CD33 targeted immunotherapies

Abstract

To provide CD33 targeted immunotherapies.SOLUTION: The present invention provides improved CD33 targeting polypeptides and compositions for adoptive T-cell therapy to treat, prevent or ameliorate the symptoms of at least one of cancers, infectious diseases, autoimmune disease, inflammatory diseases and immune disorders, or conditions associated therewith. The present invention relates generally to, in part, VHH-based dimerizer-regulated immunoreceptor complexes (DARIC) and VHH-based chimeric antigens receptors (CAR) directed against CD33, polynucleotides encoding the same, compositions thereof, and methods of making and using the same for treating cancers.SELECTED DRAWING: Figure 1A

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. 119(e) of U.S. Provisional Patent Application No. 62 / 898,392, filed September 10, 2019, and U.S. Provisional Patent Application No. 62 / 845,304, filed May 8, 2019, each of which is incorporated herein by reference in its entirety.

[0002] Sequence Listing Statement The sequence listing for this application has been submitted in text format in lieu of paper and is incorporated herein by reference. The text file containing the sequence listing is named BLBD_119_02WO_ST25.txt. The text file is 302 KB, was created on May 5, 2020, and is being submitted electronically via EFS-Web simultaneously with the filing of this application.

[0003] The present disclosure relates to improved adoptive cell therapy directed against CD33. More particularly, the present disclosure relates to anti-CD33 VHH-containing chemically regulated signaling molecules, anti-CD33 VHH-containing chimeric antigen receptors, cells, and related therapeutic methods using the same. [Background technology]

[0004] Description of related fields Cancer incidence doubled worldwide between 1975 and 2000. It 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 bronchial cancer, prostate cancer, colon and rectum cancer, bladder cancer, melanoma of the skin, non-Hodgkin's lymphoma, thyroid cancer, kidney and renal pelvis cancer, endometrial cancer, leukemia, and pancreatic cancer. The number of new cancer cases is expected to rise to 22 million within the next 20 years.

[0005] Adoptive cell therapy has emerged as a powerful paradigm for transducing complex biological signals to treat cancer. In contrast to small molecule drugs and biopharmaceuticals, adoptive cell therapy has the potential to exert unique therapeutic tasks through a wide variety of sensory and reactive programs and further defined gene regulatory mechanisms. Existing methods have primarily focused on scFv-based chimeric antigen receptors (CARs). CAR T cell therapy has achieved limited success due to insufficient CAR expression, in vivo expansion of CAR T cells, rapid cell loss after infusion, disappointing clinical activity, and antigen escape.

[0006] There is a need to engineer immune effector cells with improved CAR architectures and / or improved mechanisms for sensing and integrating chemical and / or biological information relevant to the local physiological environment. Summary of the Invention [Means for solving the problem]

[0007] The present disclosure relates generally, in part, to VHH-based dimerizer-regulated immune receptor complexes (DARICs) and VHH-based chimeric antigen receptors (CARs) directed against CD33, polynucleotides encoding same, compositions thereof, and methods of making and using same for the treatment of cancer.

[0008] In certain embodiments, the VHH DARIC or VHH CAR binds to full-length CD33. In certain embodiments, the VHH DARIC or VHH CAR binds to a CD33 splice variant. In certain embodiments, the CD33 splice variant lacks the 124 amino acids encoded by exon 2 of the human CD33 gene (CD33 C2 variant). In certain embodiments, the CD33 splice variant lacks the 54 carboxy-terminal amino acids due to an early translation stop signal present in exon 7a. In certain embodiments, the CD33 splice variant lacks the 124 amino acids encoded by exon 2 and the 54 carboxy-terminal amino acids due to an early translation stop signal present in exon 7a.

[0009] In certain embodiments, the VHH DARIC or VHH CAR binds to both full-length CD33 and CD33 splice variants.

[0010] In various embodiments, the non-native cell comprises a first polypeptide comprising an FRB multimerization domain polypeptide or a variant thereof, a CD8α transmembrane domain or a CD4 transmembrane domain, a CD137 costimulatory domain, and / or a CD3ζ primary signaling domain, and an anti-CD33 polypeptide having the amino acid sequence set forth in any one of SEQ ID NOs: 2-21. The complex comprises a second polypeptide comprising a VHH antibody, an FKBP multimerization domain polypeptide or a variant thereof, and a CD4 transmembrane domain or a CD8α transmembrane domain, and a cross-linking agent that promotes the formation of a polypeptide complex on the surface of the non-native cell, wherein the cross-linking agent associates with and is positioned between the multimerization domains of the first and second polypeptides.

[0011] In a particular embodiment, the anti-CD33 VHH antibody has the amino acid sequence set forth in SEQ ID NO:10.

[0012] In a specific embodiment, the anti-CD33 VHH antibody has the amino acid sequence set forth in SEQ ID NO:20.

[0013] In a specific embodiment, the FKBP multimerization domain is FKBP12.

[0014] In some embodiments, the FRB polypeptide is FRB T2098L.

[0015] In certain embodiments, the cross-linking agent is selected from the group consisting of: AP21967, sirolimus, everolimus, novolimus, pimecrolimus, ridaforolimus, tacrolimus, temsirolimus, umirolimus, and zotarolimus.

[0016] In various embodiments, the first polypeptide comprises a signal peptide, a CD8α transmembrane domain, a CD137 costimulatory domain, and a CD3ζ primary signaling domain.

[0017] In certain embodiments, the second polypeptide comprises a signal peptide and a CD4 transmembrane domain.

[0018] In a further embodiment, the second polypeptide comprises a costimulatory domain.

[0019] In some embodiments, the costimulatory domain of the second polypeptide is 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 (CARD11), CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD94, CD134 (OX40), CD137 (4-1BB), CD27 and a costimulatory molecule selected from the group consisting of ICOS, DNAX-activating protein 10 (DAP10), linker for activation of T-cell family member 1 (LAT), SH2 domain-containing leukocyte protein of 76 kD (SLP76), T-cell receptor-associated transmembrane adaptor 1 (TRAT1), TNFR2, TNFRS14, TNFRS18, TNRFS25, and the zeta chain of T-cell receptor-associated protein kinase 70 (ZAP70).

[0020] In additional embodiments, the costimulatory domain of the second polypeptide is a costimulatory domain isolated from OX40 or TNFR2.

[0021] In a further embodiment, the second polypeptide comprises the sequence set forth in any one of SEQ ID NOs: 22-31.

[0022] In certain embodiments, the second polypeptide comprises the sequence set forth in SEQ ID NO:30.

[0023] In a preferred embodiment, the first polypeptide comprises the amino acid sequence set forth in SEQ ID NO:82.

[0024] In various embodiments, the non-native cell comprises a first polypeptide comprising an FRB multimerization domain polypeptide or a variant thereof, a CD8α transmembrane domain or a CD4 transmembrane domain, a CD137 costimulatory domain, and / or a CD3ζ primary signaling domain, and an anti-CD33 polypeptide having the amino acid sequence set forth in any one of SEQ ID NOs: 2-21. The polypeptide complex comprises a second polypeptide comprising a VHH antibody, an FKBP multimerization domain polypeptide or a variant thereof, and a CD4 transmembrane domain or a CD8α transmembrane domain, and a cross-linking factor associated with and positioned between the multimerization domains of the first and second polypeptides.

[0025] In a particular embodiment, the anti-CD33 VHH antibody has the amino acid sequence set forth in SEQ ID NO:10.

[0026] In a specific embodiment, the anti-CD33 VHH antibody has the amino acid sequence set forth in SEQ ID NO:20.

[0027] In a specific embodiment, the FKBP multimerization domain is FKBP12.

[0028] In certain embodiments, the FRB polypeptide is FRB T2098L.

[0029] In some embodiments, the cross-linking agent is selected from the group consisting of: AP21967, sirolimus, everolimus, novolimus, pimecrolimus, ridaforolimus, tacrolimus, temsirolimus, umirolimus, and zotarolimus.

[0030] In additional embodiments, the first polypeptide comprises a signal peptide, a CD8α transmembrane domain, a CD137 costimulatory domain, and a CD3ζ primary signaling domain.

[0031] In certain embodiments, the second polypeptide comprises a signal peptide and a CD4 transmembrane domain.

[0032] In some embodiments, the second polypeptide comprises a costimulatory domain.

[0033] In various embodiments, the costimulatory domain of the second polypeptide is 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 (CARD11), CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD94, CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), DNAX-activation protein 10 (DAP10), linker for activation of T-cell family member 1 (LAT), 76 kD SH2 domain-containing leukocyte protein (SLP76), T-cell receptor-associated transmembrane adaptor 1 (TRAT1), TNFR2, TNFRS14, TNFRS18, TNRFS25, and the zeta chain of T-cell receptor-associated protein kinase 70 (ZAP70).

[0034] In additional embodiments, the costimulatory domain of the second polypeptide is a costimulatory domain isolated from OX40 or TNFR2.

[0035] In a further embodiment, the second polypeptide comprises the sequence set forth in any one of SEQ ID NOs: 22-31.

[0036] In certain embodiments, the second polypeptide comprises the sequence set forth in SEQ ID NO:30.

[0037] In a preferred embodiment, the first polypeptide comprises the amino acid sequence set forth in SEQ ID NO:82.

[0038] In certain embodiments, the cells are hematopoietic cells.

[0039] In certain embodiments, the cell is a T cell, an αβ T cell, or a γδ T cell.

[0040] In further embodiments, the cells are CD3+, CD4+, and / or CD8+ cells.

[0041] In some embodiments, the cell is an immune effector cell.

[0042] In some embodiments, the cell is a cytotoxic T lymphocyte (CTL), a tumor-infiltrating lymphocyte (TIL), or a helper T cell.

[0043] In additional embodiments, the cells are natural killer (NK) cells or natural killer T (NKT) cells.

[0044] In various embodiments, the source of the cells is peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue, or a tumor.

[0045] In certain embodiments, the FRB multimerization domain and the FKBP multimerization domain are localized extracellularly when the first polypeptide and the second polypeptide are expressed.

[0046] In some embodiments, the fusion polypeptide comprises a first polypeptide comprising an FRB multimerization domain polypeptide or a variant thereof, a CD8α transmembrane domain or a CD4 transmembrane domain, a CD137 costimulatory domain, and / or a CD3ζ primary signaling domain, and a polypeptide cleavage signal; an anti-CD33 VHH antibody having the amino acid sequence set forth in any one of SEQ ID NOs: 2 to 21, a FKBP multimerization domain polypeptide or a variant thereof, and a CD4 transmembrane domain or a CD8α transmembrane domain.

[0047] In a particular embodiment, the anti-CD33 VHH antibody has the amino acid sequence set forth in SEQ ID NO:10.

[0048] In a specific embodiment, the FKBP multimerization domain is FKBP12.

[0049] In certain embodiments, the FRB polypeptide is FRB T2098L.

[0050] In some embodiments, the cross-linking agent is selected from the group consisting of: AP21967, sirolimus, everolimus, novolimus, pimecrolimus, ridaforolimus, tacrolimus, temsirolimus, umirolimus, and zotarolimus.

[0051] In additional embodiments, the first polypeptide comprises a signal peptide, a CD8α transmembrane domain, a CD137 costimulatory domain, and a CD3ζ primary signaling domain.

[0052] In certain embodiments, the second polypeptide comprises a signal peptide and a CD4 transmembrane domain.

[0053] In certain embodiments, the fusion polypeptide comprises the sequence set forth in any one of SEQ ID NOs: 32-41.

[0054] In a specific embodiment, the fusion polypeptide comprises the sequence set forth in SEQ ID NO:40.

[0055] In a further embodiment, the second polypeptide comprises a costimulatory domain.

[0056] In various embodiments, the costimulatory domain of the second polypeptide is 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 (CARD11), CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD94, CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), DNAX-activation protein 10 (DAP10), linker for activation of T-cell family member 1 (LAT), 76 kD SH2 domain-containing leukocyte protein (SLP76), T-cell receptor-associated transmembrane adaptor 1 (TRAT1), TNFR2, TNFRS14, TNFRS18, TNRFS25, and the zeta chain of T-cell receptor-associated protein kinase 70 (ZAP70).

[0057] In additional embodiments, the costimulatory domain of the second polypeptide is a costimulatory domain isolated from OX40 or TNFR2.

[0058] In certain embodiments, the polypeptide cleavage signal is a viral self-cleaving polypeptide.

[0059] In certain embodiments, the polypeptide cleavage signal is a viral auto-cleaving 2A polypeptide.

[0060] In various embodiments, the polypeptide cleavage signal is a viral self-cleaving polypeptide selected from the group consisting of: foot-and-mouth disease virus (FMDV) (F2A) peptide, equine rhinitis A virus (ERAV) (E2A) peptide, Thosea asigna virus (TaV) (T2A) peptide, porcine teschovirus-1 (PTV-1) (P2A) peptide, tylovirus 2A peptide, and encephalomyocarditis virus 2A peptide.

[0061] In certain embodiments, the fusion polypeptide comprises the sequence set forth in any one of SEQ ID NOs: 42-61.

[0062] In certain embodiments, the fusion polypeptide comprises the sequence set forth in any one of SEQ ID NOs:50 or 60.

[0063] In a further embodiment, the FRB multimerization domain and the FKBP multimerization domain are localized extracellularly when the first polypeptide and the second polypeptide are expressed.

[0064] In various embodiments, the polypeptide complex comprises a first polypeptide comprising an FRB multimerization domain polypeptide or a variant thereof, a CD8α transmembrane domain or a CD4 transmembrane domain, a CD137 costimulatory domain, and / or a CD3ζ primary signaling domain; a second polypeptide comprising an anti-CD33 VHH antibody having the amino acid sequence set forth in any one of SEQ ID NOs: 2-21; an FKBP multimerization domain polypeptide or a variant thereof; and a CD4 transmembrane domain or a CD8α transmembrane domain; and a cross-linking agent associated with and positioned between the multimerization domains of the first and second polypeptides.

[0065] In a particular embodiment, the anti-CD33 VHH antibody has the amino acid sequence set forth in SEQ ID NO:10.

[0066] In a specific embodiment, the FKBP multimerization domain is FKBP12.

[0067] In additional embodiments, the FRB polypeptide is FRB T2098L.

[0068] In certain embodiments, the cross-linking agent is selected from the group consisting of: AP21967, sirolimus, everolimus, novolimus, pimecrolimus, ridaforolimus, tacrolimus, temsirolimus, umirolimus, and zotarolimus.

[0069] In certain embodiments, the first polypeptide comprises a CD8α transmembrane domain, a CD137 costimulatory domain, and a CD3ζ primary signaling domain.

[0070] In various embodiments, the second polypeptide comprises a CD4 transmembrane domain.

[0071] In a further embodiment, the second polypeptide comprises a costimulatory domain.

[0072] In some embodiments, the costimulatory domain of the second polypeptide is 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 (CARD11), CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD94, CD134 (OX40), CD137 (4-1BB), CD27 and a costimulatory molecule selected from the group consisting of ICOS, DNAX-activating protein 10 (DAP10), linker for activation of T-cell family member 1 (LAT), SH2 domain-containing leukocyte protein of 76 kD (SLP76), T-cell receptor-associated transmembrane adaptor 1 (TRAT1), TNFR2, TNFRS14, TNFRS18, TNRFS25, and the zeta chain of T-cell receptor-associated protein kinase 70 (ZAP70).

[0073] In certain embodiments, the costimulatory domain of the second polypeptide is a costimulatory domain isolated from OX40 or TNFR2.

[0074] In certain embodiments, the cells are hematopoietic cells.

[0075] In various embodiments, the cell is a T cell, an αβ T cell, or a γδ T cell.

[0076] In various embodiments, the cells are CD3+, CD4+, and / or CD8+ cells.

[0077] In additional embodiments, the cell is an immune effector cell.

[0078] In some embodiments, the cell is a cytotoxic T lymphocyte (CTL), a tumor-infiltrating lymphocyte (TIL), or a helper T cell.

[0079] In certain embodiments, the cells are natural killer (NK) cells or natural killer T (NKT) cells.

[0080] In additional embodiments, the source of the cells is peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, or a tumor.

[0081] In a further embodiment, the FRB multimerization domain and the FKBP multimerization domain are localized extracellularly when the first polypeptide and the second polypeptide are expressed.

[0082] In a preferred embodiment, the first polypeptide comprises the amino acid sequence set forth in SEQ ID NO:82.

[0083] In certain embodiments, the chimeric antigen receptor (CAR) comprises an anti-CD33 VHH antibody having an amino acid sequence set forth in any one of SEQ ID NOs: 2-21, a hinge domain, a transmembrane domain, one or more intracellular costimulatory signaling domains, and / or a primary signaling domain.

[0084] In a particular embodiment, the anti-CD33 VHH antibody has the amino acid sequence set forth in SEQ ID NO:10.

[0085] In a specific embodiment, the anti-CD33 VHH antibody has the amino acid sequence set forth in SEQ ID NO:20.

[0086] In various embodiments, the CAR comprises an anti-CD33 VHH antibody having an amino acid sequence set forth in any one of SEQ ID NOs: 2-21, a hinge domain, a transmembrane domain, one or more intracellular costimulatory signaling domains, and / or a primary signaling domain 5' to 3'.

[0087] In a particular embodiment, the anti-CD33 VHH antibody has the amino acid sequence set forth in SEQ ID NO:10.

[0088] In a specific embodiment, the anti-CD33 VHH antibody has the amino acid sequence set forth in SEQ ID NO:20.

[0089] In specific embodiments, the hinge domain and transmembrane domain are isolated from CD8α, CD27, CD28, CD33, CD37, CD45, CD64, CD71, CD80, CD86, CD134, CD137, CD152, CD154, AMN, and PD1.

[0090] In additional embodiments, the one or more costimulatory signaling domains are isolated from a costimulatory molecule selected from the group consisting of: CD28, CD134, CD137, and CD278.

[0091] In certain embodiments, the CAR comprises a CD8α signal peptide, a CD8α hinge and transmembrane domain, a CD134 costimulatory domain, and a CD3ζ primary signaling domain.

[0092] In a further embodiment, the CAR comprises the amino acid sequence set forth in any one of SEQ ID NOs: 62-81.

[0093] In a further embodiment, the CAR comprises the amino acid sequence set forth in any one of SEQ ID NOs: 70 or 80.

[0094] In some embodiments, a polynucleotide encoding a first or second polypeptide, a fusion polypeptide, or a CAR as contemplated herein is provided.

[0095] In various embodiments, a cDNA encoding a first or second polypeptide, a fusion polypeptide, or a CAR contemplated herein is provided.

[0096] In various embodiments, a first or second polypeptide, a fusion polypeptide, or an RNA encoding a CAR contemplated herein is provided.

[0097] In various embodiments, vectors are provided that include the polynucleotides contemplated herein.

[0098] In certain embodiments, the vector is an expression vector.

[0099] In certain embodiments, the vector is a transposon.

[0100] In a further embodiment, the vector is a piggyBAC transposon or a Sleeping Beauty transposon.

[0101] In certain embodiments, the vector is a viral vector.

[0102] In certain embodiments, the vector is an adenoviral vector, an adeno-associated viral (AAV) vector, a herpes viral vector, a vaccinia viral vector, or a retroviral vector.

[0103] In additional embodiments, the retroviral vector is a lentiviral vector.

[0104] In various embodiments, the lentiviral vector is selected from the group consisting of human immunodeficiency virus 1 (HIV-1), human immunodeficiency virus 2 (HIV-2), Visna-Maedi virus (VMV); caprine arthritis-encephalitis virus (CAEV), equine infectious anemia virus (EIAV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), and simian immunodeficiency virus (SIV).

[0105] In a further embodiment, a cell comprising the first or second polypeptide, fusion polypeptide, or CAR contemplated herein is provided.

[0106] In certain embodiments, the cells are hematopoietic cells.

[0107] In certain embodiments, the cell is an immune effector cell.

[0108] In various embodiments, the cell is a T cell, an αβ T cell, or a γδ T cell.

[0109] In some embodiments, the cells express CD3+, CD4+, CD8+, or a combination thereof.

[0110] In some embodiments, the cell is a cytotoxic T lymphocyte (CTL), a tumor-infiltrating lymphocyte (TIL), or a helper T cell.

[0111] In further embodiments, the cells are natural killer (NK) cells or natural killer T (NKT) cells.

[0112] In certain embodiments, the composition comprises a cell as contemplated herein.

[0113] In certain embodiments, the composition comprises a physiologically acceptable carrier and a cell as contemplated herein.

[0114] In an additional embodiment, a method of treating a subject in need thereof, comprising administering to the subject an effective amount of a composition contemplated herein.

[0115] In certain embodiments, a method for treating, preventing, or alleviating at least one symptom of cancer, infectious disease, autoimmune disease, inflammatory disease, and immune deficiency, or a condition related thereto, comprises administering to a subject an effective amount of a composition contemplated herein.

[0116] In some embodiments, the method of treating a solid tumor comprises administering to a subject an effective amount of a composition contemplated herein.

[0117] In various embodiments, the solid cancer is selected from the group consisting of lung cancer, liver cancer, gastric cancer, colorectal cancer, head and neck cancer, urothelial cancer, prostate cancer, endometrial cancer, pancreatic cancer, breast cancer, cervical cancer, ovarian cancer, skin cancer and melanoma.

[0118] In certain embodiments, a method of treating a hematological malignancy comprises administering to a subject an effective amount of a composition contemplated herein.

[0119] In various embodiments, the hematological malignancy is leukemia, lymphoma, or multiple myeloma.

[0120] In certain embodiments, the malignant hematological disease is acute myeloid leukemia (AML). [Brief explanation of the drawings]

[0121] [Figure 1A] FIG. 1A shows a diagram of a VHH-DARIC polypeptide complex. [Figure 1B] FIG. 1B shows a depiction of the CD33 VHH DARIC architecture. [Figure 2A] Figure 2A shows the expression of CD33 VHH1-5 DARIC on transduced T cells detected by anti-VHH staining (top row) and CD33-Fc binding (bottom row). [Figure 2B]Figure 2B shows the expression of CD33 VHH9-10 DARIC on transduced T cells detected by CD33-Fc binding. [Figure 3A] Figure 3A shows the phenotype of T cells transduced with CD33 VHH1-5 DARIC or control. [Figure 3B] Figure 3B shows the phenotype of T cells transduced with CD33 VHH9-10 DARIC or control. [Figure 4A] Figure 4A shows IFNγ secretion from CD33 VHH1-5 DARIC or control cells cultured with CD33+ THP-1 cells at an E:T ratio of 1:1 in the presence or absence of AP21967 for 24 hours. [Figure 4B] Figure 4B shows IFNγ secretion from CD33 VHH9-10 DARIC or control cells cultured with CD33+ THP-1 cells at an E:T ratio of 1:1 in the presence or absence of AP21967 for 24 hours. [Figure 4C] Figure 4C shows IFNγ secretion from CD33 VHH9-10 DARIC or control cells cultured with engineered 293T cells expressing full-length CD33 (CD33M) or CD33 splice variant (CD33m, C2) at an E:T ratio of 1:1 in the presence or absence of AP21967 for 24 h. [Figure 5A] FIG. 5A shows CD33 expression on MV4-11 cells engineered to knock out the CD33 gene (CD33-KO cells) and in unstained controls. [Figure 5B] Figure 5B shows IFNγ secretion from anti-CD33 VHH9 DARIC or UTD T cells cocultured with MV4-11 or CD33-KO cells at an E:T ratio of 1:1 in the presence or absence of AP21967 for 24 hours. [Figure 5C]Figure 5C shows IFNγ secretion from UTD T cells, anti-CD33 CAR T cells, or anti-CD33 VHH DARIC T cells, MV4-11 cells (left panel) or CD33-KO cells engineered to express the CD33m splice variant (CD33-KO-C2 cells, right panel) at an E:T ratio of 1:1 in the presence or absence of AP21967 for 24 hours. [Figure 6] Figure 6 shows IFNγ secretion from anti-CD33 VHH DARIC T cells co-cultured with CD33+ THP-1 cells at an E:T ratio of 1:1 in the presence or absence of soluble CD33 (CD33-Fc) and AP21967 for 24 hours. [Figure 7] Figure 7 shows IFNγ secretion from anti-CD33 VHH DARIC T cells co-cultured with CD33neg 293T cells transfected with different amounts of mRNA encoding CD33 at an E:T ratio of 1:1 in the presence or absence of AP21967 for 24 h. [Figure 8A] Figure 8A shows tumor growth measured as a function of luminescence in immunocompromised NSG mice inoculated with HL60 AML-expressing tumor cells expressing a luciferase reporter and treated with UTD T cells or anti-CD33 VHH DARIC T cells in the absence of rapamycin 10 days after inoculation (day 0). [Figure 8B] Figure 8B shows tumor growth measured as a function of luminescence in immunocompromised NSG mice inoculated with HL60 AML-expressing tumor cells expressing a luciferase reporter and treated 10 days post-inoculation (day 0) with UTD T cells or anti-CD33 VHH DARIC T cells and 1 mg / kg rapamycin.

[0122] Brief description of sequence identifiers SEQ ID NO: 1 sets forth the amino acid sequence of full-length human CD33. SEQ ID NOs: 2 to 21 set forth the amino acid sequences of anti-CD33 VHH domains. SEQ ID NOs: 22-31 set forth the amino acid sequences of anti-CD33 VHH DARIC binding components. SEQ ID NOs: 32-41 set forth the amino acid sequences of anti-CD33 VHH DARIC fusion proteins. SEQ ID NOs: 42-51 set forth the amino acid sequences of the anti-CD33 VHH DARIC.OX40 fusion proteins. SEQ ID NOs: 52-61 set forth the amino acid sequences of anti-CD33 VHH DARIC.TNFR2 fusion proteins. SEQ ID NOs: 62-81 set forth the amino acid sequences of anti-CD33 VHH CARs. SEQ ID NO: 82 sets forth the amino acid sequence of the anti-CD33 VHH DARIC signaling component. SEQ ID NO: 83 sets forth the polynucleotide sequence of the Kozak sequence. SEQ ID NOs: 84 to 94 list the amino acid sequences of various linkers. SEQ ID NOs: 95 to 119 set forth the amino acid sequences of the protease cleavage site and the cleavage site of the self-cleaving polypeptide.

[0123] In the above sequences, Xaa, when present, may refer to any amino acid or the absence of an amino acid. In a preferred embodiment, XaaXaa refers to the amino acid sequence SS or KP. DETAILED DESCRIPTION OF THE INVENTION

[0124] A. Overview Cancer is one of the leading causes of death worldwide. Approximately 10% of cancers are hematologic malignancies, including leukemia, lymphoma, and myeloma. Acute myeloid leukemia (AML) is the most common and fatal hematologic malignancy in adults. Despite significant scientific discoveries and novel therapies over the past 40 years, treatment outcomes for AML, especially in adult patient populations, remain dismal. While standard chemotherapy can induce complete remission in selected patients, the majority of patients eventually relapse and succumb to the disease. In 2012, the global incidence of AML was approximately 351,965, and approximately 265,461 deaths were attributed to AML.

[0125] CD33 is expressed on the majority of acute myeloid leukemia (AML) leukemia blasts and possibly on leukemia stem cells. CD33 is a difficult target due to its low expression and slow internalization, characteristics that limit antibody-dependent cell-mediated cytotoxicity and intracellular drug accumulation, and consequently the activity of unlabeled and toxin-carrying antibodies.

[0126] The present disclosure generally relates to improved compositions and methods for controlling the spatial and temporal control of adoptive cell therapy using dimerization agent-regulated immune receptor complexes (DARICs) that bind to CD33. While not wishing to be bound by any particular theory, the DARIC compositions and methods contemplated herein offer numerous benefits over existing CAR T cell therapies in the art, including, but not limited to, both spatial and temporal control over the signal-transducing binding and signaling activity of immune effector cells. Temporal control of DARIC triggers the DARIC mechanism of signal transduction through crosslinker-mediated association of the DARIC binding component with the DARIC signaling component. Spatial control of DARIC engages the signaling mechanism through recognition of CD33 by the DARIC binding domain of the DARIC binding component. In this way, DARIC immune effector cells become activated when both CD33-expressing target cells and crosslinkers are present.

[0127] The present disclosure also relates to improved anti-CD33 CAR architectures that overcome potential limitations of existing CAR T therapies, including, but not limited to, tonic or antigen-independent signaling, poor expression and / or subtherapeutic activity.

[0128] In various embodiments, the present disclosure contemplates, for example, anti-CD33 VHH DARICs or anti-CD33 VHH CARs that generate an anti-cancer response against cancers, e.g., AML, that express CD33, e.g., full-length CD33 and / or CD33 splice variants.

[0129] In certain embodiments, DARIC comprises a polypeptide comprising a multimerization domain polypeptide or a variant thereof, a transmembrane domain, a costimulatory domain, and / or a primary signaling domain (DARIC signaling component), and an anti-CD33 VHH, a polypeptide comprising a multimerization domain polypeptide or a variant thereof (DARIC binding component), a transmembrane domain, and optionally a costimulatory domain. In the presence of a cross-linking agent, the DARIC binding component and the DARIC signaling component associate with each other through the cross-linking agent to form a functionally active DARIC that targets cells expressing CD33.

[0130] In certain embodiments, the multimerization domain of the DARIC binding component and the DARIC signal transduction component is arranged extracellularly.The extracellular arrangement of the multimerization domain provides many advantages over intracellular arrangement, including but not limited to, more efficient arrangement of the anti-CD33 VHH domain, higher temporal sensitivity to cross-linking agent control, and lower toxicity due to the use of non-immunosuppressive doses of certain cross-linking agents.

[0131] Contemplated herein are polynucleotides encoding DARIC, DARIC binding components, and DARIC signaling components; DARIC binding components, DARIC signaling components, DARIC protein complexes, DARIC fusion proteins; cells containing and / or expressing polynucleotides encoding DARIC, DARIC binding components, and DARIC signaling components; and methods of using them to treat immune disorders.

[0132] Recombinant (i.e., engineered) DNA, peptide and oligonucleotide synthesis, immunoassays, tissue culture, transformation (e.g., electroporation, lipofection), enzymatic reactions, purification, and related techniques and procedures may generally be performed as described in various general and more specific references in microbiology, molecular biology, biochemistry, molecular genetics, cell biology, virology, and immunology, cited and discussed throughout this specification. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY;Current Protocols in Molecular Biology(John Wiley and Sons, updated July 2008);Short Protocols in Molecular Biology:A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub.Associates and Wiley-Interscience;Glover, DNA Cloning:A Practical Approach, vol.I & II(IRL Press,Oxford Univ.Press USA,1985);Current Protocols in Immunology(Edited by:John E.Coligan,Ada M.Kruisbeek,David H.Margulies,Ethan M.Shevach,Warren Strober 2001 John Wiley & Sons,NY,NY);Real-Time PCR:Current Technology and Applications,Edited by Julie Logan,Kirstin Edwards and Nick Saunders,2009,Caister Academic Press,Norfolk,UK;Anand,Techniques for the Analysis of Complex Genomes,(Academic Press,New York,1992);Guthrie and Fink,Guide to Yeast Genetics and Molecular Biology(Academic Press,New York,1991);Oligonucleotide Synthesis(N.Gait,Ed.,1984);Nucleic Acid The Hybridization(B.Hames & S.Higgins,Eds.,1985);Transcription and Translation(B.Hames & S.Higgins,Eds.,1984);Animal Cell Culture(R.Freshney,Ed.,1986);Perbal,A Practical Guide to Molecular Cloning(1984);Next-Generation Genome Sequencing(Janitz,2008 Wiley-VCH);PCR Protocols(Methods in Molecular Biology)(Park,Ed.,3rd Edition,2010 Humana Press);Immobilized Cells And Enzymes(IRL Press,1986);the treatise,Methods In Enzymology(Academic Press,Inc.,N.Y.);Gene Transfer Vectors For Mammalian Cells(J.H.Miller and M.P.Calos eds.,1987,Cold Spring Harbor Laboratory);Harlow and Lane,Antibodies,(Cold Spring Harbor Laboratory Press,Cold Spring Harbor,N.Y.,1998);Immunochemical Methods In Cell And Molecular Biology(Mayer and Walker,eds.,Academic Press,London,1987);Handbook Of Experimental. Immunology,Volumes I-IV(DMWeir and CC Blackwell, eds., 1986); Roitt, Essential Immunology, 6th Edition, (Blackwell Scientific Publications, Oxford, 1988); Current Protocols in Immunology (QE Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach and W. Strober, eds., 1991); Annual Review of Immunology; and research articles in journals such as, for example, Advances in Immunology.

[0133] B. Definition Before describing this disclosure in more detail, it may be helpful to an understanding thereof to provide definitions of certain terms to be used herein.

[0134] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used to practice or test particular embodiments, preferred compositions, methods and materials embodiments are disclosed herein. For purposes of this disclosure, the following terms are defined below.

[0135] The articles "a," "an," and "the" are used herein to refer to one or to more than one (i.e., to at least one, or to one or more) of the grammatical object of the article. By way of example, "an element" means one element or one or more elements.

[0136] The use of the alternative (eg, "or") should be understood to mean either one, both, or any combination of the alternatives.

[0137] The term "and / or" should be understood to mean either one or both of the alternatives.

[0138] As used herein, the term "about" or "approximately" refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that varies by up to 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% relative to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length. In one embodiment, the term "about" or "approximately" refers to a range of ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1% of the quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length relative to the reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length.

[0139] In one embodiment, a range, for example, 1 to 5, about 1 to 5, or about 1 to about 5, refers to each value subsumed within the range. For example, in one non-limiting, merely exemplary embodiment, the range "1 to 5" is equivalent to: 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.

[0140] As used herein, 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 more compared to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length. In one embodiment, "substantially the same" refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that produces approximately the same effect, e.g., a physiological effect, as the reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length.

[0141] Throughout this specification, unless otherwise required, the terms "comprise" and "comprising" should be understood to imply the inclusion of the recited step or element or group of steps or elements, but not the exclusion of any other step or element or group of steps or elements. "Consisting of" means including, but limited to, what follows the words "consisting of." Thus, the words "consisting of" indicate that the listed elements are necessary or mandatory, and that no other elements may be present. "Consisting essentially of" means including any elements listed after the words, and any elements limited to other elements that do not interfere with or contribute to the activity or function specified in this disclosure for the listed elements. Thus, the words "consisting essentially of" indicate that the listed elements are necessary or mandatory, but that there are no other elements that materially affect the activity or function of the listed elements.

[0142] References throughout this specification to "one embodiment," "an embodiment," "a particular embodiment," "a related embodiment," "an embodiment," "an additional embodiment," or "a further embodiment," or combinations thereof, mean that the particular feature, structure, or characteristic described in connection with that embodiment is included in at least one embodiment. Thus, the appearances of such phrases in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments. Furthermore, it should be understood that the affirmative recitation of a feature in one embodiment serves as grounds for the exclusion of that feature in certain embodiments.

[0143] "Antigen (Ag)" refers to a compound, composition, or substance that can stimulate antibody production or a T-cell response in an animal, including compositions (e.g., compositions containing a cancer-specific protein) that are injected into or absorbed into an animal. Examples of antigens include, but are not limited to, lipids, carbohydrates, polysaccharides, glycoproteins, peptides, or nucleic acids. Antigens react with the products of specific humoral or cellular immunity, including those induced by heterologous antigens, such as the antigens disclosed herein.

[0144] "Target antigen" or "target antigen of interest" refers to the portion of CD33 to which a binding domain contemplated herein is designed to bind. In certain embodiments, the target antigen is an epitope of the amino acid sequence set forth in SEQ ID NO:1.

[0145] "CD33" refers to a cell surface receptor also known as sialic acid-binding immunoglobulin-like lectin 3 (SIGLEC-3) or GP67. The CD33 gene is located on chromosome 19 and produces a glycosylated protein of approximately 67 kD. CD33 contains two Ig-like domains, one V-set domain and one C2-set domain. CD33 mediates cell-cell interactions and plays a role in maintaining immune cells in a quiescent state. CD33 recognizes and binds alpha-2,3- and more avidly alpha-2,6-linked sialic acid-bearing glycans. Upon binding of ligands such as C1q or sialylated glycoproteins, two immunoreceptor tyrosine-based inhibitory motifs (ITIMs) located on the CD33 cytoplasmic tail are phosphorylated by Src-like kinases such as LCK. These phosphorylations provide docking sites for the recruitment and activation of the protein-tyrosine phosphatases PTPN6 / SHP-1 and PTPN11 / SHP-2. CD33 also has at least three identified splice variants. ΔE2 The splice variant lacks the amino acid sequence encoded by exon 2 of the human CD33 gene (amino acids 13 to 139 of full-length CD33, e.g., NP_001076087.1, C2). 7a The splice variant lacks 54 carboxy-terminal amino acids due to a premature translation stop signal present in exon 7a (e.g., NP_001171079.1). ΔE2 / 7a It lacks the amino acids encoded by exon 2 and the 54 carboxy-terminal amino acids. CD33 is normally expressed on normal B cells, activated T cells, and a subset of natural killer cells, but not on hematopoietic stem cells or outside the hematopoietic system. Both full-length CD33 and / or CD33 splice variants are also expressed on acute myeloid leukemia (AML) blast cells in the majority of AML patients.

[0146] "Antibody" refers to a binding agent that is a polypeptide containing at least a light or heavy chain immunoglobulin variable region that specifically recognizes and binds to an epitope of an antigen, such as a lipid, carbohydrate, polysaccharide, glycoprotein, peptide, or nucleic acid, containing an antigenic determinant recognized by an immune cell.

[0147] References to "VH" or "VH" refer to the variable region of an immunoglobulin heavy chain or antigen-binding fragment thereof.

[0148] "Heavy chain antibodies" consist of two V H It refers to an antibody that contains two V domains and does not contain a light chain (Riechmann L. et al., J. Immunol. Methods 231:25-38 (1999); WO94 / 04678; WO94 / 25591; U.S. Patent No. 6,005,079). H "Humanized VHH" or "humanized camelid-like antibody" refers to a non-human VHH or camelid-like antibody that has undergone humanization to reduce the potential immunogenicity of the antibody in a human recipient.

[0149] As used herein, "V H H," "V H H antibody" or "V H "H domain" refers to 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)).

[0150] "Linker" refers to a number of amino acid residues between various polypeptide domains, added for proper spacing and conformation of the molecule. In certain embodiments, the linker separates one or more VHH domains, hinge domains, multimerization domains, transmembrane domains, costimulatory domains, and / or primary signaling domains.

[0151] Exemplary linkers suitable for use in certain embodiments contemplated herein include, but are not limited to, the following amino acid sequences: GGG; DGGGS (SEQ ID NO: 84); TGEKP (SEQ ID NO: 85) (see, e.g., Liu et al., PNAS 5525-5530 (1997)); GGRR (SEQ ID NO: 86) (Pomerantz et al. 1995, supra); (GGGGS) n , where n=1, 2, 3, 4, or 5 (SEQ ID NO: 87) (Kim et al., PNAS 93, 1156-1160 (1996); EGKSSGSGSESKVD (SEQ ID NO: 88) (Chaudhary et al., 1990, Proc. Natl. Acad. Sci. USA 87:1066-1070); KESGSVSSSEQLAQFRSLD (SEQ ID NO: 89) (Bird et al., 1988, Science 242:423-426), GGRRGGGS (SEQ ID NO: 90); LQRDGERP (SEQ ID NO: 91); LRQKDGGGSERP (SEQ ID NO: 92); LRQKD(GGGS)2ERP (SEQ ID NO: 93). Alternatively, flexible linkers can be modeled using a computer program (Desjarlais & Berg, PNAS 93) that can model both the DNA binding site and the peptide itself. 90:2256-2260 (1993), PNAS 91:11099-11103 (1994), or rationally designed by phage display. In one embodiment, the linker comprises the following amino acid sequence: GSTSGSGKPGSGEGSTKG (SEQ ID NO: 94) (Cooper et al., Blood, 101(4):1637-1644 (2003)).

[0152] A "spacer domain" refers to a polypeptide that separates two domains. In one embodiment, the spacer domain separates the VHH domain from the surface of the effector cell, allowing for proper cell-cell contact, antigen binding, and activation (Patel et al., Gene Therapy, 1999;6:412-419). In certain embodiments, the spacer domain separates one or more VHH domains, a multimerization domain, a transmembrane domain, a costimulatory domain, and / or a primary signaling domain. The spacer domain may be derived from natural, synthetic, semisynthetic, or recombinant sources. In certain embodiments, the spacer domain is an immunoglobulin portion containing one or more heavy chain constant regions, such as CH2 and CH3. The spacer domain may comprise the amino acid sequence of a natural immunoglobulin hinge region or a modified immunoglobulin hinge region.

[0153] "Hinge domain" refers to a polypeptide that positions an antigen-binding domain away from the surface of an effector cell and plays an important role in proper cell-to-cell contact, antigen binding, and activation. In certain embodiments, a polypeptide 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 from any natural, synthetic, semi-synthetic, or recombinant source. The hinge domain may comprise the amino acid sequence of a native immunoglobulin hinge region or a modified immunoglobulin hinge region.

[0154] As used herein, a "multimerization domain" refers to a polypeptide that preferentially interacts or associates with another distinct polypeptide, either directly or via a bridging molecule, such as a chemically induced dimerization agent, where the interaction of the distinct multimerization domains significantly contributes to or effectively promotes multimerization (i.e., the formation of dimers, trimers, or multimerized complexes, which may be homodimers, heterodimers, homotrimers, heterotrimers, homomultimers, or heteromultimers). Multimerization domains may be derived from natural, synthetic, semi-synthetic, or recombinant sources.

[0155] Exemplary multimerization domains suitable for use in certain embodiments contemplated herein include an FK506-binding protein (FKBP) polypeptide or variant thereof, an FKBP-rapamycin-binding (FRB) polypeptide or variant thereof, a calcineurin polypeptide or variant thereof, a cyclophilin polypeptide or variant thereof, a bacterial dihydrofolate reductase (DHFR) polypeptide or variant thereof, a PYR1-like 1 (PYL1) polypeptide or variant thereof, an abscisic acid insensitive 1 (ABI1) polypeptide or variant thereof, a GIB1 polypeptide or variant thereof, or a GAI polypeptide or variant thereof.

[0156] As used herein, the term "FKBP-rapamycin binding polypeptide" refers to an FRB polypeptide. In certain embodiments, the FRB polypeptide is an FKBP12-rapamycin binding polypeptide. FRB polypeptides suitable for use in certain embodiments contemplated herein generally contain at least about 85 to about 100 amino acid residues. In certain embodiments, the FRB polypeptide comprises the 93-amino acid sequence from Ile-2021 to Lys-2113, as defined by GenBank accession number L34075.1, and includes the T2098L mutation. The FRB polypeptides contemplated herein bind to FKBP polypeptides via a cross-linking agent, thereby forming a ternary complex.

[0157] As used herein, the term "FK506-binding protein" refers to an FKBP polypeptide. In certain embodiments, the FKBP polypeptide is an FKBP12 polypeptide or an FKBP12 polypeptide containing an F36V mutation. In some embodiments, the FKBP domain may also be referred to as a "rapamycin-binding domain." Information regarding the nucleotide sequences, cloning, and other aspects of various FKBP species is known in the art (see, for example, Staendart et al., Nature 346:671, 1990 (human FKBP12); Kay, Biochem. J. 314:361, 1996). The FKBP polypeptide contemplated herein binds to an FRB polypeptide via a cross-linking agent, thereby forming a ternary complex.

[0158] A "cross-linking agent" refers to a molecule that associates with and is positioned between two or more multimerization domains. In certain embodiments, a multimerization domain significantly contributes to or efficiently promotes the formation of a polypeptide complex only in the presence of a cross-linking agent. In certain embodiments, a multimerization domain does not contribute to or efficiently promote the formation of a polypeptide complex in the absence of a cross-linking agent. Examples of cross-linking agents suitable for use in certain 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)-FKBP synthetic ligand (SLF) or a derivative thereof, or any combination thereof.

[0159] Rapamycin analogs (rapalogs) include, but are not limited to, those described in U.S. Patent No. 6,649,595. Rapalog structures are incorporated herein by reference in their entirety. In certain embodiments, the cross-linking agent is a rapalog that has significantly reduced immunosuppressive effects compared to rapamycin. In preferred embodiments, the rapalog is AP21967 (also known as C-16-(S)-7-methylindole rapamycin, IC 50 =10 nM, a chemically modified, non-immunosuppressive rapamycin analog). Other examples of rapalogs suitable for use in certain embodiments contemplated herein include, but are not limited to, everolimus, novolimus, pimecrolimus, ridaforolimus, tacrolimus, temsirolimus, umirolimus, and zotarolimus.

[0160] A "significantly reduced immunosuppressive effect" refers to an immunosuppressive effect that is at least 0.1 to 0.005 times less than that observed or predicted for the same dose, as measured clinically or appropriately in vitro (e.g., inhibition of T-cell proliferation), or as measured in an in vivo surrogate of human immunosuppressive activity.

[0161] A "transmembrane domain" or "TM domain" is a domain that anchors a polypeptide to a cell membrane. TM domains may be derived from either natural, synthetic, semi-synthetic, or recombinant sources.

[0162] The term "effector function" or "effector cell function" refers to the specialized functions of immune effector cells. Effector functions include, but are not limited to, activation, cytokine production, proliferation, and cytotoxic activity, including release of cytotoxic factors, or other cellular responses triggered by antigen binding to receptors expressed on immune effector cells.

[0163] "Intracellular signaling domain" or "endodomain" refers to the portion of a protein that transmits an effector function signal, instructing the cell to perform a specialized function. Typically, the entire intracellular signaling domain can be employed, although 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 a truncated portion may be used in place of the entire domain, so long as the effector function signal is transmitted. The term intracellular signaling domain is intended to include any truncated portion of the intracellular signaling domain that is necessary or sufficient to transmit the effector function signal.

[0164] It is known that signals generated by the TCR alone are insufficient for full activation of T cells; secondary, or costimulatory, signals are also required. Thus, T cell activation can be said to be mediated by two distinct classes of intracellular signaling domains: primary signaling domains, which initiate antigen-dependent primary activation by the TCR (e.g., the TCR / CD3 complex), and costimulatory signaling domains, which act in an antigen-dependent manner to provide secondary, or costimulatory, signals.

[0165] A "primary signaling domain" refers to an intracellular signaling domain that controls the primary activation of the TCR complex, either stimulatory or inhibitory. Primary signaling domains that act in a stimulatory manner may contain signaling motifs known as immunoreceptor tyrosine-based activation motifs, or ITAMs. Examples of ITAM-containing primary signaling domains suitable for use in certain embodiments include, but are not limited to, those derived from FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d.

[0166] As used herein, the term "costimulatory signaling domain" or "costimulatory domain" refers to the intracellular signaling domain of a costimulatory molecule. A costimulatory molecule is a cell surface molecule other than an antigen receptor or an Fc receptor that, upon binding to an antigen, provides a second signal required for the efficient activation and function of T lymphocytes. Illustrative examples of costimulatory molecules from which costimulatory domains can be isolated include Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, caspase recruitment domain family member 11 (CARD11), CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD94, CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), DNAX-activation protein 10 (DAP10), Linker for activation of T-cell family member 1 (LAT), 76-kD SH2 domain-containing leukocyte protein (SLP76), T-cell receptor-associated transmembrane adaptor 1 (TRAT1), TNFR2, TNF receptor superfamily member 14 (TNFRS14; HVEM), TNF receptor superfamily member 18 (TNFRS18; GITR), TNF receptor superfamily member 25 (TNFRS25; DR3), and the zeta chain of T-cell receptor-associated protein kinase 70 (ZAP70).

[0167] "Immune disorder" refers to a disease that provokes a response from the immune system. In certain embodiments, the term "immune disorder" refers to cancer, an autoimmune disease, or an immune deficiency.

[0168] As used herein, the term "cancer" generally relates to certain diseases or conditions in which abnormal cells divide uncontrollably and can invade nearby tissues.

[0169] As used herein, the term "malignant" refers to a cancer in which tumor cells exhibit one or more of the following: uncontrolled growth (i.e., division beyond normal limits), invasion (i.e., invasion and destruction of adjacent tissues), and metastasis (i.e., spread to other parts of the body via the lymph or blood). As used herein, the term "metastasizing" refers to the spread of cancer from one part of the body to another. A tumor formed by spread cells is called a "metastatic tumor" or "metastatic cancer." A metastatic tumor contains cells similar to those of the original tumor (primary tumor).

[0170] As used herein, the terms "benign" or "non-malignant" refer to tumors that can grow large but do not spread to other parts of the body. Benign tumors are self-limited and often do not invade or metastasize.

[0171] "Cancer cell" refers to an individual cell of a cancerous growth or cancerous tissue. Cancer cells include both solid and liquid cancers. "Tumor" or "tumor cell" generally refers to an expansion due to abnormal cell proliferation or a lesion formed by abnormal cell proliferation, which may be benign, precancerous, or malignant. Most cancers form tumors, but liquid cancers, such as leukemia, do not necessarily form tumors. For cancers that form tumors, the terms cancer (cell) and tumor (cell) are used interchangeably. The amount of tumor in an individual is the "tumor burden," which can be measured as the number, volume, or weight of tumors.

[0172] The term "recurrence" refers to the diagnosis of a return of cancer, or the diagnosis of signs and symptoms of a return of cancer, after a period of improvement or remission.

[0173] "Remission," also called "clinical remission," includes both partial and complete remission. In a partial remission, some, but not all, signs and symptoms of cancer disappear. In a complete remission, all signs and symptoms of cancer disappear, although cancer may still be present in the body.

[0174] "Refractory" refers to a cancer that is resistant or unresponsive to therapy with a particular therapeutic agent. A cancer may be refractory from the start of treatment (i.e., unresponsive to initial exposure to the therapeutic agent) or may become refractory as a result of developing resistance to the therapeutic agent either during the initial treatment period or during subsequent treatment periods.

[0175] As used herein, the terms "individual" and "subject" are often used interchangeably and refer to any animal that exhibits symptoms of cancer or other immune disorders that can be treated with the compositions and methods otherwise contemplated herein. Suitable subjects (e.g., patients) include experimental animals (e.g., mice, rats, rabbits, or guinea pigs), farm animals, domestic animals, or pets (e.g., cats or dogs). Non-human primates, preferably human patients, are also included. Typical subjects include human patients who have, are at risk of, or have been diagnosed with cancer or another immune disorder.

[0176] As used herein, the term "patient" refers to a subject diagnosed with cancer or another immune disorder that can be treated with the compositions and methods otherwise disclosed herein.

[0177] As used herein, "treatment" or "treating" includes any beneficial or desired effect on the symptoms or pathology of a disease or condition, and may include even a minimal reduction in one or more measurable markers of the disease or condition being treated. Treatment may optionally include a reduction in the disease or condition, or a delay in the progression of the disease or condition, e.g., a delay in tumor growth. "Treatment" does not necessarily indicate a complete elimination or cure of the disease or condition, or its associated symptoms.

[0178] As used herein, "prevent" and similar terms, such as "prevented" and "preventing," refer to an approach aimed at preventing, inhibiting, or reducing the likelihood of 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 onset or recurrence of symptoms of a disease or condition. As used herein, "prevention" and similar terms also include reducing the intensity, effects, symptoms, and / or burden of a disease or condition before the onset or recurrence of the disease or condition.

[0179] As used herein, "amelioration of at least one symptom of" refers to a reduction in one or more symptoms of the disease or condition being treated in a subject. In certain embodiments, the disease or condition being treated is cancer, in which case the one or more symptoms that are improved include, but are not limited to, weakness, fatigue, shortness of breath, easy bruising and bleeding, frequent infections, enlarged lymph nodes, abdominal distension or pain (caused by distended abdominal organs), bone or joint pain, broken bones, unexpected weight loss, loss of appetite, night sweats, persistent low-grade fever, and decreased urination (caused by impaired kidney function).

[0180] "Enhance," "promote," "increase," or "expand" generally refer to the ability of a composition contemplated herein to produce, elicit, or generate a greater physiological response (i.e., downstream effect) compared to the response induced by a vehicle or control molecule / composition. Measurable physiological responses include, among others, increased T cell expansion, activation, persistence, cytokine secretion, and / or cancer cell killing capacity, as would be apparent from an understanding of the art and the present disclosure. An "increased" or "enhanced" amount is typically a "statistically significant" amount and may include an increase of 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, or more (e.g., 500-fold, 1000-fold) (including all integers and decimal points between and above 1, e.g., 1.5, 1.6, 1.7, 1.8, etc.) over the response induced by a vehicle or control composition.

[0181] "Decrease," or "lower," or "reduce," or "diminish," or "attenuate" generally refers to the ability of a composition contemplated herein to produce, elicit, or generate a physiological response (i.e., a downstream effect) that is smaller than the response induced by a vehicle or control molecule / composition. The amount of "reduction" or "decreased" is typically a "statistically significant" amount and can include a decrease of 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, or more (e.g., 500-fold, 1000-fold) (including all integers and decimal points in between and above 1, e.g., 1.5, 1.6, 1.7, 1.8, etc.) of the response induced by a vehicle, a control composition, or a response in a particular cell line.

[0182] "Maintain," or "preserve," or "maintain," or "no change," or "no substantial change," or "no substantial decrease" generally refer to the ability of a composition contemplated herein to produce, elicit, or produce a substantially similar or equivalent physiological response (i.e., downstream effect) in a cell compared to the response produced by a vehicle, a control molecule / composition, or the response in a particular cell line. An equivalent response is one that is not substantially different, or not measurably different, from the reference response.

[0183] Additional definitions are set forth throughout this disclosure.

[0184] C.CD33 VHH DARIC In certain embodiments, DARIC receptors are contemplated that include anti-CD33 VHH domains that direct the cytotoxicity of immune effector cells to cancer cells that express CD33. As used herein, the terms "CD33 VHH DARIC receptor," "anti-CD33 VHH DARIC receptor,” “CD33 VHH DARIC,” or “anti-CD33 VHH "DARIC" is used interchangeably and refers to one or more non-naturally occurring polypeptides that, upon exposure to target cells expressing full-length CD33 or a CD33 splice variant and a multimerizing or cross-linking agent, transduce an immunostimulatory signal in immune effector cells, e.g., stimulating immune effector cell activity and function, increasing the production and / or secretion of pro-inflammatory cytokines. In a preferred embodiment, a CD33 VHH DARIC is a multi-chain chimeric receptor comprising a DARIC signaling component and a DARIC binding / transduction component comprising a VHH domain that recognizes full-length CD33 and / or a CD33 splice variant.

[0185] In one embodiment, the DARIC signaling component and the DARIC binding component are expressed from the same cell.In another embodiment, the DARIC signaling component and the DARIC binding component are expressed from different cells.In certain embodiments, the DARIC signaling component is expressed from a cell, and the DARIC binding component is exogenously provided as a polypeptide.In one embodiment, the DARIC binding component preloaded with a cross-linking agent is exogenously provided to the cell that expresses the DARIC signaling component.

[0186] 1. CD33 DARIC signaling component The terms "DARIC signaling component," "CD33 DARIC signaling component," "DARIC signaling polypeptide," or "CD33 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. In certain embodiments, a DARIC signaling component comprises a multimerization domain, a transmembrane domain, a costimulatory domain, and / or a primary signaling domain. In certain embodiments, a DARIC signaling component comprises a first multimerization domain, a first transmembrane domain, a first costimulatory domain, and / or a primary signaling domain.

[0187] In certain embodiments, the DARIC signaling component comprises one or more multimerization domains.

[0188] Examples of multimerization domains suitable for use in certain CD33 DARIC signaling components contemplated herein include, but are not limited to, an FK506-binding protein (FKBP) polypeptide or variant thereof, an FKBP-rapamycin-binding (FRB) polypeptide or variant thereof, a calcineurin polypeptide or variant thereof, a cyclophilin polypeptide or variant thereof, a bacterial dihydrofolate reductase (DHFR) polypeptide or variant thereof, a PYR1-like 1 (PYL1) polypeptide or variant thereof, and an abscisic acid insensitive 1 (ABI1) polypeptide or variant thereof.

[0189] In certain embodiments, the CD33 DARIC signaling component comprises an FRB polypeptide.

[0190] In particularly preferred embodiments, the CD33 DARIC signaling component comprises an FRB polypeptide comprising a T2098L mutation or a variant thereof. In certain preferred embodiments, the CD33 DARIC signaling component comprises an FKBP12 polypeptide or a variant thereof.

[0191] In some embodiments, the CD33 VHH DARIC signaling component comprises a hinge domain.

[0192] Exemplary hinge domains suitable for use in the CD33 VHH DARIC signaling components described herein include hinge regions derived from the extracellular regions of type 1 membrane proteins such as CD28, CD8α and CD4, which may be the wild-type hinge regions of these molecules or may be modified.

[0193] In certain embodiments, the DARIC signaling component comprises a transmembrane domain.

[0194] In certain embodiments, the DARIC signaling component comprises a hinge domain and a transmembrane domain.

[0195] Illustrative examples of transmembrane domains suitable for use in certain CD33 DARIC signaling components contemplated herein include, but are not limited to, the transmembrane regions of the alpha, beta, gamma, or delta chains of the T cell receptor, CD3ε, CD3ζ, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD71, CD80, CD86, CD134, CD137, CD152, CD154, amnionless (AMN), and programmed cell death 1 (PDCD1). In a preferred embodiment, the CD33 DARIC signaling component comprises a CD4 transmembrane domain. In a preferred embodiment, the CD33 DARIC signaling component comprises a CD8α transmembrane domain.

[0196] In certain embodiments, the DARIC signaling component includes a linker connecting the C-terminus of the transmembrane domain and the N-terminus of the intracellular signaling domain. In various preferred embodiments, a short oligopeptide or polypeptide linker, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in length, connects the transmembrane domain and the intracellular signaling domain. Glycine-serine based linkers provide particularly suitable linkers.

[0197] Specific embodiments contemplated herein include DARIC signaling components containing one or more intracellular signaling domains. In one embodiment, the CD33 DARIC signaling component includes one or more costimulatory signaling domains and / or primary signaling domains. In one embodiment, the intracellular signaling domain contains an immunoreceptor tyrosine activation motif (ITAM).

[0198] Examples of ITAM-containing primary signaling domains suitable for use in the specific CD33 DARIC signaling components contemplated herein include, but are not limited to, those derived from FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d. In preferred embodiments, the CD33 DARIC signaling component comprises a CD3ζ primary signaling domain and one or more costimulatory signaling domains. The primary and costimulatory signaling domains can be linked in tandem, in any order, to the carboxyl terminus of the transmembrane domain.

[0199] Examples of costimulatory domains suitable for use in the particular CD33 DARIC signaling components contemplated herein include, but are not limited to, domains isolated from the following costimulatory molecules: Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, caspase recruitment domain family member 11 (CARD11), CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD94, CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), DNA X-activation protein 10 (DAP10), linker for activation of T-cell family member 1 (LAT), 76-kD SH2 domain-containing leukocyte protein (SLP76), T-cell receptor-associated transmembrane adaptor 1 (TRAT1), TNFR2, TNF receptor superfamily member 14 (TNFRS14; HVEM), TNF receptor superfamily member 18 (TNFRS18; GITR), TNF receptor superfamily member 25 (TNFRS25; DR3), and the zeta chain of T-cell receptor-associated protein kinase 70 (ZAP70).

[0200] In certain embodiments, the CD33 DARIC signaling components contemplated herein include a signal peptide. Examples of signal peptides suitable for use in particular CD33 DARIC signaling components include, but are not limited to, an IgG1 heavy chain signal polypeptide, an Igκ light chain signal polypeptide, a CD8α signal polypeptide, or a human GM-CSF receptor alpha signal polypeptide. In various preferred embodiments, the CD33 DARIC signaling component includes a CD8α signal polypeptide.

[0201] In certain embodiments, the CD33 DARIC signaling component comprises one or more costimulatory signaling domains selected from the group consisting of CD28, CD137, and CD134. In certain embodiments, the CD33 DARIC signaling component comprises one or more costimulatory signaling domains selected from the group consisting of CD28, CD137, and CD134 and a CD3ζ primary signaling domain. In certain embodiments, the CD33 DARIC signaling component comprises a CD137 costimulatory domain and a CD3ζ primary signaling domain.

[0202] In a preferred embodiment, the CD33 DARIC signaling component contains the FRB T2098L multimerization domain, the CD8α transmembrane domain, the CD137 costimulatory domain, and the CD3ζ primary signaling domain.

[0203] In a preferred embodiment, the CD33 VHH DARIC signaling component comprises the amino acid sequence set forth in SEQ ID NO:82.

[0204] 2.CD33 DARIC binding component "DARIC-binding component," "DARIC-binding polypeptide," "CD33 VHH The terms "DARIC binding component" or "CD33 VHH DARIC binding polypeptide" are used interchangeably and refer to a polypeptide comprising an anti-CD33 VHH domain and one or more multimerization domains. In certain embodiments, a CD33 VHH DARIC binding component comprises an anti-CD33 VHH domain, a multimerization domain, and a transmembrane domain. In certain embodiments, a CD33 VHH DARIC binding component comprises an anti-CD33 VHH domain, a second multimerization domain, and a second transmembrane domain. In other specific embodiments, a CD33 VHH DARIC binding component comprises an anti-CD33 VHH domain, a multimerization domain, a transmembrane domain, and one or more intracellular signaling domains. In certain embodiments, a CD33 VHH DARIC binding component comprises an anti-CD33 VHH domain, a second multimerization domain, a second transmembrane domain, and a second costimulatory domain.

[0205] In certain embodiments, the CD33 VHH DARIC binding entity comprises one or more anti-CD33 VHH domains.

[0206] In certain embodiments, the anti-CD33 VHH domain is a humanized camelid VHH. In certain embodiments, the anti-CD33 VH domain is a humanized camelid VHH that binds one or more epitopes of full-length CD33 (e.g., SEQ ID NO: 1) or one or more epitopes of a CD33 splice variant. In certain embodiments, the anti-CD33 VHH domain is a humanized camelid VHH that binds the same one or more epitopes expressed on both full-length CD33 and a CD33 splice variant.

[0207] In a particularly preferred embodiment, the anti-CD33 VHH domain is a humanized camelid-like VHH comprising the amino acid sequence set forth in any one of SEQ ID NOs: 3 to 6, 10 to 11, 13 to 16, and 20 to 21. In a particularly preferred embodiment, the anti-CD33 VHH domain is a humanized camelid-like VHH comprising the amino acid sequence set forth in SEQ ID NOs: 10. In a particularly preferred embodiment, the anti-CD33 VHH domain is a humanized camelid-like VHH comprising the amino acid sequence set forth in SEQ ID NOs: 20.

[0208] In certain embodiments, the DARIC binding entity comprises one or more multimerization domains.

[0209] Examples of multimerization domains suitable for use in the specific CD33 VHH DARIC binding components contemplated herein include, but are not limited to, an FKBP polypeptide or variant thereof, an FRB polypeptide or variant thereof, a calcineurin polypeptide or variant thereof, a cyclophilin polypeptide or variant thereof, a DHFR polypeptide or variant thereof, a PYL1 polypeptide or variant thereof, and an ABI1 polypeptide or variant thereof.

[0210] In certain embodiments, the CD33 VHH DARIC binding component comprises an FRB polypeptide or a variant thereof, and the DARIC signaling component comprises an FKBP polypeptide or a variant thereof. In preferred embodiments, the CD33 VHH DARIC binding component comprises an FRB polypeptide or a variant thereof comprising a T2098L mutation, and the DARIC signaling component comprises an FKBP12 polypeptide or a variant thereof.

[0211] In certain embodiments, the CD33 VHH DARIC binding component comprises an FKBP polypeptide or a variant thereof, and the DARIC signaling component comprises an FRB polypeptide or a variant thereof. In preferred embodiments, the CD33 VHH DARIC binding component comprises an FKBP12 polypeptide or a variant thereof, and the DARIC signaling component comprises an FRB polypeptide or a variant thereof comprising a T2098L mutation.

[0212] In some embodiments, the CD33 VHH DARIC binding transduction component comprises a hinge domain.

[0213] Exemplary hinge domains suitable for use in the CD33 VHH DARIC signal-binding components described herein include hinge regions derived from the extracellular regions of type 1 membrane proteins such as CD28, CD8α and CD4, which may be the wild-type hinge regions of these molecules or may be modified.

[0214] In certain embodiments, the DARIC binding component comprises a transmembrane domain. In certain embodiments, the DARIC binding transduction component comprises a hinge domain and a transmembrane domain. In one embodiment, the transmembrane domain may be the same as the transmembrane domain used in the DARIC signaling component. In one embodiment, the transmembrane domain may be different from the transmembrane domain used in the DARIC signaling component.

[0215] Illustrative examples of transmembrane domains suitable for use in the specific CD33 VHH DARIC binding components contemplated herein include, but are not limited to, the transmembrane regions of the alpha, beta, gamma, or delta chains of the T cell receptor, CD3ε, CD3ζ, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD71, CD80, CD86, CD134, CD137, CD152, CD154, amnionless (AMN), and programmed cell death 1 (PDCD1). In a preferred embodiment, the CD33 DARIC binding component comprises a CD8α transmembrane domain. In a preferred embodiment, the CD33 DARIC binding component comprises a CD4 transmembrane domain.

[0216] In various preferred embodiments, a short oligopeptide or polypeptide linker, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in length, connects the transmembrane domain and the intracellular signaling domain. Glycine-serine based linkers provide particularly suitable linkers.

[0217] DARIC binding entities contemplated in certain embodiments herein include one or more intracellular signaling domains.

[0218] In other specific embodiments, the CD33 VHH DARIC binding entities contemplated herein comprise one or more intracellular signaling domains. In preferred embodiments in which a CD33 VHH DARIC binding entity comprises one or more intracellular signaling domains, these domains are distinct from the intracellular signaling domains present in the cognate CD33 DARIC signaling entity. In one embodiment, a CD33 VHH DARIC binding entity comprises a costimulatory signaling domain.

[0219] Examples of costimulatory domains suitable for use in the specific CD33 VHH DARIC binding transduction components contemplated herein include, but are not limited to, domains isolated from the following costimulatory molecules: Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, caspase recruitment domain family member 11 (CARD11), CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD94, CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), DNAX. Activation protein 10 (DAP10), linker for activation of T-cell family member 1 (LAT), 76 kD SH2 domain-containing leukocyte protein (SLP76), T-cell receptor-associated transmembrane adaptor 1 (TRAT1), TNFR2, TNF receptor superfamily member 14 (TNFRS14; HVEM), TNF receptor superfamily member 18 (TNFRS18; GITR), TNF receptor superfamily member 25 (TNFRS25; DR3), and the zeta chain of T-cell receptor-associated protein kinase 70 (ZAP70). In a preferred embodiment, the costimulatory domain is derived from, obtained from, or isolated from TNFR2 or OX40.

[0220] In certain embodiments, the DARIC binding components contemplated herein include a signal peptide. Examples of signal peptides suitable for use with specific CD33 VHH DARIC binding components include, but are not limited to, an IgG1 heavy chain signal polypeptide, an Igκ light chain signal polypeptide, a CD8α signal polypeptide, or a human GM-CSF receptor alpha signal polypeptide. In various preferred embodiments, the CD33 VHH DARIC binding component contains a CD8α signal polypeptide.

[0221] In a specific embodiment, the CD33 VHH DARIC binding entity contains a VHH domain that binds to CD33, an FKBP12 multimerization domain, a CD4 transmembrane domain, and optionally a costimulatory domain.

[0222] In certain embodiments, the CD33 VHH DARIC binding component comprises a VHH that binds to CD33 and an FKBP12 multimerization domain.

[0223] In some embodiments, the CD33 VHH DARIC binding component comprises a VHH domain comprising the amino acid sequence set forth in SEQ ID NOs: 2-21, an FKBP12 multimerization domain, and a CD4 transmembrane domain, and optionally a costimulatory domain.

[0224] In some embodiments, the CD33 VHH DARIC binding component comprises a VHH domain comprising the amino acid sequence set forth in SEQ ID NO: 10 or SEQ ID NO: 20, an FKBP12 multimerization domain, and a CD4 transmembrane domain, and optionally a costimulatory domain.

[0225] In some embodiments, the CD33 VHH DARIC binding entity comprises a VHH domain comprising the amino acid sequence set forth in any one of SEQ ID NOs: 2-21, and an FKBP12 multimerization domain.

[0226] In a specific embodiment, the CD33 VHH DARIC binding entity comprises a VHH domain comprising the amino acid sequence set forth in SEQ ID NO: 10 or SEQ ID NO: 20, and an FKBP12 multimerization domain.

[0227] In some embodiments, the CD33 VHH DARIC binding member comprises the amino acid sequence set forth in any one of SEQ ID NOs: 22-31.

[0228] In some embodiments, the CD33 VHH DARIC binding component comprises the amino acid sequence set forth in SEQ ID NO:30.

[0229] 3. Crosslinking factor In certain embodiments contemplated herein, a cross-linking agent mediates or promotes the association of a CD33 DARIC signaling component with a CD33 VHH DARIC binding component through the multimerization domains in each component. The cross-linking agent is associated with and positioned between the multimerization domains to promote the association of a CD33 DARIC signaling component with a CD33 VHH DARIC binding component. In the presence of the cross-linking agent, the CD33 VHH DARIC binding component and the CD33 DARIC signaling component associate, initiating immune effector cell activity against the target cell when the CD33 VHH DARIC binding component binds to CD33 expressed on the target cell. In the absence of the cross-linking agent, the CD33 VHH DARIC binding component does not associate with the CD33 DARIC signaling component, and the CD33 VHH DARIC is inactive.

[0230] In certain embodiments, the CD33 DARIC signaling component and the CD33 VHH DARIC binding component 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 PYR1-like 1 (PYL1) and abscisic acid insensitive 1 (ABI 1).

[0231] In one embodiment, the multimerization domains of the CD33 VHH DARIC signaling component and the DARIC binding component are associated with a cross-linking agent 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 a synthetic ligand (SLF) of trimethoprim (Tmp)-FK506 binding protein (FKBP) or a derivative thereof.

[0232] In certain embodiments, the CD33 DARIC signaling component and the CD33 VHH DARIC binding component comprise one or more FRB and / or FKBP multimerization domains, or variants thereof. In certain embodiments, the CD33 DARIC DARIC signaling component contains an FRB multimerization domain, or variant thereof, and the CD33 VHH DARIC binding component comprises an FKBP multimerization domain, or variant thereof. In certain preferred embodiments, the CD33 DARIC signaling component comprises an FRB T2098L multimerization domain, or variant thereof, and the CD33 VHH DARIC binding component comprises an FKBP12 or FKBP12 F36V multimerization domain, or variant thereof.

[0233] Examples of cross-linking agents suitable for use in certain embodiments contemplated herein include, but are not limited to, AP1903, AP20187, AP21967 (also known as C-16-(S)-7-methylindole rapamycin), everolimus, novolimus, pimecrolimus, ridaforolimus, tacrolimus, temsirolimus, umirolimus, and zotarolimus. In certain preferred embodiments, the cross-linking agent is AP21967. In certain preferred embodiments, the cross-linking agent is a non-immunosuppressive dose of sirolimus (rapamycin).

[0234] D. Anti-CD33 Chimeric Antigen Receptor In certain embodiments, the immune effector cells contemplated herein are anti-CD33 Chimeric antigen receptors (CARs) include VHH CARs. Chimeric antigen receptors (CARs) are molecules that combine the specificity of an antibody system for a target antigen (e.g., a tumor antigen) with a T cell receptor activating intracellular domain to generate chimeric proteins that exhibit specific anti-tumor cell-mediated immune activity. As used herein, the term "chimera" refers to something that is composed of different protein or DNA portions derived from different sources.

[0235] In certain embodiments, T cells are engineered by introducing a polynucleotide encoding an anti-CD33 VHH CAR.

[0236] In certain embodiments, T cells are engineered by introducing a vector comprising a polynucleotide encoding an anti-CD33 VHH CAR.

[0237] In various embodiments, the anti-CD33 CAR comprises a VHH domain that binds to CD33, a transmembrane domain, and one or more intracellular signaling domains. A key feature of CARs is their ability to redirect the specificity of immune effector cells, eliciting the production of molecules that can mediate proliferation, cytokine production, phagocytosis, or cell death of target antigen-expressing cells in a major histocompatibility complex (MHC)-independent manner, eliciting the cell-specific targeting capabilities of monoclonal antibodies, soluble ligands, or cell-specific co-receptors.

[0238] In some embodiments, the anti-CD33 VHH CAR comprises a spacer domain. In certain embodiments, the spacer domain comprises the CH2 and CH3 of IgG1, IgG4, or IgD.

[0239] Exemplary hinge domains suitable for use in the anti-CD33 VHH CARs described herein include hinge regions derived from the extracellular regions of type 1 membrane proteins such as CD28, CD8α, and CD4, which may be the wild-type hinge regions of these molecules or may be modified. In another embodiment, the hinge domain contains a CD8α hinge region.

[0240] The transmembrane (TM) domain of the CAR connects the extracellular binding moiety and the intracellular signaling domain and anchors the CAR to the cell membrane of the immune effector cell. The TM domain may be derived from either natural, synthetic, semi-synthetic, or recombinant sources.

[0241] Exemplary TM domains can be derived from (i.e., include) at least the transmembrane region of the alpha, beta, gamma, or delta chain of the T cell receptor, CD3ε, CD3ζ, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD71, CD80, CD86, CD134, CD137, CD152, CD154, AMN, and PDCD1.

[0242] In one embodiment, the anti-CD33 VHH CAR comprises a TM domain derived from CD8α. In another embodiment, the CARs contemplated herein contain a TM domain derived from CD8α and a short oligo- or polypeptide linker, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in length, which links the TM domain and the intracellular signaling domain of the CAR. A glycine-serine linker provides a particularly suitable linker.

[0243] In a preferred embodiment, the anti-CD33 VHH CAR comprises an intracellular signaling domain comprising one or more costimulatory signaling domains and a primary signaling domain.

[0244] The primary stimulatory signaling domain may contain signaling motifs known as immunoreceptor tyrosine-based activation motifs or ITAMs.

[0245] Exemplary ITAM-containing primary signaling domains suitable for use in anti-CD33 VHH CARs contemplated in certain embodiments include those derived from FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d. In particularly preferred embodiments, the CAR comprises a CD3ζ primary signaling domain and one or more costimulatory signaling domains. The intracellular primary signaling domain and the costimulatory signaling domain can be linked in tandem to the carboxyl terminus of the transmembrane domain in any order.

[0246] In certain embodiments, the anti-CD33 VHH CAR comprises one or more costimulatory signaling domains to enhance the efficacy and expansion of T cells expressing the CAR receptor.

[0247] Illustrative examples of such costimulatory molecules suitable for use in anti-CD33 VHH CARs contemplated in certain embodiments include, but are not limited to, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD94, CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), DAP10, LAT, SLP76, TRAT1, TNFR2, and ZAP70. In one embodiment, the CAR comprises one or more costimulatory signaling domains selected from the group consisting of CD28, CD137, and CD134, and a CD3ζ primary signaling domain.

[0248] In various embodiments, the anti-CD33 VHH CAR comprises a VHH that binds to CD33, a transmembrane domain isolated from a polypeptide selected from the group consisting of CD4, CD8α, CD154, and PD-1, one or more intracellular costimulatory signaling domains isolated from a polypeptide selected from the group consisting of CD28, CD134, and CD137, and a signaling domain isolated from a polypeptide selected from the group consisting of FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d.

[0249] In various embodiments, the anti-CD33 VHH CAR comprises a VHH that binds to CD33, a transmembrane domain isolated from a polypeptide selected from the group consisting of CD4, CD8α, CD154, and PD-1, one or more intracellular costimulatory signaling domains isolated from a polypeptide selected from the group consisting of CD28, CD134, and CD137, and a signaling domain isolated from a polypeptide selected from the group consisting of FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d.

[0250] In a preferred embodiment, the anti-CD33 VHH CAR comprises a VHH comprising the amino acid sequence set forth in any one of SEQ ID NOs: 2 to 21, a CD8α hinge domain, a CD8α transmembrane domain, a 4-1BB costimulatory domain, and a CD3ζ primary signaling domain.

[0251] In a preferred embodiment, the anti-CD33 VHH CAR comprises a VHH comprising the amino acid sequence set forth in SEQ ID NO: 10, a CD8α hinge domain, a CD8α transmembrane domain, a 4-1BB costimulatory domain, and a CD3ζ primary signaling domain.

[0252] In a specific embodiment, the anti-CD33 VHH CAR comprises a sequence set forth in any one of SEQ ID NOs: 62-81.

[0253] In certain embodiments, the anti-CD33 VHH CAR comprises the sequence set forth in SEQ ID NO: 70 or SEQ ID NO: 80.

[0254] E. Polypeptides Various polypeptides are contemplated herein, including, but not limited to, CD33 VHH DARIC, CD33 VHH DARIC binding components, CD33 VHH DARIC signaling components, anti-CD33 VHH CARs, and fragments thereof. In preferred embodiments, the polypeptide comprises the amino acid sequence set forth in any one of SEQ ID NOS: 2-82. The terms "polypeptide," "peptide," and "protein" are used interchangeably and follow their conventional meanings unless otherwise specified, i.e., as used in reference to amino acid sequences. In one embodiment, "polypeptide" includes fusion polypeptides and other variants. Polypeptides can be prepared using any of a variety of known recombinant and / or synthetic techniques. Polypeptides are not limited to a particular length. For example, polypeptides may contain the full-length protein sequence, fragments of the full-length protein, or fusion proteins. Polypeptides may include post-translational modifications of the polypeptide, such as glycosylation, acetylation, phosphorylation, and other natural and non-natural modifications known in the art. In certain preferred embodiments, fusion polypeptides, polypeptides, fragments, and other variants thereof are prepared, obtained, or isolated from one or more human polypeptides.

[0255] As used herein, "isolated peptide" or "isolated polypeptide" or the like refers to the in vitro isolation and / or purification of a peptide or polypeptide molecule from its cellular environment and from association with other components of a cell, i.e., substantially free from association with in vivo substances. In certain embodiments, the isolated polypeptide is a synthetic polypeptide, a semi-synthetic polypeptide, or a polypeptide obtained or derived from a recombinant source.

[0256] Polypeptides include "polypeptide variants." Polypeptide variants may differ from naturally occurring polypeptides by one or more substitutions, deletions, additions, and / or insertions. Such variants may be natural, e.g., splice variants, or may be synthetically produced, e.g., by modifying one or more of the polypeptide sequences described above. For example, in certain embodiments, it may be desirable to improve the binding affinity and / or other biological properties of a polypeptide by introducing one or more substitutions, deletions, additions, and / or insertions into the polypeptide. In certain embodiments, polypeptides include those 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, and typically, such variants retain at least one biological activity of the reference sequence. In certain embodiments, the biological activity is binding affinity. In certain embodiments, the biological activity is cytolytic activity.

[0257] Polypeptide variants include biologically active "polypeptide fragments." Examples of biologically active polypeptide fragments include anti-CD33 VHH domains, intracellular signaling domains, and the like. As used herein, the terms "biologically active fragment" or "minimal biologically active fragment" refer 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 activity of the native polypeptide. In certain embodiments, a polypeptide fragment may contain an amino acid chain of at least 5 to about 1700 amino acids in length. In certain embodiments, the fragment comprises 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 in length.

[0258] In certain embodiments, the polypeptides described herein may comprise one or more amino acids designated as "X" or "Xaa," which are used interchangeably. When present in an amino acid SEQ ID NO:, "X" refers to any one or more amino acids. In certain embodiments, a SEQ ID NO: representing a fusion protein contains a sequence of consecutive X residues that cumulatively represent any amino acid sequence. In certain embodiments, "XX" represents any two amino acid combinations. In certain embodiments, "XX" represents two serine residues, SS. In certain embodiments, "XX" represents any two amino acid combinations that reduce immunogenicity.

[0259] In a preferred embodiment, "XX" represents the amino acid KP.

[0260] As mentioned above, polypeptides may be modified in various ways, including amino acid substitution, deletion, truncation, and insertion. Methods for such manipulations are generally known in the art. For example, amino acid sequence variants of a reference polypeptide can be generated by mutations in DNA. Methods for mutagenesis and nucleotide sequence modification are known in the art. See, for example, Kunkel (1985, Proc. Natl. Acad. Sci. USA. 82:488-492), Kunkel et al., (1987, Methods in Enzymol, 154:367-382), U.S. Patent No. 4,873,192, Watson, J.D. et al., (Molecular Biology of See, for example, the Gene, Fourth Edition, Benjamin / Cummings, Menlo Park, Calif., 1987), and the references cited therein. Guidance regarding appropriate amino acid substitutions that do not affect the biological activity of the protein of interest can be found in the model Dayhoff et al., (1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, DC).

[0261] In some embodiments, a polypeptide variant contains one or more conservative substitutions. A "conservative substitution" is one in which an amino acid is replaced with another amino acid with similar properties, and one skilled in the art of peptide chemistry would predict that such a substitution would not substantially alter the secondary structure and hydrophilic properties of the polypeptide. In certain embodiments, modifications may be made to the expected polynucleotide and polypeptide structures while still obtaining functional molecules encoding variant or derivative polypeptides with desired characteristics. If it is desired to alter the amino acid sequence of a polypeptide to generate an equivalent or improved variant polypeptide, one skilled in the art can change one or more codons in the encoding DNA sequence, for example, according to Table 1. [Table 1]

[0262] Guidance for determining which amino acid residues can be substituted, inserted, or deleted without abolishing biological activity can be found using computer programs known in the art, such as DNASTAR, DNA Strider, Geneious, MacVector, or Vector NTI software. Preferably, the amino acid changes in the protein variants disclosed herein are conservative amino acid changes, i.e., substitutions of similarly charged or uncharged amino acids. Conservative amino acid changes include substitutions of members of a family of amino acids that are related by their side chains. Natural amino acids are generally divided into four families: acidic amino acids (aspartic acid, glutamic acid), basic amino acids (lysine, arginine, histidine), nonpolar amino acids (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged polar amino acids (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine). Phenylalanine, tryptophan, and tyrosine are sometimes collectively classified as aromatic amino acids. In peptides or proteins, suitable conservative substitutions of amino acids are known to those skilled in the art and can generally be made without altering the biological activity of the resulting molecule. Those skilled in the art recognize that single amino acid substitutions in non-essential regions of a polypeptide generally do not significantly alter biological activity (see, for example, Watson et al., Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin / Cummings Pub. Co., p. 224).

[0263] In one embodiment, when expression of more than one polypeptide is desired, the polynucleotide sequences encoding them may be separated by an IRES sequence or polynucleotide sequences as otherwise disclosed herein.

[0264] In certain embodiments, contemplated polypeptides include fusion polypeptides. In certain embodiments, fusion polypeptides and polynucleotides encoding the fusion polypeptides are provided. Fusion polypeptides and fusion proteins refer to polypeptides having at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more polypeptide segments. In preferred embodiments, the fusion polypeptide comprises one or more CD33 VHH DARIC components. In other preferred embodiments, the fusion polypeptide comprises one or more CD33 VHH DARIC components.

[0265] In another embodiment, two or more CD33 VHH DARIC components and / or polypeptides may be expressed as a fusion protein comprising one or more self-cleaving peptide sequences between the polypeptides disclosed elsewhere herein.

[0266] In certain embodiments, the fusion polypeptide comprises a CD33 DARIC signaling component, a self-cleaving polypeptide sequence or a ribosomal skipping sequence, and a CD33 VHH Contains a DARIC binding component.

[0267] In certain embodiments, the fusion polypeptide comprises a CD33 DARIC signaling component, a self-cleaving polypeptide sequence or ribosomal skipping sequence, a CD33 VHH DARIC binding component, another self-cleaving polypeptide sequence or ribosomal skipping sequence, and another DARIC binding component directed against another target antigen.

[0268] Fusion polypeptides may contain one or more polypeptide domains or segments, including, but not limited to, signal peptides, cell permeable peptide domains (CPPs), binding domains, signaling domains, epitope tags (e.g., maltose binding protein (MBP), glutathione S-transferase (GST), HIS6, MYC, FLAG, V5, VSV-G, and HA), polypeptide linkers, and polypeptide cleavage signals. Fusion polypeptides are typically linked C-terminally to N-terminally, but can also be linked C-terminally to C-terminally, N-terminally to N-terminally, or M-terminally to C-terminally. In certain embodiments, the polypeptides of the fusion protein may be in any order. Fusion polypeptides or fusion proteins may include conservatively modified variants, polymorphic variants, alleles, mutants, subsequences, and interspecies homologs, provided that the desired activity of the fusion polypeptide is preserved. Fusion polypeptides may be made by chemical synthesis or by chemical conjugation between two moieties, or may be generally prepared using other standard methods. The ligated DNA sequence comprising the fusion polypeptide is operably linked to suitable transcriptional or translational control elements as otherwise disclosed herein.

[0269] Fusion polypeptides may optionally contain one or more linkers, which can be used to link 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 allow each polypeptide to fold into its appropriate secondary and tertiary structure, thereby enabling the polypeptide domain to perform its desired function. Such peptide linker sequences are incorporated into fusion polypeptides using standard techniques in the art. An appropriate peptide linker sequence may be selected based on the following factors: (1) the ability to adopt a flexible, extended conformation; (2) the ability to avoid secondary structures that could interact with functional epitopes on the first and second polypeptides; and (3) the absence of hydrophobic or charged residues that could interact with the functional epitopes of the polypeptides. In certain embodiments, preferred peptide linker sequences contain Gly, Asn, and Ser residues. Other closely related neutral amino acids, such as Thr and Ala, may also be used in linker sequences. Amino acid sequences that can be usefully employed as linkers include those described in Maratea et al., Gene 40:39-46, 1985; Murphy et al., Proc. Natl. Acad. Sci. USA 83:8258-8262, 1986; U.S. Pat. Nos. 4,935,233 and 4,751,180. A linker sequence is not necessary if a particular fusion polypeptide segment contains a non-essential N-terminal amino acid region that can be used to separate the functional domains and prevent steric interference. In certain embodiments, preferred linkers are typically flexible amino acid subsequences that are synthesized as part of the recombinant fusion protein. Linker polypeptides can be 1 to 200 amino acids in length, 1 to 100 amino acids in length, or 1 to 50 amino acids in length, including all integer values ​​between those values.

[0270] Examples of 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).

[0271] Suitable protease cleavage sites and self-cleaving peptides are known to those skilled in the art (see, for example, Ryan et al., 1997. J. Gener. Virol. 78, 699-722; Scymczak et al. (2004) Nature Biotech. 5, 589-594). Examples of protease cleavage sites include, but are not limited to, cleavage sites for potyvirus NIa protease (e.g., tobacco etch virus protease), potyvirus HC protease, potyvirus P1 (P35) protease, byovirus NIa protease, biovirus RNA-2-encoded protease, aphthovirus L protease, enterovirus 2A protease, rhinovirus 2A protease, picorna 3C protease, comovirus 24K protease, nepovirus 24K protease, RTSV (Waika virus) 3C-like protease, PYVF (parsnip yellow mottle virus) 3C-like protease, heparin, thrombin, factor Xa, and enterokinase. Due to their high cleavage stringency, in one embodiment TEV (tobacco etch virus) protease cleavage sites, e.g., EXXYXQ(G / S) (SEQ ID NO: 95), such as ENLYFQG (SEQ ID NO: 96) and ENLYFQS (SEQ ID NO: 97), are preferred, where X represents any amino acid (TEV cleavage occurs between Q and G or between Q and S).

[0272] In certain embodiments, the polypeptide cleavage signal is a viral self-cleaving peptide or a ribosomal skipping sequence.

[0273] Examples of ribosomal skipping sequences include, but are not limited to, 2A or 2A-like sites, sequences, or domains (see Donnelly et al., 2001, J. Gen. Virol. 82:1027-1041). In certain embodiments, the viral 2A peptide is an aphthovirus 2A peptide, a potyvirus 2A peptide, or a cardiovirus 2A peptide.

[0274] In one embodiment, the viral 2A peptide is selected from the group consisting of a foot-and-mouth disease virus (FMDV) 2A peptide, an equine rhinitis A virus (ERAV) 2A peptide, a zosea signavirus (TaV) 2A peptide, a porcine teschovirus-1 (PTV-1) 2A peptide, a tylovirus 2A peptide, and an encephalomyocarditis virus 2A peptide.

[0275] Examples of 2A sites are provided in Table 2. [Table 2]

[0276] In a preferred embodiment, the polypeptide or fusion polypeptide comprises one or more CD33 VHH DARIC components, CD33 VHH DARIC or anti-CD33 VHH CAR.

[0277] In a preferred embodiment, the fusion polypeptide comprises a CD33 DARIC signaling component and a CD33 VHH DARIC binding component separated by a self-cleaving polypeptide sequence.

[0278] In certain embodiments, the fusion polypeptide comprises the sequence set forth in any one of SEQ ID NOs: 32 to 61. In certain embodiments, the fusion polypeptide comprises the sequence set forth in any one of SEQ ID NOs: 40, 50, or 60.

[0279] In certain embodiments, the fusion polypeptide comprises a FRB T2098L multimerization domain, a CD8α transmembrane domain, a CD33 DARIC signaling component comprising a CD137 costimulatory domain and a CD3ζ primary signaling domain, a viral self-cleaving 2A polypeptide, and a CD33 VHH DARIC binding component comprising an anti-CD33, an FKBP12 multimerization domain polypeptide, and a CD4 transmembrane domain.

[0280] In certain embodiments, the fusion polypeptide comprises the sequence set forth in any one of SEQ ID NOs: 32 to 41. In certain embodiments, the fusion polypeptide comprises the sequence set forth in SEQ ID NO: 40.

[0281] In certain embodiments, the fusion polypeptide comprises a FRB T2098L multimerization domain, a CD8α transmembrane domain, a CD33 DARIC signaling component comprising a CD137 costimulatory domain and a CD3ζ primary signaling domain, a viral self-cleaving 2A polypeptide, and an anti-CD33 VHH, a CD4 transmembrane domain, and optionally a CD27, CD28, TNFRS14, TNFRS18, TNFRS25, OX40, or TNFR2 costimulatory domain.

[0282] In certain embodiments, the fusion polypeptide comprises the sequence set forth in any one of SEQ ID NOs: 42 to 51. In certain embodiments, the fusion polypeptide comprises the sequence set forth in SEQ ID NO: 50.

[0283] In certain embodiments, the fusion polypeptide comprises the sequence set forth in any one of SEQ ID NOs: 52 to 61. In certain embodiments, the fusion polypeptide comprises the sequence set forth in SEQ ID NO: 60.

[0284] F. Polynucleotides In certain embodiments, polynucleotides are provided that encode CD33 VHH DARIC, CD33 VHH DARIC binding components, CD33 DARIC signaling components, anti-CD33 VHH CARs, and fragments thereof. As used herein, the term "polynucleotide" or "nucleic acid" refers to deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and DNA / RNA hybrids. Polynucleotides may be single-stranded or double-stranded, and may be 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. Polynucleotide refers to a polymeric form of nucleotides having a length 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, including ribonucleotides or deoxyribonucleotides, or modified forms of either type of nucleotide, as well as all intermediate lengths. In this context, "intermediate length" will be readily understood to mean any length between the recited values, such as 6, 7, 8, 9, etc., 101, 102, 103, etc., 151, 152, 153, etc., 201, 202, 203, etc. In certain embodiments, a polynucleotide or variant has 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.

[0285] As used herein, "isolated polynucleotide" refers to a polynucleotide that has been purified from sequences that naturally flank it, e.g., a DNA fragment that has been removed from sequences that normally flank it. In certain embodiments, "isolated polynucleotide" also refers to a complementary DNA (cDNA), recombinant DNA, or other polynucleotide that is not found in nature and has been created by man. In certain embodiments, an isolated polynucleotide is a synthetic polynucleotide, a semi-synthetic polynucleotide, or a polynucleotide obtained or derived from a recombinant source.

[0286] In various embodiments, the polynucleotide comprises an mRNA encoding a polypeptide contemplated herein, hi some embodiments, the mRNA comprises a cap, one or more nucleotides, and a poly(A) tail.

[0287] In certain embodiments, a polynucleotide encoding one or more CD33 VHH DARIC antibodies may be codon-optimized. As used herein, the term "codon-optimized" refers to the substitution of codons in a polynucleotide encoding a polypeptide to improve 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 in codon bias 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 with context; (iv) variation of codons with their decoding tRNAs; (v) variation of codons with GC % either overall or at any single position in a triplet; (vi) variation in the degree of similarity to a reference sequence, e.g., a naturally occurring sequence; (vii) variation in codon frequency cutoff; (viii) structural properties of mRNA transcribed from a DNA sequence; (ix) prior knowledge of the function of the DNA sequence on which the design of the codon substitution set is based; (x) systematic variation of the codon set for each amino acid; and / or (xi) isolated removal of spurious translation start sites.

[0288] As used herein, the term "nucleotide" refers to a heterocyclic nitrogenous base in N-glycosidic linkage with a phosphorylated sugar. Nucleotide is understood to include natural bases and a wide variety of modified bases recognized in the art. Such bases are generally located at the 1' position of the nucleotide sugar moiety. Nucleotides generally contain a base, a sugar, and a phosphate group. In ribonucleic acid (RNA), the sugar is ribose, and in deoxyribonucleic acid (DNA), the sugar is deoxyribose. That is, deoxyribose is a sugar lacking the hydroxyl group present in ribose.

[0289] Examples of polynucleotides include, but are not limited to, polynucleotides that encode the polypeptides set forth in SEQ ID NOs: 2-82.

[0290] In various embodiments, polynucleotides contemplated herein include, but are not limited to, polynucleotides encoding one or more CD33 VHH DARIC components, CD33 VHH DARIC receptors, anti-CD33 VHH CARs, fusion polypeptides, expression vectors, viral vectors, and transfer plasmids comprising the polynucleotides contemplated herein.

[0291] As used herein, terms such as "polynucleotide variant" and "variant" refer to a polynucleotide that exhibits substantial sequence identity with reference polynucleotide sequence, or hybridizes with reference sequence under stringent conditions as defined herein.These terms also include polynucleotides that are distinguished from reference polynucleotides by the addition, deletion, substitution or modification of at least one nucleotide.Therefore, the terms "polynucleotide variant" and "variant" include polynucleotides in which one or more nucleotides are added, deleted, or modified, or replaced with another nucleotide.In this regard, it is understood in the art that certain modifications, including mutations, additions, deletions and substitutions, can be made to reference polynucleotides, and modified polynucleotides can retain the biological function or biological activity of reference polynucleotides.

[0292] As used herein, "sequence identity," or phrases such as "a sequence 50% identical to," refers to the degree to which sequences are identical nucleotide-by-nucleotide or amino acid-by-amino acid over a comparison window. Thus, "percentage of sequence identity" can be calculated by: comparing two optimally aligned sequences over a comparison window; determining the number of positions where the same nucleic acid base (e.g., A, T, C, G, I) or the same amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) is present in both sequences; calculating the number of matching positions; dividing the number of matching positions by the total number of positions in the comparison window (i.e., window size); and multiplying the result by 100 to calculate the percentage of sequence identity. Included are 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.

[0293] Polynucleotides contemplated herein, regardless of the length of the coding sequence itself, may be combined with other DNA sequences disclosed elsewhere herein or known in the art, such as promoters and / or enhancers, untranslated regions (UTRs), signal sequences, Kozak sequences, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, internal ribosome entry sites (IRES), recombinase recognition sites (e.g., LoxP, FRT, and Att sites), stop codons, transcription termination signals, and polynucleotides encoding self-cleaving polypeptides, epitope tags, etc., so that their overall length may vary considerably. It is therefore expected that polynucleotide fragments of almost any length may be employed, with the overall length preferably being limited by ease of preparation and use in the intended recombinant DNA protocol.

[0294] Polynucleotides may be prepared, manipulated, expressed, and / or delivered using any of a variety of established techniques known and available in the art. To express a desired polypeptide, a nucleotide sequence encoding the polypeptide may be inserted into an appropriate vector.

[0295] Examples of vectors include, but are not limited to, plasmids, autonomously replicating sequences, and transposable elements, such as Sleeping Beauty and PiggyBac.

[0296] Additional examples of vectors include, but are not limited to, plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs), bacteriophages such as lambda phage or M13 phage, and animal viruses.

[0297] Examples of viruses useful as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papilloma viruses, and papovaviruses (e.g., SV40).

[0298] Exemplary expression vectors include, but are not limited to, pClneo vector (Promega) for expression in mammalian cells, and pLenti4 / V5-DEST™, pLenti6 / V5-DEST™, and pLenti6.2 / V5-GW / lacZ (Invitrogen) for lentiviral-mediated gene transfer and expression in mammalian cells. In certain embodiments, the coding sequences for the polypeptides disclosed herein may be ligated into such expression vectors for expression of the polypeptides in mammalian cells.

[0299] In certain embodiments, the vector is an episomal vector, or a vector that is maintained extrachromosomally. As used herein, the term "episomal" refers to a vector that can replicate without being integrated into the chromosomal DNA of a host, and is not gradually reduced with the division of the host cell, which also means that the vector replicates extrachromosomally or episomally.

[0300] "Expression control sequences," "control elements," or "regulatory sequences" present in an expression vector are untranslated regions of the vector, including origins of replication, selection cassettes, promoters, enhancers, translation initiation signals (Shine Dalgarno or Kozak sequences), introns, polyadenylation sequences, and 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. Depending on the vector system and host utilized, any number of suitable transcription and translation elements, including ubiquitous and inducible promoters, may be used.

[0301] In certain embodiments, the polynucleotide comprises a vector, including, but not limited to, an expression vector and a viral vector. The vector may contain one or more exogenous, endogenous, or heterologous regulatory sequences, such as promoters and / or enhancers. An "endogenous regulatory sequence" is a sequence naturally linked to a given gene in the genome. An "exogenous regulatory sequence" is a sequence placed in juxtaposition to a gene by genetic engineering (i.e., molecular biological techniques), such that transcription of the gene is induced by the linked enhancer / promoter. A "heterologous regulatory sequence" is an exogenous sequence derived from a species different from the cell being genetically engineered. A "synthetic" regulatory sequence may contain one or more endogenous and / or exogenous sequence elements, and / or sequence elements determined in vitro or in silico, to provide optimal promoter and / or enhancer activity for a particular therapy.

[0302] As used herein, the term "promoter" refers to a recognition site in a polynucleotide (DNA or RNA) to which an RNA polymerase binds. The RNA polymerase initiates transcription of a polynucleotide operably linked to the promoter. In certain embodiments, promoters that operate in mammalian cells contain an AT-rich region located approximately 25-30 bases upstream from the site where transcription is initiated, and / or a separate sequence, a CNCAAT region, located 70-80 bases upstream from the transcription start site, where N can be any nucleotide.

[0303] The term "enhancer" refers to a DNA segment containing a sequence that can provide enhanced transcription, and in some cases can function regardless of orientation relative to another regulatory sequence. Enhancers can function cooperatively or additively with promoter elements and / or other enhancer elements. The term "promoter / enhancer" refers to a DNA segment containing a sequence that can provide both promoter and enhancer functions.

[0304] The term "operably linked" refers to a juxtaposition wherein the described components are in a relationship permitting them to function in their intended manner. In one embodiment, the term refers to the functional linkage between a nucleic acid expression control sequence (e.g., a promoter and / or enhancer) and a second polynucleotide sequence, e.g., a polynucleotide of interest, where the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence.

[0305] As used herein, the term "structural expression control sequence" refers to a promoter, enhancer, or promoter / enhancer that permits continuous or sequential transcription of an operably linked sequence. A structural expression control sequence may be a "ubiquitous" promoter, enhancer, or promoter / enhancer that permits expression in a variety of cell and tissue types, or it may be a "cell-specific," "cell type-specific," "cell line-specific," or "tissue-specific" promoter, enhancer, or promoter / enhancer that permits expression in restricted cell and tissue types, respectively.

[0306] Examples of ubiquitous expression control sequences suitable for use in certain embodiments include, but are not limited to, the cytomegalovirus (CMV) immediate early promoter, the viral Simian Virus 40 (SV40) (e.g., early or late), Moloney Murine Leukemia Virus (MoMLV) LTR promoter, Rous Sarcoma Virus (RSV) LTR, herpes simplex virus (HSV) (thymidine kinase) promoter, the H5, P7.5, and P11 promoters from vaccinia virus, elongation factor 1 alpha (EF1a) promoter, early growth response protein 1 (EGR1), ferritin H (FerH), ferritin L (FerL), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), eukaryotic translation initiation factor 4A1 (EIF4A1), heat shock 70 kDa protein 5 (HSPA5), heat shock protein 90 kDa, and the like. beta, member 1 (HSP90B1), heat shock protein 70 kDa (HSP70), beta-kinesin (β-KIN), human ROSA 26 locus (Irions et al., Nature Biotechnology 25, 1477-1482 (2007)), ubiquitin C promoter (UBC), phosphoglycerate kinase-1 (PGK) promoter, cytomegalovirus enhancer / chicken beta-actin (CAG) promoter, beta-actin promoter and myeloproliferative sarcoma virus enhancer, negative control region deleted, dl587rev primer binding site substitution (MND) U3 promoter (Haas et al. Journal of Virology. 2003;77(17):9439-9450).

[0307] In one embodiment, the vector contains the MNDU3 promoter.

[0308] In one embodiment, the vector contains the EF1a promoter including the first intron of the human EF1a gene.

[0309] In one embodiment, the vector contains the EF1a promoter lacking the first intron of the human EF1a gene.

[0310] In certain embodiments, it may be desirable to use cell-, cell type-, cell line-, or tissue-specific expression control sequences to achieve cell-type-, cell line-, or tissue-specific expression of a desired polynucleotide sequence (e.g., expressing a nucleic acid encoding a particular polypeptide only in a subset of cell types, cell lines, or tissues, or only at a particular stage of development).

[0311] In certain embodiments, it may be desirable to express the polynucleotide from a T cell specific promoter.

[0312] As used herein, "conditional expression" can refer to any type of conditional expression, including, but not limited to, inducible expression, repressible expression, and expression in cells or tissues that are in a particular physiological, biological, or disease state. This definition is not intended to exclude cell-type or tissue-specific expression. Certain embodiments provide for conditional expression of a polynucleotide of interest, e.g., expression controlled by exposing a cell, tissue, or organism to a treatment or condition that results in expression of the polynucleotide or that increases or decreases expression of a polypeptide encoded by the polynucleotide of interest.

[0313] Examples of inducible promoters / systems include, but are not limited to, steroid-inducible promoters, such as promoters of genes encoding glucocorticoid receptors or estrogen receptors (inducible by treatment with the corresponding hormones), metallothionein promoters (inducible by treatment with various heavy metals), MX-1 promoters (inducible by interferon), and the "GeneSwitch" mifepristone-regulated system (Sirin et al. al., 2003, Gene, 323:67), cumate-inducible gene switches (WO2002 / 088346), and tetracycline-dependent regulatory systems. Inducers include, but are not limited to, glucocorticoids, estrogens, mifepristone (RU486), metals, interferons, small molecules, cumate, tetracycline, doxycycline, and variants thereof.

[0314] As used herein, "internal ribosome entry site" or "IRES" refers to a factor that promotes direct entry of an internal ribosome at an initiation codon, such as ATG, of a cistron (protein-coding region), resulting in cap-independent gene translation. See, e.g., Jackson et al., 1990. Trends Biochem. Sci 15(12):477-83 and Jackson and Kaminski. 1995. RNA 1(10):985-1000. Examples of IRES commonly employed by those skilled in the art include those described in U.S. Pat. No. 6,692,736. Further examples of "IRES" known in the art include, but are not limited to, IRES obtainable from picornaviruses (Jackson et al., 1990), and IRES obtainable from viral or cellular mRNA sources, such as immunoglobulin heavy chain binding protein (BiP), vascular endothelial growth factor (VEGF) (Huez et al. 1998. Mol. Cell. Biol. 18(11):6178-6190), fibroblast growth factor 2 (FGF-2), and insulin-like growth factor (IGFII), the translation initiation factor eIF4G, and the yeast transcription factors TFIID and HAP4, and the IRES from encephalomyocarditis virus (EMCV) (Duke, available from Novagen). et al., 1992. J. Virol 66(3):1602-9) and VEGF IRES (Huez et al., 1998. Mol Cell Biol 18(11):6178-90). IRESs have also been reported in viral genomes of species from the Picornaviridae, Dicistroviridae, and Flaviviridae families, as well as in HCV, Friend murine leukemia virus (FrMLV), and Moloney murine leukemia virus (MoMLV).

[0315] In one embodiment, the IRES used in the polynucleotides contemplated herein is the EMCV IRES.

[0316] In certain embodiments, the polynucleotide comprises a consensus Kozak sequence. As used herein, the term "Kozak sequence" refers to a short nucleotide sequence that significantly promotes the initial binding of mRNA to the small ribosomal subunit, increasing 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).

[0317] Factors that induce efficient termination and polyadenylation of heterologous nucleic acid transcripts increase heterologous gene expression. Transcription termination signals are generally located downstream of polyadenylation signals. In certain embodiments, vectors contain a polyadenylation sequence 3' to the polynucleotide encoding the polypeptide to be expressed. As used herein, the terms "polyA site" or "polyA sequence" refer to a DNA sequence that induces both termination and polyadenylation of the nascent RNA transcript by RNA polymerase II. Polyadenylation sequences promote mRNA stability by adding a polyA tail to the 3' end of the coding sequence, thereby contributing to enhanced translation efficiency. Cleavage and polyadenylation are induced by poly(A) sequences in the RNA. The core poly(A) sequence of mammalian pre-mRNAs has two recognition factors flanking the cleavage-polyadenylation site. Typically, a nearly invariant AAUAAA hexamer is present 20-50 nucleotides upstream of a more variable factor rich in U or GU residues. Cleavage of the initial transcript occurs between these two elements, adding up to 250 adenosines to the 5' cleavage product. In certain embodiments, the core poly(A) sequence is a poly(A) sequence of choice (e.g., AATAAA, ATTAAA, AGTAAA). In certain embodiments, the poly(A) sequence is the SV40 poly(A) sequence, bovine growth hormone poly(A) sequence (BGHpA), rabbit β-globin poly(A) sequence (rβgpA), variants thereof, or another suitable heterologous or endogenous poly(A) sequence known in the art. In certain embodiments, the poly(A) sequence is synthetic.

[0318] In certain embodiments, polynucleotides encoding one or more polypeptides or fusion polypeptides may be introduced into immune effector cells, such as T cells, by both non-viral and viral methods. In certain embodiments, delivery of one or more polynucleotides may be provided by the same or different methods and / or on the same or different vectors.

[0319] The term "vector" is used herein to refer to a nucleic acid molecule capable of transferring or transporting another nucleic acid molecule. The transferred nucleic acid is generally inserted into, for example, a vector nucleic acid molecule. A vector may contain sequences that direct autonomous replication within a cell, or may contain sequences sufficient to allow integration into host cell DNA. In certain embodiments, non-viral vectors are used to deliver one or more polynucleotides contemplated herein to T cells.

[0320] Examples of non-viral vectors include, but are not limited to, plasmids (eg, DNA or RNA plasmids), transposons, cosmids, and bacterial artificial chromosomes.

[0321] Non-viral methods of polynucleotide delivery contemplated in certain embodiments include, but are not limited to, electroporation, sonoporation, lipofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, nanoparticles, polycation or lipid:nucleic acid complexes, naked DNA, artificial virions, DEAE-dextran mediated transfer, gene guns, and heat shock.

[0322] Examples of polynucleotide delivery systems suitable for use in certain contemplated embodiments include, but are not limited to, systems provided by Amaxa Biosystems, Maxcyte, Inc., BTX Molecular Delivery Systems, and Copernicus Therapeutics Inc. Lipofection reagents are commercially available (e.g., Transfectam™ and Lipofectin™). Cationic and neutral lipids 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 et al. (2011) Journal of of Drug Delivery. 2011:1-12. Antibody-targeted delivery, bacteria-directed delivery, and non-biological nanocell-based delivery are also contemplated in certain embodiments.

[0323] In certain embodiments, viral vectors containing contemplated polynucleotides can be delivered in vivo by administration to an individual patient, typically by systemic administration (e.g., intravenous, intraperitoneal, intramuscular, subcutaneous, or intracranial infusion) or local application as described below. Alternatively, vectors can be delivered ex vivo to cells, such as cells explanted from an individual patient (e.g., mobilized peripheral blood, lymphocytes, bone marrow aspirate, tissue biopsy, etc.) or hematopoietic stem cells of a universal donor, followed by reimplantation of the cells into the patient.

[0324] In one embodiment, a viral vector containing a polynucleotide contemplated herein is administered directly to an organism and transduced into cells in vivo. Alternatively, naked DNA may be administered. Administration may be by any of the routes commonly used to introduce molecules into blood or tissue cells for ultimate contact, including, but not limited to, injection, infusion, topical application, and electroporation. While suitable methods for administering such nucleic acids are available and known to those skilled in the art, multiple routes may be used to administer a particular composition, and certain routes can often provide a more immediate and effective response than other routes.

[0325] Examples of viral vector systems suitable for use in certain contemplated embodiments include, but are not limited to, adeno-associated virus (AAV), retrovirus, herpes simplex virus, adenovirus, and vaccinia virus vectors.

[0326] In various embodiments, one or more polynucleotides encoding one or more CD33 VHH DARIC components and / or other polypeptides contemplated herein are introduced into immune effector cells, such as T cells, by transducing the cells with a recombinant adeno-associated virus (rAAV) containing the one or more polynucleotides.

[0327] AAV is a small (approximately 26 nm), replication-incompetent, primarily episomal, non-enveloped virus. AAV can infect both dividing and non-dividing cells and may integrate its genome into the genome of the host cell. Recombinant AAV (rAAV) typically consists, at a minimum, of a transgene and its regulatory sequences, as well as 5' and 3' AAV inverted terminal repeats (ITRs). The ITR sequences are approximately 145 bp in length. In a specific embodiment, the rAAV contains ITRs and capsid sequences isolated from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10.

[0328] In some embodiments, chimeric rAAVs are used. The ITR sequences are isolated from one AAV serotype, and the capsid sequence is isolated from another AAV serotype. For example, an rAAV containing ITR sequences from AAV2 and a capsid sequence from AAV6 is called AAV2 / AAV6. In certain embodiments, the rAAV vector may contain ITRs from AAV2 and a capsid protein from any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10. In a preferred embodiment, the rAAV contains ITR sequences from AAV2 and a capsid sequence from AAV6. In a preferred embodiment, the rAAV contains ITR sequences from AAV2 and a capsid sequence from AAV2.

[0329] In some embodiments, AAV capsids may be subjected to methods of engineering and selection to increase the probability that they will transduce cells of interest.

[0330] The construction, production, and purification of rAAV vectors are disclosed, for example, in U.S. Patent Nos. 9,169,494, 9,169,492, 9,012,224, 8,889,641, 8,809,058, and 8,784,799, which are incorporated herein by reference in their entireties.

[0331] In various embodiments, one or more polynucleotides encoding one or more CD33 VHH DARIC components and / or other polypeptides contemplated herein are introduced into immune effector cells, e.g., T cells, by transducing the cells with a retrovirus, e.g., a lentivirus, containing the one or more polynucleotides.

[0332] As used herein, the term "retrovirus" refers to an RNA virus that reverse transcribes its genomic RNA into a linear double-stranded DNA copy and then covalently integrates its genomic DNA into the host genome. Examples of retroviruses suitable for use in certain embodiments include, but are not limited to, Moloney murine leukemia virus (M-MuLV), Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus (HaMuSV), mouse 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.

[0333] As used herein, the term "lentivirus" refers to a group (or genus) of complex retroviruses. Examples of lentiviruses include, but are not limited to, HIV (including human immunodeficiency virus, HIV type 1 and HIV type 2), Visna-Maedi virus (VMV), Caprine Arthritis-Encephalitis Virus (CAEV), Equine Infectious Anemia Virus (EIAV), Feline Immunodeficiency Virus (FIV), Bovine Immunodeficiency Virus (BIV), and Simian Immunodeficiency Virus (SIV). In one embodiment, an HIV-based vector backbone (i.e., HIV cis-acting sequence elements) is preferred.

[0334] In various embodiments, lentiviral vectors contemplated herein contain one or more LTRs and one or more or all of the following accessory elements: cPPT / FLAP, Psi (Ψ) packaging signal, export element, poly(A) sequence, and optionally, a WPRE or HPRE, an insulator element, a selectable marker, and a cell suicide gene, as otherwise discussed herein.

[0335] In certain embodiments, the lentiviral vector contemplated herein can be an integrative, non-integrative, or integration-defective lentivirus.As used herein, the term "integration-defective lentivirus" or "IDLV" refers to a lentivirus that has an integrase that is deficient in integrating viral genome into host cell genome.Integration-defective viral vectors are described in patent application WO2006 / 010834, which is incorporated herein by reference in its entirety.

[0336] Examples of mutations in the HIV-1 pol gene that are suitable for reducing 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, D116 N, D1161, D116A, N120G, N1201, N120E, E152G, E152A, D35E, K156E, K156A, E 157A, K159E, K159A, K160A, R166A, D167A, E170A, H171A, K173A, K186Q, K1 86T, K188T, E198A, R199c, R199T, R199A, D202A, K211A, Q214L, Q216L, Q221 L, W235F, W235E, K236S, K236A, K246A, G247W, D253A, R262A, R263A and K264H.

[0337] The term "long terminal repeat (LTR)" refers to a domain of base pairs located at the end of retroviral DNA; in the native sequence, the LTR is a direct repeat and contains the U3, R and U5 regions.

[0338] As used herein, the term "FLAP element" or "cPPT / FLAP" refers to a nucleic acid whose sequence includes the central polypurine tract and central termination sequences (cPPT and CTS) of retroviruses such as HIV-1 and HIV-2. Suitable FLAP elements are described in U.S. Patent No. 6,682,907 and Zennou, et al., 2000, Cell, 101:173.

[0339] As used herein, the term "packaging signal" or "packaging sequence" refers to the psi [Ψ] sequence located in the retroviral genome, which is required for the insertion of viral RNA into the viral capsid or viral particle. See, for example, Clever et al., 1995. J. of Virology, Vol. 69, No. 4; pp. 2101-2109.

[0340] The term "export factor" refers to a cis-acting post-transcriptional regulatory element that controls the transport of RNA transcripts from the nucleus to the cytoplasm of a cell. Examples of RNA export factors include, but are not limited to, the rev-responsive element (RRE) of human immunodeficiency virus (HIV) (see, e.g., Cullen et al., 1991. J. Virol. 65:1053; and Cullen et al., 1991. Cell 58:423) and the post-transcriptional regulatory element (HPRE) of hepatitis B virus.

[0341] In certain embodiments, expression of heterologous sequences in viral vectors is increased by incorporating post-transcriptional regulatory elements, efficient polyadenylation sites, and optionally, transcription termination signals into the vector. Various post-transcriptional regulatory elements can increase the expression of heterologous nucleic acids into proteins. For example, the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE; Zufferey et al., 1999, J. Virol., 73:2886); post-transcriptional regulatory element (HPRE) present in hepatitis B virus (Huang et al., Mol. Cell. Biol., 5:3864); and the like (Liu et al., 1995, Genes Dev., 9:1766).

[0342] Lentiviral vectors preferably contain several safety enhancements as a result of the LTR modifications. A "self-inactivating" (SIN) vector refers to a vector lacking replication competence, such as a retroviral or lentiviral vector, in which the right (3') LTR enhancer-promoter region, known as the U3 region, has been modified (e.g., by deletion or substitution) to inhibit viral transcription beyond the first round of viral replication. Self-inactivation is preferably achieved by introducing a deletion into the U3 region of the 3' LTR of the vector DNA, i.e., the DNA used to generate vector RNA. This deletion is then transferred to the 5' LTR of the proviral DNA during reverse transcription. In certain embodiments, it is desirable to remove the U3 sequence sufficiently to greatly reduce or completely abolish the transcriptional activity of the LTR, thereby greatly reducing or abolishing the production of full-length vector RNA in transduced cells. In the case of HIV-based lentivectors, it has been shown that such vectors can tolerate large U3 deletions, including removal of the LTR TATA box (e.g., deletion of -418 to -18), without significant loss of vector titer.

[0343] Additional safety enhancements are provided by replacing the U3 region of the 5'LTR with a heterologous promoter that drives transcription of the viral genome during viral particle production. Examples of heterologous promoters that can be used include Simian Virus 40 (SV40) (e.g., early or late), cytomegalovirus (CMV) (e.g., immediate early), Moloney Murine Leukemia Virus (MoMLV), Rous Sarcoma Virus (RSV), and Herpes Simplex Virus (HSV) (thymidine kinase) promoters.

[0344] As used herein, the term "pseudotype" or "pseudotyped" refers to a virus whose viral envelope protein has been replaced with the envelope of another virus that has favorable properties. For example, the HIV envelope protein (encoded by the env gene) is normally encoded by the CD4 + Although targeting the virus to presentation cells, pseudotyping HIV with the vesicular stomatitis virus G-protein (VSV-G) envelope protein allows HIV to infect a broad range of cells.

[0345] In one embodiment, lentiviral vectors are produced by known methods, see, e.g., Kutner et al., BMC Biotechnol. 2009;9:10. doi:10.1186 / 1472-6750-9-10; Kutner et al. Nat. Protoc. 2009;4(4):495-505. doi:10.1038 / nprot.2009.22.

[0346] According to certain embodiments contemplated herein, most or all of the viral vector backbone sequences are derived from lentiviruses, such as HIV-1. However, it should be understood that many different sources of retroviral and / or lentiviral sequences can be used, or that numerous combined substitutions and modifications of a particular lentiviral sequence can be accommodated without impairing the transfer vector's ability to perform the functions described herein. Furthermore, a variety of lentiviral vectors are known in the art. See Naldini et al., (1996a, 1996b, and 1998); Zufferey et al., (1997); Dull et al., 1998, U.S. Patent Nos. 6,013,516 and 5,994,136. Many of these can be adapted to produce the viral vectors or transfer plasmids contemplated herein.

[0347] In various embodiments, one or more polynucleotides encoding one or more CD33 VHH DARIC components and / or other polypeptides contemplated herein are introduced into immune effector cells by transducing the cells with an adenovirus containing the one or more polynucleotides.

[0348] Adenovirus-based vectors can transduce many cell types with very high efficiency and do not require cell division. Such vectors can achieve high titers and high levels of expression. They can be produced in large quantities using a relatively simple system. Most adenovirus vectors are engineered so that the transgene replaces the Ad E1a, E1b, and / or E3 genes. The resulting replication-deficient vectors are amplified in human 293 cells, which then supply the missing gene function in trans. Ad vectors can transduce multiple tissue types in vivo, including non-dividing and differentiated cells, such as those found in the liver, kidney, and muscle. Conventional Ad vectors have a large carrying capacity.

[0349] The generation and amplification of current replication-deficient adenoviral vectors can utilize a unique helper cell line called 293, which is derived from human embryonic kidney cells and expresses Ad5 Adenoviral vectors have been transformed with DNA fragments and constitutively express the E1 protein (Graham et al., 1977). Because the E3 region of the adenoviral genome is not essential (Jones & Shenk, 1978), current adenoviral vectors, supported by 293 cells, carry foreign DNA in the E1, D3, or both regions (Graham & Prevec, 1991). Adenoviral vectors have been used to express eukaryotic genes (Levrero et al., 1991; Gomez-Foix, 1992). Recombinant adenoviruses have been used in various tissues for the development of vaccines (Grunhaus & Horwitz, 1992; Graham & Prevec, 1992). Ad vectors have been administered intramuscularly (i.e., intravenously) (Herz & Gerard, 1993), intravenously (i.e., intravenous injection) (Herz & Gerard, 1993), and stereotactically (i.e., intracerebral inoculation) (Le Gal La Salle et al., 1993). Clinical trials using Ad vectors include intramuscular polynucleotide therapy for antitumor immunization (Sterman et al., Hum. Gene Ther. 7:1083-9 (1998)).

[0350] In various embodiments, one or more polynucleotides encoding one or more CD33 VHH DARIC components and / or other polypeptides contemplated herein are introduced into immune effector cells by transducing the cells with a herpes simplex virus, e.g., HSV-1 or HSV-2, containing the one or more polynucleotides.

[0351] Mature HSV virions consist of an enveloped icosahedral capsid containing a viral genome consisting of a 152 kb linear double-stranded DNA molecule. In one embodiment, the HSV-based viral vector lacks one or more essential or non-essential HSV genes. In one embodiment, the HSV-based viral vector is replication-defective. Most replication-defective HSV vectors contain deletions that remove one or more intermediate-early, early, or late HSV genes, inhibiting replication. For example, the HSV vector may lack an immediate-early gene selected from the group consisting of ICP4, ICP22, ICP27, ICP47, and combinations thereof. The advantages of HSV vectors are their ability to enter late stages and generate long-term DNA expression, and their large viral DNA genome, which can accommodate exogenous DNA inserts of up to 25 kb. HSV-based vectors are described, for example, in U.S. Pat. Nos. 5,837,532, 5,846,782, and 5,804,413, and International Patent Applications WO91 / 02788, WO96 / 04394, WO98 / 15637, and WO99 / 06583, which are incorporated herein by reference in their entireties.

[0352] G. Genetically Modified Cells In various embodiments, cells are modified to express a CD33 VHH DARIC, one or more CD33 VHH DARIC components, an anti-CD33 VHH CAR, and / or a fusion protein contemplated herein for use in cancer therapy. Cells may be non-genetically modified to express one or more of the polypeptides contemplated herein, or in certain preferred embodiments, cells may be genetically modified to express one or more of the polypeptides contemplated herein. As used herein, the terms "genetically engineered" or "genetically modified" refer to the addition of additional genetic material in the form of DNA or RNA to the total genetic material in a cell. The terms "genetically modified cell," "modified cell," and "redirected cell" are used interchangeably in certain embodiments.

[0353] In certain embodiments, one or more CD33 VHH DARIC components or anti-CD33 VHH CARs contemplated herein are introduced into and expressed in immune effector cells to improve the efficacy of the immune effector cells.

[0354] An "immune effector cell" is any cell of the immune system that has one or more effector functions (e.g., cytotoxic cell-killing activity, cytokine secretion, induction of ADCC and / or CDC, etc.). Exemplary immune effector cells contemplated herein are T lymphocytes, including, but not limited to, cytotoxic T cells (CTL; ​​CD8 + T cells), TILs, and helper T cells (HTLs; CD4 + In certain embodiments, the cells include αβ T cells. In certain embodiments, the cells include γδ T cells. In one embodiment, the immune effector cells include natural killer (NK) cells. In one embodiment, the immune effector cells include natural killer T (NKT) cells. Immune effector cells may be self or non-self (e.g., allogeneic, syngeneic, or xenogeneic).

[0355] As used herein, "autologous" refers to cells derived from the same subject. As used herein, "allogeneic" refers to cells of the same species that are genetically different from the compared cells. As used herein, "syngeneic" refers to cells of a different subject that are genetically identical to the compared cells. As used herein, "xenogeneic" refers to cells of a different species from the compared cells. In a preferred embodiment, the cells are human autoimmune effector cells.

[0356] Examples of immune effector cells suitable for the introduction of one or more CD33 VHH DARIC components or anti-CD33 VHH CARs contemplated herein include T lymphocytes. The term "T cell" or "T lymphocyte" is art-recognized and is intended to include thymocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes. T cells may be T helper (Th) cells, such as T helper 1 (Th1) cells or T helper 2 (Th2) cells. T cells may be helper T cells (HT1; CD4 + T cells), cytotoxic T cells (CTL:CD8 + T cells), CD4 + CD8 + T cells, CD4 - CD8 - The T cells may be T cells or any other subset of T cells. In certain embodiments, the T cells express a T cell receptor. A T cell receptor contains two subunits, an alpha and beta chain subunit (αβ TCR) or a gamma and delta chain subunit (γδ TCR), each of which is a unique protein generated by a recombination event in each T cell's genome. In certain embodiments, the T cells are αβ TCR T cells (αβ T cells). In certain embodiments, the T cells are γδ TCR T cells (γδ T cells). Other examples of T cell populations suitable for use in certain embodiments include naive T cells and memory T cells.

[0357] As will be understood by those skilled in the art, other cells can also be used as immune effector cells comprising one or more CD33 VHH DARIC components or anti-CD33 VHH CARs contemplated herein. In certain embodiments, immune effector cells also include NK cells, NKT cells, neutrophils, and macrophages. Immune effector cells also include precursors of effector cells, where such precursor cells may be induced to differentiate into immune effector cells in vivo or in vitro. Thus, in certain embodiments, immune effector cells include precursors of immune effector cells, such as hematopoietic stem cells (HSCs) contained within the CD34+ population of cells derived from, for example, umbilical cord blood, bone marrow, or mobilized peripheral blood, which differentiate into mature immune effector cells when administered to a subject or may be induced to differentiate into mature immune effector cells in vitro.

[0358] As used herein, the term "CD34+ cells" refers to cells that express CD34 protein on their cell surface. As used herein, "CD34" refers to a cell surface glycoprotein (e.g., sialomucin protein) that often acts as a cell-cell adhesion factor and is involved in the entry of T cells into lymph nodes. CD34+ cell populations contain hematopoietic stem cells (HSCs), which, when administered to patients, differentiate and give rise to all hematopoietic lineages, including T cells, NK cells, NKT cells, neutrophils, and cells of the monocyte / macrophage lineage.

[0359] A method for generating immune effector cells expressing one or more CD33 VHH DARIC components or anti-CD33 VHH CARs contemplated herein is presented in certain embodiments. In one embodiment, the method comprises transfecting or transducing immune effector cells isolated from an individual, wherein the immune effector cells have one or more nucleic acids and / or vectors, e.g., lentiviral vectors, encoding one or more CD33 VHH DARIC components or anti-CD33 VHH CARs contemplated herein. In one embodiment, the method comprises transfecting or transducing immune effector cells isolated from an individual, wherein the immune effector cells express one or more CD33 VHH DARIC components or anti-CD33 VHH CARs contemplated herein. In certain embodiments, the immune effector cells are isolated from an individual and genetically modified without further in vitro manipulation. Such cells may then be directly readministered to the individual. In a further embodiment, the immune effector cells are first activated in vitro and stimulated to proliferate before being genetically modified. In this regard, the immune effector cells may be cultured before and / or after being genetically modified.

[0360] In certain embodiments, a source of cells is obtained from a subject prior to in vitro manipulation or genetic modification of the immune effector cells described herein, hi certain embodiments, the modified immune effector cells comprise T cells.

[0361] T cells can be obtained from many sources, including, but not limited to, peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In one embodiment, T cells can be obtained from a unit of blood drawn from a subject using any number of techniques known to those skilled in the art, such as sedimentation, for example, FICOLL™ separation.

[0362] In other embodiments, isolated or purified T cell populations are used. In some embodiments, after isolation of PBMCs, both cytotoxic and helper T lymphocytes may be sorted into naive, memory, and effector T cell subpopulations before or after activation, expansion, and / or genetic modification.

[0363] In one embodiment, the isolated or purified T cell population is CD3 + , CD4 + , CD8 + The cells express one or more of the markers, including, but not limited to, α- and β-actin, ...

[0364] In one embodiment, T cells are isolated from an individual and first activated and stimulated in vitro to expand and then engineered to express one or more CD33 VHH DARIC components or anti-CD33 VHH CARs.

[0365] To obtain a sufficient therapeutic dose of a T cell composition, T cells often undergo one or more rounds of stimulation, activation, and / or expansion. In certain embodiments, T cells may generally be activated and expanded using methods described, for example, in U.S. Patent Nos. 6,352,694, 6,534,055, 6,905,680, 6,692,964, 5,858,358, 6,887,466, 6,905,681, 7,144,575, 7,067,318, 7,172,869, 7,232,566, 7,175,843, 5,883,223, 6,905,874, 6,797,514, and 6,867,041. These patents are incorporated herein by reference in their entirety. In certain embodiments, T cells are activated and expanded for about 6 hours, about 12 hours, about 18 hours, or about 24 hours, and then transfected with a vector or polynucleotide encoding one or more CD33 VHH DARIC components or anti-CD33 VHH CARs as contemplated herein.

[0366] H. Compositions and Formulations Compositions contemplated herein may include one or more CD33 VH DARIC constructs or anti-CD33 VHH CARs, polynucleotides encoding one or more CD33 VH DARIC constructs or anti-CD33 VHH CARs, vectors comprising the above, genetically modified immune effector cells, cross-linking agents, etc. Compositions include, but are not limited to, pharmaceutical compositions. A "pharmaceutical composition" refers to a composition formulated for administration to a cell or animal in a pharmaceutically or physiologically acceptable solution, alone or in combination with one or more other therapeutic modalities. It should also be understood that, if desired, the composition may be administered in combination with other agents, such as cytokines, growth factors, hormones, small molecules, chemotherapeutic agents, prodrugs, drugs, antibodies, or various other pharmaceutically active agents. There is virtually no limit to the other components that may be included in the composition, provided that the added agents do not adversely affect the ability of the composition to deliver the intended therapy.

[0367] As used herein, the phrase "pharmaceutically acceptable" is employed to refer to those compounds, substances, compositions and / or dosage forms that 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.

[0368] The term "pharmaceutically acceptable carrier" refers to a diluent, adjuvant, excipient, or vehicle with which a cross-linking agent, polypeptide, polynucleotide, vector containing the same, or genetically modified immune effector cells is administered. Examples of pharmaceutical carriers may 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, and sesame oil. Saline solutions and dextrose and glycerol solutions may also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients in certain embodiments 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.

[0369] In one embodiment, the composition comprising a pharmaceutically acceptable carrier is suitable for administration to a subject. In certain embodiments, the composition comprising a carrier is suitable for parenteral administration, such as intravascular (intravenous or intraarterial), intraperitoneal, or intramuscular administration. In certain embodiments, the composition comprising a pharmaceutically acceptable carrier is suitable for intracerebroventricular, intrathecal, 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 known in the art. Except insofar as any conventional media or agent is incompatible with the cross-linking agent, polypeptide, polynucleotide, vector containing same, or genetically modified immune effector cells, its use in the pharmaceutical composition is contemplated.

[0370] In certain embodiments, the compositions contemplated herein comprise genetically modified T cells and a pharmaceutically acceptable carrier. Compositions containing the cell-based compositions contemplated herein may be administered enterally or parenterally, separately, or in combination with other appropriate compounds to achieve a desired therapeutic purpose.

[0371] In certain embodiments, compositions contemplated herein comprise a cross-linking agent and a pharmaceutically acceptable carrier.

[0372] Pharmaceutically acceptable carriers must be of sufficiently high purity and sufficiently low toxicity to be suitable for administration to the human subject being treated. They must also maintain or enhance the stability of the composition. Pharmaceutically acceptable carriers may be liquid or solid and are selected to provide the desired volume and concentration when mixed with the other components of the composition, taking into account the intended mode of administration. For example, pharmaceutically acceptable carriers may be, but are not limited to, binders (e.g., pregelatinized corn starch, polyvinylpyrrolidone, or hydroxypropylmethylcellulose), fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethylcellulose, polyacrylates, calcium hydrogen phosphate, etc.), lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metal stearates, hydrogenated vegetable oils, corn starch, polyethylene glycol, sodium benzoate, sodium acetate, etc.), disintegrants (e.g., starch, sodium starch glycolate, etc.), or wetting agents (e.g., sodium lauryl sulfate, etc.). Other pharmaceutically acceptable carriers suitable for the compositions contemplated herein include, but are not limited to, water, salt solutions, alcohol, polyethylene glycol, gelatin, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone, etc.

[0373] Such carrier solutions may include buffers, diluents, and other suitable additives. As used herein, the term "buffer" refers to a solution or liquid whose chemical composition neutralizes acids or bases without causing a significant change in pH. Examples of buffers contemplated herein include, but are not limited to, Dulbecco's phosphate buffered saline (PBS), Ringer's solution, 5% dextrose solution (D5W), normal / physiological saline (0.9% NaCl).

[0374] The pharmaceutically acceptable carrier may be present in an amount sufficient to maintain the pH of the composition at about 7. Alternatively, the composition has a pH in the range of about 6.8 to about 7.4, e.g., 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, and 7.4. In yet another embodiment, the composition has a pH of about 7.4.

[0375] The composition anticipated herein may contain a non-toxic pharmaceutically acceptable medium.The composition may be a suspension.As used herein, the term "suspension" refers to a non-adherent state in which cells do not bind to a solid support.For example, cells maintained as a suspension may be stirred or agitated, and do not adhere to a support such as a culture dish.

[0376] In certain embodiments, compositions contemplated herein are formulated in a suspension, wherein the modified T cells are dispersed in an acceptable liquid medium or solution, such as in an intravenous (IV) bag, such as in a saline or serum-free medium. Acceptable diluents include, but are not limited to, water, PlasmaLyte, Ringer's solution, isotonic sodium chloride solution (saline), serum-free cell culture medium, and medium suitable for cryogenic storage, such as Cryostor® medium.

[0377] In certain embodiments, the pharmaceutically acceptable carrier is substantially free of naturally occurring proteins of human or animal origin and is suitable for storing compositions comprising engineered T cell populations. Therapeutic compositions are intended for administration to human patients and therefore are substantially free of cell culture components such as bovine serum albumin, horse serum, and fetal bovine serum.

[0378] In some embodiments, the composition is formulated in a pharmaceutically acceptable cell culture medium. Such compositions are suitable for administration to human subjects. In certain embodiments, the pharmaceutically acceptable cell culture medium is a serum-free medium.

[0379] Serum-free media have several advantages over serum-containing media, including a simpler and more transparent composition, reduced contaminant load, elimination of potential sources of infectious agents, and reduced cost. In various embodiments, serum-free media are animal-free and optionally protein-free. Optionally, the media may contain biopharmaceutical-acceptable recombinant proteins. "Animal-free" media refers to media whose components are derived from non-animal sources. Recombinant proteins replace natural animal proteins in animal-free media, and the nutrients are obtained from synthetic, plant, or microbial sources. "Protein-free" media, in contrast, are defined as being substantially protein-free.

[0380] Examples of serum-free media for use in certain compositions include, but are not limited to, QBSF-60 (Quality Biological, Inc.), StemPro-34 (Life Technologies), and X-VIVO 10.

[0381] In one embodiment, the composition comprising the modified T cells is formulated in PlasmaLyte.

[0382] In various embodiments, compositions comprising modified T cells are formulated in a cryopreservation medium. For example, a cryopreservation medium containing a cryopreservative may be used to maintain high cell viability after thawing. Exemplary cryopreservation media for use in particular compositions include, but are not limited to, CryoStor CS10, CryoStor CS5, and CryoStor CS2.

[0383] In one embodiment, the composition is formulated in a solution comprising 50:50 PlasmaLyte A and CryoStor CS10.

[0384] In certain embodiments, the compositions are substantially free of mycoplasma, endotoxin, and microbial contamination. "Substantially free," with respect to endotoxin, means lower endotoxin per cell dose than permitted by the FDA for biologics, which is 5 EU / kg body weight total endotoxin per day, or 350 EU per total cell dose for an average 70 kg human. In certain embodiments, the 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.

[0385] In certain embodiments, the formulation of pharmaceutically acceptable carrier solutions is known in the art, as is the development of appropriate dosing and treatment regimens for use of the particular compositions described herein in a variety of treatment regimens, including, for example, enteral and parenteral, e.g., intravascular, intravenous, intraarterial, intraosseous, intraventricular, intracerebral, intracranial, intrathecal, intrathecal, and intramedullary administration and formulations. Those of skill in the art will appreciate that certain embodiments contemplated herein are known, for example, in the pharmaceutical arts, and can be found in, for example, Remington: The Science and Practice of Pharmacy, volume I and volume II. 22 ndEdition. Edited by Loyd It will be understood that other formulations may be included, such as those described in V. Allen Jr. Philadelphia, PA: Pharmaceutical Press; 2012, which is incorporated herein by reference in its entirety.

[0386] In certain embodiments, a composition comprises an amount of immune effector cells comprising a polynucleotide encoding one or more CD33 VHH DARIC components or anti-CD33 VHH CARs contemplated herein. In certain embodiments, a composition comprises an amount of immune effector cells expressing one or more CD33 VHH DARIC components or anti-CD33 VHH CARs contemplated herein. As used herein, the term "amount" refers to an "effective amount" or "effective dose" of cells comprising one or more CD33 VHH DARIC components or anti-CD33 VHH CARs contemplated herein that, in the presence of a cross-linking agent, achieves a beneficial or desired prophylactic or therapeutic result, including a clinical result.

[0387] A "prophylactically effective amount" refers to an amount of cells comprising one or more CD33 VHH DARIC components or anti-CD33 VHH CARs contemplated herein that, in the presence of a cross-linking agent, is effective to achieve a desired prophylactic result. Typically, but not necessarily, a prophylactically effective amount is less than the therapeutically effective amount, since a prophylactic dose is used prior to or in subjects at an early stage of disease.

[0388] A "therapeutically effective amount" refers to an amount of cells comprising one or more CD33 VHH DARIC components or anti-CD33 VHH CARs as described herein that is effective in the presence of a cross-linking agent to "treat" a subject (e.g., a patient). When a therapeutic amount is indicated, the exact amount of composition, cells, cross-linking agent, etc. to be administered can be determined by a physician, taking into account individual differences in the patient's (subject's) age, weight, tumor size, extent of infection or metastasis, and condition.

[0389] Generally, pharmaceutical compositions comprising the immune effector cells described herein are administered in a 10 2 ~10 10 cells / kg body weight, preferably 10 5 ~10 6 It can be said that a dose of cells / kg body weight can be administered, including all integer values ​​within the range. The number of cells depends on the intended end use of the composition and the type of cells contained in the composition. For the uses presented herein, the cells are generally in a volume of liter or less, and can be 500 ml or less, or even 250 ml or 100 ml or less. Therefore, the desired cell density is often 10 6 cells / ml, typically greater than 10 7 cells / ml, typically >10 8 Clinically relevant immune cell counts are greater than 10 cells / ml, which may be divided into multiple infusions and cumulatively reach 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 or 10 12 In some embodiments, all infused cells are specifically redirected to a specific target antigen, resulting in more than 10 6 / kilogram (10 per patient) 6 -10 11 ) may be administered.

[0390] If desired, treatment may also include administration of mitogens (e.g., PHA) or lymphokines, cytokines and / or chemokines (e.g., IFN-γ, IL-2, IL-12, TNF-alpha, IL-18, and TNF-beta, GM-CSF, IL-4, IL-13, Flt3-L, RANTES, MIP1α, etc.) as described herein to enhance the induction of an immune response.

[0391] Generally, compositions comprising cells activated and expanded as described herein may be utilized for the treatment and prevention of diseases occurring in immunocompromised individuals. In particular, the compositions contemplated herein are used to treat cancer. In certain embodiments, immune effector cells may be administered alone or in a pharmaceutical composition with a carrier, diluent, excipient, and / or other components, such as IL-2 or other cytokines or other cells.

[0392] In certain embodiments, the pharmaceutical composition comprises a quantity of genetically modified T cells in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients.

[0393] In certain embodiments, a pharmaceutical composition contains an amount of a cross-linking agent in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients.

[0394] In certain embodiments, compositions contain an effective amount of immune effector cells comprising one or more CD33 VHH DARIC components or anti-CD33 VHH CARs contemplated herein, alone or in combination with a cross-linking agent 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. Such therapeutic agents may be art-accepted as standard treatments for certain disease states described herein, such as certain cancers. Examples of contemplated therapeutic agents include cytokines, growth factors, steroids, NSAIDs, DMARDs, anti-inflammatory agents, chemotherapeutic agents, radiation therapy, therapeutic antibodies, or other active adjunctive agents.

[0395] In certain embodiments, a composition comprising an effective amount of immune effector cells comprising a polynucleotide encoding one or more CD33 VHH DARIC components or anti-CD33 VHH CARs contemplated herein is administered to a subject, and a composition comprising an effective amount of a cross-linking agent is administered to the subject prior to, during, in combination with, or following the cell composition, and optionally repeatedly administered to the subject.

[0396] In certain embodiments, compositions comprising immune effector cells comprising polynucleotides encoding one or more CD33 VHH DARIC components or anti-CD33 VHH CARs contemplated herein may be administered in conjunction with any number of anti-inflammatory agents, chemotherapeutic agents, or therapeutic antibiotics, etc.

[0397] I. Treatment method Immune effector cells engineered to express a polynucleotide encoding one or more CD33 VHH DARIC components or anti-CD33 VHH CARs contemplated herein provide improved methods of adoptive immunotherapy for use in the prevention, treatment and amelioration of at least one symptom associated with an immune disorder, e.g., cancer.

[0398] Immune effector cells comprising a CD33 DARIC signaling entity, a CD33 VHH DARIC binding entity, or an anti-CD33 VHH CAR provide improved methods of adoptive immunotherapy or methods of preventing, treating, or ameliorating at least one symptom associated with an immune disorder, e.g., cancer.

[0399] In certain embodiments, immune effector cells engineered to express CD33 VHH DARIC provide an improved adoptive immunotherapy approach to stabilize the safety and efficacy of cytotoxic responses against target cells, e.g., tumor cells, that express the target antigen, while reducing the risk of on-target, off-target cytotoxic activity (recognizing the target antigen on normal, non-target cells).

[0400] In certain embodiments, a method for preventing, treating, or ameliorating at least one symptom of cancer comprises administering to a subject a therapeutically effective amount of engineered immune effector cells or T cells comprising one or more components of a CD33 VHH DARIC or anti-CD33 VHH CAR to redirect the cells to target cells. Genetically engineered cells are a highly effective and safe cellular immunotherapy in that they deliver chemically controllable immunostimulatory signals.

[0401] In certain embodiments, one or more immune effector cells, e.g., T cells, are engineered to express both a CD33 VHH DARIC binding component and a CD33 DARIC signaling component. The engineered cells are then administered to a subject in need thereof and directed to target cells via interaction between the CD33 VHH binding component expressed on the immune effector cells and CD33 expressed on the target cells. A cross-linking agent is administered to the subject before, at about the same time as, or after the engineered cells are administered to the subject. In the presence of the cross-linking agent, a ternary complex is formed between the CD33 VHH DARIC binding component, the cross-linking agent, and the CD33 DARIC signaling component. Upon formation of the ternary complex, the CD33 VHH DARIC transmits an immunostimulatory signal to the immune effector cells, which then initiate a cytotoxic response against the target cells.

[0402] In certain embodiments, one or more immune effector cells, e.g., T cells, are modified to express a CD33 DARIC signaling component. In this case, the modified cells are administered to a subject in need thereof. The CD33 VHH DARIC binding component can be administered to a subject before, approximately simultaneously with, or after the modified cells are administered to a subject. Additionally, the CD33 VHH DARIC binding component can be administered to a subject in a preformed complex with a cross-linking agent, simultaneously with but in a separate composition from the cross-linking agent, or at a different time than the cross-linking agent. The CD33 VHH binding component binds to CD33 expressed on target cells either in the presence or absence of the cross-linking agent. In the presence of the cross-linking agent, a ternary complex is formed between the CD33 VHH DARIC binding component, the cross-linking agent, and the CD33 DARIC signaling component. Upon formation of the ternary complex, CD33 VHH DARIC transmits immunostimulatory signals to immune effector cells, which then elicit a cytotoxic response against target cells.

[0403] In certain embodiments, one or more immune effector cells, e.g., T cells, are modified to express a CD33 DARIC signaling component. In this case, the modified cells are administered to a subject in need thereof. The CD33 VHH DARIC binding component can be administered to a subject before, approximately simultaneously with, or after the modified cells are administered to a subject. In addition, the CD33 VHH DARIC binding component can be administered to a subject in a preformed complex with a cross-linking agent, simultaneously with but in a separate composition from the cross-linking agent, or at a different time than the cross-linking agent. The CD33 binding component binds to a target antigen expressed on a target cell either in the presence or absence of a cross-linking agent. In the presence of a cross-linking agent, a ternary complex is formed between the CD33 VHH DARIC binding component, the cross-linking agent, and the CD33 DARIC signaling component. Upon formation of the ternary complex, the CD33 VHH DARIC transmits immunostimulatory signals to immune effector cells, which then initiate a cytotoxic response against target cells. In certain embodiments, CD33 VHH DARIC activation can be induced when remission or regression is incomplete and the condition relapses or becomes refractory to treatment.

[0404] In certain preferred embodiments, the specificity of primary T cells is redirected against tumor or cancer cells expressing CD33 by genetically modifying T cells, e.g., primary T cells, with one or more CD33 VHH components.

[0405] In certain preferred embodiments, the specificity of primary T cells is redirected to tumor or cancer cells expressing CD33 by genetically modifying the T cells, e.g., primary T cells, with an antigen receptor engineered to redirect them to the target antigen and one or more CD33 VHH DARIC components.

[0406] In certain embodiments, the engineered immune effector cells contemplated herein are used to treat solid tumors or cancers.

[0407] In certain embodiments, the engineered immune effector cells contemplated herein are selected from, but are not limited to, adrenal carcinoma, adrenocortical carcinoma, anal cancer, appendiceal cancer, astrocytoma, atypical teratoid tumor / atypical rhabdomyoid tumor, basal cell carcinoma, bile duct carcinoma, bladder cancer, bone cancer, brain / CNS cancer, breast cancer, bronchial tumor, cardiac tumor, cervical cancer, bile duct carcinoma, chondrosarcoma, chordoma, colon cancer, colorectal cancer, craniopharyngioma, ductal carcinoma in situ (DCIS), endometrial cancer, epithelial carcinoma, esophageal cancer, nasal neuroblastoma, Ewing's sarcoma, cranial Extragonadal germ cell tumor, extragonadal germ cell tumor, eye cancer, fallopian tube cancer, fibrous tissue sarcoma, fibrosarcoma, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), germ cell tumor, glioma, glioblastoma, head and neck cancer, hemangioblastoma, hepatocellular carcinoma, hypopharyngeal cancer, intraocular melanoma, Kaposi's sarcoma, kidney cancer, laryngeal cancer, leiomyosarcoma, tongue cancer, liposarcoma, liver cancer, lung cancer, non-small cell lung cancer, embryonal carcinoid tumor, malignant mesothelioma, medullary carcinoma, medulloblastoma, meningioma, melanoma, Merkel cell carcinoma, midline carcinoma, oral cancer Used to treat solid tumors or cancers, including uterine cancer, myxosarcoma, myelodysplastic syndrome, myeloproliferative neoplasms, cancer of the nasal cavity and paranasal sinuses, nasopharyngeal carcinoma, neuroblastoma, oligodendroglioma, oral cancer (oral cavity cancer), oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, islet cell tumor, papillary carcinoma, paraganglioma, parathyroid carcinoma, penile cancer, pharyngeal carcinoma, pheochromocytoma, pinealoma, pituitary tumor, pleuropulmonary blastoma, primary pleurima, prostate cancer, rectal cancer, retinoblastoma, renal cell carcinoma, cancer of the renal pelvis and ureter, rhabdomyosarcoma, salivary gland cancer, sebaceous gland carcinoma, skin cancer, soft tissue sarcoma, squamous cell carcinoma, small cell lung cancer, small intestine cancer, gastric cancer, sweat gland carcinoma, synovial tumor, testicular cancer, pharyngeal cancer, thymic carcinoma, thyroid cancer, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, vascular cancer, vulvar cancer, and Wilms' tumor

[0408] In certain embodiments, the modified immune effector cells contemplated herein are used in the treatment of solid tumors or cancers, including, but not limited to, non-small cell lung cancer, 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, glioma, glioblastoma, and oligodendroglioma.

[0409] In certain embodiments, the modified immune effector cells contemplated herein are used in the treatment of solid tumors or cancers, including, but not limited to, non-small cell lung cancer, metastatic colorectal cancer, glioblastoma, head and neck cancer, pancreatic cancer, and breast cancer.

[0410] In certain embodiments, the modified immune effector cells contemplated herein are used in the treatment of glioblastoma.

[0411] In certain embodiments, the engineered immune effector cells contemplated herein are used to treat liquid or hematological cancers.

[0412] In certain embodiments, the engineered immune effector cells contemplated herein are used to treat B-cell malignancies, including but not limited to, leukemia, lymphoma, and multiple myeloma.

[0413] In certain embodiments, the engineered immune effector cells contemplated herein are directed against leukemias, lymphomas, and multiple myeloma, including, but not limited to, acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), myeloblastic leukemia, promyelocytic leukemia, myelomonocytic leukemia, monocytic leukemia, erythroleukemia, hairy cell leukemia (HCL), chronic lymphocytic leukemia (CLL), and chronic myelogenous leukemia (CML), chronic myelomonocytic leukemia (CMML), and polycythemia vera, Hodgkin's lymphoma, nodular lymphocyte-predominant Hodgkin's lymphoma, It is used to treat liquid cancers including Burkitt's lymphoma, small lymphocytic lymphoma (SLL), diffuse large B-cell lymphoma, follicular lymphoma, immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, mantle cell lymphoma, marginal zone lymphoma, mycosis fungoides, anaplastic large cell lymphoma, Sézary syndrome, precursor T-lymphoblastic lymphoma, multiple myeloma, overt multiple myeloma, smoldering multiple myeloma, plasma cell leukemia, non-secretory myeloma, IgD myeloma, osteosclerotic myeloma, solitary bone plasmacytoma, and extramedullary plasmacytoma.

[0414] In certain embodiments, the modified immune effector cells contemplated herein are used in the treatment of acute myeloid leukemia (AML).

[0415] Preferred cells for use in the methods contemplated herein include self cells, preferably hematopoietic cells, more preferably T cells, and more preferably immune effector cells.

[0416] In certain embodiments, the methods also include administering a therapeutically effective amount of modified immune effector cells expressing one or more CD33 VHH DARIC components to a patient in need thereof, and administering a cross-linking agent to the subject. In certain embodiments, the cells are used to treat a patient at risk of developing an immune disorder. Accordingly, certain embodiments include treating or preventing or alleviating at least one symptom of an immune disorder, e.g., cancer, comprising administering a therapeutically effective amount of modified immune effector cells and a cross-linking agent as contemplated herein to a subject in need thereof.

[0417] In certain embodiments, the method comprises administering a therapeutically effective amount of modified immune effector cells expressing an anti-CD33 VHH CAR to a patient in need thereof. In certain embodiments, the cells are used to treat a patient at risk of developing an immune disorder. Accordingly, certain embodiments include treating or preventing or alleviating at least one symptom of an immune disorder, such as cancer, comprising administering a therapeutically effective amount of the modified immune effector cells and cross-linking agents contemplated herein to a subject in need of such treatment or prevention or alleviation.

[0418] In certain embodiments, the method comprises administering to a patient in need thereof a therapeutically effective amount of engineered immune effector cells expressing a CD33 DARIC signaling component, or a composition comprising them, and also administering a CD33 VHH DARIC binding component and a cross-linking agent, wherein the CD33 VHH DARIC binding component is conjugated to a cross-linking agent prior to administration to the subject. In certain embodiments, the cells are used to treat a patient at risk of developing an immune disorder. Accordingly, certain embodiments include treating or preventing or alleviating at least one symptom of an immune disorder, e.g., cancer, comprising administering to a subject in need thereof a therapeutically effective amount of engineered immune effector cells expressing a CD33 DARIC signaling component, and optionally an engineered antigen receptor or another DARIC binding component, a CD33 VHH DARIC binding component, and a cross-linking agent.

[0419] The amount and frequency of administration of the modified immune effector cells, CD33 DARIC VHH binding components, and / or cross-linking agents will be determined by factors such as the condition of the patient and the type and severity of the patient's disease, although appropriate doses and administration schedules may be determined through clinical trials.

[0420] In one exemplary embodiment, the effective amount of engineered immune effector cells provided to a subject is at least 2 x 10 6 Cells / kg, at least 3x10 6 cells / kg, at least 4x10 6 Cells / kg, at least 5x10 6 cells / kg, at least 6x10 6 Cells / kg, at least 7x10 6 Cells / kg, at least 8x10 6 Cells / kg, at least 9x10 6 cells / kg, or at least 10x10 6 cells / kg, or more, including all cell doses in between.

[0421] In another exemplary embodiment, the effective amount of engineered immune effector cells provided to a subject is about 2x10 6 cells / kg, approximately 3x10 6 cells / kg, approximately 4x10 6 cells / kg, approximately 5x10 6 cells / kg, approximately 6x10 6 cells / kg, approximately 7x10 6 cells / kg, approximately 8x10 6 cells / kg, approximately 9x10 6 cells / kg, or approximately 10x10 6 cells / kg, or more, including all cell doses in between.

[0422] In another exemplary embodiment, the effective amount of engineered immune effector cells provided to a subject is about 2x10 6 cells / kg ~ approx. 10x10 6 cells / kg, approximately 3x10 6 cells / kg ~ approx. 10x10 6 cells / kg, approximately 4x10 6 cells / kg ~ approx. 10x10 6 cells / kg, approximately 5x10 6 cells / kg ~ approx. 10x10 6 cells / kg, 2x10 6 cells / kg ~ approx. 6x10 6 cells / kg, 2x10 6 cells / kg ~ approx. 7x10 6 cells / kg, 2x10 6 cells / kg ~ approx. 8x10 6 cells / kg, 3x10 6 cells / kg ~ approx. 6x10 6 cells / kg, 3x10 6 cells / kg ~ approx. 7x10 6 cells / kg, 3x10 6 cells / kg ~ approx. 8x10 6 cells / kg, 4x10 6 cells / kg ~ approx. 6x10 6 cells / kg, 4x10 6 cells / kg ~ approx. 7x10 6 cells / kg, 4x10 6 cells / kg ~ approx. 8x10 6 cells / kg, 5x10 6cells / kg ~ approx. 6x10 6 cells / kg, 5x10 6 cells / kg ~ approx. 7x10 6 cells / kg, 5x10 6 cells / kg ~ approx. 8x10 6 cells / kg, or 6x10 6 cells / kg ~ approx. 8x10 6 cells / kg and includes all cell doses in between.

[0423] Those skilled in the art will recognize that multiple administrations of the contemplated compositions may be required in certain embodiments to achieve the desired therapeutic effect. For example, the compositions may be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more times over a period 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. The engineered immune effector cells, CD33 VHH DARIC components, and cross-linking agents may be administered in the same or different compositions, simultaneously in one or more compositions, or in multiple compositions at different times. The engineered immune effector cells, CD33 VHH DARIC components, and cross-linking agents may be administered via the same or different routes of administration.

[0424] In some embodiments, it may be desirable to administer activated T cells to a subject, then withdraw blood from the subject (or perform apheresis), activate the T cells derived therefrom, and reinfuse these activated and expanded T cells back into the patient. This process may be performed multiple times, every few weeks. In some embodiments, T cells may be activated from a blood draw of 10 cc to 400 cc. In some embodiments, T cells are activated from a blood draw of 20 cc, 30 cc, 40 cc, 50 cc, 60 cc, 70 cc, 80 cc, 90 cc, 100 cc, 150 cc, 200 cc, 250 cc, 300 cc, 350 cc, or 400 cc or more. Without being bound by theory, the use of multiple blood draws / multiple reinfusion protocols may aid in the selection of certain populations of T cells.

[0425] In one embodiment, a method of treating a subject diagnosed with cancer comprises collecting immune effector cells from the subject, modifying the immune effector cells by introducing into the cells one or more vectors encoding one or more CD33 VHH DARIC components, generating a population of modified immune effector cells, and administering to the subject the population of modified immune effector cells. In a preferred embodiment, the immune effector cells comprise T cells.

[0426] In one embodiment, a method of treating a subject diagnosed with cancer comprises harvesting immune effector cells from the subject, modifying the immune effector cells by introducing into the cells one or more vectors encoding one or more anti-CD33 VHH CARs, generating a population of modified immune effector cells, and administering to the subject the population of modified immune effector cells. In a preferred embodiment, the immune effector cells comprise T cells.

[0427] In certain embodiments, contemplated methods of administering cell compositions include any method effective to result in the reintroduction of engineered immune effector cells ex vivo, or any method effective to result in the reintroduction of engineered precursors of immune effector cells that differentiate into mature immune effector cells upon introduction into a subject. One method involves modifying peripheral blood T cells ex vivo by introducing into the cells one or more vectors encoding one or more CD33 VHH DARIC components or anti-CD33 VHH CARs and returning the transduced cells to the subject.

[0428] All publications, patent applications, and issued patents cited in this specification are herein incorporated by reference to the same extent as if each individual publication, patent application, or issued patent was specifically and individually indicated to be incorporated by reference. Although the foregoing embodiments have been described in detail in the figures and examples for purposes of clarity and understanding, it will be readily apparent to those skilled in the art in light of the teachings contemplated herein that certain changes and modifications can be made without departing from the spirit or scope of the appended claims. The following examples are provided for illustrative purposes only and not for limitation. Those skilled in the art will readily recognize a variety of non-critical parameters that can be changed or modified in specific embodiments to yield essentially similar results. [Example]

[0429] Example 1 CD33 VHH DARIC T cells demonstrate anti-tumor responses Anti-CD33 VHH DARIC binding and signaling components were designed, constructed, and demonstrated. CD33-specific VHH DARIC lentiviral vectors were constructed containing the MNDU3 promoter operably linked to polynucleotides encoding the following: DARIC signaling component (CD8α signal peptide, FRB variant (T82L), CD8α transmembrane domain, intracellular 4-1BB costimulatory domain, and CD3 zeta signaling domain); P2A sequence; and DARIC binding component (Igκ signal peptide, CD33-specific VHH binding domain (camelid or humanized), G4S linker, FKBP12 domain, CD4 transmembrane domain with truncated intracellular domain (Figure 1B). See, for example, SEQ ID NOs: 32-41. Anti-CD33 T cells transduced with the DARIC lentiviral vector express the membrane-associated polypeptide shown in Figure 1 A. Anti-CD33 scFv CAR or DARIC designs were used as controls.

[0430] T cells from three donors were transduced with LVVs encoding different CD33-specific VHH DARIC, anti-CD33scFv DARIC, or anti-CD33scFv CAR constructs and expanded for 10 days. Untransduced T cells, T cells transduced with an anti-CD33scFv control construct, or anti-CD33 VHH DARIC T cells were stained with recombinant CD33-Fc reagent. Only control CAR and DARIC T cells stained positively with CD33-Fc (Figure 2A, bottom panel; Figure 2B). However, when analyzed with a monoclonal antibody specific for the VHH domain, the majority of anti-CD33 VHH DARIC T cells stained positive, whereas control CAR or DARIC T cells did not (Figure 2A, upper panel). Both control CAR and DARIC T cells and anti-CD33 VHH DARIC T cells had similar T cell phenotypes, determined in part by CD62L and CD45RA staining (FIGS. 3A and 3B).

[0431] Non-transduced T cells, T cells transduced with an anti-CD33 scFv control construct, or anti-CD33 VHH DARIC T cells were transduced with CD33 at a 1:1 E:T ratio in the presence or absence of AP21967 for 24 hours. + Co-cultured with THP-1 cells. Anti-CD33 scFv CAR control cells had strong cytokine production both in the presence and absence of rapalog. Anti-CD33scFv DARIC T cells and anti-CD33 VHH DARIC T cells exhibited robust cytokine responses only when cultured with THP-1 cells in the presence of AP21967 (Figures 4A and 4B). Minimal cytokine production was detected in the non-transduced control.

[0432] Furthermore, the specificity of VHH9 and VHH10 DARIC was evaluated against splice variants expressing full-length CD33 (CD33M) and the shorter truncated CD33 (CD33m). Human 293T cells were electroporated with mRNA encoding either full-length CD33M or the splice variant CD33m (Figure 4C). DARIC T cells were cocultured with modified 293T cells at a 1:1 E:T ratio in the presence or absence of AP21967 for 24 hours and evaluated for activation, as measured by cytokine secretion (Figure 4C). VHH9 DARIC T cells exhibited robust cytokine responses to either CD33M or CD33m 293T cells, whereas VHH10 DARIC T cells were activated only in the presence of CD33M.

[0433] Example 2 CD33 VHH DARIC T cells specifically respond to the CD33 antigen Anti-CD33 VHH DARIC T cells were generated as described in Example 1. T cells from three donors were transduced with LVVs encoding different anti-CD33-specific VHH DARICs and expanded for 10 days. Controls included untransduced (UTD) T cells and T cells transduced with a CD33 CAR. The AML cell line MV4-11 normally expresses CD33. MV4-11 cells were engineered to knock out the CD33 gene (CD33-KO cells). The resulting CD33-KO cell line lacked CD33 expression on the cell surface. Figure 5A. Anti-CD33 VHH DARIC T cells were cocultured with MV4-11 or CD33-KO cells at a 1:1 E:T ratio for 24 hours in the presence or absence of a dimerizing agent. Anti-CD33 VHH DARIC T cells produced cytokines in the presence of MV4-11 target cells but not in the presence of CD33-KO cells (Figure 5B).

[0434] A CD33-KO cell line was engineered to express the CD33m splice variant (CD33-KO-C2). MV4-11 and CD33-KO-C2 cells were cocultured with UTD cells, anti-CD33 CAR T cells, or anti-CD33 VHH DARIC T cells in the presence or absence of dimerization agents, and cytokine production was analyzed 24 hours later. Anti-CD33 VHH9 DARIC recognizes both normal CD33 and the CD33m splice variant and produced cytokines when cocultured with MV4-11 or CD33-KO-C2 cells (Figure 5C). Anti-CD33 CAR T cells or anti-CD33 VHH2 DARIC control T cells were only active against MV4-11 target cells.

[0435] Example 3 CD33 VHH DARIC T cells are not inhibited by soluble CD33 protein Anti-CD33 VHH DARIC T cells were generated as described in Example 1. T cells from three donors were transduced with LVV encoding different anti-CD33-specific VHH DARIC and expanded for 10 days. T cells were transfected with CD33 at a 1:1 E:T ratio in the presence or absence of rapamycin. + THP-1 cells were co-cultured for 24 hours. Various amounts of recombinant CD33-Fc protein were added during the co-culture period. Anti-CD33 VHH DARIC T cells demonstrated robust cytokine responses in the presence of rapamycin, both in the presence and absence of recombinant soluble CD33 protein. (Figure 6)

[0436] Example 4 CD33 VHH DARIC T cells respond to low levels of CD33 antigen Anti-CD33 VHH DARIC T cells were generated as described in Example 1. T cells from three donors were transduced with LVV encoding different anti-CD33-specific VHH DARIC and expanded for 10 days. T cells were transfected with the AP21967 dimerizer and different amounts of mRNA encoding CD33. neg Anti-CD33 VHH DARIC T cells were co-cultured with 293T cells. Supernatants were collected after 24 hours and analyzed for cytokine production. Anti-CD33 VHH DARIC T cells showed a dose-dependent increase in IFNγ production after co-culture with CD33-transfected target cells, even at very low mRNA concentrations (Figure 7).

[0437] Example 5 CD33 VHH DARIC T cells control tumor growth in vivo Anti-CD33 VHH DARIC T cells were generated as described in Example 1. CD33-expressing tumors were established in immunodeficient NSG mice by inoculating them with HL60 AML tumor cells expressing a luciferase reporter. Tumor growth was monitored by luminescence. After 10 days, mice were inoculated with 10 × 10 6 Anti-CD33 VHH DARIC T cells were administered in combination with vehicle or rapamycin. Controls included mice receiving rapamycin alone or untransduced (UTD) T cells. Tumor growth was comparable in both treatment and control groups (Figure 8A). Mice treated with anti-CD33 VHH DARIC T cells and rapamycin showed increased tumor growth compared to mice treated with UTD T cells and rapamycin (Figure 8B).

[0438] In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and the claims, but should be construed to include all possible embodiments along with the full scope of equivalents to which such claims are entitled. Accordingly, the claims are not limited by the present disclosure. The present invention provides, for example, the following items. (Item 1) A non-native cell, (a) a first polypeptide comprising an FRB multimerization domain polypeptide or a variant thereof, a CD8α transmembrane domain or a CD4 transmembrane domain, a CD137 costimulatory domain, and / or a CD3ζ primary signaling domain; (b) an anti-CD33 VHH antibody having an amino acid sequence set forth in any one of SEQ ID NOs: 2 to 21, an FKBP multimerization domain polypeptide or a variant thereof, and a second polypeptide comprising a CD4 transmembrane domain or a CD8α transmembrane domain; A non-native cell, wherein a cross-linking agent promotes the formation of a polypeptide complex on the surface of the non-native cell, wherein the cross-linking agent is associated with and positioned between the multimerization domains of the first and second polypeptides. (Item 2) 2. The non-naturally occurring cell of item 1, wherein the FKBP multimerization domain is FKBP12. (Item 3) 3. The non-naturally occurring cell of claim 1 or 2, wherein the FRB polypeptide is FRB T2098L. (Item 4) 4. The non-native cell of any one of items 1 to 3, wherein the cross-linking agent is selected from the group consisting of AP21967, sirolimus, everolimus, novolimus, pimecrolimus, ridaforolimus, tacrolimus, temsirolimus, umirolimus, and zotarolimus. (Item 5) 5. The non-naturally occurring cell of any one of items 1 to 4, wherein the first polypeptide comprises a signal peptide, a CD8α transmembrane domain, a CD137 costimulatory domain, and a CD3ζ primary signaling domain. (Item 6) 6. The non-naturally occurring cell of any one of items 1 to 5, wherein the second polypeptide comprises a signal peptide and a CD4 transmembrane domain. (Item 7) 7. The non-naturally occurring cell of any one of items 1 to 6, wherein the second polypeptide comprises a costimulatory domain. (Item 8) The costimulatory domain of the second polypeptide is capable of activating Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, caspase recruitment domain family member 11 (CARD11), CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD94, CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), DNAX-activation protein 10 (DAP10), T cell family member 1 (TCR1), TCR2, TCR3, TCR4, TCR5, TCR6, TCR7, TCR8, TCR ...CR10, TCR11, TCR2, TCR3, TCR4, TCR5, TCR6, TCR7, TCR8, TCR9, TCR10, TCR11, TCR2, TCR3, TCR4, TCR5, TCR6, TCR7, TCR8, TCR9, TCR10, TCR11, TCR12, TCR13, TCR14, TCR15, TCR16, TCR17, TCR18, TCR19, TCR20, TCR21, TCR22, TCR23, TCR24, TCR25, T 8. The non-native cell of item 7, wherein the costimulatory molecule is selected from the group consisting of linker for activation (LAT), 76 kD SH2 domain-containing leukocyte protein (SLP76), T cell receptor-associated transmembrane adaptor 1 (TRAT1), TNFR2, TNFRS14, TNFRS18, TNRFS25, and zeta chain of T cell receptor-associated protein kinase 70 (ZAP70). (Item 9) 9. The non-naturally occurring cell of paragraph 7 or paragraph 8, wherein the costimulatory domain of the second polypeptide is a costimulatory domain isolated from OX40 or TNFR2. (Item 10) 10. The non-naturally occurring cell according to any one of items 1 to 9, wherein the second polypeptide comprises a sequence set forth in any one of SEQ ID NOs: 22 to 31. (Item 11) A non-native cell, (a) a first polypeptide comprising an FRB multimerization domain polypeptide or a variant thereof, a CD8α transmembrane domain or a CD4 transmembrane domain, a CD137 costimulatory domain, and / or a CD3ζ primary signaling domain; (b) a second polypeptide comprising an anti-CD33 VHH antibody having an amino acid sequence set forth in any one of SEQ ID NOs: 2 to 21, an FKBP multimerization domain polypeptide or a variant thereof, and a CD4 transmembrane domain or a CD8α transmembrane domain; (c) a non-native cell comprising a polypeptide complex comprising a cross-linking agent associated with and positioned between the multimerization domains of said first and second polypeptides. (Item 12) 12. The non-naturally occurring cell of item 11, wherein the FKBP multimerization domain is FKBP12. (Item 13) 13. The non-naturally occurring cell of claim 11 or 12, wherein the FRB polypeptide is FRB T2098L. (Item 14) 14. The non-native cell of any one of items 11 to 13, wherein the cross-linking agent is selected from the group consisting of AP21967, sirolimus, everolimus, novolimus, pimecrolimus, ridaforolimus, tacrolimus, temsirolimus, umirolimus, and zotarolimus. (Item 15) 15. The non-naturally occurring cell of any one of paragraphs 11 to 14, wherein the first polypeptide comprises a signal peptide, a CD8α transmembrane domain, a CD137 costimulatory domain, and a CD3ζ primary signaling domain. (Item 16) 16. The non-naturally occurring cell of any one of items 11 to 15, wherein the second polypeptide comprises a signal peptide and a CD4 transmembrane domain. (Item 17) 17. The non-naturally occurring cell of any one of items 11 to 16, wherein the second polypeptide comprises a costimulatory domain. (Item 18) The costimulatory domain of the second polypeptide is 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 (CARD11), CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD94, CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), DNAX-activation protein 10 (DAP10), linker for activation of T-cell family member 1 (LAT), 76 kD SH2 domain-containing leukocyte protein (S 18. The non-native cell of item 17, wherein the costimulatory molecule is selected from the group consisting of T-cell receptor-associated transmembrane adaptor 1 (TRAT1), TNFR2, TNFRS14, TNFRS18, TNRFS25, and the zeta chain of T-cell receptor-associated protein kinase 70 (ZAP70). (Item 19) 19. The non-naturally occurring cell of paragraph 17 or paragraph 18, wherein the costimulatory domain of the second polypeptide is a costimulatory domain isolated from OX40 or TNFR2. (Item 20) 20. The non-naturally occurring cell according to any one of Items 11 to 19, wherein the second polypeptide comprises a sequence set forth in any one of SEQ ID NOs: 22 to 31. (Item 21) 21. The non-naturally occurring cell according to any one of items 1 to 20, wherein the cell is a hematopoietic cell. (Item 22) 22. The non-naturally occurring cell of any one of items 1 to 21, wherein the cell is a T cell, an αβ T cell, or a γδ T cell. (Item 23) The cells are CD3 + , CD4 + and / or CD8 + 23. The non-naturally occurring cell of any one of items 1 to 22, which is a cell. (Item 24) 24. The non-naturally occurring cell according to any one of items 1 to 23, wherein the cell is an immune effector cell. (Item 25) 25. The non-native cell of any one of items 1 to 24, wherein the cell is a cytotoxic T lymphocyte (CTL), a tumor-infiltrating lymphocyte (TIL), or a helper T cell. (Item 26) 26. The non-natural cell of any one of items 1 to 25, wherein the cell is a natural killer (NK) cell or a natural killer T (NKT) cell. (Item 27) 27. The non-native cell of any one of items 1 to 26, wherein the source of the cell is peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue or a tumor. (Item 28) 28. The non-naturally occurring cell of any one of items 1 to 27, wherein the FRB multimerization domain and the FKBP multimerization domain are localized extracellularly when the first polypeptide and the second polypeptide are expressed. (Item 29) A fusion polypeptide comprising: (a) a first polypeptide comprising an FRB multimerization domain polypeptide or a variant thereof, a CD8α transmembrane domain or a CD4 transmembrane domain, a CD137 costimulatory domain, and / or a CD3ζ primary signaling domain; (b) a polypeptide cleavage signal, and (c) A fusion polypeptide comprising an anti-CD33 VHH antibody having the amino acid sequence set forth in any one of SEQ ID NOs: 2 to 21, an FKBP multimerization domain polypeptide or a variant thereof, and a second polypeptide comprising a CD4 transmembrane domain or a CD8α transmembrane domain. (Item 30) 30. The fusion polypeptide of item 29, wherein the FKBP multimerization domain is FKBP12. (Item 31) Item 29 or 30, wherein the FRB polypeptide is FRB T2098L. The fusion polypeptide described. (Item 32) 32. The fusion polypeptide of any one of items 29 to 31, wherein the cross-linking agent is selected from the group consisting of AP21967, sirolimus, everolimus, novolimus, pimecrolimus, ridaforolimus, tacrolimus, temsirolimus, umirolimus, and zotarolimus. (Item 33) 33. The fusion polypeptide of any one of items 29 to 32, wherein the first polypeptide comprises a signal peptide, a CD8α transmembrane domain, a CD137 costimulatory domain, and a CD3ζ primary signaling domain. (Item 34) 34. The fusion polypeptide of any one of items 29 to 33, wherein the second polypeptide comprises a signal peptide and a CD4 transmembrane domain. (Item 35) 35. The fusion polypeptide according to any one of Items 29 to 34, wherein the fusion polypeptide comprises a sequence set forth in any one of SEQ ID NOs: 32 to 41. (Item 36) 36. The fusion polypeptide of any one of items 29 to 35, wherein the second polypeptide comprises a costimulatory domain. (Item 37) The costimulatory domain of the second polypeptide is 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 (CARD11), CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD94, CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), DNAX activity, and the like. 37. The fusion polypeptide of item 36, wherein the co-stimulatory molecule is selected from the group consisting of phosphodiesterase inhibitor (PDK)-dependent activating protein 10 (DAP10), linker for activation of T-cell family member 1 (LAT), SH2 domain-containing leukocyte protein of 76 kD (SLP76), T-cell receptor-associated transmembrane adaptor 1 (TRAT1), TNFR2, TNFRS14, TNFRS18, TNRFS25, and the zeta chain of T-cell receptor-associated protein kinase 70 (ZAP70). (Item 38) 38. The fusion polypeptide of claim 36 or 37, wherein the costimulatory domain of the second polypeptide is a costimulatory domain isolated from OX40 or TNFR2. (Item 39) 39. The fusion polypeptide of any one of items 29 to 38, wherein the polypeptide cleavage signal is a viral self-cleaving polypeptide. (Item 40) 40. The fusion polypeptide of any one of items 29 to 39, wherein the polypeptide cleavage signal is a viral self-cleaving 2A polypeptide. (Item 41) 41. The fusion polypeptide of any one of Items 29 to 40, wherein the polypeptide cleavage signal is a viral self-cleaving polypeptide selected from the group consisting of foot-and-mouth disease virus (FMDV) (F2A) peptide, equine rhinitis A virus (ERAV) (E2A) peptide, Thosea asigna virus (TaV) (T2A) peptide, porcine teschovirus-1 (PTV-1) (P2A) peptide, tylovirus 2A peptide, and encephalomyocarditis virus 2A peptide. (Item 42) 42. The fusion polypeptide according to any one of Items 29 to 41, wherein the fusion polypeptide comprises a sequence set forth in any one of SEQ ID NOs: 42 to 61. (Item 43) 43. The fusion polypeptide according to any one of Items 29 to 42, wherein the FRB multimerization domain and the FKBP multimerization domain are localized extracellularly when the first polypeptide and the second polypeptide are expressed. (Item 44) 1. A polypeptide complex comprising: (a) a first polypeptide comprising an FRB multimerization domain polypeptide or a variant thereof, a CD8α transmembrane domain or a CD4 transmembrane domain, a CD137 costimulatory domain, and / or a CD3ζ primary signaling domain; (b) a second polypeptide comprising an anti-CD33 VHH antibody having an amino acid sequence set forth in any one of SEQ ID NOs: 2 to 21, an FKBP multimerization domain polypeptide or a variant thereof, and a CD4 transmembrane domain or a CD8α transmembrane domain; (c) a cross-linking agent associated with and positioned between the multimerization domains of the first and second polypeptides. (Item 45) 45. The polypeptide complex of item 44, wherein the FKBP multimerization domain is FKBP12. (Item 46) 46. ​​The polypeptide complex of claim 44 or 45, wherein the FRB polypeptide is FRB T2098L. (Item 47) 47. The polypeptide complex of any one of items 44 to 46, wherein the cross-linking agent is selected from the group consisting of AP21967, sirolimus, everolimus, novolimus, pimecrolimus, ridaforolimus, tacrolimus, temsirolimus, umirolimus, and zotarolimus. (Item 48) 48. The polypeptide complex of any one of items 44 to 47, wherein the first polypeptide comprises a CD8α transmembrane domain, a CD137 costimulatory domain, and a CD3ζ primary signaling domain. (Item 49) 49. The polypeptide complex of any one of Items 44 to 48, wherein the second polypeptide comprises a CD4 transmembrane domain. (Item 50) 50. The polypeptide complex of any one of items 44 to 49, wherein the second polypeptide comprises a costimulatory domain. (Item 51) The costimulatory domain of the second polypeptide is 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 (CARD11), CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD94, CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), DNAX activity, and the like. 51. The polypeptide complex of item 50, wherein the co-stimulatory molecule is selected from the group consisting of phosphodiesterase inhibitor (PDK)-dependent activating protein 10 (DAP10), linker for activation of T-cell family member 1 (LAT), SH2 domain-containing leukocyte protein of 76 kD (SLP76), T-cell receptor-associated transmembrane adaptor 1 (TRAT1), TNFR2, TNFRS14, TNFRS18, TNRFS25, and the zeta chain of T-cell receptor-associated protein kinase 70 (ZAP70). (Item 52) 52. The polypeptide complex of claim 50 or 51, wherein the costimulatory domain of the second polypeptide is a costimulatory domain isolated from OX40 or TNFR2. (Item 53) 53. The polypeptide complex according to any one of items 44 to 52, wherein the cell is a hematopoietic cell. (Item 54) 54. The polypeptide complex of any one of items 44 to 53, wherein the cell is a T cell, an αβ T cell, or a γδ T cell. (Item 55) The cells are CD3 + , CD4 + , and / or CD8 + 55. The polypeptide complex according to any one of items 44 to 54, which is a cell. (Item 56) 56. The polypeptide complex according to any one of items 44 to 55, wherein the cell is an immune effector cell. (Item 57) 57. The polypeptide conjugate of any one of items 44 to 56, wherein the cell is a cytotoxic T lymphocyte (CTL), a tumor-infiltrating lymphocyte (TIL), or a helper T cell. (Item 58) 58. The polypeptide complex of any one of items 44 to 57, wherein the cell is a natural killer (NK) cell or a natural killer T (NKT) cell. (Item 59) 59. The polypeptide complex of any one of items 44 to 58, wherein the source of the cells is peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue, or a tumor. (Item 60) 60. The polypeptide complex according to any one of Items 44 to 59, wherein the FRB multimerization domain and the FKBP multimerization domain are localized extracellularly when the first polypeptide and the second polypeptide are expressed. (Item 61) A chimeric antigen receptor (CAR), a) an anti-CD33 VHH antibody having an amino acid sequence set forth in any one of SEQ ID NOs: 2 to 21; b) hinge domain, c) a transmembrane domain, d) one or more intracellular costimulatory signaling domains, and / or e) Chimeric antigen receptors (CARs) containing primary signaling domains. (Item 62) The CAR is positioned 5' to 3' as follows: a) an anti-CD33 VHH antibody having an amino acid sequence set forth in any one of SEQ ID NOs: 2 to 21; b) hinge domain, c) a transmembrane domain, d) one or more intracellular costimulatory signaling domains, and / or e) The CAR of item 61, comprising a primary signaling domain. (Item 63) 63. The CAR of paragraph 61 or paragraph 62, wherein the hinge domain and transmembrane domain are isolated from CD8α, CD27, CD28, CD33, CD37, CD45, CD64, CD71, CD80, CD86, CD134, CD137, CD152, CD154, AMN, and PD1. (Item 64) 63. The one or more costimulatory signaling domains are isolated from a costimulatory molecule selected from the group consisting of CD28, CD134, CD137, and CD278. CAR according to any one of the preceding claims. (Item 65) 65. A CAR according to any one of 61 to 64, wherein the CAR comprises a CD8α signal peptide, a CD8α hinge and transmembrane domain, a CD134 costimulatory domain, and a CD3ζ primary signaling domain. (Item 66) A CAR comprising an amino acid sequence set forth in any one of SEQ ID NOs: 62 to 81. (Item 67) 67. A polynucleotide encoding the first or second polypeptide of any one of items 1 to 28, the fusion polypeptide of any one of items 29 to 43, or the CAR of any one of items 61 to 66. (Item 68) A cDNA encoding the first or second polypeptide of any one of items 1 to 28, the fusion polypeptide of any one of items 29 to 43, or the CAR of any one of items 61 to 66. (Item 69) 67. An RNA encoding the first or second polypeptide of any one of items 1 to 28, the fusion polypeptide of any one of items 29 to 43, or the CAR of any one of items 61 to 66. (Item 70) 70. A vector comprising the polynucleotide according to any one of Items 67 to 69. (Item 71) 71. The vector according to item 70, wherein the vector is an expression vector. (Item 72) 71. The vector according to item 70, wherein the vector is a transposon. (Item 73) 73. The vector of item 72, wherein the vector is a piggyBAC transposon or a Sleeping Beauty transposon. (Item 74) 71. The vector according to item 70, wherein the vector is a viral vector. (Item 75) 75. The vector of item 74, wherein the vector is an adenovirus vector, an adeno-associated virus (AAV) vector, a herpes virus vector, a vaccinia virus vector, or a retrovirus vector. (Item 76) 76. The vector of item 75, wherein the retroviral vector is a lentiviral vector. (Item 77) 77. The vector of item 76, wherein the lentiviral vector is selected from the group consisting of human immunodeficiency virus 1 (HIV-1), human immunodeficiency virus 2 (HIV-2), Visna-Maedi virus (VMV), caprine arthritis-encephalitis virus (CAEV), equine infectious anemia virus (EIAV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), and simian immunodeficiency virus (SIV). (Item 78) 67. A cell comprising the first or second polypeptide of any one of items 1 to 28, the fusion polypeptide of any one of items 29 to 43, or the CAR of any one of items 61 to 66. (Item 79) 79. The cell of item 78, wherein the cell is a hematopoietic cell. (Item 80) 80. The cell of item 78 or 79, wherein the cell is an immune effector cell. (Item 81) 81. The cell according to any one of items 78 to 80, wherein the cell is a T cell, an αβ T cell, or a γδ T cell. (Item 82) The cells are CD3+ , CD4 + , CD8 + 82. The cell of any one of items 78 to 81, expressing a nucleotide sequence encoding a nucleotide sequence encoding a nucleotide sequence of ... (Item 83) 83. The cell of any one of items 78 to 82, wherein the cell is a cytotoxic T lymphocyte (CTL), a tumor-infiltrating lymphocyte (TIL), or a helper T cell. (Item 84) 84. The cell of any one of items 78 to 83, wherein the cell is a natural killer (NK) cell or a natural killer T (NKT) cell. (Item 85) A composition comprising the cells according to any one of items 1 to 28 and 78 to 84. (Item 86) A composition comprising a physiologically acceptable carrier and the cells according to any one of items 1 to 28 and 78 to 84. (Item 87) 87. A method of treating a subject in need thereof, comprising administering to the subject an effective amount of the composition of item 85 or item 86. (Item 88) 87. A method for treating, preventing, or ameliorating at least one symptom of cancer, infectious disease, autoimmune disease, inflammatory disease, and immune deficiency, or a condition associated therewith, comprising administering to the subject an effective amount of the composition of item 85 or item 86. (Item 89) A method for treating a solid tumor, comprising administering to the subject an effective amount of the composition of item 85 or item 86. (Item 90) 90. The method of claim 89, wherein 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. (Item 91) 91. The method of claim 89 or 90, wherein the solid cancer is non-small cell lung cancer, 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, glioma, glioblastoma, or oligodendroglioma. (Item 92) 87. A method for treating a malignant hematological disease, comprising administering to the subject an effective amount of the composition of item 85 or item 86. (Item 93) Item 93. The method of item 92, wherein the malignant blood disease is leukemia, lymphoma, or multiple myeloma. (Item 94) Item 93. The method of item 92, wherein the malignant blood disease is acute myeloid leukemia (AML).

Claims

[Claim 1] The invention described in the specification.

Images