WO2022099176A1 - Immunothérapies ciblant aml - Google Patents

Immunothérapies ciblant aml Download PDF

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Publication number
WO2022099176A1
WO2022099176A1 PCT/US2021/058562 US2021058562W WO2022099176A1 WO 2022099176 A1 WO2022099176 A1 WO 2022099176A1 US 2021058562 W US2021058562 W US 2021058562W WO 2022099176 A1 WO2022099176 A1 WO 2022099176A1
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Prior art keywords
polypeptide
domain
cell
seq
transmembrane domain
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PCT/US2021/058562
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English (en)
Inventor
Jordan JARJOUR
Mark POGSON
Wai-Hang LEUNG
Alexander ASTRAKHAN
Kyle S. JONES
William Crago
Angelica SANABRIA
Andrew HOLLANDS
Jacob GANO
Milton MA
John C. Timmer
Brendan P. Eckelman
Lucas Rascon
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2Seventy Bio, Inc.
Inhibrx, Inc.
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Publication of WO2022099176A1 publication Critical patent/WO2022099176A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4631—Chimeric Antigen Receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • 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
    • A61K39/464411—Immunoglobulin superfamily
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • 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
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • 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
    • C07K16/2803—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 immunoglobulin superfamily
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • 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
    • C07K16/2851—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 lectin superfamily, e.g. CD23, CD72
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/90—Isomerases (5.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y502/00—Cis-trans-isomerases (5.2)
    • C12Y502/01—Cis-trans-Isomerases (5.2.1)
    • C12Y502/01008—Peptidylprolyl isomerase (5.2.1.8), i.e. cyclophilin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • 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
    • C07K2317/626—Diabody or triabody
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells

Definitions

  • the present disclosure relates to improved adoptive cell therapies directed against acute myeloid leukemia (AML). More particularly, the disclosure relates to dual targeting chemically regulated signaling molecules, cells, and related methods of treatment using the same.
  • AML acute myeloid leukemia
  • Cancer The global burden of cancer doubled between 1975 and 2000. Cancer is the second leading cause of morbidity and mortality worldwide, with approximately 14.1 million new cases and 8.2 million cancer related deaths in 2012.
  • the most common cancers are breast cancer, lung and bronchus cancer, prostate cancer, colon and rectum cancer, bladder cancer, melanoma of the skin, non-Hodgkin lymphoma, thyroid cancer, kidney and renal pelvis cancer, endometrial cancer, leukemia, and pancreatic cancer.
  • the number of new cancer cases is projected to rise to 22 million within the next two decades.
  • CAR T cell therapy has met with limited success due to poor CAR expression, in vivo expansion of CAR T cells, rapid disappearance of the cells after infusion, disappointing clinical activity, and antigen escape.
  • the present disclosure generally relates, in part, to dimerizing agent regulated immunoreceptor complexes (DARICs) that target one or more antigens expressed on an acute myeloid leukemia (AML) cell, polynucleotides encoding the same, compositions thereof, and methods of making and using the same to treat cancer.
  • DARICs dimerizing agent regulated immunoreceptor complexes
  • a DARIC targets CD33 and CLL-1.
  • a DARIC binds a CD33 splice variant.
  • the CD33 splice variant lacks thel24 amino acids encoded by exon 2 of the human CD33 gene (CD33 C2 variant).
  • the CD33 splice variant lacks 54 carboxyterminal amino acids due to an early translation stop signal residing in exon 7a.
  • the CD33 splice variant lacks the 124 amino acids encoded by exon 2 and 54 carboxy-terminal amino acids due to an early translation stop signal residing in exon 7a.
  • a DARIC binds both full-length CD33 and a CD33 splice variant and CLL-1.
  • a non-natural cell comprises: a first polypeptide comprising an FRB multimerization domain polypeptide or variant thereof, a first transmembrane domain, a co-stimulatory domain, and/or a primary signaling domain; and a second polypeptide comprising an anti-CD33 VHH antibody that has an amino acid sequence set forth in SEQ ID NO: 3, a polypeptide linker, an anti-CLL-1 VHH antibody that has an amino acid sequence set forth in any one of SEQ ID NOs: 4-5, an FKBP multimerization domain polypeptide or variant thereof, and a second transmembrane domain; wherein a bridging factor promotes the formation of a polypeptide complex on the non-natural cell surface with the bridging factor associated with and disposed between the multimerization domains of the first and second polypeptides.
  • a non-natural cell comprises: a first polypeptide comprising an FKBP multimerization domain polypeptide or variant thereof, a first transmembrane domain, a co-stimulatory domain, and/or a primary signaling domain; and a second polypeptide comprising an anti-CD33 VHH antibody that has an amino acid sequence set forth in SEQ ID NO: 3, a polypeptide linker, an anti-CLL-1 VHH antibody that has an amino acid sequence set forth in any one of SEQ ID NOs: 4-5, an FRB multimerization domain polypeptide or variant thereof, and a second transmembrane domain; wherein a bridging factor promotes the formation of a polypeptide complex on the non-natural cell surface with the bridging factor associated with and disposed between the multimerization domains of the first and second polypeptides.
  • a non-natural cell comprises: a first polypeptide comprising an FRB multimerization domain polypeptide or variant thereof, a first transmembrane domain, a co-stimulatory domain, and/or a primary signaling domain; and a second polypeptide comprising an anti-CLL-1 VHH antibody that has an amino acid sequence set forth in any one of SEQ ID NOs: 4-5, a polypeptide linker, an anti-CD33 VHH antibody that has an amino acid sequence set forth in SEQ ID NO: 3, an FKBP multimerization domain polypeptide or variant thereof, and a second transmembrane domain; wherein a bridging factor promotes the formation of a polypeptide complex on the non-natural cell surface with the bridging factor associated with and disposed between the multimerization domains of the first and second polypeptides.
  • a non-natural cell comprises: a first polypeptide comprising an FKBP multimerization domain polypeptide or variant thereof, a first transmembrane domain, a co-stimulatory domain, and/or a primary signaling domain; and a second polypeptide comprising an anti-CLL-1 VHH antibody that has an amino acid sequence set forth in any one of SEQ ID NOs: 4-5, a polypeptide linker, an anti-CD33 VHH antibody that has an amino acid sequence set forth in SEQ ID NO: 3, an FRB multimerization domain polypeptide or variant thereof, and a second transmembrane domain; wherein a bridging factor promotes the formation of a polypeptide complex on the non-natural cell surface with the bridging factor associated with and disposed between the multimerization domains of the first and second polypeptides.
  • the FKBP multimerization domain polypeptide is FKBP12.
  • the FRB multimerization domain polypeptide is FRB T2098L.
  • the bridging factor is selected from the group consisting of: AP21967, sirolimus, everolimus, novolimus, pimecrolimus, ridaforolimus, tacrolimus, temsirolimus, umirolimus, and zotarolimus.
  • the FRB multimerization domain polypeptide is FRB T2098L
  • the FKBP multimerization domain polypeptide is FKBP 12
  • the bridging factor is sirolimus or AP21967.
  • the first transmembrane domain and the second transmembrane domain are independently selected from a polypeptide selected from the group consisting of: alpha, beta, gamma, or delta chain of the T-cell receptor, CD2, CD35, CD3s, CD3y, CD3 ⁇ , CD4, CD5, CD8a, CD9, CD 16, CD22, CD27, CD28, CD33, CD37, CD40, CD45, CD64, CD80, CD86, CD 134, CD137, CD152, CD154, CD244, CD278, amnionless (AMN), programmed cell death 1 (PDCD1), TNRF2, TNFRS14, TNFRS25, TLR2, TLR4, TLR5, TIM1, SlamFl, SlamF5, and SlamF6.
  • the first transmembrane domain and the second transmembrane domain are independently selected from the group consisting of: a CD4 transmembrane domain and a CD8a transmembrane domain.
  • the first transmembrane domain and the second transmembrane domain are different.
  • the first polypeptide comprises a CD8a transmembrane domain and the second polypeptide comprises a CD4 transmembrane domain.
  • the CD8a transmembrane domain comprises the amino acid sequence as set forth in SEQ ID NO: 40.
  • the CD8a transmembrane domain comprises the amino acid sequence as set forth in SEQ ID NO: 41.
  • the CD8a transmembrane domain comprises the amino acid sequence as set forth in SEQ ID NO: 42.
  • the first polypeptide comprises a co-stimulatory domain selected from a co-stimulatory molecule selected from the group consisting of: Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, caspase recruitment domain family member 11 (CARD 11), CD2, CD4, CD7, CD27, CD28, CD28-YMFM, CD28-H, CD30, CD40, CD54 (ICAM), CD83, CD94, CD134 (0X40), CD137 (4-1BB), CD278 (ICOS), DNAX- Activation Protein 10 (DAP10), Linker for activation of T-cells family member 1 (LAT), interleukin 2 receptor beta (IL2RP), SH2 Domain-Containing Leukocyte Protein Of 76 ED (SLP76), T cell receptor associated transmembrane adaptor 1 (TRAT1), TNFR2, TNF receptor superfamily member 14 (TNFRS14; HVE
  • the second polypeptide comprises a co-stimulatory domain selected from a co-stimulatory molecule selected from the group consisting of: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD4, CD7, CD27, CD28, CD28-YMFM, CD28-H, CD30, CD40, CD54 (ICAM), CD83, CD94, CD134 (0X40), CD137 (4-1BB), CD278 (ICOS), DAP10, IL2Rp, LAT, SLP76, TRAT1, TNFR2, TNFRS14 (HVEM), TNFRS18 (GITR), TNFRS25 (DR3), ZAP70, HAVCR1 (TIM1), SLAMF1, SLAMF5, SLAMF6, and MYD88.
  • a co-stimulatory molecule selected from the group consisting of: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR
  • the first polypeptide comprises a co-stimulatory domain selected from CD 137.
  • the second polypeptide comprises a co-stimulatory domain selected from TNFR2, 0X40, CD28, IL2Rp, or ICOS.
  • the primary signaling domain isolated from a polypeptide selected from the group consisting of: FcRy, FcRp, CD3y, CD35, CD3e, CD3 ⁇ CD22, CD79a, CD79b, and CD66d.
  • the primary signaling domain is isolated from a CD3( ⁇ polypeptide.
  • the anti-CD33 VHH antibody comprises an amino acid sequence set forth in SEQ ID NO: 3.
  • the anti-CLL-1 VHH antibody comprises an amino acid sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 5.
  • the linker polypeptide comprises a (GGGGS)I-4 polypeptide or GGS polypeptide.
  • the linker polypeptide comprises a (GGGGS)i (SEQ ID NO: 43) polypeptide.
  • the linker polypeptide comprises a (GGGGS)2 (SEQ ID NO: 44) polypeptide.
  • the linker polypeptide comprises a (GGGGSf (SEQ ID NO: 45) polypeptide.
  • the linker polypeptide comprises a (GGGGS)4 (SEQ ID NO: 46) polypeptide.
  • the first polypeptide comprises a hinge or spacer domain polypeptide disposed between the multimerization domain and the first transmembrane domain.
  • the second polypeptide comprises a hinge or spacer domain polypeptide disposed between the multimerization domain and the second transmembrane domain.
  • the hinge of the first polypeptide and the hinge of the second polypeptide are independently selected from a polypeptide selected from the group consisting of: alpha, beta, gamma, or delta chain of the T-cell receptor, CD2, CD35, CD3e, CD3y, CD3 , CD4, CD5, CD8a, CD9, CD 16, CD22, CD27, CD28, CD33, CD37, CD40, CD45, CD64, CD80, CD86, CD 134, CD137, CD152, CD154, CD244, CD278, amnionless (AMN), programmed cell death 1 (PDCD1), TNRF2, TNFRS14, TNFRS25, TLR2, TLR4, TLR5, TIM1, SlamFl, SlamF5, and SlamF6.
  • PDCD1 programmed cell death 1
  • PDCD1 programmed cell death 1
  • PDCD1
  • the spacer domain is a GGS spacer. In certain embodiments, the spacer domain is a GGR spacer.
  • a non-natural cell comprises: a first polypeptide comprising an FRB T2098L multimerization domain polypeptide, a CD8a transmembrane domain, a CD137co-stimulatory domain, and/or a CD3( ⁇ primary signaling domain; and a second polypeptide comprising an anti-CD33 VHH antibody comprising SEQ ID NO: 3, (GGGGS)I-4 (SEQ ID NOs: 43-46) polypeptide linker, an anti-CLL-1 VHH antibody comprising SEQ ID NO: 4 or SEQ ID NO: 5, an FKBP12 multimerization domain polypeptide, and a CD4 transmembrane domain; wherein a bridging factor promotes the formation of a polypeptide complex on the non-natural cell surface with the bridging factor associated with and disposed between the multimerization domains of the first and second polypeptides.
  • a non-natural cell comprises: a first polypeptide comprising an FRB T2098L multimerization domain polypeptide, a CD8a transmembrane domain, a CD137co-stimulatory domain, and/or a CD3( ⁇ primary signaling domain; and a second polypeptide comprising an anti-CLL-1 VHH antibody comprising SEQ ID NO: 4 or SEQ ID NO: 5, (GGGGS) (SEQ ID NOs: 43-46) polypeptide linker, an anti-CD33 VHH antibody comprising SEQ ID NO: 3, an FKBP12 multimerization domain polypeptide, and a CD4 transmembrane domain; wherein a bridging factor promotes the formation of a polypeptide complex on the non-natural cell surface with the bridging factor associated with and disposed between the multimerization domains of the first and second polypeptides.
  • the cell comprises a polypeptide complex comprising the first polypeptide, the second polypeptide, and a bridging factor
  • the bridging factor is selected from the group consisting of: AP21967, sirolimus, everolimus, novolimus, pimecrolimus, ridaforolimus, tacrolimus, temsirolimus, umirolimus, and zotarolimus.
  • the cell is a hematopoietic cell.
  • the cell is a T cell, an ccp T cell, or a y5 T cell.
  • the cell is a CD3+, CD4+, and/or CD8+ cell.
  • the cell is an immune effector cell.
  • the cell is a cytotoxic T lymphocytes (CTLs), a tumor infiltrating lymphocytes (TILs), or a helper T cell.
  • CTLs cytotoxic T lymphocytes
  • TILs tumor infiltrating lymphocytes
  • helper T cell a helper T cell.
  • the cell is a natural killer (NK) cell or natural killer T (NKT) cell.
  • NK natural killer
  • NKT natural killer T
  • the source of the cell is peripheral blood mononuclear cells, bone marrow, lymph nodes tissue, cord blood, thymus issue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, or tumors.
  • the FRB multimerization domain polypeptide or variant thereof and the FKBP multimerization domain polypeptide or variant thereof localize extracellularly when the first polypeptide and the second polypeptide are expressed.
  • a fusion polypeptide comprise: a first polypeptide comprising an FRB multimerization domain polypeptide or variant thereof, a first transmembrane domain, a co- stimulatory domain, and/or a primary signaling domain; a polypeptide cleavage signal; and a second polypeptide comprising an anti-CD33 VHH antibody that has an amino acid sequence set forth in SEQ ID NO: 3, a polypeptide linker, an anti-CLL-1 VHH antibody that has an amino acid sequence set forth in any one of SEQ ID NOs: 4-5, an FKBP multimerization domain polypeptide or variant thereof, and a second transmembrane domain.
  • a fusion polypeptide comprises: a first polypeptide comprising an FKBP multimerization domain polypeptide or variant thereof, a first transmembrane domain, a co- stimulatory domain, and/or a primary signaling domain; a polypeptide cleavage signal; and a second polypeptide comprising an anti-CD33 VHH antibody that has an amino acid sequence set forth in SEQ ID NO: 3, a polypeptide linker, an anti-CLL-1 VHH antibody that has an amino acid sequence set forth in any one of SEQ ID NOs: 4-5, an FRB multimerization domain polypeptide or variant thereof, and a second transmembrane domain.
  • a fusion polypeptide comprises: a first polypeptide comprising an FRB multimerization domain polypeptide or variant thereof, a first transmembrane domain, a co- stimulatory domain; and/or a primary signaling domain; a polypeptide cleavage signal; and a second polypeptide comprising an anti-CLL-1 VHH antibody that has an amino acid sequence set forth in any one of SEQ ID NOs: 4-5, a polypeptide linker, an anti-CD33 VHH antibody that has an amino acid sequence set forth in SEQ ID NO: 3, an FKBP multimerization domain polypeptide or variant thereof, and a second transmembrane domain.
  • a fusion polypeptide comprises: a first polypeptide comprising an FKBP multimerization domain polypeptide or variant thereof, a first transmembrane domain, a co- stimulatory domain, and/or a primary signaling domain; a polypeptide cleavage signal; and a second polypeptide comprising an anti-CLL-1 VHH antibody that has an amino acid sequence set forth in any one of SEQ ID NOs: 4-5, a polypeptide linker, an anti-CD33 VHH antibody that has an amino acid sequence set forth in SEQ ID NO: 3, an FRB multimerization domain polypeptide or variant thereof, and a second transmembrane domain.
  • the FKBP multimerization domain is FKBP12.
  • the FKBP multimerization domain polypeptide is FKBP12.
  • the FRB multimerization domain polypeptide is FRB
  • the bridging factor is selected from the group consisting of: AP21967, sirolimus, everolimus, novolimus, pimecrolimus, ridaforolimus, tacrolimus, temsirolimus, umirolimus, and zotarolimus.
  • the FRB multimerization domain polypeptide is FRB T2098L
  • the FKBP multimerization domain polypeptide is FKBP12
  • the bridging factor is sirolimus or AP21967.
  • the first transmembrane domain and the second transmembrane domain are independently selected from a polypeptide selected from the group consisting of: alpha, beta, gamma, or delta chain of the T-cell receptor, CD2, CD36, CD3e, CD3y, CD3 ⁇ , CD4, CD5, CD8a, CD9, CD 16, CD22, CD27, CD28, CD33, CD37, CD40, CD45, CD64, CD80, CD86, CD 134, CD137, CD152, CD154, CD244, CD278, amnionless (AMN), programmed cell death 1 (PDCD1), TNRF2, TNFRS14, TNFRS25, TLR2, TLR4, TLR5, TIM1, SlamFl, SlamF5, and SlamF6.
  • a polypeptide selected from the group consisting of: alpha, beta, gamma, or delta chain of the T-cell receptor, CD2, CD36, CD3e, CD3y, CD3 ⁇ , CD4, CD5, CD8a, CD9, CD
  • the first transmembrane domain and the second transmembrane domain are independently selected from the group consisting of: a CD4 transmembrane domain and a CD8a transmembrane domain.
  • the first transmembrane domain and the second transmembrane domain are different.
  • the first polypeptide comprises a CD8a transmembrane domain and the second polypeptide comprises a CD4 transmembrane domain.
  • the CD8a transmembrane domain comprises the amino acid sequence as set forth in SEQ ID NO: 40.
  • the CD8a transmembrane domain comprises the amino acid sequence as set forth in SEQ ID NO: 41.
  • the CD8a transmembrane domain comprises the amino acid sequence as set forth in SEQ ID NO: 42.
  • the first polypeptide comprises a co-stimulatory domain selected from a co-stimulatory molecule selected from the group consisting of: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD4, CD7, CD27, CD28, CD28-YMFM, CD28-H, CD30, CD40, CD54 (ICAM), CD83, CD94, CD134 (0X40), CD137 (4-1BB), CD278 (ICOS), DAP10, IL2R0, LAT, SLP76, TRAT1, TNFR2, TNFRS14 (HVEM), TNFRS18 (GITR), TNFRS25 (DR3), ZAP70, HAVCR1 (TIM1), SLAMF1, SLAMF5, SLAMF6, and MYD88.
  • a co-stimulatory molecule selected from the group consisting of: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR
  • the second polypeptide comprises a co-stimulatory domain selected from a co-stimulatory molecule selected from the group consisting of: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD4, CD7, CD27, CD28, CD28-YMFM, CD28-H, CD30, CD40, CD54 (ICAM), CD83, CD94, CD134 (0X40), CD137 (4-1BB), CD278 (ICOS), DAP10, IL2R , LAT, SLP76, TRAT1, TNFR2, TNFRS14 (HVEM), TNFRS18 (GITR), TNFRS25 (DR3), ZAP70, HAVCR1 (TIM1), SLAMF1, SLAMF5, SLAMF6, and MYD88.
  • a co-stimulatory molecule selected from the group consisting of: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR
  • the first polypeptide comprises a co-stimulatory domain selected from CD 137.
  • the second polypeptide comprises a co-stimulatory domain selected from TNFR2, 0X40, CD28, IL2Rp, or ICOS.
  • the primary signaling domain is isolated from a polypeptide selected from the group consisting of: FcRy, FcRp, CD3y, CD35, CD3s, CD3 ⁇ CD22, CD79a, CD79b, and CD66d.
  • the primary signaling domain is isolated from a CD3( ⁇ polypeptide.
  • the anti-CD33 VHH antibody comprises an amino acid sequence set forth in SEQ ID NO: 2.
  • the anti-CLL-1 VHH antibody comprises an amino acid sequence set forth in SEQ ID NO: 22 or SEQ ID NO: 23.
  • the linker polypeptide comprises a (GGGGS)I-4 polypeptide or GGS polypeptide.
  • the linker polypeptide comprises a (GGGGS)i (SEQ ID NO: 43) polypeptide.
  • the linker polypeptide comprises a (GGGGS)2 (SEQ ID NO: 44) polypeptide.
  • the linker polypeptide comprises a (GGGGS)3 (SEQ ID NO: 45) polypeptide.
  • the linker polypeptide comprises a (GGGGS)4 (SEQ ID NO: 46) polypeptide.
  • the first polypeptide comprises a hinge or spacer domain polypeptide disposed between the multimerization domain and the first transmembrane domain.
  • the second polypeptide comprises a hinge or spacer domain polypeptide disposed between the multimerization domain and the second transmembrane domain.
  • the hinge of the first polypeptide and the hinge of the second polypeptide are independently selected from a polypeptide selected from the group consisting of: alpha, beta, gamma, or delta chain of the T-cell receptor, CD2, CD36, CD3e, CD3y, CD3 ⁇ CD4, CD5, CD8a, CD9, CD 16, CD22, CD27, CD28, CD33, CD37, CD40, CD45, CD64, CD80, CD86, CD 134, CD137, CD152, CD154, CD244, CD278, amnionless (AMN), programmed cell death 1 (PDCD1), TNRF2, TNFRS14, TNFRS25, TLR2, TLR4, TLR5, TIM1, SlamFl, SlamF5, and SlamF6.
  • a polypeptide selected from the group consisting of: alpha, beta, gamma, or delta chain of the T-cell receptor, CD2, CD36, CD3e, CD3y, CD3 ⁇ CD4, CD5, CD8a, CD9, CD 16, CD
  • the spacer domain is a GGS spacer. In certain embodiments, the spacer domain is a GGR spacer.
  • a fusion polypeptide cell comprises: a first polypeptide comprising an FRB T2098L multimerization domain polypeptide, a CD8a transmembrane domain, a CD137co-stimulatory domain, and/or a CD3( ⁇ primary signaling domain; a polypeptide cleavage signal; and a second polypeptide comprising an anti-CD33 VHH antibody comprising SEQ ID NO: 3 a (GGGGS)I-4 (SEQ ID NOs: 43-46) polypeptide linker, an anti-CLL-1 VHH antibody comprising SEQ ID NO: 4 or SEQ ID NO: 5, an FKBP12 multimerization domain polypeptide, and a CD4 transmembrane domain; wherein a bridging factor promotes the formation of a polypeptide complex on the non-natural cell surface with the bridging factor associated with and disposed between the multimerization domains of the first and second polypeptides.
  • a fusion polypeptide cell comprises: a first polypeptide comprising an FRB T2098L multimerization domain polypeptide, a CD8a transmembrane domain, a CD137co-stimulatory domain, and/or a CD3( ⁇ primary signaling domain; a polypeptide cleavage signal; and a second polypeptide comprising an anti-CLL-1 VHH antibody comprising SEQ ID NO: 4 or SEQ ID NO: 5, a (GGGGS)I-4 (SEQ ID NOs: 43- 46) polypeptide linker, an anti-CD33 VHH antibody comprising SEQ ID NO: 3, an FKBP12 multimerization domain polypeptide, and a CD4 transmembrane domain; wherein a bridging factor promotes the formation of a polypeptide complex on the non-natural cell surface with the bridging factor associated with and disposed between the multimerization domains of the first and second polypeptides.
  • a fusion polypeptide contemplated herein comprises a polypeptide cleavage signal is a viral self-cleaving polypeptide.
  • polypeptide cleavage signal is a viral self-cleaving 2A polypeptide.
  • the polypeptide cleavage signal is a viral self-cleaving polypeptide selected from the group consisting of: a foot-and-mouth disease virus (FMDV) (F2A) peptide, an equine rhinitis A virus (ERAV) (E2A) peptide, a Thosea asigna virus (TaV) (T2A) peptide, a porcine teschovirus-1 (PTV-1) (P2A) peptide, a Theilovirus 2A peptide, and an encephalomyocarditis virus 2A peptide.
  • FMDV foot-and-mouth disease virus
  • E2A equine rhinitis A virus
  • TaV Thosea asigna virus
  • PTV-1 porcine teschovirus-1
  • P2A porcine teschovirus-1
  • Theilovirus 2A peptide a Theilovirus 2A peptide
  • a polynucleotide encodes a first or second polypeptide or s fusion polypeptide contemplated herein.
  • a vector comprising a polynucleotide contemplated.
  • the vector is an expression vector, a transposon, a piggyBAC transposon, a Sleeping Beauty transposon, a viral vector, an adenoviral vector, an adeno-associated viral (AAV) vector, a herpes virus vector, a vaccinia virus vector, a retroviral vector, or a lentiviral vector.
  • a transposon a piggyBAC transposon
  • a Sleeping Beauty transposon a viral vector
  • an adenoviral vector an adeno-associated viral (AAV) vector
  • AAV adeno-associated viral
  • herpes virus vector a vaccinia virus vector
  • retroviral vector a retroviral vector
  • lentiviral vector lentiviral vector
  • a cell comprising a fusion polypeptide, a polynucleotide, or a vector contemplated herein.
  • the cell is a hematopoietic cell.
  • the cell is an immune effector cell.
  • the cell is a T cell, an ap T cell, or a y5 T cell.
  • the cell expresses CD3+, CD4+, CD8+, or a combination thereof.
  • the cell is a cytotoxic T lymphocyte (CTL), a tumor infiltrating lymphocyte (TIL), or a helper T cell.
  • CTL cytotoxic T lymphocyte
  • TIL tumor infiltrating lymphocyte
  • helper T cell a helper T cell.
  • the cell is a natural killer (NK) cell or natural killer T (NKT) cell.
  • NK natural killer
  • NKT natural killer T
  • composition comprises a cell contemplated herein.
  • a composition comprises a physiologically acceptable carrier and a cell contemplated herein.
  • a non-natural cell comprising: a first polypeptide comprising an FRB multimerization domain polypeptide or variant thereof, a first transmembrane domain, a co-stimulatory domain, and/or a primary signaling domain; and a second polypeptide comprising an anti-CD33 VHH antibody that has an amino acid sequence set forth in SEQ ID NO: 3, an FKBP multimerization domain polypeptide or variant thereof, and a second transmembrane domain; a third polypeptide comprising an anti-CLL-1 VHH antibody that has an amino acid sequence set forth in any one of SEQ ID NOs: 4-5, an FKBP multimerization domain polypeptide or variant thereof, and a third transmembrane domain; wherein a bridging factor promotes the formation of a polypeptide complex on the non-natural cell surface with the bridging factor associated with and disposed between the multimerization domains of the first and second polypeptides and/or the first and third polypeptides.
  • a non-natural cell comprises: a first polypeptide comprising an FKBP multimerization domain polypeptide or variant thereof, a first transmembrane domain, a co-stimulatory domain, and/or a primary signaling domain; and a second polypeptide comprising an anti-CD33 VHH antibody that has an amino acid sequence set forth in SEQ ID NO: 3, an FRB multimerization domain polypeptide or variant thereof, and a second transmembrane domain; a third polypeptide comprising an anti-CLL-1 VHH antibody that has an amino acid sequence set forth in any one of SEQ ID NOs: 4-5, an FRB multimerization domain polypeptide or variant thereof, and a third transmembrane domain; wherein a bridging factor promotes the formation of a polypeptide complex on the non-natural cell surface with the bridging factor associated with and disposed between the multimerization domains of the first and second polypeptides and/or the first and third polypeptides.
  • the FKBP multimerization domain polypeptide is FKBP12.
  • the FRB multimerization domain polypeptide is FRB T2098L.
  • the bridging factor is selected from the group consisting of: AP21967, sirolimus, everolimus, novolimus, pimecrolimus, ridaforolimus, tacrolimus, temsirolimus, umirolimus, and zotarolimus.
  • the FRB multimerization domain polypeptide is FRB T2098L
  • the FKBP multimerization domain polypeptide is FKBP 12
  • the bridging factor is sirolimus or AP21967.
  • the first transmembrane domain, the second transmembrane domain, and the third transmembrane domain are independently selected from a polypeptide selected from the group consisting of: alpha, beta, gamma, or delta chain of the T-cell receptor, CD2, CD35, CD3e, CD3y, CD31 , CD4, CD5, CD8a, CD9, CD 16, CD22, CD27, CD28, CD33, CD37, CD40, CD45, CD64, CD80, CD86, CD 134, CD137, CD152, CD154, CD244, CD278, amnionless (AMN), programmed cell death 1 (PDCD1), TNRF2, TNFRS14, TNFRS25, TLR2, TLR4, TLR5, TIM1, SlamFl, SlamF5, and SlamF6.
  • a polypeptide selected from the group consisting of: alpha, beta, gamma, or delta chain of the T-cell receptor, CD2, CD35, CD3e, CD3y, CD31 , CD4,
  • the first transmembrane domain, the second transmembrane domain, and the third transmembrane domain are independently selected from the group consisting of: a CD4 transmembrane domain and a CD8a transmembrane domain.
  • the second transmembrane domain and the third transmembrane domain are the same.
  • the first polypeptide comprises a CD8a transmembrane domain and the second polypeptide and the third polypeptide each comprise a CD4 transmembrane domain.
  • the CD8a transmembrane domain comprises the amino acid sequence as set forth in SEQ ID NO: 40.
  • the CD8a transmembrane domain comprises the amino acid sequence as set forth in SEQ ID NO: 41.
  • the CD8a transmembrane domain comprises the amino acid sequence as set forth in SEQ ID NO: 42.
  • the first polypeptide comprises a co-stimulatory domain selected from a co-stimulatory molecule selected from the group consisting of: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD4, CD7, CD27, CD28, CD28-YMFM, CD28-H, CD30, CD40, CD54 (ICAM), CD83, CD94, CD134 (0X40), CD137 (4-1BB), CD278 (ICOS), DAP10, IL2R0, LAT, SLP76, TRAT1, TNFR2, TNFRS14 (HVEM), TNFRS18 (GITR), TNFRS25 (DR3), ZAP70, HAVCR1 (TIM1), SLAMF1, SLAMF5, SLAMF6, and MYD88.
  • a co-stimulatory molecule selected from the group consisting of: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR
  • the second polypeptide and/or the third polypeptide comprises a co-stimulatory domain independently selected from a co-stimulatory molecule selected from the group consisting of: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD4, CD7, CD27, CD28, CD28-YMFM, CD28-H, CD30, CD40, CD54 (ICAM), CD83, CD94, CD134 (0X40), CD137 (4-1BB), CD278 (ICOS), DAP10, IL2RP, LAT, SLP76, TRAT1, TNFR2, TNFRS14 (HVEM), TNFRS18 (GITR), TNFRS25 (DR3), ZAP70, HAVCR1 (TIM1), SLAMF1, SLAMF5, SLAMF6, and MYD88.
  • a co-stimulatory molecule selected from the group consisting of: TLR1, TLR2, TLR3, TLR4, TLR5, T
  • the first polypeptide comprises a co-stimulatory domain selected from CD 137.
  • the second polypeptide and/or third polypeptide comprises a co-stimulatory domain selected from TNFR2, 0X40, CD28, IL2RP, or ICOS.
  • the primary signaling domain is isolated from a polypeptide selected from the group consisting of: FcRy, FcRp, CD3y, CD35, CD3s, CD3 ⁇ CD22, CD79a, CD79b, and CD66d.
  • the primary signaling domain is isolated from a CD3( ⁇ polypeptide.
  • the anti-CD33 VHH antibody comprises an amino acid sequence set forth in SEQ ID NO: 3.
  • the anti-CLL-1 VHH antibody comprises an amino acid sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 5.
  • the first polypeptide comprises a hinge or spacer domain polypeptide disposed between the multimerization domain and the first transmembrane domain.
  • the second polypeptide comprises a hinge or spacer domain polypeptide disposed between the multimerization domain and the second transmembrane domain.
  • the second polypeptide comprises a hinge or spacer domain polypeptide disposed between the multimerization domain and the second transmembrane domain.
  • the hinge of the first polypeptide, the hinge of the second polypeptide, and the hinge of the third polypeptide are independently selected from a polypeptide selected from the group consisting of: alpha, beta, gamma, or delta chain of the T-cell receptor, CD2, CD38, CD3s, CD3y, CD3 ⁇ , CD4, CD5, CD8a, CD9, CD 16, CD22, CD27, CD28, CD33, CD37, CD40, CD45, CD64, CD80, CD86, CD 134, CD137, CD 152, CD 154, CD244, CD278, amnionless (AMN), programmed cell death 1 (PDCD1), TNRF2, TNFRS14, TNFRS25, TLR2, TLR4, TLR5, TIM1, SlamFl, SlamF5, and SlamF6.
  • a polypeptide selected from the group consisting of: alpha, beta, gamma, or delta chain of the T-cell receptor, CD2, CD38, CD3s, CD3y, CD3 ⁇ ,
  • the spacer domain is a GGS spacer. In certain embodiments, the spacer domain is a GGR spacer.
  • a non-natural cell comprises: a first polypeptide comprising an FRB T2098L multimerization domain polypeptide or variant thereof, a CD8a transmembrane domain, a CD137co-stimulatory domain, and/or a CD3q primary signaling domain; and a second polypeptide comprising an anti-CD33 VHH antibody comprising SEQ ID NO: 3, an FKBP12 multimerization domain polypeptide or variant thereof, and a CD4 transmembrane domain; a third polypeptide comprising an anti-CLL-1 VHH antibody comprising SEQ ID NO: 4 or SEQ ID NO: 5, an FKBP12 multimerization domain polypeptide or variant thereof, and a CD4 transmembrane domain; wherein a bridging factor promotes the formation of a polypeptide complex on the non-natural cell surface with the bridging factor associated with and disposed between the multimerization domains of the first and second polypeptides and/or the first and third polypeptides.
  • a non-natural cell comprises: a first polypeptide comprising an FKBP12 multimerization domain polypeptide or variant thereof, a CD8a transmembrane domain, a CD137co-stimulatory domain, and/or a CD3( ⁇ primary signaling domain; and a second polypeptide comprising an anti-CD33 VHH antibody comprising SEQ ID NO: 3, an FRB T2098L multimerization domain polypeptide or variant thereof, and a CD4 transmembrane domain; a third polypeptide comprising an anti-CLL-1 VHH antibody comprising SEQ ID NO: 4 or SEQ ID NO: 5, an FRB T2098L multimerization domain polypeptide or variant thereof, and a CD4 transmembrane domain; wherein a bridging factor promotes the formation of a polypeptide complex on the non-natural cell surface with the bridging factor associated with and disposed between the multimerization domains of the first and second polypeptides and/or the first and third polypeptides
  • the cell comprises a polypeptide complex comprising the first polypeptide and the second polypeptide and/or the third polypeptide, and a bridging factor associated with and disposed between the multimerization domains of the first and second polypeptides and/or the first and third polypeptides.
  • the bridging factor is selected from the group consisting of: AP21967, sirolimus, everolimus, novolimus, pimecrolimus, ridaforolimus, tacrolimus, temsirolimus, umirolimus, and zotarolimus.
  • the cell is a hematopoietic cell.
  • the cell is a T cell, an ap T cell, or a y5 T cell.
  • the cell is a CD3+, CD4+, and/or CD8+ cell.
  • the cell is an immune effector cell.
  • the cell is a cytotoxic T lymphocytes (CTLs), a tumor infiltrating lymphocytes (TILs), or a helper T cell.
  • CTLs cytotoxic T lymphocytes
  • TILs tumor infiltrating lymphocytes
  • helper T cell a helper T cell.
  • the cell is a natural killer (NK) cell or natural killer T (NKT) cell.
  • the source of the cell is peripheral blood mononuclear cells, bone marrow, lymph nodes tissue, cord blood, thymus issue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, or tumors.
  • the FRB multimerization domain polypeptide or variant thereof and the FKBP multimerization domain polypeptide or variant thereof localize extracellularly when the first polypeptide, the second polypeptide, and the third polypeptide are expressed.
  • a fusion polypeptide comprises: a first polypeptide comprising an FRB multimerization domain polypeptide or variant thereof, a first transmembrane domain, a co- stimulatory domain, and/or a primary signaling domain; a first polypeptide cleavage signal; a second polypeptide comprising an anti-CD33 VHH antibody that has an amino acid sequence set forth in SEQ ID NO: 3, an FKBP multimerization domain polypeptide or variant thereof, and a second transmembrane domain; a second polypeptide cleavage signal; a third polypeptide comprising an anti-CLL- 1 VHH antibody that has an amino acid sequence set forth in any one of SEQ ID NOs: 4-5, an FKBP multimerization domain polypeptide or variant thereof, and a third transmembrane domain.
  • a fusion polypeptide comprises: a first polypeptide comprising an FRB multimerization domain polypeptide or variant thereof, a first transmembrane domain, a co- stimulatory domain, and/or a primary signaling domain; a first polypeptide cleavage signal; a second polypeptide comprising an anti-CLL-1 VHH antibody that has an amino acid sequence set forth in any one of SEQ ID NOs: 4-5, an FKBP multimerization domain polypeptide or variant thereof, and a third transmembrane domain; a second polypeptide cleavage signal; a third polypeptide comprising an anti- CD33 VHH antibody that has an amino acid sequence set forth in SEQ ID NO: 3, an FKBP multimerization domain polypeptide or variant thereof, and a second transmembrane domain.
  • a fusion polypeptide comprises: a first polypeptide comprising an FKBP multimerization domain polypeptide or variant thereof, a first transmembrane domain, a co- stimulatory domain, and/or a primary signaling domain; a first polypeptide cleavage signal; a second polypeptide comprising an anti-CD33 VHH antibody that has an amino acid sequence set forth in SEQ ID NO: 3, an FRB multimerization domain polypeptide or variant thereof, and a second transmembrane domain; a second polypeptide cleavage signal; a third polypeptide comprising an anti-CLL- 1 VHH antibody that has an amino acid sequence set forth in any one of SEQ ID NOs: 4-5, an FRB multimerization domain polypeptide or variant thereof, and a third transmembrane domain.
  • a fusion polypeptide comprises: a first polypeptide comprising: an FKBP multimerization domain polypeptide or variant thereof, a first transmembrane domain, a co- stimulatory domain, and/or a primary signaling domain; a first polypeptide cleavage signal; a second polypeptide comprising an anti-CLL-1 VHH antibody that has an amino acid sequence set forth in any one of SEQ ID NOs: 4-5, an FRB multimerization domain polypeptide or variant thereof, and a third transmembrane domain; a second polypeptide cleavage signal; a third polypeptide comprising an anti-CD33 VHH antibody that has an amino acid sequence set forth in SEQ ID NO: 3, an FRB multimerization domain polypeptide or variant thereof, and a second transmembrane domain.
  • the FKBP multimerization domain is FKBP12.
  • the FKBP multimerization domain polypeptide is FKBP12.
  • the FRB multimerization domain polypeptide is FRB T2098L.
  • the bridging factor is selected from the group consisting of: AP21967, sirolimus, everolimus, novolimus, pimecrolimus, ridaforolimus, tacrolimus, temsirolimus, umirolimus, and zotarolimus.
  • the FRB multimerization domain polypeptide is FRB T2098L
  • the FKBP multimerization domain polypeptide is FKBP12
  • the bridging factor is sirolimus or AP21967.
  • the first transmembrane domain, the second transmembrane domain, and the third transmembrane domain are independently selected from a polypeptide selected from the group consisting of: alpha, beta, gamma, or delta chain of the T-cell receptor, CD2, CD36, CD3s, CD3y, CD3 ⁇ , CD4, CD5, CD8a, CD9, CD 16, CD22, CD27, CD28, CD33, CD37, CD40, CD45, CD64, CD80, CD86, CD 134, CD137, CD 152, CD 154, CD244, CD278, amnionless (AMN), programmed cell death 1 (PDCD1), TNRF2, TNFRS14, TNFRS25, TLR2, TLR4, TLR5, TIM1, SlamFl, SlamF5, and SlamF6.
  • a polypeptide selected from the group consisting of: alpha, beta, gamma, or delta chain of the T-cell receptor, CD2, CD36, CD3s, CD3y, CD3 ⁇ ,
  • the first transmembrane domain, the second transmembrane domain, and the third transmembrane domain are independently selected from the group consisting of: a CD4 transmembrane domain and a CD8a transmembrane domain.
  • the second transmembrane domain and the transmembrane domain are the same.
  • the first polypeptide comprises a CD8a transmembrane domain and the second polypeptide and the third polypeptide each comprise a CD4 transmembrane domain.
  • the CD8a transmembrane domain comprises the amino acid sequence as set forth in SEQ ID NO: 40. In some embodiments, the CD8a transmembrane domain comprises the amino acid sequence as set forth in SEQ ID NO: 41. In some embodiments, the CD8a transmembrane domain comprises the amino acid sequence as set forth in SEQ ID NO: 42.
  • the first polypeptide comprises a co-stimulatory domain selected from a co-stimulatory molecule selected from the group consisting of: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD4, CD7, CD27, CD28, CD28-YMFM, CD28-H, CD30, CD40, CD54 (ICAM), CD83, CD94, CD134 (0X40), CD137 (4-1BB), CD278 (ICOS), DAP10, IL2R0, LAT, SLP76, TRAT1, TNFR2, TNFRS14 (HVEM), TNFRS18 (GITR), TNFRS25 (DR3), ZAP70, HAVCR1 (TIM1), SLAMF1, SLAMF5, SLAMF6, and MYD88.
  • a co-stimulatory molecule selected from the group consisting of: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR
  • the second polypeptide and/or the third polypeptide comprises a co-stimulatory domain independently selected from a co-stimulatory molecule selected from the group consisting of: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD4, CD7, CD27, CD28, CD28-YMFM, CD28-H, CD30, CD40, CD54 (ICAM), CD83, CD94, CD134 (0X40), CD137 (4-1BB), CD278 (ICOS), DAP10, IL2RP, LAT, SLP76, TRAT1, TNFR2, TNFRS14 (HVEM), TNFRS18 (GITR), TNFRS25 (DR3), ZAP70, HAVCR1 (TIM1), SLAMF1, SLAMF5, SLAMF6, and MYD88.
  • a co-stimulatory molecule selected from the group consisting of: TLR1, TLR2, TLR3, TLR4, TLR5, T
  • the first polypeptide comprises a co-stimulatory domain selected from CD 137.
  • the second polypeptide and/or third polypeptide comprises a co-stimulatory domain selected from TNFR2, 0X40, CD28, IL2RP, or ICOS.
  • the primary signaling domain is isolated from a polypeptide selected from the group consisting of: FcRy, FcRp, CD3y, CD35, CD3s, CD3 ⁇ CD22, CD79a, CD79b, and CD66d.
  • the primary signaling domain is isolated from a CD3( ⁇ polypeptide.
  • the anti-CD33 VHH antibody comprises an amino acid sequence set forth in SEQ ID NO: 3.
  • the anti-CLL-1 VHH antibody comprises an amino acid sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 5.
  • the first polypeptide comprises a hinge or spacer domain polypeptide disposed between the multimerization domain and the first transmembrane domain.
  • the second polypeptide comprises a hinge or spacer domain polypeptide disposed between the multimerization domain and the second transmembrane domain.
  • the second polypeptide comprises a hinge or spacer domain polypeptide disposed between the multimerization domain and the second transmembrane domain.
  • the hinge of the first polypeptide, the hinge of the second polypeptide, and the hinge of the third polypeptide are independently selected from a polypeptide selected from the group consisting of: alpha, beta, gamma, or delta chain of the T-cell receptor, CD2, CD36, CD3s, CD3y, CD3 ⁇ , CD4, CD5, CD8a, CD9, CD 16, CD22, CD27, CD28, CD33, CD37, CD40, CD45, CD64, CD80, CD86, CD 134, CD137, CD 152, CD 154, CD244, CD278, amnionless (AMN), programmed cell death 1 (PDCD1), TNRF2, TNFRS14, TNFRS25, TLR2, TLR4, TLR5, TIM1, SlamFl, SlamF5, and SlamF6.
  • a polypeptide selected from the group consisting of: alpha, beta, gamma, or delta chain of the T-cell receptor, CD2, CD36, CD3s, CD3y, CD3 ⁇ ,
  • the spacer domain is a GGS spacer. In certain embodiments, the spacer domain is a GGR spacer.
  • a fusion polypeptide cell comprises: a first polypeptide comprising an FRB T2098L multimerization domain polypeptide, a CD8a transmembrane domain, a CD137co-stimulatory domain, and/or a CD3( ⁇ primary signaling domain; a first polypeptide cleavage signal; a second polypeptide comprising, an anti-CD33 VHH antibody comprising SEQ ID NO: 3, an FKBP12 multimerization domain polypeptide, and a CD4 transmembrane domain; a second polypeptide cleavage signal; a third polypeptide comprising an anti-CLL-1 VHH antibody comprising SEQ ID NO: 4 or SEQ ID NO: 5, an FKBP12 multimerization domain polypeptide, and a CD4 transmembrane domain; wherein a bridging factor promotes the formation of a polypeptide complex on the non-natural cell surface with the bridging factor associated with and disposed between the multimerization domains of the first and second
  • a fusion polypeptide cell comprises: a first polypeptide comprising an FRB T2098L multimerization domain polypeptide, a CD8a transmembrane domain, a CD137co-stimulatory domain, and/or a CD3( ⁇ primary signaling domain; a polypeptide cleavage signal; and a second polypeptide comprising an anti-CLL-1 VHH antibody comprising SEQ ID NO: 4 or SEQ ID NO: 5, an FKBP12 multimerization domain polypeptide, and a CD4 transmembrane domain; a second polypeptide cleavage signal; a third polypeptide comprising an anti-CD33 VHH antibody comprising SEQ ID NO: 3, an FKBP12 multimerization domain polypeptide, and a CD4 transmembrane domain; wherein a bridging factor promotes the formation of a polypeptide complex on the non-natural cell surface with the bridging factor associated with and disposed between the multimerization domains of the first and second poly
  • a fusion polypeptide cell comprises: a first polypeptide comprising an FKBP12 multimerization domain polypeptide, a CD8a transmembrane domain, a CD137co-stimulatory domain, and/or a CD3 ⁇ primary signaling domain; a first polypeptide cleavage signal; a second polypeptide comprising an anti-CD33 VHH antibody comprising SEQ ID NO: 4, an FRB T2098L multimerization domain polypeptide, and a CD4 transmembrane domain; a second polypeptide cleavage signal; a third polypeptide comprising an anti-CLL-1 VHH antibody comprising SEQ ID NO: 4 or SEQ ID NO: 5, an FRB T2098L multimerization domain polypeptide, and a CD4 transmembrane domain; wherein a bridging factor promotes the formation of a polypeptide complex on the nonnatural cell surface with the bridging factor associated with and disposed between the multimerization domains of the first and second poly
  • a fusion polypeptide cell comprises: a first polypeptide comprising an FKBP12 multimerization domain polypeptide, a CD8a transmembrane domain, a CD137co-stimulatory domain, and/or a CD3( ⁇ primary signaling domain; a polypeptide cleavage signal; and a second polypeptide comprising an anti-CLL-1 VHH antibody comprising SEQ ID NO: 4 or SEQ ID NO: 5, an FRB T2098L multimerization domain polypeptide, and a CD4 transmembrane domain; a second polypeptide cleavage signal; a third polypeptide comprising an anti-CD33 VHH antibody comprising SEQ ID NO: 3, an FRB T2098L multimerization domain polypeptide, and a CD4 transmembrane domain; wherein a bridging factor promotes the formation of a polypeptide complex on the non-natural cell surface with the bridging factor associated with and disposed between the multimerization domains of the first and
  • first polypeptide cleavage signal and the second polypeptide cleavage signal is each a viral self-cleaving polypeptide. In additional embodiments, the first polypeptide cleavage signal and the second polypeptide cleavage signal is each a viral self-cleaving 2A polypeptide.
  • the first polypeptide cleavage signal and the second polypeptide cleavage signal is each a viral self-cleaving polypeptide selected from the group consisting of: a foot-and-mouth disease virus (FMDV) (F2A) peptide, an equine rhinitis A virus (ERAV) (E2A) peptide, a Thosea asigna virus (TaV) (T2A) peptide, a porcine teschovirus-1 (PTV-1) (P2A) peptide, a Theilovirus 2A peptide, and an encephalomyocarditis virus 2A peptide.
  • FMDV foot-and-mouth disease virus
  • E2A equine rhinitis A virus
  • TaV Thosea asigna virus
  • PTV-1 porcine teschovirus-1
  • P2A porcine teschovirus-1
  • Theilovirus 2A peptide a Theilovirus 2A peptide
  • a fusion polypeptide comprises an amino acid sequence set forth in any one of SEQ ID NOs: 7-16.
  • a fusion polypeptide comprises an amino acid sequence set forth in any one of SEQ ID NOs: 20-27.
  • a polynucleotide encodes a first polypeptide, a second polypeptide, and/or third polypeptide or a fusion polypeptide contemplated herein.
  • a vector comprises a polynucleotide contemplated herein.
  • the vector is an expression vector, a transposon, a piggyBAC transposon, a Sleeping Beauty transposon, a viral vector, an adenoviral vector, an adeno-associated viral (AAV) vector, a herpes virus vector, a vaccinia virus vector, a retroviral vector, or a lentiviral vector.
  • a transposon a piggyBAC transposon
  • a Sleeping Beauty transposon a viral vector
  • an adenoviral vector an adeno-associated viral (AAV) vector
  • AAV adeno-associated viral
  • herpes virus vector a vaccinia virus vector
  • retroviral vector a retroviral vector
  • lentiviral vector lentiviral vector
  • a cell comprises a fusion polypeptide, a polynucleotide, or a vector contemplated herein.
  • the cell is a hematopoietic cell.
  • the cell is an immune effector cell.
  • the cell is a T cell, an a T cell, or a y5 T cell.
  • the cell expresses CD3+, CD4+, CD8+, or a combination thereof.
  • the cell is a cytotoxic T lymphocyte (CTL), a tumor infiltrating lymphocyte (TIL), or a helper T cell.
  • CTL cytotoxic T lymphocyte
  • TIL tumor infiltrating lymphocyte
  • helper T cell a cell that kills TIL
  • NK natural killer
  • NKT natural killer T
  • composition comprises a cell contemplated herein.
  • a composition comprises a physiologically acceptable carrier and a cell contemplated herein.
  • a method of treating a subject in need thereof comprises administering the subject an effective amount of a composition contemplated herein.
  • a method of treating, preventing, or ameliorating at least one symptom of a cancer, infectious disease, autoimmune disease, inflammatory disease, and immunodeficiency, or condition associated therewith comprises administering to the subject an effective amount of a composition contemplated herein.
  • a method of treating a solid cancer comprises administering to the subject an effective amount of a composition contemplated herein.
  • the solid cancer is selected from the group consisting of: lung cancer, squamous cell carcinoma, colorectal cancer, pancreatic cancer, breast cancer, thyroid cancer, bladder cancer, cervical cancer, esophageal cancer, ovarian cancer, gastric cancer endometrial cancer, or brain cancer.
  • the solid cancer is a non-small cell lung carcinoma, head and neck squamous cell carcinoma, colorectal cancer, pancreatic cancer, breast cancer, thyroid cancer, bladder cancer, cervical cancer, esophageal cancer, ovarian cancer, gastric cancer endometrial cancer, gliomas, glioblastomas, or oligodendroglioma.
  • a method of treating a hematological malignancy comprises administering to the subject an effective amount of a composition contemplated herein.
  • the hematological malignancy is a leukemia, lymphoma, or multiple myeloma.
  • the hematological malignancy is acute myelogenous leukemia (AML).
  • AML acute myelogenous leukemia
  • Figure 1 shows a schematic of dual and tandem DARIC designs.
  • Figure 2 shows the expression of DARIC components in T cells transduced with a tandem DARIC, containing a DARIC signaling component and a DARIC binding component directed against CD33 and CLL-1. Expression was detected by staining cells with CD33-Fc, CLL-l-Fc, and anti-FRB antibody.
  • Figure 3 shows IFNy secretion from tandem DARIC T cells containing a DARIC signaling component and a DARIC binding component directed against CD33 and CLL-1 or control cells cultured with A549 cells modified to express CD33 or CLL-1, A549 cells modified to express CD33 and CLL-1, CD33- and CLL-1 -expressing tumor cell lines (MV4-11 and M0LM14), and unmodified A549 cells that express neither CD33 nor CLL- 1, at an E:T ratio of 1 : 1 in the presence of absence of rapamycin for 24 hours.
  • Figure 4 shows cytotoxicity of tandem DARIC T cells containing a DARIC signaling component and a DARIC binding component directed against CD33 and CLL-1 or control cells cultured with A549 cells modified to express CD33 or CLL-1, at an E:T ratio of 1 : 1 in the presence of absence of rapamycin for 24 hours.
  • Figure 5 shows the expression of DARIC components in T cells transduced with a dual DARIC, containing a DARIC signaling component, a DARIC binding component directed against CD33, and a DARIC binding component directed against CLL-1. Expression was detected by staining cells with CD33-Fc, CLL-l-Fc, and anti-FRB antibody.
  • Figure 6 shows IFNy secretion from dual DARIC T cells containing a DARIC signaling component, a DARIC binding component directed against CD33, and a DARIC binding component directed against CLL-1 or control cells cultured with unmodified A549 cells that express neither CD33 nor CLL-1, A549 cells modified to express CD33 or CLL- 1, and CD33- and CLL-1 -expressing tumor cell lines (AML- 193, U-937, and HL60), and at an E:T ratio of 1 : 1 in the presence of absence of rapamycin for 24 hours.
  • Figure 7 shows cytotoxicity of dual DARIC T cells containing a DARIC signaling component, a DARIC binding component directed against CD33, and a DARIC binding component directed against CLL-1 or control cells cultured with A549 cells modified to express CD33 or CLL-1, at an E:T ratio of 1 :3 in the presence of absence of rapamycin for 24 hours.
  • Figure 8A shows a cartoon of a tandem DARIC design with hinge and transmembrane elements identified for optimization.
  • Figure 8B shows a cartoon of a tandem DARIC design with the VHH/VHH linker element identified for optimization.
  • Figure 8C shows a cartoon of a tandem DARIC design with the VHH/VHH linker (SEQ ID NOs: 45 and 43) and transmembrane domain elements identified for optimization.
  • Figure 9 shows a sequence alignment of a parental tandem DARIC and various DARICs with hinge and transmembrane modifications identified (SEQ ID NOs: 23, 28, 29, and 33).
  • Figures 10A and 10B show the expression of hinge and transmembrane CD33- CLL1 tandem VHH DARIC variants in transduced T cells, as detected by CD33-Fc, CLL1- Fc, and anti-FRB binding.
  • Figures 11A and 11B show IFNg secretion from hinge and transmembrane tandem VHH DARIC variants or control cells cultured with antigen positive cell lines at an E:T ratio of 1 : 1 in the presence of absence of rapamycin for 24 hours.
  • Figures 12 shows a sequence alignment of tandem DARICs having varying VHH/VHH linker lengths (SEQ ID NOs: 84-88).
  • Figures 13A and 13B show the expression of CD33-CLL1 tandem VHH DARIC variants in transduced T cells, as detected by CD33-Fc, CLLl-Fc, and anti-FRB binding.
  • Figures 14A and 14B show IFNg secretion from tandem VHH DARIC linker variants or control cells cultured with antigen positive cell lines at an E:T ratio of 1 : 1 in the presence of absence of rapamycin for 24 hours.
  • Figures 15 shows a sequence alignment of tandem DARICs having a MH or LYC containing transmembrane sequence and varying VHH/VHH linker lengths (SEQ ID NOs: 21, 31, 34, and 39).
  • Figures 16A and 16B show the expression of combined hinge, transmembrane and linker CD33-CLL1 tandem VHH DARIC variants in transduced T cells, as detected by CD33-Fc, CLLl-Fc, and anti-FRB binding.
  • Figures 17A and 17B show IFNg secretion from combined hinge, transmembrane and linker tandem VHH DARIC variants or control cells cultured with antigen positive cell lines at an E:T ratio of 1 : 1 in the presence of absence of rapamycin for 24 hours.
  • SEQ ID NO: 1 sets forth the amino acid sequence for full-length human CD33.
  • SEQ ID NO: 2 sets forth the amino acid sequence for full-length human CD33.
  • SEQ ID NO: 3 sets forth the amino acid sequence for a CD33 VHH.
  • SEQ ID NO: 4 sets forth the amino acid sequence for a CLL-1 VHH (CLL-1.1).
  • SEQ ID NO: 5 sets forth the amino acid sequence for a CLL-1 VHH (CLL-1.2).
  • SEQ ID NO: 6 sets forth the amino acid sequence for a DARIC signaling component.
  • SEQ ID NOs: 7-10 set forth the amino acid sequences of tandem DARIC binding components directed against CD33 and CLL-1.
  • SEQ ID Nos: 11-12 set forth the amino acid sequences of DARIC binding components directed against CD33.
  • SEQ ID NOs: 13-16 set forth the amino acid sequences of DARIC binding components directed against CLL-1.
  • SEQ ID NO: 17 sets forth the amino acid sequence of a CD33 VHH DARIC fusion protein.
  • SEQ ID NO: 18 sets forth the amino acid sequence of a CLL-1.1 VHH DARIC fusion protein.
  • SEQ ID NO: 19 sets forth the amino acid sequence of a CLL-1.2 VHH DARIC fusion protein.
  • SEQ ID NOs: 20-23 set forth the amino acid sequences of exemplary DARIC fusion proteins that contain a DARIC signaling component and a tandem DARIC binding component directed against CD33 and CLL-1.
  • SEQ ID NOs: 24-27 set forth the amino acid sequences of exemplary DARIC fusion proteins that contain tandem DARIC binding components directed against CD33 and CLL-1.
  • SEQ ID NOs: 28-39 set forth the amino acid sequences for representative tandem DARIC fusion polypeptides.
  • SEQ ID Nos: 40-42 set forth the amino acid sequences for representative CD8a transmembrane variants.
  • SEQ ID NOs: 43 -57 set for the amino acid sequences for representative linkers.
  • SEQ ID NOs: 58-82 set for the amino acid sequences for representative selfcleaving peptides.
  • Xaa if present, may refer to any amino acid or the absence of an amino acid. In preferred embodiments, XaaXaa refers to the amino acid sequence SS or KP.
  • AML Acute myeloid leukemia
  • CD33 is expressed on the majority of acute myeloid leukemia (AML) leukemic blasts and, possibly, leukemic stem cells. CD33 is a challenging target because of its low expression and slow internalization; these characteristics limit antibody-dependent cell- mediated cytotoxicity and intracellular drug accumulation and, consequently, the activity of unlabeled and toxin-carrying antibodies.
  • AML acute myeloid leukemia
  • C-type lectin-like molecule-1 (CLL-1) is a type II transmembrane glycoprotein, and its expression is restricted to myeloid cells, AML blasts, and leukemia stem cells (LSCs). CLL-1 expression is absent in hematopoietic stem cells (HSCs) and bone marrow stem cells. CLL-1 is also expressed on leukemic stem cell (LSC), which possesses the ability to indefinitely self-renew and produce plenty of daughter blast cells with a specific phenotype of CLL-1, CD123, CD44, CD96, CD90, CD32, CD25, and TIM-3, acting as one of most important reasons of leukemia relapse.
  • LSC leukemic stem cell
  • the disclosure generally relates to improved compositions and methods for regulating the spatial and temporal control of adoptive cell therapies using dimerizing agent regulated immunoreceptor complexes (DARICs) that bind CD33 and CLL-1.
  • DARIC compositions targeting one or more antigens and methods contemplated herein provide numerous advantages over single antigen targeting T cell therapies existing in the art, including but not limited to, both spatial and temporal control over immune effector cell signal transduction binding and signaling activities as well as expanding the targeting capabilities of the immunotherapy, potentially increasing depth and durability of response.
  • DARIC temporal control primes the DARIC machinery for signaling through bridging factor mediated association of one or more DARIC binding components to a DARIC signaling component.
  • DARIC spatial control engages the signaling machinery through recognition of CD33 and/or CLL-1 by the DARIC binding domain(s) of the DARIC binding component. In this manner, DARIC immune effector cells become activated when both a target cell expressing CD33 and/or CLL-1 and a bridging factor are present.
  • a DARIC includes a DARIC signaling component and a tandemly formatted DARIC binding component that comprises an anti-CD33 an anti-CD33 antibody or antigen binding fragment thereof and an anti-CLL-1 antibody or antigen binding fragment thereof.
  • a DARIC includes a DARIC signaling component and dual formatted DARIC binding components wherein one DARIC binding component comprises an anti-CD33 an anti-CD33 antibody or antigen binding fragment thereof and another DARIC binding component comprises an anti-CLL-1 antibody or antigen binding fragment thereof.
  • a DARIC includes a DARIC signaling component that comprises a multimerization domain polypeptide or variant thereof, a transmembrane domain, a co-stimulatory domain; and/or a primary signaling domain; and one or more DARIC binding components that comprises an anti-CD33 antibody or antigen binding fragment thereof, e.g., a VHH antibody, a polypeptide linker, an anti-CLL-1 antibody or antigen binding fragment thereof, e.g., a VHH antibody, a multimerization domain polypeptide or variant thereof, a transmembrane domain; and optionally a co-stimulatory domain.
  • DARIC binding and DARIC signaling components may independently comprise a hinge domain. In the presence of a bridging factor, the DARIC binding and signaling components associate with one another through the bridging factor to form a functionally active DARIC that targets cells expressing CD33 and/or CLL- 1.
  • a DARIC includes a DARIC signaling component that comprises a multimerization domain polypeptide or variant thereof, a transmembrane domain, a co-stimulatory domain; and/or a primary signaling domain; a CD33 DARIC binding component that comprises an anti-CD33 antibody or antigen binding fragment thereof, e.g., a VHH antibody, a multimerization domain polypeptide or variant thereof, a transmembrane domain, and optionally a co-stimulatory domain; and a CLL-1 DARIC binding component that comprises an anti-CLL-1 antibody or antigen binding fragment thereof, e.g., a VHH antibody, a multimerization domain polypeptide or variant thereof, a transmembrane domain, and optionally a co-stimulatory domain.
  • DARIC binding and DARIC signaling components may independently comprise a hinge domain.
  • the DARIC binding and signaling components associate with one another through the bridging factor to form a functionally active DARIC that targets cells expressing CD33 and/or CLL-1.
  • the multimerization domains of the DARIC binding and DARIC signaling components are positioned extracellularly.
  • Extracellular position of the multimerization domains provides numerous advantages over intracellular positioning including, but not limited to, more efficient positioning of the anti-CD33 and anti-CLL-1 binding domains, higher temporal sensitivity to bridging factor regulation, and less toxicity due to ability to use non-immunosuppressive doses of particular bridging factors.
  • Techniques for recombinant (z.e., engineered) DNA, peptide and oligonucleotide synthesis, immunoassays, tissue culture, transformation (e.g., electroporation, lipofection), enzymatic reactions, purification and related techniques and procedures may be generally performed as described in various general and more specific references in microbiology, molecular biology, biochemistry, molecular genetics, cell biology, virology and immunology as cited and discussed throughout the present specification.
  • the term “about” or “approximately” refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.
  • the term “about” or “approximately” refers a range of quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length ⁇ 15%, ⁇ 10%, ⁇ 9%, ⁇ 8%, ⁇ 7%, ⁇ 6%, ⁇ 5%, ⁇ 4%, ⁇ 3%, ⁇ 2%, or ⁇ 1% about a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.
  • a range e.g., 1 to 5, about 1 to 5, or about 1 to about 5, refers to each numerical value encompassed by the range.
  • the range “1 to 5” is equivalent to the expression 1, 2, 3, 4, 5; or 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, or 5.0; or 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.0.
  • the term “substantially” refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher compared to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.
  • “substantially the same” refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that produces an effect, e.g., a physiological effect, that is approximately the same as a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.
  • an “antigen (Ag)” refers to a compound, composition, or substance that can stimulate the production of antibodies or a T cell response in an animal, including compositions (such as one that includes a cancer-specific protein) that are injected or absorbed into an animal.
  • exemplary antigens include but are not limited to lipids, carbohydrates, polysaccharides, glycoproteins, peptides, or nucleic acids.
  • An antigen reacts with the products of specific humoral or cellular immunity, including those induced by heterologous antigens, such as the disclosed antigens.
  • target antigen refers to a portion of CD33 or CLL-1, that a binding domain contemplated herein, is designed to bind.
  • the target antigen is an epitope of the amino acid sequence set forth in SEQ ID NO: 1 (CD33) or SEQ ID NO: 2 (CLL-1).
  • CD33 refers to a cell surface receptor also known as sialic-acid-binding immunoglobulin-like lectin 3 (SIGLEC-3) or GP67.
  • SIGLEC-3 sialic-acid-binding immunoglobulin-like lectin 3
  • GP67 a cell surface receptor also known as sialic-acid-binding immunoglobulin-like lectin 3 (SIGLEC-3) or GP67.
  • SIGLEC-3 sialic-acid-binding immunoglobulin-like lectin 3
  • CD33 has two Ig-like domains, one V-set domain and one C2-set domain.
  • CD33 plays a role in mediating cellcell interactions and in maintaining immune cells in a resting state.
  • CD33 recognizes and binds alpha-2,3- and more avidly alpha-2, 6-linked sialic acid-bearing glycans.
  • CD33 Upon engagement of ligands such as Clq or sialylated glycoproteins, two immunoreceptor tyrosine-based inhibitory motifs (ITIMs) located in CD33 cytoplasmic tail are phosphorylated by Src-like kinases such as LCK. These phosphorylations provide docking sites for the recruitment and activation of protein-tyrosine phosphatases PTPN6/SHP-1 and PTPN1 l/SHP-2.
  • CD33 also has at least three identified splice variants.
  • CDSS ⁇ 2 splice variant lacks the amino acid sequence encoded by exon 2 of the human CD33 gene (amino acids 13-139 of full-length CD33; e.g., NP_001076087.1, C2).
  • CD33 7a splice variant lacks 54 carboxy-terminal amino acids due to an early translation stop signal residing in exon 7a e.g., NP_001171079.1).
  • CD33 AE2/7a lacks the amino acids encoded by exon 2 and 54 carboxy-terminal amino acids.
  • CD33 is normally expressed on subsets of normal B cells and activated T cells and natural killer cells but is not expressed on hematopoietic stem cells or outside the hematopoietic system. Both full-length CD33 and/or CD33 splice variants are also expressed in acute myeloid leukemia (AML) blast cells in a majority of AML patients.
  • AML acute myeloid leukemia
  • C-Type Lectin-Like Molecule 1 is a member of the C-type lectin/C-type lectin-like domain (CTL/CTLD) superfamily.
  • CTL/CTLD C-type lectin-like domain
  • the gene encoding CLL-1 is located on chromosome 12.
  • CLL-1 is also known as C-Type lectin domain family 12 member A (CLEC12A), myeloid inhibitory C-Type lectin-like receptor (MICL), dendritic cell-associated lectin 2 (DCAL-2, DCAL2), and CD371.
  • the predominant isoform of CLL-1 is a 265-amino acid type II transmembrane protein with a calculated molecular mass of 31 kD.
  • CLL-1 contains an N-terminal cytoplasmic tail with an immunoreceptor tyrosine-based inhibitory motif (ITIM), a transmembrane domain, a stalk/neck region, and a C-terminal C-type lectin domain (CTLD).
  • ITIM immunoreceptor tyrosine-based inhibitory motif
  • CTL-1 can inhibit cellular activation through its cytoplasmic ITIM and negatively regulate granulocyte and monocyte function.
  • Representative CLL-1 polynucleotide sequences include NM_001207010.1, NM_001300730.1, NM_138337.5, NM_201623.3, NM_201625.1, ENST00000304361, ENST00000350667, ENST00000355690, ENST00000396507, ENST00000434319, ENST00000449959, and ENST00000543839.
  • Representative CLL-1 polypeptide sequences include Q5QGZ9, ENSP00000405244, ENSP00000345448, ENSP00000347916, ENSP00000302804, ENSP00000379764, XP_011518873.2, XP_006719099.1, NP_001193939.1, NP_612210.4, XP_011518872.1, XP_011518875.1, NP_001287659.1, XP_005253381.1, NP_963917.2, XP_006719096.1, and
  • an “antibody” refers to a binding agent that is a polypeptide comprising at least a light chain or heavy chain immunoglobulin variable region which specifically recognizes and binds an epitope of an antigen, such as a lipid, carbohydrate, polysaccharide, glycoprotein, peptide, or nucleic acid containing an antigenic determinant, such as those recognized by an immune cell.
  • an antigen such as a lipid, carbohydrate, polysaccharide, glycoprotein, peptide, or nucleic acid containing an antigenic determinant, such as those recognized by an immune cell.
  • VH refers to the variable region of an immunoglobulin heavy chain or antigen binding fragments thereof.
  • a “heavy chain 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; WO94/25591; U.S. Patent No. 6,005,079).
  • a “camelid antibody” refers to an antibody isolated from a Camel, Alpaca, or Llama that contains two VH domains and no light chains.
  • a “humanized VHH” or “humanized camelid antibody” refers to a nonhuman VHH or camelid antibody that has undergone humanization to reduce potential immunogenicity of the antibody in human recipients.
  • VHH refers an antibody fragment that contains the smallest known antigen-binding unit of the variable region of a heavy chain antibody (Koch-Nolte, et al, FASEB J., 21 : 3490-3498 (2007)).
  • linker refers to a plurality of amino acid residues between the various polypeptide domains added for appropriate spacing and conformation of the molecule.
  • a linker separates one or more VHH domains, hinge domains, multimerization domains, transmembrane domains, costimulatory domains, and/or primary signaling domains.
  • linkers suitable for use in particular embodiments contemplated herein include, but are not limited to the following amino acid sequences: GGG; DGGGS (SEQ ID NO: 48); TGEKP (SEQ ID NO: 49) (see, e.g., Liu et al., PNAS 5525-5530 (1997)); GGRR (SEQ ID NO: 50) (Pomerantz et al.
  • KESGSVSSEQLAQFRSLD (SEQ ID NO: 52) (Bird et al., 1988, Science 242:423-426), GGRRGGGS (SEQ ID NO: 53); LRQRDGERP (SEQ ID NO: 54); LRQKDGGGSERP (SEQ ID NO: 55); LRQKD(GGGS) 2 ERP (SEQ ID NO: 56).
  • flexible linkers can be rationally designed using a computer program capable of modeling both DNA-binding sites and the peptides themselves (Desjarlais & Berg, PNAS 90:2256-2260 (1993), PNAS 91: 11099-11103 (1994) or by phage display methods.
  • the linker comprises the following amino acid sequence: GSTSGSGKPGSGEGSTKG (SEQ ID NO: 57) (Cooper et al., Blood, 101(4): 1637-1644 (2003)).
  • the linker polypeptide comprises a (GGGGS)i (SEQ ID NO: 43) polypeptide.
  • the linker polypeptide comprises a (GGGGS) 2 (SEQ ID NO: 44) polypeptide.
  • the linker polypeptide comprises a (GGGGS)s (SEQ ID NO: 45) polypeptide.
  • the linker polypeptide comprises a (GGGGS)4 (SEQ ID NO: 46) polypeptide.
  • a “spacer domain,” refers to a polypeptide that separates two domains.
  • a spacer domain moves a VHH domain away from the effector cell surface to enable proper cell/cell contact, antigen binding and activation (Patel et al., Gene Therapy, 1999; 6: 412-419).
  • a spacer domain separates one or more VHH domains, multimerization domains, transmembrane domains, co-stimulatory domains, and/or primary signaling domains.
  • the spacer domain may be derived either from a natural, synthetic, semi-synthetic, or recombinant source.
  • a spacer domain is a portion of an immunoglobulin, including, but not limited to, one or more heavy chain constant regions, e.g., CH2 and CH3.
  • the spacer domain can include the amino acid sequence of a naturally occurring immunoglobulin hinge region or an altered immunoglobulin hinge region.
  • Illustrative examples of spacer domains suitable for use in particular embodiments contemplated herein include but are not limited to GGS and GGR.
  • the spacer domain is a GGS spacer.
  • the spacer domain is a GGR spacer.
  • polypeptides refers to a polypeptide that plays a role in positioning the antigen binding domain away from the effector cell surface to enable proper cell/cell contact, antigen binding and activation.
  • polypeptides may comprise one or more hinge domains between the binding domain and the multimerization domain, between the binding domain and the transmembrane domain (TM), or between the multimerization domain and the transmembrane domain.
  • the hinge domain may be derived either from a natural, synthetic, semi-synthetic, or recombinant source.
  • the hinge domain can include the amino acid sequence of a naturally occurring immunoglobulin hinge region or an altered immunoglobulin hinge region.
  • hinge domains suitable for use in particular embodiments contemplated herein include but are not limited to a alpha, beta, gamma, or delta chain of the T-cell receptor, CD2, CD36, CD3e, CD3y, CD3 CD4, CD5, CD8a, CD9, CD 16, CD22, CD27, CD28, CD33, CD37, CD40, CD45, CD64, CD80, CD86, CD 134, CD137, CD152, CD154, CD244, CD278, amnionless (AMN), programmed cell death 1 (PDCD1), TNRF2, TNFRS14, TNFRS25, TLR2, TLR4, TLR5, TIM1, SlamFl, SlamF5, and SlamF6.
  • APN amnionless
  • a “multimerization domain,” as used herein, refers to a polypeptide that preferentially interacts or associates with another different polypeptide directly or via a bridging molecule, e.g., a chemically inducible dimerizer, wherein the interaction of different multimerization domains substantially contributes to or efficiently promotes multimerization (z.e., the formation of a dimer, trimer, or multipartite complex, which may be a homodimer, heterodimer, homotrimer, heterotrimer, homomultimer, heteromultimer).
  • a multimerization domain may be derived either from a natural, synthetic, semi-synthetic, or recombinant source.
  • multimerization domains suitable for use in particular embodiments contemplated herein include an FK506 binding protein (FKBP) polypeptide or variants thereof, an FKBP-rapamycin binding (FRB) polypeptide or variants thereof, a calcineurin polypeptide or variants thereof, a cyclophilin polypeptide or variants thereof, a bacterial dihydrofolate reductase (DHFR) polypeptide or variants thereof, a PYRl-like 1 (PYL1) polypeptide or variants thereof, an abscisic acid insensitive 1 (ABI1) polypeptide or variants thereof, a GIB 1 polypeptide or variants thereof, or a GAI polypeptide or variants thereof.
  • FKBP FK506 binding protein
  • a calcineurin polypeptide or variants thereof a cyclophilin polypeptide or variants thereof
  • DHFR bacterial dihydrofolate reductase
  • FKBP-rapamycin binding polypeptide refers to an FRB polypeptide.
  • the FRB polypeptide is an FKBP12-rapamycin binding polypeptide.
  • FRB polypeptides suitable for use in particular embodiments contemplated herein generally contain at least about 85 to about 100 amino acid residues.
  • the FRB polypeptide comprises a 93 amino acid sequence Ile-2021 through Lys -2113 and a mutation of T2098L (T82L is equivalent position in 93 amino acid FRB polypeptide), with reference to GenBank Accession No. L34075.1.
  • An FRB polypeptide contemplated herein binds to an FKBP polypeptide through a bridging factor, thereby forming a ternary complex.
  • FK506 binding protein refers to an FKBP polypeptide.
  • the FKBP polypeptide is an FKBP12 polypeptide or an FKBP12 polypeptide comprising an F36V mutation.
  • an FKBP domain may also be referred to as a “rapamycin binding domain”.
  • Information concerning the nucleotide sequences, cloning, and other aspects of various FKBP species is known in the art (see, e.g., Staendart el al., Nature 346.611, 1990 (human FKBP12); Kay, Biochem. J. 314:361, 1996).
  • An FKBP polypeptide contemplated herein binds to an FRB polypeptide through a bridging factor, thereby forming a ternary complex.
  • a “bridging factor” refers to a molecule that associates with and that is disposed between two or more multimerization domains.
  • multimerization domains substantially contribute to or efficiently promote formation of a polypeptide complex only in the presence of a bridging factor.
  • multimerization domains do not contribute to or do not efficiently promote formation of a polypeptide complex in the absence of a bridging factor.
  • bridging factors suitable for use in particular embodiments contemplated herein include, but are not limited to AP21967, rapamycin (sirolimus) or a rapalog thereof, coumermycin or a derivative thereof, gibberellin or a derivative thereof, abscisic acid (ABA) or a derivative thereof, methotrexate or a derivative thereof, cyclosporin A or a derivative thereof, FKCsA or a derivative thereof, trimethoprim (Tmp)-synthetic ligand for FKBP (SLF) or a derivative thereof, or any combination thereof.
  • AP21967 rapamycin (sirolimus) or a rapalog thereof, coumermycin or a derivative thereof, gibberellin or a derivative thereof, abscisic acid (ABA) or a derivative thereof, methotrexate or a derivative thereof, cyclosporin A or a derivative thereof, FKCsA or a derivative thereof, trimethoprim (
  • Rapamycin analogs include, but are not limited to, those disclosed in U.S. Pat. No. 6,649,595, which rapalog structures are incorporated herein by reference in their entirety.
  • a bridging factor is a rapalog with substantially reduced immunosuppressive effect as compared to rapamycin.
  • rapalogs suitable for use in particular embodiments contemplated herein include, but are not limited to, everolimus, novolimus, pimecrolimus, ridaforolimus, tacrolimus, temsirolimus, umirolimus, and zotarolimus.
  • a “substantially reduced immunosuppressive effect” refers to at least less than 0.1 to 0.005 times the immunosuppressive effect observed or expected for the same dose measured either clinically or in an appropriate in vitro (e.g., inhibition of T cell proliferation) or in vivo surrogate of human immunosuppressive activity.
  • a “transmembrane domain” or “TM domain” is a domain that anchors a polypeptide to the plasma membrane of a cell.
  • the TM domain may be derived either from a natural, synthetic, semi-synthetic, or recombinant source.
  • transmembrane domains that are suitable for use in particular embodiments include, but are not limited to those derived from the alpha, beta, gamma, or delta chain of the T-cell receptor, CD2, CD38, CD3s, CD3y, CD3 , CD4, CD5, CD8a, CD9, CD 16, CD22, CD27, CD28, CD33, CD37, CD40, CD45, CD64, CD80, CD86, CD 134, CD137, CD152, CD154, CD244, CD278, amnionless (AMN), programmed cell death 1 (PDCD1), TNRF2, TLR2, TLR4, TLR5, Timl, SlamFl, SlamF5, SlamF6, HVEM, and DR3
  • APN amnionless
  • effector function refers to a specialized function of an immune effector cell. Effector function includes, but is not limited to, activation, cytokine production, proliferation and cytotoxic activity, including the release of cytotoxic factors, or other cellular responses elicited with antigen binding to the receptor expressed on the immune effector cell.
  • intracellular signaling domain refers to the portion of a protein which transduces the effector function signal and that directs the cell to perform a specialized function. While usually the entire intracellular signaling domain can be employed, in many cases it is not necessary to use the entire domain. To the extent that a truncated portion of an intracellular signaling domain is used, such truncated portion may be used in place of the entire domain as long as it transduces an effector function signal.
  • intracellular signaling domain is meant to include any truncated portion of an intracellular signaling domain necessary or sufficient to transduce an effector function signal.
  • T cell activation can be said to be mediated by two distinct classes of intracellular signaling domains: primary signaling domains that initiate antigen-dependent primary activation through the TCR (e.g., a TCR/CD3 complex) and co-stimulatory signaling domains that act in an antigen-independent manner to provide a secondary or co-stimulatory signal.
  • primary signaling domains that initiate antigen-dependent primary activation through the TCR (e.g., a TCR/CD3 complex)
  • co-stimulatory signaling domains that act in an antigen-independent manner to provide a secondary or co-stimulatory signal.
  • a “primary signaling domain” refers to an intracellular signaling domain that regulates the primary activation of the TCR complex either in a stimulatory way, or in an inhibitory way.
  • Primary signaling domains that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine-based activation motifs or ITAMs.
  • ITAM containing primary signaling domains include, but are not limited to those derived from FcRy, FcRp, CD3y, CD38, CD3s, CD31 CD22, CD79a, CD79b, and CD66d.
  • co-stimulatory signaling domain refers to an intracellular signaling domain of a co-stimulatory molecule.
  • Co- stimulatory molecules are cell surface molecules other than antigen receptors or Fc receptors that provide a second signal required for efficient activation and function of T lymphocytes upon binding to antigen.
  • TLR1 Toll-like receptor 1
  • TLR2 TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, caspase recruitment domain family member 11 (CARD11), CD2, CD4, CD7, CD27, CD28, CD28-YMFM, CD28-H, CD30, CD40, CD54 (ICAM), CD83, CD94, CD134 (0X40), CD137 (4-1BB), CD278 (ICOS), DNAX-Activation Protein 10 (DAP10), interleukin 2 receptor beta (IL2RP), Linker for activation of T-cells family member 1 (LAT), SH2 Domain- Containing Leukocyte Protein Of 76 kD (SLP76), T cell receptor associated transmembrane adaptor 1 (TRAT1), TNFR2, TNF receptor superfamily member 14 (TNFRS14; HVEM), TNF receptor super
  • TLR1 Toll-like receptor 1
  • TLR2 TLR2, TLR3, TLR4, TLR5, T
  • an “immune disorder” refers to a disease that evokes a response from the immune system.
  • the term “immune disorder” refers to a cancer, an autoimmune disease, or an immunodeficiency.
  • cancer relates generally to a class of diseases or conditions in which abnormal cells divide without control and can invade nearby tissues.
  • malignant refers to a cancer in which a group of tumor cells display one or more of uncontrolled growth (i.e., division beyond normal limits), invasion (i.e., intrusion on and destruction of adjacent tissues), and metastasis (i.e., spread to other locations in the body via lymph or blood).
  • metastasis i.e., spread to other locations in the body via lymph or blood.
  • metastasize refers to the spread of cancer from one part of the body to another.
  • a tumor formed by cells that have spread is called a “metastatic tumor” or a “metastasis.”
  • the metastatic tumor contains cells that are like those in the original (primary) tumor.
  • Benign or “non-malignanf ’ refers to tumors that may grow larger but do not spread to other parts of the body. Benign tumors are self-limited and typically do not invade or metastasize.
  • a “cancer cell” refers to an individual cell of a cancerous growth or tissue. Cancer cells include both solid cancers and liquid cancers.
  • a “tumor” or “tumor cell” refers generally to a swelling or lesion formed by an abnormal growth of cells, which may be benign, pre-malignant, or malignant. Most cancers form tumors, but liquid cancers, e.g, leukemia, do not necessarily form tumors. For those cancers that form tumors, the terms cancer (cell) and tumor (cell) are used interchangeably.
  • the amount of a tumor in an individual is the “tumor burden” which can be measured as the number, volume, or weight of the tumor.
  • relapse refers to the diagnosis of return, or signs and symptoms of return, of a cancer after a period of improvement or remission.
  • Remission is also referred to as “clinical remission,” and includes both partial and complete remission. In partial remission, some, but not all, signs and symptoms of cancer have disappeared. In complete remission, all signs and symptoms of cancer have disappeared, although cancer still may be in the body.
  • Refractory refers to a cancer that is resistant to, or non-responsive to, therapy with a particular therapeutic agent.
  • a cancer can be refractory from the onset of treatment (z.c., non-responsive to initial exposure to the therapeutic agent), or as a result of developing resistance to the therapeutic agent, either over the course of a first treatment period or during a subsequent treatment period.
  • Suitable subjects include laboratory animals (such as mouse, rat, rabbit, or guinea pig), farm animals, and domestic animals or pets (such as a cat or dog).
  • Non-human primates and, preferably, human patients are included.
  • Typical subjects include human patients that have, have been diagnosed with, or are at risk or having, cancer or another immune disorder.
  • subjects are humans that have, have been diagnosed with, or are at risk of having acute myeloid leukemia (AML).
  • AML acute myeloid leukemia
  • a patient refers to a subject that has been diagnosed with cancer or another immune disorder that can be treated with the compositions and methods disclosed elsewhere herein.
  • a patient has AML.
  • treatment includes any beneficial or desirable effect on the symptoms or pathology of a disease or pathological condition, and may include even minimal reductions in one or more measurable markers of the disease or condition being treated. Treatment can involve optionally either the reduction of the disease or condition, or the delaying of the progression of the disease or condition, e.g., delaying tumor outgrowth. “Treatment” does not necessarily indicate complete eradication or cure of the disease or condition, or associated symptoms thereof.
  • prevention indicates an approach for preventing, inhibiting, or reducing the likelihood of the occurrence or recurrence of, a disease or condition. It also refers to delaying the onset or recurrence of a disease or condition or delaying the occurrence or recurrence of the symptoms of a disease or condition. As used herein, “prevention” and similar words also includes reducing the intensity, effect, symptoms and/or burden of a disease or condition prior to onset or recurrence of the disease or condition.
  • the phrase “ameliorating at least one symptom of’ refers to decreasing one or more symptoms of the disease or condition for which the subject is being treated.
  • the disease or condition being treated is a cancer, wherein the one or more symptoms ameliorated include, but are not limited to, weakness, fatigue, shortness of breath, easy bruising and bleeding, frequent infections, enlarged lymph nodes, distended or painful abdomen (due to enlarged abdominal organs), bone or joint pain, fractures, unplanned weight loss, poor appetite, night sweats, persistent mild fever, and decreased urination (due to impaired kidney function).
  • “enhance” or “promote,” or “increase” or “expand” refers generally to the ability of a composition contemplated herein to produce, elicit, or cause a greater physiological response (i.e., downstream effects) compared to the response caused by either vehicle or a control molecule/composition.
  • a measurable physiological response may include an increase in T cell expansion, activation, persistence, cytokine secretion, and/or an increase in cancer cell killing ability, among others apparent from the understanding in the art and the description herein.
  • An “increased” or “enhanced” amount is typically a “statistically significant” amount, and may include an increase that is 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more times (e.g., 500, 1000 times) (including all integers and decimal points in between and above 1, e.g., 1.5, 1.6, 1.7. 1.8, etc.) the response produced by vehicle or a control composition.
  • a “decrease” or “lower,” or “lessen,” or “reduce,” or “abate” refers generally to the ability of composition contemplated herein to produce, elicit, or cause a lesser physiological response (z.e., downstream effects) compared to the response caused by either vehicle or a control molecule/composition.
  • a “decrease” or “reduced” amount is typically a “statistically significant” amount, and may include a decrease that is 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more times (e.g., 500, 1000 times) (including all integers and decimal points in between and above 1, e.g., 1.5, 1.6, 1.7. 1.8, etc.) the response (reference response) produced by vehicle, a control composition, or the response in a particular cell lineage.
  • ком ⁇ онент or “preserve,” or “maintenance,” or “no change,” or “no substantial change,” or “no substantial decrease” refers generally to the ability of a composition contemplated herein to produce, elicit, or cause a substantially similar or comparable physiological response ( .e., downstream effects) in a cell, as compared to the response caused by either vehicle, a control molecule/composition, or the response in a particular cell lineage.
  • a comparable response is one that is not significantly different or measurable different from the reference response.
  • DARICs that recognize one or more target antigens offer numerous advantages over immunotherapies targeting single antigens including but not limited to increased efficacy in the depth and durability of treatment.
  • the DARIC architecture provides for advantages in temporal and spatial control of the immunotherapy.
  • a DARIC receptor that binds one or more target antigens comprises one or more DARIC binding components that comprises an anti-CD33 antibody or antigen binding fragment thereof and an anti-CLL-1 antibody or antigen binding fragment thereof, wherein expression of the binding component s) redirects cytotoxicity of immune effector cells toward cancer cells expressing CD33 and/or CLL-1.
  • DARIC receptor or “DARIC” are used interchangeably and refer to one or more non-naturally occurring polypeptides that transduces an immunostimulatory signal in an immune effector cell upon exposure to a target cell expressing full-length CD33 or a CD33 splice variant and/or CLL-1 and a multimerizing agent or bridging factor, e.g., stimulating immune effector cell activity and function, increasing production and/or secretion of proinflammatory cytokines.
  • a multimerizing agent or bridging factor e.g., stimulating immune effector cell activity and function, increasing production and/or secretion of proinflammatory cytokines.
  • a DARIC contemplated herein comprises a multi-chain chimeric receptor comprising a DARIC signaling component and one or more DARIC binding components comprising a VHH domain that binds full-length CD33 and/or a CD33 splice variant and/or a VHH domain that binds CLL-1.
  • a DARIC contemplated herein comprises a multi-chain chimeric receptor comprising a DARIC signaling component and dual formatted DARIC binding components, a DARIC binding comprising a VHH domain that binds full-length CD33 and/or a CD33 splice variant and a DARIC binding component comprising a VHH domain that binds CLL-1.
  • a DARIC contemplated herein comprises a multi-chain chimeric receptor comprising a DARIC signaling component and a tandem formatted DARIC binding component comprising a VHH domain that binds full-length CD33 and/or a CD33 splice variant and a VHH domain that binds CLL-1.
  • a DARIC signaling component and one or more DARIC binding components are expressed from the same cell. In another embodiment, a DARIC signaling component and one or more DARIC binding components are expressed from different cells. In a particular embodiment, a DARIC signaling component is expressed from a cell and one or more DARIC binding components are supplied exogenously, as a polypeptide. In one embodiment, one or more DARIC binding components are pre-loaded with a bridging factor and supplied exogenously to a cell expressing a DARIC signaling component.
  • a DARIC comprises the amino acid sequence set forth in any one or more of SEQ ID NOs: 6-16. In some embodiments, a DARIC comprises a DARIC signaling component comprising the amino acid sequence set forth in SEQ ID NO: 6 and a tandem DARIC binding component comprising an amino acid sequence set forth in any one of SEQ ID NOs: 7-10.
  • a DARIC comprises a DARIC signaling component comprising the amino acid sequence set forth in SEQ ID NO: 6, a DARIC binding component comprising an amino acid sequence set forth in any one of SEQ ID NOs: 11-12, and a DARIC binding component comprising an amino acid sequence set forth in any one of SEQ ID NOs: 13-16.
  • DARIC signaling component or “DARIC signaling polypeptide” are used interchangeably and refer to a polypeptide comprising one or more multimerization domains, a transmembrane domain, and one or more intracellular signaling domains.
  • a DARIC signaling component comprises a multimerization domain, a transmembrane domain, a co-stimulatory domain and/or a primary signaling domain.
  • a DARIC signaling component comprises a first multimerization domain, a first transmembrane domain, a first co-stimulatory domain and/or a primary signaling domain.
  • a DARIC signaling component comprises one or more multimerization domains.
  • DARIC signaling components contemplated herein include, but are not limited to, an FK506 binding protein (FKBP) polypeptide or variants thereof, an FKBP-rapamycin binding (FRB) polypeptide or variants thereof, a calcineurin polypeptide or variants thereof, a cyclophilin polypeptide or variants thereof, a bacterial dihydrofolate reductase (DHFR) polypeptide or variants thereof, a PYRl-like 1 (PYL1) polypeptide or variants thereof and an abscisic acid insensitive 1 (ABI1) polypeptide or variants thereof.
  • FKBP FK506 binding protein
  • a calcineurin polypeptide or variants thereof a cyclophilin polypeptide or variants thereof
  • DHFR bacterial dihydrofolate reductase
  • PYL1 PYRl-like 1
  • ABSI1 abscisic acid insensitive 1
  • a DARIC signaling component comprises an FRB polypeptide comprising a T2098L mutation, or variant thereof. In certain preferred embodiments, a DARIC signaling component comprises an FKBP12 polypeptide or variant thereof.
  • a DARIC signaling component comprises a hinge domain.
  • a DARIC binding component also comprises a hinge, it can be the same hinge as the DARIC signaling component or a different hinge than the one used in the DARIC signaling component.
  • Illustrative hinge domains suitable for use in a DARIC signaling component described herein include the hinge region derived from the extracellular regions of alpha, beta, gamma, or delta chain of the T-cell receptor, CD2, CD35, CD3s, CD3y, CD3 ⁇ , CD4, CD5, CD8a, CD9, CD 16, CD22, CD27, CD28, CD33, CD37, CD40, CD45, CD64, CD80, CD86, CD 134, CD137, CD152, CD154, CD244, CD278, amnionless (AMN), programmed cell death 1 (PDCD1), TNRF2, TNFRS14, TNFRS25, TLR2, TLR4, TLR5, TIM1, SlamFl, SlamF5, and SlamF6, which may be wild-type hinge regions from these molecules or may be altered.
  • APN amnionless
  • hinge domains suitable for use in a DARIC signaling component described herein include the hinge region derived from the extracellular regions of type 1 membrane proteins such as CD28, CD8a, and CD4, which may be wild-type hinge regions from these molecules or may be altered.
  • a DARIC signaling component comprises a spacer domain.
  • the spacer domain is 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in length.
  • the spacer comprises the amino acid sequence GGS or GGR.
  • a DARIC signaling component comprises a transmembrane domain.
  • a DARIC signaling component comprises a hinge domain and a transmembrane domain.
  • transmembrane domains suitable for use in particular DARIC signaling components contemplated herein include, but are not limited to, the transmembrane region(s) of the alpha, beta, gamma, or delta chain of the T-cell receptor, CD2, CD38, CD3s, CD3y, CD3 CD4, CD5, CD8a, CD9, CD 16, CD22, CD27, CD28, CD33, CD37, CD40, CD45, CD64, CD80, CD86, CD 134, CD137, CD152, CD154, CD244, CD278, amnionless (AMN), programmed cell death 1 (PDCD1), TNRF2, TNFRS14, TNFRS25, TLR2, TLR4, TLR5, TIM1, SlarnFl, SlamF5, and SlamF6.
  • PDCD1 programmed cell death 1
  • PDCD1 programmed cell death 1
  • PDCD1 programmed cell death 1
  • PDCD1 programmed cell death 1
  • PDCD1 programmed cell death
  • a DARIC signaling component comprises a CD4 transmembrane domain. In a preferred embodiment, a DARIC signaling component comprises a CD8a transmembrane domain. In some embodiments, the CD8a transmembrane domain comprises the amino acid sequence as set forth in SEQ ID NO: 40. In some embodiments, the CD8a transmembrane domain comprises the amino acid sequence as set forth in SEQ ID NO: 41. In some embodiments, the CD8a transmembrane domain comprises the amino acid sequence as set forth in SEQ ID NO: 42.
  • a DARIC signaling component comprises a linker that links the C-terminus of the transmembrane domain to the N-terminus of an intracellular signaling domain.
  • a short oligo- or poly-peptide linker preferably between 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in length links the transmembrane domain and an intracellular signaling domain.
  • a glycine-serine based linker provides a particularly suitable linker.
  • DARIC signaling components contemplated in particular embodiments herein comprise one or more intracellular signaling domains.
  • a DARIC signaling component comprises one or more co-stimulatory signaling domains and/or a primary signaling domain.
  • the intracellular signaling domain comprises an immunoreceptor tyrosine activation motif (IT AM).
  • DARIC signaling components that are suitable for use in particular DARIC signaling components contemplated herein include, but are not limited to those derived from FcRy, FcRfl, CD3y, CD36, CD3e, CD3 CD22, CD79a, CD79b, and CD66d.
  • a DARIC signaling component comprises a CD3 ⁇ primary signaling domain and one or more co-stimulatory signaling domains.
  • the primary signaling and co-stimulatory signaling domains may be linked in any order in tandem to the carboxyl terminus of the transmembrane domain.
  • co-stimulatory domains suitable for use in particular DARIC signaling components contemplated herein include, but are not limited to those domains isolated from the following co-stimulatory molecules: Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, caspase recruitment domain family member 11 (CARD11), CD2, CD4, CD7, CD27, CD28, CD28- YMFM, CD28-H, CD30, CD40, CD54 (ICAM), CD83, CD94, CD134 (0X40), CD137 (4- 1BB), CD278 (ICOS), DN AX- Activation Protein 10 (DAP10), interleukin 2 receptor beta (IL2RP), Linker for activation of T-cells family member 1 (LAT), SH2 Domain-Containing Leukocyte Protein Of 76 kD (SLP76), T cell receptor associated transmembrane adaptor 1 (TRAT1), TN
  • TLR1
  • a DARIC signaling component contemplated herein comprises a signal peptide.
  • signal peptides suitable for use in particular DARIC signaling components include but are not limited to an IgGl heavy chain signal polypeptide, an IgK light chain signal polypeptide, a CD8a signal polypeptide, or a human GM-CSF receptor alpha signal polypeptide.
  • a DARIC signaling component comprises a CD8ct signal polypeptide.
  • the signal peptide is cleaved and the mature polypeptide comprises an amino acid sequence lacking the signal peptide.
  • a DARIC signaling component comprises one or more co-stimulatory signaling domains selected from the group consisting of CD28, CD 137, and CD134. In particular embodiments, a DARIC signaling component comprises one or more co-stimulatory signaling domains selected from the group consisting of CD28, CD 137, and CD134, and a CD3( ⁇ primary signaling domain. In a particular embodiment, a DARIC signaling component comprises a CD137 co-stimulatory domain and a CD3( ⁇ primary signaling domain.
  • a DARIC signaling component comprises an FRB T2098L multimerization domain, a CD8a transmembrane domain, a CD 137 co-stimulatory domain and a CD3q primary signaling domain.
  • a DARIC signaling component comprises an amino acid sequence set forth in SEQ ID NO: 6.
  • a “DARIC binding component” or “DARIC binding polypeptide” refers to one or more polypeptides comprising an anti-CD33 antibody or antigen binding fragment thereof and an anti-CLL-1 antibody or antigen binding fragment thereof that redirect an immune effector cell to a target cell expressing CD33 and/or CLL-1.
  • the DARIC binding component can be in either a tandem or dual format.
  • a “tandem formatted DARIC binding component,” “tandem formatted DARIC binding polypeptide,” “tandem DARIC binding component,” or “tandem DARIC binding polypeptide,” refers to a polypeptide comprising an anti-CD33 antibody or antigen binding fragment thereof and an anti-CLL-1 antibody or antigen binding fragment thereof that redirect an immune effector cell to a target cell expressing CD33 and/or CLL-1.
  • the tandem DARIC binding component comprises an anti-CD33 antibody or antigen binding fragment thereof, a polypeptide linker, and an anti-CLL-1 antibody or antigen binding fragment thereof.
  • the tandem DARIC binding component comprises an anti-CLL-1 antibody or antigen binding fragment thereof, a polypeptide linker, and an anti-CD33 antibody or antigen binding fragment thereof.
  • dual formatted DARIC binding component refers to embodiments comprising more than one DARIC binding component.
  • dual formatted DARIC binding components comprise a DARIC binding component comprising an anti-CD33 antibody or antigen binding fragment thereof and a DARIC binding component comprising an anti-CLL-1 antibody or antigen binding fragment thereof.
  • a tandem DARIC binding component comprises an anti- CD33 antibody or antigen binding fragment thereof, a polypeptide linker, an anti-CLL-1 antibody or antigen binding fragment thereof, a multimerization domain and a transmembrane domain.
  • a tandem DARIC binding component comprises an anti-CD33 antibody or antigen binding fragment thereof, a polypeptide linker, an anti-CLL-1 antibody or antigen binding fragment thereof, a second multimerization domain, and a second transmembrane domain.
  • a tandem DARIC binding component comprises an anti-CD33 antibody or antigen binding fragment thereof, a polypeptide linker, an anti-CLL-1 antibody or antigen binding fragment thereof, a multimerization domain, a transmembrane domain and one or more intracellular signaling domains.
  • a tandem DARIC binding component comprises an anti-CD33 antibody or antigen binding fragment thereof, a polypeptide linker, an anti-CLL-1 antibody or antigen binding fragment thereof, a second multimerization domain, a second transmembrane domain, and a second co-stimulatory domain.
  • a tandem DARIC binding component comprises an anti- CLL-1 antibody or antigen binding fragment thereof, a polypeptide linker, an anti-CD33 antibody or antigen binding fragment thereof, a multimerization domain and a transmembrane domain.
  • a tandem DARIC binding component comprises an anti-CLL-1 antibody or antigen binding fragment thereof, a polypeptide linker, an anti-CD33 antibody or antigen binding fragment thereof, a second multimerization domain, and a second transmembrane domain.
  • a tandem DARIC binding component comprises an anti-CLL-1 antibody or antigen binding fragment thereof, a polypeptide linker, an anti-CD33 antibody or antigen binding fragment thereof, a multimerization domain, a transmembrane domain and one or more intracellular signaling domains.
  • a tandem DARIC binding component comprises an anti-CLL-1 antibody or antigen binding fragment thereof, a polypeptide linker, an anti-CD33 antibody or antigen binding fragment thereof, a second multimerization domain, a second transmembrane domain, and a second co-stimulatory domain.
  • dual DARIC binding components comprise a DARIC binding component comprising an anti-CD33 antibody or antigen binding fragment thereof, a multimerization domain and a transmembrane domain; and a DARIC binding component comprising an anti-CLL-1 antibody or antigen binding fragment thereof, a multimerization domain and a transmembrane domain.
  • the multimerization domains of both DARIC binding components are different.
  • the multimerization domains of both DARIC binding components are the same.
  • the transmembrane domains of both DARIC binding components are different.
  • the transmembrane domains of both DARIC binding components are the same.
  • dual DARIC binding components comprise a DARIC binding component comprising an anti-CD33 antibody or antigen binding fragment thereof, a second multimerization domain and a second transmembrane domain; and a DARIC binding component comprising an anti-CLL-1 antibody or antigen binding fragment thereof, a third multimerization domain and a third transmembrane domain.
  • the second and third multimerization domains are different.
  • the second and third multimerization domains are the same.
  • the second and third transmembrane domains are different.
  • the second and third transmembrane domains are the same.
  • dual DARIC binding components comprise a DARIC binding component comprising an anti-CD33 antibody or antigen binding fragment thereof, a multimerization domain, a transmembrane domain, and one or more intracellular signaling domains; and a DARIC binding component comprising an anti-CLL-1 antibody or antigen binding fragment thereof, a multimerization domain, a transmembrane domain, and one or more intracellular signaling domains.
  • the multimerization domains of both DARIC binding components are different.
  • the multimerization domains of both DARIC binding components are the same.
  • the transmembrane domains of both DARIC binding components are different.
  • the transmembrane domains of both DARIC binding components are the same.
  • the one or more intracellular signaling domains of both DARIC binding components are different.
  • the one or more intracellular signaling domains of both DARIC binding components are the same.
  • dual DARIC binding components comprise a DARIC binding component comprising an anti-CD33 antibody or antigen binding fragment thereof, a second multimerization domain, a second transmembrane domain, and a second costimulatory domain; and a DARIC binding component comprising an anti-CLL-1 antibody or antigen binding fragment thereof, a third multimerization domain, a third transmembrane domain, and a third co-stimulatory domain.
  • the second and third multimerization domains are different.
  • the second and third multimerization domains are the same.
  • the second and third transmembrane domains are different.
  • the second and third transmembrane domains are the same.
  • the second and third co-stimulatory domains are different.
  • the second and third stimulatory domains are the same.
  • a DARIC binding component comprises a
  • a DARIC binding component comprises one or more anti-CD33 VHH domains.
  • a DARIC binding component comprises one or more anti-CLL VHH domains.
  • a DARIC binding component comprises one or more anti-CD33 VHH domains and one or more anti- CLL VHH domains.
  • a tandem DARIC binding component comprises a humanized camelid anti-CD33 VHH that binds one or more epitopes of full-length CD33 (e.g, SEQ ID NO: 1) and/or one or more epitopes of a CD33 splice variant, a polypeptide linker, and a humanized camelid anti-CLL- 1 VHH that binds one or more epitopes of CLL- 1 (e.g, SEQ ID NO: 2).
  • a tandem DARIC binding component comprises a humanized camelid anti-CLL-1 VHH that binds one or more epitopes of CLL-1 (e.g., SEQ ID NO: 2) and/or one or more epitopes of a CD33 splice variant, a polypeptide linker, and a humanized camelid anti-CD33 VHH that binds one or more epitopes of full-length CD33 (e.g., SEQ ID NO: 1).
  • dual DARIC binding components comprise a DARIC binding component comprising a humanized camelid anti-CD33 VHH that binds one or more epitopes of full-length CD33 e.g., SEQ ID NO: 1) and/or one or more epitopes of a CD33 splice variant; and a DARIC binding component comprising a humanized camelid anti-CLL-1 VHH that binds one or more epitopes of CLL-1 (e.g., SEQ ID NO: 2).
  • the anti-CD33 VHH domain is a humanized camelid VHH comprising amino acid sequence set forth in SEQ ID NO: 3.
  • the anti-CLL-1 VHH domain is a humanized camelid VHH comprising amino acid sequence set forth in any one of SEQ ID NOs: 4-5. In certain preferred embodiments, the anti-CLL-1 VHH domain is a humanized camelid VHH comprising amino acid sequence set forth in SEQ ID NO: 4. In certain preferred embodiments, the anti-CLL-1 VHH domain is a humanized camelid VHH comprising amino acid sequence set forth in SEQ ID NO: 5. In particular embodiments, one or more DARIC binding components comprise one or more multimerization domains. In particular embodiments, a DARIC comprises a DARIC binding component comprising a second multimerization domain. In particular embodiments, a DARIC comprises a DARIC binding component comprising a second multimerization domain and a DARIC binding component comprising a third multimerization domain.
  • multimerization domains suitable for use in particular DARIC binding components contemplated herein include, but are not limited to, an FKBP polypeptide or variants thereof, an FRB polypeptide or variants thereof, a calcineurin polypeptide or variants thereof, a cyclophilin polypeptide or variants thereof, a DHFR polypeptide or variants thereof, a PYL1 polypeptide or variants thereof and an ABI1 polypeptide or variants thereof.
  • a tandem DARIC binding component comprises an anti- CD33 antibody or antigen binding fragment thereof (e.g., a humanized VHH), a polypeptide linker, an anti-CLL-1 antibody or antigen binding fragment thereof (e.g., a humanized VHH), an FRB polypeptide or variant thereof; and a DARIC signaling component comprises an FKBP polypeptide or variant thereof.
  • a tandem DARIC binding component comprises an anti- CLL-1 antibody or antigen binding fragment thereof (e.g., a humanized VHH), a polypeptide linker, an anti-CD33 antibody or antigen binding fragment thereof (e.g., a humanized VHH), an FRB polypeptide or variant thereof; and a DARIC signaling component comprises an FKBP polypeptide or variant thereof.
  • a tandem DARIC binding component comprises an anti- CD33 antibody or antigen binding fragment thereof (e.g., a humanized VHH), a polypeptide linker, an anti-CLL-1 antibody or antigen binding fragment thereof (e.g., a humanized VHH), an FKBP polypeptide or variant thereof; and a DARIC signaling component comprises an FRB polypeptide or variant thereof.
  • a tandem DARIC binding component comprises an anti- CLL-1 antibody or antigen binding fragment thereof (e.g., a humanized VHH), a polypeptide linker, an anti-CD33 antibody or antigen binding fragment thereof (e.g., a humanized VHH), an FKBP polypeptide or variant thereof; and a DARIC signaling component comprises an FRB polypeptide or variant thereof.
  • dual DARIC binding components comprise a DARIC binding component comprising an anti-CD33 antibody or antigen binding fragment thereof (e.g., a humanized VHH) and an FRB polypeptide or variant thereof; a DARIC binding component comprising an anti-CLL-1 antibody or antigen binding fragment thereof (e.g., a humanized VHH) and an FRB polypeptide or variant thereof; and a DARIC signaling component comprising an FKBP polypeptide or variant thereof.
  • a DARIC binding component comprises an FRB polypeptide comprising a T2098L mutation, or variant thereof and a DARIC signaling component comprises an FKBP 12 polypeptide or variant thereof.
  • dual DARIC binding components comprise a DARIC binding component comprising an anti-CD33 antibody or antigen binding fragment thereof (e.g., a humanized VHH) and an FKBP polypeptide or variant thereof; a DARIC binding component comprising an anti-CLL-1 antibody or antigen binding fragment thereof (e.g., a humanized VHH) and an FKBP polypeptide or variant thereof; and a DARIC signaling component comprising an FRB polypeptide or variant thereof.
  • a DARIC binding component comprises an FKBP12 polypeptide or variant thereof and a DARIC signaling component comprises an FRB polypeptide comprising a T2098L mutation, or variant thereof.
  • one or more DARIC binding components comprise a hinge domain.
  • the DARIC signaling component also comprises a hinge, it can be the same hinge as a DARIC binding component or a different hinge than the one used in a DARIC binding component.
  • Illustrative hinge domains suitable for use in a DARIC signaling component described herein include the hinge region derived from the extracellular regions of alpha, beta, gamma, or delta chain of the T-cell receptor, CD2, CD38, CD3s, CD3y, CD3 ⁇ , CD4, CD5, CD8a, CD9, CD 16, CD22, CD27, CD28, CD33, CD37, CD40, CD45, CD64, CD80, CD86, CD 134, CD137, CD152, CD154, CD244, CD278, amnionless (AMN), programmed cell death 1 (PDCD1), TNRF2, TNFRS14, TNFRS25, TLR2, TLR4, TLR5, TIM1, SlamFl, SlamF5, and SlamF6, which may be wild-type hinge regions from these molecules or may be altered.
  • APN amnionless
  • hinge domains suitable for use in one or more DARIC binding components described herein include the hinge region derived from the extracellular regions of type 1 membrane proteins such as CD28, CD8a, and CD4, which may be wildtype hinge regions from these molecules or may be altered.
  • a CD33 VHH DARIC binding component comprises a spacer domain.
  • the spacer domain is 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in length.
  • the spacer comprises the amino acid sequence GGS or GGR.
  • a tandem DARIC binding component comprises a transmembrane domain.
  • a tandem DARIC binding component comprises a hinge domain and a transmembrane domain.
  • the transmembrane domain may be the same as the transmembrane domain used in the DARIC signaling component.
  • the transmembrane domain may be different from the transmembrane domain used in the DARIC signaling component.
  • dual DARIC binding components comprise a DARIC binding component comprising one transmembrane domain and a DARIC binding component comprising the same or different transmembrane domain.
  • dual DARIC binding components comprise a DARIC binding component comprising a hinge and transmembrane domain and a DARIC binding component comprising another hinge and transmembrane domain.
  • the hinge and transmembrane domains of either or both of the DARIC binding components may be the same or different with respect to each other and/or with respect to hinge and/or transmembrane domains of a DARIC signaling component.
  • transmembrane domains suitable for use in particular one or more DARIC binding components contemplated herein include, but are not limited to, the transmembrane region(s) of the alpha, beta, gamma, or delta chain of the T-cell receptor, CD2, CD35, CD3e, CD3y, CD3 ⁇ CD4, CD5, CD8a, CD9, CD 16, CD22, CD27, CD28, CD33, CD37, CD40, CD45, CD64, CD80, CD86, CD 134, CD137, CD152, CD154, CD244, CD278, amnionless (AMN), programmed cell death 1 (PDCD1), TNRF2, TNFRS14, TNFRS25, TLR2, TLR4, TLR5, TIM1, SlamFl, SlamF5, and SlamF6.
  • one or more DARIC binding components comprise a CD8a transmembrane domain.
  • one or more DARIC binding components comprise a CD4 transmembrane domain
  • the CD8a transmembrane domain comprises the amino acid sequence as set forth in SEQ ID NO: 40. In some embodiments, the CD8a transmembrane domain comprises the amino acid sequence as set forth in SEQ ID NO: 41. In some embodiments, the CD8a transmembrane domain comprises the amino acid sequence as set forth in SEQ ID NO: 42.
  • a short oligo- or poly-peptide linker preferably between 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in length links the transmembrane domain and the intracellular signaling domain.
  • a glycine-serine based linker provides a particularly suitable linker.
  • DARIC binding components contemplated in particular embodiments herein do not comprise one or more intracellular signaling domains.
  • one or more DARIC binding components contemplated herein comprise one or more intracellular signaling domains.
  • a DARIC binding component comprises one or more intracellular signaling domains that are different than the intracellular signaling domains present in the DARIC signaling component.
  • one or more DARIC binding components comprise a co-stimulatory signaling domain.
  • co-stimulatory domains suitable for use in one or more DARIC binding components contemplated herein include, but are not limited to those domains isolated from the following co-stimulatory molecules: Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, caspase recruitment domain family member 11 (CARD11), CD2, CD4, CD7, CD27, CD28, CD28- YMFM, CD28-H, CD30, CD40, CD54 (ICAM), CD83, CD94, CD134 (0X40), CD137 (4- 1BB), CD278 (ICOS), DN AX- Activation Protein 10 (DAP10), interleukin 2 receptor beta (IL2R0), Linker for activation of T-cells family member 1 (LAT), SH2 Domain-Containing Leukocyte Protein Of 76 kD (SLP76), T cell receptor associated transmembrane adaptor 1 (TRAT1),
  • TLR1
  • a DARIC signaling component contemplated herein comprises a signal peptide.
  • signal peptides suitable for use in particular DARIC signaling components include but are not limited to signal polypeptides derived from IgG heavy chain (e.g, from IgGl, IgG2), an IgK light chain (e.g., IgKVIII, IgKB7), CD8a, CD33, IL-2, IFNy, GM-CSF, osteonectin (BM40, SPARC), SECRECON, tissue polypeptide antigen (TPA), CHYMOTRSYPSINOGEN, TRYPSINOGEN-2, Argininosuccinate synthase (ASS), INSULIN, human serum albumin (HSA), or oncostatin M (OSM).
  • IgG heavy chain e.g, from IgGl, IgG2
  • IgK light chain e.g., IgKVIII, IgKB7
  • CD8a CD33
  • one or more DARIC binding component comprises a CD8a signal polypeptide.
  • the signal peptide is cleaved and the mature polypeptide comprises an amino acid sequence lacking the signal peptide.
  • a tandem DARIC binding component comprises a VHH domain that binds to CD33, a polypeptide linker, a VHH domain that binds to CLL-1, an FKBP12 multimerization domain, and a CD4 transmembrane domain and optionally, a costimulatory domain.
  • a tandem DARIC binding component comprises a VHH domain that binds to CLL-1, a polypeptide linker, a VHH domain that binds to CD33, an FKBP12 multimerization domain, and a CD4 transmembrane domain and optionally, a costimulatory domain.
  • dual DARIC binding components comprise a DARIC binding component that comprises a VHH domain that binds to CD33, an FKBP12 multimerization domain, and a CD4 transmembrane domain and optionally, a costimulatory domain; and a DARIC binding component that comprises a VHH domain that binds to CLL-1, an FKBP12 multimerization domain, and a CD4 transmembrane domain and optionally, a co-stimulatory domain.
  • a tandem DARIC binding component comprises a VHH domain that binds to CD33, a polypeptide linker, a VHH domain that binds to CLL-1, and an FKBP12 multimerization domain.
  • a tandem DARIC binding component comprises a VHH domain that binds to CLL-1, a polypeptide linker, a VHH domain that binds to CD33,and an FKBP12 multimerization domain.
  • dual DARIC binding components comprise a DARIC binding component that comprises a VHH domain that binds to CD33 and an FKBP12 multimerization domain; and a DARIC binding component that comprises a VHH domain that binds to CLL-1 and an FKBP12 multimerization domain.
  • a tandem DARIC binding component comprises an anti- CD33 VHH domain comprising the amino acid sequence set forth in SEQ ID NO: 3, a polypeptide linker, an anti-CLL-1 VHH domain comprising the amino acid sequence set forth in any one of SEQ ID NOs: 4-5, an FKBP12 multimerization domain, and a CD4 transmembrane domain and optionally, a co-stimulatory domain.
  • a tandem DARIC binding component comprises an anti- CLL-1 VHH domain comprising the amino acid sequence set forth in any one of SEQ ID NOs: 4-5, a polypeptide linker, an anti-CD33 VHH domain comprising the amino acid sequence set forth in SEQ ID NO: 3, an FKBP12 multimerization domain, and a CD4 transmembrane domain and optionally, a co-stimulatory domain.
  • dual DARIC binding components comprise a DARIC binding component comprising an anti-CD33 VHH domain comprising the amino acid sequence set forth in SEQ ID NO: 3, an FKBP12 multimerization domain, and a CD4 transmembrane domain and optionally, a co-stimulatory domain; and a DARIC binding component comprising an anti-CLL-1 VHH domain comprising the amino acid sequence set forth in any one of SEQ ID NOs: 4-5, an FKBP12 multimerization domain, and a CD4 transmembrane domain and optionally, a co-stimulatory domain.
  • a tandem DARIC binding component comprises an anti- CD33 VHH domain comprising the amino acid sequence set forth in SEQ ID NO: 3, a polypeptide linker, an anti-CLL-1 VHH domain comprising the amino acid sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 5, an FKBP12 multimerization domain, and a CD4 transmembrane domain and optionally, a co-stimulatory domain.
  • a tandem DARIC binding component comprises an anti- CLL-1 VHH domain comprising the amino acid sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 5, a polypeptide linker, an anti-CD33 VHH domain comprising the amino acid sequence set forth in SEQ ID NO: 3, an FKBP12 multimerization domain, and a CD4 transmembrane domain and optionally, a co-stimulatory domain.
  • dual DARIC binding components comprise a DARIC binding component comprising an anti-CD33 VHH domain comprising the amino acid sequence set forth in SEQ ID NO: 3, an FKBP12 multimerization domain, and a CD4 transmembrane domain and optionally, a co-stimulatory domain; and a DARIC binding component comprising an anti-CLL-1 VHH domain comprising the amino acid sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 5, an FKBP12 multimerization domain, and a CD4 transmembrane domain and optionally, a co-stimulatory domain.
  • a tandem DARIC binding component comprises an anti- CD33 VHH domain comprising the amino acid sequence set forth in SEQ ID NO: 3, a polypeptide linker, an anti-CLL-1 VHH domain comprising the amino acid sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 5, and an FKBP12 multimerization domain.
  • a tandem DARIC binding component comprises an anti- CLL-1 VHH domain comprising the amino acid sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 5, a polypeptide linker, an anti-CD33 VHH domain comprising the amino acid sequence set forth in SEQ ID NO: 3, and an FKBP12 multimerization domain.
  • dual DARIC binding components comprise a DARIC binding component comprising an anti-CD33 VHH domain comprising the amino acid sequence set forth in SEQ ID NO: 3 and an FKBP12 multimerization domain; and a DARIC binding component comprising an anti-CLL-1 VHH domain comprising the amino acid sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 5 and an FKBP12 multimerization domain.
  • a DARIC binding component comprises the amino acid sequence set forth in any one of SEQ ID NOs: 7-16.
  • a tandem DARIC binding component comprises the amino acid sequence set forth in any one of SEQ ID NOs: 7-10.
  • dual DARIC binding components comprise the amino acid sequences set forth in any one of SEQ ID NOs: 11-16.
  • Bridging factors contemplated in particular embodiments herein mediate or promote the association of a DARIC signaling component with a DARIC binding component through multimerization domains in the respective components.
  • a bridging factor associates with and is disposed between the multimerization domains to promote association of a DARIC signaling component and one or more DARIC binding components.
  • one or more DARIC binding components associate with the DARIC signaling component and initiate immune effector cell activity against a target cell when the one or more DARIC binding components bind CD33 and/or CLL-1 expressed on the target cell.
  • a DARIC binding component does not associate with the DARIC signaling component and the DARIC is inactive.
  • a DARIC signaling component and one or more DARIC binding components comprise a cognate pair of multimerization domains selected from the group consisting of: FKBP and FKBP12-rapamycin binding (FRB), FKBP and calcineurin, FKBP and cyclophilin, FKBP and bacterial dihydrofolate reductase (DHFR), calcineurin and cyclophilin, and PYRl-like 1 (PYL1) and abscisic acid insensitive 1 (ABI1).
  • FKBP and FKBP12-rapamycin binding FKBP and calcineurin, FKBP and cyclophilin, FKBP and bacterial dihydrofolate reductase (DHFR), calcineurin and cyclophilin, and PYRl-like 1 (PYL1) and abscisic acid insensitive 1 (ABI1).
  • the multimerization domains of DARIC signaling and binding components associate with a bridging factor selected from the group consisting of: rapamycin or a rapalog thereof, coumermycin or a derivative thereof, gibberellin or a derivative thereof, abscisic acid (ABA) or a derivative thereof, methotrexate or a derivative thereof, cyclosporin A or a derivative thereof, FK506/cyclosporin A (FKCsA) or a derivative thereof, and trimethoprim (Tmp)-synthetic ligand for FK506 binding protein (FKBP) (SLF) or a derivative thereof.
  • a bridging factor selected from the group consisting of: rapamycin or a rapalog thereof, coumermycin or a derivative thereof, gibberellin or a derivative thereof, abscisic acid (ABA) or a derivative thereof, methotrexate or a derivative thereof, cyclosporin A or a derivative thereof, FK50
  • a DARIC signaling component and a tandem DARIC binding component comprise one or more FRB and/or FKBP multimerization domains or variants thereof.
  • a DARIC signaling component comprises an FRB multimerization domain or variant thereof and a tandem DARIC binding component comprises an FKBP multimerization domain or variant thereof.
  • a DARIC signaling component comprises an FRB T2098L multimerization domain or variant thereof and a tandem DARIC binding component comprises an FKBP12 or FKBP12 F36V multimerization domains or variant thereof.
  • a DARIC signaling component and dual DARIC binding components comprise one or more FRB and/or FKBP multimerization domains or variants thereof.
  • a DARIC signaling component comprises an FRB multimerization domain or variant thereof and dual DARIC binding components comprise more than one DARIC binding components that each comprise an FKBP multimerization domain or variant thereof.
  • a DARIC signaling component comprises an FRB T2098L multimerization domain or variant thereof and each of the dual DARIC binding components comprise an FKBP12 or FKBP12 F36V multimerization domains or variant thereof.
  • bridging factors suitable for use in particular embodiments contemplated herein include, but are not limited to, AP1903, AP20187, AP21967 (also known as C-16-(S)-7-methylindolerapamycin), everolimus, novolimus, pimecrolimus, ridaforolimus, tacrolimus, temsirolimus, umirolimus, and zotarolimus.
  • the bridging factor is AP21967.
  • the bridging factor is a non-immunosuppressive dose of sirolimus (rapamycin).
  • polypeptides are contemplated herein, including, but not limited to, tandem DARIC binding components, dual DARIC binding components, and DARIC signaling components, and fragments thereof.
  • a polypeptide comprises an amino acid sequence set forth in any one of SEQ ID NOs: 3-27.
  • Polypeptide,” “peptide” and “protein” are used interchangeably, unless specified to the contrary, and according to conventional meaning, i.e., as a sequence of amino acids.
  • a “polypeptide” includes fusion polypeptides and other variants. Polypeptides can be prepared using any of a variety of well-known recombinant and/or synthetic techniques.
  • Polypeptides are not limited to a specific length, e.g., they may comprise a full-length protein sequence, a fragment of a full-length protein, or a fusion protein, and may include post-translational modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations and the like, as well as other modifications known in the art, both naturally occurring and non-naturally occurring.
  • fusion polypeptides, polypeptides, fragments and other variants thereof are prepared, obtained, or isolated from one or more human polypeptides.
  • an isolated polypeptide is a synthetic polypeptide, a semi -synthetic polypeptide, or a polypeptide obtained or derived from a recombinant source.
  • Polypeptides include “polypeptide variants.” Polypeptide variants may differ from a naturally occurring polypeptide in one or more substitutions, deletions, additions and/or insertions. Such variants may be naturally occurring, e.g., a splice variant, or may be synthetically generated, for example, by modifying one or more of the above polypeptide sequences. For example, in particular embodiments, it may be desirable to improve the binding affinity and/or to humanized or otherwise improve the biological properties of a polypeptide by introducing one or more substitutions, deletions, additions and/or insertions the polypeptide.
  • polypeptides include polypeptides having at least about 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 86%, 97%, 98%, or 99% amino acid identity to any of the reference sequences contemplated herein, typically where the variant maintains at least one biological activity of the reference sequence.
  • the biological activity is binding affinity.
  • the biological activity is cytolytic activity.
  • Polypeptide variants include biologically active “polypeptide fragments.”
  • biologically active polypeptide fragments include anti-CD33 VHH domains and anti-CLL-1 VHH domains, intracellular signaling domains, and the like.
  • biologically active fragment or minimal biologically active fragment refers to a polypeptide fragment that retains at least 100%, at least 90%, at least 80%, at least 70%, at least 60%, at least 50%, at least 40%, at least 30%, at least 20%, at least 10%, or at least 5% of the naturally occurring polypeptide activity.
  • a polypeptide fragment can comprise an amino acid chain at least 5 to about 1700 amino acids long.
  • fragments are at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700 or more amino acids long.
  • the polypeptides set forth herein may comprise one or more amino acids denoted as “X” or “Xaa ,” which are used interchangeably.
  • X if present in an amino acid SEQ ID NO, refers to any one or more amino acids.
  • SEQ ID NOs denoting a fusion protein comprise a sequence of continuous X residues that cumulatively represent any amino acid sequence.
  • XX represent any two amino acid combination.
  • XX represents two serines, SS.
  • “XX” represents any two amino acid combination that reduces immunogenicity.
  • XX represents the amino acids KP.
  • polypeptides comprising C-terminal truncations of a KP amino acid sequence are contemplated.
  • polypeptides may be altered in various ways including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known in the art.
  • amino acid sequence variants of a reference polypeptide can be prepared by mutations in the DNA. Methods for mutagenesis and nucleotide sequence alterations are well known in the art. See, for example, Kunkel (1985, Proc. Natl. Acad. Sci. USA. 82: 488-492), Kunkel etal., (1987 , Methods inEnzymol, 154: 367- 382), U.S. Pat. No. 4,873,192, Watson, J. D.
  • a polypeptide variant comprises one or more conservative substitutions.
  • a “conservative substitution” is one in which an amino acid is substituted for another amino acid that has similar properties, such that one skilled in the art of peptide chemistry would expect the secondary structure and hydropathic nature of the polypeptide to be substantially unchanged. Modifications may be made in the structure of the polynucleotides and polypeptides contemplated in particular embodiments and still obtain a functional molecule that encodes a variant or derivative polypeptide with desirable characteristics.
  • amino acid changes in the protein variants disclosed herein are conservative amino acid changes, i.e., substitutions of similarly charged or uncharged amino acids.
  • a conservative amino acid change involves substitution of one of a family of amino acids which are related in their side chains.
  • Naturally occurring amino acids are generally divided into four families: acidic (aspartate, glutamate), basic (lysine, arginine, histidine), non-polar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged polar (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine) amino acids. Phenylalanine, tryptophan, and tyrosine are sometimes classified jointly as aromatic amino acids. In a peptide or protein, suitable conservative substitutions of amino acids are known to those of skill in this art and generally can be made without altering a biological activity of a resulting molecule.
  • the polynucleotide sequences encoding them can be separated by an IRES sequence or a polynucleotide sequence encoding a ribosomal skip sequence as disclosed elsewhere herein.
  • Polypeptides contemplated in particular embodiments include fusion polypeptides.
  • Illustrative examples of fusion polypeptides are set forth in any one of SEQ ID NOs: 7-27.
  • a fusion polypeptide comprises an amino acid sequence set forth in any one of SEQ ID NOs: 20-27.
  • Fusion polypeptides and polynucleotides encoding fusion polypeptides are provided.
  • Fusion polypeptides and fusion proteins refer to a polypeptide having at least two, three, four, five, six, seven, eight, nine, or ten polypeptide segments.
  • a fusion polypeptide comprises one or more DARIC components.
  • the fusion polypeptide comprises one or more VHH DARICs.
  • two or more DARIC components and/or other polypeptides can be expressed as a fusion protein that comprises one or more self-cleaving peptide sequences between the polypeptides as disclosed elsewhere herein.
  • a fusion polypeptide comprises a DARIC signaling component, a self-cleaving polypeptide sequence or ribosomal skip sequence, and a tandem DARIC binding component.
  • a fusion polypeptide comprises a tandem DARIC binding component, a self-cleaving polypeptide sequence or ribosomal skip sequence, and a DARIC signaling component.
  • a fusion polypeptide comprises a DARIC signaling component, a first self-cleaving polypeptide sequence or ribosomal skip sequence, a DARIC binding component that binds CD33, a second self-cleaving polypeptide sequence or ribosomal skip sequence, and a DARIC binding component that binds CLL-1.
  • a fusion polypeptide comprises a DARIC signaling component, a first self-cleaving polypeptide sequence or ribosomal skip sequence, a DARIC binding component that binds CLL-1, a second self-cleaving polypeptide sequence or ribosomal skip sequence, and a DARIC binding component that binds CD33.
  • a fusion polypeptide comprises a DARIC binding component that binds CD33, a first self-cleaving polypeptide sequence or ribosomal skip sequence, a DARIC binding component that binds CLL-1, a second self-cleaving polypeptide sequence or ribosomal skip sequence, and a DARIC signaling component.
  • a fusion polypeptide comprises a DARIC binding component that binds CLL-1, a first self-cleaving polypeptide sequence or ribosomal skip sequence, a DARIC binding component that binds CD33, a second self-cleaving polypeptide sequence or ribosomal skip sequence, and a DARIC signaling component.
  • a fusion polypeptide comprises a DARIC signaling component comprising an FRB multimerization domain polypeptide or variant thereof, a first transmembrane domain, a co-stimulatory domain, and/or a primary signaling domain; a polypeptide cleavage signal; and a tandem DARIC binding component comprising an anti-CD33 VHH antibody that has an amino acid sequence set forth in SEQ ID NO: 3; a polypeptide linker (e.g., a (GGGGS)I-4 polypeptide [SEQ ID NOs: 43-46]); an anti-CLL-1 VHH antibody that has an amino acid sequence set forth in any one of SEQ ID NOs: 4-5, an FKBP multimerization domain polypeptide or variant thereof, and a second transmembrane domain.
  • a DARIC signaling component comprising an FRB multimerization domain polypeptide or variant thereof, a first transmembrane domain, a co-stimulatory domain, and/or a primary signaling domain
  • a fusion polypeptide comprises a DARIC signaling component comprising an FRB multimerization domain polypeptide or variant thereof, a first transmembrane domain, a co-stimulatory domain, and/or a primary signaling domain; a polypeptide cleavage signal; and a tandem DARIC binding component comprising an anti-CLL-1 VHH antibody that has an amino acid sequence set forth in any one of SEQ ID NOs: 4-5; a polypeptide linker (e.g., a (GGGGS)I-4 polypeptide [SEQ ID NOs: 43-46]); an anti-CD33 VHH antibody that has an amino acid sequence set forth in SEQ ID NO: 3, an FKBP multimerization domain polypeptide or variant thereof, and a second transmembrane domain.
  • a DARIC signaling component comprising an FRB multimerization domain polypeptide or variant thereof, a first transmembrane domain, a co-stimulatory domain, and/or a primary signaling domain
  • a fusion polypeptide comprises a tandem DARIC binding component comprising an anti-CD33 VHH antibody that has an amino acid sequence set forth in SEQ ID NO: 3; a polypeptide linker (e.g., a (GGGGS)I-4 polypeptide [SEQ ID NOs: 43-46]); an anti-CLL-1 VHH antibody that has an amino acid sequence set forth in any one of SEQ ID NOs: 4-5, an FKBP multimerization domain polypeptide or variant thereof, and a second transmembrane domain; a polypeptide cleavage signal; and a DARIC signaling component comprising an FRB multimerization domain polypeptide or variant thereof, a first transmembrane domain, a co-stimulatory domain, and/or a primary signaling domain.
  • a polypeptide linker e.g., a (GGGGS)I-4 polypeptide [SEQ ID NOs: 43-46]
  • an anti-CLL-1 VHH antibody that has an amino acid
  • a fusion polypeptide comprises a tandem DARIC binding component comprising an anti-CLL-1 VHH antibody that has an amino acid sequence set forth in any one of SEQ ID NOs: 4-5; a polypeptide linker (e.g., a (GGGGS)I-4 polypeptide [SEQ ID NOs: 43-46]); an anti-CD33 VHH antibody that has an amino acid sequence set forth in SEQ ID NO: 3, an FKBP multimerization domain polypeptide or variant thereof, and a second transmembrane domain; and a DARIC signaling component comprising an FRB multimerization domain polypeptide or variant thereof, a first transmembrane domain, a co-stimulatory domain, and/or a primary signaling domain; a polypeptide cleavage signal.
  • a polypeptide linker e.g., a (GGGGS)I-4 polypeptide [SEQ ID NOs: 43-46]
  • an anti-CD33 VHH antibody that has an amino acid sequence set
  • a fusion polypeptide comprises a DARIC signaling component comprising an FKBP multimerization domain polypeptide or variant thereof, a first transmembrane domain, a co-stimulatory domain, and/or a primary signaling domain; a polypeptide cleavage signal; and a tandem DARIC binding component comprising an anti-CD33 VHH antibody that has an amino acid sequence set forth in SEQ ID NO: 3; a polypeptide linker (e.g., a (GGGGS)I-4 polypeptide [SEQ ID NOs: 43-46]); an anti-CLL-1 VHH antibody that has an amino acid sequence set forth in any one of SEQ ID NOs: 4-5, an FRB multimerization domain polypeptide or variant thereof, and a second transmembrane domain.
  • a DARIC signaling component comprising an FKBP multimerization domain polypeptide or variant thereof, a first transmembrane domain, a co-stimulatory domain, and/or a primary signaling
  • a fusion polypeptide comprises a DARIC signaling component comprising an FKBP multimerization domain polypeptide or variant thereof, a first transmembrane domain, a co-stimulatory domain, and/or a primary signaling domain; a polypeptide cleavage signal; and a tandem DARIC binding component comprising an anti-CLL-1 VHH antibody that has an amino acid sequence set forth in any one of SEQ ID NOs: 4-5; a polypeptide linker (e.g., a (GGGGS)I-4 polypeptide [SEQ ID NOs: 43-46]); an anti-CD33 VHH antibody that has an amino acid sequence set forth in SEQ ID NO: 3, an FRB multimerization domain polypeptide or variant thereof, and a second transmembrane domain.
  • a DARIC signaling component comprising an FKBP multimerization domain polypeptide or variant thereof, a first transmembrane domain, a co-stimulatory domain, and/or a primary signaling domain
  • a fusion polypeptide comprises a tandem DARIC binding component comprising an anti-CD33 VHH antibody that has an amino acid sequence set forth in SEQ ID NO: 3; a polypeptide linker (e.g., a (GGGGS)I-4 polypeptide [SEQ ID NOs: 43-46]); an anti-CLL-1 VHH antibody that has an amino acid sequence set forth in any one of SEQ ID NOs: 4-5, an FRB multimerization domain polypeptide or variant thereof, and a second transmembrane domain; a polypeptide cleavage signal; and a DARIC signaling component comprising an FKBP multimerization domain polypeptide or variant thereof, a first transmembrane domain, a co-stimulatory domain, and/or a primary signaling domain.
  • a polypeptide linker e.g., a (GGGGS)I-4 polypeptide [SEQ ID NOs: 43-46]
  • an anti-CLL-1 VHH antibody that has an amino acid
  • a fusion polypeptide comprises a tandem DARIC binding component comprising an anti-CLL-1 VHH antibody that has an amino acid sequence set forth in any one of SEQ ID NOs: 4-5; a polypeptide linker (e.g., a (GGGGS)I-4 polypeptide [SEQ ID NOs: 43-46]); an anti-CD33 VHH antibody that has an amino acid sequence set forth in SEQ ID NO: 3, an FRB multimerization domain polypeptide or variant thereof, and a second transmembrane domain; and a DARIC signaling component comprising an FKBP multimerization domain polypeptide or variant thereof, a first transmembrane domain, a co-stimulatory domain, and/or a primary signaling domain; a polypeptide cleavage signal.
  • a polypeptide linker e.g., a (GGGGS)I-4 polypeptide [SEQ ID NOs: 43-46]
  • an anti-CD33 VHH antibody that has an amino acid sequence set
  • a fusion polypeptide comprises a DARIC signaling component comprising an FRB multimerization domain polypeptide or variant thereof, a first transmembrane domain, a co-stimulatory domain, and/or a primary signaling domain; a first polypeptide cleavage signal; a DARIC binding component comprising an anti-CD33 VHH antibody that has an amino acid sequence set forth in SEQ ID NO: 3, an FKBP multimerization domain polypeptide or variant thereof, and a second transmembrane domain; a second polypeptide cleavage signal; and a DARIC binding component comprising an anti-CLL-1 VHH antibody that has an amino acid sequence set forth in any one of SEQ ID NOs: 4-5, an FKBP multimerization domain polypeptide or variant thereof, and a third transmembrane domain.
  • a fusion polypeptide comprises a DARIC signaling component comprising an FRB multimerization domain polypeptide or variant thereof, a first transmembrane domain, a co-stimulatory domain, and/or a primary signaling domain; a first polypeptide cleavage signal; a DARIC binding component comprising an anti-CLL- 1 VHH antibody that has an amino acid sequence set forth in any one of SEQ ID NOs: 4-5, an FKBP multimerization domain polypeptide or variant thereof, and a third transmembrane domain, and a second transmembrane domain; a second polypeptide cleavage signal; and a DARIC binding component comprising an anti-CD33 VHH antibody that has an amino acid sequence set forth in SEQ ID NO: 3, an FKBP multimerization domain polypeptide or variant thereof.
  • a fusion polypeptide comprises a DARIC binding component comprising an anti-CD33 VHH antibody that has an amino acid sequence set forth in SEQ ID NO: 3, an FKBP multimerization domain polypeptide or variant thereof, and a second transmembrane domain; a first polypeptide cleavage signal; a DARIC binding component comprising an anti-CLL-1 VHH antibody that has an amino acid sequence set forth in any one of SEQ ID NOs: 4-5, an FKBP multimerization domain polypeptide or variant thereof, and a third transmembrane domain; a second polypeptide cleavage signal; and a DARIC signaling component comprising an FRB multimerization domain polypeptide or variant thereof, a first transmembrane domain, a co-stimulatory domain, and/or a primary signaling domain.
  • a fusion polypeptide comprises a DARIC binding component comprising an anti-CLL-1 VHH antibody that has an amino acid sequence set forth in any one of SEQ ID NOs: 4-5, an FKBP multimerization domain polypeptide or variant thereof, and a second transmembrane domain; a first polypeptide cleavage signal; a DARIC binding component comprising an anti-CD33 VHH antibody that has an amino acid sequence set forth in SEQ ID NO: 3, an FKBP multimerization domain polypeptide or variant thereof, and a third transmembrane domain; a second polypeptide cleavage signal; and a DARIC signaling component comprising an FRB multimerization domain polypeptide or variant thereof, a first transmembrane domain, a co-stimulatory domain, and/or a primary signaling domain.
  • a fusion polypeptide comprises a DARIC signaling component comprising an FKBP multimerization domain polypeptide or variant thereof, a first transmembrane domain, a co-stimulatory domain, and/or a primary signaling domain; a first polypeptide cleavage signal; a DARIC binding component comprising an anti-CD33 VHH antibody that has an amino acid sequence set forth in SEQ ID NO: 3, an FRB multimerization domain polypeptide or variant thereof, and a second transmembrane domain; a second polypeptide cleavage signal; and a DARIC binding component comprising an anti-CLL-1 VHH antibody that has an amino acid sequence set forth in any one of SEQ ID NOs: 4-5, an FRB multimerization domain polypeptide or variant thereof, and a third transmembrane domain.
  • a fusion polypeptide comprises a DARIC signaling component comprising an FKBP multimerization domain polypeptide or variant thereof, a first transmembrane domain, a co-stimulatory domain, and/or a primary signaling domain; a first polypeptide cleavage signal; a DARIC binding component comprising an anti-CLL- 1 VHH antibody that has an amino acid sequence set forth in any one of SEQ ID NOs: 4-5, an FRB multimerization domain polypeptide or variant thereof, and a third transmembrane domain, and a second transmembrane domain; a second polypeptide cleavage signal; and a DARIC binding component comprising an anti-CD33 VHH antibody that has an amino acid sequence set forth in SEQ ID NO: 3, an FRB multimerization domain polypeptide or variant thereof.
  • a fusion polypeptide comprises a DARIC binding component comprising an anti-CD33 VHH antibody that has an amino acid sequence set forth in SEQ ID NO: 3, an FRB multimerization domain polypeptide or variant thereof, and a second transmembrane domain; a first polypeptide cleavage signal; a DARIC binding component comprising an anti-CLL-1 VHH antibody that has an amino acid sequence set forth in any one of SEQ ID NOs: 4-5, an FRB multimerization domain polypeptide or variant thereof, and a third transmembrane domain; a second polypeptide cleavage signal; and a DARIC signaling component comprising an FKBP multimerization domain polypeptide or variant thereof, a first transmembrane domain, a co-stimulatory domain, and/or a primary signaling domain.
  • a fusion polypeptide comprises a DARIC binding component comprising an anti-CLL-1 VHH antibody that has an amino acid sequence set forth in any one of SEQ ID NOs: 4-5, an FRB multimerization domain polypeptide or variant thereof, and a second transmembrane domain; a first polypeptide cleavage signal; a DARIC binding component comprising an anti-CD33 VHH antibody that has an amino acid sequence set forth in SEQ ID NO: 3, an FRB multimerization domain polypeptide or variant thereof, and a third transmembrane domain; a second polypeptide cleavage signal; and a DARIC signaling component comprising an FKBP multimerization domain polypeptide or variant thereof, a first transmembrane domain, a co-stimulatory domain, and/or a primary signaling domain.
  • the FRB multimerization domain polypeptide is FRB T2098L and the FKBP multimerization domain polypeptide is FKBP12.
  • Fusion polypeptides can comprise one or more polypeptide domains or segments including, but are not limited to signal peptides, antibodies and antigen binding domains, co- stimulatory domains, primary signaling domains, polypeptide linkers, and polypeptide cleavage signals. Fusion polypeptides are typically linked C-terminus to N-terminus, although they can also be linked C-terminus to C-terminus, N-terminus to N-terminus, or N-terminus to C- terminus. In particular embodiments, the polypeptides of the fusion protein can be in any order.
  • Fusion polypeptides or fusion proteins can also include conservatively modified variants, polymorphic variants, alleles, mutants, subsequences, and interspecies homologs, so long as the desired activity of the fusion polypeptide is preserved. Fusion polypeptides may be produced by chemical synthetic methods or by chemical linkage between the two moieties or may generally be prepared using other standard techniques. Ligated DNA sequences comprising the fusion polypeptide are operably linked to suitable transcriptional or translational control elements as disclosed elsewhere herein.
  • Fusion polypeptides may optionally comprise one or more linkers that can be used to link the one or more polypeptides or domains within a polypeptide.
  • a peptide linker sequence may be employed to separate any two or more polypeptide components by a distance sufficient to ensure that each polypeptide folds into its appropriate secondary and tertiary structures so as to allow the polypeptide domains to exert their desired functions.
  • Such a peptide linker sequence is incorporated into the fusion polypeptide using standard techniques in the art.
  • Suitable peptide linker sequences may be chosen based on the following factors: (1) their ability to adopt a flexible extended conformation; (2) their inability to adopt a secondary structure that could interact with functional epitopes on the first and second polypeptides; and (3) the lack of hydrophobic or charged residues that might react with the polypeptide functional epitopes.
  • preferred peptide linker sequences contain Gly, Asn and Ser residues. Other near neutral amino acids, such as Thr and Ala may also be used in the linker sequence.
  • Amino acid sequences which may be usefully employed as linkers include those disclosed in Maratea et al., Gene 40:39-46, 1985; Murphy et al., Proc. Natl. Acad.
  • Linker sequences are not required when a particular fusion polypeptide segment contains non-essential N-terminal amino acid regions that can be used to separate the functional domains and prevent steric interference.
  • preferred linkers are typically flexible amino acid subsequences which are synthesized as part of a recombinant fusion protein.
  • Linker polypeptides can be between 1 and 200 amino acids in length, between 1 and 100 amino acids in length, or between 1 and 50 amino acids in length, including all integer values in between.
  • Exemplary polypeptide cleavage signals include polypeptide cleavage recognition sites such as protease cleavage sites, nuclease cleavage sites (e.g., rare restriction enzyme recognition sites, self-cleaving ribozyme recognition sites), and self-cleaving viral oligopeptides (see deFelipe and Ryan, 2004. Traffic, 5(8); 616-26).
  • polypeptide cleavage recognition sites such as protease cleavage sites, nuclease cleavage sites (e.g., rare restriction enzyme recognition sites, self-cleaving ribozyme recognition sites), and self-cleaving viral oligopeptides (see deFelipe and Ryan, 2004. Traffic, 5(8); 616-26).
  • Suitable protease cleavages sites and self-cleaving peptides are known to the skilled person (.see, e.g., in Ryan et al., 1997. J. Gener. Virol. 78, 699-722; Scymczak et al. (2004) Nature Biotech. 5, 589-594).
  • Exemplary protease cleavage sites include, but are not limited to the cleavage sites of potyvirus NIa proteases (e.g., tobacco etch virus protease), potyvirus HC proteases, potyvirus Pl (P35) proteases, byovirus NIa proteases, byovirus RNA-2-encoded proteases, aphthovirus L proteases, enterovirus 2A proteases, rhinovirus 2A proteases, picoma 3C proteases, comovirus 24K proteases, nepovirus 24K proteases, RTSV (rice tungro spherical virus) 3 C-like protease, PYVF (parsnip yellow fleck virus) 3 C-like protease, heparin, thrombin, factor Xa and enterokinase.
  • potyvirus NIa proteases e.g., tobacco etch virus protease
  • potyvirus Pl (P35) proteases by
  • TEV tobacco etch virus protease cleavage sites
  • EXXYXQ(G/S) SEQ ID NO: 58
  • ENLYFQG SEQ ID NO: 59
  • ENLYFQS SEQ ID NO: 60
  • X represents any amino acid (cleavage by TEV occurs between Q and G or Q and S).
  • the polypeptide cleavage signal is a viral self-cleaving peptide or ribosomal skipping sequence.
  • ribosomal skipping sequences include, but are not limited to: a 2A or 2A-like site, sequence or domain (Donnelly etal., 2001. J. Gen. Virol. 82:1027-1041).
  • the viral 2A peptide is an aphthovirus 2A peptide, a potyvirus 2A peptide, or a cardiovims 2A peptide.
  • the viral 2A peptide is selected from the group consisting of: a foot-and-mouth disease virus (FMDV) 2A peptide, an equine rhinitis A vims (ERAV) 2A peptide, a Thosea asigna vims (TaV) 2A peptide, a porcine teschovims-1 (PTV-1) 2A peptide, a Theilovims 2A peptide, and an encephalomyocarditis vims 2A peptide.
  • FMDV foot-and-mouth disease virus
  • EAV equine rhinitis A vims
  • TaV Thosea asigna vims
  • PTV-1 porcine teschovims-1
  • a polypeptide or fusion polypeptide comprises one or more DARIC binding components and a DARIC signaling component in any suitable order, wherein a polypeptide cleavage signal separates each of the components.
  • a fusion polypeptide comprises a DARIC signaling component; a self-cleaving polypeptide sequence; and a tandem DARIC binding component, wherein the binding component binds CD33 and CLL-1.
  • a fusion polypeptide comprises a DARIC signaling component; a first self-cleaving polypeptide sequence; a DARIC binding component; a second self-cleaving polypeptide sequence; and another DARIC binding component, wherein each of the binding components binds CD33 or CLL-1.
  • the each self-cleaving polypeptide is independently selected from the group consisting of: a foot-and-mouth disease virus (FMDV) (F2A) peptide, an equine rhinitis A virus (ERAV) (E2A) peptide, a Thosea asigna virus (TaV) (T2A) peptide, a porcine teschovirus-1 (PTV-1) (P2A) peptide, a Theilovirus 2A peptide, and an encephalomyocarditis virus 2A peptide.
  • FMDV foot-and-mouth disease virus
  • F2A equine rhinitis A virus
  • E2A equine rhinitis A virus
  • TaV Thosea asigna virus
  • PTV-1 porcine teschovirus-1
  • P2A porcine teschovirus-1
  • Theilovirus 2A peptide a Theilovirus 2A peptide
  • a fusion polypeptide comprises the sequence set forth in any one of SEQ ID NOs: 20-27.
  • tandem DARIC fusion polypeptide comprises the sequence set forth in any one of SEQ ID NOs: 20-23.
  • a dual DARIC fusion polypeptide comprises the sequence set forth in any one of SEQ ID NOs: 24-27.
  • polynucleotide encoding tandem DARIC binding components, dual DARIC binding components, and DARIC signaling components, and fragments thereof.
  • a polynucleotide encodes a polypeptide comprising an amino acid sequence set forth in any one of SEQ ID NOs: 3-27.
  • polynucleotide or “nucleic acid” refer to deoxyribonucleic acid (DNA), ribonucleic acid (RNA) and DNA/RNA hybrids. Polynucleotides may be single-stranded or double-stranded and either recombinant, synthetic, or isolated.
  • Polynucleotides include, but are not limited to: pre-messenger RNA (pre-mRNA), messenger RNA (mRNA), RNA, synthetic RNA, synthetic mRNA, genomic DNA (gDNA), PCR amplified DNA, complementary DNA (cDNA), synthetic DNA, or recombinant DNA.
  • pre-mRNA pre-messenger RNA
  • mRNA messenger RNA
  • RNA synthetic RNA
  • mRNA genomic DNA
  • gDNA genomic DNA
  • cDNA complementary DNA
  • synthetic DNA or recombinant DNA.
  • Polynucleotides refer to a polymeric form of nucleotides of at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, at least 1000, at least 5000, at least 10000, or at least 15000 or more nucleotides in length, either ribonucleotides or deoxyribonucleotides or a modified form of either type of nucleotide, as well as all intermediate lengths.
  • intermediate lengths in this context, means any length between the quoted values, such as 6, 7, 8, 9, etc., 101, 102, 103, etc , 151, 152, 153, etc:, 201, 202, 203, etc.
  • polynucleotides or variants have at least or about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a reference sequence.
  • isolated polynucleotide refers to a polynucleotide that has been purified from the sequences which flank it in a naturally-occurring state, e.g., a DNA fragment that has been removed from the sequences that are normally adjacent to the fragment.
  • an “isolated polynucleotide” also refers to a complementary DNA (cDNA), a recombinant DNA, or other polynucleotide that does not exist in nature and that has been made by the hand of man.
  • an isolated polynucleotide is a synthetic polynucleotide, a semi-synthetic polynucleotide, or a polynucleotide obtained or derived from a recombinant source.
  • a polynucleotide comprises an mRNA encoding a polypeptide contemplated herein.
  • the mRNA comprises a cap, one or more nucleotides, and a poly(A) tail.
  • polynucleotides encoding one or more DARIC components may be codon-optimized.
  • codon-optimized refers to substituting codons in a polynucleotide encoding a polypeptide in order to increase the expression, stability and/or activity of the polypeptide.
  • Factors that influence codon optimization include, but are not limited to one or more of: (i) variation of codon biases between two or more organisms or genes or synthetically constructed bias tables, (ii) variation in the degree of codon bias within an organism, gene, or set of genes, (iii) systematic variation of codons including context, (iv) variation of codons according to their decoding tRNAs, (v) variation of codons according to GC %, either overall or in one position of the triplet, (vi) variation in degree of similarity to a reference sequence for example a naturally occurring sequence, (vii) variation in the codon frequency cutoff, (viii) structural properties of mRNAs transcribed from the DNA sequence, (ix) prior knowledge about the function of the DNA sequences upon which design of the codon substitution set is to be based, (x) systematic variation of codon sets for each amino acid, and/or (xi) isolated removal of spurious translation initiation sites.
  • nucleotide refers to a heterocyclic nitrogenous base in N- glycosidic linkage with a phosphorylated sugar. Nucleotides are understood to include natural bases, and a wide variety of art-recognized modified bases. Such bases are generally located at the 1 ' position of a nucleotide sugar moiety. Nucleotides generally comprise a base, sugar and a phosphate group. In ribonucleic acid (RNA), the sugar is a ribose, and in deoxyribonucleic acid (DNA) the sugar is a deoxyribose, i.e., a sugar lacking a hydroxyl group that is present in ribose.
  • RNA ribonucleic acid
  • DNA deoxyribonucleic acid
  • polynucleotides contemplated herein include, but are not limited to polynucleotides encoding one or more DARIC binding components, a DARIC signaling component, fusion polypeptides, and expression vectors, viral vectors, and transfer plasmids comprising polynucleotides contemplated herein.
  • polynucleotide variant and “variant” and the like refer to polynucleotides displaying substantial sequence identity with a reference polynucleotide sequence or polynucleotides that hybridize with a reference sequence under stringent conditions that are defined hereinafter. These terms also encompass polynucleotides that are distinguished from a reference polynucleotide by the addition, deletion, substitution, or modification of at least one nucleotide. Accordingly, the terms “polynucleotide variant” and “variant” include polynucleotides in which one or more nucleotides have been added or deleted, or modified, or replaced with different nucleotides.
  • sequence identity or, for example, comprising a “sequence 50% identical to,” as used herein, refer to the extent that sequences are identical on a nucleotide- by-nucleotide basis or an amino acid-by-amino acid basis over a window of comparison.
  • a “percentage of sequence identity” may be calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base e.g., A, T, C, G, I) or the identical amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Vai, Leu, He, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gin, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (/. ⁇ ?., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.
  • the identical nucleic acid base e.g., A, T, C, G, I
  • the identical amino acid residue e.g., Ala, Pro, Ser, Thr, Gly, Vai, Leu, He, Phe, Tyr, Trp, Lys,
  • nucleotides and polypeptides having at least about 50%, 55%, 60%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 86%, 97%, 98%, or 99% sequence identity to any of the reference sequences described herein.
  • polynucleotides contemplated herein may be combined with other DNA sequences, such as promoters and/or enhancers, untranslated regions (UTRs), signal sequences, Kozak sequences, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, internal ribosomal entry sites (IRES), recombinase recognition sites (e.g., LoxP, FRT, and Att sites), termination codons, transcriptional termination signals, and polynucleotides encoding self-cleaving polypeptides, epitope tags, as disclosed elsewhere herein or as known in the art, such that their overall length may vary considerably. It is therefore contemplated that a polynucleotide fragment of almost any length may be employed, with the total length preferably being limited by the ease of preparation and use in the intended recombinant DNA protocol.
  • Polynucleotides can be prepared, manipulated, expressed and/or delivered using any of a variety of well-established techniques known and available in the art.
  • a nucleotide sequence encoding the polypeptide can be inserted into appropriate vector.
  • vectors include, but are not limited to plasmid, autonomously replicating sequences, and transposable elements, e.g., Sleeping Beauty, PiggyBac.
  • vectors include, without limitation, plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC), or Pl -derived artificial chromosome (PAC), bacteriophages such as lambda phage or M13 phage, and animal viruses.
  • artificial chromosomes such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC), or Pl -derived artificial chromosome (PAC)
  • bacteriophages such as lambda phage or M13 phage
  • animal viruses include, without limitation, plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC), or Pl -derived artificial chromosome (PAC), bacteriophages such as lambda phage or M13 phage, and animal viruses.
  • viruses useful as vectors include, without limitation, retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpesvirus (e.g, herpes simplex virus), poxvirus, baculovirus, papillomavirus, and papovavirus (e.g., SV40).
  • retrovirus including lentivirus
  • adenovirus e.g., adeno-associated virus
  • herpesvirus e.g, herpes simplex virus
  • poxvirus baculovirus
  • papillomavirus papillomavirus
  • papovavirus e.g., SV40
  • expression vectors include, but are not limited to, pClneo vectors (Promega) for expression in mammalian cells; pLenti4/V5-DESTTM, pLenti6/V5- DESTTM, and pLenti6.2/V5-GW/lacZ (Invitrogen) for lentivirus-mediated gene transfer and expression in mammalian cells.
  • coding sequences of polypeptides disclosed herein can be ligated into such expression vectors for the expression of the polypeptides in mammalian cells.
  • the vector is an episomal vector or a vector that is maintained extrachromosomally.
  • episomal vector refers to a vector that is able to replicate without integration into host’s chromosomal DNA and without gradual loss from a dividing host cell also meaning that said vector replicates extrachromosomally or episomally.
  • “Expression control sequences,” “control elements,” or “regulatory sequences” present in an expression vector are those non-translated regions of the vector including an origin of replication, selection cassettes, promoters, enhancers, translation initiation signals (Shine Dalgamo sequence or Kozak sequence) introns, a polyadenylation sequence, 5' and 3' untranslated regions, all of which interact with host cellular proteins to carry out transcription and translation.
  • Such elements may vary in their strength and specificity.
  • any number of suitable transcription and translation elements including ubiquitous promoters and inducible promoters may be used.
  • a polynucleotide comprises a vector, including but not limited to expression vectors and viral vectors.
  • a vector may comprise one or more exogenous, endogenous, or heterologous control sequences such as promoters and/or enhancers.
  • An “endogenous control sequence” is one which is naturally linked with a given gene in the genome.
  • An “exogenous control sequence” is one which is placed in juxtaposition to a gene by means of genetic manipulation (i.e., molecular biological techniques) such that transcription of that gene is directed by the linked enhancer/promoter.
  • a “heterologous control sequence” is an exogenous sequence that is from a different species than the cell being genetically manipulated.
  • a “synthetic” control sequence may comprise elements of one more endogenous and/or exogenous sequences, and/or sequences determined in vitro or in silico that provide optimal promoter and/or enhancer activity for the particular therapy.
  • promoter refers to a recognition site of a polynucleotide (DNA or RNA) to which an RNA polymerase binds.
  • An RNA polymerase initiates and transcribes polynucleotides operably linked to the promoter.
  • promoters operative in mammalian cells comprise an AT-rich region located approximately 25 to 30 bases upstream from the site where transcription is initiated and/or another sequence found 70 to 80 bases upstream from the start of transcription, a CNCAAT region where N may be any nucleotide.
  • enhancer refers to a segment of DNA which contains sequences capable of providing enhanced transcription and in some instances can function independent of their orientation relative to another control sequence.
  • An enhancer can function cooperatively or additively with promoters and/or other enhancer elements.
  • promoter/enhancer refers to a segment of DNA which contains sequences capable of providing both promoter and enhancer functions.
  • operably linked refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner.
  • the term refers to a functional linkage between a nucleic acid expression control sequence (such as a promoter, and/or enhancer) and a second polynucleotide sequence, e.g., a polynucleotide-of-interest, wherein the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence.
  • constitutive expression control sequence refers to a promoter, enhancer, or promoter/enhancer that continually or continuously allows for transcription of an operably linked sequence.
  • a constitutive expression control sequence may be a “ubiquitous” promoter, enhancer, or promoter/enhancer that allows expression in a wide variety of cell and tissue types or a “cell specific,” “cell type specific,” “cell lineage specific,” or “tissue specific” promoter, enhancer, or promoter/enhancer that allows expression in a restricted variety of cell and tissue types, respectively.
  • Illustrative ubiquitous expression control sequences suitable for use in particular embodiments include, but are not limited to, a cytomegalovirus (CMV) immediate early promoter, a viral simian virus 40 (SV40) (e.g., early or late), a Moloney murine leukemia virus (MoMLV) LTR promoter, a Rous sarcoma virus (RSV) LTR, a herpes simplex virus (HSV) (thymidine kinase) promoter, H5, P7.5, and Pl 1 promoters from vaccinia virus, an elongation factor 1 -alpha (EFla) promoter, early growth response 1 (EGR1), ferritin H (FerH), ferritin L (FerL), Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), eukaryotic translation initiation factor 4A1 (EIF4A1), heat shock 70kDa protein 5 (HSPA5), heat shock protein 90kDa beta
  • a vector comprises an MNDU3 promoter.
  • a vector comprises an EFla promoter comprising the first intron of the human EFla gene.
  • a vector comprises an EFla promoter that lacks the first intron of the human EFla gene.
  • a cell, cell type, cell lineage or tissue specific expression control sequence may be desirable to use to achieve cell type specific, lineage specific, or tissue specific expression of a desired polynucleotide sequence (e g., to express a particular nucleic acid encoding a polypeptide in only a subset of cell types, cell lineages, or tissues or during specific stages of development).
  • an “internal ribosome entry site” or “IRES” refers to an element that promotes direct internal ribosome entry to the initiation codon, such as ATG, of a cistron (a protein encoding region), thereby leading to the cap-independent translation of the gene. See, e.g., Jackson et a!., 1990. Trends Biochem Sci 15(12):477-83) and Jackson and Kaminski. 1995. RNA l(10):985-1000. Examples of IRES generally employed by those of skill in the art include those described in U.S. Pat. No. 6,692,736.
  • IRES immunoglobulin heavy-chain binding protein
  • VEGF vascular endothelial growth factor
  • FGF-2 fibroblast growth factor 2
  • IGFII insulinlike growth factor
  • eIF4G translational initiation factor eIF4G and yeast transcription factors TFIID and HAP4
  • EMCV encephelomy carditis virus
  • IRES have also been reported in viral genomes of Picomaviridae, Dicistroviridae and Flaviviridae species and in HCV, Friend murine leukemia virus (FrMLV) and Moloney murine leukemia virus (MoMLV).
  • the IRES used in polynucleotides contemplated herein is an EMCV IRES.
  • the polynucleot ides a consensus Kozak sequence.
  • Kozak sequence refers to a short nucleotide sequence that greatly facilitates the initial binding of mRNA to the small subunit of the ribosome and increases translation.
  • the consensus Kozak sequence is (GCC)RCCATGG (SEQ ID NO: 83), where R is a purine (A or G) (Kozak, 1986. Cell. 44(2):283-92, and Kozak, 1987. Nucleic Acids Res. 15(20):8125- 48).
  • vectors comprise a polyadenylation sequence 3' of a polynucleotide encoding a polypeptide to be expressed.
  • polyA site or “polyA sequence” as used herein denotes a DNA sequence which directs both the termination and polyadenylation of the nascent RNA transcript by RNA polymerase II.
  • Polyadenylation sequences can promote mRNA stability by addition of a polyA tail to the 3' end of the coding sequence and thus, contribute to increased translational efficiency.
  • Cleavage and polyadenylation are directed by a poly(A) sequence in the RNA.
  • the core poly(A) sequence for mammalian pre-mRNAs has two recognition elements flanking a cleavage-polyadenylation site. Typically, an almost invariant AAUAAA hexamer lies 20-50 nucleotides upstream of a more variable element rich in U or GU residues. Cleavage of the nascent transcript occurs between these two elements and is coupled to the addition of up to 250 adenosines to the 5' cleavage product.
  • the core poly(A) sequence is an ideal polyA sequence (e.g, AATAAA, ATTAAA, AGTAAA).
  • the poly(A) sequence is an SV40 polyA sequence, a bovine growth hormone polyA sequence (BGHpA), a rabbit p-globin polyA sequence (rPgpA), variants thereof, or another suitable heterologous or endogenous polyA sequence known in the art.
  • the poly(A) sequence is synthetic.
  • polynucleotides encoding one or more polypeptides, or fusion polypeptides may be introduced into immune effector cells, e.g., T cells, by both non-viral and viral methods.
  • delivery of one or more polynucleotides may be provided by the same method or by different methods, and/or by the same vector or by different vectors.
  • vector is used herein to refer to a nucleic acid molecule capable transferring or transporting another nucleic acid molecule.
  • the transferred nucleic acid is generally linked to, e.g., inserted into, the vector nucleic acid molecule.
  • a vector may include sequences that direct autonomous replication in a cell, or may include sequences sufficient to allow integration into host cell DNA.
  • non-viral vectors are used to deliver one or more polynucleotides contemplated herein to a T cell.
  • non-viral vectors include, but are not limited to plasmids (e.g., DNA plasmids or RNA plasmids), transposons, cosmids, and bacterial artificial chromosomes.
  • plasmids e.g., DNA plasmids or RNA plasmids
  • transposons e.g., DNA plasmids or RNA plasmids
  • cosmids e.g., cosmids, and bacterial artificial chromosomes.
  • Illustrative methods of non-viral delivery of polynucleotides contemplated in particular embodiments include, but are not limited to: electroporation, sonoporation, lipofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, nanoparticles, polycation or lipid:nucleic acid conjugates, naked DNA, artificial virions, DEAE-dextran-mediated transfer, gene gun, and heat-shock.
  • polynucleotide delivery systems suitable for use in particular embodiments contemplated in particular embodiments include, but are not limited to those provided by Amaxa Biosystems, Maxcyte, Inc., BTX Molecular Delivery Systems, and Copernicus Therapeutics Inc.
  • Lipofection reagents are sold commercially (e.g., TransfectamTM and LipofectinTM). Cationic and neutral lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides have been described in the literature. See e.g., Liu et al. (2003) Gene Therapy. 10: 180-187; and Balazs etal. (2011) Journal of Drug Delivery. 2011 : 1-12.
  • Antibody-targeted, bacterially derived, non-living nanocell-based delivery is also contemplated in particular embodiments.
  • Viral vectors comprising polynucleotides contemplated in particular embodiments can be delivered in vivo by administration to an individual patient, typically by systemic administration (e.g., intravenous, intraperitoneal, intramuscular, subdermal, or intracranial infusion) or topical application, as described below.
  • vectors can be delivered to cells ex vivo, such as cells explanted from an individual patient (e.g., mobilized peripheral blood, lymphocytes, bone marrow aspirates, tissue biopsy, etc.) or universal donor hematopoietic stem cells, followed by reimplantation of the cells into a patient.
  • viral vectors comprising polynucleotides contemplated herein are administered directly to an organism for transduction of cells in vivo.
  • naked DNA can be administered.
  • Administration is by any of the routes normally used for introducing a molecule into ultimate contact with blood or tissue cells including, but not limited to, injection, infusion, topical application and electroporation. Suitable methods of administering such nucleic acids are available and well known to those of skill in the art, and, although more than one route can be used to administer a particular composition, a particular route can often provide a more immediate and more effective reaction than another route.
  • viral vector systems suitable for use in particular embodiments contemplated in particular embodiments include, but are not limited to, adeno- associated virus (AAV), retrovirus, herpes simplex virus, adenovirus, and vaccinia virus vectors.
  • AAV adeno-associated virus
  • retrovirus retrovirus
  • herpes simplex virus adenovirus
  • vaccinia virus vectors adeno-associated virus vectors.
  • one or more polynucleotides encoding one or more DARIC binding components, a DARIC signaling component, and/or other polypeptides contemplated herein are introduced into an immune effector cell, e.g., T cell, by transducing the cell with a recombinant adeno-associated virus (rAAV), comprising the one or more polynucleotides.
  • rAAV recombinant adeno-associated virus
  • AAV is a small (-26 nm) replication-defective, primarily episomal, non-enveloped virus. AAV can infect both dividing and non-dividing cells and may incorporate its genome into that of the host cell.
  • Recombinant AAV rAAV
  • rAAV are typically composed of, at a minimum, a transgene and its regulatory sequences, and 5' and 3' AAV inverted terminal repeats (ITRs).
  • the ITR sequences are about 145 bp in length.
  • the rAAV comprises ITRs and capsid sequences isolated from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10.
  • a chimeric rAAV is used the ITR sequences are isolated from one AAV serotype and the capsid sequences are isolated from a different AAV serotype.
  • a rAAV with ITR sequences derived from AAV2 and capsid sequences derived from AAV6 is referred to as AAV2/AAV6.
  • the rAAV vector may comprise ITRs from AAV2, and capsid proteins from any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10.
  • the rAAV comprises ITR sequences derived from AAV2 and capsid sequences derived from AAV6.
  • the rAAV comprises ITR sequences derived from AAV2 and capsid sequences derived from AAV2.
  • engineering and selection methods can be applied to AAV capsids to make them more likely to transduce cells of interest.
  • one or more DARIC binding components, a DARIC signaling component, and/or other polypeptides contemplated herein are introduced into an immune effector cell, e.g., T cell, by transducing the cell with a retrovirus, e.g., lentivirus, comprising the one or more polynucleotides.
  • a retrovirus e.g., lentivirus
  • the term “retrovirus” refers to an RNA virus that reverse transcribes its genomic RNA into a linear double-stranded DNA copy and subsequently covalently integrates its genomic DNA into a host genome.
  • Illustrative retroviruses suitable for use in particular embodiments include, but are not limited to: Moloney murine leukemia virus (M- MuLV), Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), gibbon ape leukemia virus (GaLV), feline leukemia virus (FLV), spumavirus, Friend murine leukemia virus, Murine Stem Cell Virus (MSCV) and Rous Sarcoma Virus (RSV)) and lentivirus.
  • M- MuLV Moloney murine leukemia virus
  • MoMSV Moloney murine sarcoma virus
  • HaMuSV Harvey murine sarcoma virus
  • MuMTV murine mammary tumor virus
  • GaLV gibbon ape leukemia virus
  • FLV feline leukemia virus
  • RSV Rous Sarcoma Virus
  • lentivirus refers to a group (or genus) of complex retroviruses.
  • Illustrative lentiviruses include, but are not limited to, HIV (human immunodeficiency virus; including HIV type 1, and HIV type 2); visna-maedi virus (VMV) virus; the caprine arthritis-encephalitis virus (CAEV); equine infectious anemia virus (EIAV); feline immunodeficiency virus (FIV); bovine immune deficiency virus (BIV); and simian immunodeficiency virus (SIV).
  • HIV based vector backbones i.e., HIV cis-acting sequence elements
  • HIV cis-acting sequence elements are preferred.
  • a lentiviral vector contemplated herein comprises one or more LTRs, and one or more, or all, of the following accessory elements: a cPPT/FLAP, a Psi (T) packaging signal, an export element, poly (A) sequences, and may optionally comprise a WPRE or HPRE, an insulator element, a selectable marker, and a cell suicide gene, as discussed elsewhere herein.
  • lentiviral vectors contemplated herein may be integrative or non-integrating or integration defective lentivirus.
  • integration defective lentivirus or “IDLV” refers to a lentivirus having an integrase that lacks the capacity to integrate the viral genome into the genome of the host cells. Integration-incompetent viral vectors have been described in patent application WO 2006/010834, which is herein incorporated by reference in its entirety.
  • Illustrative mutations in the HIV-1 pol gene suitable to reduce integrase activity include, but are not limited to: H12N, H12C, H16C, H16V, S81 R, D41A, K42A, H51A, Q53C, D55V, D64E, D64V, E69A, K71A, E85A, E87A, DI 16N, DI 161, DI 16A, N120G, N1201, N120E, E152G, E152A, D35E, K156E, K156A, E157A, K159E, K159A, K160A, R166A, D167A, E170A, H171A, K173A, K186Q, K186T, K188T, E198A, R199c, R199T, R199A, D202A, K211A, Q214L, Q216L, Q221 L, W235F, W235E, K236S, K236A, K246A, G247W, D25
  • LTR long terminal repeat
  • FLAP element refers to a nucleic acid whose sequence includes the central polypurine tract and central termination sequences (cPPT and CTS) of a retrovirus, e.g, HIV-1 or HIV-2.
  • cPPT and CTS central polypurine tract and central termination sequences
  • Suitable FLAP elements are described in U.S. Pat. No. 6,682,907 and in Zennou, etal, 2000, Cell, 101:173.
  • packaging signal or “packaging sequence” refers to psi [ ] sequences located within the retroviral genome which are required for insertion of the viral RNA into the viral capsid or particle, see e.g., Clever el al, 1995. J. of Virology, Vol. 69, No. 4; pp. 2101-2109.
  • RNA export element refers to a cis-acting post-transcriptional regulatory element which regulates the transport of an RNA transcript from the nucleus to the cytoplasm of a cell.
  • RNA export elements include, but are not limited to, the human immunodeficiency virus (HIV) rev response element (RRE) (see e.g., Cullen et al., 1991. J. Virol. 65: 1053; and Cullen et al., 1991. Cell 58: 423), and the hepatitis B virus post- transcriptional regulatory element (HPRE).
  • HCV human immunodeficiency virus
  • RRE hepatitis B virus post- transcriptional regulatory element
  • expression of heterologous sequences in viral vectors is increased by incorporating posttranscriptional regulatory elements, efficient polyadenylation sites, and optionally, transcription termination signals into the vectors.
  • posttranscriptional regulatory elements can increase expression of a heterologous nucleic acid at the protein, e.g, woodchuck hepatitis vims posttranscriptional regulatory element (WPRE; Zufferey et al., 1999, J. Virol., 73:2886); the posttranscriptional regulatory element present in hepatitis B vims (HPRE) (Huang et al., Mol. Cell. Biol., 5:3864); and the like (Liu et al., 1995, Genes Dev., 9:1766).
  • WPRE woodchuck hepatitis vims posttranscriptional regulatory element
  • HPRE posttranscriptional regulatory element present in hepatitis B vims
  • Lentiviral vectors preferably contain several safety enhancements as a result of modifying the LTRs.
  • Self-inactivating (SIN) vectors refers to replication-defective vectors, e.g., retroviral or lentiviral vectors, in which the right (3') LTR enhancer-promoter region, known as the U3 region, has been modified (e.g., by deletion or substitution) to prevent viral transcription beyond the first round of viral replication.
  • Self-inactivation is preferably achieved through in the introduction of a deletion in the U3 region of the 3' LTR of the vector DNA, i.e., the DNA used to produce the vector RNA. Thus, during reverse transcription, this deletion is transferred to the 5' LTR of the proviral DNA.
  • the U3 sequence it is desirable to eliminate enough of the U3 sequence to greatly diminish or abolish altogether the transcriptional activity of the LTR, thereby greatly diminishing or abolishing the production of full-length vector RNA in transduced cells.
  • HIV based lentivectors it has been discovered that such vectors tolerate significant U3 deletions, including the removal of the LTR TATA box (e.g., deletions from -418 to -18), without significant reductions in vector titers.
  • heterologous promoters which can be used include, for example, viral simian virus 40 (SV40) (e.g., early or late), cytomegalovirus (CMV) (e.g., immediate early), Moloney murine leukemia vims (MoMLV), Rous sarcoma vims (RSV), and herpes simplex vims (HSV) (thymidine kinase) promoters.
  • SV40 viral simian virus 40
  • CMV cytomegalovirus
  • MoMLV Moloney murine leukemia vims
  • RSV Rous sarcoma vims
  • HSV herpes simplex vims
  • HIV can be pseudotyped with vesicular stomatitis vims G-protein (VSV-G) envelope proteins, which allows HIV to infect a wider range of cells because HIV envelope proteins (encoded by the env gene) normally target the vims to CD4 + presenting cells.
  • VSV-G vesicular stomatitis vims G-protein
  • lentiviral vectors are produced according to known methods. See e.g., Kutner etal., BMC Biotechnol. 2009;9: 10. doi: 10.1186/1472-6750-9-10; Kutnere/a/. Nat. Protoc. 2009;4(4):495-505. doi: 10.1038/nprot.2009.22.
  • most or all of the viral vector backbone sequences are derived from a lentivims, e.g., HIV-1.
  • a lentivims e.g., HIV-1.
  • many different sources of retroviral and/or lentiviral sequences can be used, or combined and numerous substitutions and alterations in certain of the lentiviral sequences may be accommodated without impairing the ability of a transfer vector to perform the functions described herein.
  • a variety of lentiviral vectors are known in the art, see Naldini etal., (1996a, 1996b, and 1998); Zufferey etal., (1997); Dull etal., 1998, U.S. Pat. Nos. 6,013,516; and 5,994,136, many of which may be adapted to produce a viral vector or transfer plasmid contemplated herein.
  • one or more DARIC binding components, a DARIC signaling component, and/or other polypeptides contemplated herein are introduced into an immune effector cell, by transducing the cell with an adenovirus comprising the one or more polynucleotides.
  • Adenoviral based vectors are capable of very high transduction efficiency in many cell types and do not require cell division. With such vectors, high titer and high levels of expression have been obtained. This vector can be produced in large quantities in a relatively simple system. Most adenovirus vectors are engineered such that a transgene replaces the Ad El a, Elb, and/or E3 genes; subsequently the replication defective vector is propagated in human 293 cells that supply deleted gene function in trans. Ad vectors can transduce multiple types of tissues in vivo, including non-dividing, differentiated cells such as those found in liver, kidney and muscle. Conventional Ad vectors have a large carrying capacity.
  • Generation and propagation of the current adenovirus vectors may utilize a unique helper cell line, designated 293, which was transformed from human embryonic kidney cells by Ad5 DNA fragments and constitutively expresses El proteins (Graham et al., 1977). Since the E3 region is dispensable from the adenovirus genome (Jones & Shenk, 1978), the current adenovirus vectors, with the help of 293 cells, carry foreign DNA in either the El, the D3 or both regions (Graham & Prevec, 1991).
  • a unique helper cell line designated 293, which was transformed from human embryonic kidney cells by Ad5 DNA fragments and constitutively expresses El proteins (Graham et al., 1977). Since the E3 region is dispensable from the adenovirus genome (Jones & Shenk, 1978), the current adenovirus vectors, with the help of 293 cells, carry foreign DNA in either the El, the D3 or both regions (Graham & Prevec, 1991).
  • Adenovirus vectors have been used in eukaryotic gene expression (Levrero et al., 1991; Gomez-Foix etal, 1992) and vaccine development (Grunhaus & Horwitz, 1992; Graham & Prevec, 1992).
  • Studies in administering recombinant adenovirus to different tissues include trachea instillation (Rosenfeld etal., 1991; Rosenfeld et al., 1992), muscle injection (Ragot et al., 1993), peripheral intravenous injections (Herz & Gerard, 1993) and stereotactic inoculation into the brain (Le Gal La Salle et al., 1993).
  • An example of the use of an Ad vector in a clinical trial involved polynucleotide therapy for antitumor immunization with intramuscular injection (Sterman et al., Hum. Gene Ther. 7:1083-9 (1998)).
  • one or more DARIC binding components, a DARIC signaling component, and/or other polypeptides contemplated herein are introduced into an immune effector cell by transducing the cell with a herpes simplex virus, e.g, HSV-1, HSV-2, comprising the one or more polynucleotides.
  • a herpes simplex virus e.g, HSV-1, HSV-2
  • the mature HSV virion consists of an enveloped icosahedral capsid with a viral genome consisting of a linear double-stranded DNA molecule that is 152 kb.
  • the HSV based viral vector is deficient in one or more essential or non-essential HSV genes.
  • the HSV based viral vector is replication deficient. Most replication deficient HSV vectors contain a deletion to remove one or more intermediate-early, early, or late HSV genes to prevent replication.
  • the HSV vector may be deficient in an immediate early gene selected from the group consisting of: ICP4, ICP22, ICP27, ICP47, and a combination thereof.
  • HSV vectors are its ability to enter a latent stage that can result in long-term DNA expression and its large viral DNA genome that can accommodate exogenous DNA inserts of up to 25 kb.
  • HSV-based vectors are described in, for example, U.S. Pat. Nos. 5,837,532, 5,846,782, and 5,804,413, and International Patent Applications WO 91/02788, WO 96/04394, WO 98/15637, and WO 99/06583, each of which are incorporated by reference herein in its entirety.
  • cells are modified to express a DARIC, one or more DARIC binding components, a DARIC signaling component, and/or fusion proteins contemplated herein, for use in the treatment of cancer.
  • Cells may be non-genetically modified to express one or more of the polypeptides contemplated herein, or in particular preferred embodiments, cells may be genetically modified to express one or more of the polypeptides contemplated herein.
  • the term “genetically engineered” or “genetically modified” refers to the addition of extra genetic material in the form of DNA or RNA into the total genetic material in a cell.
  • the terms, “genetically modified cells,” “modified cells,” and “redirected cells,” are used interchangeably in particular embodiments.
  • a DARIC DARIC
  • one or more DARIC binding components DARIC binding components
  • a DARIC signaling component DARIC signaling component
  • fusion proteins contemplated herein are introduced and expressed in immune effector cells to improve the efficacy of the immune effector cells.
  • an “immune effector cell,” is any cell of the immune system that has one or more effector functions (e.g, cytotoxic cell killing activity, secretion of cytokines, induction of ADCC and/or CDC).
  • the illustrative immune effector cells contemplated herein are T lymphocytes, including but not limited to cytotoxic T cells (CTLs; CD8 + T cells), TILs, and helper T cells (HTLs; CD4 + T cells).
  • the cells comprise a.p T cells.
  • the cells comprise 76 T cells.
  • immune effector cells include natural killer (NK) cells.
  • immune effector cells include natural killer T (NKT) cells.
  • Immune effector cells can be autologous/autogeneic (“self’) or non-autologous (“non-self,” e.g., allogeneic, syngeneic or xenogeneic).
  • “Autologous,” as used herein, refers to cells from the same subject. “Allogeneic,” as used herein, refers to cells of the same species that differ genetically to the cell in comparison. “Syngeneic,” as used herein, refers to cells of a different subject that are genetically identical to the cell in comparison. “Xenogeneic,” as used herein, refers to cells of a different species to the cell in comparison. In preferred embodiments, the cells are human autologous immune effector cells.
  • T lymphocytes suitable for introducing a DARIC, one or more DARIC binding components, a DARIC signaling component, and/or fusion proteins contemplated herein include T lymphocytes.
  • T cell or “T lymphocyte” are art- recognized and are intended to include thymocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes.
  • a T cell can be a T helper (Th) cell, for example a T helper 1 (Thl) or a T helper 2 (Th2) cell.
  • the T cell can be a helper T cell (HTL; CD4 + T cell), a cytotoxic T cell (CTL; CD8 + T cell), CD4 + CD8 + T cell, CD4'CD8‘ T cell, or any other subset of T cells.
  • the T cell expresses a T cell receptor.
  • T cell receptors comprise two subunits, an alpha chain and a beta chain subunit (apTCR), or a gamma chain and a delta chain subunit (ySTCR), each of which is a unique protein produced by recombination event in each T cell’s genome.
  • a T cell is an aPTCR T cell (an T cell).
  • a T cell is a ySTCR T cell (a y5 T cell).
  • Other illustrative populations of T cells suitable for use in particular embodiments include naive T cells and memory T cells.
  • immune effector cells comprising a DARIC, one or more DARIC binding components, a DARIC signaling component, and/or fusion proteins contemplated herein.
  • immune effector cells also include NK cells, NKT cells, neutrophils, and macrophages.
  • Immune effector cells also include progenitors of effector cells wherein such progenitor cells can be induced to differentiate into immune effector cells in vivo or in vitro.
  • immune effector cells include progenitors of immune effectors cells such as hematopoietic stem cells (HSCs) contained within the CD34 + population of cells derived from cord blood, bone marrow or mobilized peripheral blood which upon administration in a subject differentiate into mature immune effector cells, or which can be induced in vitro to differentiate into mature immune effector cells.
  • HSCs hematopoietic stem cells
  • CD34 + cell refers to a cell expressing the CD34 protein on its cell surface.
  • CD34 refers to a cell surface glycoprotein (e.g., sialomucin protein) that often acts as a cell-cell adhesion factor and is involved in T cell entrance into lymph nodes.
  • the CD34 + cell population contains hematopoietic stem cells (HSC), which upon administration to a patient differentiate and contribute to all hematopoietic lineages, including T cells, NK cells, NKT cells, neutrophils and cells of the monocyte/macrophage lineage.
  • HSC hematopoietic stem cells
  • the method comprises transfecting or transducing immune effector cells isolated from an individual such that the immune effector cells with one or more nucleic acids and/or vectors, e.g., a lentiviral vector comprising a nucleic acid encoding a DARIC, one or more DARIC binding components, a DARIC signaling component, and/or fusion proteins contemplated herein.
  • nucleic acids and/or vectors e.g., a lentiviral vector comprising a nucleic acid encoding a DARIC, one or more DARIC binding components, a DARIC signaling component, and/or fusion proteins contemplated herein.
  • the method comprises transfecting or transducing immune effector cells isolated from an individual such that the immune effector cells express a DARIC, one or more DARIC binding components, a DARIC signaling component, and/or fusion proteins contemplated herein.
  • the immune effector cells are isolated from an individual and genetically modified without further manipulation in vitro. Such cells can then be directly re-administered into the individual.
  • the immune effector cells are first activated and stimulated to proliferate in vitro prior to being genetically modified.
  • the immune effector cells may be cultured before and/or after being genetically modified.
  • the source of cells is obtained from a subject.
  • the modified immune effector cells comprise T cells.
  • T cells can be obtained from a number of sources including, but not limited to, peripheral blood mononuclear cells, bone marrow, lymph nodes tissue, cord blood, thymus issue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors.
  • T cells can be obtained from a unit of blood collected from a subject using any number of techniques known to the skilled person, such as sedimentation, e.g., FICOLLTM separation.
  • an isolated or purified population of T cells is used.
  • both cytotoxic and helper T lymphocytes can be sorted into naive, memory, and effector T cell subpopulations either before or after activation, expansion, and/or genetic modification.
  • an isolated or purified population of T cells expresses one or more of the markers including, but not limited to a CD3 + , CD4 + , CD8 + , or a combination thereof.
  • the T cells are isolated from an individual and first activated and stimulated to proliferate in vitro prior to being modified to express a DARIC, one or more DARIC binding components, a DARIC signaling component, and/or fusion proteins contemplated herein.
  • T cells are often subjected to one or more rounds of stimulation, activation and/or expansion.
  • T cells can be activated and expanded generally using methods as described, for example, in U.S.
  • T cells are activated and expanded for about 6 hours, about 12 hours, about 18 hours or about 24 hours prior to introduction of vectors or polynucleotides encoding a DARIC, one or more DARIC binding components, a DARIC signaling component, and/or fusion proteins contemplated herein.
  • compositions contemplated herein may comprise a DARIC, one or more DARIC binding components, a DARIC signaling component, fusion proteins, polynucleotides encoding a DARIC, one or more DARIC binding components, a DARIC signaling component, fusion proteins, vectors comprising the same, genetically modified immune effector cells, bridging factors, etc.
  • Compositions include, but are not limited to, pharmaceutical compositions.
  • a “pharmaceutical composition” refers to a composition formulated in pharmaceutically-acceptable or physiologically-acceptable solutions for administration to a cell or an animal, either alone, or in combination with one or more other modalities of therapy.
  • compositions may be administered in combination with other agents as well, such as, e.g., cytokines, growth factors, hormones, small molecules, chemotherapeutics, pro-drugs, drugs, antibodies, or other various pharmaceutically-active agents.
  • agents such as, e.g., cytokines, growth factors, hormones, small molecules, chemotherapeutics, pro-drugs, drugs, antibodies, or other various pharmaceutically-active agents.
  • agents e.g., cytokines, growth factors, hormones, small molecules, chemotherapeutics, pro-drugs, drugs, antibodies, or other various pharmaceutically-active agents.
  • additional agents do not adversely affect the ability of the composition to deliver the intended therapy.
  • phrases “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • pharmaceutically acceptable carrier refers to a diluent, adjuvant, excipient, or vehicle with which the bridging factors, polypeptides, polynucleotides, vectors comprising same, or genetically modified immune effector cells are administered.
  • pharmaceutical carriers can be sterile liquids, such as cell culture media, water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.
  • Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
  • a composition comprising a pharmaceutically acceptable carrier is suitable for administration to a subject.
  • a composition comprising a carrier is suitable for parenteral administration, e.g., intravascular (intravenous or intraarterial), intraperitoneal or intramuscular administration.
  • a composition comprising a pharmaceutically acceptable carrier is suitable for intraventricular, intraspinal, or intrathecal administration.
  • Pharmaceutically acceptable carriers include sterile aqueous solutions, cell culture media, or dispersions. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the bridging factors, polypeptides, polynucleotides, vectors comprising same, or genetically modified immune effector cells, use thereof in the pharmaceutical compositions is contemplated.
  • compositions contemplated herein comprise genetically modified T cells and a pharmaceutically acceptable carrier.
  • a composition comprising a cell-based composition contemplated herein can be administered separately by enteral or parenteral administration methods or in combination with other suitable compounds to effect the desired treatment goals.
  • compositions contemplated herein comprise a bridging factor and a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier must be of sufficiently high purity and of sufficiently low toxicity to render it suitable for administration to the human subject being treated. It further should maintain or increase the stability of the composition.
  • the pharmaceutically acceptable carrier can be liquid or solid and is selected, with the planned manner of administration in mind, to provide for the desired bulk, consistency, etc., when combined with other components of the composition.
  • the pharmaceutically acceptable carrier can be, without limitation, a binding agent (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.), a filler (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates, calcium hydrogen phosphate, etc.), a lubricant (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycols, sodium benzoate, sodium acetate, etc.), a disintegrant (e.g., starch, sodium starch glycolate, etc.), or a wetting agent (e.g., sodium lauryl sulfate, etc.).
  • a binding agent e.g., pregelatinized maize starch, poly
  • compositions contemplated herein include, but are not limited to, water, salt solutions, alcohols, polyethylene glycols, gelatins, amyloses, magnesium stearates, talcs, silicic acids, viscous paraffins, hydroxymethylcelluloses, polyvinylpyrrolidones and the like.
  • buffers refers to a solution or liquid whose chemical makeup neutralizes acids or bases without a significant change in pH.
  • buffers contemplated herein include, but are not limited to, Dulbecco's phosphate buffered saline (PBS), Ringer’s solution, 5% dextrose in water (D5W), normal/physiologic saline (0.9% NaCl).
  • the pharmaceutically acceptable carriers may be present in amounts sufficient to maintain a pH of the composition of about 7.
  • the composition has a pH in a range from about 6.8 to about 7.4, e.g., 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, and 7.4.
  • the composition has a pH of about 7.4.
  • compositions contemplated herein may comprise a nontoxic pharmaceutically acceptable medium.
  • the compositions may be a suspension.
  • the term “suspension” as used herein refers to non-adherent conditions in which cells are not attached to a solid support. For example, cells maintained as a suspension may be stirred or agitated and are not adhered to a support, such as a culture dish.
  • compositions contemplated herein are formulated in a suspension, where the modified T cells are dispersed within an acceptable liquid medium or solution, e.g., saline or serum-free medium, in an intravenous (IV) bag or the like.
  • acceptable liquid medium or solution e.g., saline or serum-free medium
  • IV intravenous
  • Acceptable diluents include, but are not limited to water, PlasmaLyte, Ringer's solution, isotonic sodium chloride (saline) solution, serum-free cell culture medium, and medium suitable for cryogenic storage, e.g, Cryostor® medium.
  • a pharmaceutically acceptable carrier is substantially free of natural proteins of human or animal origin, and suitable for storing a composition comprising a population of modified T cells.
  • the therapeutic composition is intended to be administered into a human patient, and thus is substantially free of cell culture components such as bovine serum albumin, horse serum, and fetal bovine serum.
  • compositions are formulated in a pharmaceutically acceptable cell culture medium. Such compositions are suitable for administration to human subjects.
  • the pharmaceutically acceptable cell culture medium is a serum free medium.
  • Serum-free medium has several advantages over serum containing medium, including a simplified and better-defined composition, a reduced degree of contaminants, elimination of a potential source of infectious agents, and lower cost.
  • the serum-free medium is animal-free, and may optionally be protein-free.
  • the medium may contain biopharmaceutically acceptable recombinant proteins.
  • “Animal-free” medium refers to medium wherein the components are derived from non-animal sources. Recombinant proteins replace native animal proteins in animal- free medium and the nutrients are obtained from synthetic, plant or microbial sources.
  • Protein-free in contrast, is defined as substantially free of protein.
  • serum-free media used in particular compositions includes, but is not limited to, QBSF-60 (Quality Biological, Inc.), StemPro-34 (Life Technologies), and X-VIVO 10.
  • compositions comprising modified T cells are formulated in PlasmaLyte.
  • compositions comprising modified T cells are formulated in a cryopreservation medium.
  • cry opreservation media with cryopreservation agents may be used to maintain a high cell viability outcome post-thaw.
  • cry opreservation media used in particular compositions includes, but is not limited to, CryoStor CS10, CryoStor CS5, and CryoStor CS2.
  • compositions are formulated in a solution comprising 50:50 PlasmaLyte A to CryoStor CS10.
  • the composition is substantially free of mycoplasma, endotoxin, and microbial contamination.
  • substantially free with respect to endotoxin is meant that there is less endotoxin per dose of cells than is allowed by the FDA for a biologic, which is a total endotoxin of 5 EU/kg body weight per day, which for an average 70 kg person is 350 EU per total dose of cells.
  • compositions contemplated herein contain about 0.5 EU/ml to about 5.0 EU/ml, or about 0.5 EU/ml, 1.0 EU/ml, 1.5 EU/ml, 2.0 EU/ml, 2.5 EU/ml, 3.0 EU/ml, 3.5 EU/ml, 4.0 EU/ml, 4.5 EU/ml, or 5.0 EU/ml.
  • formulation of pharmaceutically-acceptable carrier solutions is well-known to those of skill in the art, as is the development of suitable dosing and treatment regimens for using the particular compositions described herein in a variety of treatment regimens, including e.g, enteral and parenteral, e.g, intravascular, intravenous, intrarterial, intraosseously, intraventricular, intracerebral, intracranial, intraspinal, intrathecal, and intramedullary administration and formulation.
  • enteral and parenteral e.g, intravascular, intravenous, intrarterial, intraosseously, intraventricular, intracerebral, intracranial, intraspinal, intrathecal, and intramedullary administration and formulation.
  • enteral and parenteral e.g, intravascular, intravenous, intrarterial, intraosseously, intraventricular, intracerebral, intracranial, intraspinal, intrathecal, and intramedullary administration and formulation.
  • particular embodiments contemplated herein may comprise other formulation
  • compositions comprise an amount of immune effector cells comprising a polynucleotide encoding a DARIC, one or more DARIC binding components, a DARIC signaling component, and/or fusion proteins contemplated herein.
  • compositions comprise an amount of immune effector cells that express a DARIC, one or more DARIC binding components, a DARIC signaling component, and/or fusion proteins contemplated herein.
  • the term “amount” refers to “an amount effective” or “an effective amount” of cells comprising a DARIC, one or more DARIC binding components, a DARIC signaling component, and/or fusion proteins contemplated herein, etc., to achieve a beneficial or desired prophylactic or therapeutic result in the presence of a bridging factor, including clinical results.
  • a “prophylactically effective amount” refers to an amount of cells comprising a DARIC, one or more DARIC binding components, a DARIC signaling component, and/or fusion proteins contemplated herein, etc., effective to achieve the desired prophylactic result in the presence of a bridging factor. Typically but not necessarily, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount is less than the therapeutically effective amount.
  • a “therapeutically effective amount” refers to an amount of cells comprising a DARIC, one or more DARIC binding components, a DARIC signaling component, and/or fusion proteins contemplated herein that is effective to “treat” a subject (e.g., a patient) in the presence of a bridging factor.
  • a therapeutic amount is indicated, the precise amount of the compositions to be administered, cells, bridging factor, etc, can be determined by a physician with consideration of individual differences in age, weight, tumor size, extent of infection or metastasis, and condition of the patient (subject).
  • a pharmaceutical composition comprising the immune effector cells described herein may be administered at a dosage of 10 2 to 10 10 cells/kg body weight, preferably 10 5 to 10 6 cells/kg body weight, including all integer values within those ranges.
  • the number of cells will depend upon the ultimate use for which the composition is intended as will the type of cells included therein.
  • the cells are generally in a volume of a liter or less, can be 500 mis or less, even 250 mis or 100 mis or less.
  • the density of the desired cells is typically greater than 10 6 cells/ml and generally is greater than 10 7 cells/ml, generally 10 8 cells/ml or greater.
  • the clinically relevant number of immune cells can be apportioned into multiple infusions that cumulatively equal or exceed 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , or 10 12 cells.
  • lower numbers of cells in the range of 10 6 /kilogram ( 10 6 - 10 11 per patient) may be administered.
  • the treatment may also include administration of mitogens (e.g., PHA) or lymphokines, cytokines, and/or chemokines (e.g., IFN-y, IL-2, IL-12, TNF-alpha, IL-18, and TNF-beta, GM-CSF, IL-4, IL-13, Flt3-L, RANTES, MIPla, etc.) as described herein to enhance induction of the immune response.
  • mitogens e.g., PHA
  • lymphokines e.g., lymphokines, cytokines, and/or chemokines (e.g., IFN-y, IL-2, IL-12, TNF-alpha, IL-18, and TNF-beta, GM-CSF, IL-4, IL-13, Flt3-L, RANTES, MIPla, etc.) as described herein to enhance induction of the immune response.
  • chemokines e.g., IFN-y
  • compositions comprising the cells activated and expanded as described herein may be utilized in the treatment and prevention of diseases that arise in individuals who are immunocompromised.
  • compositions contemplated herein are used in the treatment of cancer.
  • the immune effector cells may be administered either alone, or as a pharmaceutical composition in combination with carriers, diluents, excipients, and/or with other components such as IL -2 or other cytokines or cell populations.
  • compositions comprise an amount of genetically modified T cells, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients.
  • compositions comprise an amount of bridging factor, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients.
  • compositions comprise an effective amount of immune effector cells comprising a DARIC, one or more DARIC binding components, a DARIC signaling component, and/or fusion proteins contemplated herein, alone or in combination with a bridging factor and/or one or more therapeutic agents, such as radiation therapy, chemotherapy, transplantation, immunotherapy, hormone therapy, photodynamic therapy, etc.
  • the compositions may also be administered in combination with antibiotics.
  • therapeutic agents may be accepted in the art as a standard treatment for a particular disease state as described herein, such as a particular cancer.
  • Exemplary therapeutic agents contemplated include cytokines, growth factors, steroids, NSAIDs, DMARDs, antiinflammatories, chemotherapeutics, radiotherapeutics, therapeutic antibodies, or other active and ancillary agents.
  • a composition comprising an effective amount of immune effector cells comprising a polynucleotide encoding a DARIC, one or more DARIC binding components, a DARIC signaling component, and/or fusion proteins contemplated herein is administered to a subject, and a composition comprising an effective amount of a bridging factor is administered to the subject, before, during, in combination with or subsequently to the cellular composition, and optionally repetitively administered to the subject.
  • compositions comprising immune effector cells comprising a polynucleotide encoding a DARIC, one or more DARIC binding components, a DARIC signaling component, and/or fusion proteins contemplated herein may be administered in conjunction with any number of anti-inflammatory agents, chemotherapeutic agents, or therapeutic antibodies, and the like.
  • Immune effector cells modified to express a polynucleotide encoding a DARIC, one or more DARIC binding components, a DARIC signaling component, and/or fusion proteins contemplated herein provide improved methods of adoptive immunotherapy for use in the prevention, treatment, and amelioration of, or for preventing, treating, or ameliorating at least one symptom associated with an immune disorder, e.g., cancer.
  • Immune effector cells comprising a DARIC, one or more DARIC binding components, a DARIC signaling component, and/or fusion proteins contemplated herein provide improved methods of adoptive immunotherapy for use in the prevention, treatment, and amelioration of, or for preventing, treating, or ameliorating at least one symptom associated with an immune disorder, e.g., cancer.
  • an immune disorder e.g., cancer.
  • immune effector cells modified to express a DARIC, one or more DARIC binding components, a DARIC signaling component, and/or fusion proteins contemplated herein provide improved methods of adoptive immunotherapy to finetune the safety and efficacy of a cytotoxic response against target cells, e.g., tumor cells, expressing target antigens while decreasing the risk of on-target antigen, off-target cell cytotoxicity (recognizing the target antigen on a normal, non-target cell).
  • target cells e.g., tumor cells, expressing target antigens while decreasing the risk of on-target antigen, off-target cell cytotoxicity (recognizing the target antigen on a normal, non-target cell).
  • a method of preventing, treating, or ameliorating at least one symptom of a cancer comprises administering the subject an effective amount of modified immune effector cells or T cells comprising one or more components of a DARIC, one or more DARIC binding components, a DARIC signaling component, and/or fusion proteins contemplated herein to redirect the cells to a target cell.
  • the genetically modified cells are a more efficacious and safe cellular immunotherapy by virtue of transducing a chemically regulatable immunostimulatory signal.
  • one or more immune effector cells are modified to express both one or more DARIC binding components and a DARIC signaling component.
  • the modified cells are administered to a subject in need thereof and home to the target cells via the interaction of the anti-CD33 VHH binding component expressed on the immune effector cell and CD33 expressed on the target cell and/or the interaction of the anti-CLL-1 VHH binding component expressed on the immune effector cell and CLL-1 expressed on the target cell.
  • a bridging factor is administered to the subject before the modified cells, about the same time as the modified cells, or after the modified cells have been administered to the subject.
  • a ternary complex forms between the CD33 VHH DARIC binding component, the bridging factor and the DARIC signaling component and/or between the CLL-1 VHH DARIC binding component, the bridging factor and the DARIC signaling component.
  • the DARIC transduces an immunostimulatory signal to the immune effector cell that in turn, elicits a cytotoxic response from the immune effector cell against the target cell.
  • one or more immune effector cells e.g., T cells, are modified to express a DARIC signaling component. In this case, the modified cells are administered to a subject in need thereof.
  • One or more DARIC binding components can be administered to the subject before the modified cells, about the same time as the modified cells, or after the modified cells have been administered to the subject.
  • the one or more DARIC binding components can be administered to the subject in a preformed complex with the bridging factor; at the same time as the bridging factor, but in a separate composition; or at a different time than the bridging factor.
  • One of the one or more DARIC binding components binds CD33 expressed on the target cell, either in the presence or absence of the bridging factor and one of the one or more DARIC binding components binds CLL-1 expressed on the target cell, either in the presence or absence of the bridging factor.
  • a ternary complex forms between the CD33 VHH DARIC binding component, the bridging factor and the DARIC signaling component and/or between the CLL-1 VHH DARIC binding component, the bridging factor and the DARIC signaling component.
  • the DARIC transduces an immunostimulatory signal to the immune effector cell that in turn, elicits a cytotoxic response from the immune effector cell against the target cell.
  • CD33 VHH DARIC activation can be induced in cases where remission or regression is incomplete and the condition relapses or becomes refractory to treatment.
  • the specificity of a primary T cell is redirected to tumor or cancer cells that express CD33 and/or CLL-1 by genetically modifying a T cell, e.g., a primary T cell, with one or more DARIC binding components designed to bind CD33 and/or CLL-1.
  • the modified immune effector cells contemplated herein are used in the treatment of solid tumors or cancers.
  • the modified immune effector cells contemplated herein are used in the treatment of solid tumors or cancers including, but not limited to: adrenal cancer, adrenocortical carcinoma, anal cancer, appendix cancer, astrocytoma, atypical teratoid/rhabdoid tumor, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain/CNS cancer, breast cancer, bronchial tumors, cardiac tumors, cervical cancer, cholangiocarcinoma, chondrosarcoma, chordoma, colon cancer, colorectal cancer, craniopharyngioma, ductal carcinoma in situ (DCIS) endometrial cancer, ependymoma, esophageal cancer, esthesioneuroblastoma, Ewing’s sarcoma, extracranial germ cell tumor, extragonadal germ cell tumor, eye cancer, fallopian tube cancer, fibrous histiosarcoma, fibrosarcom
  • the modified immune effector cells contemplated herein are used in the treatment of solid tumors or cancers including, without limitation, non-small cell lung carcinoma, head and neck squamous cell carcinoma, colorectal cancer, pancreatic cancer, breast cancer, thyroid cancer, bladder cancer, cervical cancer, esophageal cancer, ovarian cancer, gastric cancer endometrial cancer, gliomas, glioblastomas, and oligodendroglioma.
  • the modified immune effector cells contemplated herein are used in the treatment of solid tumors or cancers including, without limitation, non-small-cell lung cancer, metastatic colorectal cancer, glioblastoma, head and neck cancer, pancreatic cancer, and breast cancer.
  • the modified immune effector cells contemplated herein are used in the treatment of glioblastoma
  • the modified immune effector cells contemplated herein are used in the treatment of liquid cancers or hematological cancers.
  • the modified immune effector cells contemplated herein are used in the treatment of B-cell malignancies, including but not limited to: leukemias, lymphomas, and multiple myeloma.
  • the modified immune effector cells contemplated herein are used in the treatment of liquid cancers including, but not limited to leukemias, lymphomas, and multiple myelomas: acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), myeloblastic, promyelocytic, myelomonocytic, monocytic, erythroleukemia, hairy cell leukemia (HCL), chronic lymphocytic leukemia (CLL), and chronic myeloid leukemia (CML), chronic myelomonocytic leukemia (CMML) and polycythemia vera, Hodgkin lymphoma, nodular lymphocyte-predominant Hodgkin lymphoma, Burkitt lymphoma, small lymphocytic lymphoma (SLL), diffuse large B-cell lymphoma, follicular lymphoma, immunoblastic large cell lymphoma, precursor B-lymphoblastic lymph
  • ALL acute
  • the modified immune effector cells contemplated herein are used in the treatment of acute myeloid leukemia (AML).
  • AML acute myeloid leukemia
  • Preferred cells for use in the methods contemplated herein include autologous/autogeneic (“self’) cells, preferably hematopoietic cells, more preferably T cells, and more preferably immune effector cells.
  • self autologous/autogeneic
  • a method comprises administering a therapeutically effective amount of modified immune effector cells that express one or more DARIC binding components and a DARIC signaling component, to a patient in need thereof, and also administering a bridging factor to the subject.
  • the cells are used in the treatment of patients at risk for developing an immune disorder.
  • particular embodiments comprise the treatment or prevention or amelioration of at least one symptom of an immune disorder, e.g., cancer, comprising administering to a subject in need thereof, a therapeutically effective amount of the modified immune effector cells contemplated herein and a bridging factor.
  • a method comprises administering a therapeutically effective amount of modified immune effector cells that express a DARIC signaling component or a composition comprising the same, to a patient in need thereof, and also administering a CD33 VHH DARIC binding component, a CLL-1 VHH DARIC binding component, and a bridging factor, optionally wherein the one or more DARIC binding components is bound to the bridging factor prior to administration, to the subject.
  • the cells are used in the treatment of patients at risk for developing an immune disorder.
  • modified immune effector cells one or more DARIC binding components, and/or bridging factor will be determined by such factors as the condition of the patient, and the type and severity of the patient's disease, although appropriate dosages and dose schedules may be determined by clinical trials.
  • the effective amount of modified immune effector cells provided to a subject is at least 2 x 10 6 cells/kg, at least 3 x 10 6 cells/kg, at least 4 x 10 6 cells/kg, at least 5 x 10 6 cells/kg, at least 6 x 10 6 cells/kg, at least 7 x 10 6 cells/kg, at least 8 x 10 6 cells/kg, at least 9 x 10 6 cells/kg, or at least 10 x 10 6 cells/kg, or more cells/kg, including all intervening doses of cells.
  • the effective amount of modified immune effector cells provided to a subject is about 2 x 10 6 cells/kg, about 3 x 10 6 cells/kg, about 4 x 10 6 cells/kg, about 5 x 10 6 cells/kg, about 6 x 10 6 cells/kg, about 7 x 10 6 cells/kg, about 8 x 10 6 cells/kg, about 9 x 10 6 cells/kg, or about 10 x 10 6 cells/kg, or more cells/kg, including all intervening doses of cells.
  • the effective amount of modified immune effector cells provided to a subject is from about 2 x 10 6 cells/kg to about 10 x 10 6 cells/kg, about 3 x 10 6 cells/kg to about 10 x 10 6 cells/kg, about 4 x 10 6 cells/kg to about 10 x 10 6 cells/kg, about 5 x 10 6 cells/kg to about 10 x 10 6 cells/kg, 2 x 10 6 cells/kg to about 6 x 10 6 cells/kg, 2 x 10 6 cells/kg to about 7 x 10 6 cells/kg, 2 x 10 6 cells/kg to about 8 x 10 6 cells/kg, 3 x 10 6 cells/kg to about 6 x 10 6 cells/kg, 3 x 10 6 cells/kg to about 7 x 10 6 cells/kg, 3 x 10 6 cells/kg to about 8 x 10 6 cells/kg, 4 x 10 6 cells/kg to about 6 x 10 6 cells/kg, 4 x 10 6 cells/kg to about 6 x 10 6 cells/kg, 4 x 10 6 cells
  • compositions contemplated in particular embodiments may be required to effect the desired therapy.
  • a composition may be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more times over a span of 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, 2 years, 5, years, 10 years, or more.
  • Modified immune effector cells that express a DARIC signaling component; one or more DARIC binding components; and bridging factor may be administered in the same or different compositions; in one or more compositions at the same time; or more than one composition at different times.
  • Modified immune effector cells that express a DARIC signaling component; one or more DARIC binding components; and bridging factor may be administered through the same route of administration or different routes.
  • T cells can be activated from blood draws of from lOcc to 400cc.
  • T cells are activated from blood draws of 20cc, 30cc, 40cc, 50cc, 60cc, 70cc, 80cc, 90cc, lOOcc, 150cc, 200cc, 250cc, 300cc, 350cc, or 400cc or more.
  • using this multiple blood draw/multiple reinfusion protocol may serve to select out certain populations of T cells.
  • a method of treating a subject diagnosed with a cancer comprises removing immune effector cells from the subject, modifying the immune effector cells by introducing one or more vectors encoding one or more DARIC binding components and a DARIC signaling component into the cell and producing a population of modified immune effector cells, and administering the population of modified immune effector cells to the same subject.
  • the immune effector cells comprise T cells.
  • the methods for administering the cell compositions contemplated in particular embodiments include any method which is effective to result in reintroduction of ex vivo modified immune effector cells or reintroduction of modified progenitors of immune effector cells that upon introduction into a subject differentiate into mature immune effector cells.
  • One method comprises modifying peripheral blood T cells ex vivoby introducing one or more vectors encoding one or more DARIC binding components and a DARIC signaling component into the cell and returning the transduced cells into the subject.
  • Tandem DARIC binding components were constructed using an anti-CD33 VHH domain (SEQ ID NO: 3), a glycine-serine linker (GGGGS)3 (SEQ ID NO:45), and one of two anti-CLL-1 VHH domains (SEQ ID NO: 4 (anti-CLL-1.1 VHH) or SEQ ID NO: 5 (anti-CLL-1.2 VHH)).
  • Lentiviral vectors were constructed comprising an MNDU3 promoter operably linked to a polynucleotide encoding a DARIC signaling component (a CD8a signal peptide, an FRB variant (T2098L or T82L), a CD8a transmembrane domain, an intracellular 4-1BB co-stimulatory domain, and a CD3 zeta signaling domain; a P2A sequence; and one of seven following DARIC binding components, (I) an IgK-signal peptide, an anti-CD33 VHH domain, a (GGGGS)s (SEQ ID NO:45) linker, an anti-CLL-
  • a DARIC signaling component a CD8a signal peptide, an FRB variant (T2098L or T82L), a CD8a transmembrane domain, an intracellular 4-1BB co-stimulatory domain, and a CD3 zeta signaling domain
  • P2A sequence and one of seven following DARIC
  • PBMCs Peripheral blood mononuclear cells
  • T cells were expanded for 10 days in culture.
  • DARIC T cells were positively stained with CD33-Fc (stains anti-CD33 VHH), CLL-l-Fc (stains anti-CLL-1.1 VHH, anti-CLL-1.2 VHH), and an anti-FRB monoclonal antibody (stains DARIC signaling component) by flow cytometry.
  • Monovalent DARIC binding components were only detected by staining based on their respective specificity while tandem DARIC binding components were detected by staining with both CD33-Fc and CLL-l-Fc. All DARIC signaling components were detected by staining with anti-FRB antibody. All constructs labeled equivalently.
  • T cells transduced with the lentiviral vectors described in paragraph two of this example were co-cultured with CD33-expressing A549 cells, CLL-1 -expressing A549 cells, CD33/CLL-1 expressing A549 cells, an CD33/CLL-1 expressing MV4-11 AML cell line, an CD33/CLL-1 expressing M0LM14 AML cell line, or antigen negative A549 cells, at an E:T ratio of 1 : 1 in the presence or absence of InM rapamycin for 24 hours. All DARIC T cells transduced with DARICs exhibited robust cytokine response when cocultured with cell lines, in the presence of rapamycin, based on the designed DARIC binding component antigen specificity.
  • DARIC T cells expressing a monovalent DARIC binding component that binds CD33 secrete IFNy in the presence of CD33-expressing A549 cells, but not in the presence of CLL-1 -expressing A549 cells.
  • Minimal IFNy secretion was detected in cultures containing untransduced T cells an antigen negative T cells.
  • T cells transduced with the lentiviral vectors described in paragraph two of this example were co-cultured with CD33-expressing A549 cells (expressing NucLight Red) or CLL-1 -expressing A549 cells (expressing NucLight Green) at an E:T of 1: 1 in the presence of absence of InM rapamycin. Cytotoxicity was monitored using an Incucyte instrument.
  • DARIC T cells expressing tandem DARICs expressing a DARIC binding component that binds CD33 and CLL-1 were cytotoxic to both CD33 -expressing and CLL-1 -expressing A549 cell lines in a rapamycin dependent fashion.
  • DARIC T cells expressing a monovalent DARIC binding component that binds CD33 or CLL-1 were only cytotoxic to A549 cells lines expressing CD33 or CLL-1, resp.
  • Figure 4
  • a DARIC strategy using dual DARIC binding components, one DARIC binding component that binds CD33 and one DARIC binding component that binds CLL-1, and a DARIC signaling component was designed, constructed, and verified.
  • Figure 1 right panel.
  • Dual DARIC binding components were constructed using an anti-CD33 VHH domain (SEQ ID NO: 3) and one of two anti-CLL-1 VHH domains (SEQ ID NO: 4 (anti- CLL-1.1 VHH) or SEQ ID NO: 5 (anti-CLL-1.2 VHH)).
  • Lentiviral vectors were constructed comprising an MNDU3 promoter operably linked to a polynucleotide encoding a DARIC signaling component, a DARIC binding component that binds CD33, and a DARIC binding component that binds CLL-1, wherein the components are separated from each other by self-cleaving viral polypeptides and the DARIC binding components were formatted in different orders (e.g., SEQ ID NOs: 24-27).
  • Lentiviral vectors encoding monovalent DARICs constructs were used as a control (e.g., SEQ ID NOs: 17-19).
  • PBMCs Peripheral blood mononuclear cells
  • T cells were expanded for 10 days in culture.
  • DARIC T cells were positively stained with CD33-Fc (stains anti-CD33 VHH), CLL-l-Fc (stains anti-CLL-1.1 VHH, anti-CLL-1.2 VHH), and an anti-FRB monoclonal antibody (stains DARIC signaling component) by flow cytometry.
  • CD33-Fc stains anti-CD33 VHH
  • CLL-l-Fc stains anti-CLL-1.1 VHH, anti-CLL-1.2 VHH
  • an anti-FRB monoclonal antibody stains DARIC signaling component
  • T cells transduced with the lentiviral vectors described in paragraph two of this example were co-cultured with CD33-expressing A549 cells, CLL-1 -expressing A549 cells, an CD33/CLL-1 expressing AML-193 AML cell line, an CD33/CLL-1 expressing U- 937 A549 cells, an CD33/CLL-1 expressing HL60 AML cell line, or antigen negative A549 cells, at an E:T ratio of 1 : 1 in the presence or absence of InM rapamycin for 24 hours. All DARIC T cells transduced with DARICs exhibited robust cytokine response when co-cultured with cell lines, in the presence of rapamycin, based on the designed DARIC binding component antigen specificity.
  • DARIC T cells expressing a monovalent DARIC binding component that binds CD33 secrete IFNy in the presence of CD33-expressing A549 cells, but not in the presence of CLL-1 -expressing A549 cells.
  • Minimal IFNy secretion was detected in cultures containing untransduced T cells an antigen negative T cells.
  • T cells transduced with the lentiviral vectors described in paragraph two of this example were co-cultured with CD33-expressing A549 cells (expressing NucLight Red) or CLL-1 -expressing A549 cells (expressing NucLight Green) at an E:T of 1:3 in the presence of absence of InM rapamycin. Cytotoxicity was monitored using an Incucyte instrument.
  • DARIC T cells expressing dual DARIC binding components that bind CD33 and CLL-1 were cytotoxic to both CD33 -expressing and CLL-1 -expressing A549 cell lines in a rapamycin dependent fashion.
  • DARIC T cells expressing a monovalent DARIC binding component that binds CD33 or CLL-1 were only cytotoxic to A549 cells lines expressing CD33 or CLL-1, resp.
  • Figure 7 EXAMPLE 3
  • Tandem dual targeting anti-CD33/anti-CLLl VHH DARIC binding and signaling components were designed, constructed, and verified.
  • the tandem dual-specificity VHH DARIC lentiviral vectors were constructed comprising an MNDU3 promoter operably linked to a polynucleotide encoding: a DARIC signaling component (CD8a-signal peptide, an FRB variant (T82L), a CD8a transmembrane domain with either an MH or LYC/LYS sequence, an intracellular 4-1BB costimulatory domain, and a CD3 zeta signaling domain); a P2A sequence; and a DARIC binding component (an IgK-signal peptide, a CD33 specific VHH binding domain, a 3xG4S linker, a CLL1 specific VHH binding domain (CLL-1.1 or CLL-1.2), a G4S linker, an FKBP12 domain, either a GGR or GGS linker/spacer,
  • Untransduced T cells T cells transduced with empty vector and single specific anti- CD33 or anti-CLLl VHH DARIC T cells were used as a control.
  • T cells from three donors were each transduced with LVVs encoding dualspecificity anti-CD33/anti-CLLl VHH DARICs ( Figure 8A) or controls and expanded for 10 days. All samples, including untransduced T cells, were stained with recombinant CLLl-Fc, CD33-Fc, or FRB-specific antibody reagents. While untransduced did not bind the staining reagent, tandem anti-CD33/anti-CLLl DARIC and controls were positively stained with all the detection reagents ( Figures 10A and 10B).
  • T cells and T cells transduced with a tandem anti-CD33/anti-CLLl VHH DARIC were co-incubated with A549 cells engineered to express either CD33 or CLL1, at an E:T ratio of 1 : 1, normalized by FRB expression, in the presence or absence of rapamycin for 24 hours.
  • T cells were also cultured in media alone or with a U- 937, a primary AML line that endogenously expresses both CD33 and CLL1. While untransduced T cells produced minimal amounts of cytokine, T cells transduced with tandem anti-CD33/anti-CLLl VHH DARIC produced IFNg when cultured with tumor cells in the presence of rapamycin.
  • the amount of cytokine produced was mostly equivalent between all constructs, and all T cells produced minimal levels of IFNg in the absence of tumor cells ( Figures 11A and 1 IB).
  • Tandem VHH DARIC binding and signaling components were designed, constructed, and verified. Tandem CAR molecules targeting multiple epitopes or antigens can be constructed by fusing two, or more, antigen targeting modalities in series. In the case of tandem molecules formed from at least two antigen binding domains the linker sequence connecting the antigen binding domains can be optimized for simplicity, flexibility, and for antigen binding and recognition ( Figures 8B and 12).
  • tandem regulatable CAR molecules constructed from monovalent, single targeting, parental molecules specific from CD33 (SEQ ID NO: 17) and CLL1 (SEQ ID NOs: 18 and 19). The parental tandem molecules were initially constructed with a standard (Gly4Ser)3 linker (SEQ ID NOs:21 and 23).
  • Optimized tandem VHH DARIC lentiviral vectors were constructed similar to the parental molecules comprising a MNDU3 promoter operably linked to a polynucleotide encoding: a DARIC signaling component (CD8a- signal peptide, an FRB variant (T82L), a CD8a transmembrane domain, an intracellular 4- 1BB costimulatory domain, and a CD3 zeta signaling domain); a P2A sequence; and a DARIC binding component (a human IgK-signal peptide, a CD33 specific VHH binding domain, a variable G4S or GGS linker, a CLL1 specific VHH binding domain, a G4S linker, an FKBP12 domain, and a CD4 transmembrane domain).
  • the tandem constructs were constructed with the CD33 specific VHH in the membrane proximal position (SEQ ID NOs: 34-38). Untransduced T cells and parental tandem constructs were used as controls.
  • DARIC T cells from three donors were each transduced with LVVs encoding CD33-CLL1 tandem constructs and expanded for 10 days.
  • DARIC T cells were positively stained with CD33-Fc, CLLl-Fc and an anti-FRB monoclonal antibody by flow cytometry ( Figure 13A and 13B). All constructs labeled equivalently with the three labeling reagents detecting components of the DARIC constructs indicating that the linker composition does not materially affect expression or antigen recognition.
  • Transduced T cells were co-cultured with CD33+ A549, CLL1+ A549, and the double positive AML cell line U937 ( Figures 14A and 14B) at an E:T ratio of 1 : 1 in the presence or absence of InM rapamycin for 24 hours. All T cells transduced with DARIC construct exhibited robust cytokine response when co-cultured with the U937 AML cell line, in the presence of rapamycin. Minimal cytokine production was detected in T cell only, no target, samples. Cytokine was detected using MSD. Tandem constructs were also capable of eliminating single antigen positive A549 cell lines at approximately the same rate when co-cultured at an E:T ratio of 1 : 1. Tumor clearance was monitored by the loss of fluorescence associated with the tumor lines as measured by Incucyte.
  • tandem regulatable CAR molecules constructed from monovalent, single targeting, parental molecules specific from CD33 (SEQ ID NO: 17) and CLL1 (SEQ ID NOs: 18 and 19) combining hinge, transmembrane, and linker sequences from Examples 3 and 4.
  • Optimized tandem VHH DARIC lentiviral vectors were constructed similar to the parental molecules comprising a MNDU3 promoter operably linked to a polynucleotide encoding: a DARIC signaling component (CD8a-signal peptide, an FRB variant (T82L), a CD8a transmembrane domain with either an MH or LYC sequence, an intracellular 4-1BB costimulatory domain, and a CD3 zeta signaling domain); a P2A sequence; and a DARIC binding component (an IgK-signal peptide, a CD33 specific VHH binding domain, a G4S or 3xG4S linker (i.e., a (G4S)s), a CLL1 specific VHH binding domain, a G4S linker, an FKBP12 domain, either a GGR linker/spacer, and a CD4 transmembrane domain) ( Figure 8C and 15). See, e.g., S
  • DARIC T cells from three donors were each transduced with LVVs encoding CD33-CLL1 tandem constructs and expanded for 10 days.
  • DARIC T cells were positively stained with CD33-Fc, CLLl-Fc and an anti-FRB monoclonal antibody by flow cytometry ( Figures 16A and 16B). All constructs labeled equivalently with the three labeling reagents detecting components of the DARIC constructs indicating that the linker composition does not materially affect expression or antigen recognition.
  • T cells and T cells transduced with a tandem anti-CD33/anti-CLLl VHH DARIC were co-incubated with A549 cells engineered to express either CD33 or CLL1, at an E:T ratio of 1 : 1, normalized by FRB expression, in the presence or absence of rapamycin for 24 hours.
  • T cells were also cultured in media alone or with a HL60, an AML cell line that endogenously expresses both CD33 and CLL1. While untransduced T cells produced minimal amounts of cytokine, T cells transduced with tandem anti-CD33/anti-CLLl VHH DARIC produced IFNg when cultured with tumor cells in the presence of rapamycin. When normalized for FRB expression, the amount of cytokine produced was mostly equivalent between all constructs, and all T cells produced minimal levels of IFNg in the absence of tumor cells ( Figures 17A and 17B).

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Abstract

La présente divulgation concerne des compositions et des polypeptides ciblant améliorés, pour des thérapies adoptives à base de lymphocytes T pour le traitement, la prévention ou l'amélioration d'au moins un symptôme d'un cancer, d'une maladie infectieuse, d'une maladie auto-immune, d'une maladie inflammatoire et d'une immunodéficience, ou d'un état associé.
PCT/US2021/058562 2020-11-09 2021-11-09 Immunothérapies ciblant aml WO2022099176A1 (fr)

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US202063111255P 2020-11-09 2020-11-09
US63/111,255 2020-11-09
US202063112023P 2020-11-10 2020-11-10
US63/112,023 2020-11-10

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020123938A1 (fr) * 2018-12-14 2020-06-18 Bluebird Bio, Inc. Complexes d'immunorécepteurs régulés par un agent de dimérisation
US20200347133A1 (en) * 2019-05-04 2020-11-05 Inhibrx, Inc. CD33-Binding Polypeptides and Uses Thereof
US20200347139A1 (en) * 2019-05-04 2020-11-05 Inhibrx, Inc. CLEC12a Binding Polypeptides and Uses Thereof

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020123938A1 (fr) * 2018-12-14 2020-06-18 Bluebird Bio, Inc. Complexes d'immunorécepteurs régulés par un agent de dimérisation
US20200347133A1 (en) * 2019-05-04 2020-11-05 Inhibrx, Inc. CD33-Binding Polypeptides and Uses Thereof
US20200347139A1 (en) * 2019-05-04 2020-11-05 Inhibrx, Inc. CLEC12a Binding Polypeptides and Uses Thereof

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