CLL-1 Targeted Immunotherapy

Abstract

The present disclosure provides improved CLL-1 targeting polypeptides and compositions for adoptive T cell therapy for treating, preventing or ameliorating at least one symptom or a condition associated therewith of cancer, infectious diseases, autoimmune diseases, inflammatory diseases and immune deficiencies.

Description

[0001] Cross - Reference to Related Applications

[0002] This application claims the benefit of U.S. Provisional Application No. 62 / 845,306, filed on May 8, 2019, which is hereby incorporated by reference in its entirety.

[0003] Statement Regarding the Sequence Listing

[0004] The sequence listing associated with this application is provided in text format in lieu of a paper copy, and is hereby incorporated by reference into the specification. The name of the text file containing the sequence listing is BLBD_120_01WO_ST25.txt. The text file is 187 KB and was created on May 5, 2020, and was electronically submitted via EFS - Web simultaneously with the filing of this specification. Background Art Technical Field

[0006] The present disclosure relates to improved adoptive cell therapies directed against C - type lectin - like molecule - 1 (CLL - 1). More specifically, the present disclosure relates to chemically - regulated signaling molecules comprising anti - CLL - 1 VHHs, chimeric antigen receptors comprising anti - CLL - 1 VHHs, cells, and related therapeutic methods using them.

[0007] Prior Art

[0008] The global cancer burden doubled between 1975 and 2000. Cancer is the second leading cause of morbidity and mortality worldwide, and there were approximately 14.1 million new cases and 8.2 million cancer - related deaths in 2012. The most common cancers are breast cancer, lung and bronchus cancer, prostate cancer, colon and rectum cancer, bladder cancer, cutaneous melanoma, 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 in the next two decades.

[0009] Adoptive cell therapy is emerging as a powerful paradigm for delivering complex biological signals to treat cancer. Compared to small molecule and biopharmaceutical compositions, adoptive cell therapy has the potential to perform unique therapeutic tasks due to its large number of sensing and response programs and increasingly well - defined genetic control mechanisms. Existing methods have mainly focused on single - chain variable fragment (scFv) - based chimeric antigen receptors (CARs). CAR T - cell therapy has had limited success due to poor CAR expression, in vivo expansion of CAR T cells, rapid disappearance of the cells after infusion, disappointing clinical activity, and antigen escape.

[0010] Improved CAR architectures (CAR structures) and / or improved mechanisms are needed to engineer immune effector cells 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 CLL-1-targeted VHH-based dimerizer-regulated immune receptor complexes (DARICs) and VHH-based chimeric antigen receptors (CARs), polynucleotides encoding them, compositions thereof, and methods of making and using them for treating cancer.

[0012] In various embodiments, the non-natural cell comprises: a first polypeptide comprising: an FRB multimerization domain polypeptide or 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-CLL-1 VHH antibody having an amino acid sequence shown in any one of SEQ ID NOs: 2-13, an FKBP multimerization domain polypeptide or variant thereof, and a CD4 transmembrane domain or a CD8α transmembrane domain; wherein a bridging factor promotes the formation of a polypeptide complex on the surface of the non-natural cell, the bridging factor associating with and positioned between the multimerization domains of the first and second polypeptides.

[0013] In certain embodiments, the FKBP multimerization domain is FKBP12.

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

[0015] In certain embodiments, the bridging factor is selected from the group consisting of: AP21967, sirolimus, everolimus, deforolimus, pimecrolimus, temsirolimus, tacrolimus, temsirolimus, umirolimus, and zotarolimus.

[0016] 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.

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

[0018] In additional embodiments, the second polypeptide comprises a co-stimulatory domain.

[0019] In some embodiments, the co-stimulatory domain of the second polypeptide is a co-stimulatory molecule selected from the group consisting of: Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, caspase recruitment domain family member 11 (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 activation of T cell family member 1 (LAT), SH2 domain-containing leukocyte protein of 76kD (SLP76), T cell receptor-associated transmembrane adaptor 1 (TRAT1), TNFR2, TNFRS14, TNFRS18, TNRFS25, and zeta chain of T cell receptor-associated protein kinase 70 (ZAP70).

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

[0021] In additional embodiments, the second polypeptide comprises a sequence shown in any one of SEQ ID NOs: 14-19.

[0022] In a preferred embodiment, the first polypeptide comprises the amino acid sequence shown in SEQ ID NO 50.

[0023] In various embodiments, the non-natural cell comprises a polypeptide complex that comprises: a first polypeptide that comprises: an 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 that comprises: an anti-CLL-1 VHH antibody having an amino acid sequence shown in any one of SEQ ID NOs: 2-13, an FKBP multimerization domain polypeptide or a variant thereof, and a CD4 transmembrane domain or a CD8α transmembrane domain; and a bridging factor that associates with the multimerization domains of the first and second polypeptides and is positioned between the multimerization domains.

[0024] In certain embodiments, the FKBP multimerization domain is FKBP12.

[0025] In some embodiments, the FRB polypeptide is FRB T2098L.

[0026] In some embodiments, the bridging factor is selected from the group consisting of: AP21967, sirolimus, everolimus, deforolimus, pimecrolimus, deltalimus, tacrolimus, temsirolimus, umirolimus, and zotarolimus.

[0027] 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.

[0028] In certain embodiments, the second polypeptide comprises a signal peptide and a CD4 transmembrane domain.

[0029] In some embodiments, the second polypeptide comprises a co-stimulatory domain.

[0030] In various embodiments, the co-stimulatory domain of the second polypeptide is a co-stimulatory molecule selected from the group consisting of: Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, caspase recruitment domain family member 11 (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 activation of T cell family member 1 (LAT), SH2 domain-containing leukocyte protein of 76kD (SLP76), T cell receptor-associated transmembrane adapter 1 (TRAT1), TNFR2, TNFRS14, TNFRS18, TNRFS25, and zeta chain of T cell receptor-associated protein kinase 70 (ZAP70).

[0031] In additional embodiments, the co-stimulatory domain of the second polypeptide is a co-stimulatory domain isolated from OX40 or TNFR2.

[0032] In additional embodiments, the second polypeptide comprises a sequence shown in any one of SEQ ID NOs: 14-19.

[0033] In preferred embodiments, the first polypeptide comprises the amino acid sequence shown in SEQ ID NO 50.

[0034] In certain embodiments, the cell is a hematopoietic cell.

[0035] In specific embodiments, the cell is a T cell, an αβ T cell, or a γδ T cell.

[0036] In additional embodiments, the cell is a CD3+, CD4+, and / or CD8+ cell.

[0037] In various embodiments, the cell is an immune effector cell.

[0038] In some embodiments, the cell is a cytotoxic T lymphocyte, a tumor infiltrating lymphocyte, or a helper T cell.

[0039] In additional embodiments, the cell is a natural killer (NK) cell or a natural killer T (NKT) cell.

[0040] In various embodiments, the source of the cell is peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infected site, ascites, pleural effusion, spleen tissue, or a tumor.

[0041] In certain embodiments, when the first polypeptide and the second polypeptide are expressed, the FRB multimerization domain and the FKBP multimerization domain localize extracellularly.

[0042] In some embodiments, the fusion polypeptide comprises: a first polypeptide comprising: an FRB multimerization domain polypeptide or a variant thereof, a CD8α transmembrane domain or a CD4 transmembrane domain, a CD137 co-stimulatory domain, and / or a CD3ζ primary signaling domain, a polypeptide cleavage signal; and a second polypeptide comprising: an anti-CLL-1 VHH antibody having an amino acid sequence shown in any one of SEQ ID NOs: 2-13, an FKBP multimerization domain polypeptide or a variant thereof, and a CD4 transmembrane domain or a CD8α transmembrane domain.

[0043] In certain embodiments, the FKBP multimerization domain is FKBP12.

[0044] In some embodiments, the FRB polypeptide is FRB T2098L.

[0045] In some embodiments, the bridging factor is selected from the group consisting of: AP21967, sirolimus, everolimus, norolimus, pimecrolimus, deforolimus, tacrolimus, temsirolimus, umirolimus, and zotarolimus.

[0046] In further embodiments, the first polypeptide comprises a signal peptide, a CD8α transmembrane domain, a CD137 co-stimulatory domain, and a CD3ζ primary signaling domain.

[0047] In certain embodiments, the second polypeptide comprises a signal peptide and a CD4 transmembrane domain.

[0048] In some embodiments, the fusion polypeptide comprises a sequence shown in any one of SEQ ID NOs: 20-25.

[0049] In additional embodiments, the second polypeptide comprises a co-stimulatory domain.

[0050] In various embodiments, the co-stimulatory domain of the second polypeptide is a co-stimulatory molecule selected from the group consisting of Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, caspase recruitment domain family member 11 (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 activation of T cell family member 1 (LAT), SH2 domain-containing leukocyte protein of 76kD (SLP76), T cell receptor-associated transmembrane adapter 1 (TRAT1), TNFR2, TNFRS14, TNFRS18, TNRFS25, and zeta chain of T cell receptor-associated protein kinase 70 (ZAP70).

[0051] In additional embodiments, the co-stimulatory domain of the second polypeptide is a co-stimulatory domain isolated from OX40 or TNFR2.

[0052] In certain embodiments, the polypeptide cleavage signal is a viral self-cleaving polypeptide.

[0053] In specific embodiments, the polypeptide cleavage signal is a viral self-cleaving 2A polypeptide.

[0054] In various embodiments, the polypeptide cleavage signal is a viral self-cleaving polypeptide selected from the group consisting of foot-and-mouth disease virus (FMDV) (F2A) peptide, equine rhinitis A virus (ERAV) (E2A) peptide, Maraba virus (TaV) (T2A) peptide, porcine teschovirus-1 (PTV-1) (P2A) peptide, Theiler's virus 2A peptide, and encephalomyocarditis virus 2A peptide.

[0055] In some embodiments, the fusion polypeptide comprises a sequence shown in any one of SEQ ID NOs: 26-37.

[0056] In additional embodiments, when the first polypeptide and the second polypeptide are expressed, the FRB oligomerization domain and the FKBP oligomerization domain are located extracellularly.

[0057] In various embodiments, the polypeptide complex comprises: a first polypeptide comprising: an FRB multimerization domain polypeptide or 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-CLL-1 VHH antibody having an amino acid sequence shown in any one of SEQ ID NOs: 2-13, an FKBP multimerization domain polypeptide or variant thereof, and a CD4 transmembrane domain or a CD8α transmembrane domain; and a bridging factor that associates with the multimerization domains of the first and second polypeptides and is positioned between the multimerization domains.

[0058] In certain embodiments, the FKBP multimerization domain is FKBP12.

[0059] In additional embodiments, the FRB polypeptide is FRB T2098L.

[0060] In certain embodiments, the bridging factor is selected from the group consisting of: AP21967, sirolimus, everolimus, novolimus, pimecrolimus, deforolimus, tacrolimus, temsirolimus, umirolimus, and zotarolimus.

[0061] In some embodiments, the first polypeptide comprises a CD8α transmembrane domain, a CD137 co-stimulatory domain, and a CD3ζ primary signaling domain.

[0062] In various embodiments, the second polypeptide comprises a CD4 transmembrane domain.

[0063] In additional embodiments, the second polypeptide comprises a co-stimulatory domain.

[0064] In some embodiments, the co-stimulatory domain of the second polypeptide is a co-stimulatory molecule selected from the group consisting of: Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, caspase recruitment domain family member 11 (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 activation of T cells family member 1 (LAT), SH2 domain-containing leukocyte protein of 76kD (SLP76), T cell receptor-associated transmembrane adaptor 1 (TRAT1), TNFR2, TNFRS14, TNFRS18, TNRFS25, and zeta chain of T cell receptor-associated protein kinase 70 (ZAP70).

[0065] In certain embodiments, the co-stimulatory domain of the second polypeptide is a co-stimulatory domain isolated from OX40 or TNFR2.

[0066] In some embodiments, the cell is a hematopoietic cell.

[0067] In various embodiments, the cell is a T cell, an αβ T cell, or a γδ T cell.

[0068] In various embodiments, the cell is a CD3+, CD4+ and / or CD8+ cell.

[0069] In additional embodiments, the cell is an immune effector cell.

[0070] In some embodiments, the cell is a cytotoxic T lymphocyte, a tumor infiltrating lymphocyte, or a helper T cell.

[0071] In certain embodiments, the cell is a natural killer (NK) cell or a natural killer T (NKT) cell.

[0072] In additional embodiments, the source of the cell is peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infected site, ascites, pleural effusion, spleen tissue, or a tumor.

[0073] In additional embodiments, when the first polypeptide and the second polypeptide are expressed, the FRB oligomerization domain and the FKBP oligomerization domain localize extracellularly.

[0074] In a preferred embodiment, the first polypeptide comprises the amino acid sequence shown in SEQ ID NO 50.

[0075] In certain embodiments, the chimeric antigen receptor (CAR) comprises: an anti-CLL-1 VHH antibody having the amino acid sequence shown in any one of SEQ ID NOs: 2-13; a hinge domain; a transmembrane domain; one or more intracellular co-stimulatory signaling domains; and / or a primary signaling domain.

[0076] In various embodiments, the CAR comprises, from 5’ to 3’: an anti-CLL-1 VHH antibody having the amino acid sequence shown in any one of SEQ ID NOs: 2-13; a hinge domain; a transmembrane domain; one or more intracellular co-stimulatory signaling domains; and / or a primary signaling domain.

[0077] In certain embodiments, the hinge domain and transmembrane domain are isolated from CD8α, CD27, CD28, CD33, CD37, CD45, CD64, CD71, CD80, CD86, CD134, CD137, CD152, CD154, AMN, and PD1.

[0078] In additional embodiments, one or more costimulatory signaling domains are isolated from costimulatory molecules selected from the group consisting of CD28, CD134, CD137, and CD278.

[0079] In certain embodiments, the CAR comprises a CD8α signal peptide, a CD8α hinge and transmembrane domain, a CD134 costimulatory domain, and a CD3ζ primary signaling domain.

[0080] In additional embodiments, the CAR comprises a sequence shown in any one of SEQ ID NOs: 38 - 49.

[0081] In some embodiments, polynucleotides encoding the first polypeptide or second polypeptide, fusion polypeptide, or CAR encompassed herein are provided.

[0082] In various embodiments, cDNAs encoding the first polypeptide or second polypeptide, fusion polypeptide, or CAR encompassed herein are provided.

[0083] In certain embodiments, RNAs encoding the first polypeptide or second polypeptide, fusion polypeptide, or CAR encompassed herein are provided.

[0084] In additional embodiments, vectors comprising the polynucleotides encompassed herein are provided.

[0085] In certain embodiments, the vector is an expression vector.

[0086] In certain embodiments, the vector is a transposon.

[0087] In additional embodiments, the vector is a piggyBAC transposon or a Sleeping Beauty transposon.

[0088] In certain embodiments, the vector is a viral vector.

[0089] In certain embodiments, the vector is an adenovirus vector, an adeno-associated virus (AAV) vector, a herpesvirus vector, a vaccinia virus vector, or a retroviral vector.

[0090] In additional embodiments, the retroviral vector is a lentiviral vector.

[0091] In various embodiments, the lentiviral vector is selected from the group consisting of: human immunodeficiency virus 1 (HIV-1); human immunodeficiency virus 2 (HIV-2), Visna-Maedi virus (VMV); caprine arthritis-encephalitis virus (CAEV); equine infectious anemia virus (EIAV); feline immunodeficiency virus (FIV); bovine immunodeficiency virus (BIV); and simian immunodeficiency virus (SIV).

[0092] In additional embodiments, cells are provided that comprise a first polypeptide and a second polypeptide, a fusion polypeptide, or a CAR as covered herein.

[0093] In certain embodiments, the cells are hematopoietic cells.

[0094] In some embodiments, the cells are immune effector cells.

[0095] In various embodiments, the cells are T cells, αβ T cells, or γδ T cells.

[0096] In some embodiments, the cells express CD3+, CD4+, CD8+, or a combination thereof.

[0097] In certain embodiments, the cells are cytotoxic T lymphocytes (CTLs), tumor-infiltrating lymphocytes (TILs), or helper T cells.

[0098] In additional embodiments, the cells are natural killer (NK) cells or natural killer T (NKT) cells.

[0099] In certain embodiments, the composition comprises cells as covered herein.

[0100] In certain embodiments, the composition comprises a physiologically acceptable carrier and cells as covered herein.

[0101] In additional embodiments, a method of treating a subject in need thereof is provided, the method comprising administering to the subject an effective amount of a composition as covered herein.

[0102] In certain embodiments, a method of treating, preventing, or ameliorating at least one symptom or condition associated with cancer, an infectious disease, an autoimmune disease, an inflammatory disease, and an immunodeficiency comprises administering to a subject an effective amount of a composition as covered herein.

[0103] In some embodiments, a method of treating solid cancer comprises administering to a subject an effective amount of a composition as covered herein.

[0104] In various embodiments, solid cancers are 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.

[0105] In certain embodiments, a method of treating a hematological malignancy comprises administering to a subject an effective amount of a composition encompassed herein.

[0106] In various embodiments, the hematological malignancy is leukemia, lymphoma, or multiple myeloma.

[0107] In a particular embodiment, the hematological malignancy is acute myeloid leukemia (AML). BRIEF DESCRIPTION OF THE DRAWINGS

[0108] Figure 1A A schematic of the VHH-DARIC polypeptide complex is shown.

[0109] Figure 1B A schematic of the CLL-1 VHH DARIC architecture is shown.

[0110] Figure 2 Expression of CLL-1 VHHDARIC in transduced T cells as detected by CLL-1-Fc binding (top row) and by anti-VHH staining (bottom row) is shown.

[0111] Figure 3 The phenotype of T cells transduced with CLL-1 VHH DARIC or a control is shown.

[0112] Figure 4 Shown are from co-culture with CLL-1 + Cytokine (IFNγ, TNFα, and IL-2) secretion of CLL-1 VHH DARIC or control cells from THP-1 cells co-cultured with CLL-1 VHH DARIC or control cells at a 1:1 E:T ratio in the presence or absence of AP21967 for 24 hours.

[0113] Figure 5 IFNγ secretion of UTD T cells or CLL-1 VHH DARIC T cells from co-culture with MV4-11 or HL60 AML cell lines expressing CLL-1 at a 1:1 E:T ratio in the presence or absence of AP21967 for 24 hours is shown.

[0114] Figure 6A CLL-1 expression in THP-1 cells, THP-1 cells engineered to knock out the CLL-1 gene (CLL-1-KO cells), and an unstained control is shown.

[0115] Figure 6B Shows IFNγ secretion from UTD T cells or CLL-1 VHH DARIC T cells co-cultured with THP-1 cells expressing CD123 at an E∶T ratio of 1∶1 for 24 hours in the presence or absence of AP21967.

[0116] Figure 6C Shows IFNγ secretion from UTD T cells or CLL-1 VHH DARIC T cells co-cultured with CLL-1-KO cells at an E∶T ratio of 1∶1 for 24 hours in the presence or absence of AP21967.

[0117] Brief description of sequence identifiers

[0118] SEQ ID NO: 1 lists the amino acid sequence of human CLL-1.

[0119] SEQ ID NOs: 2 - 13 list the amino acid sequences of anti-CLL-1 VHH domains.

[0120] SEQ ID NOs: 14 - 19 list the amino acid sequences of anti-CLL-1 VHH DARIC binding components.

[0121] SEQ ID NOs: 20 - 25 list the amino acid sequences of anti-CLL-1 VHH DARIC fusion proteins.

[0122] SEQ ID NOs: 26 - 31 list the amino acid sequences of anti-CLL-1 VHH DARIC.OX40 fusion proteins.

[0123] SEQ ID NOs: 32 - 37 list the amino acid sequences of anti-CLL-1 VHH DARIC.TNFR2 fusion proteins.

[0124] SEQ ID NOs: 38 - 49 list the amino acid sequences of anti-CLL-1 VHH CARs.

[0125] SEQ ID NO: 50 lists the amino acid sequence of the anti-CLL-1 VHH DARIC signaling component.

[0126] SEQ ID NO: 51 lists the polynucleotide sequence of the Kozak sequence.

[0127] SEQ ID NOs: 52 - 62 list the amino acid sequences of various linkers.

[0128] SEQ ID NOs: 63 - 87 list the amino acid sequences of protease cleavage sites and self-cleaving polypeptide cleavage sites.

[0129] In the foregoing sequences, Xaa, if present, can refer to any amino acid or the absence of an amino acid. In a preferred embodiment, XaaXaa refers to the amino acid sequences SS or KP. Specific embodiments

[0130] A. Overview

[0131] Cancer is one of the leading causes of death worldwide. Approximately 10% of cancers are hematological malignancies, which include leukemia, lymphoma, and myeloma. Acute myeloid leukemia (AML) is the most common and fatal hematological malignancy in adults. Despite significant scientific discoveries and new therapies in the past four decades, the treatment outcomes for AML, especially in the adult patient population, remain dismal. Standard chemotherapy can induce complete remission in selected patients; however, most patients ultimately relapse and die from the disease. In 2012, the global incidence of AML was approximately 351,965 people, and approximately 265,461 people died from AML.

[0132] C-type lectin-like molecule-1 (CLL-1) is a type II transmembrane glycoprotein whose expression is restricted to myeloid cells, AML blasts, and leukemia stem cells (LSCs). CLL-1 expression is absent in hematopoietic stem cells (HSCs) and bone marrow stem cells. CLL-1 is also expressed on leukemia stem cells (LSCs), which are one of the most important causes of leukemia relapse and have the ability to self-renew indefinitely and generate a large number of daughter blasts with a specific phenotype of CLL-1, CD123, CD44, CD96, CD90, CD32, CD25, and TIM-3.

[0133] The present disclosure generally relates to improved compositions and methods for modulating the spatial and temporal control of adoptive cell therapy using dimerizer-regulated immune receptor complexes (DARICs) that bind CLL-1. Without being bound by any particular theory, the DARIC compositions and methods encompassed herein offer many advantages over existing CAR T cell therapies in the art, including but not limited to control of both the spatial and temporal aspects of immune effector cell signal transduction binding and signaling activity. DARIC temporal control initiates the signaling DARIC mechanism by bridging the association between the DARIC binding component and the DARIC signaling component mediated by a bridging factor. DARIC spatial control participates in the signaling mechanism by the DARIC binding domain of the DARIC binding component recognizing CLL-1. In this way, DARIC immune effector cells are activated when both target cells expressing CLL-1 and the bridging factor are present.

[0134] The present disclosure also relates to improved anti-CLL-1 CAR architectures that overcome potential limitations of existing CAR T therapies, including but not limited to tonic signaling or antigen-dependent signaling, weak expression, and / or sub-therapeutic activity.

[0135] In various embodiments, the present disclosure encompasses anti-CLL-1 VHH DARIC or anti-CLL-1 VHH CARs that generate an anti-cancer response against cancers expressing CLL-1 (e.g., AML).

[0136] In certain embodiments, the DARIC comprises a polypeptide (DARIC signaling component) that includes a multimerization domain polypeptide or variant thereof, a transmembrane domain, a co-stimulatory domain, and / or a primary signaling domain; and a polypeptide (DARIC binding component) that includes an anti-CLL-1 VHH, a multimerization domain polypeptide or variant thereof, a transmembrane domain, and optionally a co-stimulatory 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 CLL-1.

[0137] In certain embodiments, the multimerization domains of the DARIC binding component and the DARIC signaling component are extracellularly located. The extracellular location of the multimerization domain provides numerous advantages compared to an intracellular location, including but not limited to more efficient localization of the anti-CLL-1 VHH domain, higher temporal sensitivity to bridging factor modulation, and less toxicity due to the ability to use non-immunosuppressive doses of certain bridging factors.

[0138] The present disclosure encompasses polynucleotides encoding DARIC, DARIC binding components, and DARIC signaling components; DARIC binding components, DARIC signaling components, DARIC protein complexes, DARIC fusion proteins; cells comprising polynucleotides encoding and / or expressing DARIC, DARIC binding components, and DARIC signaling components; and methods of using the same to treat immune disorders.

[0139] Techniques for recombinant (i.e., engineered) DNA, peptide, and oligonucleotide synthesis, immunoassays, tissue culture, transformation (e.g., electroporation, liposome transfection), enzymatic reactions, purification, and related techniques and procedures can generally be performed as described in a variety of general and more specific references in microbiology, molecular biology, biochemistry, molecular genetics, cell biology, virology, and immunology cited and discussed throughout this specification. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Current Protocols in Molecular Biology (updated July 2008, John Wiley and Sons); Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Glover, DNA Cloning: A Practical Approach, vol. I & II (IRL Press, Oxford Univ. Press USA, 1985); Current Protocols in Immunology (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.(eds. Calos, 1987, Cold Spring Harbor Laboratory); Harlow and Lane, Antibodies, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1998); Immunochemical Methods In Cell And Molecular Biology (eds. Mayer and Walker, Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (eds. D.M. Weir and C.C. Blackwell, 1986); Roitt, Essential Immunology, 6th Edition, (Blackwell Scientific Publications, Oxford, 1988); Current Protocols in Immunology (eds. Q.E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach and W. Strober, 1991); Annual Review of Immunology; and monographs in journals such as Advances in Immunology.

[0140] B. Definitions

[0141] Before elaborating on the present disclosure in more detail, providing definitions of certain terms to be used herein may aid in understanding the present disclosure.

[0142] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used to practice or test the particular embodiments, the preferred embodiments of the compositions, methods and materials are described herein. For the purposes of the present disclosure, the following terms are defined below.

[0143] As used herein, the article "a / an" refers to one or more than one (i.e., at least one) of the grammatical object of the article. By way of example, "an element" means one element or one or more elements.

[0144] The use of alternative selections (e.g., "or") should be understood to mean one, both, or any combination of the alternative selections.

[0145] The term "and / or" shall be understood to mean either or both of the alternative choices.

[0146] 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" means that the quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length is within the range of approximately the reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2% or ±1%.

[0147] In one embodiment, a range, such as 1 to 5, about 1 to 5 or about 1 to about 5, refers to each numerical value encompassed by the range. For example, in one non-limiting and merely illustrative embodiment, the range "1 to 5" is equivalent to the statements 1, 2, 3, 4, 5; or 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5 or 5.0; or 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9 or 5.0.

[0148] As used herein, the term "substantially" means that a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or a higher percentage of the reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length. In one embodiment, "substantially the same" means that the effect produced by a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length, such as a physiological effect, is substantially the same as that of the reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.

[0149] Throughout this specification, unless the context requires otherwise, the word "comprise" and its variations "comprises" and "comprising" are to be interpreted as having an inclusive meaning, i.e. to mean including the stated step or element or group of steps or elements but not excluding any other step or element or group of steps or elements. "Consisting of" is intended to include and be limited to the matter following the phrase "consisting of". Thus, the phrase "consisting of" indicates that the listed elements are essential or required and that no other elements can be present. "Consisting essentially of" is intended to include any element listed after the phrase and to be limited to other elements that do not interfere with or affect the activity or action described in the disclosure of the listed elements. Thus, the phrase "consisting essentially of" indicates that the listed elements are essential or required and that no other elements substantially affect the activity or action of the listed elements.

[0150] References throughout this specification to "one embodiment", "a particular embodiment", "a related embodiment", "an embodiment", "an additional embodiment" or "another embodiment" or combinations thereof mean that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the foregoing phrases appearing 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 the feature in a particular embodiment.

[0151] "Antigen (Ag)" refers to a compound, composition or substance that can stimulate antibody production or a T cell response in an animal, including compositions (such as compositions containing cancer-specific proteins) that are injected or absorbed into the animal. Exemplary antigens include, but are not limited to, lipids, carbohydrates, polysaccharides, glycoproteins, peptides or nucleic acids. Antigens react with products having specific humoral or cellular immunity, including products induced by heterologous antigens such as the disclosed antigens.

[0152] "Target antigen" or "target antigen of interest" refers to the portion of CLL-1 to which the binding domain covered herein is designed to bind. In a particular embodiment, the target antigen is SEQ ID NO: 1.

[0153] "C-type lectin-like molecule 1", "CLL-1", or "CLL-1" is a member of the C-type lectin / C-type lectin-like domain (CTL / CTLD) superfamily. The gene encoding CLL-1 is located on chromosome 12. CLL-1 is also known as C-type lectin domain family 12 member A (CLEC12A), myeloid inhibitory C-type lectin-like receptor (MICL), dendritic cell-associated lectin 2 (DCAL-2, DCAL2), and CD371. The major isoform of CLL-1 is a type II transmembrane protein of 265 amino acids with a calculated molecular weight of 31 kD. CLL-1 contains an N-terminal cytoplasmic tail region as well as an immunoreceptor tyrosine-based inhibitory motif (ITIM), a transmembrane domain, a stalk / neck region, and a C-terminal C-type lectin domain (CTLD). CLL-1 can inhibit cell activation through its cytoplasmic ITIM and negatively regulate granulocyte and monocyte functions. Representative CLL-1 polynucleotide sequences include NM_001207010.1, NM_001300730.1, NM_138337.5, NM_201623.3, NM_201625.1, ENST00000304361, ENST00000350667, ENST00000355690, ENST00000396507, ENST00000434319, ENST00000449959, and ENST00000543839. Representative CLL-1 polypeptide sequences include Q5QGZ9, ENSP00000405244, ENSP00000345448, ENSP00000347916, ENSP00000302804, ENSP00000379764, XP_011518873.2, XP_006719099.1, NP_001193939.1, NP_612210.4, XP_011518872.1, XP_011518875.1, NP_001287659.1, XP_005253381.1, NP_963917.2, XP_006719096.1, and XP_006719098.1.

[0154] "Antibody" refers to an agent belonging to a polypeptide that includes at least the variable region of an immunoglobulin light or heavy chain, the variable region of the immunoglobulin light or heavy chain specifically recognizing and binding to an epitope of a target, such as a lipid, carbohydrate, polysaccharide, glycoprotein, peptide, or nucleic acid containing an antigenic determinant, such as those recognized by immune cells.

[0155] Reference to "VH" or "VH" refers to the variable region of an immunoglobulin heavy chain or an antigen-binding fragment thereof.

[0156] "Heavy chain antibody" refers to an antibody containing two V H domains and lacking a light chain (Riechmann L. et al., J. Immunol. Methods 231: 25-38 (1999); W094 / 04678; WO94 / 25591; U.S. Patent 6,005,079). "Camelid antibody" refers to an antibody isolated from camel, alpaca or llama containing two V H domains and lacking a light chain.

[0157] As used herein, "V H H", "V H H antibody" or "V H H domain" refers to an antibody fragment containing the smallest known antigen-binding unit of the variable region of a heavy chain antibody (Koch-Nolte et al., FASEB J., 21: 3490-3498 (2007)).

[0158] "Linker" refers to multiple amino acid residues between individual polypeptide domains added for proper spacing and conformation of the molecule. In certain embodiments, the linker separates one or more VHH domains, hinge domains, multimerization domains, transmembrane domains, co-stimulatory domains and / or primary signaling domains.

[0159] Illustrative examples of linkers suitable for use in the specific embodiments covered herein include, but are not limited to, the following amino acid sequences: GGG; DGGGS (SEQ ID NO: 52); TGEKP (SEQ ID NO: 53) (see, e.g., Liu et al., PNAS 5525-5530 (1997)); GGRR (SEQ ID NO: 54) (Pomerantz et al. 1995, supra); (GGGGS) n, where n = 1, 2, 3, 4 or 5 (SEQ ID NO: 55) (Kim et al., PNAS 93, 1156-1160 (1996)); EGKSSGSGSESKVD (SEQ ID NO: 56) (Chaudhary et al., 1990, Proc. Natl. Acad. Sci. U.S.A. 87: 1066-1070); KESGSVSSEQLAQFRSLD (SEQ ID NO: 57) (Bird et al., 1988, Science 242: 423-426), GGRRGGGS (SEQ ID NO: 58); LRQRDGERP (SEQ ID NO: 59); LRQKDGGGSERP (SEQ ID NO: 60); LRQKD(GGGS)2ERP (SEQ ID NO: 61). Alternatively, a computer program capable of modeling the DNA binding site and the peptide itself can be used (Desjarlais & Berg, PNAS 90: 2256-2260 (1993), PNAS 91: 11099-11103 (1994)) or a flexible linker can be rationally designed by phage display methods. In one embodiment, the linker comprises the following amino acid sequence: GSTSGSGKPGSGEGSTKG (SEQ ID NO: 62) (Cooper et al., Blood, 101(4): 1637-1644 (2003)).

[0160] "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 proper cell / cell contact, antigen binding and activation (Patel et al., Gene Therapy, 1999; 6: 412-419). In certain embodiments, the spacer domain separates one or more VHH domains, multimerization domains, transmembrane domains, co-stimulatory domains and / or primary signaling domains. The spacer domain can be derived from natural, synthetic, semi-synthetic or recombinant sources. In some embodiments, the spacer domain is part of an immunoglobulin, including but not limited to one or more heavy chain constant regions, such as CH2 and CH3. The spacer domain can include the amino acid sequence of a naturally occurring immunoglobulin hinge region or an altered immunoglobulin hinge region.

[0161] "Hinge domain" refers to a polypeptide that functions to position an antigen-binding domain away from the effector cell surface to achieve proper 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 natural, synthetic, semi-synthetic, or recombinant sources. The hinge domain may include the amino acid sequence of a naturally occurring immunoglobulin hinge region or an altered immunoglobulin hinge region.

[0162] As used herein, "multimerization domain" refers to a polypeptide that preferentially interacts or associates with another, different polypeptide, either directly or via a bridging molecule, e.g., a chemically inducible dimer, wherein the interaction of different multimerization domains substantially contributes to or efficiently facilitates multimerization (i.e., the formation of dimers, trimers, or multi-component complexes, which may be homodimers, heterodimers, homotrimers, heterotrimers, homopolymers, or heteropolymers). The multimerization domain may be derived from natural, synthetic, semi-synthetic, or recombinant sources.

[0163] Illustrative examples of multimerization domains suitable for use in certain embodiments encompassed 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.

[0164] As used herein, the term "FKBP-rapamycin binding polypeptide" refers to an FRB polypeptide. In certain embodiments, the FRB polypeptide is an FKBP12-rapamycin binding polypeptide. FRB polypeptides suitable for use in certain embodiments encompassed herein typically contain at least about 85 to about 100 amino acid residues. In certain embodiments, with reference to GenBank accession number L34075.1, the FRB polypeptide contains the 93 amino acid sequence from Ile-2021 to Lys-2113 and the mutation T2098L. The FRB polypeptides encompassed herein bind to FKBP polypeptides via a bridging factor, thereby forming a ternary complex.

[0165] 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 the F36V mutation. In some embodiments, the FKBP domain may also be referred to as the "rapamycin binding domain". Information regarding the nucleotide sequences, cloning, and other aspects of various FKBP species is 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.

[0166] A "bridging factor" refers to a molecule that associates with two or more multimerization domains and is positioned between the two or more multimerization domains. In certain embodiments, the multimerization domains substantially contribute to or efficiently promote the formation of a polypeptide complex only in the presence of the bridging factor. In certain embodiments, the multimerization domains do not contribute to or do not efficiently promote the formation of a polypeptide complex in the absence of the bridging factor. Illustrative examples of bridging factors suitable for use in the specific embodiments encompassed herein include, but are not limited to, AP21967, rapamycin (sirolimus) or rapamycin analogs, coumermycin or its derivatives, gibberellin or its derivatives, abscisic acid (ABA) or its derivatives, methotrexate or its derivatives, cyclosporin A or its derivatives, FKCsA or its derivatives, trimethoprim (Tmp)-FKBP synthetic ligand (SLF) or its derivatives, or any combination thereof.

[0167] Rapamycin analogs (rapalogs) include, but are not limited to, those disclosed in U.S. Patent No. 6,649,595, the rapalog structures of which are incorporated herein by reference in their entirety. In some embodiments, the bridging factor is a rapamycin analog that has significantly lower immunosuppressive effects compared to rapamycin. In a preferred embodiment, the rapamycin analog is AP21967 (also known as C-16-(S)-7-methylindolyl rapamycin, IC 50 = 10 nM, a chemically modified non-immunosuppressive rapamycin analog). Other illustrative rapamycin analogs suitable for use in the specific embodiments encompassed herein include, but are not limited to, everolimus, deforolimus, pimecrolimus, delpomolimus, tacrolimus, temsirolimus, umirolimus, and zotarolimus.

[0168] "Significantly reduced immunosuppressive effect" means at least less than 0.1-fold to 0.005-fold of the immunosuppressive effect observed or expected at the same dose measured in a clinical setting or in a suitable in vitro (e.g., inhibition of T cell proliferation) or in vivo surrogate of human immunosuppressive activity.

[0169] "Transmembrane domain" or "TM domain" is a domain that anchors a polypeptide to the plasma membrane of a cell. The TM domain can be derived from natural, synthetic, semi-synthetic, or recombinant sources.

[0170] 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 triggered by antigen binding to receptors expressed on immune effector cells.

[0171] "Intracellular signaling domain" or "intracellular domain" refers to the part of a protein that transduces signals for effector functions and directs the cell to perform specialized functions. Although the entire intracellular signaling domain can generally be employed, it is not necessary to use the entire domain in many cases. To the extent that a truncated portion of the intracellular signaling domain is used, such a truncated portion can be used in place of the entire domain as long as it transduces signals for effector functions. The term intracellular signaling domain means any truncated portion that is essential or sufficient to transduce signals for effector functions and that contains the intracellular signaling domain.

[0172] It is known that the signal generated solely through the TCR is not sufficient to fully activate T cells, and a secondary signal or co-stimulatory signal is also required. Thus, it can be said that T cell activation is mediated by two different classes of intracellular signaling domains: a primary signaling domain that initiates antigen-dependent primary activation through the TCR (e.g., the TCR / CD3 complex); and a co-stimulatory signaling domain that acts in an antigen-independent manner to provide a secondary signal or co-stimulatory signal.

[0173] "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 signaling motifs called immunoreceptor tyrosine-based activation motifs or ITAMs. 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.

[0174] As used herein, the term "costimulatory signaling domain" or "costimulatory domain" refers to the intracellular signaling domain of a costimulatory molecule. A costimulatory molecule is a cell surface molecule other than an antigen receptor or Fc receptor that provides a second signal required for the effective activation and function of T lymphocytes upon binding to an antigen. Illustrative examples of such costimulatory molecules from which a costimulatory domain 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 (ICOS), DNAX-activating protein 10 (DAP10), linker for activation of T cells family member 1 (LAT), SH2 domain-containing leukocyte protein of 76kD (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).

[0175] An "immune disorder" refers to a disease that elicits an immune system response. In certain embodiments, the term "immune disorder" refers to cancer, an autoimmune disease, or an immunodeficiency.

[0176] 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.

[0177] As used herein, the term "malignant" refers to a cancer in which a group of tumor cells exhibits 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 lymph or blood). As used herein, the term "metastasis" refers to the spread of cancer from one part of the body to another. A tumor formed by cells that have spread is called a "metastatic tumor" or "metastasis". Metastatic tumors contain cells similar to those in the original (primary) tumor.

[0178] 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-limiting and generally do not invade or metastasize.

[0179] "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 abnormal growth of cells, which can be benign, pre-malignant or malignant. Most cancers form tumors, but liquid cancers such as leukemia do not necessarily form tumors. For those cancers that form tumors, the terms cancer (cell) and tumor (cell) can be used interchangeably. The amount of tumor in an individual is the "tumor burden" that can be measured as the number, volume or weight of tumors.

[0180] The term "relapse" refers to the diagnosis of a recurrence of cancer or the recurrence of signs and symptoms after a period of improvement or remission.

[0181] "Remission" is also referred to as "clinical remission" and includes both partial remission and complete remission. In partial remission, some but not all cancer signs and symptoms have disappeared. In complete remission, all cancer signs and symptoms have disappeared, although cancer may still be present in the body.

[0182] "Refractory" means that cancer is resistant or unresponsive to therapy with a specific therapeutic agent. Cancer can be refractory at the start of treatment (i.e., unresponsive to initial exposure to the therapeutic agent), or become refractory due to the development of resistance to the therapeutic agent during the first treatment period or during subsequent treatment periods.

[0183] 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 covered elsewhere in this document. Suitable subjects (e.g., patients) include laboratory animals (such as mice, rats, rabbits or guinea pigs), farm animals and domestic animals or pets (such as cats or dogs). Include non-human primates and preferably human patients. Typical subjects include human patients who have, have been diagnosed with, or are at risk of having cancer or another immune condition.

[0184] 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 in this document.

[0185] As used herein, "treatment" includes any beneficial or desired effect on the symptoms or pathology of a disease or pathological condition and can even include a minimal reduction in one or more measurable markers of the disease or disorder being treated. Optionally, treatment can 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 disorder or its related symptoms.

[0186] As used herein, "prevent" and words such as "prevented / preventing" indicate the use for preventing, inhibiting or reducing the likelihood of the occurrence or recurrence of a disease or disorder. Prevention also refers to delaying the onset or recurrence of a disease or disorder or delaying the occurrence or recurrence of the symptoms of a disease or disorder. As used herein, "prevention" and similar words also include reducing the intensity, effect, symptoms and / or burden of a disease or disorder before the onset or recurrence of the disease or disorder.

[0187] As used herein, the phrase "alleviate at least one symptom of" refers to reducing one or more symptoms of a disease or disorder in a subject being treated. In certain embodiments, the disease or condition being treated is cancer, and the one or more symptoms alleviated include, but are not limited to, weakness, fatigue, shortness of breath, easy bruising and bleeding, frequent infections, enlarged lymph nodes, abdominal swelling or pain (due to enlarged abdominal organs), bone or joint pain, fractures, unintended weight loss, loss of appetite, night sweats, persistent low-grade fever, and decreased urination (due to impaired kidney function).

[0188] "Enhance" or "promote" or "increase" or "amplify" generally refers to the ability of the compositions encompassed herein to produce, elicit or cause a greater physiological response (i.e., downstream effect) compared to the response caused by a vehicle or control molecule / composition. A measurable physiological response can include T cell expansion, activation, persistence, increased cytokine secretion, and / or increased cancer cell killing ability, as well as other aspects that are apparent from the understanding in the art and the description herein. An "increased" or "enhanced" amount is generally a "statistically significant" amount and can include an increase that is 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 therebetween and above 1, e.g., 1.5, 1.6, 1.7, 1.8, etc.) of the response produced by a vehicle or control composition.

[0189] "Reduce" or "attenuate" or "lessen" or "decrease" or "alleviate" generally refers to the ability of the compositions covered herein to produce, elicit or cause less of a response (i.e., a physiological response) compared to the response caused by a vehicle or a control molecule / composition. A "reduced" or "decreased" amount is generally a "statistically significant" amount and can include a reduction that is 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 therebetween and above 1, such as 1.5, 1.6, 1.7, 1.8, etc.) of the response produced by a vehicle, control composition or response in a particular cell lineage (reference response).

[0190] "Maintain" (or "maintenance") or "preserve" or "no change" or "no substantial change" or "no substantial reduction" generally refers to the ability of the compositions covered herein to produce, elicit or cause a substantially similar or comparable physiological response (i.e., downstream effect) in cells compared to the response caused by a vehicle, control molecule / composition or response in a particular cell lineage. A comparable response is one that is not significantly or measurably different from the reference response.

[0191] Additional definitions are set forth throughout this disclosure.

[0192] C.CLL-1 VHH DARIC

[0193] In certain embodiments, DARIC receptors comprising an anti-CLL-1 VHH domain are encompassed that redirect the cytotoxicity of immune effector cells towards cancer cells expressing CLL-1. As used herein, the terms "CLL-1 VHH DARIC receptor", "anti-CLL-1 VHH DARIC receptor", "CLL-1 VHH DARIC" or "anti-CLL-1 VHH DARIC" are used interchangeably and refer to one or more non-naturally occurring polypeptides that transduce an immune-stimulatory signal in immune effector cells upon exposure to an antigen and a multimerizing agent or bridging factor, such as stimulating immune effector cell activity and function, increasing the production and / or secretion of pro-inflammatory cytokines. In a preferred embodiment, the CLL-1 VHH DARIC is a multi-chain chimeric receptor that comprises a DARIC signaling component and a DARIC binding component comprising a VHH domain that recognizes CLL-1.

[0194] In one embodiment, the DARIC signaling component and the DARIC binding component are expressed from the same cell. In another embodiment, the DARIC signaling component and the DARIC binding component are expressed from different cells. In certain embodiments, the DARIC signaling component is expressed from a cell, and the DARIC binding component is exogenously supplied as a polypeptide. In one embodiment, the DARIC binding component pre-loaded with a bridging factor is exogenously supplied to a cell expressing the DARIC signaling component.

[0195] CLL-1 DARIC signaling component

[0196] The terms "DARIC signaling component", "CLL-1 DARIC signaling component", "DARIC signaling polypeptide", or "DARIC signaling polypeptide" are used interchangeably and refer to a polypeptide comprising one or more multimerization domains, transmembrane domains, and one or more intracellular signaling domains. In certain embodiments, the DARIC signaling component comprises a multimerization domain, a transmembrane domain, a co-stimulatory 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 co-stimulatory domain, and / or a primary signaling domain.

[0197] In certain embodiments, the DARIC signaling component comprises one or more multimerization domains.

[0198] Illustrative examples of multimerization domains suitable for use in certain CLL-1 DARIC signaling components encompassed 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.

[0199] In certain embodiments, the CLL-1 DARIC signaling component comprises an FRB polypeptide.

[0200] In a particularly preferred embodiment, the CLL-1 DARIC signaling component comprises an FRB polypeptide or a variant thereof comprising the T2098L mutation. In certain preferred embodiments, the CLL-1 DARIC signaling component comprises an FKBP12 polypeptide or a variant thereof.

[0201] In some embodiments, the CLL-1 VHH DARIC signaling component comprises a hinge domain.

[0202] Exemplary hinge domains suitable for use in the CLL-1 VHH DARIC signaling components described herein include hinge regions derived from the extracellular regions of type I membrane proteins such as CD28, CD8α, and CD4, which can be wild-type hinge regions from these molecules or can be altered.

[0203] In certain embodiments, the DARIC signaling component includes a transmembrane domain.

[0204] In certain embodiments, the DARIC signaling component includes a hinge domain and a transmembrane domain.

[0205] Exemplary examples of transmembrane domains suitable for use in the specific CLL-1 DARIC signaling components encompassed herein include, but are not limited to, the transmembrane regions of the α-chain, β-chain, γ-chain, or δ-chain of the T cell receptor, CD3ε, CD3ζ, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CLL-1, CD37, CD45, CD64, CD71, CD80, CD86, CD134, CD137, CD152, CD154, amnionless protein (AMN), and programmed cell death 1 (PDCD1). In a preferred embodiment, the CLL-1 DARIC signaling component includes a CD4 transmembrane domain. In a preferred embodiment, the CLL-1 DARIC signaling component includes a CD8α transmembrane domain.

[0206] 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.

[0207] The DARIC signaling components encompassed in certain embodiments herein include one or more intracellular signaling domains. In one embodiment, the CLL-1 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).

[0208] Illustrative examples of ITAM primary signaling domains suitable for use in the specific CLL-1 DARIC signaling components encompassed 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 CLL-1 DARIC signaling component includes a CD3ζ primary signaling domain and one or more co-stimulatory signaling domains. The primary signaling domain and the co-stimulatory signaling domain can be tandemly linked to the carboxyl terminus of the transmembrane domain in any order.

[0209] Illustrative examples of co-stimulatory domains suitable for use in the specific CLL-1 DARIC signaling components encompassed herein include, but are not limited to, those domains isolated from the following co-stimulatory molecules: Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, caspase recruitment domain family member 11 (CARD11), CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD94, CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), DNAX-activating protein 10 (DAP10), linker for activation of T cells family member 1 (LAT), SH2 domain-containing leukocyte protein of 76 kDa (SLP76), T cell receptor-associated transmembrane adapter 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).

[0210] In certain embodiments, the CLL-1 DARIC signaling components encompassed herein include a signal peptide. Illustrative examples of signal peptides suitable for use in specific CLL-1 DARIC signaling 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 CLL-1 DARIC signaling component includes a CD8α signal polypeptide.

[0211] In certain embodiments, the CLL-1 DARIC signaling component comprises one or more co-stimulatory signaling domains selected from the group consisting of CD28, CD137, and CD134. In certain embodiments, the CLL-1 DARIC signaling component comprises one or more co-stimulatory signaling domains selected from the group consisting of CD28, CD137, and CD134 and a CD3ζ primary signaling domain. In certain embodiments, the CLL-1 DARIC signaling component comprises a CD137 co-stimulatory domain and a CD3ζ primary signaling domain.

[0212] In a preferred embodiment, the CLL-1 DARIC signaling component comprises an FRB T2098L multimerization domain, a CD8α transmembrane domain, a CD137 co-stimulatory domain, and a CD3ζ primary signaling domain.

[0213] In a preferred embodiment, the CLL-1 VHH DARIC signaling component comprises the amino acid sequence set forth in SEQ ID NO: 50.

[0214] 1. CLL-1 DARIC Binding Component

[0215] The “DARIC binding component”, “DARIC binding polypeptide”, “CLL-1 VHH DARIC binding component”, or “CLL-1 VHH DARIC binding polypeptide” are used interchangeably and refer to a polypeptide comprising an anti-CLL-1 VHH domain and one or more multimerization domains. In certain embodiments, the CLL-1 VHH DARIC binding component comprises an anti-CLL-1 VHH domain, a multimerization domain, and a transmembrane domain. In certain embodiments, the CLL-1 VHH DARIC binding component comprises an anti-CLL-1 VHH domain, a second multimerization domain, and a second transmembrane domain. In other certain embodiments, the CLL-1 VHH DARIC binding component comprises an anti-CLL-1 VHH domain, a multimerization domain, a transmembrane domain, and one or more intracellular signaling domains. In certain embodiments, the CLL-1 VHH DARIC binding component comprises an anti-CLL-1 VHH domain, a second multimerization domain, a second transmembrane domain, and a second co-stimulatory domain.

[0216] In certain embodiments, the CLL-1 VHH DARIC binding component comprises one or more anti-CLL-1 VHH domains.

[0217] In certain embodiments, the anti-CLL-1 VHH domain is a humanized camelid VHH that binds to one or more epitopes of CLL-1 (e.g., SEQ ID NO: 1).

[0218] In a particularly preferred embodiment, the anti-CLL-1 VHH domain is a humanized camelid VHH comprising the amino acid sequence shown in SEQ ID NO: 2-13, i.e., any one of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13.

[0219] In certain embodiments, the DARIC binding component comprises one or more multimerization domains.

[0220] Illustrative examples of multimerization domains suitable for use in the specific CLL-1 VHH DARIC binding components encompassed 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.

[0221] In certain embodiments, the CLL-1 VHH DARIC binding component comprises an FRB polypeptide or variant thereof, and the DARIC signaling component comprises an FKBP polypeptide or variant thereof. In a preferred embodiment, the CLL-1 VHH DARIC binding component comprises an FRB polypeptide or variant thereof comprising the T2098L mutation, and the DARIC signaling component comprises an FKBP12 polypeptide or variant thereof.

[0222] In certain embodiments, the CLL-1 VHH DARIC binding component comprises an FKBP polypeptide or variant thereof, and the DARIC signaling component comprises an FRB polypeptide or variant thereof. In a preferred embodiment, the CLL-1 VHH DARIC binding component comprises an FKBP12 polypeptide or variant thereof, and the DARIC signaling component comprises an FRB polypeptide or variant thereof comprising the T2098L mutation.

[0223] In some embodiments, the CLL-1 VHH DARIC binding component comprises a hinge domain.

[0224] Illustrative hinge domains suitable for use in the CLL-1 VHH DARIC binding components described herein include hinge regions derived from the extracellular regions of type I membrane proteins such as CD28, CD8α, and CD4, which may be the wild-type hinge regions from these molecules or may be altered.

[0225] In certain embodiments, the DARIC binding component includes a transmembrane domain. In certain embodiments, the DARIC binding component includes 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.

[0226] Exemplary transmembrane domains suitable for use in the specific CLL-1 VHH DARIC binding components encompassed 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, CLL-1, CD37, CD45, CD64, CD71, CD80, CD86, CD134, CD137, CD152, CD154, amelogenin (AMN), and programmed cell death 1 (PDCD1). In a preferred embodiment, the CLL-1 DARIC binding component includes the CD8α transmembrane domain. In a preferred embodiment, the CLL-1 VHH DARIC binding component includes the CD4 transmembrane domain.

[0227] 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.

[0228] The DARIC binding components encompassed in the specific embodiments herein do not include one or more intracellular signaling domains.

[0229] In other specific embodiments, the CLL-1 VHH DARIC binding components encompassed herein include one or more intracellular signaling domains. In a preferred embodiment, where the CLL-1 VHH DARIC binding component includes one or more intracellular signaling domains, those domains are different from the intracellular signaling domains present in the cognate CLL-1 DARIC signaling component. In one embodiment, the CLL-1 VHH DARIC binding component includes a co-stimulatory signaling domain.

[0230] Illustrative examples of co-stimulatory domains suitable for use in the specific CLL-1 VHH DARIC binding components covered herein include, but are not limited to, those domains isolated from the following co-stimulatory molecules: Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, caspase recruitment domain family member 11 (CARD11), CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD94, CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), DNAX-activating protein 10 (DAP10), linker for activation of T cells family member 1 (LAT), SH2 domain-containing leukocyte protein of 76 kDa (SLP76), T cell receptor-associated transmembrane adapter 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 a preferred embodiment, the co-stimulatory domain is derived, obtained, or isolated from TNFR2 or OX40.

[0231] In certain embodiments, the DARIC binding components covered herein include a signal peptide. Illustrative examples of signal peptides suitable for use in the specific CLL-1 VHH DARIC binding components include, but are not limited to, the IgG1 heavy chain signal polypeptide, the Igκ light chain signal polypeptide, the CD8α signal polypeptide, or the human GM-CSF receptor α signal polypeptide. In various preferred embodiments, the CLL-1 VHH DARIC binding component includes the CD8α signal polypeptide.

[0232] In certain embodiments, the CLL-1 VHH DARIC binding component includes a VHH domain that binds CLL-1, an FKBP12 multimerization domain, a CD4 transmembrane domain, and optionally a co-stimulatory domain.

[0233] In some embodiments, the CLL-1 VHH DARIC binding component includes a VHH that binds CLL-1 and an FKBP12 multimerization domain.

[0234] In some embodiments, the CLL-1 VHH DARIC binding component includes a VHH domain having an amino acid sequence shown in any one of SEQ ID NOs: 2-13, an FKBP12 multimerization domain, a CD4 transmembrane domain, and optionally a co-stimulatory domain.

[0235] In some embodiments, the CLL-1 VHH DARIC binding component comprises a VHH domain and an FKBP12 multimerization domain comprising the amino acid sequence shown in any one of SEQ ID NOs: 2-13.

[0236] In some embodiments, the CLL-1 VHH DARIC binding component comprises the sequence shown in any one of SEQ ID NOs: 14-19.

[0237] 2. Bridging factor

[0238] The bridging factors encompassed in certain embodiments herein mediate or facilitate the association of the CLL-1 DARIC signaling component with the CLL-1 VHH DARIC binding component through the multimerization domains in the respective components. The bridging factor associates with the multimerization domains and is positioned between the multimerization domains to facilitate the association of the CLL-1 DARIC signaling component with the CLL-1 VHH DARIC binding component. In the presence of the bridging factor, when the CLL-1 VHH DARIC binding component binds to CLL-1 expressed on a target cell, the CLL-1 VHH DARIC binding component associates with the CLL-1 DARIC signaling component and triggers immune effector cell activity against the target cell. In the absence of the bridging factor, the CLL-1 VHH DARIC binding component does not associate with the CLL-1 DARIC signaling component, and the CLL-1 VHH DARIC is inactive.

[0239] In certain embodiments, the CLL-1 DARIC signaling component and the CLL-1 VHH DARIC binding component comprise a homologous 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).

[0240] In certain embodiments, the multimerization domains of the CLL-1 VHH DARIC signaling and binding components associate with a bridging factor selected from the group consisting of: rapamycin or a rapamycin analogue, coumermycin or a derivative thereof, gibberellin or a derivative thereof, abscisic acid (ABA) or a derivative thereof, methotrexate or a derivative thereof, cyclosporin A or a derivative thereof, FK506 / cyclosporin A (FKCsA) or a derivative thereof, trimethoprim (Tmp)-FK506 binding protein (FKBP) synthetic ligand (SLF) or a derivative thereof.

[0241] In certain embodiments, the CLL-1 DARIC signaling component and the CLL-1 VHH DARIC binding component include one or more FRB and / or FKBP multimerization domains or variants thereof. In some embodiments, the CLL-1 DARIC signaling component includes an FRB multimerization domain or a variant thereof, and the CLL-1 VHH DARIC binding component includes an FKBP multimerization domain or a variant thereof. In particularly preferred embodiments, the CLL-1 DARIC signaling component includes an FRB T2098L multimerization domain or a variant thereof, and the CLL-1 VHH DARIC binding component includes an FKBP12 or an FKBP12 F36V multimerization domain or a variant thereof.

[0242] Illustrative examples of bridging factors suitable for use in the specific embodiments encompassed herein include, but are not limited to, AP1903, AP20187, AP21967 (also known as C-16-(S)-7-methylrapamycin), everolimus, deforolimus, pimecrolimus, temsirolimus, umirolimus, and zotarolimus. In certain preferred embodiments, the bridging factor is AP21967. In some preferred embodiments, the bridging factor is a non-immunosuppressive dose of sirolimus (rapamycin).

[0243] D. Anti-CLL-1 Chimeric Antigen Receptor

[0244] In certain embodiments, the immune effector cells encompassed herein comprise an anti-CLL-1 VHH CAR. A chimeric antigen receptor (CAR) is a molecule that combines the specificity of an antibody for a target antigen (e.g., a tumor antigen) with an intracellular domain that activates a T cell receptor to produce a chimeric protein that exhibits specific anti-tumor cell immune activity. As used herein, the term "chimeric" describes being composed of different protein or DNA moieties from different sources.

[0245] In certain embodiments, T cells are engineered by introducing a polynucleotide encoding an anti-CLL-1 VHH CAR.

[0246] In certain embodiments, T cells are engineered by introducing a vector comprising a polynucleotide encoding an anti-CLL-1 VHH CAR.

[0247] In various embodiments, the anti-CLL-1 CAR comprises a VHH domain that binds CLL-1, a transmembrane domain, and one or more intracellular signaling domains. The main characteristic of the CAR is its ability to re-direct immune effector cell specificity using the cell-specific targeting ability of monoclonal antibodies, soluble ligands, or cell-specific co-receptors, 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.

[0248] In some embodiments, the anti-CLL-1 VHH CAR comprises a spacer domain. In certain embodiments, the spacer domain comprises the CH2 and CH3 of IgG1, IgG4, or IgD.

[0249] In some embodiments, the anti-CLL-1 VHH CAR comprises a hinge domain.

[0250] Exemplary hinge domains suitable for use in the anti-CLL-1 VHH CARs described herein include hinge regions derived from the extracellular regions of type I membrane proteins such as CD28, CD8α, and CD4, which can be the wild-type hinge regions from these molecules or can be altered. In another embodiment, the hinge domain comprises the CD8α hinge region.

[0251] 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, semi-synthetic, or recombinant sources.

[0252] Exemplary TM domains can be derived from (i.e., include at least one or more transmembrane regions of) the α-chain, β-chain, γ-chain, or δ-chain of the T cell receptor, CD3ε, CD3ζ, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CLL-1, CD37, CD45, CD64, CD71, CD80, CD86, CD134, CD137, CD152, CD154, AMN, and PDCD1.

[0253] In one embodiment, the anti-CLL-1 VHH CAR comprises a TM domain derived from CD8α. In another embodiment, the CARs encompassed herein comprise a TM domain derived from CD8α and a short oligopeptide or polypeptide linker having a length preferably between 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, which linker connects the TM domain of the CAR and the intracellular signaling domain. A glycine-serine linker provides a particularly suitable linker.

[0254] In a preferred embodiment, the anti-CLL-1 VHH CAR comprises an intracellular signaling domain, said intracellular signaling domain comprising one or more "costimulatory signaling domains" and a "primary signaling domain".

[0255] The primary signaling domain that functions in a stimulatory manner may contain a signaling motif, said signaling motif being called an immunoreceptor tyrosine-based activation motif or ITAM.

[0256] Illustrative examples of ITAM-containing primary signaling domains suitable for use in the anti-CLL-1 VHH CARs encompassed by a particular embodiment include those derived from FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d. In a particular preferred embodiment, the CAR comprises a CD3ζ primary signaling domain and one or more costimulatory signaling domains. The intracellular primary signaling domain and costimulatory signaling domains can be tandemly linked to the carboxyl terminus of the transmembrane domain in any order.

[0257] In a particular embodiment, the anti-CLL-1 VHH CAR comprises one or more costimulatory signaling domains for enhancing the efficacy and expansion of T cells expressing the CAR receptor.

[0258] Illustrative examples of such costimulatory molecules suitable for use in the anti-CLL-1 VHH CARs encompassed by a particular embodiment include, but are not limited to, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD94, CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), DAP10, LAT, SLP76, TRAT1, TNFR2, and ZAP70. In one embodiment, the CAR comprises one or more costimulatory signaling domains selected from the group consisting of CD28, CD137, and CD134, and a CD3 primary signaling domain.

[0259] In various embodiments, the anti-CLL-1 VHH CAR comprises: a VHH that binds to CLL-1; 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.

[0260] In various embodiments, the anti-CLL-1 VHH CAR comprises: a VHH that binds to CLL-1; 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.

[0261] In a preferred embodiment, the anti-CLL-1 VHH CAR includes a VHH comprising the amino acid sequence shown in SEQ ID NO: 2-13, i.e., any one of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13, a CD8α hinge domain, a CD8α transmembrane domain, a 4-1BB co-stimulatory domain, and a CD3ζ primary signaling domain.

[0262] In a specific embodiment, the anti-CLL-1 VHH CAR comprises the sequence shown in SEQ ID NO: 38-49, i.e., any one of 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, and 49.

[0263] E. Polypeptide

[0264] This document covers various polypeptides, including but not limited to CLL-1 VHH DARIC, CLL-1 VHH DARIC binding components, CLL-1 DARIC signaling components, anti-CLL-1 VHH CARs, and fragments thereof. In a preferred embodiment, the polypeptide comprises a sequence shown in any one of SEQ ID NOs: 2-50. Unless otherwise stated, "polypeptide", "peptide", and "protein" are used interchangeably and are defined, according to their conventional meaning, as an amino acid sequence. In one embodiment, "polypeptide" includes fusion polypeptides and other variants. The polypeptides can be prepared using any of a variety of well-known recombinant and / or synthetic techniques. The polypeptides are not limited to a specific length, for example, they can comprise a full-length protein sequence, a fragment of a full-length protein, or a fusion protein, and can include post-translational modifications of the polypeptide, such as glycosylation, acetylation, phosphorylation, etc., as well as other modifications known in the art, both naturally occurring and non-naturally occurring. In a particular preferred embodiment, the fusion polypeptides, polypeptides, fragments thereof, and other variants are prepared, obtained, or isolated from one or more human polypeptides.

[0265] As used herein, "isolated peptide" or "isolated polypeptide", etc., refers to a peptide or polypeptide molecule that is isolated and / or purified in vitro from the cellular environment and from its association with other components of the cell, i.e., the peptide or polypeptide molecule is not significantly associated with substances in vivo. In a particular embodiment, the isolated polypeptide is a synthetic polypeptide, a semi-synthetic polypeptide, or a polypeptide obtained or derived from a recombinant source.

[0266] Polypeptides include "polypeptide variants". A polypeptide variant can differ from a naturally occurring polypeptide by one or more substitutions, deletions, additions, and / or insertions. Such variants can be naturally occurring or can be generated synthetically, for example, by modifying one or more of the above polypeptide sequences. For example, in a particular embodiment, it may be desirable to improve the binding affinity and / or other biological properties of the polypeptide by introducing one or more substitutions, deletions, additions, and / or insertions into the polypeptide. In a particular embodiment, 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 one of the reference sequences covered herein, typically wherein the variant maintains at least one biological activity of the reference sequence. In a particular embodiment, the biological activity is binding affinity. In a particular embodiment, the biological activity is cytolytic activity.

[0267] Polypeptide variants include bioactive "polypeptide fragments". Illustrative examples of bioactive polypeptide fragments include anti-CLL-1 VHH domains, intracellular signaling domains, etc. As used herein, the term "bioactive fragment" or "minimal bioactive 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 the naturally occurring polypeptide. In certain embodiments, the polypeptide fragment may comprise an amino acid chain that is at least 5 to about 1700 amino acids in length. It should be understood that in certain embodiments, the 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, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700 or more amino acids in length.

[0268] In certain embodiments, the polypeptides listed herein may comprise one or more amino acids represented 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, the 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 any combination of two amino acids. In some embodiments, "XX" represents two serines, i.e., SS. In some embodiments, "XX" represents any combination of two amino acids that reduces immunogenicity.

[0269] In a preferred embodiment, "XX" represents the amino acids KP.

[0270] As described above, polypeptides can be modified in various ways, including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known in the art. For example, variants of the amino acid sequence of a reference polypeptide can be prepared by mutagenesis of the DNA. Methods for mutagenesis and nucleotide sequence alteration 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, J.D. et al. (Molecular Biology of the Gene, Fourth Edition, Benjamin / Cummings, Menlo Park, Calif., 1987) and references cited therein. Guidance regarding appropriate amino acid substitutions that do not affect the biological activity of the protein of interest can be found in the model of Dayhoff et al. (1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, D.C.).

[0271] In certain embodiments, polypeptide variants include one or more conservative substitutions. A "conservative substitution" is a substitution of an amino acid with another amino acid having similar properties such that the secondary structure and hydrophilic nature of the polypeptide are expected to remain substantially unchanged by one of ordinary skill in the art of peptide chemistry. Modifications can be made to the structure of the polynucleotides and polypeptides covered in a particular embodiment and still obtain a functional molecule that encodes a variant or derivative polypeptide having the desired properties. When it is desired to alter the amino acid sequence of a polypeptide to produce an equivalent or even improved variant polypeptide, one of ordinary skill in the art can, for example, alter one or more codons of the encoding DNA sequence, such as according to Table 1.

[0272] Table 1 - Amino Acid Codons

[0273]

[0274] Computer programs well known in the art such as DNASTAR, DNA Strider, Geneious, MacVector or Vector NTI software can be used to find guidelines on determining which amino acid residues can be substituted, inserted or deleted without eliminating biological activity. Preferably, the amino acid changes of the protein variants disclosed herein are conservative amino acid changes, i.e., substitutions of amino acids with similar or no charge. Conservative amino acid changes involve the substitution of one of a group of amino acids with related side chains. Naturally occurring amino acids are generally divided into four groups: acidic amino acids (aspartic acid, glutamic acid), basic amino acids (lysine, arginine, histidine), nonpolar amino acids (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan) and uncharged polar amino acids (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine). Phenylalanine, tryptophan and tyrosine are sometimes grouped together as aromatic amino acids. In a peptide or protein, suitable conservative amino acid substitutions are known to those skilled in the art and can generally be made without altering the biological activity of the resulting molecule. Those skilled in the art will recognize that, generally speaking, a single amino acid substitution in a non-essential region of a polypeptide will substantially not change biological activity (see, for example, Watson et al., Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin / Cummings Pub. Co., p. 224).

[0275] In one embodiment, in the case where it is desired to express two or more polypeptides, as disclosed elsewhere herein, the polynucleotide sequences encoding the two or more polypeptides can be separated by an IRES sequence or a polynucleotide sequence encoding a ribosome skipping sequence.

[0276] The polypeptides covered in certain embodiments include fusion polypeptides. In certain embodiments, fusion polypeptides and polynucleotides encoding the fusion polypeptides are provided. Fusion polypeptides and fusion proteins refer to polypeptides having at least two, three, four, five, six, seven, eight, nine or ten polypeptide segments. In a preferred embodiment, the fusion polypeptide comprises one or more CLL-1 VHH DARIC components. In other preferred embodiments, the fusion polypeptide comprises one or more CLL-1 VHHDARIC.

[0277] In another embodiment, two or more CLL-1 VHH DARIC components and / or other polypeptides can be expressed as a fusion protein that contains one or more self-cleaving peptide sequences between the polypeptides disclosed elsewhere herein.

[0278] In certain embodiments, the fusion polypeptide comprises a CLL-1 DARIC signaling component, a self-cleaving polypeptide sequence or ribosomal skipping sequence, and a CLL-1 VHH DARIC binding component.

[0279] In certain embodiments, the fusion polypeptide comprises a CLL-1 DARIC signaling component, a self-cleaving polypeptide sequence or ribosomal skipping sequence, a CLL-1 VHH DARIC binding component, another self-cleaving polypeptide sequence or ribosomal skipping sequence, and another DARIC binding component directed against another target antigen.

[0280] The fusion polypeptide can include: one or more polypeptide domains or fragments, including but not limited to a signal peptide, a cell-penetrating peptide domain (CPP), a binding domain, a signaling domain, an epitope tag (e.g., maltose binding protein (“MBP”), glutathione S-transferase (GST), HIS6, MYC, FLAG, V5, VSV-G, and HA); a polypeptide linker; and a polypeptide cleavage signal. The fusion polypeptides are typically C-terminally linked to the N-terminus, although 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. The fusion polypeptide or fusion protein can also include conservatively modified variants, polymorphic variants, alleles, mutants, subsequences, and interspecies homologs, so long as the desired activity of the fusion polypeptide is preserved. The fusion polypeptide can be made by chemical synthesis methods or by chemical bonding between the two moieties, or generally can be prepared using other standard techniques. The DNA sequences encoding the linkages making up the fusion polypeptide are operably linked to suitable transcriptional or translational control elements as disclosed elsewhere herein.

[0281] The fusion polypeptide may optionally include one or more linkers that can be used to link one or more polypeptides or domains within the polypeptide. Peptide linker sequences can be used to space any two or more polypeptide components a distance sufficient to ensure that each polypeptide folds into its appropriate secondary and tertiary structure, 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: (1) their ability to assume a flexible extended conformation; (2) their inability to assume a secondary structure that can interact with the functional epitopes on the first and second polypeptides; and (3) the lack of hydrophobic or charged residues that might react with the polypeptide functional epitopes. In certain embodiments, preferred peptide linker sequences contain Gly, Asn, and Ser residues. Other near-neutral amino acids, such as Thr and Ala, can also be used in the linker sequence. Amino acid sequences that can be usefully employed as linkers include those of Maratea et al., Gene 40:39-46, 1985; Murphy et al., Proc. Natl. Acad. Sci. USA 83:8258-8262, 1986; U.S. Patent No. 4,935,233 and U.S. Patent No. 4,751,180. When a particular fusion polypeptide segment contains a non-essential N-terminal amino acid region that can be used to space the functional domains and prevent steric interference, a linker sequence is not required. In certain embodiments, the preferred linker is typically a flexible amino acid subsequence that is synthesized as part of a recombinant fusion protein. The linker polypeptide can be of a length between 1 and 200 amino acids, between 1 and 100 amino acids, or between 1 and 50 amino acids, including all integer values therebetween.

[0282] 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).

[0283] Suitable protease cleavage sites and self-cleaving peptides are known to those skilled in the art (see, e.g., Ryan et al., 1997. J. Gen. Virol. 78, 699-722; Scymczak et al. (2004) Nature Biotech. 5, 589-594). Exemplary protease cleavage sites include but are not limited to potyvirus NIa protease (e.g., tobacco etch virus protease), potyvirus HC protease, potyvirus P1 (P35) protease, byovirus NIa protease, protease encoded by byovirus RNA-2, aphthovirus L protease, enterovirus 2A protease, rhinovirus 2A protease, picornavirus 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 cleavage sites for enterokinase. In one embodiment, the TEV (tobacco etch virus) protease cleavage site is preferred due to its high cleavage stringency, e.g., EXXYXQ(G / S) (SEQ ID NO: 63), e.g., ENLYFQG (SEQ ID NO: 64) and ENLYFQS (SEQ ID NO: 65), where X represents any amino acid (TEV cleavage occurs between Q and G or Q and S).

[0284] In certain embodiments, the polypeptide cleavage signal is a viral self-cleaving peptide or a ribosomal skipping sequence.

[0285] 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 aphthovirus 2A peptide, a potyvirus 2A peptide, or a cardiovirus 2A peptide.

[0286] In one embodiment, the viral 2A peptide is selected from the group consisting of: foot-and-mouth disease virus (FMDV) 2A peptide, equine rhinitis A virus (ERAV) 2A peptide, Taura syndrome virus (TaV) 2A peptide, porcine teschovirus-1 (PTV-1) 2A peptide, Theilovirus 2A peptide, and encephalomyocarditis virus 2A peptide.

[0287] Exemplary examples of 2A sites are provided in Table 2.

[0288] Table 2:

[0289] SEQ ID NO: 66 GSGATNFSLLKQAGDVEENPGP SEQ ID NO: 67 ATNFSLLKQAGDVEENPGP SEQ ID NO: 68 LLKQAGDVEENPGP SEQ ID NO: 69 GSGEGRGSLLTCGDVEENPGP SEQ ID NO: 70 EGRGSLLTCGDVEENPGP SEQ ID NO: 71 LLTCGDVEENPGP SEQ ID NO: 72 GSGQCTNYALLKLAGDVESNPGP SEQ ID NO: 73 QCTNYALLKLAGDVESNPGP SEQ ID NO: 74 LLKLAGDVESNPGP SEQ ID NO: 75 GSGVKQTLNFDLLKLAGDVESNPGP SEQ ID NO: 76 VKQTLNFDLLKLAGDVESNPGP SEQ ID NO: 77 LLKLAGDVESNPGP SEQ ID NO: 78 LLNFDLLKLAGDVESNPGP SEQ ID NO: 79 TLNFDLLKLAGDVESNPGP SEQ ID NO: 80 LLKLAGDVESNPGP SEQ ID NO: 81 NFDLLKLAGDVESNPGP SEQ ID NO: 82 QLLNFDLLKLAGDVESNPGP SEQ ID NO: 83 APVKQTLNFDLLKLAGDVESNPGP SEQ ID NO: 84 VTELLYRMKRAETYCPRPLLAIHPTEARHKQKIVAPVKQT SEQ ID NO: 85 LNFDLLKLAGDVESNPGP SEQ ID NO: 86 LLAIHPTEARHKQKIVAPVKQTLNFDLLKLAGDVESNPGP SEQ ID NO: 87 EARHKQKIVAPVKQTLNFDLLKLAGDVESNPGP

[0290] In a preferred embodiment, the polypeptide or fusion polypeptide comprises one or more CLL-1 VHH DARIC components, CLL-1 VHH DARIC, or anti-CLL-1 VHH CAR.

[0291] In a preferred embodiment, the fusion polypeptide comprises a CLL-1 DARIC signaling component and a CLL-1 VHH DARIC binding component separated by a self-cleaving polypeptide sequence.

[0292] In a specific embodiment, the fusion polypeptide comprises SEQ ID NOs: 20-37, i.e., the sequence shown by any one of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, or 37.

[0293] In a specific embodiment, the fusion polypeptide comprises: a CLL-1 DARIC signaling component, which includes an FRBT2098L 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 CLL-1 VHH DARIC binding component, which includes an anti-CLL-1 VHH, an FKBP12 multimerization domain polypeptide, and a CD4 transmembrane domain.

[0294] In a specific embodiment, the fusion polypeptide comprises SEQ ID NOs: 20-25, i.e., the sequence shown by any one of 20, 21, 22, 23, 24, or 25.

[0295] In a specific embodiment, the fusion polypeptide comprises: a CLL-1 DARIC signaling component, which includes an FRBT2098L 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-CLL-1 VHH, a CD4 transmembrane domain; and optionally a CD27, CD28, TNFRS14, TNFRS18, TNFRS25, OX40, or TNFR2 co-stimulatory domain.

[0296] In a specific embodiment, the fusion polypeptide comprises SEQ ID NOs: 26-31, i.e., the sequence shown by any one of 26, 27, 28, 29, 30, or 31.

[0297] In certain embodiments, the fusion polypeptide comprises SEQ ID NO: 32-37, i.e., the sequence shown in any one of 32, 33, 34, 35, 36, or 37.

[0298] F. Polynucleotide

[0299] In certain embodiments, polynucleotides encoding CLL-1 VHH DARIC, CLL-1 VHH DARIC binding components, CLL-1 DARIC signaling components, anti-CLL-1 VHH CAR, and fragments thereof are provided. As used herein, the terms "polynucleotide" or "nucleic acid" refer to deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and DNA / RNA hybrids. Polynucleotides can be single-stranded or double-stranded and are 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 refers to 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, and all intermediate lengths of nucleotides, i.e., polymeric forms of ribonucleotides or deoxyribonucleotides or modified forms of either type of nucleotide. It is readily understood that, in this context, "intermediate length" 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.

[0300] As used herein, "isolated polynucleotide" refers to a polynucleotide that has been purified from the sequences that flank it in its natural state, e.g., a DNA fragment that has been removed from the sequences that normally flank it. In certain embodiments, "isolated polynucleotide" also refers to complementary DNA (cDNA), recombinant DNA, or other polynucleotides that do not exist in nature and have been made by hand. In certain embodiments, an isolated polynucleotide is a synthetic polynucleotide, a semi-synthetic polynucleotide, or a polynucleotide obtained or derived from recombinant sources.

[0301] In various embodiments, the polynucleotide includes mRNA encoding a polypeptide covered herein. In certain embodiments, the mRNA comprises a cap, one or more nucleotides, and a polyadenylation tail.

[0302] In certain embodiments, the polynucleotide encoding one or more CLL-1 VHH DARIC components can be codon-optimized. As used herein, the term "codon-optimized" refers to substituting codons in a polynucleotide encoding a polypeptide to increase the expression, stability, and / or activity of the polypeptide. Factors affecting 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 a synthetically constructed preference table; (ii) variation in the degree of codon preference within an organism, gene, or set of genes; (iii) systematic variation of codons including environmental; (iv) codon variation based on decoding tRNA; (v) codon variation based on GC% in an overall or triplet position; (vi) variation in similarity to a reference sequence, e.g., a naturally occurring sequence; (vii) variation in codon frequency cut-off; (viii) structural properties of the mRNA transcribed from the DNA sequence; (ix) prior knowledge of the function of the DNA sequence that is the basis for designing a set of codon substitutions; (x) systematic variation of the codon subset for each amino acid; and / or (xi) isolation and removal of pseudo-translation initiation sites.

[0303] As used herein, the term "nucleotide" refers to a heterocyclic nitrogenous base in an N-glycosidic linkage with a phosphorylated sugar. Nucleotides are to be 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 moiety. 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 lacks the hydroxyl group present in ribose.

[0304] Illustrative examples of polynucleotides include, but are not limited to, those encoding the sequences shown in any of SEQ ID NOs: 2-50.

[0305] In various illustrative embodiments, the polynucleotides covered herein include, but are not limited to, polynucleotides encoding one or more CLL-1 VHH DARIC components, CLL-1 VHH DARIC receptors, anti-CLL-1 VHH CARs, fusion polypeptides, as well as expression vectors, viral vectors, and transfer plasmids containing the polynucleotides covered herein.

[0306] As used herein, terms such as "polynucleotide variant" and "variant" refer to polynucleotides that exhibit a substantial degree of sequence identity with a reference polynucleotide sequence or polynucleotides that hybridize to the reference sequence under the stringent conditions defined below. These terms also encompass polynucleotides that differ from the 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 alterations, including mutations, additions, deletions, and substitutions, can be made to a reference polynucleotide such that the altered polynucleotide retains the biological function or activity of the reference polynucleotide.

[0307] As used herein, the recitation "sequence identity" or, for example, "a sequence that is 50% identical to" refers to the degree of identity of a sequence on a nucleotide-by-nucleotide basis or on an amino acid-by-amino acid basis within a comparison window. Thus, the "percent sequence identity" can be calculated by comparing two optimally aligned sequences within the comparison window, determining the number of positions at which the 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, Ash, 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., window size), and multiplying the result by 100 to obtain the percent sequence identity. 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 one of the reference sequences described herein.

[0308] 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, polylinker sites, internal ribosome entry sites (IRESs), 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, such that the total length of the polynucleotide can vary significantly. Thus, polynucleotide fragments of almost any length are encompassed, the total length of which is preferably limited by ease of preparation and use in the intended recombinant DNA protocol.

[0309] The polynucleotides can be prepared, manipulated, expressed, and / or delivered using any of a variety of well-known and available techniques in the art. To express the desired polypeptide, the nucleotide sequence encoding the polypeptide can be inserted into an appropriate vector.

[0310] Exemplary instances of vectors include, but are not limited to, plasmids, autonomously replicating sequences, and transposable elements such as Sleeping Beauty, PiggyBac.

[0311] Additional illustrative instances 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 λ phage or M13 phage, and animal viruses.

[0312] Exemplary instances of viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and polyomaviruses (e.g., SV40).

[0313] Exemplary instances of expression vectors include, but are not limited to, the pClneo vector (Promega) for expression in mammalian cells; pLenti4 / V5-DEST for lentivirus-mediated gene transfer and expression in mammalian cells TM and pLenti6 / V5-DEST TM and pLenti6.2 / V5-GW / lacZ (Invitrogen). In certain embodiments, the coding sequence of the polypeptides disclosed herein can be ligated into such expression vectors to express the polypeptides in mammalian cells.

[0314] In certain embodiments, the vector is an episomal vector or a vector maintained episomally. As used herein, the term "episomal" refers to a vector that is capable of replicating without integrating into the chromosomal DNA of the host and without being gradually lost from dividing host cells, which also means that the vector replicates extrachromosomally or episomally.

[0315] "Expression control sequences", "control elements", or "regulatory sequences" present in an expression vector are those non-translated regions of the vector, including the origin of replication, selection cassette, promoter, enhancer, translation initiation signals (Shine Dalgarno sequence or Kozak sequence), introns, polyadenylation sequences, 5' and 3' untranslated regions, which interact with host cell proteins for transcription and translation. These elements may vary in their strength and specificity. Depending on the vector system and host utilized, a variety of suitable transcriptional and translational elements may be used, including ubiquitous promoters and inducible promoters.

[0316] In certain embodiments, the polynucleotide includes a vector, which includes but is not limited to an expression vector and a viral vector. The vector may include one or more exogenous, endogenous, or heterologous control sequences, such as a promoter and / or enhancer. An "endogenous control sequence" is a sequence that is naturally linked to a given gene in the genome. An "exogenous control sequence" is a sequence that is placed in juxtaposition with a gene by genetic manipulation (i.e., molecular biology techniques) such that transcription of this gene is directed by the linked enhancer / promoter. A "heterologous control sequence" is an exogenous sequence from a different species than the cell in which the gene is being manipulated. A "synthetic" control sequence may include one or more endogenous and / or exogenous sequences and / or elements of sequences determined in vitro or in silico to provide optimal promoter and / or enhancer activity for a particular therapy.

[0317] As used herein, the term "promoter" refers to an identification site of a polynucleotide (DNA or RNA) to which RNA polymerase binds. RNA polymerase initiates and transcribes the polynucleotide operably linked to the promoter. In certain embodiments, promoters that function in mammalian cells include an AT-rich region located approximately 25 to 30 bases upstream of the transcription start site and / or another sequence found 70 to 80 bases upstream of the transcription start, i.e., the CNCAAT region where N can be any nucleotide.

[0318] The term "enhancer" refers to a segment of DNA that contains sequences capable of providing enhanced transcription and in some cases can function independently of its orientation relative to another control sequence. Enhancers can act synergistically or additively with promoters and / or other enhancer elements. The term "promoter / enhancer" refers to a segment of DNA that contains sequences capable of providing the functions of both a promoter and an enhancer.

[0319] The term "operably linked" refers to an association of the described components that permits them to function in their intended manner. In one embodiment, the term refers to a functional linkage between a nucleic acid expression control sequence, such as a promoter and / or enhancer, and a second polynucleotide sequence, e.g., a polynucleotide of interest, wherein the expression control sequence directs transcription of a nucleic acid corresponding to the second sequence.

[0320] As used herein, the term "constitutive expression control sequence" refers to a promoter, enhancer, or promoter / enhancer that continuously or constantly 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 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.

[0321] Exemplary ubiquitous expression control sequences suitable for use in a particular embodiment 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-α (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 β member 1 (HSP90B1), heat shock protein 70 kDa (HSP70), β-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 replaced with dl587rev primer binding site (MND) U3 promoter (Haas et al., Journal of Virology. 2003;77(17):9439-9450).

[0322] In one embodiment, the vector includes the MND U3 promoter.

[0323] In one embodiment, the vector comprises an EF1a promoter comprising the first intron of the human EF1a gene.

[0324] In one embodiment, the vector comprises an EF1a promoter lacking the first intron of the human EF1a gene.

[0325] In certain embodiments, it may be desirable to use cell-, cell type-, cell lineage-, or tissue-specific expression control sequences to effect 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 cell types, cell lineages, or tissues or during a particular stage of development).

[0326] In certain embodiments, it may be desirable to express a polynucleotide as a T cell-specific promoter.

[0327] 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 particular physiological, biological, or disease state, etc. This definition is not intended to exclude cell type- or tissue-specific expression. Certain embodiments provide for conditional expression of a polynucleotide of interest, e.g., by subjecting a cell, tissue, organism, etc. to a treatment or condition that results in expression of the polynucleotide or in an increase or decrease in the expression of a polypeptide encoded by the polynucleotide of interest.

[0328] Exemplary examples of inducible promoter / systems include but are not limited to: steroid-inducible promoters such as the promoters of genes encoding glucocorticoid or estrogen receptors (inducible by treatment with the corresponding hormone), 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), the cumate-inducible gene switch (WO 2002 / 088346), the tetracycline-dependent regulatory system, etc. Inducers include but are not limited to glucocorticoids, estrogens, mifepristone (RU486), metals, interferons, small molecules, cumate, tetracycline, doxycycline, and variants thereof.

[0329] As used herein, "internal ribosome entry site" or "IRES" refers to an element that promotes direct entry of ribosomes into a cistron (protein-coding region) at an initiation codon such as ATG, thereby resulting in cap-independent translation of a gene. See, e.g., Jackson et al., 1990. Trends Biochem Sci 15(12):477-83) and Jackson and Kaminski. 1995. RNA 1(10):985-1000. Examples of IRESs commonly employed by those of ordinary skill in the art include those described in U.S. Patent No. 6,692,736. Other examples of "IRESs" known in the art include, but are not limited to, IRESs obtainable from picornaviruses (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 (IGFII), translation initiation factor eIF4G, and yeast transcription factors TFIID and HAP4, the encephalomyocarditis virus (EMCV) commercially available from Novagen (Duke et al., 1992. J. Virol 66(3):1602-9), and the VEGF IRES (Huez et al., 1998. Mol Cell Biol 18(11):6178-90). IRESs have been reported in the viral genomes of species of the Picornaviridae, Dicistroviridae, and Flaviviridae families, as well as in HCV, Friend murine leukemia virus (FrMLV), and Moloney murine leukemia virus (MoMLV).

[0330] In one embodiment, the IRES used in the polynucleotides encompassed herein is the EMCV IRES.

[0331] 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 facilitates the initial binding of mRNA to the small subunit of the ribosome and increases translation. The consensus Kozak sequence is (GCC)RCCATGG (SEQ ID NO: 51), 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).

[0332] Elements that direct efficient termination and polyadenylation of heterologous nucleic acid transcripts will increase expression of the heterologous gene. Transcription termination signals are generally found downstream of the polyadenylation signal. In certain 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 "polyAsite" or "polyA sequence" refers to a DNA sequence that directs termination and polyadenylation of the primary RNA transcript by RNA polymerase II. The polyadenylation sequence can promote mRNA stability by adding a polyA tail to the 3' end of the coding sequence and thus contribute to increased translation efficiency. Cleavage and polyadenylation are directed by the polyadenylation sequence in the RNA. The core polyadenylation sequence of mammalian mRNA precursors (pre-mRNAs) flanks the cleavage-polyadenylation site with two recognition elements. Typically, the nearly invariant AAUAAA hexamer is located 20-50 nucleotides upstream of a more variable element rich in U or GU residues. Cleavage of the primary transcript occurs between these two elements and is coupled to add up to 250 adenosines to the 5' cleavage product. In certain embodiments, the core polyadenylation sequence is the desired polyadenylation sequence (e.g., AATAAA, ATTAAA, AGTAAA). In certain embodiments, the polyadenylation sequence is the SV40 polyadenylation sequence, the bovine growth hormone polyadenylation sequence (BGHpA), the rabbit β-globin polyadenylation sequence (rβgpA), variants thereof, or another suitable heterologous or endogenous polyadenylation sequence known in the art. In certain embodiments, the polyadenylation sequence is synthetic.

[0333] In certain embodiments, the polynucleotide 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 certain embodiments, delivery of one or more polynucleotides can be provided by the same method or by different methods and / or by the same vector or by different vectors.

[0334] The term "vector" as used herein refers to a nucleic acid molecule capable of transferring or delivering another nucleic acid molecule. The nucleic acid being transferred is generally linked to the vector nucleic acid molecule, for example inserted into the vector nucleic acid molecule. A vector can include sequences that direct autonomous replication in a cell, or can include sequences sufficient to allow integration into the host cell DNA. In certain embodiments, non-viral vectors are used to deliver one or more polynucleotides encompassed herein into T cells.

[0335] Exemplary instances of non-viral vectors include, but are not limited to, plasmids (e.g., DNA plasmids or RNA plasmids), transposons, cosmids, and bacterial artificial chromosomes.

[0336] Exemplary methods for non-viral delivery of polynucleotides encompassed in certain embodiments include, but are not limited to: electroporation, sonoporation, liposome transfection, microinjection, gene gun, virus-like particles, liposomes, immunoliposomes, nanoparticles, polycations or lipid:nucleic acid conjugates, naked DNA, artificial virus-like particles, DEAE-dextran-mediated transfer, gene gun, and heat shock.

[0337] Illustrative examples of polynucleotide delivery systems suitable for use in certain embodiments encompassed herein include, but are not limited to, those provided by Amaxa Biosystems, Maxcyte, Inc., BTX Molecular Delivery Systems, and Copernicus Therapeutics Inc. Lipid transfection reagents are commercially available (e.g., Transfectam TM and Lipofectin TM ). Cationic and neutral lipids suitable for efficient receptor recognition lipid transfection of polynucleotides have been described in the literature. See, e.g., Liu et al. (2003) Gene Therapy. 10:180-187; and Balazs et al. (2011) Journal of Drug Delivery. 2011:1-12. Antibody-targeted, bacterially-derived, inanimate nanocell-based delivery is also encompassed in certain embodiments.

[0338] Viral vectors containing polynucleotides encompassed in certain embodiments can be delivered by administration to an individual patient, typically by systemic administration (e.g., intravenous, intraperitoneal, intramuscular, subcutaneous, or intracranial infusion) or local 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, and then the cells are re-implanted into the patient.

[0339] In one embodiment, a viral vector comprising a polynucleotide covered herein is directly administered to an organism to transduce cells in vivo. Alternatively, naked DNA can be administered. Administration is via any route commonly used to bring a molecule into eventual contact with blood or tissue cells, including but not limited to injection, infusion, topical administration, and electroporation. Suitable methods of administering such nucleic acids are available to and well known to those skilled in the art, and although more than one route can be used to administer a particular composition, a particular route will generally provide a more direct and more effective response than another route.

[0340] Illustrative examples of viral vector systems suitable for use in the particular embodiments covered in a particular embodiment include but are not limited to adeno-associated virus (AAV), retrovirus, herpes simplex virus, adenovirus, and vaccinia virus vectors.

[0341] In various embodiments, one or more polynucleotides encoding one or more CLL-1 VHH DARIC components and / or other polypeptides covered herein are introduced into immune effector cells, such as T cells, by transducing the cells with a recombinant adeno-associated virus (rAAV) comprising one or more polynucleotides.

[0342] AAV is a small (about 26 nm) replication-defective, mainly episomal, non-enveloped virus. AAV can infect both dividing and non-dividing cells and can integrate its genome into the genome of the host cell. Recombinant AAV (rAAV) generally consists of at least a transgene and its regulatory sequences and 5' and 3' AAV inverted terminal repeats (ITRs). The ITR sequences are about 145 bp in length. In certain embodiments, the rAAV includes ITR and capsid sequences isolated from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10.

[0343] In some embodiments, chimeric rAAV is used, with the ITR sequence isolated from one AAV serotype and the capsid sequence isolated from a different AAV serotype. For example, an rAAV having an ITR sequence derived from AAV2 and a capsid sequence derived from AAV6 is referred to as AAV2 / AAV6. In certain embodiments, the rAAV vector can include the ITR from AAV2 and a capsid protein from any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10. In a preferred embodiment, the rAAV includes an ITR sequence derived from AAV2 and a capsid sequence derived from AAV6. In a preferred embodiment, the rAAV includes an ITR sequence derived from AAV2 and a capsid sequence derived from AAV2.

[0344] In some embodiments, engineering and selection methods can be applied to AAV capsids to make them more likely to transduce cells of interest.

[0345] 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.

[0346] In various embodiments, one or more polynucleotides encoding one or more CLL-1 VHH DARIC components and / or other polypeptides covered herein are introduced into immune effector cells, such as T cells, by transducing the cells with a retrovirus, such as a lentivirus, comprising one or more polynucleotides.

[0347] As used herein, the term "retrovirus" refers to an RNA virus that reverse transcribes its genomic RNA into a linear double-stranded DNA copy and then covalently integrates its genomic DNA into the host genome. Exemplary retroviruses suitable for particular embodiments include, but are not limited to: Moloney murine leukemia virus (M-MuLV), Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), gibbon ape leukemia virus (GaLV), feline leukemia virus (FLV), foamy virus, Friend murine leukemia virus, murine stem cell virus (MSCV), and Rous sarcoma virus (RSV), as well as lentiviruses.

[0348] As used herein, the term "lentivirus" refers to a group (or genus) of complex retroviruses. Illustrative lentiviruses include, but are not limited to: HIV (human immunodeficiency virus; including HIV type 1 and HIV type 2); visna-maedi virus (VMV); 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.

[0349] In various embodiments, the lentiviral vectors covered herein include one or more LTRs, and one or more or all of the following accessory elements: cPPT / FLAP, Psi (Ψ) packaging signal, export element, poly(A) sequence, and may optionally include WPRE or HPRE, insulator elements, selectable markers, and cell suicide genes, as discussed elsewhere herein.

[0350] In certain embodiments, the lentiviral vectors encompassed herein can be integrative or non-integrative or integration-defective lentiviruses. As used herein, the term "integration-defective lentivirus" or "IDLV" refers to a lentivirus having an integrase that lacks the ability to integrate the viral genome into the genome of a host cell. Viral vectors lacking integrative capacity have been described in patent application WO 2006 / 010834, which is incorporated herein by reference in its entirety.

[0351] Exemplary mutations in the HIV-1 pol gene that are suitable for reducing integrase activity include, but are not limited to: H12N, H12C, H16C, H16V, S81R, D41A, K42A, H51A, Q53C, D55V, D64E, D64V, E69A, K71A, E85A, E87A, D116N, D116I, D116A, N120G, N120I, N120E, E152G, E152A, D35E, K156E, K156A, E157A, K159E, K159A, K160A, R166A, D167A, E170A, H171A, K173A, K186Q, K186T, K188T, E198A, R199C, R199T, R199A, D202A, K211A, Q214L, Q216L, Q221L, W235F, W235E, K236S, K236A, K246A, G247W, D253A, R262A, R263A, and K264H.

[0352] The term "long terminal repeat (LTR)" refers to a domain of base pairs located at the ends of retroviral DNA that is a direct repeat in its native sequence context and contains U3, R, and U5 regions.

[0353] As used herein, the term "FLAP element" or "cPPT / FLAP" refers to a nucleic acid sequence containing the central polypurine tract and central termination sequence (cPPT and CTS) of a retrovirus (e.g., HIV-1 and HIV-2). Suitable FLAP elements are described in U.S. Patent No. 6,682,907 and Zennou et al., 2000, Cell, 101:173.

[0354] As used herein, the term "packaging signal" or "packaging sequence" refers to the psi [Ψ] sequence located within the retroviral genome, which 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.

[0355] The term "export element" refers to a cis - acting post - transcriptional regulatory element that modulates the transport of RNA transcripts from the cell nucleus to the cytoplasm. 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).

[0356] In certain embodiments, expression of a heterologous sequence in a viral vector is increased by incorporating a post - transcriptional regulatory element, an efficient polyadenylation site, and optionally a transcription termination signal into the vector. A variety of post - transcriptional regulatory elements can increase the expression of heterologous nucleic acids in 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); etc. (Liu et al., 1995, Genes Dev., 9:1766).

[0357] Due to modification of the LTR, lentiviral vectors preferably contain several safety enhancements. A "self - inactivating" (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, called the U3 region, has been modified (e.g., by deletion or substitution) to prevent viral transcription beyond the first round of viral replication. Self - inactivation is preferably achieved by introducing a deletion in the U3 region of the 3' LTR of the vector DNA (i.e., the DNA used to generate vector RNA). Thus, during reverse transcription, this deletion is transferred to the 5' LTR of the proviral DNA. In certain embodiments, it is desirable to eliminate sufficient U3 sequence to greatly reduce or completely eliminate the transcriptional activity of the LTR, thereby greatly reducing or eliminating the production of full - length vector RNA in transduced cells. In the case of HIV - based lentiviral vectors, it has been found that such vectors tolerate significant U3 deletions, including removal of the LTR TATA box (e.g., a deletion from - 418 to - 18), without a significant reduction in vector titer.

[0358] Additional safety enhancements are 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, the 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.

[0359] As used herein, the term "pseudotyped" or "pseudotyping" refers to a virus in which the viral envelope proteins have been replaced with viral envelope proteins of another virus having 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.

[0360] In certain embodiments, lentiviral vectors are produced 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.

[0361] 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 should be understood that many different sources of retroviral and / or lentiviral sequences can be used or combined, or that various substitutions and alterations of certain lentiviral sequences within the lentiviral sequences can be tolerated without impairing 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, 5,994,136, many of which can be adapted to produce the viral vectors or transfer plasmids encompassed herein.

[0362] In various embodiments, one or more polynucleotides encoding one or more CLL-1 VHH DARIC components and / or other polypeptides encompassed herein are introduced into immune effector cells by transducing the cells with an adenovirus comprising one or more polynucleotides.

[0363] Adenovirus-based vectors are capable of extremely high transduction efficiency in many cell types and do not require cell division. Using such vectors, high titers and high expression levels have been obtained. This vector can be produced in large quantities in a relatively simple system. Most adenoviral vectors are engineered such that the transgene replaces the AdE1a, E1b, and / or E3 genes; subsequently, replication-deficient vectors are propagated in human 293 cells that provide the missing gene functions in trans. Ad vectors can transduce a variety of types of tissues in vivo, including non-dividing differentiated cells found in, for example, the liver, kidney, and muscle. Conventional Ad vectors have a large carrying capacity.

[0364] The production and propagation of current adenoviral vectors with replication defects can utilize a unique helper cell line named 293, which is transformed from human embryonic kidney cells by Ad5 DNA fragments and constitutively expresses the E1 protein (Graham et al., 1977). Since the E3 region can be deleted from the adenoviral genome (Jones and Shenk, 1978), current adenoviral vectors carry foreign DNA in the E1, E3 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 adenoviruses to different tissues include intratracheal instillation (Rosenfeld et al., 1991; Rosenfeld et al., 1992), intramuscular injection (Ragot et al., 1993), intravenous injection (Herz & Gerard, 1993), and stereotactic intracerebral inoculation (Le Gal La Salle et al., 1993). Examples of the use of Ad vectors in clinical trials are described in a polynucleotide therapy for anti-tumor immunity associated with intramuscular injection (Sterman et al., Hum. Gene Ther. 7: 1083-9 (1998)).

[0365] In various embodiments, one or more polynucleotides encoding one or more CLL-1 VHH DARIC components and / or other polypeptides encompassed herein are introduced into immune effector cells by transducing the cells with a herpes simplex virus, such as HSV-1, HSV-2, comprising one or more polynucleotides.

[0366] Mature HSV virions consist of an enveloped icosahedral capsid, wherein the viral genome consists of a 152 kb linear double-stranded DNA molecule. In one embodiment, the HSV-based viral vector lacks one or more essential or non-essential HSV genes. In one embodiment, the HSV-based viral vector is replication-deficient. Most replication-deficient HSV vectors contain deletions to remove one or more immediate-early, early, or late HSV genes to prevent replication. For example, the HSV vector may lack immediate-early genes selected from the group consisting of ICP4, ICP22, ICP27, ICP47, and combinations thereof. Advantages of HSV vectors are their ability to enter a latent state that can result in long-term DNA expression, and their large viral DNA genome that can accommodate exogenous DNA inserts of up to 25 kb. 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.

[0367] G. Genetically modified cells

[0368] In various embodiments, cells are modified to express a CLL-1 VHH DARIC, one or more CLL-1 VHH DARIC components, an anti-CLL-1 VHH CAR, and / or a fusion protein as covered herein for the treatment of cancer. Cells can be non-genetically modified to express one or more of the polypeptides covered herein, or in certain preferred embodiments, cells can be genetically modified to express one or more of the polypeptides covered herein. As used herein, the terms "genetically engineered" or "genetically modified" refer to the addition of extra genetic material in the form of DNA or RNA to the total genetic material in a cell. In certain embodiments, the terms "genetically modified cell", "modified cell", and "redirected cell" are used interchangeably.

[0369] In certain embodiments, one or more CLL-1 VHH DARIC components or anti-CLL-1 VHH CARs covered herein are introduced into and expressed in immune effector cells to improve the efficacy of the immune effector cells.

[0370] 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 covered herein are T lymphocytes, including but not limited to cytotoxic T cells (CTL; CD8+ T cells), TIL, and helper T cells (HTL; CD4 + cells). 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 allogeneic ("non-self", e.g., allogeneic, syngeneic, or xenogeneic).

[0371] 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 different subjects that are genetically identical to the comparison cells. As used herein, "xenogeneic" refers to cells from a different species than the comparison cells. In a preferred embodiment, the cells are human autologous immune effector cells.

[0372] Exemplary immune effector cells suitable for introduction of one or more CLL-1 VHH DARIC components or anti-CLL-1 VHH CARs encompassed herein include T lymphocytes. The terms "T cell" or "T lymphocyte" are well recognized in the art and are intended to include thymocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes. T cells can be T helper (Th) cells, such as T helper 1 (Th1) or T helper 2 (Th2) cells. T cells can be helper T cells (HTL; CD4 + T cells), cytotoxic T cells (CTL; CD8 + T cells), CD4 + CD8 + T cells, CD4 - CD8 - T cells, or any other T cell subset. Other exemplary T cell populations suitable for certain embodiments include naive T cells and memory T cells.

[0373] As will be understood by those skilled in the art, other cells can also be used as immune effector cells comprising one or more CLL-1 VHH DARIC components or anti-CLL-1 VHH CARs covered herein. In certain embodiments, the immune effector cells further comprise NK cells, NKT cells, neutrophils, and macrophages. The immune effector cells further comprise progenitors of effector cells, wherein such progenitors can be induced to differentiate into immune effector cells in vivo or in vitro. Thus, in certain embodiments, the immune effector cells comprise progenitors of immune effector cells, such as hematopoietic stem cells (HSCs) contained within a population of CD34+ cells derived from umbilical cord blood, bone marrow, or mobilized peripheral blood, which differentiate into mature immune effector cells after administration to a subject, or the HSCs can be induced to differentiate into mature immune effector cells in vitro.

[0374] As used herein, the term "CD34+ cell" refers to a cell that expresses the CD34 protein on its cell surface. As used herein, "CD34" refers to a cell surface glycoprotein (e.g., sialomucin) that generally acts as a cell-cell adhesion factor and is involved in the entry of T cells into lymph nodes. A population of CD34+ cells contains hematopoietic stem cells (HSCs) that, when administered to a patient, differentiate and contribute to all hematopoietic lineages, including cells of the T cell, NK cell, NKT cell, neutrophil, and monocyte / macrophage lineages.

[0375] In certain embodiments, provided are methods for preparing immune effector cells expressing one or more CLL-1 VHHDARIC components or anti-CLL-1 VHH CARs covered herein. In one embodiment, the method comprises transfecting or transducing immune effector cells isolated from an individual such that the immune effector cells have one or more nucleic acids and / or vectors, such as a lentiviral vector comprising a nucleic acid encoding one or more CLL-1 VHH DARIC components or anti-CLL-1 VHH CARs covered herein. In one embodiment, the method comprises transfecting or transducing immune effector cells isolated from an individual such that the immune effector cells express one or more CLL-1 VHH DARIC components or anti-CLL-1 VHH CARs covered herein. In certain embodiments, the immune effector cells are isolated from an individual and genetically modified without further manipulation in vitro. Then, such cells can be directly administered again to the individual. In additional embodiments, the immune effector cells are first activated and stimulated to proliferate in vitro prior to genetic modification. In this regard, the immune effector cells can be cultured before and / or after genetic modification.

[0376] In certain embodiments, a cell source is obtained from a subject prior to in vitro manipulation or genetic modification of the immune effector cells described herein. In certain embodiments, the modified immune effector cells include T cells.

[0377] 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 an infected site, ascites, pleural effusion, spleen tissue, and tumors. In some embodiments, any number of techniques known to those of skill in the art, such as sedimentation (e.g., FICOLL TM separation), can be used to obtain T cells from a unit of blood collected from a subject.

[0378] In other embodiments, isolated or purified populations of T cells are used. In some embodiments, after isolation of PBMCs, cytotoxic and helper T lymphocytes can be sorted into naive, memory, and effector T cell subsets before or after activation, expansion, and / or genetic modification.

[0379] In one embodiment, the isolated or purified population of T cells expresses one or more of the markers including but not limited to: CD3 + 、CD4 + 、CD8 + or combinations thereof.

[0380] In certain embodiments, T cells are isolated from an individual and first activated and stimulated in vitro to proliferate prior to being modified to express one or more CLL-1 VHH DARIC components or an anti-CLL-1 VHH CAR.

[0381] To achieve a therapeutically sufficient dose of the T cell composition, the T cells are typically stimulated, activated, and / or expanded in one or more rounds. In certain embodiments, the T cells can be activated and expanded using methods such as those described in, for example, the following U.S. patents: U.S. Pat. No. 6,352,694; U.S. Pat. No. 6,534,055; U.S. Pat. No. 6,905,680; U.S. Pat. No. 6,692,964; U.S. Pat. No. 5,858,358; U.S. Pat. No. 6,887,466; U.S. Pat. No. 6,905,681; U.S. Pat. No. 7,144,575; U.S. Pat. No. 7,067,318; U.S. Pat. No. 7,172,869; U.S. Pat. No. 7,232,566; U.S. Pat. No. 7,175,843; U.S. Pat. No. 5,883,223; U.S. Pat. No. 6,905,874; U.S. Pat. No. 6,797,514; and U.S. Pat. No. 6,867,041, each of which is incorporated herein by reference in its entirety. In certain embodiments, the T cells are activated and expanded for about 6 hours, about 12 hours, about 18 hours, or about 24 hours after introduction of a vector or polynucleotide encoding one or more CLL-1 VHH DARIC components or anti-CLL-1 VHH CARs encompassed herein.

[0382] H. Compositions and Formulations

[0383] The compositions encompassed herein can include one or more CLL-1 VHH DARIC components or anti-CLL-1 VHHCARs, polynucleotides encoding one or more CLL-1 VHH DARIC components or anti-CLL-1 VHH CARs, vectors containing them, 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 a cell or an animal, either alone or in combination with one or more other therapeutic modalities. It should also be understood that, if desired, the compositions can also be administered in combination with other agents, such as cytokines, growth factors, hormones, small molecules, chemotherapeutic agents, prodrugs, drugs, antibodies, or various other pharmaceutically active agents. There are virtually no limitations on the other components that can be included in the compositions, provided that the additional agents do not adversely affect the ability of the composition to deliver the intended therapy.

[0384] The phrase "pharmaceutically acceptable" is used herein to refer to those compounds, materials, compositions, and / or dosage forms that, within the scope of sound medical judgment, are suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other problems or complications and are commensurate with a reasonable benefit / risk ratio.

[0385] The term "pharmaceutically acceptable carrier" refers to a diluent, adjuvant, excipient, or vehicle for the administration of a bridging factor, polypeptide, polynucleotide, a vector comprising the same, or a genetically modified immune effector cell. Illustrative examples of pharmaceutical carriers can be sterile liquids such as cell culture media, water, and oils, including oils of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. A saline solution and aqueous dextrose as well as glycerol solutions can also be used as liquid carriers, especially for injectable solutions. In certain embodiments, suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene, ethylene glycol, water, ethanol, and the like. Their use in pharmaceutical compositions is covered except in cases where any conventional media or agents are incompatible with the active ingredient. Supplementary active ingredients can also be incorporated into the compositions.

[0386] In one embodiment, a composition comprising a pharmaceutically acceptable carrier is suitable for administration to a subject. In certain embodiments, a composition comprising a carrier is suitable for parenteral administration, such as intravascular (intravenous or intraarterial), intraperitoneal, or intramuscular administration. In certain embodiments, a composition comprising a pharmaceutically acceptable carrier is suitable for intraventricular, intraspinal, or intrathecal administration. Pharmaceutically acceptable carriers include sterile aqueous solutions, cell culture media, or dispersions. The use of such media and agents for pharmaceutical active substances is well known in the art. Their use in pharmaceutical compositions is covered except in cases where any conventional media or agents are incompatible with a bridging factor, polypeptide, polynucleotide, a vector comprising the same, or a genetically modified immune effector cell.

[0387] In certain embodiments, the compositions covered herein include genetically modified T cells and a pharmaceutically acceptable carrier. Compositions comprising cell-based compositions covered herein can be administered, either alone or in combination with other suitable compounds, by enteral or parenteral administration methods to achieve the desired therapeutic effect.

[0388] In certain embodiments, the compositions covered herein include a bridging factor and a pharmaceutically acceptable carrier.

[0389] A pharmaceutically acceptable carrier must have a sufficiently high purity and a sufficiently low toxicity to render it suitable for administration to the human subject being treated. The pharmaceutically acceptable carrier should maintain or increase the stability of the composition. The pharmaceutically acceptable carrier can be liquid or solid and is selected, in the context of covering the intended mode of administration, to provide the desired volume, identity, etc. when combined with the other components of the composition. For example, the pharmaceutically acceptable carrier can be, but is not limited to, binders (e.g., pregelatinized corn starch, polyvinylpyrrolidone, or hydroxypropylmethylcellulose, etc.), fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethylcellulose, polyacrylate, calcium hydrogen phosphate, etc.), lubricants (e.g., magnesium stearate, talc, silica, colloidal silica, stearic acid, metal stearates, hydrogenated vegetable oil, corn starch, polyethylene glycol, sodium benzoate, sodium acetate, etc.), disintegrants (e.g., starch, sodium carboxymethyl starch, etc.) or wetting agents (e.g., sodium lauryl sulfate, etc.). Other suitable pharmaceutically acceptable carriers for the compositions covered herein include, but are not limited to, water, salt solutions, alcohols, polyethylene glycol, gelatin, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone, and the like.

[0390] Such carrier solutions can also contain buffers, diluents, and other suitable additives. As used herein, the term "buffer" refers to a solution or liquid whose chemical constituents neutralize an acid or a base without significantly changing the pH. Examples of buffers covered herein include, but are not limited to, Dulbecco’s phosphate buffered saline (PBS), Ringer’s solution, 5% aqueous glucose solution (D5W), normal / physiologic saline (0.9% NaCl).

[0391] The amount in which the pharmaceutically acceptable carrier can be present is 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, such as 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.

[0392] The compositions covered herein can include a non-toxic pharmaceutically acceptable culture medium. The composition can be a suspension. As used herein, the term "suspension" refers to a non-adherent condition in which the cells are not attached to a solid support. For example, cells maintained as a suspension can be stirred or agitated and the cells are not adhered to a support, such as a culture dish.

[0393] In certain embodiments, the compositions encompassed herein are formulated in suspension, wherein the modified T cells are dispersed within an acceptable liquid medium or solution, such as saline or serum-free medium, in an intravenous (IV) bag or the like. Acceptable diluents include, but are not limited to, water, PlasmaLyte, Ringer's solution, isotonic sodium chloride (saline) solution, serum-free cell culture medium, and media suitable for cryopreservation, such as media.

[0394] In some embodiments, the pharmaceutically acceptable carrier is substantially free of natural proteins of human or animal origin and is suitable for storing a composition comprising a modified T cell population. The pharmaceutical composition is intended for administration to a human patient and is thus substantially free of cell culture components such as bovine serum albumin, horse serum, and fetal bovine serum.

[0395] In some embodiments, the composition is formulated in a pharmaceutically acceptable cell culture medium. Such compositions are suitable for administration to a human subject. In certain embodiments, the pharmaceutically acceptable cell culture medium is a serum-free medium.

[0396] Serum-free media have several advantages over media containing serum, including a simplified and better-defined composition, reduced levels of contaminants, elimination of potential sources of infectious agents, and cost reduction. In various embodiments, the serum-free medium is animal-free and may optionally be protein-free. Optionally, the medium may contain a biopharmaceutically acceptable recombinant protein. An “animal-free” medium refers to a medium derived from non-animal sources. Recombinant proteins replace natural animal proteins in the animal-free medium, and nutrients are obtained from synthetic, plant, or microbial sources. In contrast, a “protein-free” medium is defined as being substantially free of proteins.

[0397] Illustrative examples of serum-free media used in certain embodiments include, but are not limited to, QBSF-60 (Quality Biological, Inc.), StemPro-34 (Life Technologies), and X-VIVO 10.

[0398] In one embodiment, the composition comprising modified T cells is formulated in PlasmaLyte.

[0399] In various embodiments, the composition comprising modified T cells is 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 media used in certain embodiments include, but are not limited to, CryoStor CS10, CryoStor CS5, and CryoStor CS2.

[0400] In one embodiment, the composition is formulated in a solution comprising 50:50 PlasmaLyte A: CryoStor CS10.

[0401] In certain embodiments, the composition is substantially free of mycoplasma, endotoxin, and microbial contamination. With respect to endotoxin, "substantially free" means that the endotoxin content per dose of cells is less than the amount permitted by the FDA for biologic agents, which is a total endotoxin of 5 EU / kg body weight per day, or 350 EU per total dose of cells for an average 70 kg human. In certain embodiments, the compositions encompassed 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.

[0402] 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 using the specific compositions described herein in a variety of therapeutic regimens, including, for example, enteral and parenteral, such as intravascular, intravenous, intraarterial, intraosseous, intraventricular, intracerebral, intracranial, intraspinal, intrathecal, and intramedullary administration and formulation. Those skilled in the art will understand that the specific embodiments encompassed herein may include other formulations, such as those well known in the pharmaceutical art and described, for example, in Remington: The Science and Practice of Pharmacy, Volumes I and II, 22nd Edition, edited by Loyd V. Allen, Jr., Philadelphia, PA: Pharmaceutical Press; 2012, each of the above U.S. patents being incorporated herein by reference in its entirety.

[0403] In certain embodiments, the composition comprises an amount of immune effector cells that comprise a polynucleotide encoding one or more CLL-1 VHH DARIC moieties or anti-CLL-1 VHH CARs as encompassed herein. In certain embodiments, the composition comprises an amount of immune effector cells that express one or more CLL-1 VHH DARIC moieties or anti-CLL-1 VHH CARs as encompassed herein. As used herein, the term "amount" refers to an "effective amount" or "effective quantity" of cells including one or more CLL-1 VHH DARIC moieties or anti-CLL-1 VHH CARs as encompassed herein to achieve a beneficial or desired prophylactic or therapeutic outcome (including clinical outcomes) in the presence of a bridging factor.

[0404] A "prophylactically effective amount" refers to an amount of cells including one or more CLL-1 VHH DARIC moieties or anti-CLL-1 VHH CARs as encompassed herein that is effective to achieve a desired prophylactic outcome. Typically, but not necessarily, since prophylactic doses are administered to a subject prior to or early in a disease, the prophylactically effective amount is less than the therapeutically effective amount.

[0405] A "therapeutically effective amount" refers to an amount of cells including one or more CLL-1 VHH DARIC moieties or anti-CLL-1 VHH CARs as encompassed herein that is effective to "treat" a subject (e.g., a patient) in the presence of a bridging factor. When indicating a therapeutic amount, the precise amount of the composition, cells, bridging factor, etc. to be administered can be determined by a physician taking into account individual differences such as age, body weight, tumor size, degree of infection or metastasis, and the condition of the patient (subject).

[0406] Generally, it can be said that a pharmaceutical composition comprising the immune effector cells described herein can be administered at a dose of 10 2 to 10 10 cells / kg body weight, preferably 10 5 to 10 6 cells / kg body weight (including all integer values within those ranges). The number of cells will depend on the desired end use of the composition, as will the type of cells contained therein. For the uses provided herein, the volume of the cells is typically one liter or less, can be 500 ml or less, even 250 ml or 100 ml or less. Thus, the density of the desired cells is typically greater than 10 6 cells / mL, and is typically greater than 10 7 cells / mL, typically 10 8 cells / mL or greater. Clinically relevant numbers of immune cells can be dispensed to a cumulative volume equal to or exceeding 10 5 、106 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 or 10 12 in multiple infusions of 10 6 / kg (10 6 -10 11 / patient). A lower number of cells within this range may be administered.

[0407] If desired, the treatment may also include administration of a mitogen (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 induction of an immune response.

[0408] Generally, a composition 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 covered herein are used to treat cancer. In certain embodiments, the 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.

[0409] In certain embodiments, the pharmaceutical composition comprises an amount of genetically modified T cells in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients.

[0410] In certain embodiments, the pharmaceutical composition comprises an amount of a bridging factor in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients.

[0411] In certain embodiments, the composition comprises an effective amount of immune effector cells, which comprise one or more CLL-1 VHHDARIC moieties or anti-CLL-1 VHH CARs, either alone or in combination with a bridging factor and / or one or more therapeutic agents, as covered herein, such as radiotherapy, chemotherapy, transplantation, immunotherapy, hormone therapy, photodynamic therapy, etc. The composition may also be administered in combination with an antibiotic. Such therapeutic agents are acceptable in the art as standard treatments for certain disease states such as certain cancers as described herein. Exemplary therapeutic agents covered include cytokines, growth factors, steroids, NSAIDs, DMARDs, anti-inflammatory agents, chemotherapeutic agents, radiotherapeutic agents, therapeutic antibodies or other active agents and adjuvants.

[0412] In certain embodiments, a composition comprising an effective amount of immune effector cells, which comprise a polynucleotide encoding one or more CLL-1 VHH DARIC moieties or anti-CLL-1 VHH CARs as covered 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 repeated administration to the subject.

[0413] In certain embodiments, a composition comprising immune effector cells, which comprise a polynucleotide encoding one or more CLL-1 VHH DARIC moieties or anti-CLL-1 VHH CARs as covered herein, may be administered in combination with any number of anti-inflammatory agents, chemotherapeutic agents, or therapeutic antibodies, etc.

[0414] I. Methods of treatment

[0415] Immune effector cells modified to express a polynucleotide encoding one or more CLL-1 VHH DARIC moieties or anti-CLL-1VHH CARs as covered herein provide an improved method of adoptive immunotherapy for preventing, treating, and ameliorating immune disorders such as cancer or preventing, treating, or ameliorating at least one symptom associated therewith.

[0416] Immune effector cells comprising a CLL-1 DARIC signaling moiety, a CLL-1 VHH DARIC binding moiety, or an anti-CLL-1 VHH CAR provide an improved method of adoptive immunotherapy for preventing, treating, and ameliorating immune disorders such as cancer or preventing, treating, or ameliorating at least one symptom associated therewith.

[0417] In certain embodiments, immune effector cells modified to express CLL-1 VHH DARIC provide an improved method of adoptive immunotherapy to fine-tune the safety and efficacy of the cytotoxic response against target cells (e.g., tumor cells) expressing a target antigen while reducing the risk of on-target antigen and off-target cytotoxicity (recognition of the target antigen on normal non-target cells).

[0418] In certain embodiments, a method of 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 CLL-1 VHH DARIC or an anti-CLL-1 VHH CAR to redirect the cells to target cells. By transducing a chemically regulatable immune-stimulatory signal, the genetically modified cells are a more effective and safer cellular immunotherapy.

[0419] In certain embodiments, one or more immune effector cells (e.g., T cells) are modified to express both a CLL-1 VHH DARIC binding component and a CLL-1 DARIC signaling component. In such cases, the modified cells are administered to a subject in need thereof and are homed to the target cells by the interaction of the CLL-1 VHH binding component expressed on the immune effector cells with CLL-1 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 have been administered to the subject. In the presence of the bridging factor, a ternary complex is formed between the CLL-1 VHH DARIC binding component, the bridging factor, and the CLL-1 DARIC signaling component. After formation of the ternary complex, the CLL-1 VHH DARIC transduces an immune-stimulatory signal to the immune effector cell, which in turn elicits a cytotoxic response from the immune effector cell against the target cells.

[0420] In certain embodiments, one or more immune effector cells (e.g., T cells) are modified to express a CLL-1 DARIC signaling component. In such cases, the modified cells are administered to a subject in need thereof. The CLL-1 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 CLL-1 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 from the bridging factor. In the presence or absence of a bridging factor, the CLL-1 VHH binding component binds to CLL-1 expressed on the target cell. In the presence of a bridging factor, a ternary complex is formed between the CLL-1 VHH DARIC binding component, the bridging factor, and the CLL-1 DARIC signaling component. After formation of the ternary complex, the CLL-1 VHH DARIC transduces an immune-stimulatory signal to the immune effector cell, which in turn elicits a cytotoxic response from the immune effector cell against the target cell.

[0421] In certain embodiments, one or more immune effector cells (e.g., T cells) are modified to express a CLL-1 DARIC signaling component. In such cases, the modified cells are administered to a subject in need thereof. The CLL-1 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 CLL-1 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 from the bridging factor. In the presence or absence of a bridging factor, the CLL-1 binding component binds to the target antigen expressed on the target cell. In the presence of a bridging factor, a ternary complex is formed between the CLL-1 VHH DARIC binding component, the bridging factor, and the CLL-1 DARIC signaling component. After formation of the ternary complex, the CLL-1 VHH DARIC transduces an immune-stimulatory signal to the immune effector cell, which in turn elicits a cytotoxic response from the immune effector cell against the target cell. In certain embodiments, CLL-1 VHH DARIC activation can be induced in cases where remission or regression is incomplete and the condition recurs or becomes refractory.

[0422] In a particularly preferred embodiment, the specificity of primary T cells is redirected to CLL-1-expressing tumor cells or cancer cells by genetically modifying the T cells (e.g., primary T cells) with one or more CLL-1 VHH DARIC components.

[0423] In a particularly preferred embodiment, the specificity of primary T cells is redirected to CLL-1-expressing tumor cells or cancer cells by genetically modifying the T cells (e.g., primary T cells) with an engineered antigen receptor against a target antigen and one or more CLL-1 VHH DARIC components.

[0424] In certain embodiments, the modified immune effector cells encompassed herein are used for the treatment of solid tumors or cancers.

[0425] In certain embodiments, the modified immune effector cells encompassed herein are used for the treatment of solid tumors or cancers, including but not limited to: adrenal cancer, adrenocortical carcinoma, anal cancer, appendiceal cancer, astrocytoma, atypical teratoid / rhabdoid tumor, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain / CNS cancer, breast cancer, bronchial tumor, cardiac tumor, cervical cancer, cholangiocarcinoma, chondrosarcoma, chordoma, colon cancer, colorectal cancer, craniopharyngioma, ductal carcinoma in situ (DCIS) 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 sarcoma, kidney cancer, laryngeal cancer, leiomyosarcoma, lip cancer, liposarcoma, liver cancer, lung cancer, non-small cell lung cancer, lung carcinoid, malignant mesothelioma, medullary carcinoma, medulloblastoma, meningioma, melanoma, Merkel cell carcinoma, midline carcinoma, oral cancer, mucosal sarcoma, myelodysplastic syndrome, myeloproliferative neoplasm, nasal and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, oligodendroglioma, oral cancer, oral cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, islet cell tumor, papillary carcinoma, paraganglioma, parathyroid carcinoma, 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 carcinoma, skin cancer, soft tissue sarcoma, squamous cell carcinoma, small cell lung cancer, small intestine cancer, stomach cancer, sweat gland cancer, synovioma, testicular cancer, throat cancer, thymic cancer, thyroid cancer, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, vascular cancer, vulvar cancer, and Wilms tumor.

[0426] 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.

[0427] 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, metastatic colorectal cancer, glioblastoma, head and neck cancer, pancreatic cancer, and breast cancer.

[0428] In certain embodiments, the modified immune effector cells encompassed herein are used to treat glioblastoma.

[0429] In certain embodiments, the modified immune effector cells encompassed herein are used to treat liquid cancers or blood cancers.

[0430] In certain embodiments, the modified immune effector cells encompassed herein are used to treat B cell malignancies, including but not limited to: leukemia, lymphoma, and multiple myeloma.

[0431] In certain embodiments, the modified immune effector cells encompassed herein are used to treat liquid cancers, including but not limited to leukemia, lymphoma, and multiple myeloma: acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), myeloblastic leukemia, promyelocytic leukemia, myelomonocytic leukemia, monocytic leukemia, erythroleukemia, hairy cell leukemia (HCL), chronic lymphocytic leukemia (CLL), and chronic myelogenous leukemia (CML), chronic myelomonocytic leukemia (CMML), and polycythemia vera, Hodgkin lymphoma, nodular lymphocyte-predominant Hodgkin lymphoma, Burkitt lymphoma, small lymphocytic lymphoma (SLL), diffuse large B-cell lymphoma, follicular lymphoma, immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, mantle cell lymphoma, marginal zone lymphoma, mycosis fungoides, anaplastic large cell lymphoma, Sézary syndrome, precursor T-lymphoblastic lymphoma, multiple myeloma, overt multiple myeloma, smoldering multiple myeloma, plasma cell leukemia, non-secretory myeloma, IgD myeloma, sclerotic myeloma, solitary plasmacytoma of bone, and extramedullary plasmacytoma.

[0432] In certain embodiments, the modified immune effector cells encompassed herein are used to treat acute myeloid leukemia (AML).

[0433] Preferred cells used in the methods covered herein include autologous / self ("self") cells, preferably hematopoietic cells, more preferably T cells, and even more preferably immune effector cells.

[0434] In certain embodiments, the method comprises administering to a patient in need thereof a therapeutically effective amount of modified immune effector cells expressing one or more CLL-1 VHH DARIC components, and also administering a bridging factor to the subject. In some embodiments, the cells are used to treat a patient at risk of developing an immune disorder. Accordingly, 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 the modified immune effector cells and bridging factor covered herein.

[0435] In certain embodiments, the method comprises administering to a patient in need thereof a therapeutically effective amount of modified immune effector cells expressing an anti-CLL-1 VHH CAR. In some embodiments, the cells are used to treat a patient at risk of developing an immune disorder. Accordingly, 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 the modified immune effector cells and bridging factor covered herein.

[0436] In certain embodiments, the method comprises administering to a patient in need thereof a therapeutically effective amount of modified immune effector cells expressing a CLL-1 DARIC signaling component or a composition comprising the same, and also administering a CLL-1 VHH DARIC binding component and a bridging factor to the subject, optionally wherein the CLL-1 VHH DARIC binding component is bound to the bridging factor prior to administration. In some embodiments, the cells are used to treat a patient at risk of developing an immune disorder. Accordingly, 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 the modified immune effector cells that express a CLL-1 DARIC signaling component and an optionally engineered antigen receptor or another DARIC binding component, a CLL-1 VHH DARIC binding component, and a bridging factor.

[0437] The amounts and frequencies of administration of the modified immune effector cells, CLL-1 DARIC VHH binding component, and / or bridging factor will be determined by factors such as the patient's condition and the type and severity of the patient's disease, even though appropriate dosages and dosing schedules can be determined through clinical trials.

[0438] In one illustrative embodiment, the effective amount of the modified immune effector cells provided to the 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 cells / kg, including all intermediate cell doses.

[0439] In another illustrative embodiment, the effective amount of the modified immune effector cells provided to the 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 about 10×10 6 cells / kg or more cells / kg, including all intermediate cell doses.

[0440] In another illustrative embodiment, the effective amount of the modified immune effector cells provided to the 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 approximately 6×10 6 cells / kg, 3×10 6 cells / kg to approximately 7×10 6 cells / kg, 3×10 6 cells / kg to approximately 8×10 6 cells / kg, 4×10 6 cells / kg to approximately 6×10 6 cells / kg, 4×10 6 cells / kg to approximately 7×10 6 cells / kg, 4×10 6 cells / kg to approximately 8×10 6 cells / kg, 5×10 6 cells / kg to approximately 6×10 6 cells / kg, 5×10 6 cells / kg to approximately 7×10 6 cells / kg, 5×10 6 cells / kg to approximately 8×10 6 cells / kg or 6×10 6 cells / kg to approximately 8×10 6 cells / kg, including all intermediate cell doses.

[0441] One of ordinary skill in the art will recognize that multiple administrations of the compositions encompassed by a particular embodiment may be required to achieve the desired therapy. For example, the compositions may be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times over a span of 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, 2 years, 5 years, 10 years or longer. The modified immune effector cells, CLL-1 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, CLL-1 VHH DARIC components, and bridging factors may be administered by the same administration route or different routes.

[0442] In certain embodiments, it may be desirable to administer activated T cells to a subject and then re-draw blood (or perform apheresis), activate T cells therefrom, and re-infuse these activated and expanded T cells into the patient. This process can be performed multiple times, every few weeks. In certain embodiments, 10 cc to 400 cc of blood may be drawn to activate T cells. In certain embodiments, 20 cc, 30 cc, 40 cc, 50 cc, 60 cc, 70 cc, 80 cc, 90 cc, 100 cc, 150 cc, 200 cc, 250 cc, 300 cc, 350 cc, or 400 cc or more of blood may be drawn to activate T cells. Without being bound by theory, use of this multiple blood draw / multiple re-infusion protocol can be used to select certain T cell populations.

[0443] 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 CLL-1 VHH DARIC components into the cells and generating a population of modified immune effector cells; and administering the population of modified immune effector cells to the same subject. In a preferred embodiment, the immune effector cells comprise T cells.

[0444] 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 an anti-CLL-1 VHH CAR into the cells and generating a population of modified immune effector cells; and administering the population of modified immune effector cells to the same subject. In a preferred embodiment, the immune effector cells comprise T cells.

[0445] Methods for administering the cell compositions encompassed in certain embodiments include any method that effectively results in the re-introduction of ex vivo-modified immune effector cells or re-introduction of modified progenitor cells of immune effector cells that differentiate into mature immune effector cells upon introduction into a subject. One method includes ex vivo modifying peripheral blood T cells by introducing one or more vectors encoding one or more CLL-1 VHH DARIC components or anti-CLL-1 VHH CAR into the cells and returning the transduced cells into the subject.

[0446] All publications, patent applications, and issued patents cited in this specification are hereby incorporated by reference in their entirety to the same extent as if each individual publication, patent application, or issued patent was specifically and individually indicated to be incorporated by reference.

[0447] Although the foregoing embodiments have been described in considerable detail for purposes of clear understanding by way of illustration and example, those of ordinary skill in the art will readily appreciate that certain changes and modifications can be made thereto without departing from the spirit or scope of the appended claims. The following examples are provided for illustration only and are not limiting. Those skilled in the art will readily recognize various non-critical parameters that can be changed or modified in a particular embodiment to produce substantially similar results.

[0448] Example

[0449] Example 1

[0450] CLL-1 VHH DARIC T cells exhibit anti-tumor responses

[0451] Design, construct, and validate an anti-CLL-1 VHH DARIC binding component and a signaling component. Construct a CLL-1-specific VHH DARIC lentiviral vector that contains an MNDU3 promoter operably linked to a polynucleotide encoding: a DARIC signaling component (CD8α-signal peptide, FRB variant (T82L), CD8α transmembrane domain, intracellular 4-1BB co-stimulatory domain, and CD3ζ signaling domain); a P2A sequence; and a DARIC binding component (Igκ-signal peptide, CLL-1-specific VHH binding domain, G4S linker, FKBP12 domain, and CD4-derived transmembrane domain with a truncated intracellular domain) ( Figure 1B ). See, for example, the sequences shown in any of SEQ ID NOs: 20-25. T cells transduced with the anti-CLL-1 DARIC lentiviral vector express Figure 1A the membrane-bound polypeptide shown in. Anti-CLL-1 scFv CAR was used as a control.

[0452] T cells from three donors were each transduced with one of four LVV encoding different CLL-1-specific VHH DARIC or anti-CLL-1 scFv CAR and amplified for 10 days. Untransduced T cells, T cells transduced with anti-CLL-1 scFv CAR, or anti-CLL-1 VHH DARIC T cells were stained with a recombinant CLL-1-Fc reagent. Control CAR and CLL-1 VHH DARIC T cells were positively stained with CLL-1-Fc staining ( Figure 2 , upper panel). However, when analyzed with a monoclonal antibody specific for the VHH domain, only anti-CLL-1 VHH DARIC T cells were positively stained, while control CAR T cells were not positively stained ( Figure 2, as shown in the figure below). As determined in part by CD62L and CD45RA staining, both control CAR T cells and anti-CLL-1 VHHDARIC T cells had a similar T cell phenotype ( Figure 3 ).

[0453] Untransduced T cells, T cells transduced with anti-CLL-1 scFv CAR, or anti-CLL-1 VHH DARIC T cells were co-cultured with CLL-1+ THP-1 cells at an E:T ratio of 1:1 for 24 hours in the presence or absence of AP21967. Anti-CLL-1 scFv CAR control cells had strong cytokine production in the presence or absence of the rapamycin analog. Anti-CLL-VHH DARIC T cells showed a robust cytokine response only when co-cultured with THP-1 cells in the presence of AP21967 ( Figure 4 ). Minimal cytokine production was detected in the untransduced control.

[0454] Example 2

[0455] CLL-1 VHH DARIC T cells respond specifically to AML cell lines

[0456] Anti-CLL-1 VHH DARIC T cells were generated as described in Example 1. T cells from three donors were transduced with LVV encoding different anti-CLL-1 VHH DARICs and expanded for 10 days. Controls included untransduced (UTD) T cells. As a response to MV4-11 or HL60 target cells in the presence of AP21967, anti-CLL-1 VHH DARIC T cells were co-cultured with MV4-11 or HL60 AML cell lines expressing CLL-1 at an E:T ratio of 1:1 for 24 hours in the presence or absence of AP21967. Figure 5 .

[0457] Example 3

[0458] CLL-1 VHH DARIC T cells specifically recognize the CLL-1 antigen

[0459] Anti-CLL-1 VHH DARIC T cells were generated as described in Example 1. T cells from three donors were transduced with LVV encoding different anti-CLL-1 VHH DARICs and expanded for 10 days. Controls included UTD T cells. The AML cell line THP-1 was modified to knockout CLL-1 expression (CLL-1-KO cells). Figure 6AThe anti-CLL-1 VHH DARIC T cells were co-cultured with THP-1 cells or CLL-1-KO cells at an E:T ratio of 1:1 in the presence or absence of AP21967 for 24 hours. The anti-CLL-1 VHH DARIC T cells produced cytokines only when co-cultured with THP-1 cells in the presence of AP21967( Figure 6B -C).

[0460] Generally, in the following claims, the terms used should not be construed as limiting the claims to the specific embodiments disclosed in the specification and the claims, but should be construed as including all possible embodiments together with the full scope of equivalents claimed by these claims. Therefore, the claims are not limited by this disclosure. Sequence Listing <110> bluebird bio, Inc. Inhibrx, Inc. Jarjour, Jordan Pogson, Mark Leung, Wai-Hang Rascon, Lucas Sanabria, Angelica Timmer, John C. Eckelman, Brendan P. <120> CLL-1 Targeted Immunotherapy <130> BLBD-120 / 01WO 315698-3172 <150> US 62 / 845,306 <151> 2019-05-08 <160> 87 <170> PatentIn version 3.5 <210> 1 <211> 265 <212> PRT <213> Homo sapiens <400> 1 Met Ser Glu Glu Val Thr Tyr Ala Asp Leu Gln Phe Gln Asn Ser Ser 1 5 10 15 Glu Met Glu Lys Ile Pro Glu Ile Gly Lys Phe Gly Glu Lys Ala Pro 20 25 30 Pro Ala Pro Ser His Val Trp Arg Pro Ala Ala Leu Phe Leu Thr Leu 35 40 45 Leu Cys Leu Leu Leu Leu Ile Gly Leu Gly Val Leu Ala Ser Met Phe 50 55 60 His Val Thr Leu Lys Ile Glu Met Lys Lys Met Asn Lys Leu Gln Asn 65 70 75 80 Ile Ser Glu Glu Leu Gln Arg Asn Ile Ser Leu Gln Leu Met Ser Asn 85 90 95 Met Asn Ile Ser Asn Lys Ile Arg Asn Leu Ser Thr Thr Leu Gln Thr 100 105 110 Ile Ala Thr Lys Leu Cys Arg Glu Leu Tyr Ser Lys Glu Gln Glu His 115 120 125 Lys Cys Lys Pro Cys Pro Arg Arg Trp Ile Trp His Lys Asp Ser Cys 130 135 140 Tyr Phe Leu Ser Asp Asp Val Gln Thr Trp Gln Glu Ser Lys Met Ala 145 150 155 160 Cys Ala Ala Gln Asn Ala Ser Leu Leu Lys Ile Asn Asn Lys Asn Ala 165 170 175 Leu Glu Phe Ile Lys Ser Gln Ser Arg Ser Tyr Asp Tyr Trp Leu Gly 180 185 190 Leu Ser Pro Glu Glu Asp Ser Thr Arg Gly Met Arg Val Asp Asn Ile 195 200 205 Ile Asn Ser Ser Ala Trp Val Ile Arg Asn Ala Pro Asp Leu Asn Asn 210 215 220 Met Tyr Cys Gly Tyr Ile Asn Arg Leu Tyr Val Gln Tyr Tyr His Cys 225 230 235 240 Thr Tyr Lys Lys Arg Met Ile Cys Glu Lys Met Ala Asn Pro Val Gln 245 250 255 Leu Gly Ser Thr Tyr Phe Arg Glu Ala 260 265 <210> 2 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Laboratory preparation - anti-CLL-1 VHH domain <400> 2 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 Leu Phe Ser Ile Tyr 20 25 30 Asp Met Asn Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Trp Val 35 40 45 Ala Gly Ile Thr Asn Asn Gly Tyr Ser Thr Ala Tyr Ala Glu Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Ile Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 His Ala Asp Leu Thr Lys Ala Tyr Asp Val Glu Tyr Ala Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Lys Pro 115 120 <210> 3 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Laboratory Preparation - Anti-CLL-1 VHH Domain <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 Phe Thr Phe Ser Val Tyr 20 25 30 Asp Met Asn Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Trp Val 35 40 45 Ser Gly Ile Ser Asn Asn Gly Phe Ser Thr Ala 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 Asn Leu Asp Val His Gly Arg Val Gly Ala Gln Gly Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Lys Pro 115 120 <210> 4 <211> 129 <212> PRT <213> Artificial Sequence <220> <223> Laboratory preparation - anti-CLL-1 VHH domain <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 Phe Thr Phe Asp Asp Tyr 20 25 30 Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Ser Thr Ile Ser Ser Ser Asp Gly Ser 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 Leu Arg Glu Gly Leu Pro Leu Leu Leu Asp Thr Leu Trp Arg Gln 100 105 110 Pro Val Glu Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Lys 115 120 125 Pro <210> 5 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> Laboratory preparation - anti-CLL-1 VHH domain <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 Leu Leu Phe Ser Ile Tyr 20 25 30 Asp Met Asn Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Trp Val 35 40 45 Ala Gly Ile Thr Asn Asn Gly Tyr Ser Thr Ala 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 His Thr Asp Glu Trp Gly Arg Glu Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Lys Pro 115 <210> 6 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Laboratory preparation - anti-CLL-1 VHH domain <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 Arg Thr Phe Glu Tyr Asp 20 25 30 Asp Met Val Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Glu Val 35 40 45 Ala Gly Ile Ser Trp Ser Gly Gly Ser Ile Tyr Tyr Ala Glu Ser Val 50 55 60 Lys Gly Arg Phe Ser Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Glu Met Asn Arg Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Asn Val Gly Ser Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Lys Pro 100 105 110 <210> 7 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> Laboratory preparation - anti-CLL-1 VHH domain <400> 7 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 Arg Ile Asn 20 25 30 Asp Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Cys Glu Met Val 35 40 45 Ala Gly Ile Ser Asn Asn Gly Phe Ser Thr Ala 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 His Arg Thr Asp Glu Gly Ala Ala His Ala Cys Ser Arg Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Lys Pro 115 <210> 8 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Laboratory preparation - anti-CLL-1 VHH domain <220> <221> MOD_RES <222> (119)..(120) <223> Xaa is any amino acid or absent <400> 8 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 Leu Phe Ser Ile Tyr 20 25 30 Asp Met Asn Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Trp Val 35 40 45 Ala Gly Ile Thr Asn Asn Gly Tyr Ser Thr Ala Tyr Ala Glu Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Ile Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 His Ala Asp Leu Thr Lys Ala Tyr Asp Val Glu Tyr Ala Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Xaa Xaa 115 120 <210> 9 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Laboratory preparation - anti-CLL-1 VHH domain <220> <221> MOD_RES <222> (120)..(121) <223> Xaa is any amino acid or absent <400> 9 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 Val Tyr 20 25 30 Asp Met Asn Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Trp Val 35 40 45 Ser Gly Ile Ser Asn Asn Gly Phe Ser Thr Ala 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 Asn Leu Asp Val His Gly Arg Val Gly Ala Gln Gly Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Xaa Xaa 115 120 <210> 10 <211> 129 <212> PRT <213> Artificial Sequence <220> <223> Laboratory preparation - anti-CLL-1 VHH domain <220> <221> MOD_RES <222> (128)..(129) <223> Xaa is any amino acid or absent <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 Gly Phe Thr Phe Asp Asp Tyr 20 25 30 Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Ser Thr Ile Ser Ser Ser Asp Gly Ser 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 Leu Arg Glu Gly Leu Pro Leu Leu Leu Asp Thr Leu Trp Arg Gln 100 105 110 Pro Val Glu Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Xaa 115 120 125 Xaa <210> 11 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> Laboratory preparation - anti-CLL-1 VHH domain <220> <221> MOD_RES <222> (115)..(116) <223> Xaa is any amino acid or absent <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 Leu Leu Phe Ser Ile Tyr 20 25 30 Asp Met Asn Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Trp Val 35 40 45 Ala Gly Ile Thr Asn Asn Gly Tyr Ser Thr Ala 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 His Thr Asp Glu Trp Gly Arg Glu Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Xaa Xaa 115 <210> 12 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Laboratory preparation - anti-CLL-1 VHH domain <220> <221> MOD_RES <222> (111)..(112) <223> Xaa is any amino acid or absent <400> 12 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 Glu Tyr Asp 20 25 30 Asp Met Val Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Glu Val 35 40 45 Ala Gly Ile Ser Trp Ser Gly Gly Ser Ile Tyr Tyr Ala Glu Ser Val 50 55 60 Lys Gly Arg Phe Ser Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Glu Met Asn Arg Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Asn Val Gly Ser Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Xaa Xaa 100 105 110 <210> 13 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> Laboratory preparation - anti-CLL-1 VHH domain <220> <221> MOD_RES <222> (118)..(119) <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 Phe Thr Phe Arg Ile Asn 20 25 30 Asp Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Cys Glu Met Val 35 40 45 Ala Gly Ile Ser Asn Asn Gly Phe Ser Thr Ala 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 His Arg Thr Asp Glu Gly Ala Ala His Ala Cys Ser Arg Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Xaa Xaa 115 <210> 14 <211> 287 <212> PRT <213> Artificial Sequence <220> <223> Laboratory Preparation - Anti-CLL-1 VHH DARIC Binding Component <400> 14 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 Leu 35 40 45 Phe Ser Ile Tyr Asp Met Asn Trp Tyr Arg Gln Ala Pro Gly Lys Glu 50 55 60 Arg Glu Trp Val Ala Gly Ile Thr Asn Asn Gly Tyr Ser Thr Ala 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 Ile Tyr Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala 100 105 110 Val Tyr Tyr Cys His Ala Asp Leu Thr Lys Ala Tyr Asp Val Glu Tyr 115 120 125 Ala 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> 15 <211> 288 <212> PRT <213> Artificial Sequence <220> <223> Laboratory preparation - anti-CLL-1 VHH DARIC binding component <400> 15 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 Val Tyr Asp Met Asn Trp Tyr Arg Gln Ala Pro Gly Lys Glu 50 55 60 Arg Glu Trp Val Ser Gly Ile Ser Asn Asn Gly Phe Ser Thr Ala 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 Asn Leu Asp Val His Gly Arg Val Gly Ala Gln Gly 115 120 125 Asp 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> 16 <211> 296 <212> PRT <213> Artificial Sequence <220> <223> Laboratory Preparation - Anti-CLL-1 VHH DARIC Binding Component <400> 16 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 Asp Asp Tyr Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu 50 55 60 Arg Glu Gly Val Ser Thr Ile Ser Ser Ser Asp Gly Ser 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 Leu Arg Glu Gly Leu Pro Leu Leu Leu Asp Thr 115 120 125 Leu Trp Arg Gln Pro Val Glu Tyr Asp Tyr Trp Gly Gln Gly Thr Gln 130 135 140 Val Thr Val Lys Pro Gly Gly Gly Gly Ser Gly Val Gln Val Glu Thr 145 150 155 160 Ile Ser Pro Gly Asp Gly Arg Thr Phe Pro Lys Arg Gly Gln Thr Cys 165 170 175 Val Val His Tyr Thr Gly Met Leu Glu Asp Gly Lys Lys Phe Asp Ser 180 185 190 Ser Arg Asp Arg Asn Lys Pro Phe Lys Phe Met Leu Gly Lys Gln Glu 195 200 205 Val Ile Arg Gly Trp Glu Glu Gly Val Ala Gln Met Ser Val Gly Gln 210 215 220 Arg Ala Lys Leu Thr Ile Ser Pro Asp Tyr Ala Tyr Gly Ala Thr Gly 225 230 235 240 His Pro Gly Ile Ile Pro Pro His Ala Thr Leu Val Phe Asp Val Glu 245 250 255 Leu Leu Lys Leu Glu Gly Gly Arg Met Ala Leu Ile Val Leu Gly Gly 260 265 270 Val Ala Gly Leu Leu Leu Phe Ile Gly Leu Gly Ile Phe Phe Cys Val 275 280 285 Arg Cys Arg His Arg Arg Arg Gln 290 295 <210> 17 <211> 283 <212> PRT <213> Artificial Sequence <220> <223> Laboratory Preparation - Anti-CLL-1 VHH DARIC Binding Component <400> 17 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 Leu Leu 35 40 45 Phe Ser Ile Tyr Asp Met Asn Trp Tyr Arg Gln Ala Pro Gly Lys Glu 50 55 60 Arg Glu Trp Val Ala Gly Ile Thr Asn Asn Gly Tyr Ser Thr Ala 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 His Thr Asp Glu Trp Gly Arg Glu Tyr 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> 18 <211> 279 <212> PRT <213> Artificial Sequence <220> <223> Laboratory preparation - anti-CLL-1 VHH DARIC binding component <400> 18 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 Glu Tyr Asp Asp Met Val Trp Phe Arg Gln Ala Pro Gly Lys Glu 50 55 60 Arg Glu Glu Val Ala Gly Ile Ser Trp Ser Gly Gly Ser Ile Tyr Tyr 65 70 75 80 Ala Glu Ser Val Lys Gly Arg Phe Ser Ile Ser Arg Asp Asn Ala Lys 85 90 95 Asn Thr Val Tyr Leu Glu Met Asn Arg Leu Lys Pro Glu Asp Thr Ala 100 105 110 Val Tyr Tyr Cys Asn Val Gly Ser Tyr Trp Gly Gln Gly Thr Leu Val 115 120 125 Thr Val Lys Pro Gly Gly Gly Gly Ser Gly Val Gln Val Glu Thr Ile 130 135 140 Ser Pro Gly Asp Gly Arg Thr Phe Pro Lys Arg Gly Gln Thr Cys Val 145 150 155 160 Val His Tyr Thr Gly Met Leu Glu Asp Gly Lys Lys Phe Asp Ser Ser 165 170 175 Arg Asp Arg Asn Lys Pro Phe Lys Phe Met Leu Gly Lys Gln Glu Val 180 185 190 Ile Arg Gly Trp Glu Glu Gly Val Ala Gln Met Ser Val Gly Gln Arg 195 200 205 Ala Lys Leu Thr Ile Ser Pro Asp Tyr Ala Tyr Gly Ala Thr Gly His 210 215 220 Pro Gly Ile Ile Pro Pro His Ala Thr Leu Val Phe Asp Val Glu Leu 225 230 235 240 Leu Lys Leu Glu Gly Gly Arg Met Ala Leu Ile Val Leu Gly Gly Val 245 250 255 Ala Gly Leu Leu Leu Phe Ile Gly Leu Gly Ile Phe Phe Cys Val Arg 260 265 270 Cys Arg His Arg Arg Arg Gln 275 <210> 19 <211> 286 <212> PRT <213> Artificial Sequence <220> <223> Laboratory Preparation - Anti-CLL-1 VHH DARIC Binding Component <400> 19 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 Arg Ile Asn Asp Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu 50 55 60 Cys Glu Met Val Ala Gly Ile Ser Asn Asn Gly Phe Ser Thr Ala 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 His Arg Thr Asp Glu Gly Ala Ala His Ala Cys Ser 115 120 125 Arg Gly Gln Gly Thr Leu Val Thr Val Lys Pro Gly Gly Gly Gly Ser 130 135 140 Gly Val Gln Val Glu Thr Ile Ser Pro Gly Asp Gly Arg Thr Phe Pro 145 150 155 160 Lys Arg Gly Gln Thr Cys Val Val His Tyr Thr Gly Met Leu Glu Asp 165 170 175 Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg Asn Lys Pro Phe Lys Phe 180 185 190 Met Leu Gly Lys Gln Glu Val Ile Arg Gly Trp Glu Glu Gly Val Ala 195 200 205 Gln Met Ser Val Gly Gln Arg Ala Lys Leu Thr Ile Ser Pro Asp Tyr 210 215 220 Ala Tyr Gly Ala Thr Gly His Pro Gly Ile Ile Pro Pro His Ala Thr 225 230 235 240 Leu Val Phe Asp Val Glu Leu Leu Lys Leu Glu Gly Gly Arg Met Ala 245 250 255 Leu Ile Val Leu Gly Gly Val Ala Gly Leu Leu Leu Phe Ile Gly Leu 260 265 270 Gly Ile Phe Phe Cys Val Arg Cys Arg His Arg Arg Arg Gln 275 280 285 <210> 20 <211> 612 <212> PRT <213> Artificial Sequence <220> <223> Laboratory Preparation - Anti-CLL-1 VHH DARIC Fusion Protein <400> 20 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 Leu Phe Ser Ile Tyr Asp Met Asn Trp Tyr Arg Gln 370 375 380 Ala Pro Gly Lys Glu Arg Glu Trp Val Ala Gly Ile Thr Asn Asn Gly 385 390 395 400 Tyr Ser Thr Ala Tyr Ala Glu Ser Val Lys Gly Arg Phe Thr Ile Ser 405 410 415 Arg Asp Asn Ala Lys Asn Thr Ile Tyr Leu Gln Met Ser Ser Leu Arg 420 425 430 Ala Glu Asp Thr Ala Val Tyr Tyr Cys His Ala Asp Leu Thr Lys Ala 435 440 445 Tyr Asp Val Glu Tyr Ala 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> 21 <211> 613 <212> PRT <213> Artificial Sequence <220> <223> Laboratory Preparation - Anti-CLL-1 VHH DARIC Fusion Protein <400> 21 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 Val Tyr Asp Met Asn Trp Tyr Arg Gln 370 375 380 Ala Pro Gly Lys Glu Arg Glu Trp Val Ser Gly Ile Ser Asn Asn Gly 385 390 395 400 Phe Ser Thr Ala 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 Asn Leu Asp Val His Gly Arg 435 440 445 Val Gly Ala Gln Gly Asp 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> 22 <211> 621 <212> PRT <213> Artificial Sequence <220> <223> Laboratory preparation - anti-CLL-1 VHH DARIC fusion protein <400> 22 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 Asp Asp Tyr Ala Ile Gly Trp Phe Arg Gln 370 375 380 Ala Pro Gly Lys Glu Arg Glu Gly Val Ser Thr Ile Ser Ser Ser Asp 385 390 395 400 Gly Ser 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 Leu Arg Glu Gly Leu Pro 435 440 445 Leu Leu Leu Asp Thr Leu Trp Arg Gln Pro Val Glu Tyr Asp Tyr Trp 450 455 460 Gly Gln Gly Thr Gln Val Thr Val Lys Pro Gly Gly Gly Gly Ser Gly 465 470 475 480 Val Gln Val Glu Thr Ile Ser Pro Gly Asp Gly Arg Thr Phe Pro Lys 485 490 495 Arg Gly Gln Thr Cys Val Val His Tyr Thr Gly Met Leu Glu Asp Gly 500 505 510 Lys Lys Phe Asp Ser Ser Arg Asp Arg Asn Lys Pro Phe Lys Phe Met 515 520 525 Leu Gly Lys Gln Glu Val Ile Arg Gly Trp Glu Glu Gly Val Ala Gln 530 535 540 Met Ser Val Gly Gln Arg Ala Lys Leu Thr Ile Ser Pro Asp Tyr Ala 545 550 555 560 Tyr Gly Ala Thr Gly His Pro Gly Ile Ile Pro Pro His Ala Thr Leu 565 570 575 Val Phe Asp Val Glu Leu Leu Lys Leu Glu Gly Gly Arg Met Ala Leu 580 585 590 Ile Val Leu Gly Gly Val Ala Gly Leu Leu Leu Phe Ile Gly Leu Gly 595 600 605 Ile Phe Phe Cys Val Arg Cys Arg His Arg Arg Arg Gln 610 615 620 <210> 23 <211> 608 <212> PRT <213> Artificial Sequence <220> <223> Laboratory Preparation - Anti-CLL-1 VHH DARIC Fusion Protein <400> 23 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 Leu Leu Phe Ser Ile Tyr Asp Met Asn Trp Tyr Arg Gln 370 375 380 Ala Pro Gly Lys Glu Arg Glu Trp Val Ala Gly Ile Thr Asn Asn Gly 385 390 395 400 Tyr Ser Thr Ala 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 His Thr Asp Glu Trp Gly Arg 435 440 445 Glu Tyr 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 Cys Val Arg Cys Arg His Arg Arg Arg Gln 595 600 605 <210> 24 <211> 604 <212> PRT <213> Artificial Sequence <220> <223> Laboratory Preparation - Anti-CLL-1 VHH DARIC Fusion Protein <400> 24 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 Glu Tyr Asp Asp Met Val Trp Phe Arg Gln 370 375 380 Ala Pro Gly Lys Glu Arg Glu Glu Val Ala Gly Ile Ser Trp Ser Gly 385 390 395 400 Gly Ser Ile Tyr Tyr Ala Glu Ser Val Lys Gly Arg Phe Ser Ile Ser 405 410 415 Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu Glu Met Asn Arg Leu Lys 420 425 430 Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn Val Gly Ser Tyr Trp Gly 435 440 445 Gln Gly Thr Leu Val Thr Val Lys Pro Gly Gly Gly Gly Ser Gly Val 450 455 460 Gln Val Glu Thr Ile Ser Pro Gly Asp Gly Arg Thr Phe Pro Lys Arg 465 470 475 480 Gly Gln Thr Cys Val Val His Tyr Thr Gly Met Leu Glu Asp Gly Lys 485 490 495 Lys Phe Asp Ser Ser Arg Asp Arg Asn Lys Pro Phe Lys Phe Met Leu 500 505 510 Gly Lys Gln Glu Val Ile Arg Gly Trp Glu Glu Gly Val Ala Gln Met 515 520 525 Ser Val Gly Gln Arg Ala Lys Leu Thr Ile Ser Pro Asp Tyr Ala Tyr 530 535 540 Gly Ala Thr Gly His Pro Gly Ile Ile Pro Pro His Ala Thr Leu Val 545 550 555 560 Phe Asp Val Glu Leu Leu Lys Leu Glu Gly Gly Arg Met Ala Leu Ile 565 570 575 Val Leu Gly Gly Val Ala Gly Leu Leu Leu Phe Ile Gly Leu Gly Ile 580 585 590 Phe Phe Cys Val Arg Cys Arg His Arg Arg Arg Gln 595 600 <210> 25 <211> 611 <212> PRT <213> Artificial Sequence <220> <223> Laboratory Preparation - Anti-CLL-1 VHH DARIC Fusion Protein <400> 25 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 Arg Ile Asn Asp Met Gly Trp Tyr Arg Gln 370 375 380 Ala Pro Gly Lys Glu Cys Glu Met Val Ala Gly Ile Ser Asn Asn Gly 385 390 395 400 Phe Ser Thr Ala 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 His Arg Thr Asp Glu Gly Ala 435 440 445 Ala His Ala Cys Ser Arg Gly Gln Gly Thr Leu Val Thr Val Lys Pro 450 455 460 Gly Gly Gly Gly Ser Gly Val Gln Val Glu Thr Ile Ser Pro Gly Asp 465 470 475 480 Gly Arg Thr Phe Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr Thr 485 490 495 Gly Met Leu Glu Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg Asn 500 505 510 Lys Pro Phe Lys Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly Trp 515 520 525 Glu Glu Gly Val Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu Thr 530 535 540 Ile Ser Pro Asp Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile Ile 545 550 555 560 Pro Pro His Ala Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu Glu 565 570 575 Gly Gly Arg Met Ala Leu Ile Val Leu Gly Gly Val Ala Gly Leu Leu 580 585 590 Leu Phe Ile Gly Leu Gly Ile Phe Phe Cys Val Arg Cys Arg His Arg 595 600 605 Arg Arg Gln 610 <210> 26 <211> 644 <212> PRT <213> Artificial Sequence <220> <223> Laboratory Preparation - Anti-CLL1 DARIC.OX40 Fusion Protein <400> 26 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 Leu Phe Ser Ile Tyr Asp Met Asn Trp Tyr Arg Gln 370 375 380 Ala Pro Gly Lys Glu Arg Glu Trp Val Ala Gly Ile Thr Asn Asn Gly 385 390 395 400 Tyr Ser Thr Ala Tyr Ala Glu Ser Val Lys Gly Arg Phe Thr Ile Ser 405 410 415 Arg Asp Asn Ala Lys Asn Thr Ile Tyr Leu Gln Met Ser Ser Leu Arg 420 425 430 Ala Glu Asp Thr Ala Val Tyr Tyr Cys His Ala Asp Leu Thr Lys Ala 435 440 445 Tyr Asp Val Glu Tyr Ala 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> 27 <211> 645 <212> PRT <213> Artificial Sequence <220> <223> Laboratory Preparation - Anti-CLL1 DARIC.OX40 Fusion Protein <400> 27 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 Val Tyr Asp Met Asn Trp Tyr Arg Gln 370 375 380 Ala Pro Gly Lys Glu Arg Glu Trp Val Ser Gly Ile Ser Asn Asn Gly 385 390 395 400 Phe Ser Thr Ala 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 Asn Leu Asp Val His Gly Arg 435 440 445 Val Gly Ala Gln Gly Asp 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> 28 <211> 653 <212> PRT <213> Artificial Sequence <220> <223> Laboratory Preparation - Anti - CLL1 DARIC.OX40 Fusion Protein <400> 28 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 Asp Asp Tyr Ala Ile Gly Trp Phe Arg Gln 370 375 380 Ala Pro Gly Lys Glu Arg Glu Gly Val Ser Thr Ile Ser Ser Ser Asp 385 390 395 400 Gly Ser 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 Leu Arg Glu Gly Leu Pro 435 440 445 Leu Leu Leu Asp Thr Leu Trp Arg Gln Pro Val Glu Tyr Asp Tyr Trp 450 455 460 Gly Gln Gly Thr Gln Val Thr Val Lys Pro Gly Gly Gly Gly Ser Gly 465 470 475 480 Val Gln Val Glu Thr Ile Ser Pro Gly Asp Gly Arg Thr Phe Pro Lys 485 490 495 Arg Gly Gln Thr Cys Val Val His Tyr Thr Gly Met Leu Glu Asp Gly 500 505 510 Lys Lys Phe Asp Ser Ser Arg Asp Arg Asn Lys Pro Phe Lys Phe Met 515 520 525 Leu Gly Lys Gln Glu Val Ile Arg Gly Trp Glu Glu Gly Val Ala Gln 530 535 540 Met Ser Val Gly Gln Arg Ala Lys Leu Thr Ile Ser Pro Asp Tyr Ala 545 550 555 560 Tyr Gly Ala Thr Gly His Pro Gly Ile Ile Pro Pro His Ala Thr Leu 565 570 575 Val Phe Asp Val Glu Leu Leu Lys Leu Glu Gly Gly Arg Met Ala Leu 580 585 590 Ile Val Leu Gly Gly Val Ala Gly Leu Leu Leu Phe Ile Gly Leu Gly 595 600 605 Ile Phe Phe Ala Leu Tyr Leu Leu Arg Arg Asp Gln Arg Leu Pro Pro 610 615 620 Asp Ala His Lys Pro Pro Gly Gly Gly Ser Phe Arg Thr Pro Ile Gln 625 630 635 640 Glu Glu Gln Ala Asp Ala His Ser Thr Leu Ala Lys Ile 645 650 <210> 29 <211> 640 <212> PRT <213> Artificial Sequence <220> <223> Laboratory Preparation - Anti-CLL1 DARIC.OX40 Fusion Protein <400> 29 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 Leu Leu Phe Ser Ile Tyr Asp Met Asn Trp Tyr Arg Gln 370 375 380 Ala Pro Gly Lys Glu Arg Glu Trp Val Ala Gly Ile Thr Asn Asn Gly 385 390 395 400 Tyr Ser Thr Ala 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 His Thr Asp Glu Trp Gly Arg 435 440 445 Glu Tyr 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> 30 <211> 636 <212> PRT <213> Artificial Sequence <220> <223> Laboratory Preparation - Anti-CLL1 DARIC.OX40 Fusion Protein <400> 30 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 Glu Tyr Asp Asp Met Val Trp Phe Arg Gln 370 375 380 Ala Pro Gly Lys Glu Arg Glu Glu Val Ala Gly Ile Ser Trp Ser Gly 385 390 395 400 Gly Ser Ile Tyr Tyr Ala Glu Ser Val Lys Gly Arg Phe Ser Ile Ser 405 410 415 Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu Glu Met Asn Arg Leu Lys 420 425 430 Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn Val Gly Ser Tyr Trp Gly 435 440 445 Gln Gly Thr Leu Val Thr Val Lys Pro Gly Gly Gly Gly Ser Gly Val 450 455 460 Gln Val Glu Thr Ile Ser Pro Gly Asp Gly Arg Thr Phe Pro Lys Arg 465 470 475 480 Gly Gln Thr Cys Val Val His Tyr Thr Gly Met Leu Glu Asp Gly Lys 485 490 495 Lys Phe Asp Ser Ser Arg Asp Arg Asn Lys Pro Phe Lys Phe Met Leu 500 505 510 Gly Lys Gln Glu Val Ile Arg Gly Trp Glu Glu Gly Val Ala Gln Met 515 520 525 Ser Val Gly Gln Arg Ala Lys Leu Thr Ile Ser Pro Asp Tyr Ala Tyr 530 535 540 Gly Ala Thr Gly His Pro Gly Ile Ile Pro Pro His Ala Thr Leu Val 545 550 555 560 Phe Asp Val Glu Leu Leu Lys Leu Glu Gly Gly Arg Met Ala Leu Ile 565 570 575 Val Leu Gly Gly Val Ala Gly Leu Leu Leu Phe Ile Gly Leu Gly Ile 580 585 590 Phe Phe Ala Leu Tyr Leu Leu Arg Arg Asp Gln Arg Leu Pro Pro Asp 595 600 605 Ala His Lys Pro Pro Gly Gly Gly Ser Phe Arg Thr Pro Ile Gln Glu 610 615 620 Glu Gln Ala Asp Ala His Ser Thr Leu Ala Lys Ile 625 630 635 <210> 31 <211> 643 <212> PRT <213> Artificial Sequence <220> <223> Laboratory Preparation - Anti-CLL1 DARIC.OX40 Fusion Protein <400> 31 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 Arg Ile Asn Asp Met Gly Trp Tyr Arg Gln 370 375 380 Ala Pro Gly Lys Glu Cys Glu Met Val Ala Gly Ile Ser Asn Asn Gly 385 390 395 400 Phe Ser Thr Ala 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 His Arg Thr Asp Glu Gly Ala 435 440 445 Ala His Ala Cys Ser Arg Gly Gln Gly Thr Leu Val Thr Val Lys Pro 450 455 460 Gly Gly Gly Gly Ser Gly Val Gln Val Glu Thr Ile Ser Pro Gly Asp 465 470 475 480 Gly Arg Thr Phe Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr Thr 485 490 495 Gly Met Leu Glu Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg Asn 500 505 510 Lys Pro Phe Lys Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly Trp 515 520 525 Glu Glu Gly Val Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu Thr 530 535 540 Ile Ser Pro Asp Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile Ile 545 550 555 560 Pro Pro His Ala Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu Glu 565 570 575 Gly Gly Arg Met Ala Leu Ile Val Leu Gly Gly Val Ala Gly Leu Leu 580 585 590 Leu Phe Ile Gly Leu Gly Ile Phe Phe Ala Leu Tyr Leu Leu Arg Arg 595 600 605 Asp Gln Arg Leu Pro Pro Asp Ala His Lys Pro Pro Gly Gly Gly Ser 610 615 620 Phe Arg Thr Pro Ile Gln Glu Glu Gln Ala Asp Ala His Ser Thr Leu 625 630 635 640 Ala Lys Ile <210> 32 <211> 776 <212> PRT <213> Artificial Sequence <220> <223> Laboratory Preparation - Anti-CLL1 DARIC.TNRF2 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 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 Leu Phe Ser Ile Tyr Asp Met Asn Trp Tyr Arg Gln 370 375 380 Ala Pro Gly Lys Glu Arg Glu Trp Val Ala Gly Ile Thr Asn Asn Gly 385 390 395 400 Tyr Ser Thr Ala Tyr Ala Glu Ser Val Lys Gly Arg Phe Thr Ile Ser 405 410 415 Arg Asp Asn Ala Lys Asn Thr Ile Tyr Leu Gln Met Ser Ser Leu Arg 420 425 430 Ala Glu Asp Thr Ala Val Tyr Tyr Cys His Ala Asp Leu Thr Lys Ala 435 440 445 Tyr Asp Val Glu Tyr Ala 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 Lys Lys Lys Pro Leu Cys 595 600 605 Leu Gln Arg Glu Ala Lys Val Pro His Leu Pro Ala Asp Lys Ala Arg 610 615 620 Gly Thr Gln Gly Pro Glu Gln Gln His Leu Leu Ile Thr Ala Pro Ser 625 630 635 640 Ser Ser Ser Ser Ser Leu Glu Ser Ser Ala Ser Ala Leu Asp Arg Arg 645 650 655 Ala Pro Thr Arg Asn Gln Pro Gln Ala Pro Gly Val Glu Ala Ser Gly 660 665 670 Ala Gly Glu Ala Arg Ala Ser Thr Gly Ser Ser Asp Ser Ser Pro Gly 675 680 685 Gly His Gly Thr Gln Val Asn Val Thr Cys Ile Val Asn Val Cys Ser 690 695 700 Ser Ser Asp His Ser Ser Gln Cys Ser Ser Gln Ala Ser Ser Thr Met 705 710 715 720 Gly Asp Thr Asp Ser Ser Pro Ser Glu Ser Pro Lys Asp Glu Gln Val 725 730 735 Pro Phe Ser Lys Glu Glu Cys Ala Phe Arg Ser Gln Leu Glu Thr Pro 740 745 750 Glu Thr Leu Leu Gly Ser Thr Glu Glu Lys Pro Leu Pro Leu Gly Val 755 760 765 Pro Asp Ala Gly Met Lys Pro Ser 770 775 <210> 33 <211> 777 <212> PRT <213> Artificial Sequence <220> <223> Laboratory Preparation - Anti-CLL1 DARIC.TNRF2 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 Phe Thr Phe Ser Val Tyr Asp Met Asn Trp Tyr Arg Gln 370 375 380 Ala Pro Gly Lys Glu Arg Glu Trp Val Ser Gly Ile Ser Asn Asn Gly 385 390 395 400 Phe Ser Thr Ala 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 Asn Leu Asp Val His Gly Arg 435 440 445 Val Gly Ala Gln Gly Asp 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 Lys Lys Lys Pro Leu 595 600 605 Cys Leu Gln Arg Glu Ala Lys Val Pro His Leu Pro Ala Asp Lys Ala 610 615 620 Arg Gly Thr Gln Gly Pro Glu Gln Gln His Leu Leu Ile Thr Ala Pro 625 630 635 640 Ser Ser Ser Ser Ser Ser Leu Glu Ser Ser Ala Ser Ala Leu Asp Arg 645 650 655 Arg Ala Pro Thr Arg Asn Gln Pro Gln Ala Pro Gly Val Glu Ala Ser 660 665 670 Gly Ala Gly Glu Ala Arg Ala Ser Thr Gly Ser Ser Asp Ser Ser Pro 675 680 685 Gly Gly His Gly Thr Gln Val Asn Val Thr Cys Ile Val Asn Val Cys 690 695 700 Ser Ser Ser Asp His Ser Ser Gln Cys Ser Ser Gln Ala Ser Ser Thr 705 710 715 720 Met Gly Asp Thr Asp Ser Ser Pro Ser Glu Ser Pro Lys Asp Glu Gln 725 730 735 Val Pro Phe Ser Lys Glu Glu Cys Ala Phe Arg Ser Gln Leu Glu Thr 740 745 750 Pro Glu Thr Leu Leu Gly Ser Thr Glu Glu Lys Pro Leu Pro Leu Gly 755 760 765 Val Pro Asp Ala Gly Met Lys Pro Ser 770 775 <210> 34 <211> 785 <212> PRT <213> Artificial Sequence <220> <223> Laboratory Preparation - Anti-CLL1 DARIC.TNRF2 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 Phe Thr Phe Asp Asp Tyr Ala Ile Gly Trp Phe Arg Gln 370 375 380 Ala Pro Gly Lys Glu Arg Glu Gly Val Ser Thr Ile Ser Ser Ser Asp 385 390 395 400 Gly Ser 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 Leu Arg Glu Gly Leu Pro 435 440 445 Leu Leu Leu Asp Thr Leu Trp Arg Gln Pro Val Glu Tyr Asp Tyr Trp 450 455 460 Gly Gln Gly Thr Gln Val Thr Val Lys Pro Gly Gly Gly Gly Ser Gly 465 470 475 480 Val Gln Val Glu Thr Ile Ser Pro Gly Asp Gly Arg Thr Phe Pro Lys 485 490 495 Arg Gly Gln Thr Cys Val Val His Tyr Thr Gly Met Leu Glu Asp Gly 500 505 510 Lys Lys Phe Asp Ser Ser Arg Asp Arg Asn Lys Pro Phe Lys Phe Met 515 520 525 Leu Gly Lys Gln Glu Val Ile Arg Gly Trp Glu Glu Gly Val Ala Gln 530 535 540 Met Ser Val Gly Gln Arg Ala Lys Leu Thr Ile Ser Pro Asp Tyr Ala 545 550 555 560 Tyr Gly Ala Thr Gly His Pro Gly Ile Ile Pro Pro His Ala Thr Leu 565 570 575 Val Phe Asp Val Glu Leu Leu Lys Leu Glu Gly Gly Arg Met Ala Leu 580 585 590 Ile Val Leu Gly Gly Val Ala Gly Leu Leu Leu Phe Ile Gly Leu Gly 595 600 605 Ile Phe Phe Lys Lys Lys Pro Leu Cys Leu Gln Arg Glu Ala Lys Val 610 615 620 Pro His Leu Pro Ala Asp Lys Ala Arg Gly Thr Gln Gly Pro Glu Gln 625 630 635 640 Gln His Leu Leu Ile Thr Ala Pro Ser Ser Ser Ser Ser Ser Leu Glu 645 650 655 Ser Ser Ala Ser Ala Leu Asp Arg Arg Ala Pro Thr Arg Asn Gln Pro 660 665 670 Gln Ala Pro Gly Val Glu Ala Ser Gly Ala Gly Glu Ala Arg Ala Ser 675 680 685 Thr Gly Ser Ser Asp Ser Ser Pro Gly Gly His Gly Thr Gln Val Asn 690 695 700 Val Thr Cys Ile Val Asn Val Cys Ser Ser Ser Asp His Ser Ser Gln 705 710 715 720 Cys Ser Ser Gln Ala Ser Ser Thr Met Gly Asp Thr Asp Ser Ser Pro 725 730 735 Ser Glu Ser Pro Lys Asp Glu Gln Val Pro Phe Ser Lys Glu Glu Cys 740 745 750 Ala Phe Arg Ser Gln Leu Glu Thr Pro Glu Thr Leu Leu Gly Ser Thr 755 760 765 Glu Glu Lys Pro Leu Pro Leu Gly Val Pro Asp Ala Gly Met Lys Pro 770 775 780 Ser 785 <210> 35 <211> 772 <212> PRT <213> Artificial Sequence <220> <223> Laboratory preparation - Anti-CLL1 DARIC.TNRF2 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 Leu Leu Phe Ser Ile Tyr Asp Met Asn Trp Tyr Arg Gln 370 375 380 Ala Pro Gly Lys Glu Arg Glu Trp Val Ala Gly Ile Thr Asn Asn Gly 385 390 395 400 Tyr Ser Thr Ala 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 His Thr Asp Glu Trp Gly Arg 435 440 445 Glu Tyr 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 Lys Lys Lys Pro Leu Cys Leu Gln Arg Glu 595 600 605 Ala Lys Val Pro His Leu Pro Ala Asp Lys Ala Arg Gly Thr Gln Gly 610 615 620 Pro Glu Gln Gln His Leu Leu Ile Thr Ala Pro Ser Ser Ser Ser Ser 625 630 635 640 Ser Leu Glu Ser Ser Ala Ser Ala Leu Asp Arg Arg Ala Pro Thr Arg 645 650 655 Asn Gln Pro Gln Ala Pro Gly Val Glu Ala Ser Gly Ala Gly Glu Ala 660 665 670 Arg Ala Ser Thr Gly Ser Ser Asp Ser Ser Pro Gly Gly His Gly Thr 675 680 685 Gln Val Asn Val Thr Cys Ile Val Asn Val Cys Ser Ser Ser Asp His 690 695 700 Ser Ser Gln Cys Ser Ser Gln Ala Ser Ser Thr Met Gly Asp Thr Asp 705 710 715 720 Ser Ser Pro Ser Glu Ser Pro Lys Asp Glu Gln Val Pro Phe Ser Lys 725 730 735 Glu Glu Cys Ala Phe Arg Ser Gln Leu Glu Thr Pro Glu Thr Leu Leu 740 745 750 Gly Ser Thr Glu Glu Lys Pro Leu Pro Leu Gly Val Pro Asp Ala Gly 755 760 765 Met Lys Pro Ser 770 <210> 36 <211> 768 <212> PRT <213> Artificial Sequence <220> <223> Laboratory Preparation - Anti-CLL1 DARIC.TNRF2 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 Arg Thr Phe Glu Tyr Asp Asp Met Val Trp Phe Arg Gln 370 375 380 Ala Pro Gly Lys Glu Arg Glu Glu Val Ala Gly Ile Ser Trp Ser Gly 385 390 395 400 Gly Ser Ile Tyr Tyr Ala Glu Ser Val Lys Gly Arg Phe Ser Ile Ser 405 410 415 Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu Glu Met Asn Arg Leu Lys 420 425 430 Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn Val Gly Ser Tyr Trp Gly 435 440 445 Gln Gly Thr Leu Val Thr Val Lys Pro Gly Gly Gly Gly Ser Gly Val 450 455 460 Gln Val Glu Thr Ile Ser Pro Gly Asp Gly Arg Thr Phe Pro Lys Arg 465 470 475 480 Gly Gln Thr Cys Val Val His Tyr Thr Gly Met Leu Glu Asp Gly Lys 485 490 495 Lys Phe Asp Ser Ser Arg Asp Arg Asn Lys Pro Phe Lys Phe Met Leu 500 505 510 Gly Lys Gln Glu Val Ile Arg Gly Trp Glu Glu Gly Val Ala Gln Met 515 520 525 Ser Val Gly Gln Arg Ala Lys Leu Thr Ile Ser Pro Asp Tyr Ala Tyr 530 535 540 Gly Ala Thr Gly His Pro Gly Ile Ile Pro Pro His Ala Thr Leu Val 545 550 555 560 Phe Asp Val Glu Leu Leu Lys Leu Glu Gly Gly Arg Met Ala Leu Ile 565 570 575 Val Leu Gly Gly Val Ala Gly Leu Leu Leu Phe Ile Gly Leu Gly Ile 580 585 590 Phe Phe Lys Lys Lys Pro Leu Cys Leu Gln Arg Glu Ala Lys Val Pro 595 600 605 His Leu Pro Ala Asp Lys Ala Arg Gly Thr Gln Gly Pro Glu Gln Gln 610 615 620 His Leu Leu Ile Thr Ala Pro Ser Ser Ser Ser Ser Ser Leu Glu Ser 625 630 635 640 Ser Ala Ser Ala Leu Asp Arg Arg Ala Pro Thr Arg Asn Gln Pro Gln 645 650 655 Ala Pro Gly Val Glu Ala Ser Gly Ala Gly Glu Ala Arg Ala Ser Thr 660 665 670 Gly Ser Ser Asp Ser Ser Pro Gly Gly His Gly Thr Gln Val Asn Val 675 680 685 Thr Cys Ile Val Asn Val Cys Ser Ser Ser Asp His Ser Ser Gln Cys 690 695 700 Ser Ser Gln Ala Ser Ser Thr Met Gly Asp Thr Asp Ser Ser Pro Ser 705 710 715 720 Glu Ser Pro Lys Asp Glu Gln Val Pro Phe Ser Lys Glu Glu Cys Ala 725 730 735 Phe Arg Ser Gln Leu Glu Thr Pro Glu Thr Leu Leu Gly Ser Thr Glu 740 745 750 Glu Lys Pro Leu Pro Leu Gly Val Pro Asp Ala Gly Met Lys Pro Ser 755 760 765 <210> 37 <211> 775 <212> PRT <213> Artificial Sequence <220> <223> Laboratory Preparation - Anti-CLL1 DARIC.TNRF2 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 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 Arg Ile Asn Asp Met Gly Trp Tyr Arg Gln 370 375 380 Ala Pro Gly Lys Glu Cys Glu Met Val Ala Gly Ile Ser Asn Asn Gly 385 390 395 400 Phe Ser Thr Ala 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 His Arg Thr Asp Glu Gly Ala 435 440 445 Ala His Ala Cys Ser Arg Gly Gln Gly Thr Leu Val Thr Val Lys Pro 450 455 460 Gly Gly Gly Gly Ser Gly Val Gln Val Glu Thr Ile Ser Pro Gly Asp 465 470 475 480 Gly Arg Thr Phe Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr Thr 485 490 495 Gly Met Leu Glu Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg Asn 500 505 510 Lys Pro Phe Lys Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly Trp 515 520 525 Glu Glu Gly Val Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu Thr 530 535 540 Ile Ser Pro Asp Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile Ile 545 550 555 560 Pro Pro His Ala Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu Glu 565 570 575 Gly Gly Arg Met Ala Leu Ile Val Leu Gly Gly Val Ala Gly Leu Leu 580 585 590 Leu Phe Ile Gly Leu Gly Ile Phe Phe Lys Lys Lys Pro Leu Cys Leu 595 600 605 Gln Arg Glu Ala Lys Val Pro His Leu Pro Ala Asp Lys Ala Arg Gly 610 615 620 Thr Gln Gly Pro Glu Gln Gln His Leu Leu Ile Thr Ala Pro Ser Ser 625 630 635 640 Ser Ser Ser Ser Leu Glu Ser Ser Ala Ser Ala Leu Asp Arg Arg Ala 645 650 655 Pro Thr Arg Asn Gln Pro Gln Ala Pro Gly Val Glu Ala Ser Gly Ala 660 665 670 Gly Glu Ala Arg Ala Ser Thr Gly Ser Ser Asp Ser Ser Pro Gly Gly 675 680 685 His Gly Thr Gln Val Asn Val Thr Cys Ile Val Asn Val Cys Ser Ser 690 695 700 Ser Asp His Ser Ser Gln Cys Ser Ser Gln Ala Ser Ser Thr Met Gly 705 710 715 720 Asp Thr Asp Ser Ser Pro Ser Glu Ser Pro Lys Asp Glu Gln Val Pro 725 730 735 Phe Ser Lys Glu Glu Cys Ala Phe Arg Ser Gln Leu Glu Thr Pro Glu 740 745 750 Thr Leu Leu Gly Ser Thr Glu Glu Lys Pro Leu Pro Leu Gly Val Pro 755 760 765 Asp Ala Gly Met Lys Pro Ser 770 775 <210> 38 <211> 367 <212> PRT <213> Artificial Sequence <220> <223> Laboratory Preparation - Anti-CLL-1 VHH Chimeric Antigen Receptor <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 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Glu 20 25 30 Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe 35 40 45 Leu Phe Ser Ile Tyr Asp Met Asn Trp Tyr Arg Gln Ala Pro Gly Lys 50 55 60 Glu Arg Glu Trp Val Ala Gly Ile Thr Asn Asn Gly Tyr Ser Thr Ala 65 70 75 80 Tyr Ala Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala 85 90 95 Lys Asn Thr Ile Tyr Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr 100 105 110 Ala Val Tyr Tyr Cys His Ala Asp Leu Thr Lys Ala Tyr Asp Val Glu 115 120 125 Tyr Ala Trp Gly Gln Gly Thr Leu Val Thr Val Lys Pro Ala Ala Ala 130 135 140 Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala 145 150 155 160 Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly 165 170 175 Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile 180 185 190 Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val 195 200 205 Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe 210 215 220 Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly 225 230 235 240 Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg 245 250 255 Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln 260 265 270 Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp 275 280 285 Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro 290 295 300 Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp 305 310 315 320 Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg 325 330 335 Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr 340 345 350 Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 355 360 365 <210> 39 <211> 368 <212> PRT <213> Artificial Sequence <220> <223> Laboratory Preparation - Anti-CLL-1 VHH Chimeric Antigen Receptor <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 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Glu 20 25 30 Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe 35 40 45 Thr Phe Ser Val Tyr Asp Met Asn Trp Tyr Arg Gln Ala Pro Gly Lys 50 55 60 Glu Arg Glu Trp Val Ser Gly Ile Ser Asn Asn Gly Phe Ser Thr Ala 65 70 75 80 Tyr Ala Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala 85 90 95 Lys Asn Thr Leu Tyr Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr 100 105 110 Ala Val Tyr Tyr Cys Asn Leu Asp Val His Gly Arg Val Gly Ala Gln 115 120 125 Gly Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Lys Pro Ala Ala 130 135 140 Ala Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile 145 150 155 160 Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala 165 170 175 Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr 180 185 190 Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu 195 200 205 Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile 210 215 220 Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp 225 230 235 240 Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu 245 250 255 Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly 260 265 270 Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr 275 280 285 Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys 290 295 300 Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys 305 310 315 320 Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg 325 330 335 Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala 340 345 350 Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 355 360 365 <210> 40 <211> 376 <212> PRT <213> Artificial Sequence <220> <223> Laboratory Preparation - Anti-CLL-1 VHH Chimeric Antigen Receptor <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 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Glu 20 25 30 Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe 35 40 45 Thr Phe Asp Asp Tyr Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys 50 55 60 Glu Arg Glu Gly Val Ser Thr Ile Ser Ser Ser Asp Gly Ser Thr Tyr 65 70 75 80 Tyr Ala Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala 85 90 95 Lys Asn Thr Val Tyr Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr 100 105 110 Ala Val Tyr Tyr Cys Ala Leu Arg Glu Gly Leu Pro Leu Leu Leu Asp 115 120 125 Thr Leu Trp Arg Gln Pro Val Glu Tyr Asp Tyr Trp Gly Gln Gly Thr 130 135 140 Gln Val Thr Val Lys Pro Ala Ala Ala Thr Thr Thr Pro Ala Pro Arg 145 150 155 160 Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg 165 170 175 Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly 180 185 190 Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr 195 200 205 Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg 210 215 220 Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro 225 230 235 240 Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu 245 250 255 Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala 260 265 270 Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu 275 280 285 Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly 290 295 300 Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu 305 310 315 320 Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser 325 330 335 Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly 340 345 350 Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu 355 360 365 His Met Gln Ala Leu Pro Pro Arg 370 375 <210> 41 <211> 363 <212> PRT <213> Artificial Sequence <220> <223> Laboratory Preparation - Anti-CLL-1 VHH Chimeric Antigen Receptor <400> 41 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 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Glu 20 25 30 Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Leu 35 40 45 Leu Phe Ser Ile Tyr Asp Met Asn Trp Tyr Arg Gln Ala Pro Gly Lys 50 55 60 Glu Arg Glu Trp Val Ala Gly Ile Thr Asn Asn Gly Tyr Ser Thr Ala 65 70 75 80 Tyr Ala Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala 85 90 95 Lys Asn Thr Val Tyr Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr 100 105 110 Ala Val Tyr Tyr Cys His Thr Asp Glu Trp Gly Arg Glu Tyr Trp Gly 115 120 125 Gln Gly Thr Leu Val Thr Val Lys Pro Ala Ala Ala Thr Thr Thr Pro 130 135 140 Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu 145 150 155 160 Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His 165 170 175 Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu 180 185 190 Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr 195 200 205 Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe 210 215 220 Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg 225 230 235 240 Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser 245 250 255 Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr 260 265 270 Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys 275 280 285 Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn 290 295 300 Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu 305 310 315 320 Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly 325 330 335 His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr 340 345 350 Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 355 360 <210> 42 <211> 359 <212> PRT <213> Artificial Sequence <220> <223> Laboratory preparation - anti-CLL-1 VHH chimeric antigen receptor <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 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Glu 20 25 30 Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg 35 40 45 Thr Phe Glu Tyr Asp Asp Met Val Trp Phe Arg Gln Ala Pro Gly Lys 50 55 60 Glu Arg Glu Glu Val Ala Gly Ile Ser Trp Ser Gly Gly Ser Ile Tyr 65 70 75 80 Tyr Ala Glu Ser Val Lys Gly Arg Phe Ser Ile Ser Arg Asp Asn Ala 85 90 95 Lys Asn Thr Val Tyr Leu Glu Met Asn Arg Leu Lys Pro Glu Asp Thr 100 105 110 Ala Val Tyr Tyr Cys Asn Val Gly Ser Tyr Trp Gly Gln Gly Thr Leu 115 120 125 Val Thr Val Lys Pro Ala Ala Ala Thr Thr Thr Pro Ala Pro Arg Pro 130 135 140 Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro 145 150 155 160 Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu 165 170 175 Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys 180 185 190 Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly 195 200 205 Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val 210 215 220 Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu 225 230 235 240 Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp 245 250 255 Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn 260 265 270 Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg 275 280 285 Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly 290 295 300 Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu 305 310 315 320 Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu 325 330 335 Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His 340 345 350 Met Gln Ala Leu Pro Pro Arg 355 <210> 43 <211> 366 <212> PRT <213> Artificial Sequence <220> <223> Laboratory preparation - anti-CLL-1 VHH chimeric antigen receptor <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 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Glu 20 25 30 Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe 35 40 45 Thr Phe Arg Ile Asn Asp Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys 50 55 60 Glu Cys Glu Met Val Ala Gly Ile Ser Asn Asn Gly Phe Ser Thr Ala 65 70 75 80 Tyr Ala Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala 85 90 95 Lys Asn Thr Val Tyr Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr 100 105 110 Ala Val Tyr Tyr Cys His Arg Thr Asp Glu Gly Ala Ala His Ala Cys 115 120 125 Ser Arg Gly Gln Gly Thr Leu Val Thr Val Lys Pro Ala Ala Ala Thr 130 135 140 Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser 145 150 155 160 Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly 165 170 175 Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp 180 185 190 Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile 195 200 205 Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys 210 215 220 Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys 225 230 235 240 Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val 245 250 255 Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn 260 265 270 Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val 275 280 285 Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg 290 295 300 Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys 305 310 315 320 Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg 325 330 335 Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys 340 345 350 Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 355 360 365 <210> 44 <211> 364 <212> PRT <213> Artificial Sequence <220> <223> Laboratory preparation - anti-CLL-1 VHH chimeric antigen receptor <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 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Glu 20 25 30 Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe 35 40 45 Leu Phe Ser Ile Tyr Asp Met Asn Trp Tyr Arg Gln Ala Pro Gly Lys 50 55 60 Glu Arg Glu Trp Val Ala Gly Ile Thr Asn Asn Gly Tyr Ser Thr Ala 65 70 75 80 Tyr Ala Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala 85 90 95 Lys Asn Thr Ile Tyr Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr 100 105 110 Ala Val Tyr Tyr Cys His Ala Asp Leu Thr Lys Ala Tyr Asp Val Glu 115 120 125 Tyr Ala Trp Gly Gln Gly Thr Leu Val Thr Val Lys Pro Thr Thr Thr 130 135 140 Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro 145 150 155 160 Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val 165 170 175 His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro 180 185 190 Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu 195 200 205 Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro 210 215 220 Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys 225 230 235 240 Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe 245 250 255 Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu 260 265 270 Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp 275 280 285 Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys 290 295 300 Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala 305 310 315 320 Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys 325 330 335 Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr 340 345 350 Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 355 360 <210> 45 <211> 365 <212> PRT <213> Artificial Sequence <220> <223> Laboratory Preparation - Anti-CLL-1 VHH Chimeric Antigen Receptor <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 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Glu 20 25 30 Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe 35 40 45 Thr Phe Ser Val Tyr Asp Met Asn Trp Tyr Arg Gln Ala Pro Gly Lys 50 55 60 Glu Arg Glu Trp Val Ser Gly Ile Ser Asn Asn Gly Phe Ser Thr Ala 65 70 75 80 Tyr Ala Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala 85 90 95 Lys Asn Thr Leu Tyr Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr 100 105 110 Ala Val Tyr Tyr Cys Asn Leu Asp Val His Gly Arg Val Gly Ala Gln 115 120 125 Gly Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Lys Pro Thr Thr 130 135 140 Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln 145 150 155 160 Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala 165 170 175 Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala 180 185 190 Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr 195 200 205 Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln 210 215 220 Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser 225 230 235 240 Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys 245 250 255 Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln 260 265 270 Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu 275 280 285 Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg 290 295 300 Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met 305 310 315 320 Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly 325 330 335 Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp 340 345 350 Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 355 360 365 <210> 46 <211> 373 <212> PRT <213> Artificial Sequence <220> <223> Laboratory preparation - Anti-CLL-1 VHH chimeric antigen receptor <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 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Glu 20 25 30 Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe 35 40 45 Thr Phe Asp Asp Tyr Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys 50 55 60 Glu Arg Glu Gly Val Ser Thr Ile Ser Ser Ser Asp Gly Ser Thr Tyr 65 70 75 80 Tyr Ala Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala 85 90 95 Lys Asn Thr Val Tyr Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr 100 105 110 Ala Val Tyr Tyr Cys Ala Leu Arg Glu Gly Leu Pro Leu Leu Leu Asp 115 120 125 Thr Leu Trp Arg Gln Pro Val Glu Tyr Asp Tyr Trp Gly Gln Gly Thr 130 135 140 Gln Val Thr Val Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr 145 150 155 160 Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala 165 170 175 Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe 180 185 190 Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val 195 200 205 Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys 210 215 220 Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr 225 230 235 240 Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu 245 250 255 Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro 260 265 270 Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly 275 280 285 Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro 290 295 300 Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr 305 310 315 320 Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly 325 330 335 Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln 340 345 350 Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln 355 360 365 Ala Leu Pro Pro Arg 370 <210> 47 <211> 360 <212> PRT <213> Artificial Sequence <220> <223> Laboratory preparation - anti-CLL-1 VHH chimeric antigen receptor <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 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Glu 20 25 30 Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Leu 35 40 45 Leu Phe Ser Ile Tyr Asp Met Asn Trp Tyr Arg Gln Ala Pro Gly Lys 50 55 60 Glu Arg Glu Trp Val Ala Gly Ile Thr Asn Asn Gly Tyr Ser Thr Ala 65 70 75 80 Tyr Ala Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala 85 90 95 Lys Asn Thr Val Tyr Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr 100 105 110 Ala Val Tyr Tyr Cys His Thr Asp Glu Trp Gly Arg Glu Tyr Trp Gly 115 120 125 Gln Gly Thr Leu Val Thr Val Lys Pro Thr Thr Thr Pro Ala Pro Arg 130 135 140 Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg 145 150 155 160 Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly 165 170 175 Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr 180 185 190 Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg 195 200 205 Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro 210 215 220 Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu 225 230 235 240 Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala 245 250 255 Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu 260 265 270 Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly 275 280 285 Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu 290 295 300 Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser 305 310 315 320 Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly 325 330 335 Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu 340 345 350 His Met Gln Ala Leu Pro Pro Arg 355 360 <210> 48 <211> 356 <212> PRT <213> Artificial Sequence <220> <223> Laboratory Preparation - Anti-CLL-1 VHH Chimeric Antigen Receptor <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 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Glu 20 25 30 Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg 35 40 45 Thr Phe Glu Tyr Asp Asp Met Val Trp Phe Arg Gln Ala Pro Gly Lys 50 55 60 Glu Arg Glu Glu Val Ala Gly Ile Ser Trp Ser Gly Gly Ser Ile Tyr 65 70 75 80 Tyr Ala Glu Ser Val Lys Gly Arg Phe Ser Ile Ser Arg Asp Asn Ala 85 90 95 Lys Asn Thr Val Tyr Leu Glu Met Asn Arg Leu Lys Pro Glu Asp Thr 100 105 110 Ala Val Tyr Tyr Cys Asn Val Gly Ser Tyr Trp Gly Gln Gly Thr Leu 115 120 125 Val Thr Val Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro 130 135 140 Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys 145 150 155 160 Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala 165 170 175 Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu 180 185 190 Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys 195 200 205 Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr 210 215 220 Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly 225 230 235 240 Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala 245 250 255 Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg 260 265 270 Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu 275 280 285 Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn 290 295 300 Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met 305 310 315 320 Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly 325 330 335 Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala 340 345 350 Leu Pro Pro Arg 355 <210> 49 <211> 356 <212> PRT <213> Artificial Sequence <220> <223> Laboratory Preparation - Anti-CLL-1 VHH Chimeric Antigen Receptor <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 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Glu 20 25 30 Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg 35 40 45 Thr Phe Glu Tyr Asp Asp Met Val Trp Phe Arg Gln Ala Pro Gly Lys 50 55 60 Glu Arg Glu Glu Val Ala Gly Ile Ser Trp Ser Gly Gly Ser Ile Tyr 65 70 75 80 Tyr Ala Glu Ser Val Lys Gly Arg Phe Ser Ile Ser Arg Asp Asn Ala 85 90 95 Lys Asn Thr Val Tyr Leu Glu Met Asn Arg Leu Lys Pro Glu Asp Thr 100 105 110 Ala Val Tyr Tyr Cys Asn Val Gly Ser Tyr Trp Gly Gln Gly Thr Leu 115 120 125 Val Thr Val Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro 130 135 140 Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys 145 150 155 160 Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala 165 170 175 Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu 180 185 190 Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys 195 200 205 Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr 210 215 220 Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly 225 230 235 240 Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala 245 250 255 Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg 260 265 270 Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu 275 280 285 Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn 290 295 300 Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met 305 310 315 320 Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly 325 330 335 Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala 340 345 350 Leu Pro Pro Arg 355 <210> 50 <211> 301 <212> PRT <213> Artificial Sequence <220> <223> Laboratory preparation - anti-CLL1 VHH DARIC signaling component <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 290 295 300 <210> 51 <211> 10 <212> DNA <213> Artificial Sequence <220> <223> Consensus Kozak sequence <400> 51 gccrccatgg 10 <210> 52 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Exemplary linker sequence <400> 52 Asp Gly Gly Gly Ser 1 5 <210> 53 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Exemplary linker sequence <400> 53 Thr Gly Glu Lys Pro 1 5 <210> 54 <211> 4 <212> PRT <213> Artificial Sequence <220&...

Claims

1. A non-natural hematopoietic cell, comprising: (a) A first polypeptide, which consists of the following in the following order: (i) An FRB polymerization domain polypeptide consisting of the FRB T2098L polymerization domain from SEQ ID NO:50; (ii) A CD8α transmembrane domain consisting of the CD8α transmembrane domain from SEQ ID NO:50; (iii) A CD137 co-stimulatory domain consisting of the CD137 co-stimulatory domain from SEQ ID NO:50; and (iv) A CD3ζ primary signaling domain consisting of the CD3ζ primary signaling domain from SEQ ID NO:50; and (b) A second polypeptide, which consists of the following in the following order: (i) An anti-CLL-1 VHH antibody consisting of the amino acid sequence shown in SEQ ID NO:2; (ii) An FKBP polymerization domain polypeptide consisting of the FKBP12 polymerization domain from SEQ ID NO:14; and (iii) A CD4 transmembrane domain consisting of the CD4 transmembrane domain from SEQ ID NO:

14.

2. The non-natural hematopoietic cell according to claim 1, further comprising a bridging factor, wherein the bridging factor promotes the formation of a polypeptide complex on the surface of the non-natural cell, wherein the bridging factor associates with the polymerization domains of the first polypeptide and the second polypeptide and is placed between the polymerization domains, and wherein the bridging factor is selected from the group consisting of: AP21967, sirolimus, everolimus, deforolimus, pimecrolimus, deltalimus, tacrolimus, temsirolimus, umirolimus, and zotarolimus.

3. The non-natural hematopoietic cell according to claim 1, wherein the first polypeptide further comprises a signal peptide from SEQ ID NO:

50.

4. The non-natural hematopoietic cell according to claim 1, wherein the second polypeptide comprises the sequence shown in SEQ ID NO:

14.

5. The non-natural hematopoietic cell according to claim 1, wherein the hematopoietic cell is a T cell.

6. The non-natural hematopoietic cell according to claim 5, wherein the T cell is an αβ T cell or a γδ T cell.

7. The non-natural hematopoietic cell according to claim 1, wherein the hematopoietic cell is CD3 + , CD4 + and / or CD8 + cells.

8. The non-natural hematopoietic cell according to claim 1, wherein the hematopoietic cell is an immune effector cell.

9. The non-natural hematopoietic cell according to claim 1, wherein the hematopoietic cell is a cytotoxic T lymphocyte (CTL), a tumor-infiltrating lymphocyte (TIL), or a helper T cell.

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

11. The non-natural hematopoietic cell according to claim 1, wherein the source of the hematopoietic cell is peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infected site, ascites, pleural effusion, spleen tissue, or a tumor.

12. The non-natural hematopoietic cell according to claim 1, wherein when the first polypeptide and the second polypeptide are expressed, the FRB multimerization domain and the FKBP multimerization domain are located extracellularly.

13. A fusion polypeptide comprising: (a) A first polypeptide, which consists of the following in the following order: (i) An FRB multimerization domain polypeptide consisting of the FRB T2098L multimerization domain from SEQ ID NO:50; (ii) A CD8α transmembrane domain consisting of the CD8α transmembrane domain from SEQ ID NO:50; (iii) A CD137 co-stimulatory domain consisting of the CD137 co-stimulatory domain from SEQ ID NO:50; and (iv) A CD3ζ primary signaling domain consisting of the CD3ζ primary signaling domain from SEQ ID NO:50; (b) A polypeptide comprising a cleavage signal, which comprises the amino acid sequence shown in any one of SEQ ID NOs: 66-87; and (c) A second polypeptide, which consists of the following in the following order: (i) An anti-CLL-1 VHH antibody consisting of the amino acid sequence shown in SEQ ID NO:2; (ii) An FKBP multimerization domain polypeptide consisting of the FKBP12 multimerization domain from SEQ ID NO:14; and (iii) A CD4 transmembrane domain consisting of the CD4 transmembrane domain from SEQ ID NO:

14.

14. The fusion polypeptide according to claim 13, wherein the first polypeptide further comprises a signal peptide from SEQ ID NO:

50.

15. The fusion polypeptide according to claim 13, wherein the polypeptide comprising a cleavage signal is a viral self-cleaving polypeptide.

16. The fusion polypeptide according to claim 13, wherein the polypeptide comprising a cleavage signal is a viral self-cleaving 2A polypeptide.

17. The fusion polypeptide according to claim 13, wherein the polypeptide comprising a cleavage signal is a viral self-cleaving polypeptide selected from the group consisting of: foot-and-mouth disease virus (FMDV) F2A peptide, equine rhinitis A virus (ERAV) E2A peptide, Mythimna separata tetravirus (TaV) T2A peptide, porcine teschovirus-1 (PTV-1) P2A peptide, Theiler's virus 2A peptide, and encephalomyocarditis virus 2A peptide.

18. The fusion polypeptide according to claim 13, wherein the polypeptide comprising a cleavage signal is a viral self-cleaving P2A peptide comprising the amino acid sequence shown in SEQ ID NO:

66.

19. The fusion polypeptide according to claim 13, wherein the fusion polypeptide consists of the sequence shown in any one of SEQ ID NOs: 20, 26, or 32.

20. The fusion polypeptide according to claim 13, wherein when the first polypeptide and the second polypeptide are expressed, the FRB multimerization domain and the FKBP multimerization domain are located extracellularly.

21. A polypeptide complex comprising: (a) A first polypeptide, which consists of the following in the following order: (i) An FRB multimerization domain polypeptide consisting of the FRB T2098L multimerization domain from SEQ ID NO:50; (ii) A CD8α transmembrane domain consisting of the CD8α transmembrane domain from SEQ ID NO:50; (iii) A D137 co-stimulatory domain consisting of the CD137 co-stimulatory domain from SEQ ID NO:50; and / or (iv) A CD3ζ primary signaling domain consisting of the CD3ζ primary signaling domain from SEQ ID NO:50; (b) A second polypeptide, which consists of the following in the following order: (i) An anti-CLL-1 VHH antibody consisting of the amino acid sequence shown in SEQ ID NO:2; (ii) An FKBP multimerization domain polypeptide consisting of the FKBP12 multimerization domain from SEQ ID NO:14; and (iii) A CD4 transmembrane domain consisting of the CD4 transmembrane domain from SEQ ID NO:14; and (c) A bridging factor that associates with the multimerization domains of the first polypeptide and the second polypeptide and is positioned between the multimerization domains.

22. The polypeptide complex according to claim 21, wherein the bridging factor is selected from the group consisting of: AP21967, sirolimus, everolimus, novolimus, pimecrolimus, deforolimus, tacrolimus, temsirolimus, umirolimus, and zotarolimus.

23. The polypeptide complex according to claim 21, wherein when the first polypeptide and the second polypeptide are expressed, the FRB multimerization domain and the FKBP multimerization domain are located extracellularly.

24. A polynucleotide encoding the first polypeptide and the second polypeptide according to any one of claims 1 to 12, or the fusion polypeptide according to any one of claims 13 to 20.

25. A cDNA encoding the first polypeptide and the second polypeptide according to any one of claims 1 to 12, or the fusion polypeptide according to any one of claims 13 to 20.

26. An RNA encoding the first polypeptide and the second polypeptide according to any one of claims 1 to 12, or the fusion polypeptide according to any one of claims 13 to 20.

27. A vector comprising the polynucleotide according to claim 24.

28. The vector according to claim 27, wherein the vector is an expression vector.

29. The vector according to claim 27, wherein the vector is a transposon.

30. The vector according to claim 29, wherein the vector is a piggyBAC transposon or a Sleeping Beauty transposon.

31. The vector according to claim 27, wherein the vector is a viral vector.

32. The vector according to claim 31, wherein the vector is an adenovirus vector, an adeno-associated virus (AAV) vector, a herpesvirus vector, a vaccinia virus vector, or a retrovirus vector.

33. The vector according to claim 32, wherein the retrovirus vector is a lentivirus vector.

34. The vector according to claim 33, wherein the lentivirus vector is selected from the group consisting of: human immunodeficiency virus 1 (HIV-1); human immunodeficiency virus 2 (HIV-2), Visna-Maedi virus (VMV); Caprine arthritis-encephalitis virus (CAEV); Equine infectious anemia virus (EIAV); Feline immunodeficiency virus (FIV); Bovine immunodeficiency virus (BIV); and Simian immunodeficiency virus (SIV).

35. A composition comprising an unnatural hematopoietic cell according to any one of claims 1-12.

36. A composition comprising a physiologically acceptable carrier and an unnatural hematopoietic cell according to any one of claims 1-12.

37. Use of the composition according to claim 35 in the preparation of a medicament for the treatment or prevention of at least one symptom of a disorder associated with abnormal CLL-1 expression, wherein the disorder associated with abnormal CLL-1 expression is selected from lung cancer, squamous cell carcinoma, colorectal cancer, pancreatic cancer, breast cancer, thyroid cancer, bladder cancer, cervical cancer, esophageal cancer, ovarian cancer, gastric cancer, endometrial cancer, brain cancer, glioma, leukemia, lymphoma, and multiple myeloma.

38. The use according to claim 37, wherein the lung cancer is non-small cell lung cancer.

39. The use according to claim 37, wherein the squamous cell carcinoma is head and neck squamous cell carcinoma.

40. The use according to claim 37, wherein the glioma is glioblastoma or oligodendroglioma.

41. The use according to claim 37, wherein the disorder associated with abnormal CLL-1 expression is leukemia.

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