CD33-targeted immunotherapy

Abstract

The present disclosure provides improved CD33-targeting polypeptides and compositions for adoptive T cell therapy for treating, preventing, or ameliorating at least one symptom of, or a condition associated with, cancer, infectious diseases, autoimmune diseases, inflammatory diseases, and immunodeficiency.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 62 / 898,392, filed on September 10, 2019, and U.S. Provisional Application No. 62 / 845,304, filed on May 8, 2019, each of which is incorporated herein by reference in its entirety.

[0003] Statement regarding sequence listing

[0004] The sequence listing associated with this application is provided in text format in lieu of a paper copy 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 was submitted electronically via EFS-Web at the same time as this specification. Background Art Technical Field

[0006] The present disclosure relates to improved adoptive cell therapy targeting CD33. More specifically, the present disclosure relates to chemically regulated signaling molecules containing anti-CD33 VHHs, chimeric antigen receptors containing anti-CD33 VHHs, cells, and related treatment methods using the same.

[0007] Existing technology

[0008] The global cancer burden doubled between 1975 and 2000. Cancer is the second leading cause of morbidity and mortality worldwide, with approximately 14.1 million new cases and 8.2 million cancer-related deaths in 2012. The most common cancers are breast cancer, lung and bronchial cancer, prostate cancer, colon and rectal cancer, bladder cancer, melanoma of the skin, non-Hodgkin lymphoma, thyroid cancer, kidney and renal pelvis cancer, endometrial cancer, leukemia, and pancreatic cancer. The number of new cancer cases is expected to increase to 22 million over the next two decades.

[0009] Adoptive cell therapy is becoming a powerful paradigm for delivering complex biological signals to treat cancer. Compared with small molecules and biopharmaceutical compositions, adoptive cell therapy has the potential to perform unique therapeutic tasks due to its extremely large number of sensory and response programs and increasingly clear genetic control mechanisms. Existing methods mainly focus on chimeric antigen receptors (CARs) based on scFv. CAR T cell therapy has achieved limited success due to poor CAR expression, in vivo expansion of CART cells, rapid disappearance of cells after infusion, disappointing clinical activity and antigen escape.

[0010] There is a need to engineer immune effector cells with improved CAR architectures (CAR structures) and / or improved mechanisms for sensing and integrating chemical and / or biological information associated with the local physiological environment. Summary of the Invention

[0011] The present disclosure generally relates in part to VHH-based dimerizer-regulated immune receptor complexes (DARICs) and VHH-based chimeric antigen receptors (CARs) directed against CD33, polynucleotides encoding the same, compositions thereof, and methods of making and using the same to treat cancer.

[0012] 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 carboxyl-terminal amino acids caused by the early translation stop signal residing in exon 7a. In certain embodiments, the CD33 splice variant lacks the 124 amino acids encoded by exon 2 and the 54 carboxyl-terminal amino acids caused by the early translation stop signal residing in exon 7a.

[0013] In certain embodiments, the VHH DARIC or VHH CAR binds to both full-length CD33 and a CD33 splice variant.

[0014] In various embodiments, the non-natural cell comprises: a first polypeptide comprising: a FRB multimerization domain polypeptide or a variant thereof, a CD8α transmembrane domain or a CD4 transmembrane domain, a CD137 co-stimulatory domain, and / or a CD3ζ primary signaling domain; and a second polypeptide comprising: an anti-CD33 VHH antibody having an amino acid sequence as set forth in any one of SEQ ID NOs: 2-21, a FKBP multimerization domain polypeptide or a variant thereof, and a CD4 transmembrane domain or a CD8α transmembrane domain; wherein formation of the polypeptide complex on the surface of the non-natural cell is facilitated by a bridging factor that associates with and is positioned between the multimerization domains of the first and second polypeptides.

[0015] In a specific embodiment, the anti-CD33 VHH antibody has the amino acid sequence shown in SEQ ID NO:10.

[0016] In a specific embodiment, the anti-CD33 VHH antibody has the amino acid sequence shown in SEQ ID NO:20.

[0017] In certain embodiments, the FKBP multimerization domain is FKBP12.

[0018] In some embodiments, the FRB polypeptide is FRB T2098L.

[0019] In certain embodiments, the bridging factor is selected from the group consisting of AP21967, sirolimus, everolimus, norflulimus, pimecrolimus, dafolimus, tacrolimus, temsirolimus, umimios, and zotarolimus.

[0020] In various embodiments, the first polypeptide comprises a signal peptide, a CD8α transmembrane domain, a CD137 co-stimulatory domain, and a CD3ζ primary signaling domain.

[0021] In certain embodiments, the second polypeptide comprises a signal peptide and a CD4 transmembrane domain.

[0022] In additional embodiments, the second polypeptide comprises a costimulatory domain.

[0023] In some embodiments, the costimulatory domain of the second polypeptide is selected from the group consisting of a costimulatory molecule 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-activating protein 10 (DAP10), linker for activated 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 T cell receptor-associated protein kinase 70 zeta chain (ZAP70).

[0024] In additional embodiments, the costimulatory domain of the second polypeptide is a costimulatory domain isolated from OX40 or TNFR2.

[0025] In further embodiments, the second polypeptide comprises the sequence set forth in any one of SEQ ID NOs: 22-31.

[0026] In certain embodiments, the second polypeptide comprises the sequence set forth in SEQ ID NO:30.

[0027] In a preferred embodiment, the first polypeptide comprises the amino acid sequence shown in SEQ ID NO 82.

[0028] In various embodiments, the non-natural cell comprises a polypeptide complex comprising: a first polypeptide comprising: a FRB multimerization domain polypeptide or a variant thereof, a CD8α transmembrane domain or a CD4 transmembrane domain, a CD137 co-stimulatory domain, and / or a CD3ζ primary signaling domain; a second polypeptide comprising: an anti-CD33 VHH antibody having an amino acid sequence as set forth in any one of SEQ ID NOs: 2-21, a FKBP multimerization domain polypeptide or a variant thereof, and a CD4 transmembrane domain or a CD8α transmembrane domain; and a bridging factor associated with and positioned between the multimerization domains of the first and second polypeptides.

[0029] In a specific embodiment, the anti-CD33 VHH antibody has the amino acid sequence shown in SEQ ID NO:10.

[0030] In a specific embodiment, the anti-CD33 VHH antibody has the amino acid sequence shown in SEQ ID NO:20.

[0031] In certain embodiments, the FKBP multimerization domain is FKBP12.

[0032] In certain embodiments, the FRB polypeptide is FRB T2098L.

[0033] In some embodiments, the bridging factor is selected from the group consisting of AP21967, sirolimus, everolimus, norflulimus, pimecrolimus, dafolimus, tacrolimus, temsirolimus, umimios, and zotarolimus.

[0034] In additional embodiments, the first polypeptide comprises a signal peptide, a CD8α transmembrane domain, a CD137 co-stimulatory domain, and a CD3ζ primary signaling domain.

[0035] In certain embodiments, the second polypeptide comprises a signal peptide and a CD4 transmembrane domain.

[0036] In some embodiments, the second polypeptide comprises a costimulatory domain.

[0037] In various embodiments, the costimulatory domain of the second polypeptide is selected from the group consisting of a costimulatory molecule: 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-activating protein 10 (DAP10), linker for activated 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 T cell receptor-associated protein kinase 70 zeta chain (ZAP70).

[0038] In additional embodiments, the costimulatory domain of the second polypeptide is a costimulatory domain isolated from OX40 or TNFR2.

[0039] In further embodiments, the second polypeptide comprises the sequence set forth in any one of SEQ ID NOs: 22-31.

[0040] In certain embodiments, the second polypeptide comprises the sequence set forth in SEQ ID NO:30.

[0041] In a preferred embodiment, the first polypeptide comprises the amino acid sequence shown in SEQ ID NO 82.

[0042] In certain embodiments, the cell is a hematopoietic cell.

[0043] In certain embodiments, the cell is a T cell, an αβ T cell, or a γδ T cell.

[0044] In further embodiments, the cells are CD3+, CD4+ and / or CD8+ cells.

[0045] In various embodiments, the cells are immune effector cells.

[0046] In some embodiments, the cell is a cytotoxic T lymphocyte, a tumor infiltrating lymphocyte, or a helper T cell.

[0047] In additional embodiments, the cell is a natural killer (NK) cell or a natural killer T (NKT) cell.

[0048] 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 a site of infection, ascites, pleural effusion, spleen tissue, or a tumor.

[0049] In certain embodiments, when the first polypeptide and the second polypeptide are expressed, the FRB multimerization domain and the FKBP multimerization domain are localized extracellularly.

[0050] In some embodiments, the fusion polypeptide comprises: a first polypeptide comprising: a FRB multimerization domain polypeptide or a variant thereof, a CD8α transmembrane domain or a CD4 transmembrane domain, a CD137 co-stimulatory domain, and / or a CD3ζ primary signaling domain, and a polypeptide cleavage signal; and a second polypeptide comprising: an anti-CD33 VHH antibody having an amino acid sequence as shown in any one of SEQ ID NOs: 2-21, a FKBP multimerization domain polypeptide or a variant thereof, and a CD4 transmembrane domain or a CD8α transmembrane domain.

[0051] In a specific embodiment, the anti-CD33 VHH antibody has the amino acid sequence shown in SEQ ID NO:10.

[0052] In certain embodiments, the FKBP multimerization domain is FKBP12.

[0053] In certain embodiments, the FRB polypeptide is FRB T2098L.

[0054] In some embodiments, the bridging factor is selected from the group consisting of AP21967, sirolimus, everolimus, norflulimus, pimecrolimus, dafolimus, tacrolimus, temsirolimus, umimios, and zotarolimus.

[0055] In additional embodiments, the first polypeptide comprises a signal peptide, a CD8α transmembrane domain, a CD137 co-stimulatory domain, and a CD3ζ primary signaling domain.

[0056] In certain embodiments, the second polypeptide comprises a signal peptide and a CD4 transmembrane domain.

[0057] In certain embodiments, the fusion polypeptide comprises the sequence set forth in any one of SEQ ID NOs: 32-41.

[0058] In certain embodiments, the fusion polypeptide comprises the sequence set forth in SEQ ID NO:40.

[0059] In additional embodiments, the second polypeptide comprises a costimulatory domain.

[0060] In various embodiments, the costimulatory domain of the second polypeptide is selected from the group consisting of a costimulatory molecule: 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-activating protein 10 (DAP10), linker for activated 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 T cell receptor-associated protein kinase 70 zeta chain (ZAP70).

[0061] In additional embodiments, the costimulatory domain of the second polypeptide is a costimulatory domain isolated from OX40 or TNFR2.

[0062] In certain embodiments, the polypeptide cleavage signal is a viral self-cleaving polypeptide.

[0063] In certain embodiments, the polypeptide cleavage signal is a viral autolytic 2A polypeptide.

[0064] 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 virus type A (ERAV) (E2A) peptide, trichotep beta-tetrasomal virus (TaV) (T2A) peptide, porcine teschovirus-1 (PTV-1) (P2A) peptide, Theiler virus 2A peptide, and encephalomyocarditis virus 2A peptide.

[0065] In some embodiments, the fusion polypeptide comprises the sequence set forth in any one of SEQ ID NOs: 42-61.

[0066] In some embodiments, the fusion polypeptide comprises the sequence set forth in either SEQ ID NO: 50 or 60.

[0067] In additional embodiments, when the first polypeptide and the second polypeptide are expressed, the FRB multimerization domain and the FKBP multimerization domain are localized extracellularly.

[0068] In various embodiments, the polypeptide complex comprises: a first polypeptide comprising: a FRB multimerization domain polypeptide or a variant thereof, a CD8α transmembrane domain or a CD4 transmembrane domain, a CD137 co-stimulatory domain, and / or a CD3ζ primary signaling domain; a second polypeptide comprising: an anti-CD33 VHH antibody having an amino acid sequence as shown in any one of SEQ ID NOs: 2-21, a FKBP multimerization domain polypeptide or a variant thereof, and a CD4 transmembrane domain or a CD8α transmembrane domain; and a bridging factor associated with the multimerization domains of the first and second polypeptides and positioned between the multimerization domains.

[0069] In a specific embodiment, the anti-CD33 VHH antibody has the amino acid sequence shown in SEQ ID NO:10.

[0070] In certain embodiments, the FKBP multimerization domain is FKBP12.

[0071] In additional embodiments, the FRB polypeptide is FRB T2098L.

[0072] In certain embodiments, the bridging factor is selected from the group consisting of AP21967, sirolimus, everolimus, norflulimus, pimecrolimus, dafolimus, tacrolimus, temsirolimus, umimios, and zotarolimus.

[0073] In certain embodiments, the first polypeptide comprises a CD8α transmembrane domain, a CD137 co-stimulatory domain, and a CD3ζ primary signaling domain.

[0074] In various embodiments, the second polypeptide comprises a CD4 transmembrane domain.

[0075] In additional embodiments, the second polypeptide comprises a costimulatory domain.

[0076] In some embodiments, the costimulatory domain of the second polypeptide is selected from the group consisting of a costimulatory molecule 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-activating protein 10 (DAP10), linker for activated 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 T cell receptor-associated protein kinase 70 zeta chain (ZAP70).

[0077] In certain embodiments, the costimulatory domain of the second polypeptide is a costimulatory domain isolated from OX40 or TNFR2.

[0078] In certain embodiments, the cell is a hematopoietic cell.

[0079] In various embodiments, the cell is a T cell, an αβ T cell, or a γδ T cell.

[0080] In various embodiments, the cells are CD3+, CD4+ and / or CD8+ cells.

[0081] In additional embodiments, the cell is an immune effector cell.

[0082] In some embodiments, the cell is a cytotoxic T lymphocyte, a tumor infiltrating lymphocyte, or a helper T cell.

[0083] In certain embodiments, the cell is a natural killer (NK) cell or a natural killer T (NKT) cell.

[0084] In other 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.

[0085] In additional embodiments, when the first polypeptide and the second polypeptide are expressed, the FRB multimerization domain and the FKBP multimerization domain are localized extracellularly.

[0086] In a preferred embodiment, the first polypeptide comprises the amino acid sequence shown in SEQ ID NO 82.

[0087] 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.

[0088] In a specific embodiment, the anti-CD33 VHH antibody has the amino acid sequence shown in SEQ ID NO:10.

[0089] In a specific embodiment, the anti-CD33 VHH antibody has the amino acid sequence shown in SEQ ID NO:20.

[0090] In various embodiments, the CAR comprises, from 5′ to 3′, an anti-CD33 VHH antibody having an amino acid sequence as shown 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.

[0091] In a specific embodiment, the anti-CD33 VHH antibody has the amino acid sequence shown in SEQ ID NO:10.

[0092] In a specific embodiment, the anti-CD33 VHH antibody has the amino acid sequence shown in SEQ ID NO:20.

[0093] In certain embodiments, the hinge domain and transmembrane domain are isolated from CD8α, CD27, CD28, CD33, CD37, CD45, CD64, CD71, CD80, CD86, CD 134, CD137, CD152, CD154, AMN, and PD1.

[0094] 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.

[0095] In a specific embodiment, the CAR comprises a CD8α signal peptide, a CD8α hinge and transmembrane domain, a CD134 co-stimulatory domain, and a CD3ζ primary signaling domain.

[0096] In additional embodiments, the CAR comprises the amino acid sequence set forth in any one of SEQ ID NOs: 62-81.

[0097] In further embodiments, the CAR comprises the amino acid sequence set forth in either SEQ ID NO: 70 or 80.

[0098] In some embodiments, polynucleotides encoding the first or second polypeptide, fusion polypeptide, or CAR contemplated herein are provided.

[0099] In various embodiments, a cDNA encoding the first or second polypeptide, fusion polypeptide, or CAR contemplated herein is provided.

[0100] In certain embodiments, RNA encoding the first or second polypeptide, fusion polypeptide, or CAR contemplated herein is provided.

[0101] In further embodiments, vectors comprising the polynucleotides contemplated herein are provided.

[0102] In certain embodiments, the vector is an expression vector.

[0103] In certain embodiments, the vector is a transposon.

[0104] In further embodiments, the vector is a piggyBAC transposon or a Sleeping Beauty transposon.

[0105] In certain embodiments, the vector is a viral vector.

[0106] 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.

[0107] In further embodiments, the retroviral vector is a lentiviral vector.

[0108] 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), Vesna-Medi virus (VMV) virus; caprine arthritis-encephalitis virus (CAEV); equine infectious anemia virus (EIAV); feline immunodeficiency virus (FIV); bovine immunodeficiency virus (BIV); and simian immunodeficiency virus (SIV).

[0109] In additional embodiments, cells are provided comprising a first polypeptide and a second polypeptide, a fusion polypeptide, or a CAR encompassed herein.

[0110] In certain embodiments, the cells are hematopoietic cells.

[0111] In certain embodiments, the cell is an immune effector cell.

[0112] In various embodiments, the cell is a T cell, an αβ T cell, or a γδ T cell.

[0113] In some embodiments, the cells express CD3+, CD4+, CD8+, or a combination thereof.

[0114] In certain embodiments, the cell is a cytotoxic T lymphocyte (CTL), a tumor infiltrating lymphocyte (TIL), or a helper T cell.

[0115] In further embodiments, the cell is a natural killer (NK) cell or a natural killer T (NKT) cell.

[0116] In certain embodiments, the compositions comprise the cells contemplated herein.

[0117] In certain embodiments, the compositions comprise a physiologically acceptable carrier and the cells contemplated herein.

[0118] In additional embodiments, methods of treating a subject in need thereof are provided, the methods comprising administering to the subject an effective amount of a composition contemplated herein.

[0119] In certain embodiments, a method of treating, preventing, or ameliorating at least one symptom of, or a condition associated with, cancer, infectious disease, autoimmune disease, inflammatory disease, and immunodeficiency comprises administering to a subject an effective amount of a composition contemplated herein.

[0120] In some embodiments, a method of treating a solid cancer comprises administering to a subject an effective amount of a composition contemplated herein.

[0121] 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, testicular cancer, endometrial cancer, pancreatic cancer, breast cancer, cervical cancer, ovarian cancer, skin cancer, and melanoma.

[0122] In certain embodiments, a method of treating a hematological malignancy comprises administering to a subject an effective amount of a composition contemplated herein.

[0123] In various embodiments, the hematological malignancy is a leukemia, lymphoma, or multiple myeloma.

[0124] In certain embodiments, the hematological malignancy is acute myeloid leukemia (AML). BRIEF DESCRIPTION OF THE DRAWINGS

[0125] Figure 1A A sketch of the VHH-DARIC polypeptide complex is shown.

[0126] Figure 1B A sketch of the CD33 VHH DARIC architecture is shown.

[0127] Figure 2A Shown is the expression of CD33VHH1-5 DARIC in transduced T cells as detected by anti-VHH staining (top row) and by CD33-Fc binding (bottom row).

[0128] Figure 2B Shown is the expression of CD33 VHH9-10 DARIC in transduced T cells as detected by CD33-Fc binding.

[0129] Figure 3A Shown are the phenotypes of T cells transduced with CD33 VHH1-5 DARIC or control.

[0130] Figure 3B Shown are the phenotypes of T cells transduced with CD33 VHH9-10 DARIC or control.

[0131] Figure 4A Shown are the results from the + IFNγ secretion from CD33 VHH1-5 DARIC or control cells cultured with THP-1 cells at an E:T ratio of 1:1 for 24 hours in the presence or absence of AP21967.

[0132] Figure 4B Shown are the results from the + IFNγ secretion from CD33 VHH9-10 DARIC or control cells cultured with THP-1 cells at an E:T ratio of 1:1 for 24 hours in the presence or absence of AP21967.

[0133] Figure 4C Shown is IFNγ secretion from CD33VHH9-10 DARIC or control cells cultured with modified 293T cells expressing full-length CD33 (CD33M) or a CD33 splice variant (CD33m, C2) at an E:T ratio of 1:1 in the presence or absence of AP21967 for 24 hours.

[0134] Figure 5A Shown are CD33 expression in MV4-11 cells, MV4-11 cells engineered to knock out the CD33 gene (CD33-KO cells), and unstained controls.

[0135] Figure 5B Shown are IFNγ secretion from anti-CD33 VHH9 DARIC T cells or UTD T cells co-cultured with MV4-11 cells or CD33-KO cells at an E:T ratio of 1:1 for 24 hours in the presence or absence of AP21967.

[0136] Figure 5C Shown is IFNγ secretion from UTD T cells, anti-CD33 CAR T cells, or anti-CD33 VHH DARIC T cells cocultured with MV4-11 cells (left panel) or CD33-KO cells engineered to express a 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.

[0137] Figure 6 Shown are the results from the + IFNγ secretion from anti-CD33 VHH DARIC T cells co-cultured with 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 h.

[0138] Figure 7 Figure 2 shows CD33 from cells transfected with different amounts of CD33 mRNA. neg IFNγ secretion by anti-CD33 VHH DARIC T cells co-cultured with 293T cells at an E:T ratio of 1:1 in the presence or absence of AP21967 for 24 hours.

[0139] Figure 8A Shown are tumor growth measured as a function of luminescence in immunodeficient NSG mice inoculated with HL60 AML tumor cells expressing a luciferase reporter gene and treated 10 days after inoculation (day 0) with UTD T cells or anti-CD33 VHH DARIC T cells in the absence of rapamycin.

[0140] Figure 8B Shown are tumor growth measured as luminescence in immunodeficient NSG mice inoculated with HL60 AML tumor cells expressing a luciferase reporter gene and treated 10 days after inoculation (day 0) with UTD T cells or anti-CD33 VHH DARIC T cells and 0.1 mg / kg rapamycin.

[0141] A brief description of sequence identifiers

[0142] SEQ ID NO: 1 sets forth the amino acid sequence of full-length human CD33.

[0143] SEQ ID NOs: 2-21 set forth the amino acid sequences of anti-CD33 VHH domains.

[0144] SEQ ID NOs: 22-31 set forth the amino acid sequences of the anti-CD33 VHH DARIC binding components.

[0145] SEQ ID NOs: 32-41 set forth the amino acid sequences of anti-CD33 VHH DARIC fusion proteins.

[0146] SEQ ID NOs: 42-51 set forth the amino acid sequences of anti-CD33 VHH DARIC.OX40 fusion proteins.

[0147] SEQ ID NOs: 52-61 set forth the amino acid sequences of anti-CD33 VHH DARIC.TNFR2 fusion proteins.

[0148] SEQ ID NOs: 62-81 set forth the amino acid sequences of anti-CD33 VHH CARs.

[0149] SEQ ID NO: 82 sets forth the amino acid sequence of the anti-CD33 VHH DARIC signaling component.

[0150] SEQ ID NO: 83 sets forth the polynucleotide sequence of the Kozak sequence.

[0151] SEQ ID NOs: 84-94 set forth the amino acid sequences of various linkers.

[0152] SEQ ID NOs: 95-119 set forth the amino acid sequences of protease cleavage sites and self-cleaving polypeptide cleavage sites.

[0153] In the foregoing sequences, Xaa, if present, may refer to any amino acid or to the absence of a particular amino acid. In preferred embodiments, XaaXaa refers to the amino acid sequence SS or KP. Specific implementation plan

[0154] A. Overview

[0155] Cancer is one of the leading causes of death worldwide. Approximately 10% of all cancers are hematological malignancies, which include leukemias, lymphomas, and myelomas. Acute myeloid leukemia (AML) is the most common and fatal hematological malignancy in adults. Despite significant scientific discoveries and new therapies over the past four decades, treatment outcomes for AML, particularly in the adult patient population, remain dismal. Standard chemotherapy can induce complete remission in selected patients; however, the majority of patients ultimately relapse and die from the disease. In 2012, the global incidence of AML was approximately 351,965, and approximately 265,461 people died from AML.

[0156] CD33 is expressed on most acute myeloid leukemia (AML) leukemic blasts and possibly leukemic stem cells. CD33 is a challenging target due to its low expression and slow internalization; these features limit antibody-dependent cell-mediated cytotoxicity and intracellular drug accumulation, and therefore restrict the activity of unlabeled and toxin-loaded antibodies.

[0157] The present disclosure generally relates to improved compositions and methods for regulating the spatial and temporal control of adoptive cell therapy using an immune receptor complex (DARIC) regulated by a dimerizing agent in conjunction with CD33. Without wishing to be bound by any particular theory, the DARIC compositions and methods encompassed herein provide many advantages over existing CAR T cell therapies in the art, including but not limited to control of both the space and time of immune effector cell signal transduction binding and signaling activity. DARIC temporal control triggers the DARIC mechanism of signal transduction by the association between the DARIC binding components and the DARIC signaling components mediated by bridging factors. DARIC spatial control participates in the signaling mechanism by recognizing CD33 by the DARIC binding domain of the DARIC binding components. In this way, when both target cells expressing CD33 and bridging factors are present, DARIC immune effector cells are activated.

[0158] 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-dependent signaling, weak expression, and / or subtherapeutic activity.

[0159] In various embodiments, the present disclosure encompasses anti-CD33 VHH DARICs or anti-CD33 VHH CARs that generate an anti-cancer response against a cancer, e.g., AML, that expresses CD33 (e.g., full-length CD33 and / or CD33 splice variants).

[0160] In certain embodiments, DARIC comprises a polypeptide (DARIC signaling component) comprising a multimerization domain polypeptide or variant thereof, a transmembrane domain, a costimulatory domain, and / or a primary signaling domain; and a polypeptide (DARIC binding component) comprising an anti-CD33 VHH, a multimerization domain polypeptide or variant thereof, a transmembrane domain, and optionally a costimulatory domain. In the presence of a bridging factor, the DARIC binding and signaling components associate with each other via the bridging factor to form a functionally active DARIC that targets cells expressing CD33.

[0161] In certain embodiments, the multimerization domains of the DARIC binding component and the DARIC signaling component are located extracellularly. Compared to intracellular localization, the extracellular location of the multimerization domains provides a number of advantages, including but not limited to more efficient localization of the anti-CD33 VHH domains, greater temporal sensitivity to modulation by bridging factors, and less toxicity due to the ability to use non-immunosuppressive doses of specific bridging factors.

[0162] 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 comprising and / or expressing polynucleotides encoding and / or expressing DARIC, DARIC binding components, and DARIC signaling components; and methods of using the same to treat immune disorders.

[0163] Techniques for 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 can generally be performed as described in various general and more specific references in microbiology, molecular biology, biochemistry, molecular genetics, cell biology, virology, and immunology that are 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, GreenePub.Associates and Wiley-Interscience; Glover, DNA Cloning: A Practical Approach, vol. I & II (IRL Press, Oxford Univ. Press USA, 1985); Current Protocols in Immunology (Editors: 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 ed., 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, ed., 1987, Cold Spring Harbor Laboratory); Harlow and Lane, Antibodies, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1998); Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell, eds., 1986); Roitt, Essential Immunology, 6th ed., (Blackwell Scientific Publications, Oxford, 1988); Current Protocols in Immunology (Q. E. Colligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, and W. Strober, eds., 1991); Annual Review of Immunology; and monographs in journals such as Advances in Immunology.

[0164] B. Definition

[0165] Before describing the present disclosure in greater detail, it may be helpful to understand the present disclosure by providing definitions of certain terms used herein.

[0166] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention belongs. Although any methods and materials similar or equivalent to the methods and materials described herein can be used to practice or test specific embodiments, preferred embodiments of compositions, methods and materials are described herein. For the purposes of this disclosure, the following terms are defined below.

[0167] As used herein, the articles "a" and "an" refer to one or more than one (i.e., at least one or one or more) the grammatical object of the article. As an example, "an element" means one element or one or more elements.

[0168] The use of alternatives such as "or" should be understood to mean one, two, or any combination of the alternatives.

[0169] The term "and / or" should be understood to mean one or both of the alternatives.

[0170] 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 quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that is within the range of approximately ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1% of a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length.

[0171] In one embodiment, a range, for example, 1 to 5, about 1 to 5, or about 1 to about 5, refers to every numerical value encompassed within the range. 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.

[0172] As used herein, the term "substantially" refers to an amount, level, value, number, frequency, percentage, dimension, size, quantity, weight, or length that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of a reference amount, level, value, number, frequency, percentage, dimension, size, quantity, weight, or length. In one embodiment, "substantially the same" refers to an amount, level, value, number, frequency, percentage, dimension, size, quantity, weight, or length that produces an effect, such as a physiological effect, that is about the same as a reference amount, level, value, number, frequency, percentage, dimension, size, quantity, weight, or length.

[0173] Throughout this specification, unless the context requires otherwise, the words "comprise / comprises / comprising" should be understood to imply the inclusion of a stated step or element or group of steps or elements, but not the exclusion of any other step or element or any other group of steps or elements. "Consisting of is intended to include and be limited to what follows the phrase "consisting of." Thus, the phrase "consisting of indicates that the listed elements are essential or required, and no other elements can be present. "Consisting essentially of is intended to include any element listed after the phrase, and is limited to other elements that do not interfere with or affect the activity or action described in the disclosure regarding the listed elements. Thus, the phrase "consisting essentially of indicates that the listed elements are essential or required, and no other elements are present that would materially affect the activity or action of the listed elements.

[0174] Reference throughout this specification to "one embodiment," "a specific embodiment," "a related embodiment," "an embodiment," "an additional embodiment," or "another embodiment" or combinations thereof means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of the aforementioned phrases throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments. It should also be understood that a positive recitation of a feature in one embodiment serves as a basis for excluding that feature from a particular embodiment.

[0175] "Antigen (Ag)" refers to a compound, composition, or substance that can stimulate antibody production or a T cell response in an animal, including compositions injected or absorbed into an animal (e.g., compositions comprising a cancer-specific protein). Exemplary antigens include, but are not limited to, lipids, carbohydrates, polysaccharides, glycoproteins, peptides, or nucleic acids. Antigens react with products that elicit specific humoral or cellular immunity, including products induced by heterologous antigens such as the disclosed antigens.

[0176] "Target antigen" or "target antigen of interest" refers to the portion of CD33 to which the binding domains contemplated herein are designed to bind. In certain embodiments, the target antigen is an epitope of the amino acid sequence set forth in SEQ ID NO:1.

[0177] "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 has two Ig-like domains, a V-set domain and a C2-set domain. CD33 plays a role in mediating cell-cell interactions and maintaining immune cells in a resting state. CD33 recognizes and binds to glycans with α-2,3-linked sialic acids and more strongly recognizes and binds to glycans with α-2,6-linked sialic acids. Upon engagement of a ligand such as C1q or a sialylated glycoprotein, two immunoreceptor tyrosine-based inhibitory motifs (ITIMs) located in the cytoplasmic tail of CD33 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 13-139 of full-length CD33; e.g., NP_001076087.1, C2). 7a The splice variant lacks the 54 carboxyl-terminal amino acids resulting from an early translation termination signal (e.g., NP_001171079.1) residing in exon 7a. ΔE2 / 7 a lacks the amino acids encoded by exon 2 and the 54 carboxyl-terminal amino acids. CD33 is normally expressed on normal B cells and a subset of activated T cells and natural killer cells, but is not expressed on hematopoietic stem cells or outside the hematopoietic system. Both full-length CD33 and / or CD33 splice variants are also expressed in acute myeloid leukemia (AML) blasts in most AML patients.

[0178] "Antibody" refers to a binding agent that is a polypeptide comprising at least a light or heavy chain immunoglobulin variable region that specifically recognizes and binds an epitope of a target, such as a lipid, carbohydrate, polysaccharide, glycoprotein, peptide, or nucleic acid containing antigenic determinants, such as those recognized by immune cells.

[0179] Reference to "VH" or "VH" refers to the variable region of an immunoglobulin heavy chain or antigen-binding fragment thereof.

[0180] A "heavy chain antibody" is one containing two V Hdomains and no light chain (Riechmann L. et al., J. Immunol. Methods 231: 25-38 (1999); WO94 / 04678; WO94 / 25591; U.S. Patent 6,005,079). "Camelid antibodies" are antibodies containing two V H An antibody isolated from a camel, alpaca, or llama that has a VHH domain and no light chain. "Humanized VHH" or "humanized camelid antibody" refers to a non-human VHH or camelid antibody that has undergone humanization to reduce the potential immunogenicity of the antibody in a human recipient.

[0181] As used herein, “V H H", "V H H antibody" or "V H The term "H domain" refers to an antibody fragment that contains the smallest known antigen-binding unit of the heavy chain antibody variable region (Koch-Nolte et al., FASEB J., 21: 3490-3498 (2007)).

[0182] "Linker" refers to a plurality of amino acid residues added between polypeptide domains 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.

[0183] Illustrative examples of linkers suitable for use in the specific 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, wherein 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); KESGSVSSEQLAQFRSLD (SEQ ID NO: 89) (Bird et al., 1988, Science 242: 423-426), GGRRGGGS (SEQ ID NO: 90); LRQRDGERP (SEQ ID NO: 91); LRQKDGGGSERP (SEQ ID NO: 92); LRQKD(GGGS)2ERP (SEQ ID NO: 93). Alternatively, flexible linkers can be rationally designed using computer programs capable of modeling the DNA binding site and the peptide itself (Desjarlais & Berg, PNAS 90: 2256-2260 (1993), PNAS 91: 11099-11103 (1994)) or by phage display methods. 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)).

[0184] "Spacer domain" refers to a polypeptide that separates two domains. In one embodiment, the spacer domain moves the VHH domain away from the effector cell surface to achieve appropriate cell / cell contact, antigen binding and activation (Patel et al., Gene Therapy, 1999; 6: 412-419). In specific embodiments, the spacer domain separates one or more VHH domains, multimerization domains, transmembrane domains, costimulatory domains and / or primary signaling domains. The spacer domain may be derived from natural, synthetic, semisynthetic or recombinant sources. In certain embodiments, the spacer domain is a part of an immunoglobulin, including but not limited to one or more heavy chain constant regions, such as CH2 and CH3. The spacer domain may include a naturally occurring immunoglobulin hinge region or an altered immunoglobulin hinge region amino acid sequence.

[0185] "Hinge domain" refers to a polypeptide that plays a role in positioning the antigen binding domain away from the effector cell surface to achieve appropriate cell / cell contact, antigen binding and activation. In certain embodiments, the polypeptide may include 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 a natural, synthetic, semisynthetic or recombinant source. The hinge domain may include the amino acid sequence of a naturally occurring immunoglobulin hinge region or an altered immunoglobulin hinge region.

[0186] As used herein, a "multimerization domain" refers to a polypeptide that preferentially interacts or associates with another different polypeptide, directly or via a bridging molecule, e.g., a chemically inducible dimer, wherein the interaction of different multimerization domains substantially facilitates or efficiently promotes multimerization (i.e., the formation of a dimer, trimer, or multimeric complex, which can be a homodimer, heterodimer, homotrimer, heterotrimer, homomultimer, or heteromultimer). A multimerization domain can be derived from natural, synthetic, semisynthetic, or recombinant sources.

[0187] Illustrative examples of multimerization domains suitable for use in the specific embodiments contemplated herein include FK506 binding protein (FKBP) polypeptides or variants thereof, FKBP-rapamycin binding (FRB) polypeptides or variants thereof, calcineurin polypeptides or variants thereof, cyclophilin polypeptides or variants thereof, bacterial dihydrofolate reductase (DHFR) polypeptides or variants thereof, PYR1-like 1 (PYL1) polypeptides or variants thereof, abscisic acid insensitive 1 (ABI1) polypeptides or variants thereof, GIB1 polypeptides or variants thereof, or GAI polypeptides or variants thereof.

[0188] As used herein, the term "FKBP-rapamycin binding polypeptide" refers to a FRB polypeptide. In certain embodiments, the FRB polypeptide is a FKBP12-rapamycin binding polypeptide. FRB polypeptides suitable for use in the specific embodiments contemplated herein generally contain at least about 85 to about 100 amino acid residues. In certain embodiments, with reference to GenBank Accession No. L34075.1, the FRB polypeptide comprises a 93 amino acid sequence of Ile-2021 to Lys-2113 and a T2098L mutation. The FRB polypeptides contemplated herein bind to the FKBP polypeptide via a bridging factor, thereby forming a ternary complex.

[0189] 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 comprising an F36V mutation. In certain embodiments, the FKBP domain may also be referred to as a "rapamycin binding domain." Nucleotide sequences, cloning, and other information regarding various FKBP species are known in the art (see, Staendart et al., Nature 346:671, 1990 (human FKBP12); Kay, Biochem. J. 314:361, 1996). The FKBP polypeptides encompassed herein bind to the FRB polypeptide via a bridging factor, thereby forming a ternary complex.

[0190] "Bridging factor" refers to a molecule that associates with two or more multimerization domains and is placed between the two or more multimerization domains. In certain embodiments, the multimerization domain substantially contributes to or efficiently promotes the formation of a polypeptide complex only in the presence of a bridging factor. In certain embodiments, the multimerization domain does not contribute to or efficiently promote the formation of a polypeptide complex in the absence of a bridging factor. Illustrative examples of bridging factors suitable for use in the specific embodiments contemplated herein include, but are not limited to, AP21967, rapamycin (sirolimus) or a rapamycin analog, kumarin 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.

[0191] Rapamycin analogs (rapalogs) include, but are not limited to, those disclosed in U.S. Patent No. 6,649,595, the structure of which is incorporated herein by reference in its entirety. In certain embodiments, the bridging factor is a rapamycin analog that has significantly lower immunosuppressive effects than rapamycin. In a preferred embodiment, the rapamycin analog is AP21967 (also known as C? 16-(S)-7-methylindorapamycin, IC 50 = 10 nM, chemically modified non-immunosuppressive rapamycin analogs). Other illustrative rapamycin analogs suitable for use in specific embodiments contemplated herein include, but are not limited to, everolimus, norflulimus, pimecrolimus, defostiolimus, tacrolimus, temsirolimus, umimios, and zotarolimus.

[0192] "Significantly reduced immunosuppressive effect" means at least 0.1-fold to 0.005-fold less than the immunosuppressive effect observed or expected for the same dose measured clinically or in an appropriate in vitro (e.g., inhibition of T cell proliferation) or in vivo surrogate of human immunosuppressive activity.

[0193] A "transmembrane domain" or "TM domain" is a domain that anchors a polypeptide to the plasma membrane of a cell. A TM domain can be derived from natural, synthetic, semisynthetic or recombinant sources.

[0194] 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 the release of cytotoxic factors, or other cellular responses initiated by antigen binding to receptors expressed on immune effector cells.

[0195] "Intracellular signaling domain" or "intracellular domain" refers to a portion of a protein that transduces an effector function signal and directs the cell to perform a specialized function. Although the entire intracellular signaling domain can often be employed, in many cases it is not necessary to use the entire domain. To the extent that a truncated portion of an intracellular signaling domain is employed, such a truncated portion can be used in place of the entire domain as long as it transduces an effector function signal. The term intracellular signaling domain means any truncated portion comprising an intracellular signaling domain that is necessary or sufficient to transduce an effector function signal.

[0196] It is known that the signal generated by TCR alone is insufficient to fully activate T cells and that secondary or co-stimulatory signals are also required. Therefore, it can be said that T cell activation is mediated by two different categories of intracellular signaling domains: primary signaling domains, which initiate antigen-dependent primary activation through TCR (e.g., TCR / CD3 complex); and co-stimulatory signaling domains, which act in an antigen-independent manner to provide secondary or co-stimulatory signals.

[0197] A "primary signaling domain" refers to an intracellular signaling domain that regulates the primary activation of the TCR complex in a stimulatory or inhibitory manner. A primary signaling domain that acts in a stimulatory manner may contain a signaling motif known as an immunoreceptor tyrosine-based activation motif or ITAM. Illustrative examples of ITAM-containing primary signaling domains suitable for use in particular embodiments include, but are not limited to, those derived from FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d.

[0198] As used herein, the term "costimulatory signaling domain" or "costimulatory domain" refers to the intracellular signaling domain of a costimulatory molecule. Costimulatory molecules are cell surface molecules other than antigen receptors or Fc receptors that provide a second signal required for the effective activation and function of T lymphocytes after binding to an antigen. Illustrative examples of such costimulatory molecules from which costimulatory domains can be isolated include, but are not limited to, 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 (IC The proteins involved in the TNF receptor kinase 70 (TNFRS70) are TNF receptors with a high affinity for TNF-α and TNF-α.

[0199] An "immune disorder" refers to a disease that causes a response from the immune system. In certain embodiments, the term "immune disorder" refers to cancer, an autoimmune disease, or an immunodeficiency.

[0200] As used herein, the term "cancer" generally refers to a class of diseases or conditions in which abnormal cells divide without control and may invade nearby tissues.

[0201] As used herein, the term "malignant" refers to a group of cancer cells that show one or more of uncontrolled growth (i.e., division beyond normal limits), invasion (i.e., invasion and destruction of adjacent tissues), and metastasis (i.e., spread to other locations in the body via the lymph or blood). As used herein, the term "metastasis" refers to the spread of cancer from one part of the body to another. Tumors formed by cells that have spread are called "metastatic tumors" or "metastasis". Metastatic tumors contain cells similar to those in the original (primary) tumor.

[0202] As used herein, the term "benign" or "non-malignant" refers to a tumor that can grow larger but does not spread to other parts of the body. Benign tumors are self-limited and usually do not invade or metastasize.

[0203] "Cancer cell" refers to a single cell of a cancerous growth or tissue. Cancer cells include solid cancers and liquid cancers. "Tumor" or "tumor cell" generally refers to a swelling or lesion formed by an abnormal growth of cells, which can be benign, pre-malignant, or malignant. Most cancers form tumors, but liquid cancers such as leukemias do not necessarily form tumors. For those 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.

[0204] The term "relapse" refers to the return of cancer diagnosis or signs and symptoms after a period of improvement or remission.

[0205] "Remission" is also called "clinical remission" and includes both partial remission and complete remission. In a partial remission, some but not all signs and symptoms of cancer have disappeared. In a complete remission, all signs and symptoms of cancer have disappeared, although the cancer may still be in the body.

[0206] "Refractory" means that the cancer is resistant or unresponsive to treatment with a particular therapeutic agent. A cancer can be refractory at the start of treatment (i.e., unresponsive to initial exposure to the therapeutic agent), or it can become refractory because it develops resistance to the therapeutic agent during the first treatment period or during subsequent treatment periods.

[0207] 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 encompassed elsewhere herein. Suitable subjects (e.g., patients) include experimental animals (such as mice, rats, rabbits, or guinea pigs), farm animals, and domestic animals or pets (such as cats or dogs). Non-human primates and preferably human patients are included. Typical subjects include human patients who have, have been diagnosed as having, or are at risk of having cancer or another immune condition.

[0208] As used herein, the term "patient" refers to a subject who has been diagnosed with cancer or another immune disorder that can be treated with the compositions and methods disclosed elsewhere herein.

[0209] As used herein, "treatment" or "treating" encompasses any beneficial or desired effect on the symptoms or pathology of a disease or pathological condition and may even encompass a minimal reduction in one or more measurable markers of the disease or condition being treated. Optionally, treatment may involve a reduction in the disease or condition or a delay in the progression of the disease or condition, such as delaying tumor growth. "Treatment" does not necessarily indicate complete eradication or cure of the disease or condition or its associated symptoms.

[0210] As used herein, "prevent" and similar words such as "prevented" or "preventing" refer to actions that prevent, inhibit, or reduce the likelihood of the occurrence or recurrence of a disease or condition. Prevention 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 words also encompass reducing the intensity, effects, symptoms, and / or burden of a disease or condition prior to the onset or recurrence of the disease or condition.

[0211] As used herein, the phrase "alleviating at least one symptom of" refers to reducing one or more symptoms of the disease or condition being treated. In certain embodiments, the disease or condition being treated is cancer, wherein the one or more symptoms alleviated include, but are not limited to, weakness, fatigue, shortness of breath, easy bruising and bleeding, frequent infections, swollen lymph nodes, abdominal swelling or pain (due to enlarged abdominal organs), bone or joint pain, bone fractures, unintentional weight loss, loss of appetite, night sweats, persistent mild fever, and decreased urination (due to impaired kidney function).

[0212] "Enhancement" or "promotion" or "increase" or "amplification" generally refers to that the compositions encompassed herein can produce, induce or cause a greater physiological response (i.e., downstream effect) compared to the response caused by a vehicle or control molecule / composition. Measurable physiological responses can include an increase in T cell expansion, activation, persistence, cytokine secretion and / or an increase in cancer cell killing ability, as well as other aspects apparent from the understanding of the art and the description herein. An "increased" or "enhanced" amount is typically a "statistically significant" amount and can include an increase of 1.1 times, 1.2 times, 1.5 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 15 times, 20 times, 30 times or more (e.g., 500 times, 1000 times) (including all integers and decimal points therebetween and above 1, such as 1.5, 1.6, 1.7, 1.8, etc.) of the response produced by a vehicle or control composition.

[0213] "Decrease" or "attenuate" or "become less" or "reduce" or "mitigate" generally refers to the ability of the compositions encompassed herein to produce, elicit or induce less of a response (i.e., a physiological response) than the response elicited by vehicle or a control molecule / composition. A "decreased" or "reduced" amount is generally a "statistically significant" amount and can include a decrease of 1.1-fold, 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold or more (e.g., 500-fold, 1000-fold) (including all integers and decimal points in between and above 1, such as 1.5, 1.6, 1.7, 1.8, etc.) compared to the response produced by vehicle, a control composition, or a response in a particular cell lineage (reference response).

[0214] "Maintain" or "maintain" or "no change" or "no substantial change" or "no substantial decrease" generally refers to the ability of the compositions contemplated herein to produce, elicit or cause a substantially similar or comparable physiological response (i.e., downstream effect) in a cell compared to the response elicited by vehicle, a control molecule / composition, or a response in a particular cell lineage. A comparable response is one that is not significantly different or measurably different from a reference response.

[0215] Additional definitions are set forth throughout this disclosure.

[0216] C.CD33 VHH DARIC

[0217] In certain embodiments, DARIC receptors comprising anti-CD33 VHH domains are contemplated that redirect immune effector cell cytotoxicity toward CD33-expressing cancer cells. As used herein, the terms "CD33 VHH DARIC receptor," "anti-CD33 VHH DARIC receptor," "CD33 VHH DARIC," or "anti-CD33 VHH DARIC" are used interchangeably and refer to one or more non-naturally occurring polypeptides that, when exposed to target cells expressing full-length CD33 or a CD33 splice variant and a multimerizing agent or bridging factor, transduce immunostimulatory signals in immune effector cells, e.g., stimulate immune effector cell activity and function, increase production and / or secretion of proinflammatory cytokines. In preferred embodiments, the CD33 VHH DARIC is a multi-chain chimeric receptor comprising a DARIC signaling component and a DARIC binding component comprising a VHH domain that recognizes full-length CD33 and / or a CD33 splice variant.

[0218] 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 a specific embodiment, the DARIC signaling component is expressed from a cell and the DARIC binding component is supplied exogenously as a polypeptide. In one embodiment, the DARIC binding component pre-loaded with the bridging factor is supplied exogenously to the cell expressing the DARIC signaling component.

[0219] 1.CD33 DARIC signaling components

[0220] 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, the DARIC signaling component comprises a multimerization domain, a transmembrane domain, a costimulatory domain, and / or a primary signaling domain. In certain embodiments, the DARIC signaling component comprises a first multimerization domain, a first transmembrane domain, a first costimulatory domain, and / or a primary signaling domain.

[0221] In certain embodiments, the DARIC signaling component includes one or more multimerization domains.

[0222] Illustrative examples of multimerization domains suitable for use in the specific CD33 DARIC signaling components contemplated herein include, but are not limited to, FK506 binding protein (FKBP) polypeptides or variants thereof, FKBP-rapamycin binding (FRB) polypeptides or variants thereof, calcineurin polypeptides or variants thereof, cyclophilin polypeptides or variants thereof, bacterial dihydrofolate reductase (DHFR) polypeptides or variants thereof, PYR1-like 1 (PYL1) polypeptides or variants thereof, and abscisic acid insensitive 1 (ABI1) polypeptides or variants thereof.

[0223] In certain embodiments, the CD33 DARIC signaling component comprises a FRB polypeptide.

[0224] In particularly preferred embodiments, the CD33 DARIC signaling component comprises a FRB polypeptide comprising a T2098L mutation, or a variant thereof. In certain preferred embodiments, the CD33 DARIC signaling component comprises a FKBP12 polypeptide, or a variant thereof.

[0225] In some embodiments, the CD33 VHH DARIC signaling component includes a hinge domain.

[0226] Illustrative 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 wild-type hinge regions from these molecules or may be altered.

[0227] In certain embodiments, the DARIC signaling component includes a transmembrane domain.

[0228] In certain embodiments, the DARIC signaling component includes a hinge domain and a transmembrane domain.

[0229] Illustrative examples of transmembrane domains suitable for use in the specific CD33 DARIC signaling components contemplated herein include, but are not limited to, the transmembrane regions of the α, β, γ, or δ chains of the T cell receptor, CD3ε, CD3ζ, CD4, CD5, CD8α, CD9, CD 16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD71, CD80, CD86, CD 134, CD137, CD152, CD 154, amnion free protein (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.

[0230] In certain embodiments, the DARIC signaling component includes a linker that connects the C-terminus of the transmembrane domain to the N-terminus of the intracellular signaling domain. In various preferred embodiments, a short oligopeptide linker or polypeptide linker, preferably between 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.

[0231] The DARIC signaling components contemplated in certain embodiments herein include one or more intracellular signaling domains. In one embodiment, the CD33 DARIC signaling component includes one or more co-stimulatory signaling domains and / or primary signaling domains. In one embodiment, the intracellular signaling domain includes an immunoreceptor tyrosine-based activation motif (ITAM).

[0232] Illustrative 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 a preferred embodiment, the CD33 DARIC signaling component comprises a CD3ζ primary signaling domain and one or more costimulatory signaling domains. The primary signaling domain and the costimulatory signaling domain can be linked in tandem to the carboxyl terminus of the transmembrane domain in any order.

[0233] Illustrative examples of costimulatory domains suitable for use in the specific CD33 DARIC signaling components contemplated herein include, but are not limited to, those 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-activating protein 10 (DAP10), adaptor for activated T cells family member 1 (LAT), SH2 domain-containing leukocyte protein of 76 kD (SLP76), T-cell receptor-associated transmembrane adaptor 1 (TRAT1), TNFR2, TNF receptor superfamily member 14 (TNFRS14; HVEM), TNF receptor superfamily member 18 (TNFRS18; GITR), TNF receptor superfamily member 25 (TNFRS25; DR3), and T-cell receptor-associated protein kinase 70 zeta chain (ZAP70).

[0234] In certain embodiments, the CD33 DARIC signaling components contemplated herein include a signal peptide. Illustrative examples of signal peptides suitable for use in specific CD33 DARIC signaling components include, but are not limited to, an IgG1 heavy chain signaling polypeptide, an Igκ light chain signaling polypeptide, a CD8α signaling polypeptide, or a human GM-CSF receptor α signaling polypeptide. In various preferred embodiments, the CD33 DARIC signaling component includes a CD8α signaling polypeptide.

[0235] 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 CD3 zeta primary signaling domain. In certain embodiments, the CD33 DARIC signaling component comprises a CD137 costimulatory domain and a CD3 zeta primary signaling domain.

[0236] In a preferred embodiment, the CD33 DARIC signaling components include the FRB T2098L multimerization domain, the CD8α transmembrane domain, the CD137 co-stimulatory domain, and the CD3ζ primary signaling domain.

[0237] In a preferred embodiment, the CD33 VHH DARIC signaling component comprises the amino acid sequence shown in SEQ ID NO:82.

[0238] 2.CD33 DARIC binding components

[0239] "DARIC binding component," "DARIC binding polypeptide," "CD33 VHH 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 specific embodiments, the CD33 VHH DARIC binding component comprises an anti-CD33 VHH domain, a multimerization domain, and a transmembrane domain. In specific embodiments, the CD33 VHH DARIC binding component comprises an anti-CD33 VHH domain, a second multimerization domain, and a second transmembrane domain. In other specific embodiments, the 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 specific embodiments, the CD33 VHH DARIC binding component comprises an anti-CD33 VHH domain, a second multimerization domain, a second transmembrane domain, and a second costimulatory domain.

[0240] In certain embodiments, the CD33 VHH DARIC binding component comprises one or more anti-CD33 VHH domains.

[0241] In a particularly preferred embodiment, the anti-CD33 VHH domain is a humanized Camelidae VHH. In a specific embodiment, the anti-CD33 VHH domain is a humanized Camelidae VHH that binds to 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 a specific embodiment, the anti-CD33 VHH domain is a humanized Camelidae VHH that binds to the same one or more epitopes displayed on both full-length CD33 and a CD33 splice variant.

[0242] In particularly preferred embodiments, the anti-CD33 VHH domain is a humanized Camelidae VHH comprising the amino acid sequence set forth in any one of SEQ ID NOs: 3-6, 10-11, 13-16, and 20-21. In certain preferred embodiments, the anti-CD33 VHH domain is a humanized Camelidae VHH comprising the amino acid sequence set forth in SEQ ID NO: 10. In certain preferred embodiments, the anti-CD33 VHH domain is a humanized Camelidae VHH comprising the amino acid sequence set forth in SEQ ID NO: 20.

[0243] In certain embodiments, a DARIC binding component comprises one or more multimerization domains.

[0244] Illustrative examples of multimerization domains suitable for use in the specific CD33 VHH DARIC binding components contemplated herein include, but are not limited to, FKBP polypeptides or variants thereof, FRB polypeptides or variants thereof, calcineurin polypeptides or variants thereof, cyclophilin polypeptides or variants thereof, DHFR polypeptides or variants thereof, PYL1 polypeptides or variants thereof, and ABI1 polypeptides or variants thereof.

[0245] In certain embodiments, the CD33 VHH DARIC binding component comprises a FRB polypeptide or a variant thereof, and the DARIC signaling component comprises a FKBP polypeptide or a variant thereof. In preferred embodiments, the CD33 VHH DARIC binding component comprises a FRB polypeptide or a variant thereof comprising a T2098L mutation, and the DARIC signaling component comprises a FKBP12 polypeptide or a variant thereof.

[0246] 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.

[0247] In some embodiments, the CD33 VHH DARIC binding component includes a hinge domain.

[0248] Illustrative hinge domains suitable for use in the CD33 VHH DARIC 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 wild-type hinge regions from these molecules or may be altered.

[0249] In certain embodiments, the DARIC binding component comprises a transmembrane domain. In certain embodiments, the DARIC binding component comprises a hinge domain and a transmembrane domain. In one embodiment, the transmembrane domain can be the same as the transmembrane domain used in the DARIC signaling component. In one embodiment, the transmembrane domain can be different from the transmembrane domain used in the DARIC signaling component.

[0250] 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 α, β, γ, or δ 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, amniotic fluid (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 VHH DARIC binding component comprises a CD4 transmembrane domain.

[0251] In various preferred embodiments, a short oligopeptide linker or polypeptide linker, preferably between 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.

[0252] The DARIC binding components contemplated in certain embodiments herein do not comprise one or more intracellular signaling domains.

[0253] In other specific embodiments, the CD33 VHH DARIC binding components contemplated herein include one or more intracellular signaling domains. In preferred embodiments, wherein the CD33 VHH DARIC binding component includes one or more intracellular signaling domains, those domains are different from the intracellular signaling domains present in the cognate CD33 DARIC signaling component. In one embodiment, the CD33 VHH DARIC binding component includes a costimulatory signaling domain.

[0254] Illustrative examples of costimulatory domains suitable for use in the specific CD33 VHH DARIC binding components contemplated herein include, but are not limited to, those 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-activating protein 10 (DAP10), linker for activated T-cell family member 1 (LAT), SH2 domain-containing leukocyte protein of 76 kD (SLP76), T-cell receptor-associated transmembrane adaptor 1 (TRAT1), TNFR2, TNF receptor superfamily member 14 (TNFRS14; HVEM), TNF receptor superfamily member 18 (TNFRS18; GITR), TNF receptor superfamily member 25 (TNFRS25; DR3), and zeta chain of T-cell receptor-associated protein kinase 70 (ZAP70). In preferred embodiments, the costimulatory domain is derived, obtained, or isolated from TNFR2 or OX40.

[0255] In certain embodiments, the DARIC binding components contemplated herein include a signal peptide. Illustrative examples of signal peptides suitable for use in specific CD33VHH DARIC binding components include, but are not limited to, IgG1 heavy chain signal polypeptide, Igκ light chain signal polypeptide, CD8α signal polypeptide, or human GM-CSF receptor α signal polypeptide. In various preferred embodiments, the CD33VHH DARIC binding component includes a CD8α signal polypeptide.

[0256] In certain embodiments, the CD33 VHH DARIC binding component comprises a CD33-binding VHH domain, a FKBP12 multimerization domain, a CD4 transmembrane domain, and optionally a costimulatory domain.

[0257] In certain embodiments, the CD33 VHH DARIC binding component comprises a CD33 binding VHH and a FKBP12 multimerization domain.

[0258] In some embodiments, the CD33 VHH DARIC binding component comprises a VHH domain comprising the amino acid sequence set forth in any one of SEQ ID NOs: 2-21, a FKBP12 multimerization domain, a CD4 transmembrane domain, and optionally a costimulatory domain.

[0259] In some embodiments, the CD33 VHH DARIC binding component includes a VHH domain comprising the amino acid sequence set forth in SEQ ID NO: 10 or SEQ ID NO: 20, a FKBP12 multimerization domain, a CD4 transmembrane domain, and optionally a costimulatory domain.

[0260] In some embodiments, the CD33 VHH DARIC binding component comprises a VHH domain comprising the amino acid sequence set forth in any one of SEQ ID NOs: 2-21 and a FKBP12 multimerization domain.

[0261] 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 and a FKBP12 multimerization domain.

[0262] In some embodiments, the CD33 VHH DARIC binding component comprises the amino acid sequence set forth in any one of SEQ ID NOs: 22-31.

[0263] In some embodiments, the CD33 VHH DARIC binding component comprises the amino acid sequence set forth in SEQ ID NO:30.

[0264] 3. Bridging Factor

[0265] The bridging factors contemplated herein, in specific embodiments, mediate or promote the association of the CD33 DARIC signaling component and the CD33 VHH DARIC binding component via the multimerization domains of each component. The bridging factor associates with and is positioned between the multimerization domains to promote the association of the CD33 DARIC signaling component and the CD33 VHH DARIC binding component. In the presence of the bridging factor, when the CD33 VHH DARIC binding component binds to CD33 expressed on a target cell, the CD33 VHH DARIC binding component associates with the CD33 DARIC signaling component and elicits immune effector cell activity against the target cell. In the absence of the bridging factor, the CD33 VHH DARIC binding component does not associate with the CD33 DARIC signaling component, and the CD33 VHH DARIC is inactive.

[0266] 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 (ABI1).

[0267] In certain embodiments, the multimerization domain of the CD33 VHH DARIC signaling and binding component is associated with a bridging factor selected from the group consisting of rapamycin or a rapamycin analog, coumarins or derivatives thereof, gibberellins or derivatives thereof, abscisic acid (ABA) or derivatives thereof, methotrexate or derivatives thereof, cyclosporin A or derivatives thereof, FK506 / cyclosporin A (FKCsA) or derivatives thereof, trimethoprim (Tmp)-FK506 binding protein (FKBP) synthetic ligand (SLF) or derivatives thereof.

[0268] 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 signaling component comprises a FRB multimerization domain or variants thereof, and the CD33 VHH DARIC binding component comprises a FKBP multimerization domain or variants thereof. In particularly preferred embodiments, the CD33 DARIC signaling component comprises a FRB T2098L multimerization domain or variants thereof, and the CD33 VHH DARIC binding component comprises an FKBP12 or FKBP12 F36V multimerization domain or variants thereof.

[0269] Illustrative examples of bridging factors suitable for use in the specific embodiments contemplated herein include, but are not limited to, AP1903, AP20187, AP21967 (also known as C? 16-(S)-7-methylindorapamycin), everolimus, norflulimus, pimecrolimus, defostiolimus, tacrolimus, temsirolimus, umimios, and zotarolimus. In certain preferred embodiments, the bridging factor is AP21967. In certain preferred embodiments, the bridging factor is a non-immunosuppressive dose of sirolimus (rapamycin).

[0270] D. Anti-CD33 Chimeric Antigen Receptor

[0271] In certain embodiments, the immune effector cells encompassed herein include anti-CD33 VHH CARs. Chimeric antigen receptors (CARs) are molecules that combine the specificity of antibodies for target antigens (e.g., tumor antigens) with T cell receptor activation intracellular domains to produce chimeric proteins that exhibit specific anti-tumor cell immune activity. As used herein, the term "chimeric" describes a protein composed of different proteins or DNA portions from different sources.

[0272] In certain embodiments, T cells are engineered by introducing a polynucleotide encoding an anti-CD33 VHH CAR.

[0273] In certain embodiments, T cells are engineered by introducing a vector comprising a polynucleotide encoding an anti-CD33 VHH CAR.

[0274] 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. The main feature of CAR is its ability to leverage the cell-specific targeting capabilities of monoclonal antibodies, soluble ligands, or cell-specific co-receptors to redirect immune effector cell specificity, thereby triggering proliferation, cytokine production, phagocytosis, or the production of molecules that can mediate cell death of target antigen-expressing cells in a major histocompatibility (MHC)-independent manner.

[0275] In some embodiments, the anti-CD33 VHH CAR comprises a spacer domain. In specific embodiments, the spacer domain comprises CH2 and CH3 of IgG1, IgG4, or IgD.

[0276] Illustrative 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 can be wild-type hinge regions from these molecules or can be altered. In another embodiment, the hinge domain comprises a CD8α hinge region.

[0277] The "transmembrane (TM) domain" of the CAR fuses the extracellular binding portion and the intracellular signaling domain and anchors the CAR to the plasma membrane of the immune effector cell. The TM domain can be derived from natural, synthetic, semisynthetic or recombinant sources.

[0278] Illustrative TM domains can be derived from (i.e., include at least one or more transmembrane regions of) the alpha, beta, gamma, or delta chain of a T cell receptor, CD3ε, CD3ζ, CD4, CD5, CD8α, CD9, CD 16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD71, CD80, CD86, CD 134, CD137, CD152, CD 154, AMN, and PDCD1.

[0279] In one embodiment, the anti-CD33 VHH CAR comprises a TM domain derived from CD8α. In another embodiment, the CAR encompassed herein comprises a TM domain derived from CD8α and a short oligopeptide or polypeptide linker preferably between 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids in length, which connects the TM domain of the CAR to the intracellular signaling domain. A glycine-serine linker provides a particularly suitable linker.

[0280] In preferred embodiments, the anti-CD33 VHH CAR includes an intracellular signaling domain comprising one or more "co-stimulatory signaling domains" and a "primary signaling domain."

[0281] The primary signaling domain, which acts in a stimulatory manner, may contain signaling motifs known as immunoreceptor tyrosine-based activation motifs or ITAMs.

[0282] Illustrative examples of ITAM-containing primary signaling domains suitable for use in the anti-CD33 VHH CARs encompassed in specific embodiments include those derived from FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d. In a specific preferred embodiment, 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.

[0283] 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.

[0284] Illustrative examples of such co-stimulatory molecules suitable for use in the anti-CD33 VHH CARs encompassed in specific 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 co-stimulatory signaling domains selected from the group consisting of CD28, CD137, and CD134, and a CD3 zeta primary signaling domain.

[0285] In various embodiments, the anti-CD33 VHH CAR comprises: a VHH that binds CD33; a transmembrane domain isolated from a polypeptide selected from the group consisting of: CD4, CD8α, CD154, and PD-1; one or more intracellular co-stimulatory 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.

[0286] In various embodiments, the anti-CD33 VHH CAR comprises: a VHH that binds CD33; a transmembrane domain isolated from a polypeptide selected from the group consisting of: CD4, CD8α, CD154, and PD-1; one or more intracellular co-stimulatory 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.

[0287] In a preferred embodiment, the anti-CD33 VHH CAR comprises a VHH comprising the amino acid sequence shown in any one of SEQ ID NOs: 2-21, a CD8α hinge domain, a CD8α transmembrane domain, a 4-1BB costimulatory domain, and a CD3ζ primary signaling domain.

[0288] In a preferred embodiment, the anti-CD33 VHH CAR comprises a VHH comprising the amino acid sequence shown in SEQ ID NO: 10, a CD8a hinge domain, a CD8a transmembrane domain, a 4-1BB co-stimulatory domain, and a CD3ζ primary signaling domain.

[0289] In a specific embodiment, the anti-CD33 VHH CAR comprises the sequence set forth in any one of SEQ ID NOs: 62-81.

[0290] In certain embodiments, the anti-CD33 VHH CAR comprises the sequence shown in SEQ ID NO:70 or SEQ ID NO:80.

[0291] E. Peptide

[0292] Various polypeptides are contemplated herein, including but not limited to CD33 VHH DARICs, CD33 VHH DARIC binding components, CD33 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. Unless otherwise indicated, "polypeptide," "peptide," and "protein" are used interchangeably and are defined according to conventional meanings, i.e., as amino acid sequences. In one embodiment, "polypeptide" includes fusion polypeptides and other variants. Polypeptides can be prepared using any of a variety of well-known recombinant and / or synthetic techniques. Polypeptides are not limited to a specific length; for example, they may comprise a full-length protein sequence, a fragment of a full-length protein, or a fusion protein, and may include post-translational modifications of the polypeptide, such as glycosylation, acetylation, phosphorylation, and the like, as well as other naturally occurring and non-naturally occurring modifications known in the art. In certain preferred embodiments, fusion polypeptides, polypeptides, fragments thereof, and other variants are prepared, obtained, or isolated from one or more human polypeptides.

[0293] As used herein, "isolated peptide" or "isolated polypeptide" and the like refer to a peptide or polypeptide molecule that has been isolated and / or purified in vitro from its cellular environment and from association with other components of the cell, i.e., the peptide or polypeptide molecule is not significantly associated with substances in the body. In certain embodiments, the isolated polypeptide is a synthetic polypeptide, a semisynthetic polypeptide, or a polypeptide obtained or derived from a recombinant source.

[0294] Polypeptides include "polypeptide variants." A polypeptide variant may differ from a naturally occurring polypeptide by one or more substitutions, deletions, additions, and / or insertions. Such variants may be naturally occurring, such as splice variants, or may be generated synthetically, such as by modifying one or more of the above-described polypeptide sequences. 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, the polypeptide comprises a polypeptide 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 encompassed herein, typically wherein the variant maintains 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.

[0295] Polypeptide variants include biologically active "polypeptide fragments". Illustrative examples of biologically active polypeptide fragments include anti-CD33 VHH domains, intracellular signaling domains, and the like. As used herein, the term "biologically active fragment" or "minimum biologically active fragment" refers to a polypeptide fragment that retains at least 100%, at least 90%, at least 80%, at least 70%, at least 60%, at least 50%, at least 40%, at least 30%, at least 20%, at least 10%, or at least 5% of the activity of a naturally occurring polypeptide. In certain embodiments, a polypeptide fragment may comprise an amino acid chain of at least 5 to about 1700 amino acids in length. It will be appreciated that in certain embodiments, a fragment is 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, 1500, 1600, 1700 or more amino acids in length.

[0296] In certain embodiments, the polypeptides listed herein may include one or more amino acids designated as "X" or "Xaa," which are used interchangeably. "X," if present in an amino acid SEQ ID NO, refers to any one or more amino acids. In certain embodiments, a SEQ ID NO representing a fusion protein includes a sequence of consecutive X residues that cumulatively represent any amino acid sequence. In certain embodiments, "XX" represents a combination of any two amino acids. In certain embodiments, "XX" represents two serines, i.e., SS. In certain embodiments, "XX" represents a combination of any two amino acids that reduces immunogenicity.

[0297] In a preferred embodiment, "XX" represents amino acids KP.

[0298] As described above, polypeptides can be altered in various ways, including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known in the art. For example, amino acid sequence variants of a reference polypeptide can be prepared by mutations in the DNA. Methods for mutagenesis and nucleotide sequence alterations are well known in the art. See, for example, Kunkel (1985, Proc. Natl. Acad. Sci. USA. 82: 488-492), Kunkel et al. (1987, Methods in Enzymol, 154: 367-382), U.S. Patent No. 4,873,192, Watson, JD et al. (Molecular Biology of the Gene, Fourth Edition, Benjamin / Cummings, Menlo Park, Calif., 1987), and references cited therein. Guidance for appropriate amino acid substitutions that do not affect the biological activity of the protein of interest can be found in the model of Dayhoff et al. (1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, DC).

[0299] In certain embodiments, polypeptide variants comprises one or more conservative replacements." conservative replacement " is that amino acid is replaced by another amino acid with similar characteristics, makes the technical staff of peptide chemistry field can expect the secondary structure of polypeptide and the replacement that hydrophilicity is constant basically.Modification can be carried out at the structure of the polynucleotide and polypeptide contained in the specific embodiment and still obtained the variant or the derivative polypeptide with desired characteristics being encoded functional molecule.When wishing to change the amino acid sequence of polypeptide to produce the variant polypeptide of equivalence or even improvement, those skilled in the art for example can change one or more codons of coding DNA sequence, for example according to Table 1.

[0300] Table 1 - Amino acid codons

[0301]

[0302]

[0303] Guidance on determining which amino acid residues can be substituted, inserted or deleted without eliminating biological activity can be found using computer programs well 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., replacements of similarly charged or uncharged amino acids. Conservative amino acid changes involve replacement of one of a group of amino acids associated with a side chain. Naturally occurring amino acids are generally divided into four groups: acidic amino acids (aspartic acid, glutamic acid), basic amino acids (lysine, arginine, histidine), non-polar 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 amino acid substitutions are known to those skilled in the art and can generally be made without changing the biological activity of the resulting molecule. Those skilled in the art will recognize that, generally speaking, single amino acid substitutions in non-essential regions of a polypeptide will not substantially alter biological activity (see, e.g., Watson et al. Molecular Biology of the Gene, 4th ed., 1987, The Benjamin / Cummings Pub. Co., p. 224).

[0304] In one embodiment, where expression of two or more polypeptides is desired, the polynucleotide sequences encoding the two or more polypeptides may be separated by an IRES sequence or a polynucleotide sequence encoding a ribosomal skipping sequence, as disclosed elsewhere herein.

[0305] Polypeptides encompassed in certain embodiments include fusion polypeptides. In certain embodiments, fusion polypeptides and polynucleotides encoding fusion polypeptides are provided. Fusion polypeptides and fusion proteins refer to polypeptides having at least two, three, four, five, six, seven, eight, nine, or ten 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.

[0306] In another embodiment, two or more CD33 VHH DARIC components and / or other polypeptides may be expressed as a fusion protein comprising one or more self-cleaving peptide sequences between the polypeptides disclosed elsewhere herein.

[0307] 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 DARIC binding component.

[0308] In certain embodiments, the fusion polypeptide comprises a CD33 DARIC signaling component, a self-cleaving polypeptide sequence or a ribosome-skipping sequence, a CD33 VHH DARIC binding component, another self-cleaving polypeptide sequence or a ribosome-skipping sequence, and another DARIC binding component to another target antigen.

[0309] Fusion polypeptides may include: one or more polypeptide domains or fragments, including but not limited to signal peptides, cell permeability 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 C-terminally linked to the N-terminus, but they can also be C-terminally linked to the C-terminus, N-terminally linked to the N-terminus, or N-terminally linked to the C-terminus. In certain embodiments, the polypeptides of the fusion protein can follow any order. Fusion polypeptides or fusion proteins may also include conservatively modified variants, polymorphic variants, alleles, mutants, subsequences, and interspecies homologs, as long as the desired activity of the fusion polypeptide is preserved. Fusion polypeptides can be made by chemical synthesis methods or by chemical linkage between the two parts, or can generally be prepared using other standard techniques. The linked DNA sequences that constitute the fusion polypeptide are operably linked to suitable transcriptional or translational control elements as disclosed elsewhere herein.

[0310] The fusion polypeptide may optionally include one or more linkers that can be used to connect one or more polypeptides or domains within the polypeptide. Peptide linker sequences can be used to separate any two or more polypeptide groups by a distance sufficient to ensure that each polypeptide folds into its appropriate secondary and tertiary structures, thereby allowing the polypeptide domains to perform their desired functions. Such peptide linker sequences are incorporated into the fusion polypeptide using standard techniques in the art. Suitable peptide linker sequences can be selected based on the following factors: (1) their ability to present a flexible extended conformation; (2) their inability to present a secondary structure that can interact with functional epitopes on the first and second polypeptides; and (3) the lack of hydrophobic or charged residues that may react with functional epitopes on the polypeptides. In certain embodiments, preferred peptide linker sequences contain Gly, Asn, and Ser residues. Other nearly neutral amino acids, such as Thr and Ala, may also be used in the linker sequence. Amino acid sequences that can be usefully used as linkers include Maratea et al., Gene 40: 39-46, 1985; Murphy et al., Proc. Natl. Acad. Sci. USA 83: 8258-8262, 1986; U.S. Pat. No. 4,935,233 and U.S. Pat. No. 4,751,180. When 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, a linker sequence is not required. In certain embodiments, preferred linkers are typically flexible amino acid subsequences that are synthesized as part of a recombinant fusion protein. The linker polypeptide can be between 1 and 200 amino acids in length, between 1 and 100 amino acids in length, or between 1 and 50 amino acids in length, including all integer values therebetween.

[0311] Exemplary polypeptide cleavage signals include polypeptide cleavage recognition sites, such as protease cleavage sites, nuclease cleavage sites (e.g., rare restriction enzyme recognition sites, self-cleaving ribozyme recognition sites), and self-cleaving viral oligopeptides (see deFelipe and Ryan, 2004. Traffic, 5(8); 616-26).

[0312] Suitable protease cleavage sites and self-cleaving peptides are known to the skilled person (see, for example, Ryan et al., 1997. J. Gener. Virol. 78, 699-722; Scymczak et al. (2004) Nature Biotech. 5, 589-594). Exemplary protease cleavage sites include, but are not limited to, cleavage sites for potyvirus NIa protease (e.g., tobacco etch virus protease), potyvirus HC protease, potyvirus PI (P35) protease, byovirus NIa protease, protease encoded by byovirus RNA-2, aphthovirus L protease, enterovirus 2A protease, rhinovirus 2A protease, picorna 3C protease, comovirus 24K protease, nepovirus 24K protease, rice tungro sphericalvirus (RTSV) 3C-like protease, parsnip yellow fleck virus (PYVF) 3C-like protease, heparin, thrombin, factor Xa, and enterokinase. In one embodiment, TEV (Tobacco Etch Virus) protease cleavage sites are preferred due to their higher cleavage stringency, such as EXXYXQ(G / S) (SEQ ID NO: 95), such as ENLYFQG (SEQ ID NO: 96) and ENLYFQS (SEQ ID NO: 97), where X represents any amino acid (TEV cleavage occurs between Q and G or Q and S).

[0313] In certain embodiments, the polypeptide cleavage signal is a viral self-cleaving peptide or a ribosomal skipping sequence.

[0314] Illustrative examples of ribosomal skipping sequences include, but are not limited to, 2A or 2A-like sites, sequences, or domains (Donnelly et al., 2001. J. Gen. Virol. 82: 1027-1041). In certain embodiments, the viral 2A peptide is an aphthous 2A peptide, a potato virus 2A peptide, or a cardiovirus 2A peptide.

[0315] In one embodiment, the viral 2A peptide is selected from the group consisting of: foot-and-mouth disease virus (FMDV) 2A peptide, equine rhinitis virus A (ERAV) 2A peptide, Tetrasomyces cerevisiae (TaV) 2A peptide, porcine Teschovirus-1 (PTV-1) 2A peptide, Theilovirus 2A peptide, and encephalomyocarditis virus 2A peptide.

[0316] Illustrative examples of 2A sites are provided in Table 2.

[0317] Table 2:

[0318]

[0319]

[0320] In preferred embodiments, the polypeptide or fusion polypeptide comprises one or more CD33 VHH DARIC components, CD33 VHH DARIC, or anti-CD33 VHH CAR.

[0321] 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.

[0322] In certain embodiments, the fusion polypeptide comprises the sequence set forth in any one of SEQ ID NOs: 32-61. In certain embodiments, the fusion polypeptide comprises the sequence set forth in any one of SEQ ID NOs: 40, 50, or 60.

[0323] In a specific embodiment, the fusion polypeptide comprises: a CD33 DARIC signaling component, which includes a FRB T2098L multimerization domain, a CD8α transmembrane domain, a CD137 co-stimulatory domain, and a CD3ζ primary signaling domain; a viral self-cleaving 2A polypeptide; and a CD33 VHH DARIC binding component, which includes an anti-CD33 VHH, a FKBP12 multimerization domain polypeptide, and a CD4 transmembrane domain.

[0324] In certain embodiments, the fusion polypeptide comprises the sequence set forth in any one of SEQ ID NOs: 32-41. In a specific embodiment, the fusion polypeptide comprises the sequence set forth in SEQ ID NO: 40.

[0325] In certain embodiments, the fusion polypeptide comprises: a CD33 DARIC signaling component, which includes a FRB T2098L multimerization domain, a CD8α transmembrane domain, a CD137 co-stimulatory 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 co-stimulatory domain.

[0326] In certain embodiments, the fusion polypeptide comprises the sequence set forth in any one of SEQ ID NOs: 42-51. In a specific embodiment, the fusion polypeptide comprises the sequence set forth in SEQ ID NO: 50.

[0327] In certain embodiments, the fusion polypeptide comprises the sequence set forth in any one of SEQ ID NOs: 52-61. In a specific embodiment, the fusion polypeptide comprises the sequence set forth in SEQ ID NO: 60.

[0328] F. Polynucleotides

[0329] In certain embodiments, polynucleotides encoding CD33 VHH DARIC, CD33 VHH DARIC binding components, CD33 DARIC signaling components, anti-CD33 VHH CARs, and fragments thereof are provided. As used herein, the term "polynucleotide" or "nucleic acid" refers to deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and DNA / RNA hybrids. Polynucleotides can be single-stranded or double-stranded and 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. A polynucleotide is 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, as well as all intermediate lengths of nucleotides, i.e., polymeric forms of ribonucleotides or deoxyribonucleotides or modified forms of either type of nucleotide. It will be readily understood that in this context, "intermediate lengths" means 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, the 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.

[0330] As used herein, an "isolated polynucleotide" refers to a polynucleotide that has been purified from its naturally occurring sequences on both sides, for example, a DNA fragment that has been removed from sequences that are usually adjacent to it. In certain embodiments, an "isolated polynucleotide" also refers to complementary DNA (cDNA), recombinant DNA, or other polynucleotides that do not exist in nature and have been manufactured by human hands. In certain embodiments, an isolated polynucleotide is a synthetic polynucleotide, a semisynthetic polynucleotide, or a polynucleotide obtained or derived from a recombinant source.

[0331] In various embodiments, the polynucleotides include mRNAs encoding the polypeptides contemplated herein. In certain embodiments, the mRNA comprises a cap, one or more nucleotides, and a poly(A) tail.

[0332] In certain embodiments, the polynucleotides encoding one or more CD33 VHH DARIC components can be codon-optimized. As used herein, the term "codon-optimized" refers to the substitution of codons in a polynucleotide encoding a polypeptide to increase the expression, stability, and / or activity of the polypeptide. Factors influencing codon optimization include, but are not limited to, one or more of the following: (i) variation in codon preference between two or more organisms or genes or synthetically constructed preference tables; (ii) variation in the degree of codon preference within an organism, gene, or gene set; (iii) systematic variation in codons, including environmental variation; (iv) variation in codons based on decoding tRNA; (v) variation in codon % overall or in a single triplet position; (vi) variation in the degree of similarity to a reference sequence, such as a naturally occurring sequence; (vii) variation in codon frequency cutoffs; (viii) structural properties of mRNA transcribed from a DNA sequence; (ix) prior knowledge of the function of the DNA sequence upon which the codon substitution set was designed; (x) systematic variation in the codon set for each amino acid; and / or (xi) isolated removal of spurious translation start sites.

[0333] As used herein, the term "nucleotide" refers to a heterocyclic nitrogenous base in an N-glycosidic linkage to a phosphorylated sugar. Nucleotides are understood to include natural bases, as well as a variety of modified bases recognized in the art. Such bases are generally located at the 1' position of the nucleotide sugar portion. Nucleotides generally comprise 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, i.e., a sugar that does not contain the hydroxyl group present in ribose.

[0334] Illustrative examples of polynucleotides include, but are not limited to, polynucleotides encoding the polypeptide set forth in any one of SEQ ID NOs: 2-82.

[0335] In various exemplary embodiments, the 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, and expression vectors, viral vectors, and transfer plasmids comprising the polynucleotides contemplated herein.

[0336] As used herein, the terms "polynucleotide variant" and "variant" and the like refer to polynucleotides that exhibit substantial sequence identity to a reference polynucleotide sequence or that hybridize to a reference sequence under stringent conditions as defined below. These terms also encompass polynucleotides that differ from a reference polynucleotide by the addition, deletion, substitution, or modification of at least one nucleotide. Thus, the terms "polynucleotide variant" and "variant" include polynucleotides in which one or more nucleotides are added or deleted or modified, or replaced with a different nucleotide. In this regard, it is well understood in the art that certain changes, including mutations, additions, deletions, and substitutions, may be made to a reference polynucleotide such that the altered polynucleotide retains the biological function or activity of the reference polynucleotide.

[0337] As used herein, the description "sequence identity" or, for example, "a sequence that is 50% identical to..." refers to the degree to which the sequences are identical on a nucleotide-by-nucleotide basis or on an amino acid-by-amino acid basis within the comparison window. Thus, the "percentage of sequence identity" can be calculated by comparing two optimally aligned sequences within the comparison window, determining the number of positions at which identical nucleic acid bases (e.g., A, T, C, G, I) or identical amino acid residues (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) occur in both sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window (i.e., the window size), and multiplying the result by 100 to obtain the percentage of sequence identity. comprising 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.

[0338] As disclosed elsewhere herein or as known in the art, regardless of the length of the coding sequence itself, the polynucleotides encompassed herein can be combined with other DNA sequences, such as promoters and / or enhancers, untranslated regions (UTRs), signal sequences, Kozak sequences, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, internal ribosome entry sites (IRES), recombinase recognition sites (e.g., LoxP sites, FRT sites, and Att sites), stop codons, transcription termination signals, and polynucleotides encoding self-cleaving polypeptides, epitope tags, so that the overall length of the polynucleotide can vary significantly. Thus, it is contemplated that polynucleotide fragments of virtually any length can be employed, with the overall length preferably being limited by ease of preparation and use in the intended recombinant DNA protocol.

[0339] Polynucleotides can be prepared, manipulated, expressed and / or delivered using any of a variety of well-established techniques known and available in the art. In order to express the desired polypeptide, the nucleotide sequence encoding the polypeptide can be inserted into an appropriate vector.

[0340] Illustrative examples of vectors include, but are not limited to, plasmids, autonomously replicating sequences, and transposable elements, such as SleepingBeauty, PiggyBac.

[0341] Additional illustrative 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.

[0342] Illustrative examples of viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (e.g., SV40).

[0343] Illustrative examples of expression vectors include, but are not limited to, pClneo vectors (Promega) for expression in mammalian cells; 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 sequence of a polypeptide disclosed herein can be linked to such expression vectors to express the polypeptide in mammalian cells.

[0344] In a specific embodiment, carrier is an episomal vector or maintains an extrachromosomal carrier. As used herein, term "episomal" refers to a carrier that can replicate and is not integrated into the host's chromosomal DNA and can not gradually lose from the host cell of division, which also means that the carrier replicates extrachromosomally or additionally.

[0345] "Expression control sequences," "control elements," or "regulatory sequences" present in an expression vector are those non-translated regions of the vector, including origin of replication, selection cassette, promoter, enhancer, translation initiation signal (Shine Dalgamo sequence or Kozak sequence), introns, polyadenylation sequence, 5' and 3' non-translated regions, which interact with host cell proteins to effect transcription and translation. These elements may vary in their strength and specificity. Depending on the vector system and host utilized, a variety of suitable transcription and translation elements may be used, including ubiquitous promoters and inducible promoters.

[0346] In a specific embodiment, polynucleotide comprises a vector, and the vector includes but is not limited to expression vectors and viral vectors. The vector may include one or more exogenous, endogenous or heterologous control sequences, such as promoters and / or enhancers. "Endogenous control sequence" is a sequence naturally connected to a given gene in the genome. "Exogenous control sequence" is a sequence that is placed in juxtaposition with a gene so that the transcription of this gene is guided by the connected enhancer / promoter by genetic manipulation (i.e., molecular biology techniques). "Heterologous control sequence" is an exogenous sequence from a different species than the cell manipulated by the gene. "Synthetic" control sequence may include one or more endogenous and / or exogenous sequences and / or elements of a sequence that provides optimal promoter and / or enhancer activity for a particular therapy as determined in vitro or in a computer.

[0347] As used herein, the term "promoter" refers to a recognition site for a polynucleotide (DNA or RNA) to which RNA polymerase binds. RNA polymerase initiates and transcribes a polynucleotide operably linked to the promoter. In a specific embodiment, a promoter that functions in mammalian cells includes an AT-rich region approximately 25 to 30 bases upstream of the start transcription site and / or another sequence found 70 to 80 bases upstream of the start of transcription, i.e., a CNCAAT region in which N can be any nucleotide.

[0348] The term "enhancer" refers to a segment of DNA that contains sequences that can provide enhanced transcription and, in some cases, can function independently of its orientation relative to another control sequence. An enhancer can function synergistically or additively with a promoter and / or other enhancer elements. The term "promoter / enhancer" refers to a segment of DNA that contains sequences that can provide both promoter and enhancer functions.

[0349] The term "operably linked" refers to a juxtaposition in which the described components are in a relationship permitting them to function in their intended manner. In one embodiment, the term refers to a 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, wherein the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence.

[0350] As used herein, the term "constitutive expression control sequence" refers to a promoter, enhancer, or promoter / enhancer that continuously or continually permits transcription of an operably linked sequence. A constitutive expression control sequence can be a "ubiquitous" promoter, enhancer, or promoter / enhancer that permits expression in a wide variety of cell and tissue types, or a "cell-specific," "cell type-specific," "cell lineage-specific," or "tissue-specific" promoter, enhancer, or promoter / enhancer that permits expression in a restricted variety of cell and tissue types, respectively.

[0351] Exemplary ubiquitous expression control sequences suitable for use in particular embodiments include, but are not limited to, cytomegalovirus (CMV) immediate early promoter, 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, H5, P7.5 promoter and P11 promoter from vaccinia virus, elongation factor 1-alpha (EF1a) promoter, early growth response 1 (EGR1), ferritin H (FerH), ferritin L (FerL), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), eukaryotic translation initiation factor 4A1 (EIF4A1), heat shock 70 kDa protein 5 (HSPA5), heat shock protein 90 kDa 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 β-actin (CAG) promoter, β-actin promoter and myeloproliferative sarcoma virus enhancer negative control region deleted and d1587rev primer binding site replaced (MND) U3 promoter (Haas et al., Journal of Virology. 2003; 77(17): 9439-9450).

[0352] In one embodiment, the vector includes the MNDU3 promoter.

[0353] In one embodiment, the vector comprises an EF1a promoter comprising the first intron of the human EF1a gene.

[0354] In one embodiment, the vector includes the EF1a promoter lacking the first intron of the human EF1a gene.

[0355] In certain embodiments, it may be desirable to use cell-, cell type-, cell lineage-, or tissue-specific expression control sequences to achieve cell type-specific, lineage-specific, or tissue-specific expression of a desired polynucleotide sequence (e.g., to express a particular nucleic acid encoding a polypeptide only in a subset of a cell type, cell lineage, or tissue, or during a particular stage of development).

[0356] In certain embodiments, it may be desirable to express the polynucleotide from a T cell-specific promoter.

[0357] As used herein, "conditional expression" can refer to any type of conditional expression, including but not limited to: inducible expression; repressible expression; expression in cells or tissues having a specific physiological, biological or disease state, etc. This definition is not intended to exclude cell type or tissue specific expression. Certain embodiments provide conditional expression of a polynucleotide of interest, for example, by controlling expression by subjecting a cell, tissue, organism, etc., to a treatment or condition that results in increased or decreased expression of the polynucleotide or a polypeptide encoded by the polynucleotide of interest.

[0358] Illustrative examples of inducible promoters / systems include, but are not limited to, steroid-inducible promoters such as promoters of genes encoding glucocorticoid 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), the "GeneSwitch" mifepristone-regulatable system (Sirin et al., 2003, Gene, 323:67), cumate-inducible gene switches (WO 2002 / 088346), tetracycline-dependent regulatory systems, etc. Inducing agents include, but are not limited to, glucocorticoids, estrogens, mifepristone (RU486), metals, interferons, small molecules, cumate, tetracycline, doxycycline, and variants thereof.

[0359] As used herein, "internal ribosome entry site" or "IRES" refers to an element that facilitates direct entry of internal ribosomes into a cistron (protein coding region) at a start codon such as ATG, thereby leading to cap-independent translation of the gene. See, for example, 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 used by those skilled in the art include those described in U.S. Patent No. 6,692,736. Other examples of "IRES" known in the art include, but are not limited to, IRESs obtainable from picorna viruses (Jackson et al., 1990) and IRESs 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 (IGF-1D), translation initiation factor eIF4G and yeast transcription factors TFIID and HAP4, Ephelomencycarditis virus (EMCV) commercially available from Novagen (Duke et al., 1992. J. Virol 66(3): 1602-9), and VEGF IRES (Huez et al., 1998. Mol Cell Biol IRESs have been reported in viral genomes of species from the families Picomaviridae, Dicistroviridae, and Flaviviridae, as well as in HCV, Friend murine leukemia virus (FrMLV), and Moloney murine leukemia virus (MoMLV).

[0360] In one embodiment, the IRES used in the polynucleotides contemplated herein is an EMCVIRES.

[0361] In certain embodiments, the polynucleotide is a consensus Kozak sequence. As used herein, the term "Kozak sequence" refers to a short nucleotide sequence that greatly promotes the initial binding of mRNA to the small subunit of the ribosome and increases translation. The consensus Kozak sequence is (GCC)RCCATGG (SEQ ID NO: 83), where R is a purine (A or G) (Kozak, 1986. Cell. 44(2): 283-92, and Kozak, 1987. Nucleic Acids Res. 15(20): 8125-48).

[0362] Elements that guide efficient termination and polyadenylation of heterologous nucleic acid transcripts will increase the expression of heterologous genes. Transcription termination signals are generally found downstream of the polyadenylation signal. In specific embodiments, the vector comprises a polyadenylation sequence at the 3' end of the polynucleotide encoding the polypeptide to be expressed. As used herein, the term "polyA site" or "polyA sequence" refers to a DNA sequence that guides the termination and polyadenylation of nascent RNA transcripts caused by RNA polymerase II. The polyadenylation sequence can promote mRNA stability by adding a polyadenylic acid tail at the 3' end of the coding sequence, thereby promoting increased translation efficiency. Cleavage and polyadenylation are guided by the polyadenylic acid sequence in the RNA. The core polyadenylic acid sequence of mammalian mRNA precursors (pre-mRNA) has two recognition elements flanking the cleavage-polyadenylation site. Typically, the nearly invariant AAUAAA hexamer is located 20-50 nucleotides upstream of the more variable element rich in U or GU residues. Cleavage of the nascent transcript occurs between these two elements and is coupled to add up to 250 adenosines to the 5' cleavage product. In specific embodiments, the core poly (A) sequence is an ideal poly (A) sequence (e.g., AATAAA, ATTAAA, AGTAAA). In specific 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 specific embodiments, the poly (A) sequence is synthetic.

[0363] In a specific embodiment, the polynucleotides encoding one or more polypeptides or fusion polypeptides can be introduced into immune effector cells, such as T cells, by non-viral and viral methods. In a specific embodiment, the delivery of one or more polynucleotides can be provided by the same method or by different methods and / or by the same carrier or by different carriers.

[0364] 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 connected to a carrier nucleic acid molecule, for example, inserted into a carrier nucleic acid molecule. The vector may include a sequence that guides autonomous replication in the cell, or may include a sequence sufficient to allow integration into the host cell DNA. In specific embodiments, non-viral vectors are used to deliver one or more polynucleotides encompassed herein into T cells.

[0365] Illustrative examples of non-viral vectors include, but are not limited to, plasmids (eg, DNA plasmids or RNA plasmids), transposons, cosmids, and bacterial artificial chromosomes.

[0366] Exemplary methods of non-viral delivery of polynucleotides encompassed in particular embodiments include, but are not limited to, electroporation, sonoporation, lipofection, microinjection, gene guns, virions, liposomes, immunoliposomes, nanoparticles, polycation or lipid:nucleic acid conjugates, naked DNA, artificial virions, DEAE-dextran mediated transfer, gene guns, and heat shock.

[0367] Illustrative examples of polynucleotide delivery systems suitable for use in the specific embodiments contemplated in the specific embodiments include, but are not limited to, those provided by Amaxa Biosystems, Maxcyte, Inc., BTX Molecular Delivery Systems, and Copemicus Therapeutics Inc. Lipofection reagents are commercially available (e.g., Transfectam® and Lipofectin®). Cationic lipids and neutral lipids suitable for efficient receptor-recognition lipofection of polynucleotides have been described in the literature. See, for example, Liu et al. (2003) Gene Therapy. 10: 180-187; and Balazs et al. (2011) Journal of Drug Delivery. 2011: 1-12. Antibody-targeted, bacteria-derived, and inanimate nanocell-based delivery are also contemplated in specific embodiments.

[0368] In certain embodiments, the viral vectors comprising polynucleotides encompassed herein can be delivered by administration to an individual patient, typically by systemic administration (e.g., intravenous, intraperitoneal, intramuscular, subcutaneous, or intracranial infusion) or topical administration, as described below. Alternatively, the vector can be delivered ex vivo to cells, such as cells transplanted from an individual patient (e.g., mobilized peripheral blood, lymphocytes, bone marrow aspirates, tissue biopsies, etc.) or universal donor hematopoietic stem cells, which are then reimplanted into the patient.

[0369] In one embodiment, the viral vector comprising the polynucleotide encompassed herein is directly administered to an organism to transduce cells in vivo. Alternatively, naked DNA can be used. Use by being generally used for any approach that introduces a molecule and finally contacts with blood or tissue cells, including but not limited to injection, infusion, topical application and electroporation. The suitable method for using this nucleic acid is obtainable and known to those skilled in the art, and although more than a kind of approach can be used to use a particular composition, a particular approach can provide more direct and more effective reaction than another approach usually.

[0370] Illustrative examples of viral vector systems suitable for use in particular embodiments contemplated herein include, but are not limited to, adeno-associated virus (AAV), retrovirus, herpes simplex virus, adenovirus, and vaccinia virus vectors.

[0371] 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) comprising the one or more polynucleotides.

[0372] AAV is a small (about 26 nm), replication-defective, primarily episomal, non-enveloped virus. AAV can infect both dividing and non-dividing cells and can incorporate its genome into the genome of the host cell. Recombinant AAV (rAAV) typically consists of at least a transgene and its regulatory sequences and 5' and 3' AAV inverted terminal repeats (ITRs). The ITR sequence is about 145 bp in length. In a specific embodiment, rAAV comprises ITRs and capsid sequences isolated from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10.

[0373] In some embodiments, chimeric rAAV is used, ITR sequences are isolated from one AAV serotype, and capsid sequences are isolated from a different AAV serotype. For example, a rAAV having ITR sequences derived from AAV2 and capsid sequences derived from AAV6 is referred to as AAV2 / AAV6. In specific embodiments, the rAAV vector can include ITRs from AAV2 and capsid proteins from any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10. In a preferred embodiment, the rAAV includes ITR sequences derived from AAV2 and capsid sequences derived from AAV6. In a preferred embodiment, the rAAV includes ITR sequences derived from AAV2 and capsid sequences derived from AAV2.

[0374] In some embodiments, engineering and selection methods can be applied to AAV capsids to make them more likely to transduce cells of interest.

[0375] The construction, preparation, and purification of rAAV vectors have been 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, each of which is incorporated herein by reference in its entirety.

[0376] 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, comprising the one or more polynucleotides.

[0377] 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 subsequently covalently integrates its genomic DNA into the host genome. Exemplary retroviruses suitable for use in specific 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), foamy virus, Friedreich's murine leukemia virus, murine stem cell virus (MSCV), and Rous sarcoma virus (RSV), as well as lentiviruses.

[0378] As used herein, the term "mantle virus" refers to a group (or genus) of complex retroviruses. Illustrative lentiviruses include, but are not limited to, HIV (human immunodeficiency virus; comprising 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.

[0379] In various embodiments, the lentiviral vectors encompassed herein include one or more LTRs, and one or more or all of the following accessory elements: cPPT / FLAP, a Psi(Ψ) packaging signal, an export element, a poly(A) sequence, and may optionally include a WPRE or HPRE, an insulator element, a selectable marker, and a cell suicide gene, as discussed elsewhere herein.

[0380] In certain embodiments, the lentiviral vectors encompassed herein can be integrating or non-integrating or integration-defective lentiviruses. As used herein, the term "integration-defective lentivirus" or "IDLV" refers to a lentivirus that lacks an integrase capable of integrating the viral genome into the host cell's genome. Integration-incompetent viral vectors have been described in patent application WO 2006 / 010834, which is incorporated herein by reference in its entirety.

[0381] Exemplary mutations in the HIV-1 pol gene 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.

[0382] The term "long terminal repeat (LTR)" refers to a domain of base pairs located at the end of retroviral DNA that is a direct repeat in its native sequence context and contains the U3, R, and U5 regions.

[0383] As used herein, the term "FLAP element" or "cPPT / FLAP" refers to a nucleic acid whose sequence comprises the central polypurine tract and central termination sequence (cPPT and CTS) of retroviruses (e.g., HIV-1 and HIV-2). Suitable FLAP elements are described in U.S. Pat. No. 6,682,907 and Zennou et al., 2000, Cell, 101: 173.

[0384] As used herein, the term "packaging signal" or "packaging sequence" refers to a psi[Ψ] sequence located within the retroviral genome that is required for insertion of viral RNA into the viral capsid or particle, see, e.g., Clever et al., 1995. J. of Virology, Vol. 69, No. 4; pp. 2101-2109.

[0385] The term "export element" refers to a cis-acting post-transcriptional regulatory element that regulates the transport of RNA transcripts from the nucleus to the cytoplasm of a cell. Examples of RNA export elements include, but are not limited to, the human immunodeficiency virus (HIV) rev response element (RRE) (see, e.g., Cullen et al., 1991. J. Virol. 65: 1053; and Cullen et al., 1991. Cell 58: 423), and the hepatitis B virus post-transcriptional regulatory element (HPRE).

[0386] In certain embodiments, expression of heterologous sequences in viral vectors is increased by incorporating into the vector a post-transcriptional regulatory element, an efficient polyadenylation site, and optionally a transcription termination signal. A variety of post-transcriptional regulatory elements can increase expression of heterologous nucleic acids on proteins, such as the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE; Zufferey et al., 1999, J. Virol., 73:2886); the post-transcriptional regulatory element present in hepatitis B virus (HPRE) (Huang et al., Mol. Cell. Biol., 5:3864); and the like (Liu et al., 1995, Genes Dev., 9:1766).

[0387] Due to the modification of LTR, lentiviral vectors preferably contain several safety enhancements." Self-inactivation" (SIN) vector refers to a replication-defective vector, such as a retroviral vector or a lentiviral vector, in which the right (3') LTR enhancer-promoter region referred to as the U3 region has been modified (e.g., by deletion or replacement) to prevent viral transcription from exceeding the first round of viral replication. Preferably, self-inactivation is achieved by introducing a deletion (i.e., DNA for producing vector RNA) in the U3 region of the 3' LTR of the vector DNA. Therefore, during reverse transcription, this deletion is transferred to the 5' LTR of the proviral DNA. In a specific embodiment, it is desirable to eliminate enough U3 sequences to greatly reduce or completely eliminate the transcriptional activity of LTR, thereby greatly reducing or eliminating the generation of full-length vector RNA in transduced cells. In the case of HIV-based lentiviral vectors, it has been found that this vector tolerates significant U3 deletions, including removal of the LTR TATA box (e.g., deletion from -418 to -18), without significantly reducing vector titer.

[0388] Additional safety enhancement is provided by replacing the U3 region of the 5' LTR with a heterologous promoter to drive transcription of the viral genome during viral particle production. Examples of heterologous promoters that can be used include, for example, viral simian virus 40 (SV40) (e.g., early or late), cytomegalovirus (CMV) (e.g., immediate early), Moloney murine leukemia virus (MoMLV), Rous sarcoma virus (RSV), and herpes simplex virus (HSV) (thymidine kinase) promoters.

[0389] As used herein, the term "pseudotype" or "pseudotyping" refers to a virus whose viral envelope protein has been replaced with a viral envelope protein of another virus with preferred properties. For example, HIV can be pseudotyped with the vesicular stomatitis virus G protein (VSV-G) envelope protein, which allows HIV to infect a wider range of cells because the HIV envelope protein (encoded by the env gene) normally targets the virus to CD4 + Presenting cells.

[0390] In certain embodiments, lentiviral vectors are generated according to known methods. See, for example, 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.

[0391] According to certain specific embodiments encompassed herein, most or all of the viral vector backbone sequences are derived from lentiviruses, such as HIV-1. However, it will be appreciated that retroviral and / or lentiviral sequences from many different sources may be used or combined, or that a variety of substitutions and alterations of certain lentiviral sequences in the lentiviral sequences may be accommodated without compromising the ability of the transfer vector to perform the functions described herein. In addition, 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 which may be suitable for producing viral vectors or transfer plasmids encompassed herein.

[0392] 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 comprising the one or more polynucleotides.

[0393] Adenovirus-based vectors are capable of extremely high transduction efficiencies in many cell types and do not require cell division. High titers and high expression levels have been achieved using these vectors. These vectors can be produced in large quantities in relatively simple systems. Most adenovirus vectors are engineered so that the transgene replaces the Ad E1a, E1b, and / or E3 genes; the replication-defective vectors are then propagated in human 293 cells that provide the missing gene function in trans. Ad vectors can transduce a variety of tissue types in vivo, including non-dividing, differentiated cells such as those found in the liver, kidney, and muscle. Conventional Ad vectors have a large carrying capacity.

[0394] The production and propagation of replication-deficient current adenoviral vectors can utilize a unique helper cell line named 293, which is transformed from human embryonic kidney cells by Ad5 DNA fragments and constitutively expresses E1 protein (Graham et al., 1977). Since the E3 region can be allocated from the adenoviral genome (Jones and Shenk, 1978), current adenoviral vectors carry foreign DNA in the E1, D3 region, or both regions with the help of 293 cells (Graham and Prevec, 1991). Adenoviral vectors have been used for eukaryotic gene expression (Levrero et al., 1991; Gomez-Foix et al., 1992) and vaccine development (Grunhaus & Horwitz, 1992; Graham & Prevec, 1992). Studies on the administration of recombinant adenovirus to various tissues include intratracheal instillation (Rosenfeld et al., 1991; Rosenfeld et al., 1992), intramuscular injection (Ragot et al., 1993), peripheral intravenous injection (Herz & Gerard, 1993), and stereotactic intracerebral inoculation (Le Gal La Salle et al., 1993). An example of the use of Ad vectors in clinical trials involves polynucleotide therapy for anti-tumor immunity with intramuscular injection (Sterman et al., Hum. Gene Ther. 7: 1083-9 (1998)).

[0395] 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, HSV-2, comprising the one or more polynucleotides.

[0396] Mature HSV virions are composed of an enveloped icosahedral capsid, wherein the viral genome is composed of a 152kb linear double-stranded DNA molecule. In one embodiment, the viral vector based on HSV lacks one or more essential or non-essential HSV genes. In one embodiment, the viral vector based on HSV is replication-defective. Most replication-defective HSV vectors contain deletions to remove one or more early, early or late HSV genes to prevent replication. For example, the HSV vector may lack the immediate early genes selected from the group consisting of: ICP4, ICP22, ICP27, ICP47 and combinations thereof. The advantage of the HSV vector is its ability to enter a latent period that can lead to long-term DNA expression, and its large viral DNA genome that can accommodate exogenous DNA inserts of up to 25kb. HSV-based vectors are described, for example, in U.S. Patent Nos. 5,837,532, 5,846,782, and 5,804,413, and International Patent Applications WO 91 / 02788, WO 96 / 04394, WO 98 / 15637, and WO 99 / 06583, each of which is incorporated herein by reference in its entirety.

[0397] G. Genetically modified cells

[0398] 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 as contemplated herein for use in treating cancer. Cells can be non-genetically modified to express one or more of the polypeptides contemplated herein, or, in certain preferred embodiments, can 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 of a cell. In certain embodiments, the terms "genetically modified cell," "modified cell," and "redirected cell" are used interchangeably.

[0399] 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.

[0400] 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). Illustrative immune effector cells encompassed herein are T lymphocytes, including but not limited to cytotoxic T cells (CTLs; CD8 +T cells), TILs, and helper T cells (HTL; 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. The immune effector cells can be autologous / self ("self") or non-autologous ("non-self", e.g., allogeneic, isogenic, or xenogeneic).

[0401] As used herein, "autologous" refers to cells from the same subject. As used herein, "allogeneic" refers to cells from the same species that are genetically different from the comparison cells. As used herein, "syngeneic" refers to cells from a different subject that are genetically identical to the comparison cells. As used herein, "xenogeneic" refers to cells from a different species than the comparison cells. In preferred embodiments, the cells are human autologous immune effector cells.

[0402] Illustrative immune effector cells suitable for introduction of one or more CD33 VHH DARIC components or anti-CD33 VHH CARs encompassed herein include T lymphocytes. The terms "T cell" or "T lymphocyte" are art-recognized and are intended to encompass thymocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes. The T cell can be a T helper (Th) cell, such as a T helper 1 (Th1) or T helper 2 (Th2) cell. The T cell can be a helper T cell (HTL; CD4 + T cells), cytotoxic T cells (CTL; CD8 + T cells), CD4 + CD8 + In certain embodiments, T cells are T cells that express t cell receptors. In certain embodiments, t cells express t cell receptors. T cell receptors include two subunits, i.e., alpha chain and beta chain subunits (αβTCR), or gamma chain and delta chain subunits (γδTCR), each of which is a unique protein produced by the recombination event in each t cell genome. In certain embodiments, t cells are αβTCR T cells (αβ T cells). In certain embodiments, t cells are γδTCR T cells (γδ T cells). Other exemplary t cell groups applicable in certain embodiments include primary t cells and memory t cells.

[0403] As will be appreciated by those skilled in the art, other cells may also be used as immune effector cells comprising one or more CD33 VHH DARIC components or anti-CD33 VHH CARs encompassed herein. In certain embodiments, immune effector cells further include NK cells, NKT cells, neutrophils, and macrophages. Immune effector cells further include progenitor cells of effector cells, wherein such progenitor cells can be induced to differentiate into immune effector cells in vivo or in vitro. Therefore, in certain embodiments, immune effector cells include progenitor cells of immune effector cells, such as hematopoietic stem cells (HSCs) contained in CD34+ cell populations derived from umbilical cord blood, bone marrow, or mobilized peripheral blood, which are differentiated into mature immune effector cells after administration in a subject, or the HSCs can be induced in vitro to differentiate into mature immune effector cells.

[0404] As used herein, the term "CD34+ cells" refers to cells expressing CD34 protein on their cell surface. As used herein, "CD34" refers to a cell surface glycoprotein (e.g., sialomucin) that typically acts as a cell-cell adhesion factor and participates in the entry of T cells into lymph nodes. The CD34+ cell population contains hematopoietic stem cells (HSCs), which differentiate and contribute to all hematopoietic lineages when administered to a patient, including cells of the T cell, NK cell, NKT cell, neutrophil, and monocyte / macrophage lineages.

[0405] In a specific embodiment, a method for preparing immune effector cells expressing one or more CD33 VHH DARIC components or anti-CD33 VHH CARs encompassed herein is provided. In one embodiment, the method includes transfecting or transducing immune effector cells isolated from an individual so that the immune effector cells have one or more nucleic acids and / or vectors, such as lentiviral vectors comprising nucleic acids encoding one or more CD33 VHH DARIC components or anti-CD33 VHH CARs encompassed herein. In one embodiment, the method includes transfecting or transducing immune effector cells isolated from an individual so that the immune effector cells express one or more CD33 VHH DARIC components or anti-CD33 VHH CARs encompassed herein. In certain embodiments, immune effector cells are isolated from an individual and genetically modified without further manipulation in vitro. Such cells can then be directly administered to the individual again. In another embodiment, the immune effector cells are first activated and stimulated to proliferate in vitro before genetic modification. In this regard, immune effector cells can be cultured before and / or after genetic modification.

[0406] In certain embodiments, prior to the in vitro manipulation or genetic modification of the immune effector cells described herein, the source of cells is obtained from a subject. In certain embodiments, the modified immune effector cells comprise T cells.

[0407] T cells can be obtained from a variety of sources, including but not limited to peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from the site of infection, ascites, pleural effusion, spleen tissue, and tumors. In certain embodiments, any number of techniques known to those skilled in the art, such as sedimentation (e.g., FICOLL) can be used. TM T cells are obtained by isolation) from a unit of blood collected from a subject.

[0408] In other embodiments, isolated or purified T cell populations are used.In some embodiments, following isolation of PBMCs, cytotoxic and helper T lymphocytes may be sorted into naive, memory, and effector T cell subsets before or after activation, expansion, and / or genetic modification.

[0409] In one embodiment, the isolated or purified T cell population expresses one or more of the following markers including but not limited to: CD3 + 、CD4 + 、CD8 + or a combination thereof.

[0410] In certain embodiments, T cells are isolated from an individual and first activated and stimulated to proliferate in vitro before being modified to express one or more CD33 VHH DARIC components or anti-CD33 VHH CARs.

[0411] To achieve a sufficient therapeutic dose of a T cell composition, the T cells are typically subjected to one or more rounds of stimulation, activation, and / or expansion. In certain embodiments, T cells may generally be activated and expanded using methods as 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, each of which is incorporated herein by reference in its entirety. In specific embodiments, T cells are activated and expanded for about 6 hours, about 12 hours, about 18 hours, or about 24 hours following the introduction of vectors or polynucleotides encoding one or more CD33 VHH ARIC components or anti-CD33 VHH CARs contemplated herein.

[0412] H. Compositions and Formulations

[0413] The compositions encompassed herein may include one or more CD33 VHH DARIC components or anti-CD33 VHH CARs, polynucleotides encoding one or more CD33 VHH DARIC components or anti-CD33 VHH CARs, vectors containing the same, genetically modified immune effector cells, bridging factors, and the like. Compositions include, but are not limited to, pharmaceutical compositions. A "pharmaceutical composition" refers to a composition formulated in a pharmaceutically acceptable or physiologically acceptable solution that is administered to cells or animals, alone or in combination with one or more other therapeutic modalities. It should also be understood that, if desired, the composition may also be administered in combination with other agents, such as cytokines, growth factors, hormones, small molecules, chemotherapeutic agents, prodrugs, drugs, antibodies, or other various pharmaceutically active agents. There is virtually no limitation on the other components that may be included in the composition, provided that the additional agents do not adversely affect the ability of the composition to deliver the intended therapy.

[0414] The phrase "pharmaceutically acceptable" is used herein to refer to those compounds, materials, compositions and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0415] The term "pharmaceutically acceptable carrier" refers to a diluent, adjuvant, excipient or vehicle for administering a bridging factor, polypeptide, polynucleotide, a carrier thereof or a genetically modified immune effector cell. Illustrative examples of pharmaceutical carriers can be sterile liquids, such as cell culture media, water and oil, including oils from petroleum, animal, plant or synthetic sources, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Physiological saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. In a particular embodiment, suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene, ethylene glycol, water, ethanol, etc. Except for the situation that any conventional culture medium or medicament is incompatible with the active ingredient, its use in the pharmaceutical composition is covered. Supplementary active ingredients can also be incorporated into the composition.

[0416] In one embodiment, the composition including a pharmaceutically acceptable carrier is suitable for administration to a subject. In a specific embodiment, the composition including a carrier is suitable for parenteral administration, such as intravascular (intravenous or intraarterial), intraperitoneal or intramuscular administration. In a specific embodiment, the composition including a pharmaceutically acceptable carrier is suitable for intraventricular, intraspinal or intrathecal administration. Pharmaceutically acceptable carriers include sterile aqueous solutions, cell culture media or dispersions. These culture media and medicaments are well known in the art for the purposes of pharmaceutically active substances. Except for any conventional culture media or medicaments and bridging factors, polypeptides, polynucleotides, carriers including them, or genetically modified immune effector cells incompatible situation, its purposes in pharmaceutical compositions are covered.

[0417] In certain embodiments, the compositions encompassed herein include genetically modified T cells and a pharmaceutically acceptable carrier. Compositions encompassed herein, including cell-based compositions, can be administered alone or in combination with other suitable compounds via enteral or parenteral administration methods to achieve the desired therapeutic purpose.

[0418] In certain embodiments, the compositions contemplated herein include a bridging factor and a pharmaceutically acceptable carrier.

[0419] Pharmaceutically acceptable carrier must have sufficiently high purity and sufficiently low toxicity so that it is suitable for being applied to the human subject being treated.Pharmaceutically acceptable carrier should keep or increase the stability of composition.Pharmaceutically acceptable carrier can be liquid or solid and is selected to provide the volume, homogeneity etc. of expectation when being combined with other components of composition when containing the mode of administration of plan.For example, pharmaceutically acceptable carrier can be but is not limited to binding agent (for example, pregelatinized corn starch, polyvinyl pyrrolidone or hydroxypropyl methylcellulose etc.), filler (for example, lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylate, calcium hydrogen phosphate etc.), lubricant (for example, magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metal stearate, hydrogenated vegetable oil, corn starch, polyethylene glycol, sodium benzoate, sodium acetate etc.), disintegrant (for example, starch, sodium starch glycolate etc.) or wetting agent (for example, sodium lauryl sulfate etc.). Other suitable pharmaceutically acceptable carriers for the compositions contemplated herein include, but are not limited to, water, saline solutions, alcohol, polyethylene glycol, gelatin, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinyl pyrrolidone, and the like.

[0420] Such carrier solutions may also contain buffers, diluents, and other suitable additives. As used herein, the term "buffer" refers to a solution or liquid whose chemical composition neutralizes an acid or base without significantly changing the pH. Examples of buffers encompassed herein include, but are not limited to, Dulbecco's phosphate-buffered saline (PBS), Ringer's solution, 5% dextrose in water (D5W), normal / physiologic saline (0.9% NaCl).

[0421] The pharmaceutically acceptable carrier may be present in an amount sufficient to maintain the pH of the composition at about 7. Alternatively, the pH of the composition ranges from about 6.8 to about 7.4, for example, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, and 7.4. In yet another embodiment, the pH of the composition is about 7.4.

[0422] The compositions encompassed herein may include non-toxic pharmaceutically acceptable culture media. The compositions may be suspensions. As used herein, the term "suspension" refers to a non-adhesive condition in which cells are not attached to a solid support. For example, cells maintained in suspension may be stirred or agitated and the cells are not adhered to a support, such as a culture dish.

[0423] In certain embodiments, the compositions contemplated herein are formulated in suspension, wherein the modified T cells are dispersed in an acceptable liquid medium or solution, such as saline or serum-free medium, in an intravenous (IV) bag, etc. Acceptable diluents include, but are not limited to, water, PlasmaLyte, Ringer's solution, isotonic sodium chloride (saline) solution, serum-free cell culture medium, and culture medium suitable for cryogenic storage, such as culture medium.

[0424] In certain embodiments, the pharmaceutically acceptable carrier is substantially free of natural proteins of human or animal origin and is suitable for storing the composition comprising the modified T cell population. The pharmaceutical composition is intended for administration to human patients and is therefore substantially free of cell culture components, such as bovine serum albumin, horse serum, and fetal bovine serum.

[0425] 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.

[0426] Serum-free culture media have several advantages over serum-containing culture media, including simplified and better-defined compositions, reduced levels of contaminants, elimination of possible infectious agent sources, and reduced costs. In various embodiments, serum-free culture media are animal-free and can optionally be protein-free. Optionally, the culture media can contain biopharmaceutically acceptable recombinant proteins. "Animal-free" culture media refers to culture media whose compositions are derived from non-animal sources. Recombinant proteins replace natural animal proteins in animal-free culture media, and nutrients are obtained from synthetic, plant, or microbial sources. In contrast, "protein-free" culture media are defined as being substantially protein-free.

[0427] Illustrative examples of serum-free culture media used in certain embodiments include, but are not limited to, QBSF-60 (Quality Biological, Inc.), StemPro-34 (Life Technologies), and X-VIVO 10.

[0428] In one embodiment, the composition comprising the modified T cells is formulated in PlasmaLyte.

[0429] In various embodiments, the compositions comprising the modified T cells are formulated in a cryopreservation medium. For example, a cryopreservation medium with a cryopreservation agent can be used to maintain high cell viability results after thawing. Illustrative examples of cryopreservation medium used in specific embodiments include, but are not limited to, CryoStor CS10, CryoStor CS5, and CryoStor CS2.

[0430] In one embodiment, the composition is formulated in a solution comprising 50:50 PlasmaLyte A:CryoStor CS10.

[0431] In certain embodiments, the compositions are substantially free of mycoplasma, endotoxins, and microbial contamination. With respect to endotoxins, "substantially free" means that the endotoxin content per dose of cells is less than the FDA-approved level for biologics, which is 5 EU / kg body weight per day of total endotoxin, or 350 EU per total dose of cells for an average 70 kg human. In certain embodiments, the compositions contemplated herein contain from 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.

[0432] In certain embodiments, the formulation of pharmaceutically acceptable carrier solutions is well known to those skilled in the art, as is the development of suitable dosing and treatment regimens for the use of the specific compositions described herein in a variety of treatment regimens, including, for example, enteral and parenteral, such as intravascular, intravenous, intraarterial, intraosseous, intraventricular, intracerebral, intracranial, intraspinal, intrathecal, and intramedullary administration and formulation. It will be understood by those skilled in the art that certain embodiments contemplated herein may include other formulations, such as those well known in the pharmaceutical arts and described, for example, in Remington: The Science and Practice of Pharmacy, Volumes I and II, 22nd Edition, ed.: Loyd V. Allen Jr. Philadelphia, PA: Pharmaceutical Press; 2012, each of which is incorporated herein by reference in its entirety.

[0433] In certain embodiments, the composition comprises an amount of immune effector cells comprising polynucleotides encoding one or more CD33 VHH DARIC components or anti-CD33 VHH CARs as contemplated herein. In certain embodiments, the composition comprises an amount of immune effector cells expressing one or more CD33 VHH DARIC components or anti-CD33 VHH CARs as contemplated herein. As used herein, the term "amount" refers to an "effective amount" or "effective amount" of cells comprising one or more CD33 VHH DARIC components or anti-CD33 VHH CARs as contemplated herein, etc., to achieve a beneficial or desired prophylactic or therapeutic outcome (including a clinical outcome) in the presence of a bridging factor.

[0434] A "prophylactically effective amount" refers to an amount of cells comprising one or more CD33 VHH DARIC components or anti-CD33 VHH CARs, etc., encompassed herein, effective to achieve the desired prophylactic result. Typically, but not necessarily, a prophylactic effective amount is less than a therapeutically effective amount because a prophylactic dose is used in a subject prior to or at an earlier stage of disease.

[0435] "Therapeutically effective amount" refers to the amount of cells comprising one or more CD33 VHH DARIC components or anti-CD33 VHH CARs encompassed herein that is effective to "treat" a subject (e.g., a patient) in the presence of a bridging factor. When a therapeutic amount is indicated, the precise amount of the composition, cells, bridging factor, etc. to be administered can be determined by a physician taking into account individual differences in age, weight, tumor size, degree of infection or metastasis, and condition of the patient (subject).

[0436] Generally speaking, the pharmaceutical composition comprising the immune effector cells described herein can be taken as 10 2 to 10 10 cells / kg body weight, preferably 10 5 to 10 6 The dosage of cells / kg body weight (including all integer values within those ranges) is preferably administered. The number of cells will depend on the desired end use of the composition, as will the type of cells contained therein. For the uses provided herein, the volume of cells is typically one liter or less, and can be 500 ml or less, or even 250 ml or 100 ml or less. Thus, the desired cell density is typically greater than 10 6 cells / mL and usually greater than 10 7 cells / mL, usually 10 8 cells / mL or greater. Clinically relevant numbers of immune cells can be allocated to cumulatively equal to or greater than 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 or 10 12 In some embodiments, particularly since all infused cells will be redirected to a specific target antigen, 10 6 / kg(10 6 -10 11 / patient) range.

[0437] If desired, treatment may also comprise administration of mitogens (e.g., PHA) or lymphokines, cytokines and / or chemokines (e.g., IFN-γ, IL-2, IL-12, TNF-α, IL-18 and TNF-β, GM-CSF, IL-4, IL-13, Flt3-L, RANTES, MIP1α, etc.) as described herein to enhance the induction of an immune response.

[0438] In general, compositions comprising activated and expanded cells as described herein can be used to treat and prevent diseases that occur in immunocompromised individuals. In particular, the compositions encompassed herein are used to treat cancer. In certain embodiments, immune effector cells can be administered alone or in combination with a carrier, diluent, excipient, and / or other components such as IL-2 or other cytokines or cell populations as a pharmaceutical composition.

[0439] In certain embodiments, the pharmaceutical composition includes an amount of genetically modified T cells in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients.

[0440] In certain embodiments, the pharmaceutical composition includes an amount of a bridging factor in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients.

[0441] In certain embodiments, the composition comprises an effective amount of immune effector cells comprising one or more CD33 VHH DARIC components or anti-CD33 VHH CARs as contemplated herein, alone or in combination with a bridging factor and / or one or more therapeutic agents, such as radiation therapy, chemotherapy, transplantation, immunotherapy, hormone therapy, photodynamic therapy, and the like. The composition may also be administered in combination with an antibiotic. Such therapeutic agents are accepted in the art as standard treatment for specific disease states, such as specific cancers, as described herein. Exemplary therapeutic agents contemplated include cytokines, growth factors, steroids, NSAIDs, DMARDs, anti-inflammatory agents, chemotherapeutic agents, radiotherapeutic agents, therapeutic antibodies, or other active agents and adjuvants.

[0442] 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 bridging factor is administered to the subject before, during, in combination with, or after the cell composition, and optionally, administered repeatedly to the subject.

[0443] 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, therapeutic antibodies, and the like.

[0444] I. Treatment Methods

[0445] Immune effector cells modified to express polynucleotides encoding one or more CD33 VHH DARIC components or anti-CD33 VHHCARs contemplated herein provide improved methods of adoptive immunotherapy for preventing, treating, and ameliorating immune disorders such as cancer or preventing, treating, or ameliorating at least one symptom associated therewith.

[0446] Immune effector cells comprising a CD33 DARIC signaling component, a CD33 VHH DARIC binding component, or an anti-CD33 VHH CAR provide improved adoptive immunotherapy methods for preventing, treating, and ameliorating immune disorders such as cancer or preventing, treating, or ameliorating at least one symptom associated therewith.

[0447] In certain embodiments, immune effector cells modified to express CD33 VHH DARICs provide an improved approach to adoptive immunotherapy to fine-tune the safety and efficacy of cytotoxic responses against target cells expressing the target antigen (e.g., tumor cells) while reducing the risk of on-target antigen, off-target cytotoxicity (recognition of the target antigen on normal non-target cells).

[0448] In certain embodiments, a method for preventing, treating, or ameliorating at least one symptom of cancer comprises administering to a subject an effective amount of modified 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. By transducing chemically regulated immune stimulatory signals, genetically modified cells are more effective and safe cellular immunotherapies.

[0449] In certain embodiments, one or more immune effector cells (e.g., T cells) are modified to express both a CD33 VHH DARIC binding component and a CD33 DARIC signaling component. In this case, the modified cells are administered to a subject in need thereof and homed to the target cells through interaction between the CD33 VHH binding component expressed on the immune effector cells and CD33 expressed on the target cells. A bridging factor is administered to the subject before, at about the same time as, or after the modified cells are administered to the subject. In the presence of the bridging factor, a ternary complex is formed between the CD33 VHH DARIC binding component, the bridging factor, and the CD33 DARIC signaling component. After the ternary complex is formed, the CD33 VHH DARIC transduces an immunostimulatory signal to the immune effector cell, which in turn triggers a cytotoxic response against the target cell from the immune effector cell.

[0450] 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 is administered to the subject before, at approximately the same time as, or after the modified cells have been administered to the subject. Alternatively, the CD33 VHH DARIC binding component can be administered to the subject in a preformed complex with a bridging factor; at the same time as the bridging factor but in a separate composition; or at a different time than the bridging factor. The CD33 VHH binding component binds to CD33 expressed on the target cell in the presence or absence of a bridging factor. In the presence of a bridging factor, a ternary complex is formed between the CD33 VHH DARIC binding component, the bridging factor, and the CD33 DARIC signaling component. After forming the ternary complex, the CD33 VHH DARIC transduces immunostimulatory signals to immune effector cells, which in turn elicit a cytotoxic response from the immune effector cells against target cells.

[0451] 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 is administered to the subject before, at about the same time as, or after the modified cells have been administered to the subject. Additionally, the CD33 VHH DARIC binding component can be administered to the subject in a preformed complex with a bridging factor; at the same time as the bridging factor, but in a separate composition; or at a different time than the bridging factor. The CD33 binding component binds to a target antigen expressed on a target cell in the presence or absence of a bridging factor. In the presence of a bridging factor, a ternary complex is formed between the CD33 VHH DARIC binding component, the bridging factor, and the CD33 DARIC signaling component. After forming the ternary complex, the CD33 VHH DARIC transduces immunostimulatory signals to immune effector cells, which in turn trigger a cytotoxic response from the immune effector cells against the target cells. In certain embodiments, CD33 VHH DARIC activation can be induced in cases where remission or resolution is incomplete and the condition relapses or becomes refractory.

[0452] In particularly preferred embodiments, the specificity of primary T cells is redirected towards CD33-expressing tumor or cancer cells by genetically modifying T cells (e.g., primary T cells) with one or more CD33 VHH DARIC components.

[0453] In particularly preferred embodiments, the specificity of primary T cells is redirected to tumor cells or cancer cells expressing CD33 by genetically modifying T cells (e.g., primary T cells) with an engineered antigen receptor for the target antigen and one or more CD33 VHH DARIC components.

[0454] In certain embodiments, the modified immune effector cells contemplated herein are used to treat solid tumors or cancers.

[0455] In certain embodiments, the modified immune effector cells encompassed herein are used to treat solid tumors or cancers, including but not limited to adrenal cancer, adrenocortical cancer, anal cancer, appendix cancer, astrocytoma, atypical teratoid / rhabdoid tumor, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain / CNS cancer, breast cancer, bronchial tumors, cardiac tumors, cervical cancer, bile duct cancer, chondrosarcoma, chordoma, colon cancer, colorectal cancer, craniopharyngioma, ductal carcinoma in situ (DCIS), and chondrosarcoma. ) Endometrial cancer, ependymoma, esophageal cancer, nasal glioma, Ewing's sarcoma, extracranial germ cell tumor, extragonadal germ cell tumor, eye cancer, fallopian tube cancer, fibrosarcoma, fibrosarcoma, gallbladder cancer, gastric cancer, gastrointestinal carcinoid, 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, lip cancer, fat meat tumors, liver cancer, lung cancer, non-small cell lung cancer, lung carcinoid, malignant mesothelioma, medullary carcinoma, medulloblastoma, meningioma, melanoma, Merkel cell carcinoma, midline cancer, oral cancer, mucosal sarcoma, myelodysplastic syndrome, myeloproliferative neoplasms, nasal cavity and sinus cancer, nasopharyngeal cancer, neuroblastoma, oligodendroglioma, oral cancer, oral cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, islet cell tumor, papillary carcinoma, paraganglioma, parathyroid cancer, penile cancer, pharyngeal cancer , pheochromocytoma, pinealoma, pituitary tumor, pleuropulmonary blastoma, primary peritoneal cancer, prostate cancer, rectal cancer, retinoblastoma, renal cell carcinoma, renal pelvis and ureter cancer, rhabdomyosarcoma, salivary gland cancer, sebaceous gland cancer, skin cancer, soft tissue sarcoma, squamous cell carcinoma, small cell lung cancer, small intestine cancer, stomach cancer, sweat gland cancer, synovioma, testicular cancer, pharyngeal cancer, thymus cancer, thyroid cancer, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, vascular cancer, vulvar cancer and Wilms tumor.

[0456] In certain embodiments, the modified immune effector cells encompassed herein are used to treat 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.

[0457] In certain embodiments, the modified immune effector cells contemplated herein are used to treat 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.

[0458] In certain embodiments, the modified immune effector cells contemplated herein are used to treat glioblastoma.

[0459] In certain embodiments, the modified immune effector cells contemplated herein are used to treat liquid or hematological cancers.

[0460] In certain embodiments, the modified immune effector cells contemplated herein are used to treat B-cell malignancies, including but not limited to leukemias, lymphomas, and multiple myeloma.

[0461] In certain embodiments, the modified immune effector cells encompassed herein are used to treat liquid cancers, including but not limited to leukemias, lymphomas, and multiple myeloma: 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 myeloid leukemia (CML), chronic myelomonocytic leukemia (CMML) and polycythemia vera, Hodgkin lymphoma, nodular lymphocyte-predominant Hodgkin lymphoma, Burkitt lymphoma, 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, nonsecretory myeloma, IgD myeloma, osteosclerotic myeloma, solitary plasmacytoma of bone, and extramedullary plasmacytoma.

[0462] In certain embodiments, the modified immune effector cells contemplated herein are used to treat acute myeloid leukemia (AML).

[0463] Preferred cells for use in the methods contemplated herein include autologous / self ("self") cells, preferably hematopoietic cells, more preferably T cells, and more preferably immune effector cells.

[0464] In certain embodiments, the methods comprise administering to a patient in need thereof a therapeutically effective amount of modified immune effector cells expressing one or more CD33 VHH DARIC components, and further administering to the subject a bridging factor. In certain embodiments, the cells are used to treat a patient at risk of developing an immune disorder. Thus, certain embodiments include treating or preventing or ameliorating at least one symptom of an immune disorder, such as cancer, comprising administering to a subject in need thereof a therapeutically effective amount of a modified immune effector cell contemplated herein and a bridging factor.

[0465] In certain embodiments, the method comprises administering to a patient in need thereof a therapeutically effective amount of a modified immune effector cell expressing an anti-CD33 VHHCAR. In certain embodiments, the cells are used to treat a patient at risk of developing an immune disorder. Thus, certain embodiments include treating or preventing or ameliorating at least one symptom of an immune disorder, such as cancer, comprising administering to a subject in need thereof a therapeutically effective amount of a modified immune effector cell and a bridging factor as contemplated herein.

[0466] In certain embodiments, the method comprises administering to a patient in need thereof a therapeutically effective amount of modified immune effector cells expressing a CD33 DARIC signaling component, or a composition comprising the same, and further administering to the subject a CD33 VHH DARIC binding component and a bridging factor, optionally wherein the CD33 VHH DARIC binding component is bound to the bridging factor prior to administration. In certain embodiments, the cells are used to treat a patient at risk of developing an immune disorder. Thus, certain embodiments include treating, preventing, or ameliorating at least one symptom of an immune disorder, such as cancer, comprising administering to a subject in need thereof a therapeutically effective amount of modified 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 bridging factor.

[0467] The amount and frequency of administration of modified immune effector cells, CD33 DARIC VHH binding components and / or bridging factors will be determined by factors such as the patient's condition and the type and severity of the patient's disease, although appropriate doses and dosing schedules can be determined through clinical trials.

[0468] In one illustrative embodiment, the effective amount of modified immune effector cells provided to a subject is at least 2×10 6 cells / kg, at least 3×10 6 cells / kg, at least 4×10 6 cells / kg, at least 5×10 6 cells / kg, at least 6×10 6 cells / kg, at least 7×10 6 cells / kg, at least 8×10 6 cells / kg, at least 9×10 6 cells / kg, or at least 10×10 6 cells / kg or more, including all intermediate doses of cells.

[0469] In another illustrative embodiment, the effective amount of modified immune effector cells provided to a subject is about 2×10 6 cells / kg, about 3×10 6 cells / kg, about 4×10 6 cells / kg, about 5×10 6 cells / kg, about 6×10 6 cells / kg, about 7×10 6 cells / kg, about 8×10 6 cells / kg, about 9×10 6 cells / kg, or approximately 10×10 6 cells / kg or more, including all intermediate doses of cells.

[0470] In another illustrative embodiment, the effective amount of modified immune effector cells provided to a subject is about 2×10 6 cells / kg to about 10×10 6 cells / kg, about 3×10 6 cells / kg to about 10×10 6 cells / kg, about 4×10 6 cells / kg to about 10×10 6 cells / kg, about 5×10 6 cells / kg to about 10×10 6 cells / kg, 2×10 6 cells / kg to about 6×10 6 cells / kg, 2×10 6 cells / kg to about 7×10 6 cells / kg, 2×10 6 cells / kg to about 8×106 cells / kg, 3×10 6 cells / kg to about 6×10 6 cells / kg, 3×10 6 cells / kg to about 7×10 6 cells / kg, 3×10 6 cells / kg to about 8×10 6 cells / kg, 4×10 6 cells / kg to about 6×10 6 cells / kg, 4×10 6 cells / kg to about 7×10 6 cells / kg, 4×10 6 cells / kg to about 8×10 6 cells / kg, 5×10 6 cells / kg to about 6×10 6 cells / kg, 5×10 6 cells / kg to about 7×10 6 cells / kg, 5×10 6 cells / kg to about 8×10 6 cells / kg or 6×10 6 cells / kg to about 8×10 6 cells / kg, including all intermediate doses.

[0471] Those of ordinary skill in the art will recognize that multiple administrations of the compositions encompassed in a particular embodiment may be required to achieve the desired therapy. For example, the composition may be administered once, twice, three times, four times, five times, six times, seven times, eight times, nine times, or ten times or more over a period of one week, two weeks, three weeks, one month, two months, three months, four months, five months, six months, one year, two years, five years, ten years, or longer. The modified immune effector cells, CD33 VHH DARIC components, and bridging factors may be administered in the same or different compositions; simultaneously in one or more compositions; or at different times in more than one composition. The modified immune effector cells, CD33 VHH DARIC components, and bridging factors may be administered by the same route of administration or by different routes.

[0472] In certain embodiments, it may be desirable to administer activated T cells to a subject, and subsequently redraw blood (or perform apheresis), activate T cells therefrom, and re-infuse these activated and amplified T cells to the patient. This process can be performed multiple times every few weeks. In certain embodiments, 10cc to 400cc of blood can be drawn to activate T cells. In certain embodiments, 20cc, 30cc, 40cc, 50cc, 60cc, 70cc, 80cc, 90cc, 100cc, 150cc, 200cc, 250cc, 300cc, 350cc, or 400cc or more of blood are drawn to activate T cells. Without being bound by theory, using this multiple blood draw / multiple re-infusion protocol can be used to select certain T cell populations.

[0473] In one embodiment, a method of treating a subject diagnosed with cancer comprises: removing immune effector cells from the subject; modifying the immune effector cells by introducing one or more vectors encoding one or more CD33 VHH DARIC components into the cells and generating a population of modified immune effector cells; and administering the modified population of immune effector cells to the same subject. In a preferred embodiment, the immune effector cells comprise T cells.

[0474] In one embodiment, a method for treating a subject diagnosed with cancer comprises: removing immune effector cells from the subject; modifying the immune effector cells by introducing one or more vectors encoding anti-CD33 VHH CARs into the cells and generating a population of modified immune effector cells; and administering the modified immune effector cell population to the same subject. In a preferred embodiment, the immune effector cells include T cells.

[0475] Methods for administering the cell compositions encompassed in specific embodiments include any method that effectively results in the reintroduction of ex vivo modified immune effector cells or modified progenitors of immune effector cells that differentiate into mature immune effector cells when introduced into a subject. One method includes ex vivo modification of peripheral blood T cells by introducing one or more vectors encoding one or more CD33 VHH DARIC components or anti-CD33 VHH CARs into cells and returning the transduced cells to the subject.

[0476] 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.

[0477] Although the foregoing embodiments have been described in considerable detail by way of illustration and example for the purpose of clear understanding, it will be readily apparent to those skilled in the art that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims, based on the teachings encompassed herein. The following examples are provided for illustration only and are not intended to be limiting. Those skilled in the art will readily recognize various noncritical parameters that may be changed or modified in particular embodiments to produce substantially similar results.

[0478] Example

[0479] Example 1

[0480] CD33 VHH DARIC T cells exhibit anti-tumor responses

[0481] Design, construction and validation of anti-CD33 VHH DARIC binding and signaling components. A CD33-specific VHH DARIC lentiviral vector was constructed, comprising an MNDU3 promoter operably linked to polynucleotides encoding: DARIC signaling components (CD8α-signal peptide, FRB variant (T82L), CD8α transmembrane domain, intracellular 4-1BB costimulatory domain, and CD3ζ signaling domain); P2A sequence; and DARIC binding components (Igκ-signal peptide, CD33-specific VHH binding domain (camelid or humanized), G4S linker, FKBP12 domain, and CD4-derived transmembrane domain with truncated intracellular domain) ( Figure 1B ). See, e.g., SEQ ID NOs: 32-41. T cells transduced with anti-CD33 DARIC lentiviral vectors express Figure 1A The membrane-bound polypeptides shown in . Anti-CD33 scFv CAR or DARIC design were used as controls.

[0482] T cells from three donors were transduced with LVV encoding different CD33-specific VHH DARIC, anti-CD33 scFv DARIC or anti-CD33 scFv CAR and expanded for 10 days. Untransduced T cells, T cells transduced with anti-CD33 scFv control construct or anti-CD33 VHH DARIC T cells were stained with recombinant CD33-Fc reagent. Only control CAR and DARIC T cells were positively stained with CD33-Fc ( Figure 2A , the figure below, Figure 2B However, when analyzed with a VHH domain-specific monoclonal antibody, the majority of anti-CD33 VHH DARIC T cells, but not control CAR or DARIC T cells, stained positive ( Figure 2A, upper panel). As determined in part by CD62L and CD45RA staining, control CAR and DARIC T cells and anti-CD33 VHH DARIC T cells all had similar T cell phenotypes ( Figure 3A and Figure 3B ).

[0483] Untransduced T cells, T cells transduced with an anti-CD33 scFv control construct, or anti-CD33 VHH DARIC T cells were co-cultured with CD33 + THP-1 cells were co-cultured in the presence or absence of AP21967 at a 1:1 E:T ratio for 24 hours. Anti-CD33 scFv CAR control cells had robust cytokine production in the presence or absence of the rapamycin analog. Anti-CD33 scFv DARICT cells and anti-CD33 VHH DARIC T cells exhibited robust cytokine responses only when cultured with THP-1 cells in the presence of AP21967 ( Figure 4A and Figure 4B ). Minimal cytokine production was detected in the untransduced control.

[0484] Additionally, the specificity of VHH9 and VHH10 DARICs was assessed against full-length CD33 (CD33M) and a splice variant expressing a shorter truncated CD33 (CD33m). Human 293T cells were electroporated with mRNA encoding full-length CD33M or the splice variant CD33m. Figure 4C DARIC T cells were co-cultured with modified 293T cells at a 1:1 E:T ratio in the presence or absence of AP21967 for 24 hours, and activation was assessed as measured by cytokine secretion ( Figure 4C VHH9 DARIC T cells displayed robust cytokine responses to either CD33M or CD33m 293T cells, whereas VHH10 DARIC T cells were activated only in the presence of CD33M.

[0485] Example 2

[0486] CD33VHH DARIC T cells specifically respond to the CD33 antigen

[0487] Anti-CD33 VHH DARIC T cells were generated as described in Example 1. T cells from three donors were each transduced with LVV encoding a different anti-CD33 specific VHH DARIC and expanded for 10 days. Controls included untransduced (UTD) T cells and T cells transduced with 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 lacks CD33 expression on the cell surface. Figure 5A Anti-CD33 VHH DARIC T cells were co-cultured with MV4-11 cells or CD33-KO cells at a 1:1 E:T ratio for 24 hours in the presence or absence of the dimerizing drug. 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 .

[0488] The CD33-KO cell line was modified to express a CD33m splice variant (CD33-KO-C2). MV4-11 cells and CD33-KO-C2 cells were co-cultured with UTD cells, anti-CD33 CAR T cells, or anti-CD33VHH DARIC T cells in the presence or absence of the dimerizing drug, and cytokine production was analyzed after 24 hours. Anti-CD33VHH9 DARIC recognizes both normal CD33 and CD33m splice variants and produces cytokines when co-cultured with MV4-11 cells or CD33-KO-C2 cells ( Figure 5C ). Anti-CD33CAR T cells or anti-CD33VHH2 DARIC control T cells were active only against MV4-11 target cells.

[0489] Example 3

[0490] CD33 VHH DARIC T cells are not inhibited by soluble CD33 protein

[0491] Anti-CD33 VHH DARIC T cells were generated as described in Example 1. T cells from three donors were each transduced with LVV encoding a different anti-CD33 specific VHH DARIC and expanded for 10 days. +THP-1 cells were co-cultured for 24 hours in the presence or absence of rapamycin at a 1:1 E:T ratio. Various amounts of recombinant CD33-Fc protein were added during the co-culture period. Anti-CD33 VHH DARIC T cells exhibited robust cytokine responses in the presence and absence of recombinant soluble CD33 protein. Figure 6 .

[0492] Example 4

[0493] CD33 VHH DARIC T cells respond to low levels of CD33 antigen

[0494] Anti-CD33 VHH DARIC T cells were generated as described in Example 1. T cells from three donors were each transduced with LVV encoding a different anti-CD33 specific VHH DARIC and expanded for 10 days. Anti-CD33 VHH DARIC T cells were incubated with the AP21967 dimerizer and CD33 transfected with different amounts of CD33 mRNA. neg 293T cells were co-cultured. Supernatants were collected after 24 h and analyzed for cytokine production. Anti-CD33 VHH DARIC T cells showed a dose-dependent increase in IFNγ production even at very low mRNA concentrations after co-culture with CD33-transfected target cells ( Figure 7 ).

[0495] Example 5

[0496] CD33 VHH DARIC T cells control tumor growth in vivo

[0497] Anti-CD33 VHH DARIC T cells were generated as described in Example 1. CD33-expressing tumors were established in immunodeficient NSG mice by inoculating mice with HL60 AML tumor cells expressing a luciferase reporter gene. Tumor growth was monitored by luminescence. Ten days later, mice were administered 10 × 10 6 The researchers tested a single-cell mouse model of the rapamycin-treated mice and found that the mice received rapamycin alone or untransduced (UTD) T cells. Tumor growth was comparable between the treated and control groups. Figure 8A Mice treated with anti-CD33 vHHDARIC T cells and rapamycin showed enhanced tumor control compared with mice treated with UTD T cells and rapamycin. Figure 8B .

[0498] 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 encompass all possible embodiments, along with the full scope of equivalents to which such claims are entitled. Therefore, the claims are not limited by this disclosure. Sequence Listing <110> bluebird bio, Inc. Inhibrx, Inc. Jarjour, Jordan Pogson, Mark Leung Wai-Hang Kyle Jones Crago, William Sanabria, Angelica Hollands, Andrew Gano, Jacob Ma, Milton Timmer, John C. Eckelman, Brendan P. <120> CD33-targeted immunotherapy <130> BLBD-119 / 02WO 315698-3173 <150> US 62 / 898,392 <151> 2019-09-10 <150> US 62 / 845,304 <151> 2019-05-08 <160> 119 <170> PatentIn version 3.5 <210> 1 <211> 364 <212> PRT <213> Homo sapiens <400> 1 Met Pro Leu Leu Leu Leu Leu Pro Leu Leu Trp Ala Gly Ala Leu Ala 1 5 10 15 Met Asp Pro Asn Phe Trp Leu Gln Val Gln Glu Ser Val Thr Val Gln 20 25 30 Glu Gly Leu Cys Val Leu Val Pro Cys Thr Phe Phe His Pro Ile Pro 35 40 45 Tyr Tyr Asp Lys Asn Ser Pro Val His Gly Tyr Trp Phe Arg Glu Gly 50 55 60 Ala Ile Ile Ser Arg Asp Ser Pro Val Ala Thr Asn Lys Leu Asp Gln 65 70 75 80 Glu Val Gln Glu Glu Thr Gln Gly Arg Phe Arg Leu Leu Gly Asp Pro 85 90 95 Ser Arg Asn Asn Cys Ser Leu Ser Ile Val Asp Ala Arg Arg Arg Asp 100 105 110 Asn Gly Ser Tyr Phe Phe Arg Met Glu Arg Gly Ser Thr Lys Tyr Ser 115 120 125 Tyr Lys Ser Pro Gln Leu Ser Val His Val Thr Asp Leu Thr His Arg 130 135 140 Pro Lys Ile Leu Ile Pro Gly Thr Leu Glu Pro Gly His Ser Lys Asn 145 150 155 160 Leu Thr Cys Ser Val Ser Trp Ala Cys Glu Gln Gly Thr Pro Pro Ile 165 170 175 Phe Ser Trp Leu Ser Ala Ala Pro Thr Ser Leu Gly Pro Arg Thr Thr 180 185 190 His Ser Ser Val Leu Ile Ile Thr Pro Arg Pro Gln Asp His Gly Thr 195 200 205 Asn Leu Thr Cys Gln Val Lys Phe Ala Gly Ala Gly Val Thr Thr Glu 210 215 220 Arg Thr Ile Gln Leu Asn Val Thr Tyr Val Pro Gln Asn Pro Thr Thr 225 230 235 240 Gly Ile Phe Pro Gly Asp Gly Ser Gly Lys Gln Glu Thr Arg Ala Gly 245 250 255 Val Val His Gly Ala Ile Gly Gly Ala Gly Val Thr Ala Leu Leu Ala 260 265 270 Leu Cys Leu Cys Leu Ile Phe Phe Ile Val Lys Thr His Arg Arg Lys 275 280 285 Ala Ala Arg Thr Ala Val Gly Arg Asn Asp Thr His Pro Thr Thr Gly 290 295 300 Ser Ala Ser Pro Lys His Gln Lys Lys Ser Lys Leu His Gly Pro Thr 305 310 315 320 Glu Thr Ser Ser Cys Ser Gly Ala Ala Pro Thr Val Glu Met Asp Glu 325 330 335 Glu Leu His Tyr Ala Ser Leu Asn Phe His Gly Met Asn Pro Ser Lys 340 345 350 Asp Thr Ser Thr Glu Tyr Ser Glu Val Arg Thr Gln 355 360 <210> 2 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> Laboratory Preparation - Anti-CD33 VHH Sequence <400> 2 Gln Val Thr Leu Arg Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Lys Gly Ser Gly Arg Ala Ile Asn Thr Tyr 20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Ala Ile Ser Trp Asn Gly Gly His Thr Arg Tyr Ala Asp Ser Val 50 55 60 Gln Gly Arg Phe Ala Ile Ser Arg Asp Asn Ala Asp Asn Thr Met Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr His Cys 85 90 95 Ala Ala Tyr Ser Asp Tyr His Arg Ile Ala Thr Met Glu Ala Asp Ala 100 105 110 Asp Ser Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 3 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Laboratory Preparation - Anti-CD33 VHH Sequence <400> 3 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Glu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Gly Tyr 20 25 30 Ile Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val 35 40 45 Ala Arg Ile Ser Gly Asn Asn Leu Ser Thr Glu Tyr Ala Glu Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Glu Tyr Asp Tyr Ser Ser Gly Asp Phe Val Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Lys Pro 115 120 <210> 4 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Laboratory preparation - anti-CD33 VHH sequence <400> 4 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Glu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Gly Phe Ser Ala Ser 20 25 30 Leu Met Ser Trp His Arg Gln Ala Pro Gly Lys Gln Arg Asp Leu Val 35 40 45 Ala Ser Ile Thr Arg Asp Gly Arg Ala Asn Tyr Val Glu Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys His 85 90 95 Ala Tyr Ser Phe Asp Tyr Pro Ile Arg Ser Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Lys Pro 115 <210> 5 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Laboratory preparation - anti-CD33 VHH sequence <400> 5 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Glu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu Trp Val 35 40 45 Ala Ala Ile Thr Thr Ser Gly Asp Thr Thr Tyr Tyr Ala Glu Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala His Arg Gly Gly Gly Val Ile Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Lys Pro 115 <210> 6 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Laboratory Preparation - Anti-CD33 VHH Sequence <400> 6 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Glu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Phe Ser Ile Ser 20 25 30 Ile Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val 35 40 45 Ala Ser Thr Thr Ser Ser Gly Thr Thr Asn Tyr Val Glu Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys His 85 90 95 Ala Tyr Ile Ala Thr Thr Thr Asp Arg Gly Tyr Arg Gly Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Lys Pro 115 120 <210> 7 <211> 124 <212> PRT <213> Artificial Sequence <220> <223> Laboratory preparation - anti-CD33 VHH sequence <400> 7 Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Ser Ser Tyr Asn 20 25 30 Val Val Gly Trp Tyr Arg Gln Leu Ser Gly Asn Glu Arg Gly Gly Arg 35 40 45 Thr Met Val Ala Gln Ile Asn Ala Tyr Gly Asp Thr Asn Tyr Ala Asn 50 55 60 Ala Val Val Gly Arg Phe Thr Ile Ser Arg Asp Asp Ala Lys Asn Thr 65 70 75 80 Val Tyr Leu His Met Ser Asn Leu Lys Pro Glu Asp Thr Gly Val Tyr 85 90 95 Tyr Cys Asn Gly Gln Arg Met Leu Glu Asn Tyr Thr Tyr Arg Asp Gln 100 105 110 Ser Trp Gly Gln Gly Thr Gln Val Thr Val Lys Pro 115 120 <210> 8 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> Laboratory preparation - anti-CD33 VHH sequence <400> 8 Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Thr Leu Ser Cys Ala Ala Ser Arg Ser Ser Gly Ile Asp Val 20 25 30 Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val Ala 35 40 45 Glu Ile Ser Gly Val Gly Asp Thr Asn Tyr Ala Ala Ser Leu Ala Asp 50 55 60 Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln 65 70 75 80 Met Lys Asn Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn Ala 85 90 95 His Ser Phe Leu Asp Leu Val Gly Ala Trp Gly Gln Gly Thr Gln Val 100 105 110 Thr Val Lys Pro 115 <210> 9 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Laboratory Preparation - Anti-CD33 VHH Sequence <400> 9 Glu Val Gln Leu Val Gln Ser Gly Gly Gly Ser Val Gln Val Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Leu Asn Ile Asp 20 25 30 His Ile Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val 35 40 45 Gly Val Ile Ser Ser Gly Ala Gly Pro Asn Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Asn Cys Asn 85 90 95 Ala Trp Ile Asp Tyr Gly Ser Gly Leu Pro Gln Asn Tyr Trp Gly Gln 100 105 110 Gly Thr Gln Val Thr Val Lys Pro 115 120 <210> 10 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> Laboratory-prepared anti-CD33 VHH sequences <400> 10 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Glu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Arg Ser Ser Gly Ile Asp Val 20 25 30 Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val Ala 35 40 45 Glu Ile Ser Gly Val Gly Asp Thr Asn Tyr Ala Ala Ser Leu Ala Asp 50 55 60 Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln 65 70 75 80 Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Asn Ala 85 90 95 His Ser Phe Leu Asp Leu Val Gly Ala Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Lys Pro 115 <210> 11 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Laboratory-prepared anti-CD33 VHH sequences <400> 11 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Glu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Leu Asn Ile Asp 20 25 30 His Ile Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val 35 40 45 Gly Val Ile Ser Ser Gly Ala Gly Pro Asn Tyr Ala Glu Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Asn 85 90 95 Ala Trp Ile Asp Tyr Gly Ser Gly Leu Pro Gln Asn Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Lys Pro 115 120 <210> 12 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> Laboratory Preparation - Anti-CD33 VHH Sequence <220> <221> MOD_RES <222> (124)..(125) <223> Xaa is any amino acid or absent <400> 12 Gln Val Thr Leu Arg Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Lys Gly Ser Gly Arg Ala Ile Asn Thr Tyr 20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Ala Ile Ser Trp Asn Gly Gly His Thr Arg Tyr Ala Asp Ser Val 50 55 60 Gln Gly Arg Phe Ala Ile Ser Arg Asp Asn Ala Asp Asn Thr Met Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr His Cys 85 90 95 Ala Ala Tyr Ser Asp Tyr His Arg Ile Ala Thr Met Glu Ala Asp Ala 100 105 110 Asp Ser Trp Gly Gln Gly Thr Gln Val Thr Val Xaa Xaa 115 120 125 <210> 13 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Laboratory-prepared anti-CD33 VHH sequences <220> <221> MOD_RES <222> (119)..(120) <223> Xaa is any amino acid or absent <400> 13 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Glu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Gly Tyr 20 25 30 Ile Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val 35 40 45 Ala Arg Ile Ser Gly Asn Asn Leu Ser Thr Glu Tyr Ala Glu Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Glu Tyr Asp Tyr Ser Ser Gly Asp Phe Val Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Xaa Xaa 115 120 <210> 14 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Laboratory-prepared anti-CD33 VHH sequences <220> <221> MOD_RES <222> (117)..(118) <223> Xaa is any amino acid or absent <400> 14 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Glu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Gly Phe Ser Ala Ser 20 25 30 Leu Met Ser Trp His Arg Gln Ala Pro Gly Lys Gln Arg Asp Leu Val 35 40 45 Ala Ser Ile Thr Arg Asp Gly Arg Ala Asn Tyr Val Glu Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys His 85 90 95 Ala Tyr Ser Phe Asp Tyr Pro Ile Arg Ser Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Xaa Xaa 115 <210> 15 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Laboratory-prepared anti-CD33 VHH sequences <220> <221> MOD_RES <222> (117)..(118) <223> Xaa is any amino acid or absent <400> 15 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Glu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu Trp Val 35 40 45 Ala Ala Ile Thr Thr Ser Gly Asp Thr Thr Tyr Tyr Tyr Ala Glu Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala His Arg Gly Gly Gly Val Ile Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Xaa Xaa 115 <210> 16 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Laboratory-prepared anti-CD33 VHH sequences <220> <221> MOD_RES <222> (120)..(121) <223> Xaa is any amino acid or absent <400> 16 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Glu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Phe Ser Ile Ser 20 25 30 Ile Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val 35 40 45 Ala Ser Thr Thr Ser Ser Gly Thr Thr Asn Tyr Val Glu Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys His 85 90 95 Ala Tyr Ile Ala Thr Thr Thr Asp Arg Gly Tyr Arg Gly Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Xaa Xaa 115 120 <210> 17 <211> 124 <212> PRT <213> Artificial Sequence <220> <223> Laboratory-prepared anti-CD33 VHH sequences <220> <221> MOD_RES <222> (123)..(124) <223> Xaa is any amino acid or absent <400> 17 Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Ser Ser Tyr Asn 20 25 30 Val Val Gly Trp Tyr Arg Gln Leu Ser Gly Asn Glu Arg Gly Gly Arg 35 40 45 Thr Met Val Ala Gln Ile Asn Ala Tyr Gly Asp Thr Asn Tyr Ala Asn 50 55 60 Ala Val Val Gly Arg Phe Thr Ile Ser Arg Asp Asp Ala Lys Asn Thr 65 70 75 80 Val Tyr Leu His Met Ser Asn Leu Lys Pro Glu Asp Thr Gly Val Tyr 85 90 95 Tyr Cys Asn Gly Gln Arg Met Leu Glu Asn Tyr Thr Tyr Arg Asp Gln 100 105 110 Ser Trp Gly Gln Gly Thr Gln Val Thr Val Xaa Xaa 115 120 <210> 18 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> Laboratory-prepared anti-CD33 VHH sequences <220> <221> MOD_RES <222> (115)..(116) <223> Xaa is any amino acid or absent <400> 18 Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Thr Leu Ser Cys Ala Ala Ser Arg Ser Ser Gly Ile Asp Val 20 25 30 Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val Ala 35 40 45 Glu Ile Ser Gly Val Gly Asp Thr Asn Tyr Ala Ala Ser Leu Ala Asp 50 55 60 Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln 65 70 75 80 Met Lys Asn Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn Ala 85 90 95 His Ser Phe Leu Asp Leu Val Gly Ala Trp Gly Gln Gly Thr Gln Val 100 105 110 Thr Val Xaa Xaa 115 <210> 19 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Laboratory-prepared anti-CD33 VHH sequences <220> <221> MOD_RES <222> (119)..(120) <223> Xaa is any amino acid or absent <400> 19 Glu Val Gln Leu Val Gln Ser Gly Gly Gly Ser Val Gln Val Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Leu Asn Ile Asp 20 25 30 His Ile Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val 35 40 45 Gly Val Ile Ser Ser Gly Ala Gly Pro Asn Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Asn Cys Asn 85 90 95 Ala Trp Ile Asp Tyr Gly Ser Gly Leu Pro Gln Asn Tyr Trp Gly Gln 100 105 110 Gly Thr Gln Val Thr Val Xaa Xaa 115 120 <210> 20 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> Laboratory-prepared anti-CD33 VHH sequences <220> <221> MOD_RES <222> (115)..(116) <223> Xaa is any amino acid or absent <400> 20 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Glu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Arg Ser Ser Gly Ile Asp Val 20 25 30 Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val Ala 35 40 45 Glu Ile Ser Gly Val Gly Asp Thr Asn Tyr Ala Ala Ser Leu Ala Asp 50 55 60 Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln 65 70 75 80 Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Asn Ala 85 90 95 His Ser Phe Leu Asp Leu Val Gly Ala Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Xaa Xaa 115 <210> twenty one <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Laboratory-prepared anti-CD33 VHH sequences <220> <221> MOD_RES <222> (119)..(120) <223> Xaa is any amino acid or absent <400> twenty one Glu Val Gln Leu Val Glu Ser Gly Gly Gly Glu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Leu Asn Ile Asp 20 25 30 His Ile Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val 35 40 45 Gly Val Ile Ser Ser Gly Ala Gly Pro Asn Tyr Ala Glu Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Asn 85 90 95 Ala Trp Ile Asp Tyr Gly Ser Gly Leu Pro Gln Asn Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Xaa Xaa 115 120 <210> twenty two <211> 292 <212> PRT <213> Artificial Sequence <220> <223> Laboratory Preparation - Anti-CD33 VHH DARIC Binding Component <400> twenty two Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Gln Val Thr Leu Arg Glu Ser Gly Gly Gly Leu Val 20 25 30 Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Lys Gly Ser Gly Arg Ala 35 40 45 Ile Asn Thr Tyr Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu 50 55 60 Arg Glu Phe Val Ala Ala Ile Ser Trp Asn Gly Gly His Thr Arg Tyr 65 70 75 80 Ala Asp Ser Val Gln Gly Arg Phe Ala Ile Ser Arg Asp Asn Ala Asp 85 90 95 Asn Thr Met Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala 100 105 110 Val Tyr His Cys Ala Ala Tyr Ser Asp Tyr His Arg Ile Ala Thr Met 115 120 125 Glu Ala Asp Ala Asp Ser Trp Gly Gln Gly Thr Gln Val Thr Val Ser 130 135 140 Ser Gly Gly Gly Gly Ser Gly Val Gln Val Glu Thr Ile Ser Pro Gly 145 150 155 160 Asp Gly Arg Thr Phe Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr 165 170 175 Thr Gly Met Leu Glu Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg 180 185 190 Asn Lys Pro Phe Lys Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly 195 200 205 Trp Glu Glu Gly Val Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu 210 215 220 Thr Ile Ser Pro Asp Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile 225 230 235 240 Ile Pro Pro His Ala Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu 245 250 255 Glu Gly Gly Arg Met Ala Leu Ile Val Leu Gly Gly Val Ala Gly Leu 260 265 270 Leu Leu Phe Ile Gly Leu Gly Ile Phe Phe Cys Val Arg Cys Arg His 275 280 285 Arg Arg Arg Gln 290 <210> twenty three <211> 287 <212> PRT <213> Artificial Sequence <220> <223> Laboratory Preparation - Anti-CD33 VHH DARIC Binding Component <400> twenty three Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Glu Val Gln Leu Val Glu Ser Gly Gly Gly Glu Val 20 25 30 Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr 35 40 45 Phe Ser Gly Tyr Ile Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu 50 55 60 Arg Glu Leu Val Ala Arg Ile Ser Gly Asn Asn Leu Ser Thr Glu Tyr 65 70 75 80 Ala Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys 85 90 95 Asn Thr Leu Tyr Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala 100 105 110 Val Tyr Tyr Cys Ala Ala Glu Tyr Asp Tyr Ser Ser Gly Asp Phe Val 115 120 125 Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Lys Pro Gly Gly Gly Gly 130 135 140 Ser Gly Val Gln Val Glu Thr Ile Ser Pro Gly Asp Gly Arg Thr Phe 145 150 155 160 Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr Thr Gly Met Leu Glu 165 170 175 Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg Asn Lys Pro Phe Lys 180 185 190 Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly Trp Glu Glu Gly Val 195 200 205 Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu Thr Ile Ser Pro Asp 210 215 220 Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile Ile Pro Pro His Ala 225 230 235 240 Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu Glu Gly Gly Arg Met 245 250 255 Ala Leu Ile Val Leu Gly Gly Val Ala Gly Leu Leu Leu Phe Ile Gly 260 265 270 Leu Gly Ile Phe Phe Cys Val Arg Cys Arg His Arg Arg Arg Gln 275 280 285 <210> twenty four <211> 285 <212> PRT <213> Artificial Sequence <220> <223> Laboratory Preparation - Anti-CD33 VHH DARIC Binding Component <400> twenty four Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Glu Val Gln Leu Val Glu Ser Gly Gly Gly Glu Val 20 25 30 Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Gly 35 40 45 Phe Ser Ala Ser Leu Met Ser Trp His Arg Gln Ala Pro Gly Lys Gln 50 55 60 Arg Asp Leu Val Ala Ser Ile Thr Arg Asp Gly Arg Ala Asn Tyr Val 65 70 75 80 Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn 85 90 95 Thr Leu Tyr Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val 100 105 110 Tyr Tyr Cys His Ala Tyr Ser Phe Asp Tyr Pro Ile Arg Ser Tyr Trp 115 120 125 Gly Gln Gly Thr Leu Val Thr Val Lys Pro Gly Gly Gly Gly Ser Gly 130 135 140 Val Gln Val Glu Thr Ile Ser Pro Gly Asp Gly Arg Thr Phe Pro Lys 145 150 155 160 Arg Gly Gln Thr Cys Val Val His Tyr Thr Gly Met Leu Glu Asp Gly 165 170 175 Lys Lys Phe Asp Ser Ser Arg Asp Arg Asn Lys Pro Phe Lys Phe Met 180 185 190 Leu Gly Lys Gln Glu Val Ile Arg Gly Trp Glu Glu Gly Val Ala Gln 195 200 205 Met Ser Val Gly Gln Arg Ala Lys Leu Thr Ile Ser Pro Asp Tyr Ala 210 215 220 Tyr Gly Ala Thr Gly His Pro Gly Ile Ile Pro Pro His Ala Thr Leu 225 230 235 240 Val Phe Asp Val Glu Leu Leu Lys Leu Glu Gly Gly Arg Met Ala Leu 245 250 255 Ile Val Leu Gly Gly Val Ala Gly Leu Leu Leu Phe Ile Gly Leu Gly 260 265 270 Ile Phe Phe Cys Val Arg Cys Arg His Arg Arg Arg Gln 275 280 285 <210> 25 <211> 285 <212> PRT <213> Artificial Sequence <220> <223> Laboratory Preparation - Anti-CD33 VHH DARIC Binding Component <400> 25 Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Glu Val Gln Leu Val Glu Ser Gly Gly Gly Glu Val 20 25 30 Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr 35 40 45 Phe Ser Ser Tyr Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly 50 55 60 Arg Glu Trp Val Ala Ala Ile Thr Thr Ser Gly Asp Thr Thr Tyr Tyr 65 70 75 80 Ala Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys 85 90 95 Asn Thr Val Tyr Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala 100 105 110 Val Tyr Tyr Cys Ala Ala His Arg Gly Gly Gly Val Ile Asp Tyr Trp 115 120 125 Gly Gln Gly Thr Leu Val Thr Val Lys Pro Gly Gly Gly Gly Ser Gly 130 135 140 Val Gln Val Glu Thr Ile Ser Pro Gly Asp Gly Arg Thr Phe Pro Lys 145 150 155 160 Arg Gly Gln Thr Cys Val Val His Tyr Thr Gly Met Leu Glu Asp Gly 165 170 175 Lys Lys Phe Asp Ser Ser Arg Asp Arg Asn Lys Pro Phe Lys Phe Met 180 185 190 Leu Gly Lys Gln Glu Val Ile Arg Gly Trp Glu Glu Gly Val Ala Gln 195 200 205 Met Ser Val Gly Gln Arg Ala Lys Leu Thr Ile Ser Pro Asp Tyr Ala 210 215 220 Tyr Gly Ala Thr Gly His Pro Gly Ile Ile Pro Pro His Ala Thr Leu 225 230 235 240 Val Phe Asp Val Glu Leu Leu Lys Leu Glu Gly Gly Arg Met Ala Leu 245 250 255 Ile Val Leu Gly Gly Val Ala Gly Leu Leu Leu Phe Ile Gly Leu Gly 260 265 270 Ile Phe Phe Cys Val Arg Cys Arg His Arg Arg Arg Gln 275 280 285 <210> 26 <211> 288 <212> PRT <213> Artificial Sequence <220> <223> Laboratory Preparation - Anti-CD33 VHH DARIC Binding Component <400> 26 Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Glu Val Gln Leu Val Glu Ser Gly Gly Gly Glu Val 20 25 30 Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile 35 40 45 Phe Ser Ile Ser Ile Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu 50 55 60 Arg Glu Leu Val Ala Ser Thr Thr Ser Ser Gly Thr Thr Asn Tyr Val 65 70 75 80 Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn 85 90 95 Thr Leu Tyr Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val 100 105 110 Tyr Tyr Cys His Ala Tyr Ile Ala Thr Thr Thr Asp Arg Gly Tyr Arg 115 120 125 Gly Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Lys Pro Gly Gly Gly 130 135 140 Gly Ser Gly Val Gln Val Glu Thr Ile Ser Pro Gly Asp Gly Arg Thr 145 150 155 160 Phe Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr Thr Gly Met Leu 165 170 175 Glu Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg Asn Lys Pro Phe 180 185 190 Lys Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly Trp Glu Glu Gly 195 200 205 Val Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu Thr Ile Ser Pro 210 215 220 Asp Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile Ile Pro Pro His 225 230 235 240 Ala Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu Glu Gly Gly Arg 245 250 255 Met Ala Leu Ile Val Leu Gly Gly Val Ala Gly Leu Leu Leu Phe Ile 260 265 270 Gly Leu Gly Ile Phe Phe Cys Val Arg Cys Arg His Arg Arg Arg Gln 275 280 285 <210> 27 <211> 291 <212> PRT <213> Artificial Sequence <220> <223> Laboratory Preparation - Anti-CD33 VHH DARIC Binding Component <400> 27 Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val 20 25 30 Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile 35 40 45 Ser Ser Tyr Asn Val Val Gly Trp Tyr Arg Gln Leu Ser Gly Asn Glu 50 55 60 Arg Gly Gly Arg Thr Met Val Ala Gln Ile Asn Ala Tyr Gly Asp Thr 65 70 75 80 Asn Tyr Ala Asn Ala Val Val Gly Arg Phe Thr Ile Ser Arg Asp Asp 85 90 95 Ala Lys Asn Thr Val Tyr Leu His Met Ser Asn Leu Lys Pro Glu Asp 100 105 110 Thr Gly Val Tyr Tyr Cys Asn Gly Gln Arg Met Leu Glu Asn Tyr Thr 115 120 125 Tyr Arg Asp Gln Ser Trp Gly Gln Gly Thr Gln Val Thr Val Lys Pro 130 135 140 Gly Gly Gly Gly Ser Gly Val Gln Val Glu Thr Ile Ser Pro Gly Asp 145 150 155 160 Gly Arg Thr Phe Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr Thr 165 170 175 Gly Met Leu Glu Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg Asn 180 185 190 Lys Pro Phe Lys Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly Trp 195 200 205 Glu Glu Gly Val Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu Thr 210 215 220 Ile Ser Pro Asp Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile Ile 225 230 235 240 Pro Pro His Ala Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu Glu 245 250 255 Gly Gly Arg Met Ala Leu Ile Val Leu Gly Gly Val Ala Gly Leu Leu 260 265 270 Leu Phe Ile Gly Leu Gly Ile Phe Phe Cys Val Arg Cys Arg His Arg 275 280 285 Arg Arg Gln 290 <210> 28 <211> 283 <212> PRT <213> Artificial Sequence <220> <223> Laboratory Preparation - Anti-CD33 VHH DARIC Binding Component <400> 28 Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val 20 25 30 Gln Ala Gly Gly Ser Leu Thr Leu Ser Cys Ala Ala Ser Arg Ser Ser 35 40 45 Gly Ile Asp Val Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg 50 55 60 Glu Leu Val Ala Glu Ile Ser Gly Val Gly Asp Thr Asn Tyr Ala Ala 65 70 75 80 Ser Leu Ala Asp Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Asn Thr 85 90 95 Val Tyr Leu Gln Met Lys Asn Leu Lys Pro Glu Asp Thr Ala Val Tyr 100 105 110 Tyr Cys Asn Ala His Ser Phe Leu Asp Leu Val Gly Ala Trp Gly Gln 115 120 125 Gly Thr Gln Val Thr Val Lys Pro Gly Gly Gly Gly Ser Gly Val Gln 130 135 140 Val Glu Thr Ile Ser Pro Gly Asp Gly Arg Thr Phe Pro Lys Arg Gly 145 150 155 160 Gln Thr Cys Val Val His Tyr Thr Gly Met Leu Glu Asp Gly Lys Lys 165 170 175 Phe Asp Ser Ser Arg Asp Arg Asn Lys Pro Phe Lys Phe Met Leu Gly 180 185 190 Lys Gln Glu Val Ile Arg Gly Trp Glu Glu Gly Val Ala Gln Met Ser 195 200 205 Val Gly Gln Arg Ala Lys Leu Thr Ile Ser Pro Asp Tyr Ala Tyr Gly 210 215 220 Ala Thr Gly His Pro Gly Ile Ile Pro Pro His Ala Thr Leu Val Phe 225 230 235 240 Asp Val Glu Leu Leu Lys Leu Glu Gly Gly Arg Met Ala Leu Ile Val 245 250 255 Leu Gly Gly Val Ala Gly Leu Leu Leu Phe Ile Gly Leu Gly Ile Phe 260 265 270 Phe Cys Val Arg Cys Arg His Arg Arg Arg Gln 275 280 <210> 29 <211> 287 <212> PRT <213> Artificial Sequence <220> <223> Laboratory Preparation - Anti-CD33 VHH DARIC Binding Component <400> 29 Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Glu Val Gln Leu Val Gln Ser Gly Gly Gly Ser Val 20 25 30 Gln Val Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr 35 40 45 Leu Asn Ile Asp His Ile Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu 50 55 60 Arg Glu Leu Val Gly Val Ile Ser Ser Gly Ala Gly Pro Asn Tyr Ala 65 70 75 80 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn 85 90 95 Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val 100 105 110 Tyr Asn Cys Asn Ala Trp Ile Asp Tyr Gly Ser Gly Leu Pro Gln Asn 115 120 125 Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Lys Pro Gly Gly Gly Gly 130 135 140 Ser Gly Val Gln Val Glu Thr Ile Ser Pro Gly Asp Gly Arg Thr Phe 145 150 155 160 Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr Thr Gly Met Leu Glu 165 170 175 Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg Asn Lys Pro Phe Lys 180 185 190 Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly Trp Glu Glu Gly Val 195 200 205 Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu Thr Ile Ser Pro Asp 210 215 220 Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile Ile Pro Pro His Ala 225 230 235 240 Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu Glu Gly Gly Arg Met 245 250 255 Ala Leu Ile Val Leu Gly Gly Val Ala Gly Leu Leu Leu Phe Ile Gly 260 265 270 Leu Gly Ile Phe Phe Cys Val Arg Cys Arg His Arg Arg Arg Gln 275 280 285 <210> 30 <211> 283 <212> PRT <213> Artificial Sequence <220> <223> Laboratory Preparation - Anti-CD33 VHH DARIC Binding Component <400> 30 Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Glu Val Gln Leu Val Glu Ser Gly Gly Gly Glu Val 20 25 30 Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Arg Ser Ser 35 40 45 Gly Ile Asp Val Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg 50 55 60 Glu Leu Val Ala Glu Ile Ser Gly Val Gly Asp Thr Asn Tyr Ala Ala 65 70 75 80 Ser Leu Ala Asp Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Asn Thr 85 90 95 Val Tyr Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 100 105 110 Tyr Cys Asn Ala His Ser Phe Leu Asp Leu Val Gly Ala Trp Gly Gln 115 120 125 Gly Thr Leu Val Thr Val Lys Pro Gly Gly Gly Gly Ser Gly Val Gln 130 135 140 Val Glu Thr Ile Ser Pro Gly Asp Gly Arg Thr Phe Pro Lys Arg Gly 145 150 155 160 Gln Thr Cys Val Val His Tyr Thr Gly Met Leu Glu Asp Gly Lys Lys 165 170 175 Phe Asp Ser Ser Arg Asp Arg Asn Lys Pro Phe Lys Phe Met Leu Gly 180 185 190 Lys Gln Glu Val Ile Arg Gly Trp Glu Glu Gly Val Ala Gln Met Ser 195 200 205 Val Gly Gln Arg Ala Lys Leu Thr Ile Ser Pro Asp Tyr Ala Tyr Gly 210 215 220 Ala Thr Gly His Pro Gly Ile Ile Pro Pro His Ala Thr Leu Val Phe 225 230 235 240 Asp Val Glu Leu Leu Lys Leu Glu Gly Gly Arg Met Ala Leu Ile Val 245 250 255 Leu Gly Gly Val Ala Gly Leu Leu Leu Phe Ile Gly Leu Gly Ile Phe 260 265 270 Phe Cys Val Arg Cys Arg His Arg Arg Arg Gln 275 280 <210> 31 <211> 287 <212> PRT <213> Artificial Sequence <220> <223> Laboratory Preparation - Anti-CD33 VHH DARIC Binding Component <400> 31 Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Glu Val Gln Leu Val Glu Ser Gly Gly Gly Glu Val 20 25 30 Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr 35 40 45 Leu Asn Ile Asp His Ile Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu 50 55 60 Arg Glu Leu Val Gly Val Ile Ser Ser Gly Ala Gly Pro Asn Tyr Ala 65 70 75 80 Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn 85 90 95 Thr Val Tyr Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val 100 105 110 Tyr Tyr Cys Asn Ala Trp Ile Asp Tyr Gly Ser Gly Leu Pro Gln Asn 115 120 125 Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Lys Pro Gly Gly Gly Gly 130 135 140 Ser Gly Val Gln Val Glu Thr Ile Ser Pro Gly Asp Gly Arg Thr Phe 145 150 155 160 Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr Thr Gly Met Leu Glu 165 170 175 Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg Asn Lys Pro Phe Lys 180 185 190 Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly Trp Glu Glu Gly Val 195 200 205 Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu Thr Ile Ser Pro Asp 210 215 220 Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile Ile Pro Pro His Ala 225 230 235 240 Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu Glu Gly Gly Arg Met 245 250 255 Ala Leu Ile Val Leu Gly Gly Val Ala Gly Leu Leu Leu Phe Ile Gly 260 265 270 Leu Gly Ile Phe Phe Cys Val Arg Cys Arg His Arg Arg Arg Gln 275 280 285 <210> 32 <211> 617 <212> PRT <213> Artificial Sequence <220> <223> Laboratory preparation - anti-CD33 VHH DARIC fusion protein <400> 32 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Gly Ser Ile Leu Trp His Glu Met Trp His Glu 20 25 30 Gly Leu Glu Glu Ala Ser Arg Leu Tyr Phe Gly Glu Arg Asn Val Lys 35 40 45 Gly Met Phe Glu Val Leu Glu Pro Leu His Ala Met Met Glu Arg Gly 50 55 60 Pro Gln Thr Leu Lys Glu Thr Ser Phe Asn Gln Ala Tyr Gly Arg Asp 65 70 75 80 Leu Met Glu Ala Gln Glu Trp Cys Arg Lys Tyr Met Lys Ser Gly Asn 85 90 95 Val Lys Asp Leu Leu Gln Ala Trp Asp Leu Tyr Tyr His Val Phe Arg 100 105 110 Arg Ile Ser Lys Ala Ser Ala Gly Thr Gly Ser Asp Ile Tyr Ile Trp 115 120 125 Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile 130 135 140 Thr Met His Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln 145 150 155 160 Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser 165 170 175 Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys 180 185 190 Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln 195 200 205 Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu 210 215 220 Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg 225 230 235 240 Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met 245 250 255 Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly 260 265 270 Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp 275 280 285 Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Ser Gly Ser 290 295 300 Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu 305 310 315 320 Asn Pro Gly Pro Ser Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu 325 330 335 Leu Leu Trp Val Pro Gly Ser Thr Gly Gln Val Thr Leu Arg Glu Ser 340 345 350 Gly Gly Gly Leu Val Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Lys 355 360 365 Gly Ser Gly Arg Ala Ile Asn Thr Tyr Ala Met Gly Trp Phe Arg Gln 370 375 380 Ala Pro Gly Lys Glu Arg Glu Phe Val Ala Ala Ile Ser Trp Asn Gly 385 390 395 400 Gly His Thr Arg Tyr Ala Asp Ser Val Gln Gly Arg Phe Ala Ile Ser 405 410 415 Arg Asp Asn Ala Asp Asn Thr Met Tyr Leu Gln Met Asn Ser Leu Lys 420 425 430 Pro Glu Asp Thr Ala Val Tyr His Cys Ala Ala Tyr Ser Asp Tyr His 435 440 445 Arg Ile Ala Thr Met Glu Ala Asp Ala Asp Ser Trp Gly Gln Gly Thr 450 455 460 Gln Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Val Gln Val Glu 465 470 475 480 Thr Ile Ser Pro Gly Asp Gly Arg Thr Phe Pro Lys Arg Gly Gln Thr 485 490 495 Cys Val Val His Tyr Thr Gly Met Leu Glu Asp Gly Lys Lys Phe Asp 500 505 510 Ser Ser Arg Asp Arg Asn Lys Pro Phe Lys Phe Met Leu Gly Lys Gln 515 520 525 Glu Val Ile Arg Gly Trp Glu Glu Gly Val Ala Gln Met Ser Val Gly 530 535 540 Gln Arg Ala Lys Leu Thr Ile Ser Pro Asp Tyr Ala Tyr Gly Ala Thr 545 550 555 560 Gly His Pro Gly Ile Ile Pro Pro His Ala Thr Leu Val Phe Asp Val 565 570 575 Glu Leu Leu Lys Leu Glu Gly Gly Arg Met Ala Leu Ile Val Leu Gly 580 585 590 Gly Val Ala Gly Leu Leu Leu Phe Ile Gly Leu Gly Ile Phe Phe Cys 595 600 605 Val Arg Cys Arg His Arg Arg Arg Gln 610 615 <210> 33 <211> 612 <212> PRT <213> Artificial Sequence <220> <223> Laboratory Preparation - Anti-CD33 VHH DARIC Fusion Protein <400> 33 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Gly Ser Ile Leu Trp His Glu Met Trp His Glu 20 25 30 Gly Leu Glu Glu Ala Ser Arg Leu Tyr Phe Gly Glu Arg Asn Val Lys 35 40 45 Gly Met Phe Glu Val Leu Glu Pro Leu His Ala Met Met Glu Arg Gly 50 55 60 Pro Gln Thr Leu Lys Glu Thr Ser Phe Asn Gln Ala Tyr Gly Arg Asp 65 70 75 80 Leu Met Glu Ala Gln Glu Trp Cys Arg Lys Tyr Met Lys Ser Gly Asn 85 90 95 Val Lys Asp Leu Leu Gln Ala Trp Asp Leu Tyr Tyr His Val Phe Arg 100 105 110 Arg Ile Ser Lys Ala Ser Ala Gly Thr Gly Ser Asp Ile Tyr Ile Trp 115 120 125 Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile 130 135 140 Thr Met His Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln 145 150 155 160 Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser 165 170 175 Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys 180 185 190 Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln 195 200 205 Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu 210 215 220 Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg 225 230 235 240 Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met 245 250 255 Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly 260 265 270 Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp 275 280 285 Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Ser Gly Ser 290 295 300 Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu 305 310 315 320 Asn Pro Gly Pro Ser Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu 325 330 335 Leu Leu Trp Val Pro Gly Ser Thr Gly Glu Val Gln Leu Val Glu Ser 340 345 350 Gly Gly Gly Glu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala 355 360 365 Ala Ser Gly Arg Thr Phe Ser Gly Tyr Ile Met Gly Trp Phe Arg Gln 370 375 380 Ala Pro Gly Lys Glu Arg Glu Leu Val Ala Arg Ile Ser Gly Asn Asn 385 390 395 400 Leu Ser Thr Glu Tyr Ala Glu Ser Val Lys Gly Arg Phe Thr Ile Ser 405 410 415 Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Ser Ser Leu Arg 420 425 430 Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala Glu Tyr Asp Tyr Ser 435 440 445 Ser Gly Asp Phe Val Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Lys 450 455 460 Pro Gly Gly Gly Gly Ser Gly Val Gln Val Glu Thr Ile Ser Pro Gly 465 470 475 480 Asp Gly Arg Thr Phe Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr 485 490 495 Thr Gly Met Leu Glu Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg 500 505 510 Asn Lys Pro Phe Lys Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly 515 520 525 Trp Glu Glu Gly Val Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu 530 535 540 Thr Ile Ser Pro Asp Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile 545 550 555 560 Ile Pro Pro His Ala Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu 565 570 575 Glu Gly Gly Arg Met Ala Leu Ile Val Leu Gly Gly Val Ala Gly Leu 580 585 590 Leu Leu Phe Ile Gly Leu Gly Ile Phe Phe Cys Val Arg Cys Arg His 595 600 605 Arg Arg Arg Gln 610 <210> 34 <211> 610 <212> PRT <213> Artificial Sequence <220> <223> Laboratory Preparation - Anti-CD33 VHH DARIC Fusion Protein <400> 34 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Gly Ser Ile Leu Trp His Glu Met Trp His Glu 20 25 30 Gly Leu Glu Glu Ala Ser Arg Leu Tyr Phe Gly Glu Arg Asn Val Lys 35 40 45 Gly Met Phe Glu Val Leu Glu Pro Leu His Ala Met Met Glu Arg Gly 50 55 60 Pro Gln Thr Leu Lys Glu Thr Ser Phe Asn Gln Ala Tyr Gly Arg Asp 65 70 75 80 Leu Met Glu Ala Gln Glu Trp Cys Arg Lys Tyr Met Lys Ser Gly Asn 85 90 95 Val Lys Asp Leu Leu Gln Ala Trp Asp Leu Tyr Tyr His Val Phe Arg 100 105 110 Arg Ile Ser Lys Ala Ser Ala Gly Thr Gly Ser Asp Ile Tyr Ile Trp 115 120 125 Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile 130 135 140 Thr Met His Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln 145 150 155 160 Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser 165 170 175 Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys 180 185 190 Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln 195 200 205 Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu 210 215 220 Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg 225 230 235 240 Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met 245 250 255 Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly 260 265 270 Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp 275 280 285 Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Ser Gly Ser 290 295 300 Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu 305 310 315 320 Asn Pro Gly Pro Ser Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu 325 330 335 Leu Leu Trp Val Pro Gly Ser Thr Gly Glu Val Gln Leu Val Glu Ser 340 345 350 Gly Gly Gly Glu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala 355 360 365 Ala Ser Gly Ser Gly Phe Ser Ala Ser Leu Met Ser Trp His Arg Gln 370 375 380 Ala Pro Gly Lys Gln Arg Asp Leu Val Ala Ser Ile Thr Arg Asp Gly 385 390 395 400 Arg Ala Asn Tyr Val Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg 405 410 415 Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Ser Ser Leu Arg Ala 420 425 430 Glu Asp Thr Ala Val Tyr Tyr Cys His Ala Tyr Ser Phe Asp Tyr Pro 435 440 445 Ile Arg Ser Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Lys Pro Gly 450 455 460 Gly Gly Gly Ser Gly Val Gln Val Glu Thr Ile Ser Pro Gly Asp Gly 465 470 475 480 Arg Thr Phe Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr Thr Gly 485 490 495 Met Leu Glu Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg Asn Lys 500 505 510 Pro Phe Lys Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly Trp Glu 515 520 525 Glu Gly Val Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu Thr Ile 530 535 540 Ser Pro Asp Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile Ile Pro 545 550 555 560 Pro His Ala Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu Glu Gly 565 570 575 Gly Arg Met Ala Leu Ile Val Leu Gly Gly Val Ala Gly Leu Leu Leu 580 585 590 Phe Ile Gly Leu Gly Ile Phe Phe Cys Val Arg Cys Arg His Arg Arg 595 600 605 Arg Gln 610 <210> 35 <211> 610 <212> PRT <213> Artificial Sequence <220> <223> Laboratory-prepared anti-CD33 VHH DARIC fusion protein <400> 35 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Gly Ser Ile Leu Trp His Glu Met Trp His Glu 20 25 30 Gly Leu Glu Glu Ala Ser Arg Leu Tyr Phe Gly Glu Arg Asn Val Lys 35 40 45 Gly Met Phe Glu Val Leu Glu Pro Leu His Ala Met Met Glu Arg Gly 50 55 60 Pro Gln Thr Leu Lys Glu Thr Ser Phe Asn Gln Ala Tyr Gly Arg Asp 65 70 75 80 Leu Met Glu Ala Gln Glu Trp Cys Arg Lys Tyr Met Lys Ser Gly Asn 85 90 95 Val Lys Asp Leu Leu Gln Ala Trp Asp Leu Tyr Tyr His Val Phe Arg 100 105 110 Arg Ile Ser Lys Ala Ser Ala Gly Thr Gly Ser Asp Ile Tyr Ile Trp 115 120 125 Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile 130 135 140 Thr Met His Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln 145 150 155 160 Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser 165 170 175 Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys 180 185 190 Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln 195 200 205 Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu 210 215 220 Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg 225 230 235 240 Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met 245 250 255 Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly 260 265 270 Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp 275 280 285 Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Ser Gly Ser 290 295 300 Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu 305 310 315 320 Asn Pro Gly Pro Ser Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu 325 330 335 Leu Leu Trp Val Pro Gly Ser Thr Gly Glu Val Gln Leu Val Glu Ser 340 345 350 Gly Gly Gly Glu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala 355 360 365 Ala Ser Gly Phe Thr Phe Ser Ser Tyr Ala Met Gly Trp Phe Arg Gln 370 375 380 Ala Pro Gly Lys Gly Arg Glu Trp Val Ala Ala Ile Thr Thr Ser Gly 385 390 395 400 Asp Thr Thr Tyr Tyr Ala Glu Ser Val Lys Gly Arg Phe Thr Ile Ser 405 410 415 Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln Met Ser Ser Leu Arg 420 425 430 Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala His Arg Gly Gly Gly 435 440 445 Val Ile Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Lys Pro Gly 450 455 460 Gly Gly Gly Ser Gly Val Gln Val Glu Thr Ile Ser Pro Gly Asp Gly 465 470 475 480 Arg Thr Phe Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr Thr Gly 485 490 495 Met Leu Glu Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg Asn Lys 500 505 510 Pro Phe Lys Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly Trp Glu 515 520 525 Glu Gly Val Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu Thr Ile 530 535 540 Ser Pro Asp Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile Ile Pro 545 550 555 560 Pro His Ala Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu Glu Gly 565 570 575 Gly Arg Met Ala Leu Ile Val Leu Gly Gly Val Ala Gly Leu Leu Leu 580 585 590 Phe Ile Gly Leu Gly Ile Phe Phe Cys Val Arg Cys Arg His Arg Arg 595 600 605 Arg Gln 610 <210> 36 <211> 613 <212> PRT <213> Artificial Sequence <220> <223> Laboratory-prepared anti-CD33 VHH DARIC fusion protein <400> 36 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Gly Ser Ile Leu Trp His Glu Met Trp His Glu 20 25 30 Gly Leu Glu Glu Ala Ser Arg Leu Tyr Phe Gly Glu Arg Asn Val Lys 35 40 45 Gly Met Phe Glu Val Leu Glu Pro Leu His Ala Met Met Glu Arg Gly 50 55 60 Pro Gln Thr Leu Lys Glu Thr Ser Phe Asn Gln Ala Tyr Gly Arg Asp 65 70 75 80 Leu Met Glu Ala Gln Glu Trp Cys Arg Lys Tyr Met Lys Ser Gly Asn 85 90 95 Val Lys Asp Leu Leu Gln Ala Trp Asp Leu Tyr Tyr His Val Phe Arg 100 105 110 Arg Ile Ser Lys Ala Ser Ala Gly Thr Gly Ser Asp Ile Tyr Ile Trp 115 120 125 Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile 130 135 140 Thr Met His Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln 145 150 155 160 Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser 165 170 175 Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys 180 185 190 Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln 195 200 205 Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu 210 215 220 Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg 225 230 235 240 Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met 245 250 255 Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly 260 265 270 Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp 275 280 285 Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Ser Gly Ser 290 295 300 Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu 305 310 315 320 Asn Pro Gly Pro Ser Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu 325 330 335 Leu Leu Trp Val Pro Gly Ser Thr Gly Glu Val Gln Leu Val Glu Ser 340 345 350 Gly Gly Gly Glu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala 355 360 365 Ala Ser Gly Ser Ile Phe Ser Ile Ser Ile Met Gly Trp Tyr Arg Gln 370 375 380 Ala Pro Gly Lys Glu Arg Glu Leu Val Ala Ser Thr Thr Ser Ser Gly 385 390 395 400 Thr Thr Asn Tyr Val Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg 405 410 415 Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Ser Ser Leu Arg Ala 420 425 430 Glu Asp Thr Ala Val Tyr Tyr Cys His Ala Tyr Ile Ala Thr Thr Thr 435 440 445 Asp Arg Gly Tyr Arg Gly Tyr Trp Gly Gln Gly Thr Leu Val Thr Val 450 455 460 Lys Pro Gly Gly Gly Gly Ser Gly Val Gln Val Glu Thr Ile Ser Pro 465 470 475 480 Gly Asp Gly Arg Thr Phe Pro Lys Arg Gly Gln Thr Cys Val Val His 485 490 495 Tyr Thr Gly Met Leu Glu Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp 500 505 510 Arg Asn Lys Pro Phe Lys Phe Met Leu Gly Lys Gln Glu Val Ile Arg 515 520 525 Gly Trp Glu Glu Gly Val Ala Gln Met Ser Val Gly Gln Arg Ala Lys 530 535 540 Leu Thr Ile Ser Pro Asp Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly 545 550 555 560 Ile Ile Pro Pro His Ala Thr Leu Val Phe Asp Val Glu Leu Leu Lys 565 570 575 Leu Glu Gly Gly Arg Met Ala Leu Ile Val Leu Gly Gly Val Ala Gly 580 585 590 Leu Leu Leu Phe Ile Gly Leu Gly Ile Phe Phe Cys Val Arg Cys Arg 595 600 605 His Arg Arg Arg Gln 610 <210> 37 <211> 616 <212> PRT <213> Artificial Sequence <220> <223> Laboratory preparation - anti-CD33 VHH DARIC fusion protein <400> 37 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Gly Ser Ile Leu Trp His Glu Met Trp His Glu 20 25 30 Gly Leu Glu Glu Ala Ser Arg Leu Tyr Phe Gly Glu Arg Asn Val Lys 35 40 45 Gly Met Phe Glu Val Leu Glu Pro Leu His Ala Met Met Glu Arg Gly 50 55 60 Pro Gln Thr Leu Lys Glu Thr Ser Phe Asn Gln Ala Tyr Gly Arg Asp 65 70 75 80 Leu Met Glu Ala Gln Glu Trp Cys Arg Lys Tyr Met Lys Ser Gly Asn 85 90 95 Val Lys Asp Leu Leu Gln Ala Trp Asp Leu Tyr Tyr His Val Phe Arg 100 105 110 Arg Ile Ser Lys Ala Ser Ala Gly Thr Gly Ser Asp Ile Tyr Ile Trp 115 120 125 Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile 130 135 140 Thr Met His Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln 145 150 155 160 Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser 165 170 175 Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys 180 185 190 Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln 195 200 205 Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu 210 215 220 Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg 225 230 235 240 Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met 245 250 255 Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly 260 265 270 Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp 275 280 285 Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Ser Gly Ser 290 295 300 Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu 305 310 315 320 Asn Pro Gly Pro Ser Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu 325 330 335 Leu Leu Trp Val Pro Gly Ser Thr Gly Gln Val Gln Leu Val Gln Ser 340 345 350 Gly Gly Gly Leu Val Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Ala 355 360 365 Ala Ser Gly Ser Ile Ser Ser Tyr Asn Val Val Gly Trp Tyr Arg Gln 370 375 380 Leu Ser Gly Asn Glu Arg Gly Gly Arg Thr Met Val Ala Gln Ile Asn 385 390 395 400 Ala Tyr Gly Asp Thr Asn Tyr Ala Asn Ala Val Val Gly Arg Phe Thr 405 410 415 Ile Ser Arg Asp Asp Ala Lys Asn Thr Val Tyr Leu His Met Ser Asn 420 425 430 Leu Lys Pro Glu Asp Thr Gly Val Tyr Tyr Cys Asn Gly Gln Arg Met 435 440 445 Leu Glu Asn Tyr Thr Tyr Arg Asp Gln Ser Trp Gly Gln Gly Thr Gln 450 455 460 Val Thr Val Lys Pro Gly Gly Gly Gly Ser Gly Val Gln Val Glu Thr 465 470 475 480 Ile Ser Pro Gly Asp Gly Arg Thr Phe Pro Lys Arg Gly Gln Thr Cys 485 490 495 Val Val His Tyr Thr Gly Met Leu Glu Asp Gly Lys Lys Phe Asp Ser 500 505 510 Ser Arg Asp Arg Asn Lys Pro Phe Lys Phe Met Leu Gly Lys Gln Glu 515 520 525 Val Ile Arg Gly Trp Glu Glu Gly Val Ala Gln Met Ser Val Gly Gln 530 535 540 Arg Ala Lys Leu Thr Ile Ser Pro Asp Tyr Ala Tyr Gly Ala Thr Gly 545 550 555 560 His Pro Gly Ile Ile Pro Pro His Ala Thr Leu Val Phe Asp Val Glu 565 570 575 Leu Leu Lys Leu Glu Gly Gly Arg Met Ala Leu Ile Val Leu Gly Gly 580 585 590 Val Ala Gly Leu Leu Leu Phe Ile Gly Leu Gly Ile Phe Phe Cys Val 595 600 605 Arg Cys Arg His Arg Arg Arg Gln 610 615 <210> 38 <211> 608 <212> PRT <213> Artificial Sequence <220> <223> Laboratory Preparation - Anti-CD33 VHH DARIC Fusion Protein <400> 38 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Gly Ser Ile Leu Trp His Glu Met Trp His Glu 20 25 30 Gly Leu Glu Glu Ala Ser Arg Leu Tyr Phe Gly Glu Arg Asn Val Lys 35 40 45 Gly Met Phe Glu Val Leu Glu Pro Leu His Ala Met Met Glu Arg Gly 50 55 60 Pro Gln Thr Leu Lys Glu Thr Ser Phe Asn Gln Ala Tyr Gly Arg Asp 65 70 75 80 Leu Met Glu Ala Gln Glu Trp Cys Arg Lys Tyr Met Lys Ser Gly Asn 85 90 95 Val Lys Asp Leu Leu Gln Ala Trp Asp Leu Tyr Tyr His Val Phe Arg 100 105 110 Arg Ile Ser Lys Ala Ser Ala Gly Thr Gly Ser Asp Ile Tyr Ile Trp 115 120 125 Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile 130 135 140 Thr Met His Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln 145 150 155 160 Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser 165 170 175 Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys 180 185 190 Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln 195 200 205 Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu 210 215 220 Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg 225 230 235 240 Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met 245 250 255 Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly 260 265 270 Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp 275 280 285 Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Ser Gly Ser 290 295 300 Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu 305 310 315 320 Asn Pro Gly Pro Ser Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu 325 330 335 Leu Leu Trp Val Pro Gly Ser Thr Gly Gln Val Gln Leu Val Gln Ser 340 345 350 Gly Gly Gly Leu Val Gln Ala Gly Gly Ser Leu Thr Leu Ser Cys Ala 355 360 365 Ala Ser Arg Ser Ser Gly Ile Asp Val Met Gly Trp Tyr Arg Gln Ala 370 375 380 Pro Gly Lys Glu Arg Glu Leu Val Ala Glu Ile Ser Gly Val Gly Asp 385 390 395 400 Thr Asn Tyr Ala Ala Ser Leu Ala Asp Arg Phe Thr Val Ser Arg Asp 405 410 415 Asn Ala Lys Asn Thr Val Tyr Leu Gln Met Lys Asn Leu Lys Pro Glu 420 425 430 Asp Thr Ala Val Tyr Tyr Cys Asn Ala His Ser Phe Leu Asp Leu Val 435 440 445 Gly Ala Trp Gly Gln Gly Thr Gln Val Thr Val Lys Pro Gly Gly Gly 450 455 460 Gly Ser Gly Val Gln Val Glu Thr Ile Ser Pro Gly Asp Gly Arg Thr 465 470 475 480 Phe Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr Thr Gly Met Leu 485 490 495 Glu Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg Asn Lys Pro Phe 500 505 510 Lys Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly Trp Glu Glu Gly 515 520 525 Val Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu Thr Ile Ser Pro 530 535 540 Asp Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile Ile Pro Pro His 545 550 555 560 Ala Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu Glu Gly Gly Arg 565 570 575 Met Ala Leu Ile Val Leu Gly Gly Val Ala Gly Leu Leu Leu Phe Ile 580 585 590 Gly Leu Gly Ile Phe Phe Cys Val Arg Cys Arg His Arg Arg Arg Gln 595 600 605 <210> 39 <211> 612 <212> PRT <213> Artificial Sequence <220> <223> Laboratory Preparation - Anti-CD33 VHH DARIC Fusion Protein <400> 39 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Gly Ser Ile Leu Trp His Glu Met Trp His Glu 20 25 30 Gly Leu Glu Glu Ala Ser Arg Leu Tyr Phe Gly Glu Arg Asn Val Lys 35 40 45 Gly Met Phe Glu Val Leu Glu Pro Leu His Ala Met Met Glu Arg Gly 50 55 60 Pro Gln Thr Leu Lys Glu Thr Ser Phe Asn Gln Ala Tyr Gly Arg Asp 65 70 75 80 Leu Met Glu Ala Gln Glu Trp Cys Arg Lys Tyr Met Lys Ser Gly Asn 85 90 95 Val Lys Asp Leu Leu Gln Ala Trp Asp Leu Tyr Tyr His Val Phe Arg 100 105 110 Arg Ile Ser Lys Ala Ser Ala Gly Thr Gly Ser Asp Ile Tyr Ile Trp 115 120 125 Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile 130 135 140 Thr Met His Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln 145 150 155 160 Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser 165 170 175 Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys 180 185 190 Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln 195 200 205 Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu 210 215 220 Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg 225 230 235 240 Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met 245 250 255 Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly 260 265 270 Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp 275 280 285 Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Ser Gly Ser 290 295 300 Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu 305 310 315 320 Asn Pro Gly Pro Ser Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu 325 330 335 Leu Leu Trp Val Pro Gly Ser Thr Gly Glu Val Gln Leu Val Gln Ser 340 345 350 Gly Gly Gly Ser Val Gln Val Gly Gly Ser Leu Arg Leu Ser Cys Ala 355 360 365 Ala Ser Gly Ser Thr Leu Asn Ile Asp His Ile Gly Trp Tyr Arg Gln 370 375 380 Ala Pro Gly Lys Glu Arg Glu Leu Val Gly Val Ile Ser Ser Gly Ala 385 390 395 400 Gly Pro Asn Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg 405 410 415 Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro 420 425 430 Glu Asp Thr Ala Val Tyr Asn Cys Asn Ala Trp Ile Asp Tyr Gly Ser 435 440 445 Gly Leu Pro Gln Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Lys 450 455 460 Pro Gly Gly Gly Gly Ser Gly Val Gln Val Glu Thr Ile Ser Pro Gly 465 470 475 480 Asp Gly Arg Thr Phe Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr 485 490 495 Thr Gly Met Leu Glu Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg 500 505 510 Asn Lys Pro Phe Lys Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly 515 520 525 Trp Glu Glu Gly Val Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu 530 535 540 Thr Ile Ser Pro Asp Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile 545 550 555 560 Ile Pro Pro His Ala Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu 565 570 575 Glu Gly Gly Arg Met Ala Leu Ile Val Leu Gly Gly Val Ala Gly Leu 580 585 590 Leu Leu Phe Ile Gly Leu Gly Ile Phe Phe Cys Val Arg Cys Arg His 595 600 605 Arg Arg Arg Gln 610 <210> 40 <211> 608 <212> PRT <213> Artificial Sequence <220> <223> Laboratory Preparation - Anti-CD33 VHH DARIC Fusion Protein <400> 40 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Gly Ser Ile Leu Trp His Glu Met Trp His Glu 20 25 30 Gly Leu Glu Glu Ala Ser Arg Leu Tyr Phe Gly Glu Arg Asn Val Lys 35 40 45 Gly Met Phe Glu Val Leu Glu Pro Leu His Ala Met Met Glu Arg Gly 50 55 60 Pro Gln Thr Leu Lys Glu Thr Ser Phe Asn Gln Ala Tyr Gly Arg Asp 65 70 75 80 Leu Met Glu Ala Gln Glu Trp Cys Arg Lys Tyr Met Lys Ser Gly Asn 85 90 95 Val Lys Asp Leu Leu Gln Ala Trp Asp Leu Tyr Tyr His Val Phe Arg 100 105 110 Arg Ile Ser Lys Ala Ser Ala Gly Thr Gly Ser Asp Ile Tyr Ile Trp 115 120 125 Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile 130 135 140 Thr Met His Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln 145 150 155 160 Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser 165 170 175 Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys 180 185 190 Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln 195 200 205 Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu 210 215 220 Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg 225 230 235 240 Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met 245 250 255 Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly 260 265 270 Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp 275 280 285 Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Ser Gly Ser 290 295 300 Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu 305 310 315 320 Asn Pro Gly Pro Ser Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu 325 330 335 Leu Leu Trp Val Pro Gly Ser Thr Gly Glu Val Gln Leu Val Glu Ser 340 345 350 Gly Gly Gly Glu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala 355 360 365 Ala Ser Arg Ser Ser Gly Ile Asp Val Met Gly Trp Tyr Arg Gln Ala 370 375 380 Pro Gly Lys Glu Arg Glu Leu Val Ala Glu Ile Ser Gly Val Gly Asp 385 390 395 400 Thr Asn Tyr Ala Ala Ser Leu Ala Asp Arg Phe Thr Val Ser Arg Asp 405 410 415 Asn Ala Lys Asn Thr Val Tyr Leu Gln Met Ser Ser Leu Arg Ala Glu 420 425 430 Asp Thr Ala Val Tyr Tyr Cys Asn Ala His Ser Phe Leu Asp Leu Val 435 440 445 Gly Ala Trp Gly Gln Gly Thr Leu Val Thr Val Lys Pro Gly Gly Gly 450 455 460 Gly Ser Gly Val Gln Val Glu Thr Ile Ser Pro Gly Asp Gly Arg Thr 465 470 475 480 Phe Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr Thr Gly Met Leu 485 490 495 Glu Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg Asn Lys Pro Phe 500 505 510 Lys Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly Trp Glu Glu Gly 515 520 525 Val Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu Thr Ile Ser Pro 530 535 540 Asp Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile Ile Pro Pro His 545 550 555 560 Ala Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu Glu Gly Gly Arg 565 570 575 甲硫氨酸 丙氨酸 亮氨酸 异亮氨酸 缬氨酸 亮氨酸 甘氨酸 甘氨酸 缬氨酸 丙氨酸 甘氨酸 亮氨酸 亮氨酸 亮氨酸 苯丙氨酸 异亮氨酸 580 585 590 甘氨酸 亮氨酸 甘氨酸 异亮氨酸 苯丙氨酸 苯丙氨酸 半胱氨酸 缬氨酸 精氨酸 半胱氨酸 精氨酸 组氨酸 精氨酸 精氨酸 精氨酸 谷氨酰胺 595 600 605 <210> 41 <211> 612 <212> PRT <213> Artificial Sequence <220> <223> Laboratory preparation - anti-CD33 VHH DARIC fusion protein <400> 41 甲硫氨酸 丙氨酸 亮氨酸 脯氨酸 缬氨酸 苏氨酸 丙氨酸 亮氨酸 亮氨酸 亮氨酸 脯氨酸 亮氨酸 丙氨酸 亮氨酸 亮氨酸 亮氨酸 1 5 10 15 组氨酸 丙氨酸 丙氨酸 精氨酸 脯氨酸 甘氨酸 丝氨酸 异亮氨酸 亮氨酸 色氨酸 组氨酸 谷氨酸 甲硫氨酸 色氨酸 组氨酸 谷氨酸 20 25 30 甘氨酸 亮氨酸 谷氨酸 谷氨酸 丙氨酸 丝氨酸 精氨酸 亮氨酸 酪氨酸 苯丙氨酸 甘氨酸 谷氨酸 精氨酸 天冬酰胺 缬氨酸 赖氨酸 35 40 45 甘氨酸 甲硫氨酸 苯丙氨酸 谷氨酸 缬氨酸 亮氨酸 谷氨酸 脯氨酸 亮氨酸 组氨酸 丙氨酸 甲硫氨酸 甲硫氨酸 谷氨酸 精氨酸 甘氨酸 50 55 60 脯氨酸 谷氨酰胺 苏氨酸 亮氨酸 赖氨酸 谷氨酸 苏氨酸 丝氨酸 苯丙氨酸 天冬酰胺 谷氨酰胺 丙氨酸 酪氨酸 甘氨酸 精氨酸 天冬氨酸 65 70 75 80 亮氨酸 甲硫氨酸 谷氨酸 丙氨酸 谷氨酰胺 谷氨酸 色氨酸 半胱氨酸 精氨酸 赖氨酸 酪氨酸 甲硫氨酸 赖氨酸 丝氨酸 甘氨酸 天冬酰胺 85 90 95 Val Lys Asp Leu Leu Gln Ala Trp Asp Leu Tyr Tyr His Val Phe Arg 100 105 110 Arg Ile Ser Lys Ala Ser Ala Gly Thr Gly Ser Asp Ile Tyr Ile Trp 115 120 125 Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile 130 135 140 Thr Met His Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln 145 150 155 160 Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser 165 170 175 Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys 180 185 190 Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln 195 200 205 Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu 210 215 220 Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg 225 230 235 240 Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met 245 250 255 Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly 260 265 270 Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp 275 280 285 Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Ser Gly Ser 290 295 300 Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu 305 310 315 320 Asn Pro Gly Pro Ser Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu 325 330 335 Leu Leu Trp Val Pro Gly Ser Thr Gly Glu Val Gln Leu Val Glu Ser 340 345 350 Gly Gly Gly Glu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala 355 360 365 Ala Ser Gly Ser Thr Leu Asn Ile Asp His Ile Gly Trp Tyr Arg Gln 370 375 380 Ala Pro Gly Lys Glu Arg Glu Leu Val Gly Val Ile Ser Ser Gly Ala 385 390 395 400 Gly Pro Asn Tyr Ala Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg 405 410 415 Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln Met Ser Ser Leu Arg Ala 420 425 430 Glu Asp Thr Ala Val Tyr Tyr Cys Asn Ala Trp Ile Asp Tyr Gly Ser 435 440 445 Gly Leu Pro Gln Asn Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Lys 450 455 460 Pro Gly Gly Gly Gly Ser Gly Val Gln Val Glu Thr Ile Ser Pro Gly 465 470 475 480 Asp Gly Arg Thr Phe Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr 485 490 495 Thr Gly Met Leu Glu Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg 500 505 510 Asn Lys Pro Phe Lys Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly 515 520 525 Trp Glu Glu Gly Val Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu 530 535 540 Thr Ile Ser Pro Asp Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile 545 550 555 560 Ile Pro Pro His Ala Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu 565 570 575 Glu Gly Gly Arg Met Ala Leu Ile Val Leu Gly Gly Val Ala Gly Leu 580 585 590 Leu Leu Phe Ile Gly Leu Gly Ile Phe Phe Cys Val Arg Cys Arg His 595 600 605 Arg Arg Arg Gln 610 <210> 42 <211> 649 <212> PRT <213> Artificial Sequence <220> <223> Laboratory Preparation - Anti-CD33 VHH DARIC.OX40 Fusion Protein <400> 42 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Gly Ser Ile Leu Trp His Glu Met Trp His Glu 20 25 30 Gly Leu Glu Glu Ala Ser Arg Leu Tyr Phe Gly Glu Arg Asn Val Lys 35 40 45 Gly Met Phe Glu Val Leu Glu Pro Leu His Ala Met Met Glu Arg Gly 50 55 60 Pro Gln Thr Leu Lys Glu Thr Ser Phe Asn Gln Ala Tyr Gly Arg Asp 65 70 75 80 Leu Met Glu Ala Gln Glu Trp Cys Arg Lys Tyr Met Lys Ser Gly Asn 85 90 95 Val Lys Asp Leu Leu Gln Ala Trp Asp Leu Tyr Tyr His Val Phe Arg 100 105 110 Arg Ile Ser Lys Ala Ser Ala Gly Thr Gly Ser Asp Ile Tyr Ile Trp 115 120 125 Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile 130 135 140 Thr Met His Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln 145 150 155 160 Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser 165 170 175 Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys 180 185 190 Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln 195 200 205 Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu 210 215 220 Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg 225 230 235 240 Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met 245 250 255 Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly 260 265 270 Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp 275 280 285 Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Ser Gly Ser 290 295 300 Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu 305 310 315 320 Asn Pro Gly Pro Ser Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu 325 330 335 Leu Leu Trp Val Pro Gly Ser Thr Gly Gln Val Thr Leu Arg Glu Ser 340 345 350 Gly Gly Gly Leu Val Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Lys 355 360 365 Gly Ser Gly Arg Ala Ile Asn Thr Tyr Ala Met Gly Trp Phe Arg Gln 370 375 380 Ala Pro Gly Lys Glu Arg Glu Phe Val Ala Ala Ile Ser Trp Asn Gly 385 390 395 400 Gly His Thr Arg Tyr Ala Asp Ser Val Gln Gly Arg Phe Ala Ile Ser 405 410 415 Arg Asp Asn Ala Asp Asn Thr Met Tyr Leu Gln Met Asn Ser Leu Lys 420 425 430 Pro Glu Asp Thr Ala Val Tyr His Cys Ala Ala Tyr Ser Asp Tyr His 435 440 445 Arg Ile Ala Thr Met Glu Ala Asp Ala Asp Ser Trp Gly Gln Gly Thr 450 455 460 Gln Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Val Gln Val Glu 465 470 475 480 Thr Ile Ser Pro Gly Asp Gly Arg Thr Phe Pro Lys Arg Gly Gln Thr 485 490 495 Cys Val Val His Tyr Thr Gly Met Leu Glu Asp Gly Lys Lys Phe Asp 500 505 510 Ser Ser Arg Asp Arg Asn Lys Pro Phe Lys Phe Met Leu Gly Lys Gln 515 520 525 Glu Val Ile Arg Gly Trp Glu Glu Gly Val Ala Gln Met Ser Val Gly 530 535 540 Gln Arg Ala Lys Leu Thr Ile Ser Pro Asp Tyr Ala Tyr Gly Ala Thr 545 550 555 560 Gly His Pro Gly Ile Ile Pro Pro His Ala Thr Leu Val Phe Asp Val 565 570 575 Glu Leu Leu Lys Leu Glu Gly Gly Arg Met Ala Leu Ile Val Leu Gly 580 585 590 Gly Val Ala Gly Leu Leu Leu Phe Ile Gly Leu Gly Ile Phe Phe Ala 595 600 605 Leu Tyr Leu Leu Arg Arg Asp Gln Arg Leu Pro Pro Asp Ala His Lys 610 615 620 Pro Pro Gly Gly Gly Ser Phe Arg Thr Pro Ile Gln Glu Glu Gln Ala 625 630 635 640 Asp Ala His Ser Thr Leu Ala Lys Ile 645 <210> 43 <211> 644 <212> PRT <213> Artificial Sequence <220> <223> Laboratory preparation - anti-CD33 VHH DARIC.OX40 fusion protein <400> 43 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Gly Ser Ile Leu Trp His Glu Met Trp His Glu 20 25 30 Gly Leu Glu Glu Ala Ser Arg Leu Tyr Phe Gly Glu Arg Asn Val Lys 35 40 45 Gly Met Phe Glu Val Leu Glu Pro Leu His Ala Met Met Glu Arg Gly 50 55 60 Pro Gln Thr Leu Lys Glu Thr Ser Phe Asn Gln Ala Tyr Gly Arg Asp 65 70 75 80 Leu Met Glu Ala Gln Glu Trp Cys Arg Lys Tyr Met Lys Ser Gly Asn 85 90 95 Val Lys Asp Leu Leu Gln Ala Trp Asp Leu Tyr Tyr His Val Phe Arg 100 105 110 Arg Ile Ser Lys Ala Ser Ala Gly Thr Gly Ser Asp Ile Tyr Ile Trp 115 120 125 Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile 130 135 140 Thr Met His Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln 145 150 155 160 Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser 165 170 175 Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys 180 185 190 Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln 195 200 205 Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu 210 215 220 Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg 225 230 235 240 Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met 245 250 255 Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly 260 265 270 Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp 275 280 285 Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Ser Gly Ser 290 295 300 Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu 305 310 315 320 Asn Pro Gly Pro Ser Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu 325 330 335 Leu Leu Trp Val Pro Gly Ser Thr Gly Glu Val Gln Leu Val Glu Ser 340 345 350 Gly Gly Gly Glu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala 355 360 365 Ala Ser Gly Arg Thr Phe Ser Gly Tyr Ile Met Gly Trp Phe Arg Gln 370 375 380 Ala Pro Gly Lys Glu Arg Glu Leu Val Ala Arg Ile Ser Gly Asn Asn 385 390 395 400 Leu Ser Thr Glu Tyr Ala Glu Ser Val Lys Gly Arg Phe Thr Ile Ser 405 410 415 Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Ser Ser Leu Arg 420 425 430 Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala Glu Tyr Asp Tyr Ser 435 440 445 Ser Gly Asp Phe Val Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Lys 450 455 460 Pro Gly Gly Gly Gly Ser Gly Val Gln Val Glu Thr Ile Ser Pro Gly 465 470 475 480 Asp Gly Arg Thr Phe Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr 485 490 495 Thr Gly Met Leu Glu Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg 500 505 510 Asn Lys Pro Phe Lys Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly 515 520 525 Trp Glu Glu Gly Val Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu 530 535 540 Thr Ile Ser Pro Asp Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile 545 550 555 560 Ile Pro Pro His Ala Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu 565 570 575 Glu Gly Gly Arg Met Ala Leu Ile Val Leu Gly Gly Val Ala Gly Leu 580 585 590 Leu Leu Phe Ile Gly Leu Gly Ile Phe Phe Ala Leu Tyr Leu Leu Arg 595 600 605 Arg Asp Gln Arg Leu Pro Pro Asp Ala His Lys Pro Pro Gly Gly Gly 610 615 620 Ser Phe Arg Thr Pro Ile Gln Glu Glu Gln Ala Asp Ala His Ser Thr 625 630 635 640 Leu Ala Lys Ile <210> 44 <211> 642 <212> PRT <213> Artificial Sequence <220> <223> Laboratory Preparation - Anti-CD33 VHH DARIC.OX40 Fusion Protein <400> 44 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Gly Ser Ile Leu Trp His Glu Met Trp His Glu 20 25 30 Gly Leu Glu Glu Ala Ser Arg Leu Tyr Phe Gly Glu Arg Asn Val Lys 35 40 45 Gly Met Phe Glu Val Leu Glu Pro Leu His Ala Met Met Glu Arg Gly 50 55 60 Pro Gln Thr Leu Lys Glu Thr Ser Phe Asn Gln Ala Tyr Gly Arg Asp 65 70 75 80 Leu Met Glu Ala Gln Glu Trp Cys Arg Lys Tyr Met Lys Ser Gly Asn 85 90 95 Val Lys Asp Leu Leu Gln Ala Trp Asp Leu Tyr Tyr His Val Phe Arg 100 105 110 Arg Ile Ser Lys Ala Ser Ala Gly Thr Gly Ser Asp Ile Tyr Ile Trp 115 120 125 Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile 130 135 140 Thr Met His Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln 145 150 155 160 Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser 165 170 175 Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys 180 185 190 Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln 195 200 205 Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu 210 215 220 Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg 225 230 235 240 Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met 245 250 255 Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly 260 265 270 Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp 275 280 285 Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Ser Gly Ser 290 295 300 Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu 305 310 315 320 Asn Pro Gly Pro Ser Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu 325 330 335 Leu Leu Trp Val Pro Gly Ser Thr Gly Glu Val Gln Leu Val Glu Ser 340 345 350 Gly Gly Gly Glu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala 355 360 365 Ala Ser Gly Ser Gly Phe Ser Ala Ser Leu Met Ser Trp His Arg Gln 370 375 380 Ala Pro Gly Lys Gln Arg Asp Leu Val Ala Ser Ile Thr Arg Asp Gly 385 390 395 400 Arg Ala Asn Tyr Val Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg 405 410 415 Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Ser Ser Leu Arg Ala 420 425 430 Glu Asp Thr Ala Val Tyr Tyr Cys His Ala Tyr Ser Phe Asp Tyr Pro 435 440 445 Ile Arg Ser Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Lys Pro Gly 450 455 460 Gly Gly Gly Ser Gly Val Gln Val Glu Thr Ile Ser Pro Gly Asp Gly 465 470 475 480 Arg Thr Phe Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr Thr Gly 485 490 495 Met Leu Glu Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg Asn Lys 500 505 510 Pro Phe Lys Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly Trp Glu 515 520 525 Glu Gly Val Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu Thr Ile 530 535 540 Ser Pro Asp Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile Ile Pro 545 550 555 560 Pro His Ala Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu Glu Gly 565 570 575 Gly Arg Met Ala Leu Ile Val Leu Gly Gly Val Ala Gly Leu Leu Leu 580 585 590 Phe Ile Gly Leu Gly Ile Phe Phe Ala Leu Tyr Leu Leu Arg Arg Asp 595 600 605 Gln Arg Leu Pro Pro Asp Ala His Lys Pro Pro Gly Gly Gly Ser Phe 610 615 620 Arg Thr Pro Ile Gln Glu Glu Gln Ala Asp Ala His Ser Thr Leu Ala 625 630 635 640 Lys Ile <210> 45 <211> 642 <212> PRT <213> Artificial Sequence <220> <223> Laboratory Preparation - Anti-CD33 VHH DARIC.OX40 Fusion Protein <400> 45 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Gly Ser Ile Leu Trp His Glu Met Trp His Glu 20 25 30 Gly Leu Glu Glu Ala Ser Arg Leu Tyr Phe Gly Glu Arg Asn Val Lys 35 40 45 Gly Met Phe Glu Val Leu Glu Pro Leu His Ala Met Met Glu Arg Gly 50 55 60 Pro Gln Thr Leu Lys Glu Thr Ser Phe Asn Gln Ala Tyr Gly Arg Asp 65 70 75 80 Leu Met Glu Ala Gln Glu Trp Cys Arg Lys Tyr Met Lys Ser Gly Asn 85 90 95 Val Lys Asp Leu Leu Gln Ala Trp Asp Leu Tyr Tyr His Val Phe Arg 100 105 110 Arg Ile Ser Lys Ala Ser Ala Gly Thr Gly Ser Asp Ile Tyr Ile Trp 115 120 125 Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile 130 135 140 Thr Met His Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln 145 150 155 160 Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser 165 170 175 Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys 180 185 190 Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln 195 200 205 Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu 210 215 220 Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg 225 230 235 240 Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met 245 250 255 Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly 260 265 270 Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp 275 280 285 Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Ser Gly Ser 290 295 300 Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu 305 310 315 320 Asn Pro Gly Pro Ser Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu 325 330 335 Leu Leu Trp Val Pro Gly Ser Thr Gly Glu Val Gln Leu Val Glu Ser 340 345 350 Gly Gly Gly Glu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala 355 360 365 Ala Ser Gly Phe Thr Phe Ser Ser Tyr Ala Met Gly Trp Phe Arg Gln 370 375 380 Ala Pro Gly Lys Gly Arg Glu Trp Val Ala Ala Ile Thr Thr Ser Gly 385 390 395 400 Asp Thr Thr Tyr Tyr Ala Glu Ser Val Lys Gly Arg Phe Thr Ile Ser 405 410 415 Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln Met Ser Ser Leu Arg 420 425 430 Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala His Arg Gly Gly Gly 435 440 445 Val Ile Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Lys Pro Gly 450 455 460 Gly Gly Gly Ser Gly Val Gln Val Glu Thr Ile Ser Pro Gly Asp Gly 465 470 475 480 Arg Thr Phe Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr Thr Gly 485 490 495 Met Leu Glu Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg Asn Lys 500 505 510 Pro Phe Lys Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly Trp Glu 515 520 525 Glu Gly Val Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu Thr Ile 530 535 540 Ser Pro Asp Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile Ile Pro 545 550 555 560 Pro His Ala Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu Glu Gly 565 570 575 Gly Arg Met Ala Leu Ile Val Leu Gly Gly Val Ala Gly Leu Leu Leu 580 585 590 Phe Ile Gly Leu Gly Ile Phe Phe Ala Leu Tyr Leu Leu Arg Arg Asp 595 600 605 Gln Arg Leu Pro Pro Asp Ala His Lys Pro Pro Gly Gly Gly Ser Phe 610 615 620 Arg Thr Pro Ile Gln Glu Glu Gln Ala Asp Ala His Ser Thr Leu Ala 625 630 635 640 Lys Ile <210> 46 <211> 645 <212> PRT <213> Artificial Sequence <220> <223> Laboratory preparation - anti-CD33 VHH DARIC.OX40 fusion protein <400> 46 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Gly Ser Ile Leu Trp His Glu Met Trp His Glu 20 25 30 Gly Leu Glu Glu Ala Ser Arg Leu Tyr Phe Gly Glu Arg Asn Val Lys 35 40 45 Gly Met Phe Glu Val Leu Glu Pro Leu His Ala Met Met Glu Arg Gly 50 55 60 Pro Gln Thr Leu Lys Glu Thr Ser Phe Asn Gln Ala Tyr Gly Arg Asp 65 70 75 80 Leu Met Glu Ala Gln Glu Trp Cys Arg Lys Tyr Met Lys Ser Gly Asn 85 90 95 Val Lys Asp Leu Leu Gln Ala Trp Asp Leu Tyr Tyr His Val Phe Arg 100 105 110 Arg Ile Ser Lys Ala Ser Ala Gly Thr Gly Ser Asp Ile Tyr Ile Trp 115 120 125 Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile 130 135 140 Thr Met His Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln 145 150 155 160 Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser 165 170 175 Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys 180 185 190 Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln 195 200 205 Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu 210 215 220 Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg 225 230 235 240 Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met 245 250 255 Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly 260 265 270 Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp 275 280 285 Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Ser Gly Ser 290 295 300 Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu 305 310 315 320 Asn Pro Gly Pro Ser Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu 325 330 335 Leu Leu Trp Val Pro Gly Ser Thr Gly Glu Val Gln Leu Val Glu Ser 340 345 350 Gly Gly Gly Glu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala 355 360 365 Ala Ser Gly Ser Ile Phe Ser Ile Ser Ile Met Gly Trp Tyr Arg Gln 370 375 380 Ala Pro Gly Lys Glu Arg Glu Leu Val Ala Ser Thr Thr Ser Ser Gly 385 390 395 400 Thr Thr Asn Tyr Val Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg 405 410 415 Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Ser Ser Leu Arg Ala 420 425 430 Glu Asp Thr Ala Val Tyr Tyr Cys His Ala Tyr Ile Ala Thr Thr Thr 435 440 445 Asp Arg Gly Tyr Arg Gly Tyr Trp Gly Gln Gly Thr Leu Val Thr Val 450 455 460 Lys Pro Gly Gly Gly Gly Ser Gly Val Gln Val Glu Thr Ile Ser Pro 465 470 475 480 Gly Asp Gly Arg Thr Phe Pro Lys Arg Gly Gln Thr Cys Val Val His 485 490 495 Tyr Thr Gly Met Leu Glu Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp 500 505 510 Arg Asn Lys Pro Phe Lys Phe Met Leu Gly Lys Gln Glu Val Ile Arg 515 520 525 Gly Trp Glu Glu Gly Val Ala Gln Met Ser Val Gly Gln Arg Ala Lys 530 535 540 Leu Thr Ile Ser Pro Asp Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly 545 550 555 560 Ile Ile Pro Pro His Ala Thr Leu Val Phe Asp Val Glu Leu Leu Lys 565 570 575 Leu Glu Gly Gly Arg Met Ala Leu Ile Val Leu Gly Gly Val Ala Gly 580 585 590 Leu Leu Leu Phe Ile Gly Leu Gly Ile Phe Phe Ala Leu Tyr Leu Leu 595 600 605 Arg Arg Asp Gln Arg Leu Pro Pro Asp Ala His Lys Pro Pro Gly Gly 610 615 620 Gly Ser Phe Arg Thr Pro Ile Gln Glu Glu Gln Ala Asp Ala His Ser 625 630 635 640 Thr Leu Ala Lys Ile 645 <210> 47 <211> 648 <212> PRT <213> Artificial Sequence <220> <223> Laboratory-prepared anti-CD33 VHH DARIC.OX40 fusion protein <400> 47 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Gly Ser Ile Leu Trp His Glu Met Trp His Glu 20 25 30 Gly Leu Glu Glu Ala Ser Arg Leu Tyr Phe Gly Glu Arg Asn Val Lys 35 40 45 Gly Met Phe Glu Val Leu Glu Pro Leu His Ala Met Met Glu Arg Gly 50 55 60 Pro Gln Thr Leu Lys Glu Thr Ser Phe Asn Gln Ala Tyr Gly Arg Asp 65 70 75 80 Leu Met Glu Ala Gln Glu Trp Cys Arg Lys Tyr Met Lys Ser Gly Asn 85 90 95 Val Lys Asp Leu Leu Gln Ala Trp Asp Leu Tyr Tyr His Val Phe Arg 100 105 110 Arg Ile Ser Lys Ala Ser Ala Gly Thr Gly Ser Asp Ile Tyr Ile Trp 115 120 125 Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile 130 135 140 Thr Met His Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln 145 150 155 160 Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser 165 170 175 Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys 180 185 190 Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln 195 200 205 Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu 210 215 220 Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg 225 230 235 240 Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met 245 250 255 Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly 260 265 270 Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp 275 280 285 Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Ser Gly Ser 290 295 300 Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu 305 310 315 320 Asn Pro Gly Pro Ser Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu 325 330 335 Leu Leu Trp Val Pro Gly Ser Thr Gly Gln Val Gln Leu Val Gln Ser 340 345 350 Gly Gly Gly Leu Val Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Ala 355 360 365 Ala Ser Gly Ser Ile Ser Ser Tyr Asn Val Val Gly Trp Tyr Arg Gln 370 375 380 Leu Ser Gly Asn Glu Arg Gly Gly Arg Thr Met Val Ala Gln Ile Asn 385 390 395 400 Ala Tyr Gly Asp Thr Asn Tyr Ala Asn Ala Val Val Gly Arg Phe Thr 405 410 415 Ile Ser Arg Asp Asp Ala Lys Asn Thr Val Tyr Leu His Met Ser Asn 420 425 430 Leu Lys Pro Glu Asp Thr Gly Val Tyr Tyr Cys Asn Gly Gln Arg Met 435 440 445 Leu Glu Asn Tyr Thr Tyr Arg Asp Gln Ser Trp Gly Gln Gly Thr Gln 450 455 460 Val Thr Val Lys Pro Gly Gly Gly Gly Ser Gly Val Gln Val Glu Thr 465 470 475 480 Ile Ser Pro Gly Asp Gly Arg Thr Phe Pro Lys Arg Gly Gln Thr Cys 485 490 495 Val Val His Tyr Thr Gly Met Leu Glu Asp Gly Lys Lys Phe Asp Ser 500 505 510 Ser Arg Asp Arg Asn Lys Pro Phe Lys Phe Met Leu Gly Lys Gln Glu 515 520 525 Val Ile Arg Gly Trp Glu Glu Gly Val Ala Gln Met Ser Val Gly Gln 530 535 540 Arg Ala Lys Leu Thr Ile Ser Pro Asp Tyr Ala Tyr Gly Ala Thr Gly 545 550 555 560 His Pro Gly Ile Ile Pro Pro His Ala Thr Leu Val Phe Asp Val Glu 565 570 575 Leu Leu Lys Leu Glu Gly Gly Arg Met Ala Leu Ile Val Leu Gly Gly 580 585 590 Val Ala Gly Leu Leu Leu Phe Ile Gly Leu Gly Ile Phe Phe Ala Leu 595 600 605 Tyr Leu Leu Arg Arg Asp Gln Arg Leu Pro Pro Asp Ala His Lys Pro 610 615 620 Pro Gly Gly Gly Ser Phe Arg Thr Pro Ile Gln Glu Glu Gln Ala Asp 625 630 635 640 Ala His Ser Thr Leu Ala Lys Ile 645 <210> 48 <211> 640 <212> PRT <213> Artificial Sequence <220> <223> Laboratory Preparation - Anti-CD33 VHH DARIC.OX40 Fusion Protein <400> 48 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Gly Ser Ile Leu Trp His Glu Met Trp His Glu 20 25 30 Gly Leu Glu Glu Ala Ser Arg Leu Tyr Phe Gly Glu Arg Asn Val Lys 35 40 45 Gly Met Phe Glu Val Leu Glu Pro Leu His Ala Met Met Glu Arg Gly 50 55 60 Pro Gln Thr Leu Lys Glu Thr Ser Phe Asn Gln Ala Tyr Gly Arg Asp 65 70 75 80 Leu Met Glu Ala Gln Glu Trp Cys Arg Lys Tyr Met Lys Ser Gly Asn 85 90 95 Val Lys Asp Leu Leu Gln Ala Trp Asp Leu Tyr Tyr His Val Phe Arg 100 105 110 Arg Ile Ser Lys Ala Ser Ala Gly Thr Gly Ser Asp Ile Tyr Ile Trp 115 120 125 Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile 130 135 140 Thr Met His Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln 145 150 155 160 Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser 165 170 175 Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys 180 185 190 Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln 195 200 205 Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu 210 215 220 Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg 225 230 235 240 Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met 245 250 255 Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly 260 265 270 Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp 275 280 285 Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Ser Gly Ser 290 295 300 Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu 305 310 315 320 Asn Pro Gly Pro Ser Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu 325 330 335 Leu Leu Trp Val Pro Gly Ser Thr Gly Gln Val Gln Leu Val Gln Ser 340 345 350 Gly Gly Gly Leu Val Gln Ala Gly Gly Ser Leu Thr Leu Ser Cys Ala 355 360 365 Ala Ser Arg Ser Ser Gly Ile Asp Val Met Gly Trp Tyr Arg Gln Ala 370 375 380 Pro Gly Lys Glu Arg Glu Leu Val Ala Glu Ile Ser Gly Val Gly Asp 385 390 395 400 Thr Asn Tyr Ala Ala Ser Leu Ala Asp Arg Phe Thr Val Ser Arg Asp 405 410 415 Asn Ala Lys Asn Thr Val Tyr Leu Gln Met Lys Asn Leu Lys Pro Glu 420 425 430 Asp Thr Ala Val Tyr Tyr Cys Asn Ala His Ser Phe Leu Asp Leu Val 435 440 445 Gly Ala Trp Gly Gln Gly Thr Gln Val Thr Val Lys Pro Gly Gly Gly 450 455 460 Gly Ser Gly Val Gln Val Glu Thr Ile Ser Pro Gly Asp Gly Arg Thr 465 470 475 480 Phe Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr Thr Gly Met Leu 485 490 495 Glu Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg Asn Lys Pro Phe 500 505 510 Lys Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly Trp Glu Glu Gly 515 520 525 Val Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu Thr Ile Ser Pro 530 535 540 Asp Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile Ile Pro Pro His 545 550 555 560 Ala Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu Glu Gly Gly Arg 565 570 575 Met Ala Leu Ile Val Leu Gly Gly Val Ala Gly Leu Leu Leu Phe Ile 580 585 590 Gly Leu Gly Ile Phe Phe Ala Leu Tyr Leu Leu Arg Arg Asp Gln Arg 595 600 605 Leu Pro Pro Asp Ala His Lys Pro Pro Gly Gly Gly Ser Phe Arg Thr 610 615 620 Pro Ile Gln Glu Glu Gln Ala Asp Ala His Ser Thr Leu Ala Lys Ile 625 630 635 640 <210> 49 <211> 644 <212> PRT <213> Artificial Sequence <220> <223> Laboratory Preparation - Anti-CD33 VHH DARIC.OX40 Fusion Protein <400> 49 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Gly Ser Ile Leu Trp His Glu Met Trp His Glu 20 25 30 Gly Leu Glu Glu Ala Ser Arg Leu Tyr Phe Gly Glu Arg Asn Val Lys 35 40 45 Gly Met Phe Glu Val Leu Glu Pro Leu His Ala Met Met Glu Arg Gly 50 55 60 Pro Gln Thr Leu Lys Glu Thr Ser Phe Asn Gln Ala Tyr Gly Arg Asp 65 70 75 80 Leu Met Glu Ala Gln Glu Trp Cys Arg Lys Tyr Met Lys Ser Gly Asn 85 90 95 Val Lys Asp Leu Leu Gln Ala Trp Asp Leu Tyr Tyr His Val Phe Arg 100 105 110 Arg Ile Ser Lys Ala Ser Ala Gly Thr Gly Ser Asp Ile Tyr Ile Trp 115 120 125 Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile 130 135 140 Thr Met His Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln 145 150 155 160 Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser 165 170 175 Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys 180 185 190 Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln 195 200 205 Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu 210 215 220 Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg 225 230 235 240 Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met 245 250 255 Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly 260 265 270 Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp 275 280 285 Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Ser Gly Ser 290 295 300 Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu 305 310 315 320 Asn Pro Gly Pro Ser Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu 325 330 335 Leu Leu Trp Val Pro Gly Ser Thr Gly Glu Val Gln Leu Val Gln Ser 340 345 350 Gly Gly Gly Ser Val Gln Val Gly Gly Ser Leu Arg Leu Ser Cys Ala 355 360 365 Ala Ser Gly Ser Thr Leu Asn Ile Asp His Ile Gly Trp Tyr Arg Gln 370 375 380 Ala Pro Gly Lys Glu Arg Glu Leu Val Gly Val Ile Ser Ser Gly Ala 385 390 395 400 Gly Pro Asn Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg 405 410 415 Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro 420 425 430 Glu Asp Thr Ala Val Tyr Asn Cys Asn Ala Trp Ile Asp Tyr Gly Ser 435 440 445 Gly Leu Pro Gln Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Lys 450 455 460 Pro Gly Gly Gly Gly Ser Gly Val Gln Val Glu Thr Ile Ser Pro Gly 465 470 475 480 Asp Gly Arg Thr Phe Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr 485 490 495 Thr Gly Met Leu Glu Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg 500 505 510 Asn Lys Pro Phe Lys Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly 515 520 525 Trp Glu Glu Gly Val Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu 530 535 540 Thr Ile Ser Pro Asp Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile 545 550 555 560 Ile Pro Pro His Ala Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu 565 570 575 Glu Gly Gly Arg Met Ala Leu Ile Val Leu Gly Gly Val Ala Gly Leu 580 585 590 Leu Leu Phe Ile Gly Leu Gly Ile Phe Phe Ala Leu Tyr Leu Leu Arg 595 600 605 Arg Asp Gln Arg Leu Pro Pro Asp Ala His Lys Pro Pro Gly Gly Gly 610 615 620 Ser Phe Arg Thr Pro Ile Gln Glu Glu Gln Ala Asp Ala His Ser Thr 625 630 635 640 Leu Ala Lys Ile <210> 50 <211> 640 <212> PRT <213> Artificial Sequence <220> <223> Laboratory Preparation - Anti-CD33 VHH DARIC.OX40 Fusion Protein <400> 50 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Gly Ser Ile Leu Trp His Glu Met Trp His Glu 20 25 30 Gly Leu Glu Glu Ala Ser Arg Leu Tyr Phe Gly Glu Arg Asn Val Lys 35 40 45 Gly Met Phe Glu Val Leu Glu Pro Leu His Ala Met Met Glu Arg Gly 50 55 60 Pro Gln Thr Leu Lys Glu Thr Ser Phe Asn Gln Ala Tyr Gly Arg Asp 65 70 75 80 Leu Met Glu Ala Gln Glu Trp Cys Arg Lys Tyr Met Lys Ser Gly Asn 85 90 95 Val Lys Asp Leu Leu Gln Ala Trp Asp Leu Tyr Tyr His Val Phe Arg 100 105 110 Arg Ile Ser Lys Ala Ser Ala Gly Thr Gly Ser Asp Ile Tyr Ile Trp 115 120 125 Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile 130 135 140 Thr Met His Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln 145 150 155 160 Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser 165 170 175 Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys 180 185 190 Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln 195 200 205 Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu 210 215 220 Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg 225 230 235 240 Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met 245 250 255 Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly 260 265 270 Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp 275 280 285 Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Ser Gly Ser 290 295 300 Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu 305 310 315 320 Asn Pro Gly Pro Ser Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu 325 330 335 Leu Leu Trp Val Pro Gly Ser Thr Gly Glu Val Gln Leu Val Glu Ser 340 345 350 Gly Gly Gly Glu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala 355 360 365 Ala Ser Arg Ser Ser Gly Ile Asp Val Met Gly Trp Tyr Arg Gln Ala 370 375 380 Pro Gly Lys Glu Arg Glu Leu Val Ala Glu Ile Ser Gly Val Gly Asp 385 390 395 400 Thr Asn Tyr Ala Ala Ser Leu Ala Asp Arg Phe Thr Val Ser Arg Asp 405 410 415 Asn Ala Lys Asn Thr Val Tyr Leu Gln Met Ser Ser Leu Arg Ala Glu 420 425 430 Asp Thr Ala Val Tyr Tyr Cys Asn Ala His Ser Phe Leu Asp Leu Val 435 440 445 Gly Ala Trp Gly Gln Gly Thr Leu Val Thr Val Lys Pro Gly Gly Gly 450 455 460 Gly Ser Gly Val Gln Val Glu Thr Ile Ser Pro Gly Asp Gly Arg Thr 465 470 475 480 Phe Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr Thr Gly Met Leu 485 490 495 Glu Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg Asn Lys Pro Phe 500 505 510 Lys Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly Trp Glu Glu Gly 515 520 525 Val Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu Thr Ile Ser Pro 530 535 540 Asp Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile Ile Pro Pro His 545 550 555 560 Ala Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu Glu Gly Gly Arg 565 570 575 Met Ala Leu Ile Val Leu Gly Gly Val Ala Gly Leu Leu Leu Phe Ile 580 585 590 Gly Leu Gly Ile Phe Phe Ala Leu Tyr Leu Leu Arg Arg Asp Gln Arg 595 600 605 Leu Pro Pro Asp Ala His Lys Pro Pro Gly Gly Gly Ser Phe Arg Thr 610 615 620 Pro Ile Gln Glu Glu Gln Ala Asp Ala His Ser Thr Leu Ala Lys Ile 625 630 635 640 <210> 51 <211> 644 <212> PRT <213> Artificial Sequence <220> <223> Laboratory Preparation - Anti-CD33 VHH DARIC.OX40 Fusion Protein <400> 51 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Gly Ser Ile Leu Trp His Glu Met Trp His Glu 20 25 30 Gly Leu Glu Glu Ala Ser Arg Leu Tyr Phe Gly Glu Arg Asn Val Lys 35 40 45 Gly Met Phe Glu Val Leu Glu Pro Leu His Ala Met Met Glu Arg Gly 50 55 60 Pro Gln Thr Leu Lys Glu Thr Ser Phe Asn Gln Ala Tyr Gly Arg Asp 65 70 75 80 Leu Met Glu Ala Gln Glu Trp Cys Arg Lys Tyr Met Lys Ser Gly Asn 85 90 95 Val Lys Asp Leu Leu Gln Ala Trp Asp Leu Tyr Tyr His Val Phe Arg 100 105 110 Arg Ile Ser Lys Ala Ser Ala Gly Thr Gly Ser Asp Ile Tyr Ile Trp 115 120 125 Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile 130 135 140 Thr Met His Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln 145 150 155 160 Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser 165 170 175 Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys 180 185 190 Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln 195 200 205 Leu Tyr Asn...

Claims

1. A non-natural cell comprising: (a) a first polypeptide comprising: (1) a FRB multimerization domain polypeptide; (2) a CD8α transmembrane domain or a CD4 transmembrane domain; (3) a CD137 costimulatory domain; and (4) a CD3ζ primary signaling domain; and (b) a second polypeptide comprising: (1) an anti-CD33 VHH antibody having an amino acid sequence as shown in any one of SEQ ID NOs: 10, 2-6, and 11; (2) an FKBP multimerization domain polypeptide; and (3) a CD4 transmembrane domain or a CD8α transmembrane domain.

2. The non-natural cell of claim 1, wherein the FKBP multimerization domain polypeptide is FKBP12.

3. The non-natural cell of claim 1 or claim 2, wherein the FRB multimerization domain polypeptide is FRB T2098L.

4. The non-natural cell of claim 1, wherein the first polypeptide comprises a signal peptide, a CD8α transmembrane domain, a CD137 co-stimulatory domain, and a CD3ζ primary signaling domain.

5. The non-natural cell of claim 1, wherein the second polypeptide comprises a signal peptide and a CD4 transmembrane domain.

6. The non-natural cell of claim 1, wherein the second polypeptide comprises a costimulatory domain.

7. The non-natural cell of claim 6, wherein the costimulatory domain of the second polypeptide is selected from the group consisting of a costimulatory molecule: 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-activating protein 10 (DAP10), linker for activated 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).

8. The non-natural cell of claim 6 or claim 7, wherein the costimulatory domain of the second polypeptide is a costimulatory domain isolated from OX40 or TNFR2.

9. The non-natural cell of claim 1, wherein the second polypeptide comprises the sequence set forth in any one of SEQ ID NOs: 30, 22-26, and 31.

10. A non-natural cell comprising a polypeptide complex comprising: (a) a first polypeptide comprising: (1) a FRB multimerization domain polypeptide; (2) a CD8α transmembrane domain or a CD4 transmembrane domain; (3) a CD137 costimulatory domain; and (4) a CD3ζ primary signaling domain; (b) a second polypeptide comprising: (1) an anti-CD33 VHH antibody having an amino acid sequence as shown in any one of SEQ ID NOs: 10, 2-6, and 11; (2) an FKBP multimerization domain polypeptide; and (3) a CD4 transmembrane domain or a CD8α transmembrane domain; and (c) a bridging factor associated with the multimerization domains of the first polypeptide and the second polypeptide and positioned between the multimerization domains.

11. The non-natural cell of claim 10, wherein the FKBP multimerization domain polypeptide is FKBP12.

12. The non-natural cell of claim 10 or claim 11, wherein the FRB multimerization domain polypeptide is FRB T2098L.

13. The non-natural cell of claim 10, wherein the bridging factor is selected from the group consisting of AP21967, sirolimus, everolimus, norflulimus, pimecrolimus, dafolimus, tacrolimus, temsirolimus, umimios, and zotarolimus.

14. The non-natural cell of claim 10, wherein the first polypeptide comprises a signal peptide, a CD8α transmembrane domain, a CD137 co-stimulatory domain, and a CD3ζ primary signaling domain.

15. The non-natural cell of claim 10, wherein the second polypeptide comprises a signal peptide and a CD4 transmembrane domain.

16. The non-natural cell of claim 10, wherein the second polypeptide comprises a costimulatory domain.

17. The non-natural cell of claim 16, wherein the costimulatory domain of the second polypeptide is selected from the group consisting of a costimulatory molecule: 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-activating protein 10 (DAP10), linker for activated 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).

18. The non-natural cell of claim 16 or claim 17, wherein the costimulatory domain of the second polypeptide is a costimulatory domain isolated from OX40 or TNFR2.

19. The non-natural cell of claim 10, wherein the second polypeptide comprises the sequence set forth in any one of SEQ ID NOs: 30, 22-26, and 31.

20. The non-naturally occurring cell of claim 1 or claim 10, wherein the cell is a hematopoietic cell.

21. The non-natural cell of claim 1 or claim 10, wherein the cell is a T cell.

22. The non-natural cell of claim 21, wherein the T cell is an αβ T cell or a γδ T cell.

23. The non-natural cell of claim 1 or claim 10, wherein the cell is a CD3 + 、CD4 + and / or CD8 + cell.

24. The non-natural cell of claim 1 or claim 10, wherein the cell is an immune effector cell.

25. The non-natural cell of claim 1 or claim 10, wherein the cell is a cytotoxic T lymphocyte (CTL), a tumor infiltrating lymphocyte (TIL), or a helper T cell.

26. The non-natural cell of claim 1 or claim 10, wherein the cell is a natural killer (NK) cell or a natural killer T (NKT) cell.

27. The non-natural cell of claim 1 or claim 10, wherein the source of the cell 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.

28. The non-natural cell of claim 1 or claim 10, wherein when the first polypeptide and the second polypeptide are expressed, the FRB multimerization domain polypeptide and the FKBP multimerization domain polypeptide are localized extracellularly.

29. A fusion polypeptide comprising: (a) a first polypeptide comprising: (1) a FRB multimerization domain polypeptide; (2) a CD8α transmembrane domain or a CD4 transmembrane domain; (3) a CD137 costimulatory domain; and (4) a CD3ζ primary signaling domain; (b) a polypeptide cleavage signal, wherein the polypeptide cleavage signal is a protease cleavage site, a nuclease cleavage site, a viral self-cleaving peptide, or a ribosomal skipping sequence; and (c) a second polypeptide comprising: (1) an anti-CD33 VHH antibody having an amino acid sequence as shown in any one of SEQ ID NOs: 10, 2-6, and 11; (2) an FKBP multimerization domain polypeptide; and (3) a CD4 transmembrane domain or a CD8α transmembrane domain.

30. The fusion polypeptide of claim 29, wherein the FKBP multimerization domain polypeptide is FKBP12.

31. The fusion polypeptide of claim 29 or claim 30, wherein the FRB multimerization domain polypeptide is FRB T2098L.

32. The fusion polypeptide of claim 29, wherein a bridging factor is associated with the multimerization domains of the first and second polypeptides, and wherein the bridging factor is selected from the group consisting of AP21967, sirolimus, everolimus, norflulimus, pimecrolimus, dafolimus, tacrolimus, temsirolimus, umimios, and zotarolimus.

33. The fusion polypeptide of claim 29, wherein the first polypeptide comprises a signal peptide, a CD8α transmembrane domain, a CD137 co-stimulatory domain, and a CD3ζ primary signaling domain.

34. The fusion polypeptide of claim 29, wherein the second polypeptide comprises a signal peptide and a CD4 transmembrane domain.

35. The fusion polypeptide of claim 29, wherein the fusion polypeptide comprises the sequence shown in any one of SEQ ID NOs: 40, 32-36, and 41.

36. The fusion polypeptide of claim 29, wherein the second polypeptide comprises a costimulatory domain.

37. The fusion polypeptide of claim 36, wherein the costimulatory domain of the second polypeptide is selected from the group consisting of a costimulatory molecule: 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-activating protein 10 (DAP10), linker for activated 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).

38. The fusion polypeptide of claim 36 or claim 37, wherein the costimulatory domain of the second polypeptide is a costimulatory domain isolated from OX40 or TNFR2.

39. The fusion polypeptide of claim 29, wherein the polypeptide cleavage signal is a viral self-cleavage polypeptide.

40. The fusion polypeptide of claim 29, wherein the polypeptide cleavage signal is a viral self-cleavage 2A polypeptide.

41. The fusion polypeptide of claim 29, 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 virus type A (ERAV) E2A peptide, trichotep beta-tetrasomal virus (TaV) T2A peptide, porcine teschovirus-1 (PTV-1) P2A peptide, Theiler virus 2A peptide, and encephalomyocarditis virus 2A peptide.

42. The fusion polypeptide of claim 29, wherein the fusion polypeptide comprises the sequence shown in any one of SEQ ID NOs: 50, 60, 42-46, 51-56, and 61.

43. The fusion polypeptide of claim 29, wherein the FRB multimerization domain polypeptide and the FKBP multimerization domain polypeptide are localized extracellularly when the first polypeptide and the second polypeptide are expressed.

44. A polypeptide complex comprising: (a) a first polypeptide comprising: (1) a FRB multimerization domain polypeptide; (2) a CD8α transmembrane domain or a CD4 transmembrane domain; (3) a CD137 costimulatory domain; and (4) a CD3ζ primary signaling domain; (b) a second polypeptide comprising: (1) an anti-CD33 VHH antibody having an amino acid sequence as shown in any one of SEQ ID NOs: 10, 2-6, and 11; (2) an FKBP multimerization domain polypeptide; and (3) a CD4 transmembrane domain or a CD8α transmembrane domain; and (c) a bridging factor associated with the multimerization domains of the first polypeptide and the second polypeptide and positioned between the multimerization domains.

45. The polypeptide complex of claim 44, wherein the FKBP multimerization domain polypeptide is FKBP12.

46. The polypeptide complex of claim 44 or claim 45, wherein the FRB multimerization domain polypeptide is FRB T2098L.

47. The polypeptide complex according to claim 44, wherein the bridging factor is selected from the group consisting of AP21967, sirolimus, everolimus, norflulimus, pimecrolimus, dafolimus, tacrolimus, temsirolimus, umimios and zotarolimus.

48. The polypeptide complex of claim 44, wherein the first polypeptide comprises a CD8α transmembrane domain, a CD137 co-stimulatory domain, and a CD3ζ primary signaling domain.

49. The polypeptide complex of claim 44, wherein the second polypeptide comprises a CD4 transmembrane domain.

50. The polypeptide complex of claim 44, wherein the second polypeptide comprises a costimulatory domain.

51. The polypeptide complex of claim 50, wherein the costimulatory domain of the second polypeptide is selected from the group consisting of a costimulatory molecule: 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-activating protein 10 (DAP10), linker for activated 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).

52. The polypeptide complex of claim 50 or claim 51, wherein the costimulatory domain of the second polypeptide is a costimulatory domain isolated from OX40 or TNFR2.

53. The polypeptide complex of claim 44, wherein when the first polypeptide and the second polypeptide are expressed, the FRB multimerization domain polypeptide and the FKBP multimerization domain polypeptide are localized extracellularly.

54. A chimeric antigen receptor (CAR), comprising: a) an anti-CD33 VHH antibody having an amino acid sequence as shown in any one of SEQ ID NOs: 10, 2-6, and 11; b) hinge domain; c) CD4 or CD8α transmembrane domain; d) CD137 intracellular co-stimulatory signaling domain; and e) CD3ζ primary signaling domain.

55. The CAR of claim 54, wherein the hinge domain is isolated from CD8α, CD27, CD28, CD33, CD37, CD45, CD64, CD71, CD80, CD86, CD 134, CD137, CD152, CD154, AMN, and PD1.

56. The CAR of claim 54 or 55, wherein the CAR comprises a CD8α signal peptide, a CD8α hinge domain and a transmembrane domain, a CD137 co-stimulatory domain, and a CD3ζ primary signaling domain.

57. A CAR comprising the sequence shown in any one of SEQ ID NOs: 70, 80, 62-66, 71-76 and 81.

58. A polynucleotide encoding the first polypeptide and the second polypeptide according to claim 1 or claim 10, the fusion polypeptide according to claim 29, the first polypeptide and the second polypeptide of the polypeptide complex according to claim 44, or the CAR according to claim 54.

59. A combination of polynucleotides comprising a first polynucleotide encoding the first polypeptide of claim 1 or claim 10 and a second polynucleotide encoding the second polypeptide of claim 1 or claim 10.

60. A cDNA encoding the first polypeptide and the second polypeptide according to claim 1 or claim 10, the fusion polypeptide according to claim 29, the first polypeptide and the second polypeptide of the polypeptide complex according to claim 44, or the CAR according to claim 54.

61. A combination of cDNAs comprising a first cDNA encoding the first polypeptide of claim 1 or claim 10 and a second cDNA encoding the second polypeptide of claim 1 or claim 10.

62. An RNA encoding the first polypeptide and the second polypeptide according to claim 1 or claim 10, the fusion polypeptide according to claim 29, the first polypeptide and the second polypeptide of the polypeptide complex according to claim 44, or the CAR according to claim 54.

63. A combination of RNA comprising a first RNA encoding the polypeptide of claim 1 or claim 10 and a second RNA encoding the polypeptide of claim 1 or claim 10.

64. A vector comprising the polynucleotide of claim 58 or a combination of the polynucleotides of claim 59.

65. The vector of claim 64, wherein the vector is an expression vector.

66. The vector of claim 64, wherein the vector is a transposon.

67. The vector of claim 66, wherein the vector is a piggyBAC transposon or a Sleeping Beauty transposon.

68. The vector of claim 64, wherein the vector is a viral vector.

69. The vector of claim 68, wherein the vector is an adenoviral vector, an adeno-associated viral (AAV) vector, a herpes viral vector, a vaccinia viral vector, or a retroviral vector.

70. The vector of claim 69, wherein the retroviral vector is a lentiviral vector.

71. The vector of claim 70, wherein the lentiviral vector is selected from the group consisting of: human immunodeficiency virus 1 (HIV-1); human immunodeficiency virus 2 (HIV-2), Vesna-Medi 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).

72. A cell comprising the CAR of claim 54.

73. The cell of claim 72, wherein the cell is a hematopoietic cell.

74. The cell of claim 72 or 73, wherein the cell is an immune effector cell.

75. The cell of claim 72, wherein the cell is a T cell.

76. The cell of claim 75, wherein the T cell is an αβ T cell or a γδ T cell.

77. The cell of claim 72, wherein the cell expresses CD3 + 、CD4 + 、CD8 + or a combination thereof.

78. The cell of claim 72, wherein the cell is a cytotoxic T lymphocyte (CTL), a tumor infiltrating lymphocyte (TIL), or a helper T cell.

79. The cell of claim 72, wherein the cell is a natural killer (NK) cell or a natural killer T (NKT) cell.

80. The cell of claim 79, wherein the source of the cell 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.

81. A composition comprising the cells of claim 1, claim 10, or claim 72.

82. A composition comprising a physiologically acceptable carrier and the cell of claim 1, claim 10 or claim 72.

83. Use of the composition of claim 81 or claim 82 in the preparation of a medicament for treating acute myeloid leukemia (AML).

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