorphenamide.
  • the stimulus is acetazolamide.
  • the present disclosure provides a method of treating a B cell malignancy in a subject, comprising (a) administering to the subject a therapeutically effective amount of the nucleic acid molecule encoding CA2 DRD-regulated IL15 and anti-BCMA CAR or a vector comprising the nucleic acid molecule; and (b) administering a therapeutically effective amount of a stimulus to the subject, wherein the hCA2 DRD is responsive to the stimulus, and wherein expression of IL15 is modulated in response to the stimulus.
  • the B cell malignancy is multiple myeloma, non-Hodgkiris lymphoma, Waldenstrom's macroglobulinemia, Burkitt lymphoma or diffuse large B-cell lymphoma. In some embodiments, the B cell malignancy is multiple myeloma.
  • the stimulus is acetazolamide, celecoxib, valdecoxib, rofecoxib, methazolamide, dorzolamide, brinzolamide, diclofenamide, ethoxzolamide, zonisamide, dansylamide or dichlorphenamide. In some embodiments, the stimulus is acetazolamide.
  • administering a therapeutically effective amount of the nucleic acid molecule encoding a CA2 DRD-regulated IL15 and an anti-BCMA CAR or a vector comprising the nucleic acid molecule further comprises (i) providing isolated autologous human T cells from the subject or providing allogeneic human T cells, (ii) transducing the autologous or allogeneic human T cells with the nucleic acid molecule or vector, and (iii) growing the transduced autologous or allogeneic human cells at least two-fold, to form a dosing T cell population, and administering the dosing T cell population into the subject.
  • the present disclosure provides a method of treating a B cell malignancy in a subject, comprising (a) administering to the subject a therapeutically effective amount of modified cells, in some embodiments a modified human T cells, or a pharmaceutical composition thereof, wherein the modified cells or modified human T cells comprise CA2 DRD- regulated IL15 and anti-BCMA CAR; and (b) administering a therapeutically effective amount of a stimulus to the subject, wherein the hCA2 DRD is responsive to the stimulus, and wherein expression of IL15 is modulated in response to the stimulus in a subject in need thereof.
  • the B cell malignancy is multiple myeloma, non-Hodgkin's lymphoma, Waldenstrom's macroglobulinemia, Burkitt lymphoma or diffuse large B-cell lymphoma. In some embodiments, the B cell malignancy is multiple myeloma.
  • the stimulus is acetazolamide, celecoxib, valdecoxib, rofecoxib, methazolamide, dorzolamide, brinzolamide, diclofenamide, ethoxzolamide, zonisamide, dansylamide or dichlorphenamide. In some embodiments, the stimulus is acetazolamide.
  • the present disclosure provides a method of treating multiple myeloma in a subject, comprising (a) administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising human T cells, wherein the human T cells comprise an anti-BCMA CAR and a CA2 DRD-IL15; and (b) administering a therapeutically effective amount of a stimulus to the subject, wherein the hCA2 DRD is responsive to the stimulus, and wherein expression of IL15 is modulated in response to the stimulus.
  • the stimulus is acetazolamide, celecoxib, valdecoxib, rofecoxib, methazolamide, dorzolamide, brinzolamide, diclofenamide, ethoxzolamide, zonisamide, dansylamide or dichlorphenamide. In some embodiments, the stimulus is acetazolamide.
  • the present disclosure provides a combination for use in immunotherapy of a first protein and a second protein.
  • the first protein is a chimeric antigen receptor (CAR) comprising (a) an antibody or antigen recognition moiety that binds to one or more epitopes of a human B-cell Maturation Antigen (BCMA) polypeptide, (b) a transmembrane domain, which may optionally further comprise a hinge domain, and (c) one or more intracellular T cell signaling domains.
  • CAR chimeric antigen receptor
  • the second protein comprises a drug responsive domain (DRD) operably linked to an IL15 payload, wherein the DRD is a polypeptide derived from human carbonic anhydrase II (CA2) and comprising one, two, three or four amino acid insertions, deletions or substitutions compared to SEQ ID NO: 1.
  • DRD drug responsive domain
  • the present disclosure provides a composition comprising a first nucleic acid molecule and a second nucleic acid molecule.
  • the first nucleic acid molecule encodes a chimeric antigen receptor (CAR) comprising (a) an antibody or antigen recognition moiety that binds to one or more epitopes of a human B-cell Maturation Antigen (BCMA) polypeptide, (b) a transmembrane domain, which may optionally further comprise a hinge domain, and (c) one or more intracellular T cell signaling domains.
  • CAR chimeric antigen receptor
  • the second nucleic acid molecule encodes the second protein comprises a drug responsive domain (DRD) operably linked to an IL15 payload, wherein the DRD is a polypeptide derived from human carbonic anhydrase II (CA2) and comprising one, two, three or four amino acid insertions, deletions or substitutions compared to SEQ ID NO:1.
  • DRD drug responsive domain
  • the present disclosure provides an anti-BCMA cell-expressing immunotherapy system comprising one or more vectors.
  • the one or more vectors comprise a first nucleic acid molecule encoding a chimeric antigen receptor (CAR) comprising (a) an antibody or antigen recognition moiety that binds to one or more epitopes of a human B-cell Maturation Antigen (BCMA) polypeptide, (b) a transmembrane domain, which may optionally further comprise a hinge domain, and (c) one or more intracellular T cell signaling domains; and a second nucleic acid molecule encoding a second protein comprising a drug responsive domain (DRD) operably linked to an ELI 5 payload, wherein the DRD is a polypeptide derived from human carbonic anhydrase II (CA2) and comprising one, two, three or four amino acid insertions, deletions or substitutions compared to SEQ ID NO: 1.
  • the first nucleic acid molecule encoding a chimeric anti
  • the present disclosure provides a combination of a first protein and a second protein, both operable to be expressed in a T-cell, wherein: (i) the first protein of the combination is a chimeric antigen receptor (CAR) comprising: (a) an antibody or antigen recognition moiety that binds to one or more epitopes of a human B-cell Maturation Antigen (BCMA) polypeptide, (b) a transmembrane domain, which may optionally further comprise a hinge domain, and (c) one or more intracellular T cell signaling domains; and (ii) the second protein of the combination comprises an IL15 polypeptide.
  • CAR chimeric antigen receptor
  • the second protein of the combination further comprises a drug responsive domain (DRD) operably linked to the IL15 polypeptide, wherein the DRD is a polypeptide derived from human carbonic anhydrase II (CA2) and comprising one, two, three or four amino acid insertions, deletions or substitutions compared to SEQ ID NO: 1.
  • DRD drug responsive domain
  • CA2 human carbonic anhydrase II
  • the present disclosure provides an engineered T-cell comprising the combination of proteins.
  • the present disclosure provides a nucleic acid molecule encoding a first protein and a second protein, wherein: (i) the first protein is a chimeric antigen receptor (CAR) comprising: (a) an antibody or antigen recognition moiety that binds to one or more epitopes of a human B-cell Maturation Antigen (BCMA) polypeptide, (b) a transmembrane domain, which may optionally further comprise a hinge domain, and (c) one or more intracellular T cell signaling domains; and (ii) the second protein comprises a IL15 polypeptide.
  • CAR chimeric antigen receptor
  • BCMA human B-cell Maturation Antigen
  • the second protein further comprises a drug responsive domain (DRD) operably linked to the IL15 polypeptide, wherein the DRD is a polypeptide derived from human carbonic anhydrase II (CA2) and comprising one, two, three or four amino acid insertions, deletions or substitutions compared to SEQ ID NO: 1.
  • the DRD is hCA2(Mldel, L156H) comprising the amino acid sequence of SEQ ID NO: 4.
  • the DRD is hCA2(Mldel, L156H) consisting of the amino acid sequence of SEQ ID NO: 4.
  • the IL15 polypeptide is a membrane-bound IL15 polypeptide.
  • the second protein comprises the amino acid sequence of SEQ ID NO: 6 or wherein the second protein consists of the amino acid sequence of SEQ ID NO: 6.
  • the first protein comprises an amino acid sequence selected from an amino acid sequence of Table 3.
  • the first protein comprises the amino acid sequence of SEQ ID NO: 34 or wherein the first protein consisting essentially of the amino acid sequence of SEQ ID NO: 34.
  • the present disclosure provides a vector comprising the nucleic acid molecule, hi some embodiments, the present disclosure provides an engineered T-cell comprising the nucleic acid molecule or the vector. [0048]
  • the present disclosure provides a pharmaceutical composition comprising a nucleic acid molecule, a vector, or an engineered T-cell according to the present disclosure, and a pharmaceutically acceptable excipient.
  • the present disclosure provides a method of producing a modified cell, said method comprising introducing into a cell a first nucleic acid molecule and a second nucleic acid molecule, wherein: (i) said first nucleic acid molecule encodes a chimeric antigen receptor (CAR) comprising: (a) an antibody or antigen recognition moiety that binds to one or more epitopes of a human B-cell Maturation Antigen (BCMA) polypeptide, (b) a transmembrane domain, which may optionally further comprise a hinge domain, and (c) one or more intracellular T cell signaling domains; and (ii) said second nucleic acid molecule encodes a second protein comprising an IL15 polypeptide.
  • CAR chimeric antigen receptor
  • BCMA human B-cell Maturation Antigen
  • the second nucleic acid further encodes a drug responsive domain (DRD) operably linked to the IL15 polypeptide, wherein the DRD is a polypeptide derived from human carbonic anhydrase II (CA2) and comprising one, two, three or four amino acid insertions, deletions or substitutions compared to SEQ ID NO: 1.
  • DRD drug responsive domain
  • the present disclosure provides a method of treating a B cell malignancy in a subject in need thereof, said method comprising: (a) administering to the subject a therapeutically effective amount of a nucleic acid molecule, a vector, an engineered T-cell, or a pharmaceutical composition of the disclosure; and (b) optionally, administering a therapeutically effective amount of a stimulus to the subject, wherein the hCA2 DRD is responsive to the stimulus, and wherein expression of IL15 polypeptide is modulated in response to the stimulus.
  • the B cell malignancy is multiple myeloma, non-Hodgkin's lymphoma, Waldenstrom's macroglobulinemia, Burkitt lymphoma or diffuse large B-cell lymphoma. In some embodiments, the B cell malignancy is multiple myeloma.
  • administering the engineered T-cell further comprises, (i) providing isolated autologous human T cells from the subject or providing allogeneic human T cells; (ii) transducing the autologous or allogeneic human T cells with a nucleic acid molecule or a vector of the disclosure; and (iii) growing the transduced autologous or allogeneic human cells at least two-fold, to form a dosing T cell population, wherein said administering to the subject the cell comprises administering the dosing T cell population into the subject.
  • the second protein encoded by the nucleic acid molecule or vector of the disclosure, or expressed by the engineered T-cell of the disclosure, or contained in the pharmaceutical composition of the disclosure comprises a drug responsive domain (DRD) operably linked to the IL15 polypeptide, wherein said DRD is a polypeptide derived from human carbonic anhydrase II (CA2) and comprising one, two, three or four amino acid insertions, deletions or substitutions compared to SEQ ID NO: 1; wherein said first protein encoded by the nucleic acid molecule or the vector, or expressed by the engineered T-cell, or contained in the pharmaceutical composition comprises: (a) an antibody or antigen recognition moiety that binds to one or more epitopes of a human B-cell Maturation Antigen (BCMA) polypeptide (b) a transmembrane domain, which may optionally further comprise a hinge domain, and (c) one or more intracellular
  • DRD drug responsive domain operably linked to the IL15 polypeptide
  • CA2 human
  • FIG. 1 illustrates constructs BCMA-IL15-007, BCMA-IL15-008 and BCMA-IL15- 009 of the present disclosure.
  • BCMA CAR refers to an anti-B cell maturation antigen chimeric antigen receptor
  • P2A refers to P2A ribosome skipping peptide
  • mbIL15 refers to membranebound IL15
  • CA2(Mldel, L156H) refers to carbonic anhydrase II (Mldel, L156H) drug responsive domain (DRD)
  • CA2(Mldel) refers to carbonic anhydrase II having an Mldel mutation.
  • FIG. 2A-FIG. 2C show expression of BCMA-CAR and mbIL15 in transduced T cells.
  • FIG. 2A shows regulated in vitro mbIL15 expression on T cells transduced with BCMA-IL15- 008 in response to increasing concentrations of acetazolamide (ACZ).
  • FIG. 2B shows expression of BCMA-CAR in T cells transduced with the indicated constructs. Cells were gated for Live, Singlet, CD3+, CD45+ cells to then quantitate the percent BCMA-CAR positive cells. Dots represent 3 different T cell donors.
  • FIG. 1A shows regulated in vitro mbIL15 expression on T cells transduced with BCMA-IL15- 008 in response to increasing concentrations of acetazolamide (ACZ).
  • FIG. 2B shows expression of BCMA-CAR in T cells transduced with the indicated constructs. Cells were gated for Live, Singlet, CD3+, CD45+ cells to then quantitate the percent BC
  • 2C shows the geometric mean fluorescence intensity (gMFI) for the expression of IL 15 as measured by a IL15Ra-Fc fusion protein reagent detected with a fluorescently labeled anti-human Fc antibody.
  • Cells were gated for Live, Singlet, CD3+, CD45+, BCMA-CAR+ cells. Dots represent 3 different T cell donors.
  • FIG. 3A-FIG. 3D show cytokine production and cytotoxicity in co-cultures of BCMA CAR-T cells and target cells.
  • FIG. 3A-FIG. 3B show interferon y expression and cytotoxicity in a co-culture of BCMA CAR-T cells and KMS11-luc target cells.
  • FIG. 3 A shows interferon y secretion from BCMA CAR-T cells in response to KMS11-luc target cells.
  • the “target only” condition represents a condition in which only the target (KMS11-luc) cells alone were analyzed without effector cells.
  • E:T refers to effector cell Target cell ratio.
  • “+_” refers to ACZ-treated. refers to vehicle-treated.
  • FIG. 3B shows cytotoxicity of BCMA CAR-T cells against KMS11-luc target cells.
  • RLU refers to relative light unit, for luciferase release from the luciferised target cells.
  • FIG. 3C shows cytokine secretion (interferon y (IFNg) and IL-2) from BCMA CAR-T cells in response to RPMI8226-luc target cells.
  • FIG. 3D shows cytotoxicity of BCMA CAR-T cells against RPMI8226-luc target cells.
  • FIG. 4A-FIG. 4C show regulated in vitro antigen-independent survival of anti-BCMA CAR T cells transduced with BCMA-IL15-008 (‘008).
  • EV refers to empty vector
  • exo IL15 refers to exogenous IL15 (also referred to as recombinant human IL15 or rhIL15)
  • ‘011” refers to BCMA-011
  • ‘“007” refers to BCMA-IL15-007.
  • dots represent the mean of 3 different T cell donors and error bars represent standard deviation.
  • Control BCMA-CAR is OT-BCMA-011 with and without rhILl 5.
  • Constant mbIL15-CAR is OT- BCMA-IL15-007.
  • Regular mbIL15-CAR is OT-BCMA-IL 15-008 treated with vehicle or ACZ.
  • Constant refers to continuous in vitro exposure to either rhIL15 or ACZ (left panel and right panel, respectively).
  • 3 ONG OFF” and “6ON/6OFF” refers to intermittent exposure of cells to a treatment cycle of either 3 days or 6 days of exposure to rhILl 5 or ACZ, then a media wash followed by a 3 day or 6 day rest period without rhILl 5 or ACZ before repeating the exposure cycle again until the end of the 14 day experiment.
  • FIG. 5 highlights the characteristics of T memory stem cells (TSCM) that sit on the path of cell differentiation from naive T cells (TN) to terminally differentiated effector T cells (TEFF).
  • TSCM T memory stem cells
  • TN naive T cells
  • TEFF terminally differentiated effector T cells
  • FIG. 6A-FIG. 6D show that at the end of an extended antigen-independent survival assay (as shown in FIG. 4A-FIG. 4B), IL15 enriches for a T stem cell memory phenotype (TSCM) and mbIL15-expressing BCMA CAR-T cells maintain their cytotoxicity and ability to produce cytokines (interferon y (IFN y) and IL-2) in response to target cells.
  • TSCM T stem cell memory phenotype
  • IFN y interferon y
  • IL-2 interferon y
  • FIG. 6A shows the percentage of T cells expressing markers of TSCM (CD27+, CD45RO-, CCR7+ and CD95+) after transduction with BCMA-IL15-007 (BCMA-007, shown as “Constitutive IL15”), BCMA-IL15- 008 (BCMA-008, shown as “Reg. IL 15”), BCMA-011 (shown as “CAR alone”) or empty vector (EV).
  • FIG. 6B and FIG. 6C show that IL 15 increases Tscm phenotype frequency over time in multiple T cell donors. Cells were gated according to a Singlet, Live, CD3+, CD45+ cells gate.
  • 6D shows cytokine production (interferon y (IFNy) and IL-2) and cytotoxicity in cocultures of BCMA CAR-T cells and target (RPMI8226-luc) cells. Dots represent 3 different T cell donors, along bars showing the mean.
  • “3ON/3OFF” and “6ON/6OFF” refers to intermittent exposure of cells to a treatment cycle of either 3 days or 6 days of exposure to rhIL15 or ACZ, then a media wash followed by a 3 day or 6 day rest period without rhIL15 or ACZ before repeating the exposure cycle again until the end of the 14 day experiment.
  • FIG. 7A illustrates constructs BCMA-IL15-004, BCMA-IL15-005, and BCMA-IL15- 006 of the present disclosure.
  • BCMA CAR refers to an anti-B cell maturation antigen chimeric antigen receptor
  • P2A refers to P2A ribosome skipping peptide
  • mbIL15 refers to membranebound IL15
  • CA2(Mldel, L156H) refers to carbonic anhydrase II (Mldel, L156H) drug responsive domain (DRD)
  • CA2(Mldel) refers to carbonic anhydrase II having an Mldel mutation.
  • FIG. 7B-FIG. 7C show interferon y expression and cytotoxicity in a co-culture of BCMA CAR-T cells and KMSl l-luc cells.
  • FIG. 7B shows interferon y (IFN-g) expression in a co-culture of BCMA CAR-T cells and KMS11-luc cells.
  • the “target only” condition represents a condition in which only the target (KMS11-luc) cells alone were analyzed without effector cells.
  • E:T refers to effector cell Target cell ratio. refers to ACZ-treated, refers to vehicle- treated.
  • FIG. 7C shows cytotoxicity of BCMA CAR-T cells in response to KMS11-luc target cells.
  • FIG. 8A-FIG. 8B show in vivo antigen-independent BCMA CAR-T expansion and mbIL15 expression.
  • FIG. 8A shows number of human CD3+, CD45+ T cells per 50 gL blood on day 7 or day 13 post CAR-T transfer.
  • FIG. 8B shows mbIL15 surface expression on T cells in the blood as measured by mean fluorescence intensity (gMFI) using a IL15-Ra-Fc fusion protein detected with a fluorescently-labeled anti-human Fc antibody (“IL15Ra-Fc”). Analysis is shown for day 13 post CAR-T transfer. Cells were gated according to a Live, Singlet, CD3+, CD45+ gate.
  • FIG. 9A-FIG. 9B show graphs of in vivo tumor growth after adoptive cell transfer of T cells in a RPMI8226-luc xenograft animal model.
  • FIG. 9A shows tumor growth in animals that received 0.3xl0 6 ; IxlO 6 ; or 3xl0 6 transferred T cells (left, middle and right panels, respectively). The T cells were transduced with the indicated constructs (BCMA-011, BCMA-IL15-007 or empty vector).
  • FIG. 9B shows tumor burden in animals that received 0.3x10 6 transferred T cells.
  • the T cells were transduced with constructs OT-BCMA-011 (“control BCMA-CAR”), OT- BCMA-IL15-007 (“constitutive mbIL15-CAR”), OT-BCMA-IL 15-008 (“regulated mbIL15- CAR”) or empty vector.
  • control BCMA-CAR control BCMA-CAR
  • OT- BCMA-IL15-007 Constitutive mbIL15-CAR
  • OT-BCMA-IL 15-008 regulated mbIL15- CAR
  • empty vector empty vector.
  • FIG. 10 shows IFNy concentration in plasma collected from animals on day 1 or day 7 after adoptive cell transfer of T cells in a RPMI8226-luc xenograft animal model.
  • the T cells were transduced with constructs OT-BCMA-011 (“control BCMA-CAR”), OT-BCMA-IL 15-007 (“Constitutive mbIL15-CAR”), OT-BCMA-IL15-008 (“Regulated mbIL15-CAR”) or empty vector.
  • FIG. 11 A-FIG. 1 ID show cytotoxicity, cell counts and cytokine production for BCMA-CARTs with or without mbIL15 in a chronic stimulation assay.
  • “Veh” refers to vehicle (DMSO)-treatment.
  • “EV” refers to empty vector.
  • FIG. 11 A shows CART cytotoxicity interrogated using the Bright-GloTM Luciferase Assay System (Promega cat # PRE2620). In brief, 100 pl of co-culture was lysed open with a luciferin containing substrate. The amount of luciferase that was generated was measured and reported as percent of target-only luciferase signal.
  • FIG. 1 IB shows the cell counts obtained by flow cytometry from a 100 pl aliquot of assay cell culture. Samples were collected with a fixed volume on a BD Fortessa flow cytometer where live, CD3+ populations were counted. These counts where then used in the re-seeding process as well as documenting growth kinetics longitudinally over 4 weeks. All constructs, with the exception of the vehicle-treated BCMA-IL-15-008 and empty vector groups, had the same growth kinetics where expansion was observed for the first 2 weeks and then all constructs started to decline at the same rate.
  • FIG. 11C-FIG. 1 ID show an MSD (Meso Scale Discovery) quantification of IFNy and IL-2 levels in the cell supernatants taken 24 hours after a new co-culture was set up. All constructs after Day 4, with the exception of empty vector cells, made a significant amount of IFNy and IL- 2 for the duration of the chronic stimulation assay with the vehicle-treated BCMA-IL- 15-008 cells starting to decline more quickly than the other BCMA CARTs.
  • MSD Meso Scale Discovery
  • FIG. 12A-FIG. 12D show cytotoxicity, cell counts and cytokine production for BCMA-CARTs with or without mbIL15 in a chronic stimulation assay.
  • “Veh” refers to vehicle (DMSO)-treatment.
  • “EV” refers to empty vector.
  • FIG. 12A shows the cytotoxicity of the effector cells against the target cells, as determined using a luciferase assay to quantitate viable RPMI8226-luc target cells. All groups, with the exception of empty vector cells, show complete cytotoxicity through 7 days. At day 11 the control BCMA-011 cells and vehicle-treated BCMA- IL-15-008 cells began to lose their cytotoxic ability.
  • FIG. 12B shows the cell counts obtained by flow cytometry.
  • FIG. 12C-FIG. 12D show an MSD (Meso Scale Discovery) quantification of IFNy and IL-2 levels in the cell supernatants taken 24 hours after a new co-culture was set up.
  • control BCMA-011 cells and vehicle-treated BCMA- IL-15-008 cells showed lower levels of IFNy and IL-2 than the constitutive mbIL15 expressing BCMA-IL15-007 cells or regulated mbIL15 expressing BCMA-IL15-008 cells treated with ACZ.
  • Cancer immunotherapy aims to induce or restore the reactivity of the immune system towards cancer.
  • Significant advances in immunotherapy research have led to the development of various strategies which may broadly be classified into active immunotherapy and passive immunotherapy. In general, these strategies may be utilized to directly kill cancer cells or to counter the immunosuppressive tumor microenvironment.
  • Active immunotherapy aims at induction of an endogenous, long-lasting tumor-antigen specific immune response. The response can further be enhanced by non-specific stimulation of immune response modifiers such as cytokines.
  • passive immunotherapy includes approaches where immune effector molecules such as tumor-antigen specific cytotoxic T cells or antibodies are administered to the host. This approach is short lived and requires multiple applications.
  • the present disclosure provides systems, compositions, immunotherapeutic agents and methods that avoid the issues of continuously dosed or expressed IL15 by providing tunable regulation of IL15 gene expression and function for cancer immunotherapy, specifically for CAR T therapy.
  • the present invention also provides DRDs operably linked to IL 15 payloads, as well as polynucleotides encoding any of the foregoing.
  • the systems, compositions, immunotherapeutic agents and other components of the invention can be controlled by a separately added stimulus, which provides a significant flexibility to regulate cancer immunotherapy.
  • compositions of the invention has the potential to improve the potency and duration of the efficacy of immunotherapies.
  • the ability to reversibly increase, decrease or silence the biological activity of adoptively transferred cells using compositions of the present invention allows maximizing the potential of cell therapy, which is not available using a “kill switch” that will terminate the therapy.
  • the present invention provides methods for fine tuning of immunotherapy after administration to patients. This in turn improves the safety and efficacy of immunotherapy and increases the subject population that may benefit from immunotherapy.
  • the CA2 DRD-regulated IL15 disclosed herein may provide a desired signaling enabling adoptively transferred anti- BCMA CAR T cells to improve cell expansion, increase durability of response by enhancing memory phenotype, prolong persistence, and overcome T cell exhaustion, thereby providing durable immune surveillance and therapeutic potential.
  • the present disclosure provides a polynucleotide or nuleic acid molecule encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen recognition moiety and a T-cell activation moiety.
  • the nucleic acid molecule is isolated or purified.
  • the nucleic acid molecule is non-naturally occurring.
  • a chimeric antigen receptor (CAR) is an artificially constructed hybrid protein or polypeptide containing an antigen binding domain of an antibody (e.g., a single chain variable fragment (scFv)) linked to T-cell signaling or T-cell activation domains.
  • CARs have the ability to redirect T-cell specificity and reactivity toward a selected target in a non-MHC-restricted manner, exploiting the antigen-binding properties of monoclonal antibodies.
  • the non-MHC-restricted antigen recognition gives T-cells expressing CARs the ability to recognize an antigen independent of antigen processing, thus bypassing a major mechanism of tumor escape.
  • CARs when expressed in T-cells, CARs advantageously do not dimerize with endogenous T-cell receptor (TCR) alpha and beta chains.
  • TCR T-cell receptor
  • the nucleic acid sequence encodes a CAR which comprises an antigen recognition moiety that is directed against B-cell Maturation Antigen (BCMA, also known as CD269).
  • BCMA is a member of the tumor necrosis factor receptor superfamily (see, e.g., Thompson et al., J. Exp. Medicine, 192(1): 129-135 (2000), and Mackay et al., Annu. Rev. Immunol., 21 : 231-264 (2003)).
  • BCMA binds B-cell activating factor (BAFF) and a proliferation inducing ligand (APRIL) (see, e.g., Mackay et al., supra, and Kalled et al., Immunological Reviews, 204: 43-54 (2005)).
  • BAFF B-cell activating factor
  • APRIL proliferation inducing ligand
  • BCMA has been reported to be expressed mostly in plasma cells and subsets of mature B-cells (see, e.g., Laabi et al., EMBO J., 11(11): 3897-3904 (1992); Laabi et al., Nucleic Acids Res., 22(7): 1147-1154 (1994); Kalled et al., supra; O'Connor et al., J. Exp. Medicine, 199(1): 91-97 (2004); and Ng et al., J.
  • mice deficient in BCMA are healthy and have normal numbers of B-cells, but the survival of long-lived plasma cells is impaired (see, e.g., O'Connor et al, supra; Xu et al., Mol. Cell. Biol., 21(12): 4067-4074 (2001); and Schiemann et al., Science, 293(5537): 2111-2114 (2001)).
  • BCMA RNA has been detected universally in multiple myeloma cells, and BCMA protein has been detected on the surface of plasma cells from multiple myeloma patients by several investigators (see, e.g., Novak et al., Blood, 103(2): 689-694 (2004); Neri et al., Clinical Cancer Research, 13(19): 5903-5909 (2007); Bellucci et al, Blood, 105(10): 3945-3950 (2005); and Moreaux et al., Blood, 103(8): 3148-3157 (2004)).
  • the nucleic acid sequence encodes a CAR which comprises a monoclonal antibody or antigen recognition moiety directed against BCMA, or an antigenbinding portion thereof.
  • a CAR which comprises a monoclonal antibody or antigen recognition moiety directed against BCMA, or an antigenbinding portion thereof.
  • monoclonal antibodies refers to antibodies that are produced by a single clone of B-cells and bind to the same epitope.
  • polyclonal antibodies refer to a population of antibodies that are produced by different B-cells and bind to different epitopes of the same antigen.
  • the term “antigen recognition moiety” of the CAR encoded by the inventive nucleic acid sequence means a polypeptide domain comprising an antibody fragment, an antigen-binding portion of an antibody or a synthetic antibody comprised of portions of anti-BCMA antibody, that retains the ability to specifically bind to BCMA.
  • a whole antibody typically consists of four polypeptides: two identical copies of a heavy (H) chain polypeptide and two identical copies of a light (L) chain polypeptide.
  • Each of the heavy chains contains one N-terminal variable (VH) region and three C-terminal constant (CHI, CH2 and CH3) regions, and each light chain contains one N-terminal variable (VL) region and one C-terminal constant (CL) region.
  • the variable regions of each pair of light and heavy chains form the antigen binding site of an antibody.
  • the VH and VL regions have the same general structure, with each region comprising four framework regions, whose sequences are relatively conserved.
  • the framework regions are connected by three complementarity determining regions (CDRs).
  • the three CDRs known as CDR1, CDR2, and CDR3, form the “hypervariable region” of an antibody, which is responsible for antigen binding.
  • the antigen recognition moiety of the CAR encoded by the nucleic acid sequence can contain any BCMA-binding antibody fragment.
  • the antibody fragment may comprise, for example, one or more CDRs, the variable region (or portions thereof), the constant region (or portions thereof), or combinations thereof.
  • antibody fragments include, but are not limited to, (i) a Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL, and CHI domains; (ii) a F(ab')2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (iv) a single chain Fv (scFv), which is a monovalent molecule consisting of the two domains of the Fv fragment (i.e., VL and VH) joined by a synthetic linker which enables the two domains to be synthesized as a single polypeptide chain and (v) a diabody, which is a dimer of polypeptide chains, wherein each polypeptide chain comprises a VH connected to a VL by a peptide linker that is too short to allow pairing between the
  • the antigen recognition moiety of the CAR encoded by the nucleic acid sequence comprises an anti-BCMA single chain Fv (scFv).
  • the antigen recognition moiety of the CAR encoded by the nucleic acid sequence may be a Camel Ig (a camelid antibody (VHH)), an Ig NAR, an F(ab)'3, a bis-scFv, an (scFv)2, a minibody, a triabody, a tetrabody, a disulfide stabilized Fv protein (“dsFv”), and a single-domain antibody (sdAb, Nanobody).
  • a "camel Ig” or “camelid VHH” refers to the smallest known antigenbinding unit of a heavy chain antibody (Koch-No Ite, et al, FASEB J., 21 : 3490- 3498 (2007)).
  • a "heavy chain antibody” or a “camelid antibody” refers to an antibody that contains two VH domains and no light chains (Riechmann L. et al, J. Immunol. Methods 231 :25-38 (1999); WO94/04678; W094/25591; U.S. Patent No. 6,005,079).
  • an “IgNAR” or “immunoglobulin new antigen receptor” refers to class of antibodies from the shark immune repertoire that consist of homodimers of one variable new antigen receptor (VNAR) domain and five constant new antigen receptor (CNAR) domains.
  • IgNARs represent some of the smallest known immunoglobulin-based protein scaffolds and are highly stable and possess efficient binding characteristics.
  • the inherent stability can be attributed to both (i) the underlying Ig scaffold, which presents a considerable number of charged and hydrophilic surface exposed residues compared to the conventional antibody VH and VL domains found in murine antibodies; and (ii) stabilizing structural features in the complementary determining region (CDR) loops including inter-loop disulphide bridges, and patterns of intraloop hydrogen bonds.
  • CDR complementary determining region
  • a “triabody” and a “tetrabody” are as described in Hudson et al, Nat. Med. 9: 129-134 (2003).
  • a “single domain antibody” or “sdAb” or “nanobody” refers to an antibody fragment that consists of the variable region of an antibody heavy chain (VH domain) or the variable region of an antibody light chain (VL domain) (Holt, L., et al, Trends in Biotechnology, 21(11): 484-490).
  • the anti-BCMA CAR comprises an antigen recognition moiety that binds to one or more epitopes of a human BCMA polypeptide and is an scFv.
  • the scFv may be a murine, human or humanized scFv.
  • VH variable region heavy chain
  • VL variable region light chain
  • the antigen recognition moiety of the CAR encoded by the nucleic acid sequence is a humanized antibody (such as a humanized monoclonal antibody) or fragment thereof that specifically binds to a human BCMA polypeptide.
  • a "humanized” antibody is an immunoglobulin including a human framework region and one or more CDRs from a non-human (for example a mouse, rat, or synthetic) immunoglobulin.
  • the non-human immunoglobulin providing the CDRs is termed a "donor,” and the human immunoglobulin providing the framework is termed an "acceptor.”
  • all the CDRs are from the donor immunoglobulin in a humanized immunoglobulin.
  • Constant regions need not be present, but if they are, they must be substantially identical to human immunoglobulin constant regions, i.e., at least about 85-90%, such as about 95% or more identical. Hence, all parts of a humanized immunoglobulin, except possibly the CDRs, are substantially identical to corresponding parts of natural human immunoglobulin sequences.
  • Humanized or other monoclonal antibodies can have additional conservative amino acid substitutions, which have substantially no effect on antigen binding or other immunoglobulin functions. Humanized antibodies can be constructed by means of genetic engineering (see for example, U.S. Patent No. 5,585,089).
  • An antigen recognition moiety that is an antigen-binding portion or fragment of a monoclonal antibody can be of any size so long as the portion binds to BCMA.
  • an antigen binding portion or fragment of the monoclonal antibody directed against BCMA may comprise between about 5 and 18 amino acids (e.g., about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or a range defined by any two of the foregoing values).
  • the nucleic acid sequence encodes an antigen recognition moiety that comprises a variable region of an anti-BCMA monoclonal antibody.
  • the antigen recognition moiety comprises a light chain variable region, a heavy chain variable region, or both a light chain variable region and a heavy chain variable region of an anti-BCMA monoclonal antibody.
  • the antigen recognition moiety of the CAR encoded by the inventive nucleic acid sequence comprises a light chain variable region and a heavy chain variable region of an anti-BCMA monoclonal antibody.
  • Heavy and light chain monoclonal antibody amino acid sequences that bind to BCMA are disclosed in, e.g., International Patent Application Publication WO 2010/104949.
  • the nucleic acid molecule of the present disclosure encode a first and a second protein, wherein: (i) the first protein is a chimeric antigen receptor (CAR) that specifically binds to BCMA (B-cell maturation antigen), also referred to as the CD269.
  • CAR chimeric antigen receptor
  • an exemplary BCMA-antigen binding domain of the present invention may be antibodies, fragments and variants thereof which are specific to a BCMA antigen, for example, a human BCMA protein antigen.
  • an illustrative BCMA protein is provided as BCMA (UNIPROT ID: Q02223) encoded by the gene, TNFRS17. BCMA is a member of the TNF receptor super family.
  • BCMA B cell activating factor
  • APRIL proliferation inducing ligand
  • the present disclosure provides a nucleic acid molecule that encodes a first protein BCMA CAR molecule comprising an antibody or antibody fragment engineered for enhanced binding to a BCMA protein.
  • the chimeric antigen receptor (CAR) comprises: (a) an antibody or antigen recognition moiety that binds to one or more epitopes of a human B-cell Maturation Antigen (BCMA) polypeptide, (b) a transmembrane domain, which may optionally further comprise a hinge domain, and (c) one or more intracellular T cell signaling domains.
  • BCMA human B-cell Maturation Antigen
  • the antibody or antigen recognition moiety that binds to one or more epitopes of a human B-cell Maturation Antigen (BCMA) polypeptide is a scFv antibody fragment.
  • such antibody fragments are functional in that they retain the equivalent binding affinity, e.g., they bind the same antigen with comparable efficacy, as the IgG antibody from which it is derived.
  • the CAR antibody or antigen recognition moiety has a lower binding affinity, e.g., it binds the same antigen with a lower binding affinity than the antibody from which it is derived, but is functional in that it provides a biological response described herein.
  • the CAR BCMA antigen binding domain comprises an antibody fragment that has a binding affinity KD of 10' 4 M to 10' 9 M, e.g., 10' 5 M to 10' 8 M, e.g., 10' 6 M or 10' 7 M, or 1 O' 8 , for the target BCMA antigen.
  • the BCMA CAR antibody or antigen recognition moiety are functional in that they provide a biological response that can include, but is not limited to, activation of an immune response, inhibition of signal-transduction origination from its target antigen, inhibition of kinase activity, and the like, as will be understood by a skilled artisan.
  • the CAR antibody or antigen recognition moiety is a scFv antibody fragment that is humanized compared to the murine sequence of the scFv from which it is derived.
  • the antibody or antigen recognition moiety is a human anti-BCMA antigen binding domain.
  • the antibody or antigen recognition moiety is a humanized antibody or antigen recognition moiety.
  • the BCMA CAR comprises an anti-BCMA binding domain (e.g., human or humanized antibody or antigen recognition moiety, for example, a scFv directed against BCMA), a transmembrane domain, and an intracellular signaling domain.
  • the anti-BCMA binding domain e.g. an scFv
  • illustrative scFv domains that bind to a human BCMA polypeptide are provided in Table 1.
  • a listing of BCMA CARs are provided in Table 3.
  • an anti-BCMA scFv is provided in SEQ ID NO: 14.
  • an anti-BCMA CAR is provided in SEQ ID NO:34.
  • the human or humanized antibody or antigen recognition moiety comprises: an amino acid sequence having at least one, two or three modifications (e.g., substitutions, deletions, or additions, e.g., conservative substitutions) but not more than 30, 20 or 10 modifications (e.g., substitutions, e.g., conservative substitutions) of an amino acid sequence provided in Table 1, or an amino acid sequence with at least 85% (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%) identity thereof.
  • modifications e.g., substitutions, deletions, or additions, e.g., conservative substitutions
  • substitutions e.g., conservative substitutions
  • Exemplary antibodies or antigen recognition moiety targeting BCMA include, but are not limited to: BCMA 50, BCMA30, C11D5.3 and C13F12.1.
  • the antibody is derived from Cl 1D5.3.
  • antibodies with high affinity may be derived from any of the BCMA antibody heavy and light chain variables described in Table 2 (or a sequence at least about 85%, 90%, 95%, 99% or more identical thereto, and/or having one, two, three or more substitutions, insertions or deletions, e.g., conserved substitutions).
  • the encoded CAR molecule comprises a full CAR amino acid sequence listed in Table 3 (or a sequence at least about 85%, 90%, 95%, 99% or more identical thereto, and/or having one, two, three or more substitutions, insertions or deletions, e.g., conserved substitutions).
  • BCMA CAR sequences of the present disclosure may be, but are not limited to those listed in Table 3.
  • BCMA CAR sequences include but are not limited to: BCMA (CD269) specific CARs disclosed in International Patent Publication Nos. WO2016/014565 and WO2016/014789.
  • the nucleic acid sequence encodes a CAR which comprises a signal sequence.
  • the signal sequence may be positioned at the amino terminus of the antigen recognition moiety (e.g., the variable region of the anti-BCMA antibody).
  • the signal sequence may comprise any suitable signal sequence.
  • the signal sequence is a human granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor sequence, IgGl heavy chain, or CD8a signal sequence.
  • GM-CSF human granulocyte-macrophage colony-stimulating factor
  • the CAR comprises a hinge sequence.
  • a hinge sequence is a short sequence of amino acids that facilitates antibody flexibility (see, e.g., Woof et al., Nat. Rev. Immunol., 4(2): 89-99 (2004)).
  • the hinge sequence may be positioned between the antigen recognition moiety (e.g., an anti-BCMA scFv) and the T-cell activation moiety.
  • the hinge sequence can be any suitable sequence derived or obtained from any suitable molecule.
  • the hinge sequence can be a sequence derived or obtained from CD28, 4- IBB, CD8 alpha, CD4, CD 19, CD3 epsilon, CD45, CD5, CD9, CD16, CD22, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD154, PD1, CTLA-4, an alpha chain of a T cell receptor, a beta chain of a T cell receptor, or a zeta chain of a T cell receptor.
  • the hinge sequence is derived from the human CD8a molecule or a CD28 molecule.
  • the nucleic acid sequence encodes an anti-BCMA CAR comprising a transmembrane domain.
  • the transmembrane domain can be any suitable transmembrane domain derived or obtained from any suitable molecule.
  • the transmembrane domain can be any transmembrane domain derived or obtained from any molecule known in the art.
  • the transmembrane domain can be obtained or derived from CD28, 4-1BB, CD8 alpha, CD4, CD19, CD3 epsilon, CD45, CD5, CD9, CD16, CD22, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD154, PD1, CTLA-4, an alpha chain of a T cell receptor, a beta chain of a T cell receptor, a zeta chain of a T cell receptor.
  • the transmembrane domain can be obtained or derived from CD28, CD8 alpha, CD4, CD45, PD1 or CTLA-4.
  • the transmembrane domain can be obtained or derived from a CD8a molecule or a CD28 molecule.
  • CD8 is a transmembrane glycoprotein that serves as a coreceptor for the T-cell receptor (TCR), and is expressed primarily on the surface of cytotoxic T- cells. The most common form of CD8 exists as a dimer composed of a CD8a and CD8[3 chain.
  • CD28 is expressed on T-cells and provides co-stimulatory signals required for T-cell activation.
  • CD28 is the receptor for CD80 (B7.1) and CD86 (B7.2).
  • the CD8a and CD28 are human.
  • the nucleic acid sequence encodes an anti-BCMA CAR comprising one or more intracellular T cell signaling domains.
  • each intracellular T cell signaling domain is independently selected from a suitable molecule.
  • the intracellular T cell signaling domains can be obtained or derived from, and are independently selected from, an intracellular signaling domain of 4-1BB, CARD 11, CD2, CD3( ⁇ , CD7, CD27, CD28, CD30, CD40, CD54, CD83, CD134, CD150, CTLA-4, CD223, CD270, CD273, CD274, CD278, DAP10, FcRy, LAT, NKD2C, SLP76, TRIM or ZAP70.
  • the intracellular T cell signaling domains can be obtained or derived from, and are independently selected from, an intracellular signaling domain of a CD28 molecule, a CD3 zeta (Q molecule or modified versions thereof, a human Fc receptor gamma (FcRy) chain, a CD27 molecule, an 0X40 molecule, a 4-1BB molecule, or other intracellular signaling molecules known in the art.
  • CD28 is a T-cell marker important in T-cell costimulation.
  • CD3 ⁇ associates with TCRs to produce a signal and contains immunoreceptor tyrosine-based activation motifs (IT AMs).
  • 4-1BB also known as CD137
  • CD137 transmits a potent costimulatory signal to T-cells, promoting differentiation and enhancing long-term survival of T lymphocytes.
  • the CD28, CD3 zeta, 4- IBB, 0X40, and CD27 are human.
  • the transmembrane domain and intracellular T cell signaling domains of the anti-BCMA CAR encoded by the nucleic acid sequence can comprise any one of aforementioned transmembrane domains and any one or more of the aforementioned intercellular T-cell signaling domains in any combination.
  • the nucleic acid sequence encodes a CAR which comprises, from 5' to 3 ', a CD8a leader sequence; an scFv directed against BCMA; a CD8a hinge domain or a CD28 hinge domain; a CD8a transmembrane domain or a CD28 hinge domain; an intracellular T cell signaling domain selected from a 4- IBB intracellular T cell signaling domain, an 0X40 intracellular T cell signaling domain, or a CD28 intracellular T cell signaling domain; and a CD3 ⁇ intracellular T cell signaling domain.
  • the nucleic acid sequence encodes a CAR which comprises, from 5' to 3 ', a CD8a leader sequence; an scFv directed against BCMA; a CD8a hinge domain or a CD28 hinge domain; a CD8a transmembrane domain or a CD28 hinge domain; an intracellular T cell signaling domain selected from a 4- IBB intracellular T cell signaling domain, an 0X40 intracellular T cell signaling domain, or a CD28 intracellular T cell signaling domain; and a CD3 ⁇ intracellular T cell signaling domain.
  • the nucleic acid sequence encodes a CAR which comprises, from 5' to 3', a CD8a leader sequence; an scFv directed against BCMA; a CD8a hinge domain; a CD8a transmembrane domain; a 4-1BB intracellular T cell signaling domain; and a CD3( ⁇ intracellular T cell signaling domain.
  • a CAR which comprises, from 5' to 3', a CD8a leader sequence; an scFv directed against BCMA; a CD8a hinge domain; a CD8a transmembrane domain; a 4-1BB intracellular T cell signaling domain; and a CD3( ⁇ intracellular T cell signaling domain.
  • the nucleic acid sequence encodes a CAR of any length, i.e., the CAR can comprise any number of amino acids, provided that the CAR retains its biological activity, e.g., the ability to specifically bind to antigen, detect diseased cells in a mammal, or treat or prevent disease in a mammal, etc.
  • the CAR can comprise 50 or more (e.g., 60 or more, 100 or more, or 500 or more) amino acids, but less than 1,000 (e.g., 900 or less, 800 or less, 700 or less, or 600 or less) amino acids.
  • the CAR is about 50 to about 700 amino acids (e.g., about 70, about 80, about 90, about 150, about 200, about 300, about 400, about 550, or about 650 amino acids), about 100 to about 500 amino acids (e.g., about 125, about 175, about 225, about 250, about 275, about 325, about 350, about 375, about 425, about 450, or about 475 amino acids), or a range defined by any two of the foregoing values.
  • amino acids e.g., about 70, about 80, about 90, about 150, about 200, about 300, about 400, about 550, or about 650 amino acids
  • about 100 to about 500 amino acids e.g., about 125, about 175, about 225, about 250, about 275, about 325, about 350, about 375, about 425, about 450, or about 475 amino acids
  • the nucleic acid sequence encodes a functional portion of the anti-BCMA CAR described herein.
  • Functional portions encompass, for example, those parts of a CAR that retain the ability to recognize target cells, or detect, treat, or prevent a disease, to a similar extent, the same extent, or to a higher extent, as the parent CAR.
  • a nucleic acid sequence encoding a functional portion of the CAR can encode a protein comprising, for example, about 10%, 25%, 30%, 50%, 68%, 80%, 90%, 95%, or more, of the parent CAR.
  • the nucleic acid sequence can encode a functional portion of a CAR that contains additional amino acids at the amino or carboxy terminus of the portion, or at both termini, which additional amino acids are not found in the amino acid sequence of the parent CAR.
  • the additional amino acids do not interfere with the biological function of the functional portion, e.g., recognize target cells, detect cancer, treat or prevent cancer, etc.
  • the additional amino acids enhance the biological activity of the CAR, as compared to the biological activity of the parent CAR.
  • the present disclosure also provides nucleic acid sequences encoding functional variants of the aforementioned anti-BCMA CAR.
  • the term “functional variant,” as used herein, refers to a CAR, a polypeptide, or a protein having substantial or significant sequence identity or similarity to the CAR encoded by the inventive nucleic acid sequence, which functional variant retains the biological activity of the CAR of which it is a variant.
  • Functional variants encompass, for example, those variants of the CAR described herein (the parent CAR) that retain the ability to recognize target cells to a similar extent, the same extent, or to a higher extent, as the parent CAR.
  • a nucleic acid sequence encoding a functional variant of the CAR can be for example, about 10% identical, about 25% identical, about 30% identical, about 50% identical, about 65% identical, about 80% identical, about 90% identical, about 95% identical, or about 99% identical to the nucleic acid sequence encoding the parent CAR.
  • a functional variant may comprise the amino acid sequence of the CAR encoded by the inventive nucleic acid sequence with at least one conservative amino acid substitution.
  • conservative amino acid substitution or “conservative mutation” refers to the replacement of one amino acid by another amino acid with a common property.
  • a functional way to define common properties between individual amino acids is to analyze the normalized frequencies of amino acid changes between corresponding proteins of homologous organisms (Schulz, G. E. and Schirmer, R. H., Principles of Protein Structure, Springer-Verlag, New York (1979)).
  • groups of amino acids may be defined where amino acids within a group exchange preferentially with each other, and therefore resemble each other most in their impact on the overall protein structure (Schulz, G. E. and Schirmer, R. H., supra).
  • conservative mutations include amino acid substitutions of amino acids within the sub-groups above, for example, lysine for arginine and vice versa such that a positive charge may be maintained; glutamic acid for aspartic acid and vice versa such that a negative charge may be maintained; serine for threonine such that a free — OH can be maintained; and glutamine for asparagine such that a free — NH2 can be maintained.
  • the functional variants may comprise the amino acid sequence of the parent CAR with at least one non-conservative amino acid substitution.
  • “Non-conservative mutations” involve amino acid substitutions between different groups, for example, lysine for tryptophan, or phenylalanine for serine, etc. In this case, it is preferable for the non-conservative amino acid substitution to not interfere with, or inhibit the biological activity of, the functional variant.
  • the non-conservative amino acid substitution may enhance the biological activity of the functional variant, such that the biological activity of the functional variant is increased as compared to the parent CAR.
  • the nucleic acid sequence can encode a CAR (including functional portions and functional variants thereof) that comprises synthetic amino acids in place of one or more naturally-occurring amino acids.
  • synthetic amino acids include, for example, aminocyclohexane carboxylic acid, norleucine, a-amino n-decanoic acid, homoserine, S-acetylaminomethyl-cysteine, trans-3- and trans-4-hydroxyproline, 4- aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, p- phenylserine P-hydroxyphenylalanine, phenylglycine, a-naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline-2-carboxylic acid, l,2,3,4-tetrahydroisoquinoline-3
  • the nucleic acid sequence can encode a CAR (including functional portions and functional variants thereof) which is glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated, cyclized via, e.g., a disulfide bridge, or converted into an acid addition salt and/or optionally dimerized or polymerized, or conjugated.
  • a CAR including functional portions and functional variants thereof
  • the present disclosure provides a nucleic acid molecule encoding a DRD operably linked to an IL15 payload.
  • the nucleic acid molecule further encodes an anti-BCMA CAR.
  • DRDs Drug responsive domains
  • a DRD is operably linked to a target protein of interest.
  • DRDs render the attached protein of interest unstable in the absence of a DRD-binding ligand.
  • a specific small molecule ligand binds its intended DRD as a ligand binding partner, the instability is reversed, and protein function is restored.
  • the conditional nature of DRD stability allows a rapid and non-perturbing switch from stable protein to unstable substrate for degradation.
  • its dependency on the concentration of its ligand further provides tunable control of degradation rates.
  • the term drug responsive domain (DRD) is interchangeable with the term destabilizing domain (DD).
  • Regions or portions or domains of wild type proteins may be utilized as DRDs in whole or in part.
  • the DRD comprises one, two, three, or four or more mutations compared to the parent CA2 protein, for example, a human CA2 having amino acids 1-260 (SEQ ID NO: 1), which is encoded by the polynucleotide having a nucleic acid sequence of SEQ ID NO: 3, or human CA2 having amino acids 2-260 (SEQ ID NO:2).
  • the phrase “derived from” as it relates to DRDs means that the DRD originates at least in part from the stated parent molecule or sequence.
  • DRD may be derived from an epitope or region of a naturally occurring protein but then have been modified in any of the ways taught herein to optimize the DRD function.
  • a transmembrane domain derived from a specified protein means that the transmembrane domain comprises all or substantially all of the amino acid sequence of the transmembrane domain of the specified protein.
  • the DRDs of the present disclosure may be derived from CA2 (SEQ ID NO: 1; Uniprot ID: P00918) which may be stabilized by ligands such as small molecule inhibitors of CA2.
  • CA2 WT refers to the human wildtype CA2 protein sequence, which is defined as SEQ ID NO: I.
  • DRDs may be derived from CA2 having amino acids 2-260 of the parent CA2 sequence (SEQ ID NO:2). This is referred to herein as an Mldel mutation.
  • the Mldel mutation may also be referred to herein as an amino acid deletion.
  • human DRD constructs disclosed herein may not comprise an N-terminal methionine corresponding to the N-terminal methionine of SEQ ID NO: 1.
  • the present disclosure identifies positions of the CA2 DRD relative to the wildtype human CA2 (Uniprot ID: P00918) of SEQ ID NO: 1, wherein reference position 1 is the N-terminal methionine of SEQ ID NO: 1.
  • a hypothetical CA2 DRD comprising a G12A mutation refers herein to a CA2 DRD construct wherein glycine (G) is mutated to alanine (A) at a position in the CA2 DRD construct that corresponds to the twelfth amino acid of SEQ ID NO: 1, regardless of whether the CA2 DRD construct itself comprises an N-terminal methionine corresponding to the N-terminal methionine of SEQ ID NO: 1.
  • the glycine (G) to alanine (A) change may also be referred to as an amino acid substitution.
  • the DRD of the present disclosure has an amino acid sequence as set forth in SEQ ID NO: 4.
  • SEQ ID NO: 4 provides the amino acid sequence of the CA2 DRD having a deletion of the first amino acid (Ml del) and a substitution of leucine at position 156 by histidine (L156H). The positions of the deleted and substituted amino acids are relative to the full length CA2 of SEQ ID NO: 1.
  • an exemplary DRD derived from CA2 that regulates an operably linked IL15 payload comprises or consists of the amino acid sequence of SEQ ID NO: 4 or is encoded by the nucleotide sequence of SEQ ID NO: 5.
  • a “payload” or “target payload” or “payload of interest (POI)” is defined as any protein whose function is to be altered. Payloads may include any protein or fragment thereof.
  • the CA2 DRD is attached, appended or associated to an IL15 payload.
  • the CA2 DRD is operably linked to the payload.
  • the payload comprises a human IL15, comprising the amino acid sequence of SEQ ID NO: 8.
  • the payload may be encoded by a nucleic acid sequence comprising the nucleotide sequence of SEQ ID NO: 9.
  • the payload may comprise the mature form of IL15.
  • a payload of the present disclosure may comprise amino acid sequences similar to the amino acid sequence of human IL15, for example, UniProtKB - P40933 (IL15 HUMAN).
  • a payload of the present disclosure may comprise an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 8.
  • the payload is a polypeptide comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 8.
  • the payload is a polypeptide comprising an amino acid sequence having at least 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:8.
  • payloads of the present disclosure may be utilized to improve expansion, survival, persistence, and potency of CAR T cells used for immunotherapy.
  • the payloads of the present disclosure may be utilized to improve expansion, survival, persistence, and potency of anti-BCMA CAR T cells used for treating a B cell malignancy in a subject in need thereof
  • the payloads of the present disclosure may be utilized to improve expansion, survival, persistence, and potency of anti- BCMA CAR T cells used for treating multiple myeloma in a subject in need thereof
  • the term “nucleic acid,” or “nucleic acid molecule” includes any compound and/or substance that comprise a polymer of nucleotides, e.g., linked nucleosides.
  • nucleic acids or polynucleotides of the disclosure include, but are not limited to, ribonucleic acids (RNAs) and deoxyribonucleic acids (DNAs).
  • RNAs ribonucleic acids
  • DNAs deoxyribonucleic acids
  • the nucleic acid molecule is DNA.
  • the nucleic acid molecule is a messenger RNA (mRNA). Nucleic acids may be single stranded or double stranded.
  • mRNA messenger RNA
  • Polynucleotides of the disclosure may be mRNA or any nucleic acid molecule and may or may not be chemically modified.
  • polynucleotides of the present disclosure may harbor 5' UTR sequences which play a role in translation initiation.
  • 5' UTR sequences may include features such as Kozak sequences which are commonly known to be involved in the process by which the ribosome initiates translation of genes, Kozak sequences have the consensus XCCR(A/G) CCAUG, where R is a purine (adenine or guanine) three bases upstream of the start codon (AUG) and X is any nucleotide.
  • the Kozak sequence is ACCGCC.
  • polynucleotides of the present disclosure may encode variant polypeptides which have a certain identity with a reference polypeptide sequence.
  • a “reference polypeptide sequence” refers to a starting polypeptide sequence. Reference sequences may be wild type sequences or any sequence to which reference is made in the design of another sequence.
  • identity refers to a relationship between two or more sequences, as determined by comparing the sequences. In the art, identity also means the degree of sequence relatedness between sequences, as determined by the number of matches between strings of two or more residues (amino acid or nucleic acid). Identity measures the percent of identical matches between two or more sequences with gap alignments (if any) addressed by a particular mathematical model or computer program (i.e., “algorithms”). Identity of related sequences can be readily calculated by known methods. Such methods include, but are not limited to, those described in Computational Molecular Biology, Lesk, A.
  • the variant sequence may have the same or a similar activity as the reference sequence.
  • the variant may have an altered activity (e.g., increased or decreased) relative to a reference sequence.
  • variants of a particular polynucleotide or polypeptide of the disclosure will have at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% but less than 100% sequence identity to that particular reference polynucleotide or polypeptide as determined by sequence alignment programs and parameters described herein and known to those skilled in the art.
  • Such tools for alignment include those of the BLAST suite (Stephen F. Altschul, Thomas L. Madrden, Alejandro A.
  • the proteins of the present disclosure may further comprise a signal sequence which regulates the distribution of the payload of interest, a cleavage and/or processing feature which facilitate cleavage of the payload from the DRD, a targeting and/or penetrating signal which can regulate the cellular localization of the regulated protein, a tag, and/or one or more linker sequences which link different components of the regulated protein.
  • regulated proteins of the disclosure may further comprise one or more additional features such as one or more signal sequences.
  • Signal sequences (sometimes referred to as signal peptides, targeting signals, target peptides, localization sequences, transit peptides, leader sequences or leader peptides) direct proteins (e.g., a protein of the present disclosure) to their designated cellular and/or extracellular locations. Protein signal sequences play a central role in the targeting and translocation of nearly all secreted proteins and many integral membrane proteins.
  • a signal sequence is a short (5-30 amino acids long) peptide present at the N-terminus of the majority of newly synthesized proteins that are destined towards a particular location.
  • Signal sequences can be recognized by signal recognition particles (SRPs) and cleaved using type I and type II signal peptide peptidases.
  • SRPs signal recognition particles
  • Signal sequences derived from human proteins can be incorporated as a regulatory module of the protein to direct the protein to a particular cellular and/or extracellular location.
  • a signal sequence may be, although not necessarily, located at the N-terminus or C-terminus of the payload, and may be, although not necessarily, cleaved off to yield a “mature” payload.
  • the signal sequence used herein may exclude the methionine at the position 1 of amino acid sequence of the signal sequence. This may be referred to as an Ml del mutation.
  • a signal sequence may be a variant modified from a known signal sequence of a protein.
  • signal sequences directing the payload of interest to the surface membrane of the target cell may be used.
  • Expression of the payload on the surface of the target cell may be useful to limit the diffusion of the payload to non-target in vivo environments, thereby potentially improving the safety profile of the payloads.
  • the membrane presentation of the payload may allow for physiologically and qualitative signaling as well as stabilization and recycling of the payload for a longer half-life.
  • Membrane sequences may be the endogenous signal sequence of the N terminal component of the payload of interest. Optionally, it may be desirable to exchange this sequence for a different signal sequence.
  • Signal sequences may be selected based on their compatibility with the secretory pathway of the cell type of interest so that the payload is presented on the surface of the T cell.
  • the signal sequence may be IgE signal sequence, CD8a signal sequence (also referred to as CD8a leader), or IL15Ra signal sequence (also referred to as IL15Ra leader) or Mldel CD8a signal sequence (also referred to as Mldel CD8 leader sequence).
  • the regulated protein comprises a cleavage and/or processing feature.
  • the regulated protein of the present disclosure may include at least one protein cleavage signal/site.
  • the protein cleavage signal/site may be located at the N- terminus, the C-terminus, at any space between the N-and the C-termini such as, but not limited to, half-way between the N-and C-termini, between the N-terminus and the half-way point, between the half-way point and the C-terminus, and combinations thereof.
  • the regulated protein comprises a linker.
  • the regulated protein of the disclosure may further comprise a linker sequence.
  • the linker region serves primarily as a spacer between two or more peptides or polypeptides within a protein.
  • the "linker” or “spacer”, as used herein, refers to a molecule or group of molecules that connects two molecules, or two parts of a molecule such as two domains of a recombinant protein.
  • Linker refers to an oligo-or polypeptide region of from about 1 to 100 amino acids in length, which links together any of the domains/regions of the polypeptide or protein (also called peptide linker).
  • an artificially designed peptide linker may be composed of a polymer of flexible residues such as Glycine (G) and Serine (S) so that the adjacent protein domains are free to move relative to one another.
  • G Glycine
  • S Serine
  • Longer linkers may be used when it is desirable to ensure that two adjacent domains do not interfere with one another.
  • the choice of a particular linker sequence may be determined based on biological activity, stability, folding, targeting and/or pharmacokinetic features of the fusion protein (e.g., a regulated protein described herein).
  • a linker sequence may be a natural linker derived from a multi-domain protein.
  • a natural linker is a short peptide sequence that separates two different domains or motifs within a protein.
  • the linker may be a BamHI site.
  • the BamHI site has the amino acid sequence GS and/or the DNA sequence GGATCC.
  • Regulated proteins of the present disclosure are triggered by one or more stimuli.
  • the stimulus is a small molecule.
  • the small molecules are cell permeable.
  • the small molecules are FDA-approved, safe and orally administered.
  • the ligands bind to carbonic anhydrases. In some embodiments, the ligand binds to and inhibits carbonic anhydrase function and is herein referred to as carbonic anhydrase inhibitor.
  • the ligand is a small molecule that binds to carbonic anhydrase 2.
  • the small molecule is CA2 inhibitor.
  • the ligand is a small molecule selected from acetazolamide, celecoxib, valdecoxib, rofecoxib, methazolamide, dorzolamide, brinzolamide, diclofenamide, ethoxzolamide, zonisamide, dansylamide, and dichlorphenamide.
  • the ligand is a small molecule selected from acetazolamide, brinzolamide, dorzolamide hydrochloride, dichlorphenamide, chlorthalidone, methazolamide, topiramate, indapamide, ambroxol hydrochloride, glimepiride, tetracaine hydrochloride and celecoxib.
  • the ligand is a small molecule selected from acetazolamide, brinzolamide, dorzolamide hydrochloride, dichlorphenamide, chlorthalidone, methazolamide or topiramate.
  • the ligand is a CA2 inhibitor selected from acetazolamide, brinzolamide, dorzolamide hydrochloride, dichlorphenamide or methazolamide. In some embodiments, the ligand is acetazolamide (ACZ). [00146] In some embodiments, ligands that do not affect the activity of the immune cell, and/or the chimeric antigen receptor, in the absence of the DRD may be selected.
  • nucleic acid molecules of the disclosure comprise a promoter.
  • the constructs of the disclosure may be placed under the transcriptional control of the human cytomegalovirus (CMV) promoter, an Elongation Factor la (EFla) promoter, HIV LTR promoter, 3 -phosphoglycerate kinase (PGK) promoter, Rous sarcoma virus long terminal repeat (RSV) promoter, spleen focus forming virus (SFFV) promoter, synthetic MND promoter, murine stem cell virus (MSCV) promoter, synthetic RPBSA promoter or a ubiquitin promoter.
  • CMV human cytomegalovirus
  • EFla Elongation Factor la
  • HIV LTR HIV LTR promoter
  • PGK 3 -phosphoglycerate kinase
  • RSV Rous sarcoma virus long terminal repeat
  • SFFV spleen focus forming virus
  • synthetic MND promoter murine stem cell virus (MSCV) promoter
  • a promoter is defined as a DNA sequence recognized by transcription machinery of the cell, required to initiate specific transcription of the polynucleotide sequence of the present disclosure.
  • Vectors can comprise native or non-native promoters operably linked to the polynucleotides of the disclosure.
  • the promoters selected may be strong, weak, constitutive, inducible, tissue specific, development stage- specific, and/or organism specific.
  • a strong constitutive promoter sequence is capable of driving high levels of expression of polynucleotide sequence that is operably linked to it. Examples of strong constitutive promoters include, without limitation, immediate early cytomegalovirus (CMV) promoter and Elongation Growth Factor-1 Alpha (EF-1 alpha).
  • CMV immediate early cytomegalovirus
  • EF-1 alpha Elongation Growth Factor-1 Alpha
  • simian virus 40 SV40
  • mouse mammary tumor virus MMTV
  • human immunodeficiency virus HIV
  • LTR long terminal repeat
  • SFFV avian leukemia virus promoter
  • MSCV murine stem cell virus
  • EGF Epstein-Barr virus immediate early promoter
  • Rous sarcoma virus promoter human gene promoters including, but not limited to, the phosphoglycerate kinase (PGK) promoter, an actin promoter, a myosin promoter, the hemoglobin promoter, the Ubiquitin C (Ubc) promoter, the human U6 small nuclear protein promoter and a creatine kinase promoter.
  • PGK phosphoglycerate kinase
  • Synthetic promoters include an MND promoter and an RPBSA promoter.
  • inducible promoters such as, but not limited to, metallothionine promoter, glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter may be used.
  • the optimal promoter may be selected based on its ability to achieve minimal expression of the DRDs and payloads of the disclosure in the absence of the ligand and detectable expression in the presence of the ligand.
  • Additional promoter elements e.g., enhancers may be used to regulate the frequency of transcriptional initiation. Such regions may be located 10-100 base pairs upstream or downstream of the start site. In some instances, two or more promoter elements may be used to cooperatively or independently activate transcription.
  • Regulated proteins of the present disclosure may comprise additional features including, but not limited to, signal sequences, linker, spacers, tags, flags, cleavage sites, and IRES. Any of the exemplary DRDs, payloads of interest, signal sequences, linker, spacers, hinges, tags, flags, cleavage sites, and IRES taught herein or known in the art may be combined to create the CA2 DRD regulated proteins of the present disclosure.
  • the CA2 DRD comprises an IL15 payload.
  • the payload of interest may be a wild-type sequence, a fragment of a wild-type sequence and/or comprise one or more mutations.
  • an IL15 payload is N-terminal to the DRD.
  • the IL15 payload comprises an amino acid sequence that is at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or at least 100% identical to the amino acid sequence of SEQ ID NO: 8.
  • the payload may be encoded by a nucleic acid sequence comprising the nucleotide sequence of SEQ ID NO: 9.
  • the payload is a membrane-bound form of ELI 5.
  • the payload is a membrane-bound form of ILL comprising a transmembrane domain and an intracellular tail.
  • the payload is a membrane-bound form of ILL comprising an ILL polypeptide component comprising the amino acid sequence of SEQ ID NO: 8, a transmembrane domain and an intracellular tail, wherein the transmembrane domain is C-terminal to the ILL polypeptide component and the intracellular tail is C-terminal to the transmembrane domain.
  • the payload is a membrane-bound form of ILL comprising a transmembrane domain, intracellular tail and one or more linkers.
  • linkers are peptide domains that may be placed between the DRD and the payload, or between different domains within the payload.
  • linkers are peptide domains comprising glycine and serine amino acid residues.
  • peptide linkers comprising glycine and serine amino acid residues may be from 2-36 amino acids in length.
  • at least one payload operably linked to the CA2 DRD is a membrane-bound form of ILL which further includes a linker (GS)15 (SEQ ID NO: 26), a B7.1 Hinge, a B7.1 transmembrane domain, a B7.1 intracellular tail, and a linker (GS).
  • the CA2 DRD-regulated polypeptide may include a payload component of a transmembrane domain and/or cytoplasmic domain from another parent protein as well as the ILL payload component.
  • one or more vectors comprise the nucleic acid molecules encoding an anti-BCMA CAR and a CA2 DRD-ILL protein.
  • a vector comprises a nucleic acid molecule encoding an anti-BCMA CAR and another vector comprises a nucleic acid molecule encoding a CA2 DRD-ILL protein.
  • a single vector comprises a nucleic acid molecule encoding both an anti-BCMA CAR and a CA2 DRD-ILL protein.
  • the nucleic acid molecules of the present disclosure may be delivered using one or more modalities.
  • Vectors of the present disclosure may be used to deliver the nucleic acid molecules to a cell, a local tissue site or a subject. These vectors may be of any kind, including DNA vectors, RNA vectors, plasmids, viral vectors and particles. Viral vector technology is well known and described in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). Viruses, which are useful as vectors include, but are not limited to lentiviral vectors, adenoviral vectors, adeno-associated viral (AAV) vectors, herpes simplex viral vectors, retroviral vectors, oncolytic viruses, and the like.
  • AAV adeno-associated viral
  • a viral vector useful for introducing one or more nucleic acid molecules exemplified herein into a cell may be derived from an adenovirus, adeno-associated virus (AAV), alphavirus, flavivirus, herpes virus, measles virus, rhabdovirus, retrovirus, lentivirus, Newcastle disease virus (NDV), poxvirus, or picomavirus.
  • AAV adeno-associated virus
  • alphavirus alphavirus
  • flavivirus herpes virus
  • measles virus measles virus
  • rhabdovirus retrovirus
  • lentivirus lentivirus
  • NDV Newcastle disease virus
  • picomavirus picomavirus
  • Nucleic acid molecules of the present disclosure may be transferred to cells by physical methods such as needles, electroporation, sonoporation, hydroporation; chemical carriers such as inorganic particles (e.g. calcium phosphate, silica, gold) and/or chemical methods.
  • chemical carriers such as inorganic particles (e.g. calcium phosphate, silica, gold) and/or chemical methods.
  • synthetic or natural biodegradable agents may be used for delivery such as cationic lipids, lipid nanoemulsions, nanoparticles, peptide-based compositions, or polymer-based compositions.
  • vectors contain an origin of replication functional in at least one organism, a promoter sequence and convenient restriction endonuclease site, and one or more selectable markers e.g. a drug resistance gene.
  • the expression vector may comprise regulatory sequences, such as transcription and translation initiation and termination codons, which are specific to the type of host cell into which the vector is to be introduced.
  • the vector of the disclosure may comprise a nucleic acid sequence encoding a CA2 DRD-regulated IL15 and a nucleic acid sequence encoding an anti- BCMA CAR.
  • the vector may comprise a nucleic acid sequence encoding one or more additional polypeptides.
  • a vector comprises a nucleic acid sequence encoding a CA2 DRD-regulated IL15 and a separate vector comprises a nucleic acid sequence encoding an anti-BCMA CAR.
  • lentiviral vehicles/particles may be used as delivery modalities.
  • Lentiviruses are subgroup of the Retroviridae family of viruses, named because reverse transcription of viral RNA genomes to DNA is required before integration into the host genome. As such, the most important features of lentiviral vehicles/particles are the integration of their genetic material into the genome of a target/host cell.
  • lentivirus examples include the Human Immunodeficiency Viruses: HIV-1 and HIV-2, the Simian Immunodeficiency Virus (SIV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), Jembrana Disease Virus (JDV), equine infectious anemia virus (EIAV), equine infectious anemia virus, visna-maedi and caprine arthritis encephalitis virus (CAEV).
  • SIV Simian Immunodeficiency Virus
  • FV feline immunodeficiency virus
  • BIV bovine immunodeficiency virus
  • JDV Jembrana Disease Virus
  • EIAV equine infectious anemia virus
  • CAEV visna-maedi and caprine arthritis encephalitis virus
  • lentiviral particles making up the gene delivery vehicle are replication defective on their own (also referred to as “self-inactivating”). Lentiviruses are able to infect both dividing and non-dividing cells by virtue of the entry mechanism through the intact host nuclear envelope (Naldini L et al., Curr. Opin. Biotechnol, 1998, 9: 457-463). Recombinant lentiviral vehicles/particles have been generated by multiply attenuating the HIV virulence genes, for example, the genes Env, Vif, Vpr, Vpu, Nef and Tat are deleted making the vector biologically safe.
  • lentiviral vehicles for example, derived from HIV-l/HIV-2 can mediate the efficient delivery, integration and long-term expression of transgenes into nondividing cells.
  • the term “recombinant” refers to a vector or other nucleic acid containing both lentiviral sequences and non-lentiviral retroviral sequences.
  • Lentiviral particles may be generated by co-expressing the virus packaging elements and the vector genome itself in a producer cell such as human HEK293T cells. These elements are usually provided in three or four separate plasmids.
  • the producer cells are co-transfected with plasmids that encode lentiviral components including the core (i.e. structural proteins) and enzymatic components of the virus, and the envelope protein(s) (referred to as the packaging systems), and a plasmid that encodes the genome including a foreign transgene, to be transferred to the target cell, the vehicle itself (also referred to as the transfer vector).
  • the plasmids or vectors are included in a producer cell line.
  • the plasmids/vectors are introduced via transfection, transduction or infection into the producer cell line.
  • Methods for transfection, transduction or infection are well known by those of skill in the art.
  • the packaging and transfer constructs can be introduced into producer cell lines by calcium phosphate transfection, lipofection or electroporation, generally together with a dominant selectable marker, such as neo, DHFR, Gin synthetase or ADA, followed by selection in the presence of the appropriate drug and isolation of clones.
  • the producer cell produces recombinant viral particles that contain the foreign gene, for example, the nucleic acid molecule of the present disclosure.
  • the recombinant viral particles are recovered from the culture media and titrated by standard methods used by those of skill in the art.
  • the recombinant lentiviral vehicles can be used to infect target cells.
  • Cells that can be used to produce high-titer lentiviral particles include, but are not limited to, HEK293T cells, 293G cells, STAR cells (Relander et al., Mol. Then, 2005, 11 : 452- 459), FreeStyleTM 293 Expression System (ThermoFisher, Waltham, MA), and other HEK293T- based producer cell lines (e.g., Stewart et al., Hum Gene Ther. 2011, 22(3):357-369; Lee et al., Biotechnol Bioeng, 2012, 10996): 1551-1560; Throm et al., Blood. 2009, 113(21): 5104-5110; the contents of each of which are incorporated herein by reference in their entirety).
  • Lentivirus vectors used may be selected from, but are not limited to pLVX, pLenti, pLenti6, pLJMl, FUGW, pWPXL, pWPI, pLenti CMV puro DEST, pLJMl-EGFP, pULTRA, p!nducer20, pHIV-EGFP, pCW57.1, pTRPE, pELPS, pRRL, and pLionll.
  • the envelope proteins may be heterologous envelope proteins from other viruses, such as the G protein of vesicular stomatitis virus (VSV G) or baculoviral gp64 envelope proteins.
  • VSV-G glycoprotein may especially be chosen among species classified in the vesiculovirus genus: Carajas virus (CJSV), Chandipura virus (CHPV), Cocal virus (COCV), Isfahan virus (ISFV), Maraba virus (MARAV), Piry virus (PIRYV), Vesicular stomatitis Alagoas virus (VSAV), Vesicular stomatitis Indiana virus (VSIV) and Vesicular stomatitis New lersey virus (VSNIV) and/or stains provisionally classified in the vesiculovirus genus as Grass carp rhabdovirus, BeAn 157575 virus (BeAn 157575), Boteke virus (BTKV), Calchaqui
  • the gp64 or other baculoviral env protein can be derived from Autographa califomica nucleopolyhedrovirus (AcMNPV), Anagrapha falcifera nuclear polyhedrosis virus, Bombyx mori nuclear polyhedrosis virus, Choristoneura fumiferana nucleopolyhedrovirus, Orgyia pseudotsugata single capsid nuclear polyhedrosis virus, Epiphyas postvittana nucleopolyhedrovirus, Hyphantria cunea nucleopolyhedrovirus, Galleria mellonella nuclear polyhedrosis virus, Dhori virus, Thogoto virus, Antheraea pemyi nucleopolyhedrovirus or Batken virus.
  • Other envelope proteins can be derived from baboon envelope (BaEV).
  • lentiviral particles may comprise retroviral LTR (long- terminal repeat) at either 5’ or 3’ terminus, a retroviral export element, optionally a lentiviral reverse response element (RRE), a promoter or active portion thereof, and a locus control region (LCR) or active portion thereof.
  • retroviral LTR long- terminal repeat
  • RRE lentiviral reverse response element
  • LCR locus control region
  • Lentiviral vectors are used for introducing transgenes into T cells (e.g., primary human T cells or Jurkat cells) for preclinical research and clinical applications, including recently approved products such as Tisagenlecleucel (KYMRIAH®) for relap sed/refractory B-cell lymphoma.
  • VSV-G pseudotyped 3rd generation lentiviral vectors offer high titers, high transduction efficiency and safety, and have become the vectors of choice for T cell engineering.
  • T cell engineering usually involves T cell activation by CD3/CD28 antibodies, followed by lentivirus transduction, and then cell expansion which can last from 5 to 30 days (e.g., 9 to 14 days or 9 to 15 days).
  • lentivirus transgene integration may take over 7 days to fully stabilize in T cells (e.g., primary human T cells or Jurkat cells).
  • vectors comprising nucleic acid sequences encoding a CA2 DRD-regulated IL15 and an anti-BCMA CAR, and compositions thereof may be introduced into cells.
  • the cells may be immune cells.
  • the cells may be human immune cells.
  • the cells may be human T cells.
  • activated primary human T cells e.g., primary human T cells activated by CD3/CD28
  • the cells may be analyzed by methods described herein and/or known in the art for viability, viral genomic integration (e.g., by using quantitative PCR), transcript levels (e.g., by using quantitative RT-PCR), and cell surface expression of the anti-BCMA CAR and/or the CA2 DRD-regulated mbIL15.
  • the cells may be analyzed prior to transduction and/or after transduction from 1 to 30 days or longer.
  • the cells may be analyzed at various time points between 3 to 15 days after transduction. As a non-limiting example, the cells may be analyzed 9 to 15 days after transduction.
  • the activated primary human T cells e.g., primary human T cells activated by CD3/CD28
  • the cells may be reactivated between 5 and 30 days or more after transduction.
  • the cells may be analyzed by methods described herein and/or known in the art for viability, viral genomic integration (e.g., by using quantitative PCR), transcript levels (e.g., by using quantitative RT- PCR), cell surface expression of the transgene, copy number, and/or mRNA levels.
  • the cell viability of activated primary human T cells transduced with lentivirus carrying a transgene is greater than 65%, 70%, 75%, 80%, 85%, 90% or 95%. As a non-limiting example, the cell viability is greater than 75%.
  • a percentage of the cultured T cells may express the transgene.
  • the percentage of culture T cells expressing the transgene may be, but is not limited to, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, or greater than 60%. As a non-limiting example, the percentage may be greater than 30%.
  • the mRNA levels from the culture may decline over the duration of the study. The decline may or may not be limited to a specific transgene and the trend may be seen across multiple classes of expressed proteins.
  • the cells may be reactivated after the mRNA levels decrease from the initial levels. The cells may be reactivated 5 to 30 days or more after transduction. As a non-limiting example, in order to increase mRNA levels in the culture, the cells may be reactivated with CD3/CD28 beads after transduction.
  • the surface expression of the CA2 DRD-regulated IL15 and/or the BCMA CAR on the transduced cells in culture may decline over a period of time.
  • the cells may be reactivated after the surface expression decrease from the initial levels.
  • the cells may be reactivated after the surface expression levels decrease from the initial levels.
  • the cells may be reactivated 5 to 30 days or more after transduction.
  • the cells in order to increase surface expression levels in the culture, the cells may be reactivated with CD3/CD28 beads 5 to 30 days or more after transduction.
  • the present disclosure provides methods for modulating protein expression, function or level by the use of a CA2 DRD operably linked to the IL 15 payload.
  • the modulation of protein expression, function or level refers to modulation of expression, function or level by at least 50%. In some embodiments, the modulation of protein expression, function or level refers to modulation of expression, function or level by at least 75%, 100%, 150%, 200%, 250% or 300%.
  • present teachings further comprise pharmaceutical compositions comprising one or nucleic acid molecules, vectors or cells of the present disclosure, and optionally at least one pharmaceutically acceptable excipient.
  • composition refers to a nucleic acid molecule, vector or small molecule, refers to a preparation of one or more of the nucleic acid molecules, vectors or small molecules described herein, or a pharmaceutically acceptable salt thereof, optionally with other chemical components such as pharmaceutically acceptable excipient.
  • a pharmaceutical composition refers to a preparation comprising one or more human T cells in a pharmaceutically acceptable excipient.
  • a pharmaceutical composition comprising a cell such as a human T cell, comprises a population of transduced human T cells in a pharmaceutically acceptable excipient.
  • a pharmaceutical composition comprising a cell such as a human T cell, comprises a cryopreservative, whether as the sole pharmaceutically acceptable excipient or as one of several pharmaceutically acceptable excipients of the pharmaceutical composition.
  • pharmaceutically acceptable excipient or “inactive ingredient” refers to an inert or inactive substance added to a pharmaceutical composition to facilitate administration, manufacture or storage of a nucleic acid molecule, vector, polypeptide or cell.
  • compositions are principally directed to pharmaceutical compositions which are suitable for administration to humans, such compositions are generally suitable for administration to other animals, e.g., to non-human animals, e.g. non-human mammals.
  • non-human mammals include, but are not limited to, agricultural animals such as cattle, horses, chickens and pigs, domestic animals such as cats and dogs, or research animals such as mice, rats, rabbits, dogs and non-human primates.
  • the compositions of the present disclosure may be formulated in any manner suitable for delivery.
  • the formulation may be, but is not limited to, nanoparticles, poly (lactic-co-glycolic acid) (PLGA) microspheres, lipidoids, lipoplex, liposome, polymers, carbohydrates (including simple sugars), cationic lipids and combinations thereof.
  • PLGA poly (lactic-co-glycolic acid)
  • pharmaceutical or other formulations may comprise at least one excipient which is an inactive ingredient.
  • the term “inactive ingredient” refers to one or more inactive agents included in formulations.
  • all, none or some of the inactive ingredients which may be used in the formulations of the present disclosure may be approved by the US Food and Drug Administration (FDA).
  • FDA US Food and Drug Administration
  • the compositions of the disclosure may be delivered to a cell or a subject through one or more routes and modalities.
  • the nucleic acid molecules and viral vectors, or pharmaceutical compositions thereof may be administered directly via gene therapy.
  • a cell transduced with a nucleic acid molecule or vector described herein is administered to the subject in need thereof via adoptive cell therapy (ACT).
  • ACT adoptive cell therapy
  • compositions comprising the nucleic acid molecules, vectors or cells described herein may be formulated to include, but are not limited to, cell penetration agents, pharmaceutically acceptable carriers, delivery agents, bioerodible or biocompatible polymers, solvents, and/or sustained-release delivery depots.
  • compositions may be formulated for direct delivery to organs or tissues in any of several ways in the art including, but not limited to, direct soaking or bathing, via a catheter, by gels, powder, ointments, creams, gels, lotions, and/or drops, by using substrates such as fabric or biodegradable materials coated or impregnated with compositions, and the like.
  • the present disclosure provides methods comprising administering a nucleic acid molecule, a vector or a cell as described herein to a subject in need thereof. These may be administered to a subject using any amount and any route of administration effective for preventing or treating a disease or disorder.
  • compositions comprising a stimulus to which the CA2 DRD is responsive.
  • the stimulus is acetazolamide, celecoxib, valdecoxib, rofecoxib, methazolamide, dorzolamide, brinzolamide, diclofenamide, ethoxzolamide, zonisamide, dansylamide or di chlorphenamide.
  • the present disclosure provides methods of administering a stimulus in accordance with the disclosure to a subject in need thereof.
  • the stimulus may be administered to a subject or to cells, using any amount and any route of administration effective for modulating the CA2 DRD- regulated IL15 of the present disclosure.
  • the exact amount required will vary from subject to subject, depending on the age and general condition of the subject, the severity of the disease, the particular composition, its mode of administration, its mode of activity, and the like.
  • Stimulus compositions in accordance with the present disclosure are typically formulated in unit dosage form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily usage of the compositions of the present disclosure may be decided by the attending physician within the scope of sound medical judgment.
  • compositions of the disclosure may be used in varying doses to improve cell expansion, increase durability of response by enhancing memory phenotype, prolong persistence, and overcome T cell exhaustion. Compositions of the disclosure also may be used to prevent cytokine release syndrome and minimize toxicity associated with immunotherapy. For example, low doses of the compositions of the present disclosure may be used to initially treat patients with high tumor burden, while patients with low tumor burden may be treated with high and repeated doses of the compositions of the disclosure to ensure recognition of a minimal tumor antigen load. In another instance, the compositions of the present disclosure may be delivered in a pulsatile fashion to reduce tonic T cell signaling and enhance persistence in vivo.
  • toxicity may be minimized by initially using low doses of the compositions of the disclosure, prior to administering high doses. Dosing may be modified if serum markers such as ferritin, serum C-reactive protein, IL6, IFN-y, and TNF-a are elevated.
  • the present disclosure provides methods for delivering to a cell or tissue any of the stimuli described herein, comprising contacting the cell or tissue with said stimuli, which can be accomplished in vitro, ex vivo, or in vivo.
  • the stimuli in accordance with the present disclosure may be administered to cells at dosage levels sufficient to deliver from about 1 nM to about 500
  • the desired dosage of the stimulus of the present disclosure may be delivered once daily, twice daily, three times a day, or on a schedule from once every two weeks to once every two months.
  • the stimulus is provided only one time.
  • the stimulus is administered on an ad hoc basis determined by the subject’s physician.
  • the desired dosage of the stimulus may be delivered using multiple administrations.
  • the stimulus of the present disclosure may be administered as a “pulse dose” or as a “continuous flow”.
  • a “pulse dose” is a series of doses of the stimulus administered with a set frequency over a period of time.
  • a “continuous flow” is a dose of the stimulus administered continuously for a period of time.
  • the nucleic acid molecules or vectors comprising the nucleic acid molecules of the present disclosure may be administered to cells ex vivo and subsequently administered to a subject.
  • Immune cells such as human T cells, can be isolated and expanded ex vivo using a variety of methods known in the art. For example, methods of isolating cytotoxic T cells are described in U.S. Pat. Nos. 6,805,861 and 6,531, 451; the contents of each of which are incorporated herein by reference in their entirety.
  • the cells may be introduced into a host organism, e.g., a mammal, in a wide variety of ways including by injection, transfusion, infusion, local instillation or implantation.
  • the cells described herein may be introduced at the site of the tumor or may be administered intravenously.
  • the number of cells that are employed will depend upon a number of circumstances, the purpose for the introduction, the lifetime of the cells, the protocol to be used, for example, the number of administrations, the ability of the cells to multiply, or the like.
  • the cells described herein are administered one time only to a subject having a disease or condition.
  • the cells described herein are administered in multiple doses to a subject having a disease or condition.
  • the cells described herein may be administrated in multiple doses to subjects having a disease or condition.
  • the nucleic acid molecules, vectors and cells, and pharmaceutical compositions thereof of the present disclosure may be administered by any route to achieve a therapeutically effective outcome.
  • routes include, without limitation, subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous or intralymphatic injection, intraperitoneally, transdermally, intranasally, buccally, sublingually, rectally, vaginally or by suppository administration.
  • the pharmaceutical composition is administered using peripheral systemic delivery by intravenous, intraperitoneal, or subcutaneous injection.
  • the nucleic acid molecules, vectors and cells of the present disclosure may be used in the development and implementation of cell therapies such as adoptive cell therapy.
  • the CA2 DRD-regulated IL15 and anti-BCMA CAR may be used to effect CAR T cell therapy and CAR NK cell therapy, any of which may be used in combination therapy with other treatment lines (e.g. radiation, cytokines).
  • the CA2 DRD-regulated IL15 and anti-BCMA CAR may be used to effect CAR T cell therapy.
  • CA2 DRD-regulated IL15 and anti-BCMA CAR nucleic acid molecules and vectors may be used to engineer immune cells including T cells such as CD8 + T cells and CD4 + T cells, cytotoxic T lymphocytes (CTLs), lymphokine activated killer (LAK) cells, memory T cells, regulatory T cells (Tregs), helper T cells, cytokine-induced killer (CIK) cells, and any combination thereof.
  • T cells such as CD8 + T cells and CD4 + T cells
  • CTLs cytotoxic T lymphocytes
  • LAK lymphokine activated killer
  • memory T cells memory T cells
  • Regs regulatory T cells
  • helper T cells cytokine-induced killer cells
  • CA2 DRD-regulated IL 15 and anti- BCMA CAR nucleic acid molecules and vectors may be used to engineer immune stimulatory cells generated from embryonic stem cell (ESC) and induced pluripotent stem cell (iPSC) that may be used for ACT.
  • ESC embryonic stem cell
  • CA2 DRD-regulated LL15 and anti-BCMA CAR nucleic acid molecules and vectors may be used to engineer autologous or allogeneic immune cells that may be used for ACT.
  • CA2 DRD-regulated IL 15 and anti- BCMA CAR nucleic acid molecules and vectors may be used to engineer T cells.
  • the present disclosure provides an anti-BCMA CAR T cell that is “armed” with a CA2 DRD-regulated IL 15 polypeptide to improve the engineered cells’ efficacy and persistence. In some embodiments, the present disclosure provides an anti-BCMA CAR T cell that is “armed” with a CA2 DRD-regulated IL 15 polypeptide to improve the engineered cells’ efficacy and persistence or prevent immune exhaustion and senescence.
  • cells of the present disclosure may be autologous, allogeneic, syngeneic, or xenogeneic in relation to a particular individual subject.
  • cells of the present disclosure may be mammalian cells, particularly human cells.
  • Cells described herein may be primary cells or immortalized cell lines.
  • the cells of the present disclosure are autologous human T cells derived from a subject in need of treatment.
  • Efficacy of treatment or amelioration of disease can be assessed, for example by measuring disease progression, disease remission, symptom severity, reduction in pain, quality of life, dose of a medication required to sustain a treatment effect, level of a disease marker or any other measurable parameter appropriate for a given disease being treated or targeted for prevention. It is well within the ability of one skilled in the art to monitor efficacy of treatment or prevention by measuring any one of such parameters, or any combination of parameters.
  • compositions of the present disclosure "effective against” for example a cancer, indicates that administration in a clinically appropriate manner results in a beneficial effect for at least a statistically significant fraction of patients, such as an improvement of symptoms, a cure, a reduction in disease load, reduction in tumor mass or cell numbers, extension of life, improvement in quality of life, or other effect generally recognized as positive by medical doctors familiar with treating the particular type of cancer.
  • a treatment or preventive effect is evident when there is a statistically significant improvement in one or more parameters of disease status, or by a failure to worsen or to develop symptoms where they would otherwise be anticipated.
  • a favorable change of at least 10% in a measurable parameter of disease, and preferably at least 20%, 30%, 40%, 50% or more can be indicative of effective treatment.
  • Efficacy for a given composition or formulation of the present disclosure can also be judged using an experimental animal model for the given disease as known in the art. When using an experimental animal model, efficacy of treatment is evidenced when a statistically significant change is observed.
  • Cancer immunotherapy aims to induce or restore the reactivity of the immune system towards cancer.
  • Adoptive cell therapy is a form of active immunotherapy that aims to induce an endogenous, long-lasting tumor-antigen specific immune response. The response may be enhanced by non-specific stimulation of immune response modifiers such as cytokines.
  • Immunotherapy may produce on target, on-tumor toxicities that emerge when tumor cells are killed in response to the immunotherapy.
  • the adverse effects include tumor lysis syndrome, cytokine release syndrome and the related macrophage activation syndrome.
  • Cytokine release syndrome (CRS) is caused by a large, rapid release of cytokines into the blood from immune cells affected by the immunotherapy.
  • CRS can often be controlled, including through administration of tocilizumab, an IL-6 receptor antagonist, it can still cause subject injury and may be more dangerous for fragile patients.
  • CRS can often be controlled, including through administration of tocilizumab, an IL-6 receptor antagonist, it can still cause subject injury and may be more dangerous for fragile patients.
  • these adverse effects may occur during the destruction of tumors, and thus even a successful on-tumor immunotherapy might result in toxicity.
  • a major risk involved in immunotherapy is the on- target but off tumor side effects resulting from T cell activation in response to normal tissue expression of the tumor associated antigen (TAA).
  • TAA tumor associated antigen
  • adoptively transferred immune cells continue to proliferate within the patient, often unpredictably, which may lead to toxicity or autoimmune diseases.
  • Approaches to regulatably control immunotherapy are thus highly desirable since they have the potential to reduce toxicity and maximize efficacy.
  • the tunable nature of the systems and compositions of the disclosure has the potential to improve the potency and duration of the efficacy of immunotherapies.
  • Reversibly silencing the biological activity of adoptively transferred cells using compositions of the present disclosure allows maximizing the potential of cell therapy without irretrievably killing and terminating the therapy.
  • the present disclosure also provides methods for fine tuning of immunotherapy after administration to patients. This in turn improves the safety and efficacy of immunotherapy and increases the subject population that may benefit from immunotherapy.
  • adoptive cell therapy is carried out by autologous transfer, wherein the cells are derived from a subject in need of a treatment and the cells, following isolation and processing are administered to the same subject.
  • ACT may involve allogenic transfer wherein the cells are isolated and/or prepared from a donor subject other than the recipient subject who ultimately receives cell therapy.
  • the donor and recipient subject may be genetically identical, or similar or may express the same HLA class or subtype.
  • cells are administered to the subject in need thereof. Methods for administration of cells for adoptive cell therapy are known and may be used in connection with the provided methods and compositions.
  • immune cells engineered with CA2-IL15 for ACT may be further modified to express one or more immunotherapeutic agents which facilitate immune cells activation, infiltration, expansion, survival and anti-tumor functions.
  • the immunotherapeutic agents may be a CAR or TCR specific to a target molecule on a tumor cell; a second cytokine or a cytokine receptor; a chimeric switch receptor that converts an inhibitory signal to a stimulatory signal; a homing receptor that guides adoptively transferred cells to a target site such as the tumor tissue; an agent that optimizes the metabolism of the immune cell; or a safety switch gene (e.g., a suicide gene) that kills activated T cells when a severe event is observed after adoptive cell transfer or when the transferred immune cells are no-longer needed.
  • a safety switch gene e.g., a suicide gene
  • immune cells used for adoptive cell transfer can be genetically manipulated to improve their persistence, cytotoxicity, tumor targeting capacity, and ability to home to disease sites in vivo, with the overall aim of further improving upon their capacity to kill tumors in cancer patients.
  • One example is to introduce nucleic acid molecules encoding a DRD- regulated IL15 into immune cells to promote immune cell proliferation and survival.
  • T cell exhaustion refers to the stepwise and progressive loss of T cell function caused by chronic T cell activation. T cell exhaustion is a major factor limiting the efficacy of antiviral and antitumor immunotherapies. Exhausted T cells have low proliferative and cytokine producing capabilities concurrent with high rates of apoptosis and high surface expression of multiple inhibitory receptors. T cell activation leading to exhaustion may occur either in the presence or absence of the antigen.
  • the CA2 DRD-regulated mbILl 5 may be utilized to prevent T cell exhaustion in the context of Chimeric Antigen Receptor-T cell therapy (CAR-T).
  • CAR-T Chimeric Antigen Receptor-T cell therapy
  • exhaustion in some instances, may be caused by the oligomerization of the scFvs of the CAR on the cell surface which leads to continuous activation of the intracellular domains of the CAR.
  • CARs of the present disclosure may include scFvs that are unable to oligomerize.
  • CARs that are rapidly internalized and re-expressed following antigen exposure may also be selected to prevent chronic scFv oligomerization on cell surface.
  • the framework region of the scFvs may be modified to prevent constitutive CAR signaling.
  • CA2 DRDs of the present disclosure may also be used to regulate the surface expression of the CAR on the T cell surface to prevent chronic T cell activation.
  • the CARs of the disclosure may also be engineered to minimize exhaustion.
  • the 4- 1-BB signaling domain may be incorporated into CAR design together with membrane bound IL15 expression regulated by CA2 DRDs to ameliorate T cell exhaustion.
  • the tunable nature of the CA2-IL15 proteins of the present disclosure may be utilized to reverse human T cell exhaustion observed with tonic CAR signaling.
  • Reversibly silencing the biological activity of adoptively transferred cells using compositions of the present disclosure may be used to reverse tonic signaling which, in turn, may reinvigorate the T cells.
  • Reversal of exhaustion may be measured by the downregulation of multiple inhibitory receptors associated with exhaustion.
  • a method of reducing a tumor volume or burden in a subject in need comprising introducing into the subject a composition of the disclosure.
  • the present disclosure also provides methods for treating a cancer in a subject, comprising administering to the subject an effective amount of an effector immune cell genetically modified to express at least one nucleic acid molecule of the disclosure.
  • cancers may be treated with pharmaceutical compositions comprising the BCMA CAR and mbIL15 of the present disclosure.
  • cancer refers to any of various malignant neoplasms characterized by the proliferation of anaplastic cells that tend to invade surrounding tissue and metastasize to new body sites and also refers to the pathological condition characterized by such malignant neoplastic growths. Cancers may be tumors or hematological malignancies,
  • compositions of the present disclosure may be used in the modulation or alteration or exploitation of the immune system to target one or more cancers.
  • This approach may also be considered with other such biological approaches, e.g., immune response modifying therapies such as the administration of interferons, interleukins, colony-stimulating factors, other monoclonal antibodies, vaccines, gene therapy, and nonspecific immunomodulating agents are also envisioned as anti-cancer therapies to be combined with the pharmaceutical compositions of the present disclosure.
  • Cancer immunotherapy refers to a diverse set of therapeutic strategies designed to induce the patient's own immune system to fight the cancer.
  • pharmaceutical compositions of the present disclosure are designed as immune-oncology therapeutics.
  • NK cells NK cells
  • TIL tumor infiltrating lymphocyte
  • CARs genetically engineered T cells bearing chimeric antigen receptors (CARs) or recombinant TCR technology.
  • the BCMA CAR T cell of the present disclosure may be an “armed” T cell that comprises a CA2 DRD-regulated mbIL15 to improve efficacy and persistence.
  • patients may also be stratified according to the immunogenic peptides presented by their immune cells and may be utilized as a parameter to determine suitable patient cohorts that may therapeutically benefit for the compositions of the disclosure.
  • cells of the disclosure may be autologous, allogeneic, syngeneic, or xenogeneic in relation to a particular individual subject.
  • cells of the disclosure may be mammalian cells, particularly human cells.
  • Cells of the disclosure may be primary cells or immortalized cell lines.
  • Engineered immune cells can be accomplished by transducing a cell composition with one or more nucleic acid molecules of the disclosure or a vector comprising said polynucleotide.
  • the vector may be a viral vector such as a lentiviral vector or gamma retroviral vector.
  • immune cells of the disclosure are genetically modified to express at least one immunotherapeutic agent of the disclosure which is tunable using a stimulus.
  • compositions of the present disclosure are disclosed in groups or in ranges. It is specifically intended that the present disclosure include each and every individual sub combination of the members of such groups and ranges. The following is a non-limiting list of term definitions.
  • the present disclosure may interchangeably identify these constructs with or without the term “OT-” at the beginning of the construct name.
  • the names “OT-BCMA-IL15-008” and “BCMA-IL15-008” refer to the same construct.
  • CAR-T CART
  • CAR-T cells CART cells
  • CART cells CART cells
  • CART cells CART cells
  • CART cells CART cells
  • CART cells CART cells
  • CART cells CART cells
  • CART cells CART cells
  • CART cells CART cells
  • CART cells CART cells
  • CART cells CART cells
  • CART cells CART cells
  • CART cells CART cells
  • CART cells CART cells
  • Activity refers to the condition in which things are happening or being done.
  • Compositions of the disclosure may have activity and this activity may involve one or more biological events.
  • biological events may include cell signaling events.
  • Adoptive cell therapy refers to a cell therapy involving in the transfer of cells into a patient, wherein cells may have originated from the patient, or from another individual, and are engineered (altered) before being transferred back into the patient.
  • the therapeutic cells may be derived from the immune system, such as effector immune cells including T cells such as CD4+ T cells and CD8+ T cells.
  • effector immune cells including T cells such as CD4+ T cells and CD8+ T cells.
  • Most commonly transferred cells are autologous anti-tumor T cells after ex vivo expansion or manipulation.
  • autologous peripheral blood lymphocytes can be genetically engineered to recognize specific tumor antigens by expressing chimeric antigen receptors (CAR).
  • CAR chimeric antigen receptors
  • the terms “associated with,” “conjugated,” “linked,” “attached,” and “tethered,” when used with respect to two or more moieties, mean that the moieties are physically associated or connected with one another, either directly or via one or more additional moieties that serve as linking agents, to form a structure that is sufficiently stable so that the moieties remain physically associated under the conditions in which the structure is used, e.g., physiological conditions.
  • An “association” need not be strictly through direct covalent chemical bonding. It may also suggest ionic or hydrogen bonding or a hybridization-based connectivity sufficiently stable such that the “associated” entities remain physically associated.
  • Autologous the term “autologous” as used herein is meant to refer to any material derived from the same individual to which it is later to be re-introduced into the individual.
  • Cancer refers a broad group of various diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division and growth results in the formation of malignant tumors that invade neighboring tissues ultimately metastasize to distant parts of the body through the lymphatic system or bloodstream.
  • Consisting essentially of The phrase “consisting essentially of’ or “consists essentially of’ when used in reference to an embodiment of the present disclosure can be interpreted as limiting the scope of that embodiment to the specified materials or steps and those that do not materially affect the basic and novel characteristic(s) of the embodiment.
  • corresponding with reference to positions of a protein, such as recitation that amino acid positions “correspond to” amino acid positions in a disclosed sequence, such as set forth in the sequence listing, refers to amino acid positions identified upon alignment with the disclosed sequence based on structural sequence alignment or using a standard alignment algorithm. By aligning the sequences, one skilled in the art can identify corresponding residues, for example, using conserved and identical amino acid residues as guides.
  • Co-stimulatory molecule As used herein, in accordance with its meaning in immune T cell activation, refers to a group of immune cell surface receptor/ligands which engage between T cells and APCs and generate a stimulatory signal in T cells which combines with the stimulatory signal in T cells that results from T cell receptor (TCR) recognition of antigen/MHC complex (pMHC) on APCs.
  • TCR T cell receptor
  • pMHC antigen/MHC complex
  • Cytokines the term “cytokines”, as used herein, refers to a family of small soluble factors with pleiotropic functions that are produced by many cell types that can influence and regulate the function of the immune system.
  • Delivery refers to the act or manner of delivering a compound, substance, entity, moiety, cargo or payload.
  • a “delivery agent” refers to any agent which facilitates, at least in part, the in vivo delivery of one or more substances (including, but not limited to a compound and/or composition of the present disclosure) to a cell, subject or other biological system cells.
  • the phrase “derived from” refers to a polypeptide or polynucleotide that originates from the stated parent molecule or region or domain thereof or the stated parent sequence (e.g., nucleic acid sequence or amino acid sequence) and retains similarity to one or more structural and/or functional characteristics of the parent molecule or region or domain thereof or parent sequence.
  • a polypeptide or polynucleotide is derived from either (i) a full-length wild-type parent molecule or sequence; or (ii) a region or domain of a full-length wild-type parent molecule or sequence and retains the structural and/or functional characteristics of either (i) the full-length wild-type parent molecule or sequence; or (ii) the region or domain thereof, respectively.
  • Structural characteristics include an amino acid sequence, a nucleic acid sequence, or a protein structure (e.g., such as a secondary protein structure, a tertiary protein structure, and/or quaternary protein structure).
  • Functional characteristics include biological activity such as catalytic activity, binding ability, and/or subcellular localization.
  • a polypeptide or polynucleotide retains similarity to a parent molecule or sequence if it has at least about 70% identity, preferably at least about 75% or 80% identity, more preferably at least about 85%, 86%, 87%, 88%, 89% or 90% identity, and further preferably at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to a parent nucleic acid sequence or amino acid sequence, over the entire length of the parent molecule or sequence.
  • a polypeptide retains similarity to a parent molecule or sequence if it comprises a region of amino acids that shares 100% identity to a parent amino acid sequence and said region ranges from 10-1,000 amino acids in length (e.g., greater than 20, 30, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, and 900 amino acids or at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, and 1,000 amino acids).
  • a polypeptide retains similarity to a parent molecule or amino acid sequence if it comprises one, two, three, four, or five amino acid mutations as compared to the parent amino acid sequence.
  • a polypeptide or polynucleotide is considered to retain similarity to a parent molecule or region or domain thereof or a parent sequence if it has substantially the same biological activity as compared to the parent molecule or region or domain thereof or the parent sequence.
  • a polypeptide or polynucleotide is considered to retain similarity to a parent molecule or region or domain thereof or a parent sequence if there is overlap of at least one biological activity as compared to the parent molecule or region or domain thereof or parent sequence.
  • a polypeptide or polynucleotide is considered to retain similarity to a parent molecule or region or domain thereof or a parent sequence if it has improvement or optimization of one or more biological activities as compared to the parent molecule or region or domain thereof or parent sequence.
  • a DRD may be derived from a domain or region of a naturally occurring protein and is modified in any of the ways taught herein to optimize DRD function.
  • biological activity may be optimized for a specified purpose, such as by retaining or enhancing certain activity while reducing or eliminating another activity as compared to a parent molecule.
  • Destabilized As used herein, the term “destabilize,” “destabilized” or “destabilizing region” means a region or molecule that is less stable than a starting, reference, wild-type or native form of the same region or molecule.
  • Engineered As used herein, embodiments of the disclosure are “engineered” when they are designed to have a feature or property, whether structural or chemical, that varies from a starting point, wild type or native molecule.
  • Formulation includes at least a compound and/or composition of the present disclosure and a delivery agent.
  • fragment refers to a portion of a molecule that is less than the entire molecule.
  • fragments of proteins may comprise polypeptides obtained by digesting full-length protein.
  • fragments of an antibody include portions of an antibody.
  • a “functional” biological molecule is a biological entity with a structure and in a form in which it exhibits a property and/or activity by which it is characterized.
  • Immune cells refers to any cell of the immune system that originates from a hematopoietic stem cell in the bone marrow, which gives rise to two major lineages, a myeloid progenitor cell (which give rise to myeloid cells such as monocytes, macrophages, dendritic cells, megakaryocytes and granulocytes) and a lymphoid progenitor cell (which give rise to lymphoid cells such as T cells, B cells and natural killer (NK) cells).
  • myeloid progenitor cell which give rise to myeloid cells such as monocytes, macrophages, dendritic cells, megakaryocytes and granulocytes
  • lymphoid progenitor cell which give rise to lymphoid cells such as T cells, B cells and natural killer (NK) cells).
  • Exemplary immune system cells include a CD4+ T cell, a CD8+ T cell, a CD4- CD8- double negative T cell, a T y5 cell, a Tap cell, a regulatory T cell, a natural killer cell, and a dendritic cell.
  • Macrophages and dendritic cells may be referred to as “antigen presenting cells” or “APCs,” which are specialized cells that can activate T cells when a major histocompatibility complex (MHC) receptor on the surface of the APC complexed with a peptide interacts with a TCR on the surface of a T cell.
  • MHC major histocompatibility complex
  • Immunotherapy refers to a type of treatment of a disease by the induction or restoration of the reactivity of the immune system towards the disease.
  • Immunotherapeutic agent refers to the treatment of disease by the induction or restoration of the reactivity of the immune system towards the disease with a biological, pharmaceutical, or chemical compound.
  • in vitro' refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, in a Petri dish, etc., rather than within an organism (e.g., animal, plant, or microbe).
  • in vivo refers to events that occur within an organism (e.g., animal, plant, or microbe or cell or tissue thereof).
  • Isolated refers to the removal of a nucleic acid molecule or a protein from its natural environment.
  • purified refers to a nucleic acid or a protein that has been increased in purity, whether the nucleic acid molecule or protein has been removed from nature (including genomic DNA, mRNA or cellular proteins), chemically or enzymatically synthesized (including cDNA and chemically synthesized peptides or proteins) or recombinantly made (including recombinantly-expressed proteins).
  • nucleic acids and proteins may be formulated with diluents or adjuvants and still for practical purposes be isolated.
  • nucleic acids typically are mixed with an acceptable carrier or diluent when used for introduction into cells.
  • Linker refers to a moiety that connects two or more domains, moieties or entities.
  • a linker may comprise 10 or more atoms.
  • a linker may comprise a group of atoms, e.g., 10-1,000 atoms.
  • a linker may comprise a polynucleotide comprising one or more nucleotides linking two polynucleotide moieties.
  • a linker may comprise a peptide comprising one or more amino acid residues linking two polypeptide moieties.
  • the linker may comprise an amino acid, peptide, polypeptide or protein.
  • Modified refers to a changed state or structure of a molecule or entity as compared with a parent or reference molecule or entity.
  • Molecules may be modified in many ways including chemically, structurally, and functionally.
  • compounds and/or compositions of the present disclosure are modified by the introduction of non-natural amino acids.
  • mutations refers to a change and/or alteration.
  • mutations may be changes and/or alterations to proteins (including peptides and polypeptides) and/or nucleic acids (including polynucleic acids).
  • mutations comprise changes and/or alterations to a protein and/or nucleic acid sequence.
  • Such changes and/or alterations may comprise the addition, substitution and or deletion of one or more amino acids (in the case of proteins and/or peptides) and/or nucleotides (in the case of nucleic acids and or polynucleic acids e.g., polynucleotides).
  • mutations comprise the addition and/or substitution of amino acids and/or nucleotides
  • such additions and/or substitutions may comprise 1 or more amino acid and/or nucleotide residues and may include modified amino acids and/or nucleotides.
  • the resulting construct, molecule or sequence of a mutation, change or alteration may be referred to herein as a mutant.
  • Non-naturally occurring when used in reference to a nucleic acid molecule or protein means a nucleic acid molecule or protein with a nucleotide sequence or amino acid sequence, respectively, that has been modified by a person.
  • Off-target refers to any unintended effect on any one or more target, gene, cellular transcript, cell, and/or tissue.
  • operbly linked refers to a functional connection between two or more molecules, constructs, transcripts, entities, moieties or the like.
  • “Operably-linked” or “functionally linked” as it refers to nucleic acid sequences and polynucleotides refers to the association of nucleic acid sequences so that the function of one is affected by the other, while the nucleic acid sequences need not necessarily be adjacent or contiguous to each other, but may have intervening sequences between them.
  • a regulatory DNA sequence is said to be “operably linked to” or “associated with” a DNA sequence that codes for an RNA or a polypeptide if the two sequences are situated such that the regulatory DNA sequence affects expression of the coding DNA sequence (i.e., that the coding sequence or functional RNA is under the transcriptional control of the promoter).
  • Coding sequences can be operably linked to regulatory sequences in sense or antisense orientation.
  • a transcriptional regulatory sequence is generally operably linked in cis with a coding sequence but need not be directly adjacent to it.
  • an enhancer is a transcriptional regulatory sequence that is operably linked to a coding sequence, even though it is not contiguous with the coding sequence.
  • a promoter is operably linked to a gene of interest if the promoter regulates or mediates transcription of the gene of interest in a cell.
  • promoter transcriptional regulatory sequences that are operably linked to a transcribed sequence are physically contiguous to the transcribed sequence, i.e., they are cisacting.
  • some transcriptional regulatory sequences, such as enhancers need not be physically contiguous or located in close proximity to the coding sequences whose transcription they enhance.
  • the term “operably linked” means that the state or function of one polypeptide in the fusion protein is affected by the other polypeptide in the fusion protein.
  • the DRD and the payload are operably linked if stabilization of the DRD with a ligand results in stabilization of the payload, while destabilization of the DRD in the absence of a ligand results in destabilization of the payload.
  • Payload or payload of interest refers to any protein or compound whose function is to be altered.
  • the POI is a component in the immune system, including both innate and adaptive immune systems.
  • Payloads of interest may be a protein, a fusion construct encoding a fusion protein, or noncoding gene, or variant and fragment thereof.
  • Payload of interest may, when amino acid based, may be referred to as a protein of interest.
  • compositions refers to any ingredient other than active agents (e.g., as described herein) present in pharmaceutical compositions and having the properties of being substantially nontoxic in subjects.
  • a pharmaceutically acceptable excipient for a particular therapeutic agent is determined by the nature of the therapeutic agent and the route of administration. For example, a pharmaceutically acceptable excipient for a modified human T cell of the present disclosure will be different than a pharmaceutically acceptable excipient for a stimulus of the present disclosure. Further, a pharmaceutically acceptable excipient for a therapeutic agent that is to be administered orally will likely be different than a pharmaceutically acceptable excipient for a therapeutic agent that is to be administered intravenously.
  • Plasmid refers to an extra-chromosomal element often carrying a gene that is not part of the central metabolism of the cell, and usually in the form of circular double-stranded DNA molecules. Such elements may be autonomously replicating sequences, genome integrating sequences, phage or nucleotide sequences, linear, circular, or supercoiled, of a single- or double-stranded DNA or RNA, derived from any source, in which a number of nucleotide sequences have been joined or recombined into a unique construction which is capable of introducing a promoter fragment and DNA sequence for a selected gene product along with appropriate 3' untranslated sequence into a cell.
  • plasmids and other cloning and expression vectors that can be used in accordance with the present disclosure are well known and readily available to those of skill in the art. Moreover, those of skill readily may construct any number of other plasmids suitable for use in the invention. The properties, construction and use of such plasmids, as well as other vectors, in the present invention will be readily apparent to those of skill from the present disclosure.
  • Polypeptide refers to a linear polymer comprised of amino acid residues covalently linked by peptide bonds, which is comprised of natural amino acid residues, unnatural amino acid residues or a combination of both. A polypeptide also may comprise other covalent bonds, such as disulfide bonds. In some embodiments, a polypeptide is comprised of natural amino acid residues. As used herein, the term “protein” may be used interchangeably with “polypeptide.”
  • Stable refers to a compound or entity that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture, and preferably capable of formulation into an efficacious therapeutic agent.
  • Stabilized As used herein, the term “stabilize”, “stabilized,” “stabilized region” means to make or become stable. In some embodiments, stability is measured relative to an absolute value. In some embodiments, stability is measured relative to a secondary status or state or to a reference compound or entity.
  • Subject refers to any organism to which a composition in accordance with the disclosure may be administered, e.g., for experimental, diagnostic, prophylactic, and/or therapeutic purposes.
  • a subject includes mammals including mice, rats, rabbits, non-human primates and humans.
  • therapeutically effective amount means an amount of an agent to be delivered (e.g., nucleic acid, vector, modified cell or stimulus) that is sufficient, when administered to a subject suffering from or susceptible to an disease, disorder, and/or condition, to treat, improve symptoms of, prevent, and/or delay the onset of the disease, disorder, and/or condition.
  • the therapeutically effective amount may be provided in a single dose or in a plurality of doses.
  • a unit dosage form may be considered to comprise a therapeutically effective amount of a particular agent or entity if it comprises an amount that is effective when administered as part of a dosage regimen.
  • treatment or treating denote an approach for obtaining a beneficial or desired result including and preferably a beneficial or desired clinical result.
  • beneficial or desired clinical results include, but are not limited to, one or more of the following: reducing the proliferation of (or destroying) cancerous cells or other diseased cells, reducing metastasis of cancerous cells found in cancers, shrinking the size of the tumor, decreasing symptoms resulting from the disease, increasing the quality of life of those suffering from the disease, decreasing the dose of other medications required to treat the disease, delaying the progression of the disease, and/or prolonging survival of individuals.
  • articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context.
  • the disclosure includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process.
  • the disclosure includes embodiments in which more than one, or the entire group members are present in, employed in or otherwise relevant to a given product or process.
  • compositions of the present disclosure and their use.
  • Embodiment 1 A nucleic acid molecule encoding a first and a second protein, wherein: (i) the first protein is a chimeric antigen receptor (CAR) comprising: (a) an antibody or antigen recognition moiety that binds to one or more epitopes of a human B-cell Maturation Antigen (BCMA) polypeptide, (b) a transmembrane domain, which may optionally further comprise a hinge domain, and (c) one or more intracellular T cell signaling domains; and (ii) the second protein comprises a drug responsive domain (DRD) operably linked to an IL 15 payload, wherein said DRD is a polypeptide derived from human carbonic anhydrase II (CA2) and comprises one, two, three or four amino acid insertions, deletions or substitutions compared to SEQ ID NO: 1.
  • CAR chimeric antigen receptor
  • BCMA human B-cell Maturation Antigen
  • the second protein comprises a drug responsive domain (DRD
  • Embodiment 8 The nucleic acid molecule of any of embodiments 1-7, wherein the IL 15 payload comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQID NO:8.
  • Embodiment 14 The nucleic acid molecule of embodiment 13, wherein the membrane-bound IL15 polypeptide comprises an ELI 5 polypeptide component comprising the amino acid sequence of SEQ ID NO: 8, a transmembrane domain and an intracellular tail, wherein the transmembrane domain is C-terminal to the ELI 5 polypeptide component and the intracellular tail is C-terminal to the transmembrane domain.
  • ELI 5 polypeptide component comprising the amino acid sequence of SEQ ID NO: 8
  • the transmembrane domain is C-terminal to the ELI 5 polypeptide component
  • the intracellular tail is C-terminal to the transmembrane domain.
  • Embodiment 15 The nucleic acid molecule of embodiment 14, wherein the membrane-bound IL 15 polypeptide further comprises a linker between the IL15 polypeptide component and the transmembrane domain.
  • Embodiment 18 The nucleic acid molecule of any one of embodiments 1-17, wherein the antibody or the antigen recognition moiety of the CAR is a camel immunoglobulin (Ig), an immunoglobulin new antigen receptor (IgNAR), an Fab fragment, an Fab' fragment, an F(ab)'2 fragment, an F(ab)'3 fragment, an Fv, a single-chain Fv antibody (scFv), a bis-scFv, an (SCFV)2, a minibody, a diabody, a triabody, a tetrabody, a disulfide-stabilized F v (dsFv), a singledomain antibody (sdAb) or a nanobody.
  • Ig camel immunoglobulin
  • IgNAR immunoglobulin new antigen receptor
  • an Fab fragment an Fab' fragment
  • an F(ab)'2 fragment an F(ab)'3 fragment
  • Fv a single-chain
  • Embodiment 23 The nucleic acid molecule of any one of embodiments 18-22, wherein the transmembrane domain and optional hinge domain of the CAR is the transmembrane domain and hinge domain of a polypeptide selected from CD28, 4-1BB, CD8 alpha, CD4, CD19, CD3 epsilon, CD45, CD5, CD9, CD16, CD22, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD154, PD1, CTLA-4, an alpha chain of a T cell receptor, a beta chain of a T cell receptor, or a zeta chain of a T cell receptor.
  • a polypeptide selected from CD28, 4-1BB, CD8 alpha, CD4, CD19, CD3 epsilon, CD45, CD5, CD9, CD16, CD22, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD154, PD1, CTLA-4, an alpha chain of a
  • each of the one or more intracellular T cell signaling domains of the CAR are independently selected from an intracellular signaling domain of 4-1BB, CARD11, CD2, CD3( ⁇ , CD7, CD27, CD28, CD30, CD40, CD54, CD83, CD134, CD150, CTLA-4, CD223, CD270, CD273, CD274, CD278, DAP10, FcRy, LAT, NKD2C, SLP76, TRIM or ZAP70.
  • each of the one or more intracellular T cell signaling domains of the CAR are independently selected from the intracellular signaling domain of CD28, CD134, CD3i ⁇ or 4-1BB.
  • Embodiment 29 The nucleic acid molecule of embodiment 28, wherein the intracellular T cell signaling domains of the CAR comprise the intracellular signaling domain of 4- IBB, consisting of the amino acid sequence of SEQ ID NO: 18, and the intracellular signaling domain of CD3( ⁇ , consisting of the amino acid sequence of SEQ ID NO:20, wherein the 4-1BB signaling domain is N-terminal to the CD3 ⁇ signaling domain.
  • (Embodiment 31) The nucleic acid molecule of any one of embodiments 18-30, wherein the CAR comprises, from N- to C-terminus, the antibody or antigen recognition moiety, the optional hinge domain, the transmembrane domain, and the one or more intracellular T cell signaling domains.
  • (Embodiment 32) The nucleic acid molecule of embodiment 31, wherein the CAR further comprises a signal sequence domain N-terminal to the antibody or the antigen recognition moiety.
  • Embodiment 33 The nucleic acid molecule of embodiment 32, wherein the signal sequence domain is selected from the signal sequence of granulocyte macrophage-colony stimulating factor (GM-CSF), CD8a or IgGl heavy chain.
  • GM-CSF granulocyte macrophage-colony stimulating factor
  • CD8a granulocyte macrophage-colony stimulating factor
  • IgGl heavy chain The nucleic acid molecule of embodiment 32, wherein the signal sequence domain is selected from the signal sequence of granulocyte macrophage-colony stimulating factor (GM-CSF), CD8a or IgGl heavy chain.
  • GM-CSF granulocyte macrophage-colony stimulating factor
  • the first protein comprises, from N-terminal to C-terminal: a CD8a leader sequence; an scFv directed against BCMA; a CD8a hinge domain or a CD28 hinge domain; a CD8a transmembrane domain or a CD28 hinge domain; an intracellular T cell signaling domain selected from a 4-1BB intracellular T cell signaling domain, an 0X40 intracellular T cell signaling domain, or a CD28 intracellular T cell signaling domain; and a CD3( ⁇ intracellular T cell signaling domain.
  • Embodiment 38 The nucleic acid molecule of embodiment 37, wherein the first protein comprises, from N-terminal to C-terminal: a CD8a leader sequence; an scFv directed against BCMA; a CD8a hinge domain; a CD8a transmembrane domain; a 4- IBB intracellular T cell signaling domain; and a CD3( ⁇ intracellular T cell signaling domain.
  • nucleic acid molecule of any one of embodiments 1-38 wherein a nucleic acid sequence encoding the first protein and a nucleic acid sequence encoding the second protein are separated by a co-expression element that promotes production of separate first proteins and second proteins.
  • coexpression element is a nucleic acid sequence encoding a cleavable linker sequence, a peptide that causes ribosome skipping, or an internal ribosome entry site (IRES).
  • Embodiment 41 The nucleic acid molecule of embodiment 40, wherein the peptide that causes ribosome skipping is a 2A peptide selected from foot-and-mouth disease virus (FMDV) 2A (F2A), equine rhinitis A virus (ERAV) 2A (E2A), porcine teschovirus-1 2A (P2A), and Thosea asigna virus 2A (T2A).
  • FMDV foot-and-mouth disease virus
  • E2A equine rhinitis A virus
  • P2A porcine teschovirus-1 2A
  • T2A Thosea asigna virus 2A
  • nucleic acid molecule of any of embodiments 1-43, wherein a nucleic acid sequence that encodes the first protein is 5’ to a nucleic acid sequence that encodes the second protein.
  • nucleic acid molecule of embodiment 44 further comprising a nucleic acid sequence encoding a P2A peptide 3’ to the nucleic acid sequence encoding the first protein and 5’ to the nucleic acid sequence encoding the second protein.
  • nucleic acid molecule of embodiment 46 further comprising a nucleic acid sequence encoding a P2A peptide 3’ to the nucleic acid sequence encoding the second protein and 5’ to the nucleic acid sequence encoding the first protein.
  • nucleic acid molecule of either of embodiments 44 or 45 further comprising a promoter operably linked to the nucleic acid sequence encoding the first protein.
  • Embodiment 50 The nucleic acid molecule of either of embodiments 46 or 47, further comprising a promoter operably linked to the nucleic acid sequence encoding the second protein.
  • Embodiment 51 The nucleic acid molecule of either of embodiments 49 or 50, wherein the promoter is selected from a CMV promoter, an EFla promoter or a PGK promoter.
  • Embodiment 52 The nucleic acid molecule of embodiment 51, wherein the promoter is the EFla promoter.
  • Embodiment 56 The vector of embodiment 55, wherein the viral vector is derived from an adenovirus, adeno-associated virus (AAV), alphavirus, flavivirus, herpes virus, measles virus, rhabdovirus, retrovirus, lentivirus, Newcastle disease virus (NDV), poxvirus or picomavirus.
  • AAV adeno-associated virus
  • alphavirus alphavirus
  • flavivirus alphavirus
  • flavivirus herpes virus
  • measles virus measles virus
  • rhabdovirus retrovirus
  • lentivirus lentivirus
  • NDV Newcastle disease virus
  • invention 57 The vector of embodiment 55, wherein the viral vector is selected from a lentiviral vector, a gamma retroviral vector, adeno-associated viral (AAV) vector, adenoviral vector, or a herpes viral vector.
  • AAV adeno-associated viral
  • Embodiment 64 The cell of embodiment 61, wherein the human T cell is a memory T cell.
  • the first protein is a chimeric antigen receptor (CAR) comprising: (a) an antibody or antigen recognition moiety that binds to one or more epitopes of a human B-cell Maturation Antigen (BCMA) polypeptide, (b) a transmembrane domain, which may optionally further comprise a hinge domain, and (c) one or more intracellular T cell signaling domains; and (ii) the second protein comprises a drug responsive domain (DRD) operably linked to an IL 15 payload, wherein said DRD is a polypeptide derived from human carbonic anhydrase II (CA2) and comprises one, two, three or four amino acid insertions, deletions or substitutions compared to SEQ ID NO: 1.
  • CAR chimeric antigen receptor
  • BCMA human B-cell Maturation Antigen
  • the second protein comprises a drug responsive domain (DRD) operably
  • Embodiment 66 The T cell of embodiment 65, wherein the T cell is a human T cell.
  • Embodiment 67 The T cell of embodiment 66, wherein the human T cell is a CD4+ T cell, a CD8+ T cell or a memory T cell.
  • Embodiment 68 The T cell of any one of embodiments 65-67, wherein the second protein comprises the DRD hCA2(Mldel, L156H) comprising the amino acid sequence of SEQ ID NO:4 and the IL15 payload is a membrane-bound IL15 (mbIL15) polypeptide N-terminal to the DRD.
  • the second protein comprises the DRD hCA2(Mldel, L156H) comprising the amino acid sequence of SEQ ID NO:4 and the IL15 payload is a membrane-bound IL15 (mbIL15) polypeptide N-terminal to the DRD.
  • Embodiment 70 The T cell of any one of embodiments 65-68, wherein the first protein comprises, from N-terminal to C-terminal: a CD8a leader sequence; an scFv directed against BCMA; a CD8a hinge domain or a CD28 hinge domain; a CD8a transmembrane domain or a CD28 hinge domain; an intracellular T cell signaling domain selected from a 4-1BB intracellular T cell signaling domain, an 0X40 intracellular T cell signaling domain, or a CD28 intracellular T cell signaling domain; and a CD3 intracellular T cell signaling domain.
  • a CD8a leader sequence an scFv directed against BCMA
  • a CD8a hinge domain or a CD28 hinge domain a CD8a transmembrane domain or a CD28 hinge domain
  • an intracellular T cell signaling domain selected from a 4-1BB intracellular T cell signaling domain, an 0X40 intracellular T cell signaling domain, or a CD28 intracellular T cell signal
  • the T cell of embodiment 70 wherein the first protein comprises, from N-terminal to C-terminal: a CD8a leader sequence; an scFv directed against BCMA; a CD8a hinge domain; a CD8a transmembrane domain; a 4-1BB intracellular T cell signaling domain; and a CD3( ⁇ intracellular T cell signaling domain.
  • Embodiment 72 The T cell of embodiment 70, wherein the first protein comprises the amino acid sequence of SEQ ID NO:34.
  • Embodiment 73 A pharmaceutical composition comprising the nucleic acid molecule of any one of embodiments 1-52, the vector of any one of embodiments 53-58, or the cell of any one of embodiments 59-64, and a pharmaceutically acceptable excipient.
  • Embodiment 76 A method of producing a modified T cell, said method comprising introducing into the T cell the nucleic acid molecule of any one of embodiments 1-52, or the vector of any one of embodiments 53-58.
  • said first nucleic acid molecule encodes a chimeric antigen receptor (CAR) comprising: (a) an antibody or antigen recognition moiety that binds to one or more epitopes of a human B-cell Maturation Antigen (BCMA) polypeptide, (b) a transmembrane domain, which may optionally further comprise a hinge domain, and (c) one or more intracellular T cell signaling domains; and
  • CAR chimeric antigen receptor
  • said second nucleic acid molecule encodes a second protein comprising a drug responsive domain (DRD) operably linked to an IL15 payload, wherein said DRD is a polypeptide derived from human carbonic anhydrase II (CA2) and comprises one, two, three or four amino acid insertions, deletions or substitutions compared to SEQ ID NO:1.
  • DRD drug responsive domain
  • Embodiment 80 The method of any one of embodiments 77-79, wherein the CAR comprises, from N-terminal to C-terminal: a CD8a leader sequence; an scFv directed against BCMA; a CD8a hinge domain or a CD28 hinge domain; a CD8a transmembrane domain or a CD28 hinge domain; an intracellular T cell signaling domain selected from a 4-1BB intracellular T cell signaling domain, an 0X40 intracellular T cell signaling domain, or a CD28 intracellular T cell signaling domain; and a CD3( ⁇ intracellular T cell signaling domain; or wherein the CAR comprises, from N-terminal to C-terminal: a CD8a leader sequence; an scFv directed against BCMA; a CD8a hinge domain; a CD8a transmembrane domain; a 4- IBB intracellular T cell signaling domain; and a CD3( ⁇ intracellular T cell signaling domain.
  • Embodiment 82 A method of producing a modified cell, said method comprising introducing into a cell a nucleic acid molecule, wherein: said nucleic acid molecule encodes a protein comprising a drug responsive domain (DRD) operably linked to an ELI 5 payload, wherein said DRD is a polypeptide derived from human carbonic anhydrase II (CA2) and comprises one, two, three or four amino acid insertions, deletions or substitutions compared to SEQ ID NO: 1; wherein said cell expresses a chimeric antigen receptor (CAR) comprising: (a) an antibody or antigen recognition moiety that binds to one or more epitopes of a human B-cell Maturation Antigen (BCMA) polypeptide, (b) a transmembrane domain, which may optionally further comprise a hinge domain, and (c) one or more intracellular T cell signaling domains.
  • DRD drug responsive domain
  • CA2 human carbonic anhydrase II
  • Embodiment 84 A method of producing a modified cell, said method comprising introducing into a cell a nucleic acid molecule, wherein: said nucleic acid molecule encodes a chimeric antigen receptor (CAR) comprising: (a) an antibody or antigen recognition moiety that binds to one or more epitopes of a human B-cell Maturation Antigen (BCMA) polypeptide, (b) a transmembrane domain, which may optionally further comprise a hinge domain, and (c) one or more intracellular T cell signaling domains; wherein said cell expresses a protein comprising a drug responsive domain (DRD) operably linked to an IL15 payload, wherein said DRD is a polypeptide derived from human carbonic anhydrase II (CA2) and comprising one, two, three or four amino acid insertions, deletions or substitutions compared to SEQ ID NO: 1.
  • CAR chimeric antigen receptor
  • BCMA human B-cell Maturation
  • Embodiment 85 The method of embodiment 84, wherein the CAR comprises, from N-terminal to C-terminal: a CD8a leader sequence; an scFv directed against BCMA; a CD8a hinge domain or a CD28 hinge domain; a CD8a transmembrane domain or a CD28 hinge domain; an intracellular T cell signaling domain selected from a 4-1BB intracellular T cell signaling domain, an 0X40 intracellular T cell signaling domain, or a CD28 intracellular T cell signaling domain; and a CD3( ⁇ intracellular T cell signaling domain; or wherein the CAR comprises, from N-terminal to C-terminal: a CD8a leader sequence; an scFv directed against BCMA; a CD8a hinge domain; a CD8a transmembrane domain; a 4- IBB intracellular T cell signaling domain; and a CD3( ⁇ intracellular T cell signaling domain.
  • (Embodiment 90) A method of treating a B cell malignancy in a subject in need thereof, said method comprising: (a) administering to the subject a therapeutically effective amount of the nucleic acid molecule of any one of embodiments 1-52, or the vector of any one of embodiments 53-58, the cell of any one of embodiments 59-64 or the pharmaceutical composition of embodiment 73; and (b) administering a therapeutically effective amount of a stimulus to the subject, wherein the hCA2 DRD is responsive to the stimulus, and wherein expression of ELI 5 is modulated in response to the stimulus.
  • (Embodiment 93) The method of any one of embodiments 90-92, wherein the stimulus is selected from acetazolamide, celecoxib, valdecoxib, rofecoxib, methazolamide, dorzolamide, brinzolamide, diclofenamide, ethoxzolamide, zonisamide, dansylamide, and dichlorphenamide.
  • (Embodiment 94) The method of embodiment 93, wherein the stimulus is acetazolamide.
  • administering the cell further comprises, (i) providing isolated autologous human T cells from the subject or providing allogeneic human T cells; (ii) transducing the autologous or allogeneic human T cells with the nucleic acid molecule of any one of embodiments 1-48 or the vector of any one of embodiments 49-54; and (iii) growing the transduced autologous or allogeneic human cells at least two-fold, to form a dosing T cell population, wherein said administering to the subject the cell comprises administering the dosing T cell population into the subject.
  • (Embodiment 96) A method of treating a B cell malignancy in a subject in need thereof, said method comprising: (a) administering to the subject a therapeutically effective amount of the T cell of any one of embodiments 65-72 or a pharmaceutical composition thereof; and (b) administering a therapeutically effective amount of a stimulus to the subject, wherein the hCA2 DRD is responsive to the stimulus, and wherein expression of IL 15 is modulated in response to the stimulus.
  • Embodiment 99 The method of any one of embodiments 96-98, wherein the stimulus is selected from acetazolamide, celecoxib, valdecoxib, rofecoxib, methazolamide, dorzolamide, brinzolamide, diclofenamide, ethoxzolamide, zonisamide, dansylamide, and dichlorphenamide.
  • the stimulus is acetazolamide.
  • administering the T cell or the pharmaceutical composition thereof further comprises, (i) providing isolated autologous human T cells from the subject or providing allogeneic human T cells; (ii) transducing the autologous or allogeneic human T-cells with the nucleic acid molecule of any one of embodiments 1-48 or the vector of any one of embodiments 49-54; and (iii) growing the transduced autologous or allogeneic human cells at least two-fold, to form a dosing T cell population, wherein said administering to the subject the cell comprises administering the dosing T cell population into the subject.
  • administering the cell further comprises, prior to (a), (i) providing isolated autologous human T cells from the subject or providing allogeneic human T cells; (ii) transducing the autologous or allogeneic human T cells with the nucleic acid molecule of any one of embodiments 1-48 or the vector of any one of embodiments 49-54; and (iii) growing the transduced autologous or allogeneic human cells at least two-fold, to form a dosing T cell population, wherein said administering to the subject the cell in (a) comprises administering the dosing T cell population into the subject.
  • Embodiment 103 A method of treating multiple myeloma in a subject in need thereof, the method comprising (a) administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising human T cells, wherein the human T cells comprise a first protein and a second protein, wherein: (i) the first protein is a chimeric antigen receptor (CAR) comprising: (1) an antibody or antigen recognition moiety that binds to one or more epitopes of a human B-cell Maturation Antigen (BCMA) polypeptide, (2) a transmembrane domain, which may optionally further comprise a hinge domain, and (3) one or more intracellular T cell signaling domains; and (ii) the second protein comprises a drug responsive domain (DRD) operably linked to an ELI 5 payload, wherein said DRD is a polypeptide derived from human carbonic anhydrase II (CA2) and comprising one, two, three or four amino acid insertions, deletions or substitutions
  • CAR
  • Embodiment 104 The method of embodiment 103, wherein the CAR comprises, from N-terminal to C-terminal: a CD8a leader sequence; an scFv directed against BCMA; a CD8a hinge domain or a CD28 hinge domain; a CD8a transmembrane domain or a CD28 hinge domain; an intracellular T cell signaling domain selected from a 4-1BB intracellular T cell signaling domain, an 0X40 intracellular T cell signaling domain, or a CD28 intracellular T cell signaling domain; and a CD3(j intracellular T cell signaling domain.
  • a CD8a leader sequence an scFv directed against BCMA
  • a CD8a hinge domain or a CD28 hinge domain a CD8a transmembrane domain or a CD28 hinge domain
  • an intracellular T cell signaling domain selected from a 4-1BB intracellular T cell signaling domain, an 0X40 intracellular T cell signaling domain, or a CD28 intracellular T cell signaling domain
  • Embodiment 110 A combination of a first protein and a second protein, said combination for use in immunotherapy, wherein: (i) the first protein of the combination is a chimeric antigen receptor (CAR) comprising: (a) an antibody or antigen recognition moiety that binds to one or more epitopes of a human B-cell Maturation Antigen (BCMA) polypeptide, (b) a transmembrane domain, which may optionally further comprise a hinge domain, and (c) one or more intracellular T cell signaling domains; and (ii) the second protein of the combination comprises a drug responsive domain (DRD) operably linked to an IL 15 payload, wherein said DRD is a polypeptide derived from human carbonic anhydrase II (CA2) and comprising one, two, three or four amino acid insertions, deletions or substitutions compared to SEQ ID NO:1.
  • CAR chimeric antigen receptor
  • BCMA human B-cell Maturation Antigen
  • a composition comprising: (a) a first nucleic acid molecule comprising: (i) a polynucleotide encoding a chimeric antigen receptor (CAR) comprising: (1) an antibody or antigen recognition moiety that binds to one or more epitopes of a human B-cell Maturation Antigen (BCMA) polypeptide, (2) a transmembrane domain, which may optionally further comprise a hinge domain, and (3) one or more intracellular T cell signaling domains; and (b) a second nucleic acid molecule comprising: (ii) a polynucleotide encoding a protein comprising a drug responsive domain (DRD) operably linked to an IL 15 payload, wherein said DRD is a polypeptide derived from human carbonic anhydrase II (CA2) and comprising one, two, three or four amino acid insertions, deletions or substitutions compared to SEQ ID NO:1; and (CAR) a chimeric antigen receptor (
  • An anti-BCMA cell-expressing immunotherapy system comprising one or more vectors comprising: a) a first nucleic acid molecule encoding a chimeric antigen receptor (CAR) comprising: (1) an antibody or antigen recognition moiety that binds to one or more epitopes of a human B-cell Maturation Antigen (BCMA) polypeptide, (2) a transmembrane domain, which may optionally further comprise a hinge domain, and (3) one or more intracellular T cell signaling domains; and (b) a second nucleic acid molecule encoding a protein comprising a drug responsive domain (DRD) operably linked to an ELI 5 payload, wherein said DRD is a polypeptide derived from human carbonic anhydrase II (CA2) and comprising one, two, three or four amino acid insertions, deletions or substitutions compared to SEQ ID NO: 1, wherein components (a) and (b) are located on the same or different
  • Example 1 T cell transduction with Anti-BCMA CAR ACZ-Regulated mbIL15 Construct
  • the present example demonstrates methods that may be used for preparing constructs comprising an anti-BCMA CAR and an acetazolamide (ACZ)-regulated membrane-bound ELI 5 (mbIL15), and methods that may be used for transduction of T cells with these constructs. Assembly of Constructs
  • OT-BCMA-IL15-007, OT-BCMA-IL15-008, and OT-BCMA-IL 15-009 were each constructed in a pELNS vector (a third-generation self-inactivating lentiviral expression vector) using standard molecular biology techniques.
  • Gene fragments encoding mbIL15 (for OT- BCMA-IL15-007), CA2 (Mldel, L156H)-mbIL15 (for OT-BCMA-IL15-008) or CA2 (Mldel)- mbIL15 (for OT-BCMA-IL 15-009) were isolated using standard PCR techniques.
  • the nucleic acid sequence for mbIL15 encodes a leader sequence, codon-optimized IL15, GS linker, B7-1 hinge, transmembrane domain and cytoplasmic tail. If a CA2 domain is present, the nucleic acid molecule also encodes a 30 amino acid GS linker.
  • Gene fragments encoding an anti-BCMA CAR which comprises a CD8a leader sequence, anti-BCMA scFv, CD8a hinge and transmembrane domain, 4-1BB intracellular domain and CD3( ⁇ intracellular domain were purchased from Quintara Biosciences using Gibson assembly (NEBuilder Hifi). Gene fragments encoding the anti-BCMA CAR and a P2A were isolated using standard PCR techniques.
  • FIG. 1 provides a schematic of OT-BCMA-IL15-008.
  • HEK293T cells were seeded on collagen coated tissue culture plates until 70% confluent.
  • Cells were transfected with pELNS transfer vector carrying OT-BCMA-IL15-007, OT-BCMA-IL15-008, and OT-BCMA-IL15-009 constructs, as well as packaging plasmids (pRSV.REV, pMDLg/p.RRE and pMD2.G) using Lipofectamine 3000 transfection reagent in Opti-MEM media. Media was replaced 6-8 hrs post-transfection with serum-free media. Supernatants containing virus were harvested 24 hr post-transfection, fresh media was added, and supernatants were harvested again at 48 hr post-transfection. Viral supernatants were filtered to remove debris and concentrated by ultracentrifugation in 20% sucrose gradient. Virus were resuspended, aliquoted and stored at -80C freezer.
  • the amino acid sequences of the constructs of OT-BCMA-IL15-007, OT-BCMA- IL15-008, and OT-BCMA-IL15-009 are SEQ ID NO: 36, SEQ ID NO: 38 and SEQ ID NO: 40, respectively.
  • the nucleic acid sequences of the constructs of OT-BCMA-IL 15-007, OT-BCMA- IL15-008, and OT-BCMA-IL15-009 are SEQ ID NO: 37, SEQ ID NO: 39 and SEQ ID NO: 41, respectively.
  • T cells were isolated from Leukopaks collected from human healthy donors. After PBMC isolation with Ficoll gradients, T cells were isolated using negative selection kit (StemCell Technologies) according to manufacturer’s protocol. T cells were resuspended in cell freezing media (Bambanker), aliquoted and stored in liquid nitrogen.
  • T cells were thawed, cells were washed and counted. T cells were mixed with CD3/CD28 beads (Invitrogen cat#l 1141D) at 3 : 1 bead to T cell ratio. 5xl0 5 cells/well were added to 24-well plates in 1 mL media. Cells were activated for 24 hrs. Next day, lentivirus was thawed and added to each well at different volumes. After 24 hrs, 1 mL of fresh media was added to wells, and cells were expanded by adding equal volume of fresh media every 2-3 days to keep cell density at 0.5-lxl0 6 /mL. Cells were analyzed by flow cytometry to confirm viability and anti-BCMA CAR and mbIL15 expression on day 5 or 6, and again at the end of expansion on day 9 or 10.
  • CD3/CD28 beads Invitrogen cat#l 1141D
  • the present example shows that ACZ can regulate mbIL15 expression in BCMA CAR T cells, demonstrating that presence of the BCMA CAR does not affect regulation of mbIL15.
  • the present example also confirms that BCMA CAR and mbIL15 are expressed after T cell expansion.
  • Human T cells were transduced with OT-BCMA-IL 15-008 and expanded in accordance with the methods described in Example 1 above.
  • FIG. 2B shows the CAR expression levels on T cells transduced with an empty vector control (pELNS-001), a control BCMA CAR construct that does not include IL15 (OT-BCMA-011), a construct expressing constitutive mbIL15 (OT-BCMA-IL15- 007), and a construct containing CA2-regulated mbIL15 (OT-BCMA-IL15-008).
  • FIG. 2C shows the mbIL15 expression as gMFI by flow cytometry within the BCMA-CAR+ T cell population.
  • the results demonstrate that with the OT-BCMA-IL15-008 construct, mbIL15 expression is regulated on BCMA CAR T cells.
  • the off-state levels of mbIL15-CA2 generated by the OT-BCMA-IL15- 008 construct in the absence of ACZ are about five-fold lower than constitutive mbIL15 levels generated by the OT-BCMA-IL 15-007 construct.
  • the peak on-state levels of mbIL15-CA2 in the presence of ACZ are about two-fold lower than the levels of mbIL15 obtained on the constitutive OT-BCMA-IL15-007 construct.
  • the off-state levels of mbIL15-CA2 in the absence of ACZ are about three-fold lower than the on-state levels of mbIL15-CA2 in the presence of ACZ.
  • the present example shows that a BCMA CAR is functional in transduced T cells expressing an ACZ-regulated mbIL15 as measured by cytotoxicity and cytokine secretion (interferon-y (IFN-y) and/or IL-2 secretion) after co-culture with BCMA-expressing multiple myeloma cell lines, KMS11 cells and RPMI-8226 cells.
  • IFN-y interferon-y
  • IL-2 secretion interferon-y
  • BCMA CAR activity is not affected in T cells that co-express mbIL15 and the CAR.
  • KMS11 cells JCRB Cell Bank, catalogue # JCRB1179
  • RPMI-8226 cells ATCC, catalogue # CCL-155
  • Redifect Red-Flue Perkin Elmer
  • Puromycin Puromycin for approximately 2 months to generate cell lines that stably express the luciferase reporter.
  • the resultant KMS11-luc and RPMI8226-luc cells were expanded and frozen.
  • poly-L-ornithine-coated 96 well plates were seeded with 40,000 KMS11-luc cells per well or 20,000 RPMI8226-luc cells per well in a 96 well plate in a volume of 50 pL or 25 pL, respectively.
  • T cells transduced and expanded as described in Example 1 were serially diluted.
  • 50 pL or 25 pL of the transduced T cells were added to the KMS11-luc cells or RPMI8226-luc cells, respectively, to achieve effector cell Target cell ratios of 0.03, 0.1, 0.3, 1 and 3.
  • T cells transduced with OT-BCMA-IL 15-008 were incubated with 10 pM acetazolamide (ACZ) or equivalent volume of DMSO as a vehicle control.
  • T cells transduced with empty vector (EV, also referred to herein as pELNS-001), OT-BCMA- 011, OT-IL15-292, OT-BCMA-IL15-007 and OT-BCMA-IL 15-009 were incubated with the DMSO vehicle control.
  • FIG. 3 A Secretion of IFN-y by transduced T cells co-cultured with KMS 11-luc cells is shown in FIG. 3 A. Secretion of IFN-y is low in T cells that do not express a BCMA CAR (EV and OT- IL15-292), whereas T cells expressing a BCMA CAR (OT-BCMA-011, OT-BCMA-IL15-007, OT-BCMA-IL15-008 and OT-BCMA-IL 15 -009) show robust secretion of IFN-y, which increases with an increasing E:T ratio.
  • BCMA CAR OT-BCMA-011, OT-BCMA-IL15-007, OT-BCMA-IL15-008 and OT-BCMA-IL 15 -009
  • T cells transduced with OT-BCMA-IL15- 008 and either treated with ACZ (“008+”) or treated with vehicle (“008_”) demonstrates that secretion of IFN-y is not affected by ACZ treatment or expression of mbIL15.
  • T cells transduced with a BCMA CAR are cytotoxic to KMS11- luc cells.
  • KMS 11-luc cell viability was unaffected in T cells transduced with EV.
  • KMS 11-luc cell viability decreased in all co-cultures in which the T cells were transduced with a BCMA CAR (OT-BCMA-011, OT-BCMA-IL 15 -007, OT-BCMA-IL15-008 and OT-BCMA- IL15-009).
  • Comparison of T cells transduced with OT-BCMA-IL 15-008 and either treated with ACZ (“008+”) or treated with vehicle (“008_”) demonstrates that cytotoxicity is not affected by ACZ treatment or expression of mbIL15.
  • FIG. 3C Secretion of IFN-y and IL-2 by transduced T cells co-cultured RPMI8226-luc cells is shown in FIG. 3C.
  • Secretion of IFN-y is low in T cells that do not express a BCMA CAR (pELNS-001), whereas T cells expressing a BCMA CAR (OT-BCMA-011, OT-BCMA-IL15- 007, and OT-BCMA-IL15-008) show robust secretion of IFN-y and IL-2, which increase with an increasing E:T ratio.
  • T cells transduced with a BCMA CAR are cytotoxic to RPMI8226-luc cells.
  • RPMI8226-luc cell viability was unaffected in T cells transduced with empty vector (pELNS-001).
  • Empty Vector T cells showed some nonspecific killing, only at high (10:1) effector cell to target cell (E:T) ratios.
  • RPMI8226-luc cell viability decreased in all co-cultures in which the T cells were transduced with a BCMA CAR (OT-BCMA-011, OT-BCMA-IL15-007, and OT-BCMA-IL15-008).
  • Comparison of T cells treated with ACZ or treated with vehicle (DMSO) demonstrates that cytotoxicity is not affected by ACZ treatment or expression of mbIL15.
  • Example 4 Evaluation of in vitro antigen-independent survival by BCMA CAR T cells expressing regulated mbIL15
  • mbIL15 confers cell expansion and persistence of BCMA-CAR T cells in the absence of antigen, and further demonstrates that this effect can be reversed in BCMA-CAR T cells expressing a regulated mbIL15.
  • Human T cells were transduced with Empty Vector (EV), OT-BCMA-011, OT- BCMA-IL15-007 or OT-BCMA-IL15-008, and expanded in accordance with the methods described in Example 1 above.
  • EV Empty Vector
  • OT-BCMA-011, OT- BCMA-IL15-007 or OT-BCMA-IL15-008 were transduced with Empty Vector (EV), OT-BCMA-011, OT- BCMA-IL15-007 or OT-BCMA-IL15-008, and expanded in accordance with the methods described in Example 1 above.
  • T cell media RPMI, 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, 1% non-essential amino acids, 1% sodium pyruvate and 1% HEPES
  • RPMI fetal bovine serum
  • penicillin/streptomycin 1% penicillin/streptomycin
  • non-essential amino acids 1% sodium pyruvate and 1% HEPES
  • T cell media was replaced every three days.
  • Cells transduced with EV or OT-BCMA-011 were cultured with 2 ng/mL recombinant human IL15 (rhIL15).
  • T cells transduced with OT-BCMA CAR-IL15-007 and OT-BCMA CAR-IL15- 008 were cultured in T cell media with 10 pM ACZ or DMSO as a control. T cells were counted using a Cellaca (Nexcelom Biosciences) automated cell counter.
  • FIG. 4A shows that T cells that do not express mbIL15 require administration of rhIL15 to proliferate and survive in the absence of antigen.
  • BCMA CAR T cells expressing mbIL15 expand without exogenously administered IL15 (FIG. 4A, right panel).
  • mbIL15 is expressed in T cells transduced with OT-BCMA CAR-IL15-008 and cells proliferate and survive. If ACZ is not present, mbIL15 is destabilized and the cells do not expand in the absence of antigen. T cells transduced with a BCMA CAR and a constitutively expressed mbIL15 also proliferate and survive in cell culture. The star on the graph indicates that the culture conditions were optimized only for a 10-day assay, so the fall off in cell numbers may be due to overcrowding of the cells.
  • This same experiment contained two additional experimental arms in which instead of continuous in vitro exposure to rhIL15 or ACZ, the control BCMA-011 or BCMA-IL15-008 cells were treated intermittently (or pulsed) with rhIL15 or ACZ, respectively.
  • the pulsed dosing consisted of either a 3 day on, 3 day off or a 6 day on, 6 day off pattern of exposure to rhIL15 or ACZ.
  • the BCMA CART cells displayed an intermediate level of expansion and persistence in this in vitro antigen-independent survival assay (FIG. 4C). This result indicates that alternative ACZ exposure regiments can be used to control expansion and persistence of BCMA-CARTs expressing regulated mbIL15.
  • Example 5 Evaluation of a stem cell-like memory T cell (TSCM) phenotype in BCMA CAR T cells expressing regulated mbIL15
  • the present example demonstrates that mbIL15 expression enriches for a stem celllike memory T cell phenotype in BCMA CAR T cells at the end of an antigen independent survival.
  • FIG. 5 highlights characteristic differences between T memory stem cells (TSCM) and terminally differentiated effector T cells (TIE), and illustrates that TSCM can differentiate into central memory T cells (TCM), effector memory T cells (TEM), and TTE cells.
  • TCM central memory T cells
  • TEM effector memory T cells
  • TTE TTE cells
  • Human T cells were transduced with EV, OT-BCMA-011, OT-BCMA-IL15-007 and OT-BCMA-IL15-008, and expanded in accordance with the methods described in Example 1, and then cultured for 14 days in an antigen-independent survival assay as described in Example 4.
  • the T cells were plated in a 96 well plate, centrifuged and resuspended in T cell culture medium containing a cell surface staining panel, which included a viability stain and fluorochrome-conjugated antibodies including CD3, BCMA, CCR7, CD45RA, CD27, CD45RO and CD95.
  • CD27+ CD45RO- cells were then further analyzed for expression of CCR7 and CD95 to identify Tsc -like cells, which are phenotypically CD27+ CD45RO- CCR7+ CD95+.
  • mbIL15 expression in BCMA CAR-T cells enriches the percentage of TscM-like cells compared to transduced cells that do not express mbIL15.
  • T cells that do not express mbIL15 including T cells transduced with empty vector or with a construct expressing only BCMA CAR exhibit a very low percentage of TscM-like cells.
  • T cells transduced with a constitutively-expressed mbIL15 (OT-BCMA-IL15-007) have a high percentage of TscM-like cells.
  • T cells transduced with OT-BCMA-IL15- 008 contain a destabilized form of mb IL-15 that limits expression on the cell surface, and thus OT-BCMA-IL-15-008 T cells exhibit a low percentage of TscM-like cells.
  • T cells transduced with OT-BCMA-IL15-008 expressed a stabilized version of mbIL15 and therefore exhibit a much higher percentage of TscM-like cells, indicative of the role that mbIL15 may play in promotion of this phenotype.
  • the mbIL15 construct had a similar ability to increase the frequency of TscM-like cells when used in its constitutive form (BCMA-IL 15-007) or in the CA2 regulated version (BCMA-IL15-008). While there was an elevated frequency of TscM-like cells with mbIL15-CA2 in the absence of ACZ, ACZ treatment tended to increase the frequency of TscM-like cells in the cultures for all three donors.
  • TscM-like cells maintain a higher proliferative and self-renewing capacity allowing for reconstitution of memory and effector subsets.
  • This multi-potent potential may permit administration of fewer engineered cells for adoptive cell therapy.
  • the significant longevity of TscM-like cells promotes continued persistence in the absence of tumor antigens, for example, in residual disease.
  • the mbIL15-expressing BCMA CART cells have also retained their effector functions of cytotoxicity and cytokine production at the end of the 14 day survival assay.
  • Example 4 mbIL15-expressing BCMA CARTs treated with intermittent exposure regimens of ACZ retained similar cytotoxicity and production of cytokines in response to target cells as IL15-BCMA-CARTs continuously treated with ACZ.
  • Example 5 demonstrate that intermittent treatment with ACZ can control IL 15- BCMA-CART cell expansion while retaining IL15-BCMA-CART function.
  • Example 6 T cell transduction with Anti-BCMA CAR ACZ-Regulated mbIL15 Construct
  • OT-BCMA-IL15-004, OT-BCMA-IL15-005, and OT-BCMA-IL 15-006, were each constructed in a pELNS vector using standard molecular biology techniques as described in Example 1.
  • Gene fragments encoding mbIL15 (for OT-BCMA-IL 15 -004), CA2 (Mldel, L156H)-mbIL15 (for OT-BCMA-IL15-005) or CA2 (Mldel)-mbIL15 (for OT-BCMA-IL15- 006) were isolated using standard PCR techniques.
  • the nucleic acid sequence for mbIL15 encodes a leader sequence, codon-optimized IL15, GS linker, B7-1 hinge, transmembrane domain and cytoplasmic tail. If a CA2 domain is present, the nucleic acid molecule also encodes a 30 amino acid GS linker.
  • Gene fragments encoding an anti-BCMA CAR which comprises a CD8a leader sequence, anti-BCMA scFv, CD8a hinge and transmembrane domain, 4- IBB intracellular domain and CD3 ⁇ intracellular domain were purchased from Quintara Biosciences using Gibson assembly (NEBuilder Hifi). Gene fragments encoding the anti-BCMA CAR and a P2A were isolated using standard PCR techniques.
  • the mbIL15, P2A and anti-BCMA CAR gene fragments were then inserted into a pELNS vector using Gibson assembly (NEBuilder Hifi).
  • the assembled plasmids were transformed into E.coli (NEB stable) for amplification and sequence confirmed before proceeding with virus production.
  • Lentivirus and T cell stocks were produced as described in Example 1. Transduction of T cells by lentivirus was performed as described in Example 1.
  • the amino acid sequences of the constructs of OT-BCMA-IL15-004, OT-BCMA- IL15-005, and OT-BCMA-IL15-006 are SEQ ID NO: 42, SEQ ID NO: 44 and SEQ ID NO: 46, respectively.
  • the nucleic acid sequences of the constructs of OT-BCMA-IL 15-004, OT-BCMA- IL15-005, and OT-BCMA-IL15-006 are SEQ ID NO: 43, SEQ ID NO: 45 and SEQ ID NO: 47, respectively.
  • FIG. 7A provides a schematic of the BCMA-IL15-005 construct.
  • Example 7 Evaluation of regulated mbIL15 expression in BCMA CAR T cells
  • the present example shows that ACZ can regulate mbIL15 expression in BCMA CAR T cells regardless of whether the BCMA CAR is N-terminal or C-terminal to the mbIL15-DRD.
  • ACZ can regulate mbIL15 expression in BCMA CAR T cells regardless of whether the BCMA CAR is N-terminal or C-terminal to the mbIL15-DRD.
  • Example 6 To evaluate the regulation of mbIL15 expression in T cells transduced with BCMA CAR and an ACZ-regulated mbIL15 as described in Example 6, the effect of different concentrations of ACZ on mbIL15 expression was tested essentially as described in Example 2 except that ACZ concentrations ranged from O.OO3-3OO pM.
  • the cells were gated on BCMA CAR+ T cells and IL15 expression was detected using either a fluorescently-labeled ELI 5 monoclonal antibody (IL 15 Ab) or an IL 15 receptor alpha-Fc reagent detected by an anti-human- Fc antibody (IL15Ra-Fc).
  • IL 15 Ab fluorescently-labeled ELI 5 monoclonal antibody
  • IL15Ra-Fc IL 15 receptor alpha-Fc reagent detected by an anti-human- Fc antibody
  • the geometric Mean Fluorescence Intensity (gMFI) was measured at different concentrations of ACZ, then the EC50 and fold change for BCMA-IL15-005 and BCMA-IL15-008 were determined.
  • Example 8 Evaluation of cytotoxicity and interferon-y production by BCMA CAR T cells [00426] The present example shows that a BCMA CAR is functional in transduced T cells expressing an ACZ-regulated mbIL15 N-terminal to the CAR as measured by cytotoxicity and interferon-y (IFN-y) secretion after co-culture with KMS11 cells.
  • IFN-y interferon-y
  • T cells transduced and expanded as described in Example 6 were serially diluted. 50 pL of the transduced T cells were added to the KMS11-luc cells to achieve effector celktarget cell ratios of 0.03, 0.1, 0.3, 1 and 3. T cells transduced with OT- BCMA-IL15-005 were incubated with acetazolamide (50 pL of 40 pM ACZ stock in DMSO) or 50 pL DMSO.
  • T cells transduced with empty vector (EV), OT-BCMA-011, OT-IL15-292, OT- BCMA-IL15-004 and OT-BCMA-IL 15-006 were incubated with 50 pL DMSO. After 72 hours, samples were collected and analyzed by MSD for IFNy as described in Example 3. To evaluate the cytotoxicity of the transduced cells on KMS11-luc cells, a Bright-Glo Luciferase Asay (Promega, catalogue number E2610) was run, per manufacturer’s instructions.
  • FIG. 7B Secretion of IFN-y by transduced T cells co-cultured with KMS11-luc cells is shown in FIG. 7B. Secretion of IFN-y is low in T cells transduced with an empty vector, whereas T cells expressing a BCMA CAR (OT-BCMA-011, OT-BCMA-IL 15-004, OT-BCMA-IL15-005 and OT-BCMA-IL15-006) show robust secretion of IFN-y, which increases with an increasing E:T ratio.
  • BCMA CAR OT-BCMA-011, OT-BCMA-IL 15-004, OT-BCMA-IL15-005 and OT-BCMA-IL15-006
  • T cells transduced with OT-BCMA-IL15-005 and either treated with ACZ (“005+”) or treated with vehicle (“005_”) demonstrates that secretion of IFN-y is not affected by ACZ treatment or expression of mbIL15.
  • T cells transduced with a BCMA CAR are cytotoxic to KMS11- luc cells.
  • KMS11-luc cell viability was unaffected in T cells transduced with EV.
  • KMS 11-luc cell viability decreased in all co-cultures in which the T cells were transduced with a BCMA CAR (OT-BCMA-011, OT-BCMA-IL15-004, OT-BCMA-IL15-005 and OT-BCMA- IL15-006).
  • Comparison of T cells transduced with OT-BCMA-IL 15-005 and either treated with ACZ (“005+”) or treated with vehicle (“005_”) demonstrates that cytotoxicity is not affected by ACZ treatment or expression of mbIL15.
  • Example 9 In vivo antigen-independent persistence of BCMA CAR T cells expressing constitutive or CA2-regulated mbIL15
  • T cells transduced with constructs encoding BCMA CAR and constitutive or CA2- regulated mbIL15 were tested for in vivo antigen-independent persistence in non-tumor-bearing eight-week old female NSG mice.
  • the experimental design is summarized in Table 5.
  • Example 1 Healthy human T cells were transduced and expanded, as described in Example 1. Cells were cryopreserved and assessed for BCMA CAR and mbIL15 expression, as described in Example 2, and functionally assessed for cytotoxicity and cytokine production as described in Example 3.
  • acetazolamide (ACZ) at 200 mg/kg were administered in vehicle (100% Lactated Ringer’s solution) starting one day prior to adoptive cell transfer.
  • vehicle (100% Lactated Ringer’s solution) Five million BCMA CARTs or empty vector transduced control T cells were injected per mouse intravenously via the tail vein. Animals were bled via submandibular puncture on day 7 and day 13 post adoptive cell transfer to monitor cell expansion and mbIL15 expression.
  • Red blood cells were lysed with ACK (Ammonium-Chloride-Potassium) lysing buffer.
  • the total number of human CD3+ CD45+ T cells were quantitated per 50 pL blood (FIG. 8A) and expression of surface mbIL15 was confirmed using IL15Ra-Fc and a fluorescently labeled anti-human Fc antibody by flow cytometry (FIG. 8B).
  • Example 10 In vivo efficacy of BCMA CAR expressing constitutive or CA2-regulated mbIL15
  • mice were imaged for bioluminescent intensity (BLI) on a Xenogen system and sorted into groups based on their tumor burden to ensure that all groups had equivalent tumor burden.
  • BLI bioluminescent intensity
  • ACZ acetazolamide
  • Indicated cell doses of 300,000; IxlO 6 ; or 3 xlO 6 CART cells were transferred into the animals via tail vein injection.
  • FIG. 9A demonstrates that BCMA CARTs expressing mbIL15 display higher antitumor efficacy than BCMA CAR only control T cells across the range of tested cell doses from 0.3-3 xlO 6 CARTs.
  • BCMA CARTs expressing mbIL15 result in a more potent product than BCMA CARTs lacking mbIL15.
  • FIG. 9B demonstrates that ACZ dosing controls the efficacy of mbIL15-CA2 expressing BCMA CARTs (OT-BCMA-IL 15-008), with peak on-state efficacy matching the efficacy level of BCMA CARTs expressing the constitutive mbIL15 (OT-BCMA-IL 15-007), and off-state efficacy matching that of the control BCMA CARTs without mbIL15 (OT-BCMA- 011).
  • the BCMA CARTs expressing constitutive mbIL15 or regulated mbIL15-CA2 dosed with ACZ were able to control the additional tumor rechallenge, demonstrating durable efficacy of the BCMA CARTs expressing mbIL15.
  • Example 11 Systemic IFNy cytokine exposure from BCMA CARTs expressing constitutive or CA2-regulated mbIL15 in a disseminated RPMI8226-luc xenograft model
  • Example 12 BCMA CAR-T cells remain cytotoxic and produce pro-inflammatory cytokines during chronic stimulation
  • a chronic stimulation assay was developed to determine whether BCMA-CARTs with or without mbIL15 would become exhausted after repeated antigen stimulation. Functional exhaustion was determined by measuring CART activity in terms of cytotoxic ability and cytokine production throughout the course of the chronic stimulation assay. Along with these capabilities, various activation and exhaustion markers were interrogated longitudinally over a 3 or 4 week period.
  • BCMA-CARTs prepared as described in Example 1 and RPMI-8226-luc target cells were thawed and allowed to rest overnight. BCMA- IL-15-008 cells were rested overnight. On day 0, a co-culture was set up at a 1 : 1 Effector : Target (E:T) ratio with 0.5xl0 A 6 cells each of BCMA-CARTs in a total volume of 1 mL in a 24- well plate. Control BCMA-011 CARTs were treated with 2 nM final rhIL-15 and regulated mbIL15 expressing BCMA-CARTs were treated with 25 pM ACZ.
  • the T cells were re-seeded based on their CD3+ cell numbers to match the original starting cell number of 0.5xl0 A 6 cells/mL. If there were not enough T cells to reach this number, the remaining unstained cells in the co-culture were spun down and re-seeded in a new 24-well plate. 0.5xl0 A 6 fresh target cells were added to the co-culture regardless of the CD3+ count results. Fresh ACZ and IL-15 was added to appropriate wells. On day 5, 50 pl of cell supernatants were collected and frozen down at -80° C. On day 7, the process repeated weekly like the prior week, and continued until day 21.
  • mbIL-15 enhanced the survival of BCMA CARTs upon repeated antigen stimulation (FIG. 1 IB and FIG. 12B).
  • the mbIL15 expressing BCMA-CARTs continued to carry out their effector functions, in terms of cytotoxicity and cytokine production (FIG. 11A, FIG. 11C-FIG. 11D, FIG. 12A, and FIG. 12C-FIG. 12D).
  • cytotoxicity and cytokine production FIG. 11A, FIG. 11C-FIG. 11D, FIG. 12A, and FIG. 12C-FIG. 12D.
  • Example 13 Illustrative sequences of the present disclosure
  • Table 8 provides sequences of various embodiments of the present disclosure.
  • An asterisk (*) in Table 8 indicates the translation of a stop codon.
  • AA Sequence indicates an amino acid sequence
  • NA Sequence indicates a nucleic acid sequence

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Abstract

La présente divulgation concerne des molécules d'acide nucléique, des polypeptides et des cellules se rapportant à des domaines sensibles à un médicament (DRD) qui modulent la stabilité de protéine de l'interleukine 15 humaine (IL15) en réponse à une petite molécule, conjointement avec des récepteurs antigéniques chimériques (CAR) anti-antigène de maturation des cellules B (BCMA), et des compositions et des procédés d'utilisation de ceux-ci.
PCT/US2021/050420 2020-09-16 2021-09-15 Compositions et procédés pour l'expression de récepteurs antigéniques chimériques anti-bcma ayant une il15 régulée par petites molécules dans des cellules t WO2022060806A1 (fr)

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CN112795543A (zh) * 2021-02-04 2021-05-14 华中农业大学 杂交瘤细胞株及其分泌的抗草鱼IL-15Rα单克隆抗体和应用
CN116042527A (zh) * 2022-09-07 2023-05-02 广州瑞臻再生医学科技有限公司 一种促进NK细胞分化的iPS细胞系及其构建方法与应用

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CN112795543B (zh) * 2021-02-04 2022-09-27 华中农业大学 杂交瘤细胞株及其分泌的抗草鱼IL-15Rα单克隆抗体和应用
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