NK conjugate molecules and methods of use thereof

Abstract

Compositions are provided for activating NK cells to stimulate an immune response for treating cancer and other disorders. In one embodiment, the invention provides a compound comprising an NK engager domain that binds to CD16; an NK activation domain operably linked to the NK engager domain; and a targeting domain that selectively binds to a target cell and is operably linked to the NK activation domain and the NK engager domain, wherein the targeting domain binds to CLEC12A.

Description

[0001] Cross - Reference to Related Applications

[0002] This application claims the benefit of priority under 35 U.S.C.§119(e) of U.S. Serial No. 62 / 747,983, filed on October 19, 2018, the entire content of which is incorporated herein by reference in its entirety.

[0003] Government Support

[0004] This invention was made with government support under grants CA111412 and CA65493 awarded by the National Institutes of Health, and grants CA36725, CA72669, CA077598, and CA197292 awarded by the National Cancer Institute, and grant CA150085 awarded by the Department of Defense. The government has certain rights in this invention.

[0005] Sequence Listing

[0006] The material in the accompanying sequence listing is hereby incorporated by reference into this application. The accompanying sequence listing text file, named GTBIO2090_1WO_Sequence_Listing.txt, was created on October 15, 2019 and is 23 kb. The file can be accessed using Microsoft Word on a computer with a Windows OS. Background of the Invention Field of the Invention

[0008] The present invention generally relates to immunotherapy and, more particularly, to compositions that can be used to engage natural killer (NK) cells in an immune response. Background Art

[0009] Natural killer (NK) cells are cytotoxic lymphocytes of the innate immune system capable of performing immune surveillance. Like cytotoxic T cells, NK cells deliver large amounts of membrane - penetrating and apoptosis - inducing granzyme and perforin granules. Unlike T cells, NK cells do not require antigen priming and, in the absence of MHC recognition, recognize targets by engaging activating receptors. NK cells express CD16, an activating receptor that binds to the Fc portion of IgG antibodies and is involved in antibody - dependent cell - mediated cytotoxicity (ADCC). NK cells are regulated by IL - 15, which can induce increased antigen - dependent cytotoxicity, lymphokine - activated killer activity, and / or mediate interferon (IFN), tumor necrosis factor (TNF), and / or granulocyte - macrophage colony - stimulating factor (GM - CSF) responses. All of these IL - 15 - activated functions contribute to improved cancer defense.

[0010] In therapy, for example, when adoptive transfer of NK cells is used in combination with lymphodepleting chemotherapy and IL-2 to stimulate the survival and in vivo expansion of NK cells, adoptive transfer of NK cells can induce remission in patients with refractory acute myeloid leukemia (AML). This therapy may be limited by the lack of antigen specificity and IL-2-mediated induction of regulatory T (Treg) cells, which inhibit the proliferation and function of NK cells. Generating a reagent that drives the specificity, expansion, and / or persistence of NK cell antigens while bypassing the negative effects of Treg inhibition can enhance NK cell-based immunotherapy. SUMMARY OF THE INVENTION

[0011] The present invention relates to compounds and compositions for activating NK cells to stimulate an immune response for the treatment of cancer and other disorders. In one embodiment, the present invention provides a compound comprising an NK engagement domain; an NK activation domain operably linked to the NK engagement domain; and a targeting domain that selectively binds to a target cell and is operably linked to the NK activation domain and the NK engagement domain, wherein the targeting domain selectively binds to CLEC12A.

[0012] In some embodiments, the NK engagement domain comprises a moiety that selectively binds to CD16. In some embodiments, the NK engagement domain moiety comprises an antibody or a binding fragment thereof or a nanobody, also known as a single domain antibody (sdAb or VHH). In some embodiments, the antibody binding fragment comprises an scFv, F(ab)2, or Fab. In some embodiments, the antibody or a binding fragment thereof or a nanobody is human or humanized. In some embodiments, the antibody or a binding fragment thereof or a nanobody is camelid.

[0013] In some embodiments, the NK activation domain comprises a cytokine or a functional fragment thereof. In some embodiments, the NK activation domain comprises IL-15 or a functional fragment thereof. In some embodiments, IL-15 comprises the amino acid sequence of SEQ ID NO:9 or a functional variant thereof. In one aspect, the functional variant of IL-15 comprises an N72D or N72A amino acid substitution compared to SEQ ID NO:9.

[0014] In some embodiments, the targeting domain moiety comprises an antibody or a binding fragment thereof or a nanobody. In some embodiments, the antibody binding fragment comprises an scFv, F(ab)2, or Fab.

[0015] In some embodiments, the NK engagement domain comprises a moiety that selectively binds to CD16, the NK activation domain comprises IL-15, and the targeting domain selectively binds to CLEC12A.

[0016] In some embodiments, the compounds and compositions described herein comprise at least one flanking sequence that links two domains. In some embodiments, the compounds and compositions described herein further comprise a second flanking sequence that links the two linked domains to a third domain. In some embodiments, the flanking sequence flanks the NK activation domain. In some embodiments, the first flanking sequence is the C-terminus of the NK engagement domain, and the second flanking sequence is the N-terminus of the anti-CLEC12A targeting domain.

[0017] In some embodiments, the present disclosure provides an isolated amino acid sequence comprising SEQ ID NO.:1. In some embodiments, the present disclosure provides an isolated DNA sequence encoding the amino acid sequence of SEQ ID NO.:1.

[0018] In some embodiments, the present disclosure provides an isolated amino acid sequence comprising SEQ ID NO.:2. In some embodiments, the present disclosure provides an isolated DNA sequence encoding the amino acid sequence of SEQ ID NO.:2.

[0019] In some embodiments, the present disclosure provides an isolated amino acid sequence comprising SEQ ID NO.:4. In some embodiments, the present disclosure provides an isolated DNA sequence encoding the amino acid sequence of SEQ ID NO.:4.

[0020] In some embodiments, the present disclosure provides a composition comprising the compounds described herein and a pharmaceutically acceptable carrier.

[0021] In some embodiments, the present disclosure provides methods comprising administering to a subject an amount of the compounds described herein effective to induce NK-mediated killing of target cells. In some embodiments, the target cells are cancer cells.

[0022] In some embodiments, the present disclosure provides methods for stimulating the expansion of NK cells in vivo, the methods comprising administering to a subject an amount of the compounds described herein effective to stimulate the expansion of NK cells in the subject.

[0023] In some embodiments, provided herein are methods of treating cancer in a subject, the methods comprising: administering to the subject an amount of a compound described herein effective to treat cancer. In some embodiments, the cancer comprises prostate cancer, lung cancer, colon cancer, rectal cancer, bladder cancer, melanoma, kidney cancer, renal cancer, oral cancer, pharyngeal cancer, pancreatic cancer, uterine cancer, thyroid cancer, skin cancer, head and neck cancer, cervical cancer, ovarian cancer, or hematological cancer. In some embodiments, the methods provided herein further comprise administering the compound before, concurrently with, or after chemotherapy, surgical resection of a tumor, or radiotherapy. In some embodiments, the chemotherapy comprises altretamine, aclarubicin, L-asparaginase, asparaginase, bleomycin, busulfan, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, cyclophosphamide, ifosfamide, cytarabine, dacarbazine, actinomycin D, daunorubicin, docetaxel, doxorubicin, epirubicin, etoposide, fluorouracil, fludarabine, fotemustine, ganciclovir, gemcitabine, hydroxyurea, idarubicin, ifosfamide, irinotecan, lomustine, melphalan, mercaptopurine, methotrexate, mitoxantrone, mitomycin C, nimustine, oxaliplatin, paclitaxel, pemetrexed, procarbazine, raltitrexed, temozolomide, teniposide, thioguanine, thiotepa, topotecan, vinblastine, vincristine, vindesine, and vinorelbine. In some embodiments, the hematological cancer is AML. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figures 1A - 1C Shows NK cell proliferation ( Figure 1A ), NK cell killing ( Figure 1B ), and functional assays ( Figure 1C ) after treatment with CLEC12A TriKE.

[0025] Figure 2 Shows the percentage of CD33 and CLEC12A surface expression on primary AML samples from 10 patients.

[0026] Figures 3A - 3B Shows CD16-IL15-CLEC12A TriKE ( Figure 3A ) and mechanism of action ( Figure 3B ).

[0027] Figure 4 Shows the binding of CD16-IL15-CLEC12A TriKE to targets expressing CLEC12A.

[0028] Figures 5A - 5B Shows the promotion of NK cell proliferation by CD16-IL15-CLEC12A TriKE.

[0029] Figures 6A - 6C Shows CD16-IL15-CLEC12A TriKE-induced degranulation ( Figure 6A ) and cytokine production ( Figure 6B -C) against AML target cells.

[0030] Figures 7A - 7B Shows CD16-IL15-CLEC12A TriKE-induced killing of AML target cells.

[0031] Figures 8A - 8D Shows CD16-IL15-CLEC12A TriKE-induced killing of primary AML targets in vitro.

[0032] Figures 9A - 9C Shows CD16-IL15-CLEC12A TriKE-induced NK cell proliferation.

[0033] Figures 10A - 10D Shows the functional verification of CD16-IL15-CLEC12A TriKE.

[0034] Figures 11A - 11C Shows CD16-IL15-CLEC12A TriKE-induced killing of target cells in a real-time imaging assay. Shows THP-1 tumor targets.

[0035] Figures 12A - 12G Shows CD16-IL15-CLEC12A TriKE-induced killing of primary AML blasts.

[0036] Figures 13A - 13G Shows CD16-IL15-CLEC12A TriKE-restricted tumor growth in vivo.

[0037] Figures 14A - 14C Shows the binding verification of CD16-IL15-CLEC12A TriKE.

[0038] Figures 15A - 15B Shows CD16-IL15-CLEC12A TriKE-induced killing of target cells in a real-time imaging assay. Shows HL-60 tumor targets.

[0039] Figures 16A - 16B Shows CD16-IL15-CLEC12A TriKE-mediated target killing. Shows target gating strategy ( Figure 16A ) and target cell killing ( Figure 16B ). Shows AML blast targets.

[0040] Figure 17Shows a gating strategy for identifying cancer stem cells in bone marrow samples from AML patients.

[0041] Figures 18A - 18B Shows the expression of CLEC12A and CD33 in the CD34pos progenitor cell compartment in bone marrow. Shows cell populations from two representative donors ( Figure 18A ) and cell colonies after treatment with the indicated TriKE ( Figure 18B ).

[0042] Figure 19 Shows a gating strategy for assaying different CD34pos progenitor cell subsets in healthy bone marrow samples. Detailed Description

[0043] Natural killer (NK) cells are cytotoxic lymphocytes of the innate immune system capable of performing immune surveillance. Like cytotoxic T cells, NK cells deliver large amounts of membrane-penetrating and apoptosis-inducing granzyme and perforin granules. Different from T cells, NK cells do not require antigen priming and, in the absence of MHC recognition, recognize targets by engaging activating receptors. NK cells express CD16, an activating receptor that binds to the Fc portion of IgG antibodies and is involved in antibody-dependent cell-mediated cytotoxicity (ADCC). NK cells are regulated by IL-15, which can induce increased antigen-dependent cytotoxicity, lymphokine-activated killer activity, and / or mediate interferon (IFN), tumor necrosis factor (TNF), and / or granulocyte-macrophage colony-stimulating factor (GM-CSF) responses. All of these IL-15-activated functions contribute to improved cancer defense.

[0044] Therapeutically, for example, when adoptive transfer of NK cells is used in combination with lymphodepleting chemotherapy and IL-2 to stimulate the survival and in vivo expansion of NK cells, adoptive transfer of NK cells can induce remission in patients with refractory acute myeloid leukemia (AML). This therapy may be limited by the lack of antigen specificity and IL-2-mediated induction of regulatory T (Treg) cells, which inhibit the proliferation and function of NK cells. Generating a reagent that drives the specificity, expansion, and / or persistence of NK cell antigens while bypassing the negative effects of Treg inhibition can enhance NK cell-based immunotherapy.

[0045] The present disclosure describes the generation of trispecific molecules that comprise two domains capable of driving NK cell-mediated killing of tumor cells (e.g., CD33+ and / or CD33− tumor cells) and an intramolecular NK activation domain capable of generating NK cell self-maintenance signals. The trispecific molecules can drive NK cell proliferation and / or enhance NK cell-driven cytotoxicity against, for example, HL-60 targets, cancer cells, or cancer cell-derived cell lines.

[0046] The present invention is based on the development of a CD16 / IL-15 / CD33 trispecific killer engager (TriKE) molecule that targets acute myeloid leukemia (AML) cells using natural killer (NK) cells. The molecule contains a camelid nanobody against CD16 that activates NK cells, a single-chain variable fragment (scFv) against CD33 that binds to the cancer target, and an IL-15 molecule that drives NK cell priming, expansion, and survival. Using an earlier version of the molecule, CD33 TriKE was shown to effectively activate NK cells against AML targets in vitro and in vivo. This preclinical data has led to the establishment of a clinical trial at the University of Minnesota in patients with refractory AML, which is scheduled to begin in the third quarter of 2018. While these previous studies have validated the use of TriKE as an effective strategy for harnessing NK cells in cancer immunotherapy, CD33 has limitations as a target antigen.

[0047] The high mortality rate and poor five-year survival rate (26%) of AML patients can be attributed to chemotherapy resistance and disease relapse. It is hypothesized that most chemotherapy-resistant leukemia stem cells (LSCs) that contribute to relapse do not express CD33. In addition, all hematopoietic stem cells and normal bone marrow cells express CD33, so targeting this antigen can lead to severe hematopoietic defects and on-target / off-tumor toxicity. To address these limitations, the development of TriKEs targeting CLEC12A or C-type lectin-like molecule 1 (CLL-1) is described herein. CLEC12A is highly expressed on AML cells, and more than 70% of CD33-negative cells express CLEC12A. It has been considered a stem cell marker in AML and is selectively overexpressed in LSCs. In regenerating bone marrow, CLEC12A is expressed by CD34+ / CD38-LSCs but not by normal CD34+ / CD38-hematopoietic stem cells, thus minimizing off-target effects. C-type lectin domain family 12 member A is a human protein encoded by the CLEC12A gene. This gene encodes a member of the C-type lectin / C-type lectin-like domain (CTL / CTLD) superfamily. Members of this family share a common protein fold and have diverse functions, such as cell adhesion, intercellular signaling, glycoprotein turnover, and roles in inflammation and immune responses. The protein encoded by this gene is a negative regulator of granulocyte and monocyte function. Several alternatively spliced transcript variants of this gene have been described, but the full-length nature of some of these variants has not been determined. This gene is closely related to other CTL / CTLD superfamily members in the natural killer gene complex region on chromosome 12p13.

[0048] BiKE and TriKE Compounds

[0049] Bispecific fusions have been performed that incorporate anti-human anti-CD16 scFv derived from human phage display library technology (McCall et al., 1999, Mol Immunol, 36:433-445). NK cells mediate antibody-dependent cell-mediated cytotoxicity (ADCC) through the CD16 (FcγRIII) receptor. Signaling through the CD16 receptor induces calcium flux and phosphorylation of ITAM, triggering the release of lytic granules and cytokines such as interferon (IFNγ) and tumor necrosis factor (TNFα). A bispecific molecule has been designed to bind to other targeting molecules to trigger the CD16 receptor (Gleason et al., Blood, 2014(19):3016-26), a so-called bispecific killer engager (BiKE). Since one scFv recognizes NK cells and the other scFv recognizes a tumor antigen, BiKE can significantly enhance cytotoxic killing against a variety of human cancers. An exemplary BiKE targets CD33 and enhances NK cell responses against acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS). MDS is a clonal heterogeneous stem cell disorder characterized by a normal or hypercellular bone marrow (BM) with peripheral blood (PB) cytopenia and an increased risk of progression to AML.

[0050] NK cells respond to a variety of cytokines, including for example IL-15, which is involved in NK cell homeostasis, proliferation, survival, activation, and / or development. For example, IL-15 can activate NK cells and can restore functional defects of transplanted NK cells after hematopoietic stem cell transplantation (HSCT). IL-15 and IL-2 share several signaling components, including IL-2 / IL-15Rβ (CD122) and the common γ chain (CD132). Different from IL-2, IL-15 does not stimulate Tregs, allowing NK cell activation while bypassing Treg suppression of the immune response. In addition to promoting NK cell homeostasis and proliferation, IL-15 can also rescue NK cell functional defects that may occur in the post-transplantation setting. IL-15 can also stimulate CD8+ T cell function, further enhancing its immunotherapeutic potential. Furthermore, according to preclinical studies, the toxicity profile of IL-15 may be more favorable than that of IL-2 at low doses. According to some embodiments, the compositions described herein can be used to activate NK cells and drive NK cell priming, expansion, and survival.

[0051] In one aspect, the present disclosure describes trispecific killer engager (TriKE) molecules, which generally comprise one or more targeting domains (which target, for example, tumor cells or virus-infected cells) and one or more cytokine NK activation domains (such as IL-15, IL-12, IL-18, IL-21 or other NK cell-enhancing cytokines, chemokines, and / or activating molecules), wherein each domain is operably linked to the other domains. As used herein, the term "operably linked" means a direct or indirect covalent linkage. Thus, two operably linked domains can be directly covalently coupled to each other. Conversely, two operably linked domains can be linked by covalently attaching to an intermediate moiety (e.g., a linker and flanking sequences) to each other. Two domains can be considered to be operably linked, for example, if they are separated by a third domain with or without one or more intervening flanking sequences.

[0052] Exemplary BiKE and TriKE molecules or compounds are described in WO2017062604, the disclosure of which is incorporated herein by reference in its entirety.

[0053] In some embodiments, the present disclosure describes compounds that comprise an NK engager domain; an NK activation domain operably linked to the NK engager domain; and a targeting domain that selectively binds to a target cell and is operably linked to the NK activation domain and the NK engager domain, wherein the targeting domain selectively binds to a target molecule. For example, the target molecule can be expressed on the surface of the target cell. For example, the target cell can be a tumor cell. In some embodiments, the targeting domain selectively binds to CLEC12A.

[0054] As used herein, the term "selectively binding" or "selectively binds" with respect to the interaction of a binding molecule or domain described herein (such as an antibody or an engager domain, activation domain, or targeting domain) with its binding partner (such as an antigen or receptor) means that the interaction depends on the presence of a specific structure (such as an epitope or antigenic determinant or amino acid sequence) on the binding partner. In other words, the binding molecule or domain preferentially binds or recognizes the binding partner even if the binding partner is present in a mixture of other molecules. The binding can be mediated by covalent or non-covalent interactions or a combination of both. The terms "selectively binding" or "selectively binds" and "specifically binding" or "specifically binds" can be used interchangeably.

[0055] The compounds described herein may or may not have a His tag. For example, a His tag allows for the purification of a protein and can be used in research applications. The His tag can be located at the C-terminus or N-terminus of the compounds or molecules described herein and can include a spacer at the N-terminus or C-terminus of the His tag. As an example, a His tag placed at the C-terminus of the compounds or molecules described herein can include a spacer located at the N-terminus of the His tag. As another example, a His tag placed at the N-terminus of the compounds or molecules described herein can include a spacer located at the C-terminus of the His tag. An exemplary His tag with a spacer is SEQ ID NO:3. SEQ ID NO:3 can be placed at the C-terminus of the compounds described herein. Those skilled in the art will understand that any number of His repeats can constitute a His tag and any spacer sequence of any length can be used or no spacer can be used.

[0056] In some embodiments, the TriKE compounds or molecules that selectively bind to CLEC12A comprise the isolated amino acid sequence of SEQ ID NO:1. In some embodiments, the TriKE compounds or molecules that selectively bind to CLEC12A comprise the isolated amino acid sequence of SEQ ID NO:2. In some embodiments, the targeting domain of the compounds described herein that selectively binds to CLEC12A comprises the isolated amino acid sequence of SEQ ID NO:4.

[0057] Also described herein are nucleic acid sequences encoding the sequences of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:4. For example, SEQ ID NO:1 can be encoded by SEQ ID NO:5, SEQ ID NO:2 can be encoded by SEQ ID NO:6, and SEQ ID NO:4 can be encoded by SEQ ID NO:7. Those skilled in the art will recognize that any functional variant of the nucleic acid molecules provided herein is encompassed by the present disclosure. A functional variant is a nucleic acid sequence that can be translated to provide an amino acid sequence that is homologous or identical to the amino acid sequence translated from the parental molecule.

[0058] NK Binding Domain

[0059] The NK engaging domain can comprise any portion that binds to and / or activates NK cells and / or any portion that blocks inhibition of NK cells. Exemplary NK cell engaging domains comprise portions that bind to, for example, CD16, CD16+CD2, CD16+DNAM, or CD16+NKp46. In some embodiments, the engaging domain comprises a portion that selectively binds to CD16. In some embodiments, the NK engaging domain activates NK cells. In some embodiments, the NK engaging domain blocks inhibition of NK cells.

[0060] In some embodiments, the NK engaging domain can comprise an antibody that selectively binds to a surface component of an NK cell. In other embodiments, the NK engaging domain can comprise a ligand or small molecule that selectively binds to a surface component of an NK cell. As used herein, the term "selectively binds" refers to the ability to distinguish between two or more alternatives, such as having any degree of differential affinity for a particular target. As used herein, an "antibody" generally refers to an immunoglobulin or fragment thereof, and thus includes monoclonal antibodies, fragments thereof (e.g., scFv, Fab, F(ab')2, Fv, or other modified forms), combinations of monoclonal antibodies and / or their fragments, and / or combinations of polyclonal antibodies. Thus, for brevity, reference to an antibody that selectively binds to a surface component of an NK cell includes any antibody fragment that exhibits the described binding properties. Similarly, reference to a ligand that selectively binds to a surface component of an NK cell includes any fragment of a ligand that exhibits the described binding properties.

[0061] In some embodiments, the NK engaging domain can selectively bind to a receptor that is at least partially located on the surface of an NK cell. In certain embodiments, the NK engaging domain can function to bind the NK cell and thereby bring the NK in spatial proximity to a target to which the targeting domain selectively binds (described in more detail below). However, in certain embodiments, the NK engaging domain can selectively bind to a receptor that activates an NK cell and thus also has an activating function. As described above, activation of the CD16 receptor can trigger antibody-dependent cell-mediated cytotoxicity. Thus, in certain embodiments, the NK engaging domain can comprise at least a portion of an anti-CD16 receptor antibody that effectively and selectively binds to the CD16 receptor. In other embodiments, the NK engager domain can interrupt the mechanism that inhibits an NK cell. In such embodiments, the NK engager domain can comprise, for example, anti-PD1 / PDL1, anti-NKG2A, anti-TIGIT, anti-killer immunoglobulin receptor (KIR), and / or any other inhibitory blocking domain.

[0062] A skilled person can design the NK engagement domain to have a desired degree of NK selectivity and thus desired immunoconjugation properties. For example, CD16 has been identified as the Fc receptors FcγRIIIa (CD16a) and FcγRIIIb (CD16b). These receptors bind to the Fc portion of IgG antibodies, which then activate NK cells for antibody-dependent cell-mediated cytotoxicity. Anti-CD16 antibodies selectively bind to NK cells but can also bind to neutrophils. Anti-CD16a antibodies selectively bind to NK cells but do not bind to neutrophils. TriKE embodiments comprising an NK engagement domain (comprising an anti-CD16a antibody) can bind to NK cells but not to neutrophils. Thus, in situations where a skilled person may want to engage NK cells but not neutrophils, the skilled person can design the NK engagement domain of the TriKE to comprise an anti-CD16a antibody.

[0063] Although described herein in the context of various embodiments in which the NK engagement domain comprises an anti-CD16 receptor scFv, the NK engagement domain can comprise any antibody or other ligand that selectively binds to the CD16 receptor. Additionally, the NK engagement domain can comprise an antibody or ligand that selectively binds to any NK cell receptor, such as, for example, the cell cytotoxicity receptor 2B4, the low affinity Fc receptor CD16, killer immunoglobulin-like receptors (KIRs), CD2, NKG2A, TIGIT, NKG2C, LIR-1, and / or DNAM-1. In one embodiment, the composition of the invention is a construct operably linked NKG2C / IL-15 / CD33. It should be understood that the positions of the moieties can be altered based on activity assays (e.g., CD33 / IL-15 / NKG2C).

[0064] In some embodiments, the NK engagement domain comprises an antibody or a binding fragment thereof or a nanobody. The antibody binding fragment can be an scFv, F(ab)2, or Fab. In some embodiments, the NK engagement domain comprises a nanobody. In some embodiments, the NK cell engager can involve the use of a humanized CD16 engager derived from an animal nanobody. While an scFv has a heavy variable chain component and a light variable chain component linked by a linker, a nanobody consists of a single monomeric variable chain (i.e., a variable heavy chain or a variable light chain) capable of specifically engaging the target. Nanobodies can be derived from antibodies of any suitable animal (such as, for example, camelids (e.g., llamas or camels) or cartilaginous fish). Compared to larger antibody fragments, nanobodies can offer excellent physical stability, the ability to bind deep grooves, and increased yields.

[0065] In an exemplary embodiment, the nanobody-based NK engager molecule can involve a humanized CD16 nanobody (GeneBank sequence EF561291; Behar et al., 2008, Protein Eng Des Sel, 21(1):1-10) derived from the published Llama nanobody, designated EF91. Llama EF91 was initially constructed as a BiKE containing CD19 to test the ability of this CD16 engager to drive NK cell activation. In a chromium release assay containing Raji targets, it showed similar functionality to rituximab-mediated killing. After confirming the functionality of the molecule, the CDRs were cloned into a humanized camelid scaffold (Vincke et al., 2009, J Biol Chem, 284(5):3273-3284) to humanize the CD16 engager, now designated HuEF91. The binding of HuEF91 was equivalent to that observed using a standard CD16 scFv, indicating that integration of the Llama nanobody variable heavy chain into the humanized backbone did not impede the specificity of the molecule. Using HuEF91 as the NK engager in the TriKE molecules described herein can increase drug production, increase stability, and / or increase NK cell-mediated ADCC potency.

[0066] Thus, according to some embodiments, the antibody or its binding fragment or nanobody is human or humanized. In some embodiments, the antibody or its binding fragment or nanobody is camelid.

[0067] NK Activation Domain

[0068] The NK activation domain can comprise an amino acid sequence that activates NK cells, promotes the maintenance of NK cells, or otherwise promotes NK cell activity. The NK activation domain can be or can be derived from one or more cytokines that can activate and / or maintain NK cells. As used herein, the term "derived from" refers to an amino acid fragment of a cytokine (e.g., IL-15) that is sufficient to provide NK cell activation and / or maintenance activity. In embodiments that include more than one NK activation domain, the NK activation domains can be provided in tandem or in any other combination. Additionally, each cytokine-based NK activation domain can comprise the full amino acid sequence of the cytokine or can be an amino acid fragment, regardless of the nature of other NK activation domains included in the TriKE molecule. Exemplary cytokines on which the NK activation domain can be based include, for example, IL-15, IL-18, IL-12, and IL-21. Thus, although described in detail herein in the context of an exemplary model embodiment in which the NK activation domain is derived from IL-15, TriKE can be designed using an NK activation domain that is or is derived from any suitable cytokine.

[0069] For brevity, in this specification, the NK activation domain that comprises the full amino acid sequence of a cytokine, any suitable amino acid fragment of a cytokine, and / or a modified version of a cytokine that contains one or more amino acid substitutions is referred to by identifying the cytokine on which the NK activation domain is based. Thus, reference to an "IL-15" NK activation domain includes an NK activation domain that comprises the full amino acid sequence of IL-15, an NK activation domain that comprises a fragment of IL-15, or an NK activation domain that contains an amino acid substitution compared to the wild-type IL-15 amino acid sequence, such as, for example, IL-15N72D or IL-15N72A.

[0070] Use of the IL-15 NK activation domain in TriKE can provide sustained NK cell activity (as demonstrated in a mouse model that showed a significant elevation of human NK cells and a reduction in cancer), even after three weeks. NK cells are activated in the mouse to produce a range of anti-cancer factors and cytokines. Additionally, the IL-15 NK activation domain can alter the chemical properties of these molecules such that they are more readily refolded and / or recovered in higher yields, making the TriKE molecule more suitable for clinical scale-up.

[0071] Thus, in some embodiments, the NK activation domain comprises a cytokine or a functional fragment thereof. In some embodiments, the activation domain comprises IL-15 or a functional fragment thereof. In some embodiments, IL-15 is wild-type IL-15. In some embodiments, IL-15 is human. In some embodiments, IL-15 is wild-type human IL-15. In some embodiments, IL-15 comprises the amino acid sequence of SEQ ID NO:9 or a functional variant thereof. In some embodiments, compared to SEQ ID NO:9, the functional variant of IL-15 comprises an N72D or N72A amino acid substitution.

[0072] As used herein, the term "functional variant" refers to a molecule (including a binding molecule) that, for example, comprises a nucleotide and / or amino acid sequence that is altered by one or more nucleotides and / or amino acids compared to the nucleotide and / or amino acid sequence of the parental molecule. For a binding molecule, the functional variant is still capable of competing with the parental binding molecule for binding to the binding partner. In other words, the modifications in the amino acid and / or nucleotide sequence of the parental binding molecule do not significantly affect or alter the binding properties of the binding molecule encoded by the nucleotide sequence or containing the amino acid sequence, i.e., the binding molecule is still capable of recognizing and binding its target. Functional variants can have conservative sequence modifications, including nucleotide and amino acid substitutions, additions, and deletions. These modifications can be introduced by standard techniques known in the art, such as site-directed mutagenesis and random PCR-mediated mutagenesis.

[0073] Functional variants can also include, but are not limited to, derivatives that are substantially similar in primary structure sequence but contain, for example, in vitro or in vivo chemical and / or biochemical modifications not found in the parental binding molecule. Such modifications include, in particular, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of a covalent cross-link, formation of cysteine, formation of pyroglutamate, formylation, γ-carboxylation, glycosylation, GPI-anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, polyethylene glycolylation, proteolytic processing, phosphorylation, prenylation, racemization, selenylation, sulfation, transfer-RNA-mediated addition of an amino acid to a protein such as arginylation, ubiquitination, etc.

[0074] Targeting Domain and Target

[0075] The targeting domain can comprise any portion that selectively binds to an intended target, such as, for example, a tumor cell, a target in the cancer stroma, a target on an inhibitory cell such as a myeloid-derived inhibitory cell that is CD33+, or a target on a virus-infected cell. Thus, the targeting domain can comprise, for example, anti-tumor antibodies such as rituximab (anti-CD20), ofatumumab (anti-CD20), trastuzumab (anti-HER2 / neu), pertuzumab (anti-HER2 / neu), lapatinib (anti-CEA), adecatumumab (anti-EpCAM), pasotuxizumab (anti-EpCAM), eculizumab (anti-EpCAM), asimadoline (anti-CEA), bevacizumab (anti-VEGF-A), cetuximab (anti-EGFR), nimotuzumab (anti-EGFR), panitumumab (anti-EGFR), zalutumumab (anti-EGFR), gemtuzumab (anti-CD33), lintuzumab (anti-CD33), edrecolomab (anti-integrin α v β 3 ), inotuzumab (anti-CD51), ipilimumab (anti-CD152), oregovomab (anti-CA-125), fotumumab (anti-tumor antigen CTAA16.88), or petumamab (anti-MUC1), anti-CD19, anti-CD22, anti-CD133, anti-CD38, anti-mesothelin, anti-ROR1, CSPG4, SS1, or IGFR1. Any tumor marker can be targeted. In some embodiments, the targeting domain or the targeted tumor-associated antigen can comprise CD133, CD20, HER2, CEA, EpCAM, VEGF-A, EGFR, CD33, integrin αVβ3, CD51, CD152, CD125, CTAA16.88, MUC1, CD19, CD22, CD38, mesothelin, ROR1, CSPG4, SS1, or IGFR1, NKG2 family members, such as including but not limited to 2A, 2B, 2C, etc., BCMA, APRIL, B7H3, and PSMA.

[0076] In some embodiments, the target cell is a tumor cell. In some embodiments, the tumor cell is CD33+. In some embodiments, the tumor cell is CD33-. In some embodiments, the tumor cell is a hematopoietic cancer cell. In some embodiments, the tumor cell is a leukemia cell. In some embodiments, the leukemia cell is an acute myeloid leukemia (AML) cell. In other embodiments, the targeting domain can selectively bind to a target on a virus-infected cell, such as the virus being, for example, EBV, HBV, HCV, and / or HPV. In some embodiments, the viral target is a tumor marker or a tumor antigen. Any viral tumor marker or viral or non-viral tumor antigen can be targeted.

[0077] As described above, the targeting domain portion can comprise an antibody or a binding fragment of an antibody or a nanobody. Antibody binding fragments can include scFv, F(ab)2 or Fab.

[0078] In certain specific embodiments, the targeting domain can comprise an anti-CLEC12A antibody. In other specific embodiments, a second targeting domain can be included. The second targeting domain can comprise a portion that can bind to any of the targets described above. In some embodiments, the second targeting domain can selectively bind to CD33.

[0079] In some embodiments, the compounds described herein comprise an NK engager domain having a portion that selectively binds to CD16, an activation domain having IL-15, and a targeting domain that selectively binds to CLEC12A. The terms "CLEC12A Trike", "1615CLEC12A TriKe", and "CD16-IL15-CLEC12A TriKE" can be used interchangeably to refer to a TriKE targeting CLEC12A, unless the context clearly indicates otherwise.

[0080] Flanking Sequences

[0081] In some embodiments, the compounds described herein can further comprise a flanking sequence or linker sequence that can connect the two domains described above. Unless the context clearly indicates otherwise, the terms "flanking sequence" and "linker sequence" can be used interchangeably. In some embodiments, the presence of the flanking sequence can further increase NK cell activation. Any amino acid sequence can be a flanking sequence or linker sequence. An exemplary flanking sequence comprises 20 amino acids of SEQ ID NO:13. Another exemplary flanking sequence comprises seven amino acids of SEQ ID NO:14. Yet other exemplary flanking sequences comprise SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:15. As yet another example, any number of repeats of an amino acid sequence can be a flanking sequence or linker sequence. For example, any number of repeats of the sequence of SEQ ID NO:15 can be a flanking sequence or linker sequence. The sequence repeats can be complete or partial, and the complete or partial repeats can be at the beginning (i.e., at the N-terminus) or end (i.e., at the C-terminus) of the flanking sequence or linker sequence. The flanking sequence can be in any orientation.

[0082] Certain embodiments (e.g., 1615 CLEC12A TriKE without His-tag, SEQ ID NO:1 or 1615 CLEC12A TriKE with His-tag, SEQ ID NO:2) can include more than one flanking sequence. As an example, SEQ ID NO:1 and / or SEQ ID NO:2 includes the flanking sequence of SEQ ID NO:11 to link the NK engaging domain (e.g., anti-CD16 receptor scFv) to the NK activating domain (e.g., IL-15). SEQ ID NO:1 and / or SEQ ID NO:2 also includes the flanking sequence of SEQ ID NO:12 to link the NK activating domain to the targeting domain (e.g., anti-CLEC12A scFv). The flanking sequences linking the domains of the linker molecule can be the same or can be different. As an example, the same or different flanking sequences can link the NK engaging domain (e.g., anti-CD16 receptor scFv) to the NK activating domain (e.g., IL-15) and link the NK activating domain to the targeting domain (e.g., anti-CLEC12A scFv). In some embodiments, constructs lacking flanking sequences exhibit reduced activity compared to constructs having flanking sequences.

[0083] In some embodiments, the compounds described herein include at least one flanking sequence linking two domains. In some embodiments, the compounds described herein further include a second flanking sequence linking the two linked domains to a third domain. In some embodiments, the flanking sequences are the same. In some embodiments, the flanking sequences are different.

[0084] In some embodiments, the flanking sequence flanks the NK activating domain. In some embodiments, the first flanking sequence is the C-terminus of the NK engaging domain. In some embodiments, the second flanking sequence is the N-terminus of the anti-CLEC12A targeting domain. In some embodiments, the first flanking sequence is the C-terminus of the NK engaging domain and the second flanking sequence is the N-terminus of the anti-CLEC12A targeting domain.

[0085] Formulations

[0086] The compounds described herein can be formulated with a pharmaceutically acceptable carrier. As used herein, "carrier" includes any solvent, dispersion medium, vehicle, coating, diluent, antibacterial and / or antifungal agent, isotonic agent, absorption delaying agent, buffer, carrier solution, suspension, colloid, etc. The use of such media and / or agents for pharmaceutical active substances is well known in the art. Their use in therapeutic compositions is also contemplated, except in cases where any conventional medium or agent is incompatible with the active ingredient. Supplementary active ingredients can also be incorporated into the compositions. As used herein, "pharmaceutically acceptable" means a material that is not biologically or otherwise undesirable, i.e., the material can be administered to an individual together with the TriKE molecule without causing any undesirable biological effects or interacting in a harmful manner with any other component of the pharmaceutical composition containing the material.

[0087] Accordingly, TriKE molecules can be formulated into pharmaceutical compositions. The pharmaceutical compositions can be formulated into a variety of forms suitable for the preferred route of administration. Thus, the compositions can be administered by known routes, including, for example, orally, parenterally (e.g., intradermal, transdermal, subcutaneous, intramuscular, intravenous, intraperitoneal, etc.) or topically (e.g., intranasal, intralung, intramammary, intravaginal, intrauterine, intradermal, transdermal, rectal, etc.). The pharmaceutical compositions can be administered to mucosal surfaces, such as by administration to, for example, the nasal or respiratory mucosa (e.g., by spray or aerosol). The compositions can also be administered by sustained release or delayed release.

[0088] Accordingly, TriKE molecules can be provided in any suitable form, including but not limited to solutions, suspensions, emulsions, sprays, aerosols, or any form of mixture. The composition can be delivered in dosage form with any pharmaceutically acceptable excipient, carrier, or vehicle. For example, the dosage form can be delivered in conventional topical forms, such as, for example, creams, ointments, aerosol formulations, non-aerosol sprays, gels, lotions, etc. The dosage form can further comprise one or more additives, which include, for example, adjuvants, skin penetration enhancers, colorants, fragrances, flavoring agents, humectants, thickening agents, etc.

[0089] The dosage form can conveniently be provided in unit dosage form and can be prepared by methods well known in the pharmaceutical art. The method of preparing a composition with a pharmaceutically acceptable carrier involves the step of associating the TriKE molecule with a carrier that constitutes one or more accessory ingredients. In general, the dosage form can be prepared by uniformly and / or intimately associating the active molecule with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product into the desired dosage form.

[0090] Methods of Treatment

[0091] In some embodiments, provided herein is a method that includes administering to a subject a compound or molecule described herein in an amount effective to induce NK-mediated killing of target cells. Any cell can be a target cell. In some embodiments, the target cell is a cancer cell. The methods described herein can include administering a TriKE molecule to a subject in an amount effective to induce NK-mediated killing of target cells in the subject. In some embodiments, the TriKE molecule is administered to treat a disease or disorder in the subject.

[0092] As used herein, "treatment" or a variant thereof refers to alleviating, limiting progression, improving, or resolving symptoms or signs associated with a disorder to any extent. As used herein, "improve" refers to any reduction in the degree, severity, frequency, and / or likelihood of the characteristics of the symptoms or clinical signs of a particular disorder; "symptom" refers to any subjective evidence of a disease or a patient's disorder; and "sign" or "clinical sign" refers to an objective physical finding related to a particular disorder that can be detected by someone other than the subject or patient.

[0093] As used herein, the term "subject" refers to any individual or patient to whom the methods disclosed herein are performed. The term "subject" can be used interchangeably with the term "individual" or "patient". A "subject" can be any animal, such as, for example, a mammal (e.g., dog, cat, horse, cow, sheep, goat, monkey, etc.). In certain embodiments, the subject can be a human.

[0094] "Treatment" can be therapeutic or prophylactic. "Therapeutic" and its variants refer to a treatment that improves one or more existing symptoms or clinical signs associated with a disorder. "Prophylactic" and its variants refer to a treatment that limits the development and / or occurrence of symptoms or clinical signs of a disorder to any extent. Generally, "therapeutic" treatment begins after a disorder has manifested in a subject, while "prophylactic" treatment begins before a disorder has manifested in a subject. Thus, in certain embodiments, the methods can include prophylactic treatment of a subject at risk of developing a disorder. "At risk" means that a subject may or may not actually have the risk. Thus, for example, a subject "at risk" of developing a particular disorder is a subject having one or more markers that increase the risk of having or developing the particular disorder, compared to an individual lacking one or more of the markers, regardless of whether the subject exhibits any symptoms or clinical signs of having or developing the disorder. Exemplary markers of a disorder can include, for example, genetic predisposition, lineage, age, gender, geographical location, lifestyle, or medical history. Treatment can also continue after symptoms have subsided, for example, to prevent or delay their recurrence.

[0095] In other embodiments, provided herein are methods for the in vivo stimulation of NK cell expansion, which comprise administering to a subject a compound or molecule described herein in an amount effective to stimulate NK cell expansion in the subject. In some embodiments, a TriKE molecule is administered to treat a disease or disorder in a subject. Using a TriKE molecule as part of an in vivo therapy can endow NK cells with antigen specificity while co-stimulation, survival enhancement, and expansion occur, which may be antigen specific. In other instances, TriKE can be used in vitro as an adjuvant for NK cell adoptive transfer therapy.

[0096] In other embodiments, provided herein are methods for treating cancer, which comprise administering to a subject a compound or molecule described herein that is effective to treat cancer. In some embodiments, the cancer is prostate cancer, lung cancer, colon cancer, rectal cancer, bladder cancer, melanoma, kidney cancer, renal cancer, oral cancer, pharyngeal cancer, pancreatic cancer, uterine cancer, thyroid cancer, skin cancer, head and neck cancer, cervical cancer, ovarian cancer, or hematopoietic cancer. In some embodiments, the hematopoietic cancer is myelodysplastic syndrome (MDS). In some embodiments, the hematopoietic cancer is lymphoma. In some embodiments, the hematopoietic cancer is leukemia. In some embodiments, the hematopoietic cancer is acute myeloid leukemia (AML).

[0097] As used herein, the term “myeloid leukemia” refers to leukemia characterized by the proliferation of myeloid tissue and an abnormal increase in the number of granulocytes, myelocytes, and promyelocytes in the circulating blood. The term is synonymous with the terms myelocytic leukemia, myelogenous leukemia, myeloid leukemia, and granulocytic leukemia. The term “myeloid leukemia” can particularly denote acute and chronic myeloid leukemia (AML and CML), acute promyelocytic leukemia (APL), chronic myelomonocytic leukemia (“CMML”), myelodysplastic syndrome, and juvenile myelomonocytic leukemia, which involve the myeloid components of the bone marrow (e.g., white blood cells, red blood cells, and megakaryocytes), and encompasses all subtypes defined by morphological, histochemical, and immunological techniques well known to those of skill in the art. Subtypes of AML include FAB-M0, FAB-M1, FAB-M2, FAB-M3, FAB-M4, FAB-M5, FAB-M6, and FAB-M7 according to the FAB classification.

[0098] As used herein, the term "myelodysplastic syndromes" encompasses a heterogeneous group of closely related clonal hematopoietic disorders that originate from early hematopoietic cells in the bone marrow. All of the disorders are characterized by impaired morphological and maturation of cells in the bone marrow (dysmyelopoiesis) and peripheral cytopenia, which is caused by ineffective hematopoiesis. In other words, mature blood cells typically die in the bone marrow before they reach full maturity and enter the bloodstream, which is the reason for the low concentration of blood cells. In patients with myelodysplastic syndromes, there may also be an accumulation of very immature bone marrow cells (referred to as leukemic blasts).

[0099] The amount of TriKE molecule administered can vary according to various factors, including but not limited to the specific TriKE molecule used, the body weight, physical condition and / or age of the subject, and / or the route of administration. Thus, the absolute weight of the TriKE molecule contained in a given unit dosage form may vary widely and depends on factors such as the species, age, body weight and physical condition of the subject, and / or the method of administration. Therefore, it is generally not practical to set forth an amount of TriKE molecule that is effective for all possible applications. However, a person of ordinary skill in the art can readily determine an appropriate amount by duly considering these factors.

[0100] In some embodiments, the method may comprise administering an amount of TriKE molecule sufficient to provide a dose to the subject, for example, of from about 100 ng / kg to about 50 mg / kg, although in some embodiments the method may be carried out by administering a dose of TriKE molecule outside of this range. In some of these embodiments, the method comprises administering an amount of TriKE molecule sufficient to provide a dose to the subject of from about 10 μg / kg to about 5 mg / kg, for example, from about 100 μg / kg to about 1 mg / kg.

[0101] Alternatively, the actual body weight obtained just prior to the start of the treatment course may be used to calculate the dose. For doses calculated in this manner, the body surface area (m2) is calculated using the Dubois method prior to the start of the treatment course: m2 = (wt kg0.425 × height cm0.725) × 0.007184.

[0102] In some embodiments, the method may comprise administering an amount of TriKE molecule sufficient to provide a dose, for example, of from about 0.01 mg / m2 to about 10 mg / m2.

[0103] In some embodiments, a single dose up to multiple doses of TriKE molecule may be administered, for example, weekly, although in some embodiments the method may be carried out by administering TriKE molecule at a frequency outside of this range. In certain embodiments, the TriKE molecule may be administered from about once a month to about five times a week.

[0104] In some embodiments, the method further comprises administering one or more additional therapeutic agents. The one or more additional therapeutic agents may be administered before, after, and / or concurrently with the administration of the TriKE molecule. The TriKE molecule and the additional therapeutic agent(s) may be co-administered. As used herein, "co-administered" refers to two or more components administered in combination such that the combined therapeutic or prophylactic effect is greater than the therapeutic or prophylactic effect of any one component administered alone. The two components may be co-administered simultaneously or sequentially. Components co-administered simultaneously may be provided in one or more pharmaceutical compositions. Sequential co-administration of two or more components includes situations in which the components are administered such that each component may be present at the site of treatment simultaneously. Alternatively, sequential co-administration of two components may include situations in which at least one component has cleared from the site of treatment, but at least one cellular effect of the administered component (e.g., cytokine production, activation of a particular cell population, etc.) persists at the site of treatment until one or more additional components are administered to the site of treatment. Thus, in certain cases, a co-administered combination may include components that never exist in a chemical mixture with each other. In other embodiments, the TriKE molecule and the additional therapeutic agent(s) may be administered as part of a mixture or admixture. In some aspects, administering the TriKE molecule may permit a lower dose of other therapeutic modalities to be effective compared to administering one or more other therapeutic agents alone, thereby reducing the likelihood, severity, and / or extent of toxicity observed when higher doses of one or more other therapeutic agents are administered.

[0105] Exemplary additional therapeutic agents include, for example, altretamine, aclarubicin, L-asparaginase, asparaginase, bleomycin, busulfan, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, cyclophosphamide, ifosfamide, cytarabine, dacarbazine, actinomycin D, daunorubicin, docetaxel, doxorubicin, epirubicin, etoposide, fluorouracil, fludarabine, fotemustine, ganciclovir, gemcitabine, hydroxyurea, idarubicin, ifosfamide, irinotecan, lomustine, melphalan, mercaptopurine, methotrexate, mitoxantrone, mitomycin C, nimustine, oxaliplatin, paclitaxel, pemetrexed, procarbazine, raltitrexed, temozolomide, teniposide, thioguanine, thiotepa, topotecan, vinblastine, vincristine, vindesine, and vinorelbine.

[0106] Thus, in some embodiments, the methods for treating cancer provided herein further comprise administering the compounds, molecules, compositions or formulations described herein before, concurrently with, or after chemotherapy, surgical resection of a tumor, or radiotherapy. Chemotherapy can include, for example, altretamine, aclarubicin, L-asparaginase, asparaginase, bleomycin, busulfan, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, cyclophosphamide, ifosfamide, cytarabine, dacarbazine, actinomycin D, daunorubicin, docetaxel, doxorubicin, epirubicin, etoposide, fluorouracil, fludarabine, fotemustine, ganciclovir, gemcitabine, hydroxyurea, idarubicin, ifosfamide, irinotecan, lomustine, melphalan, mercaptopurine, methotrexate, mitoxantrone, mitomycin C, nimustine, oxaliplatin, paclitaxel, pemetrexed, procarbazine, raltitrexed, temozolomide, teniposide, thioguanine, thiotepa, topotecan, vinblastine, vincristine, vindesine, and vinorelbine.

[0107] In some embodiments, the methods provided herein can include administering a sufficient amount of a TriKE molecule as described herein and administering at least one additional therapeutic agent, wherein administering the TriKE molecule and the at least one additional therapeutic agent demonstrates a therapeutic synergy. In some aspects of the methods of the invention, a measurement of the response to treatment observed after administering a TriKE molecule as described herein and an additional therapeutic agent is improved over the same measurement of the response to treatment observed after administering the TriKE molecule or the additional therapeutic agent alone. In some embodiments, the additional therapeutic agent can include an additional agent that targets EpCAM, including, for example, an EpCAM-specific monoclonal antibody, such as, for example, catumaxomab, a monoclonal hybrid antibody that targets EpCAM and CD3.

[0108] As used herein, the term "and / or" refers to one or all of the listed elements or any combination of two or more of the listed elements; the terms "comprises", "comprising", and variations thereof should be construed as open-ended, i.e., additional elements or steps are optional and may or may not be present.

[0109] As used herein, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Unless otherwise stated, "a", "an", "the", and "at least one" can be used interchangeably and can mean one or more than one. Thus, for example, reference to "the method" includes one or more methods and / or steps of the type described herein, which will be apparent to those skilled in the art after reading this disclosure and the like.

[0110] As used herein, the recitation of a numerical range by endpoints includes all numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).

[0111] When referring to measurable values such as amounts, periods of time, etc., "about" as used herein means a variation of ±20%, or ±10%, or ±5%, or even ±1% of the specified value, because such variations are appropriate for the disclosed methods or for carrying out the disclosed methods.

[0112] 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.

[0113] As used herein, the term "protein" refers to any polymer chain of amino acids. The terms "peptide" and "polypeptide" may be used interchangeably with the term "protein" and also refer to a polymer chain of amino acids. The term "protein" includes natural or artificial proteins, protein fragments, and polypeptide analogs of protein sequences. Proteins can be monomeric or polymeric. The term "protein" includes its fragments and variants (including fragments of variants), unless the context is otherwise contradictory.

[0114] As used herein, the term "nucleic acid" refers to any deoxyribonucleic acid (DNA) molecule, ribonucleic acid (RNA) molecule, or nucleic acid analog. DNA or RNA molecules can be double-stranded or single-stranded and can be of any size. Exemplary nucleic acids include, but are not limited to, chromosomal DNA, plasmid DNA, cDNA, cell-free DNA (cfDNA), mRNA, tRNA, rRNA, siRNA, microRNA (miRNA or miR), hnRNA. Exemplary nucleic acid analogs include peptide nucleic acid, morpholino, and locked nucleic acid, glycol nucleic acid, and threose nucleic acid.

[0115] In the foregoing description, specific embodiments may be described separately for clarity. Unless otherwise explicitly specified that the features of a particular embodiment are incompatible with those of another embodiment, certain embodiments may include combinations of compatible features described herein in connection with one or more embodiments.

[0116] For any method disclosed herein that includes discrete steps, those steps may be carried out in any feasible order. Additionally, where appropriate, any combination of two or more steps may be carried out simultaneously.

[0117] The present invention is illustrated by the following examples. It should be understood that the specific examples, materials, amounts, and procedures should be construed broadly in accordance with the scope and spirit of the present invention as described herein.

[0118] Examples

[0119] Example 1

[0120] This example describes the development of CD16-IL15-CLEC12A TriKE.

[0121] 1615CLEC12A TriKE was developed in a mammalian cell system to ensure the presence of appropriate post-translational modifications. Specific binding of TriKE to HL 60 and THP 1 target cells expressing CLEC12A was confirmed compared to Raji cells that do not express CLEC12A. Treatment of peripheral blood mononuclear cells (PBMC) with 1615CLEC12A TriKE drove a significant increase in NK cell-specific proliferation over 7 days compared to treatment with either CLEC12A scFv or IL-15 alone, as measured by CellTrace dilution (69.7 ± 6.7% / 11.9 ± 2.5% / 38.4 ± 7.3%) ( Figure 1A ). To measure NK cell killing, an IncuCyte Zoom assay was performed. Here, HL-60 target cells were labeled with caspase 3 / 7 reagent, where a color change indicates target cell death. 1615CLEC12A TriKE was able to induce more target cell killing than CLEC12A scFv or IL-15, as determined by the number of live target cells at the end of the 48-hour assay (53.9 ± 1.9% / 103.3 ± 3.4% / 71.1 ± 1.4%). 1615CLEC12A TriKE induced an increase in NK cell degranulation compared to treatment with either CLEC12A scFv or IL-15 alone, as measured by CD107a expression against HL-60 AML tumor targets in a 4-hour functional assay (62.3 ± 1.1% / 19.4 ± 3.8% / 27.5 ± 4.9%). In this assay, there was also an increase in cytokine production, measured by IFNg and TNFa respectively (16.7 ± 4.2% / 2.3 ± 1.5% / 4.7 ± 1.9% and 18.0 ± 5.1% / 2.5 ± 1.7% / 4.6 ± 2.5%) ( Figure 1B)。Similar enhanced functional responses were observed against the THP-1 AML tumor target. In these functional assays, treatment with 1615CLEC12A TriKE produced less background activation compared to CD33 TriKE, indicating a smaller off-target effect on PBMCs. To confirm the clinical relevance of this molecule, the potency of 1615CLEC12A TriKE against primary AML targets was tested. AML blasts were identified as SSC-low, CD45-intermediate, and CD34-high cells. Among the 9 AML samples tested, 7 expressed high levels of CD33 (70.4 ± 6.3%) and CLEC 12a (78.1 ± 5.2%). In functional assays performed on these samples, 1615CLEC12A TriKE was able to induce higher CD107a and IFNγ expression and enhanced killing of tumor targets as measured by the live / dead staining method ( Figure 1C )。In these assays, the potency of 1615CLEC12A TriKE was comparable to that of CD33 TriKE. These data indicate that 1615CLEC12A TriKE drives NK cell-specific proliferation, degranulation, cytokine secretion, and killing of tumor targets in vitro. In addition to AML, CLEC12A is also expressed on cancer cells and LSCs from patients with myelodysplastic syndromes (MDS). These findings highlight the clinical potential of 1615CLEC12A TriKE alone or in combination with CD33 TriKE for the treatment of MDS and AML.

[0122] Example 2

[0123] This example describes the expression of CD33 and CLEC12A on AML cells.

[0124] Most of all deaths from hematopoietic malignancies are caused by acute myeloid leukemia (AML), which has a poor five-year survival rate of 26%, highlighting the need for new therapies. The most common antigen used to target AML cells is CD33. However, there are many limitations to the development of anti-CD33 therapies. For example, not all cancer cells express CD33, including cancer cells in patients with refractory AML. In addition, all cells of the myeloid lineage and some cells of the lymphoid lineage such as activated NK cells and T cells express CD33, resulting in off-target toxicity. Furthermore, cancer stem cells, which are thought to contribute to relapse, do not express CD33.

[0125] A new antigen called C-type lectin-like molecule 1 (CLL-1) or CLEC12A aims to address the above limitations.

[0126] Figure 2Shows the percentage of CD33 and CLEC12A surface expression measured by flow cytometry analysis of primary AML samples from 10 patients. CLEC12A is highly expressed on AML cells. Approximately 70% of CD33-negative cells express CLEC12A. The expression of CLEC12A is restricted to a subset of myeloid cells, limiting off-target toxicity. CLEC12A is present on leukemia stem cells but not on hematopoietic stem cells.

[0127] These data establish CLEC12A as a surface marker on primary AML cells expressing CD33 and lacking CD33 expression. Thus, according to some embodiments, CLEC12A can be targeted by trispecific killer engager (TriKE) molecules on AML and other cells expressing or lacking CD33.

[0128] Example 3

[0129] This example describes a trispecific killer engager (TriKE) molecule targeting CLEC12A.

[0130] To target cancer cells using natural killer (NK) cells, a trispecific killer engager (TriKE) molecule was developed that contains an anti-CD16 heavy chain antibody that activates NK cells, an IL-15 molecule that drives NK cell priming, expansion, and survival, and an anti-CLEC12A single-chain variable fragment (scFv) that engages the cancer target. A schematic of the CD16-IL15-CLEC12A TriKE and mechanism of action is shown in Figure 3A -B.

[0131] TriKE contains an anti-CD16 heavy chain antibody ( HH ) constructed by integrating the CDRs of a camelid anti-CD16 V HH into a humanized V Figure 3A backbone. This is linked to a wild-type IL-15 molecule, which is connected to the scFv from an anti-CLEC12A antibody. TriKE (SEQ ID NO.:1) was produced in a mammalian system containing Expi-293 cells and contains a His tag for purification of the molecule. According to some embodiments, the TriKE molecule may lack the His tag. The TriKE molecule lacking the His tag may be suitable for clinical applications, although TriKE containing the His tag can also be used. TriKE forms an immunological synapse between CLEC12A+ tumor cells and NK cells, promoting the release of cytotoxic granules and the secretion of cytokines that kill target cells ( Figure 3B ).

[0132] In addition to the scFv from the anti-CLEC12A antibody (SEQ ID NO.: 4; SC02-357 corresponding to U.S. Patent No. 7,741,443) as an illustrative example above, the TriKE targeting domain can also include any sequence capable of targeting or binding to CLEC12A, such as scFvs SC02-378 and SC02-161 and any derivatives of scFvs SC02-357, SC02-378, and SC02-161. scFvs SC02-357, SC02-378, and SC02-161 are described in U.S. Patent No. 7,741,443, the disclosure of which is incorporated herein in its entirety, particularly with respect to the sequences of scFvs SC02-357, SC02-378, and SC02-161.

[0133] Example 4

[0134] This example describes the verification of the binding of CD16-IL15-CLEC12A TriKE to target cells.

[0135] CLEC12A+HL-60 and CLEC12A-Raji targets were incubated with equimolar concentrations of 1615CLEC12A TriKE or scFv. Binding was evaluated by an anti-His antibody that binds to the His tag on the TriKE or scFv. A secondary streptavidin antibody detected by flow cytometry was used. Figure 4 The data in show that 1615CLEC12A TriKE binds to the HL-60 target but not to the Raji target.

[0136] ELISAs directed against CD16 ( Figure 14A ), IL15 receptor α ( Figure 14B ), and the extracellular domain (ED) of CLEC12A ( Figure 14C ) were used to test the binding of the different components of 1615CLEC12A TriKE. Unless otherwise stated, TriKE was tested in 3-fold serial dilutions from 900 nM to 0.4 nM and had the highest binding at 30 nM, which was used for subsequent experiments.

[0137] These data indicate that each component of 1615CLEC12A TriKE binds to its respective target molecule and that CD16-IL15-CLEC12A TriKE specifically binds to targets expressing CLEC12A.

[0138] Example 5

[0139] This example describes the NK cell proliferation induced by CD16-IL15-CLEC12A TriKE.

[0140] PBMCs were labeled with Cell Trace and incubated with equimolar concentrations of IL-15, CLEC12A scFv, or CLEC12A TriKE for 7 days ( Figure 5A ). NK cell populations were evaluated by estimating the dilution of the Cell Trace dye in the CD56+CD3- population using flow cytometry ( Figure 5B ). The percentage of proliferating NK cells was calculated using FlowJo Analyzer. Statistical data reflect significant differences between groups calculated by one-way AVONA, *P<0.05, **P<0.005, N = 10. Higher percentages of proliferating NK cells were observed in the presence of CLEC12A TriKE compared to IL-15 or CLEC12A scFv.

[0141] In other experiments, PBMCs were isolated from fresh healthy donor samples (n = 6), labeled with CellTrace Violet, and incubated for 7 days with 30 nM of 1615 CLEC12A TriKE or control treatment as described above. After the incubation period, cells were harvested and NK cell (CD3-, CD56+) proliferation was estimated by flow cytometry.

[0142] Pooled data ( Figure 9A ) and representative histograms ( Figure 9B ) show NK cell proliferation (by CellTrace dilution) in different treatment groups. Figure 9C Mixed NK cell counts at harvest (at a constant rate of 45 seconds) are shown. One-way analysis of variance (ANOVA) with repeated measures was used to calculate differences compared to the 1615 CLEC12A group. Error bars represent + / - standard error of the mean. Statistical significance was determined as **P<0.005, ****P<0.0001. Significantly higher percentages of proliferating NK cells were observed after treatment with CLEC12A TriKE compared to no treatment, treatment with IL-15, or treatment with CLEC12A scFv.

[0143] These data indicate that 1615 CLEC12A TriKE induces potent NK cell proliferation.

[0144] Example 6

[0145] This example describes the functional validation of CD16-IL15-CLEC12A TriKE.

[0146] In the presence of equimolar concentrations of IL-15, CLEC12A scFv, or CLEC12A TriKE, PBMC were incubated with CLEC12A+HL-60 and THP1 cells at an effector-to-target ratio of 2:1. Surface CD107a was evaluated on CD56 + CD3 - NK cells to estimate degranulation ( Figure 6A ), intracellular IFNg ( Figure 6B ), and TNFa to estimate the production of inflammatory cytokines ( Figure 6C ). Statistical data comparing treatment with CLEC12A TriKE to treatment with IL-15 or CLEC12A scFv controls reflected significant differences between groups calculated as using one-way ANOVA, **P<0.005, N = 6. Higher percentages of surface CD107a staining and higher percentages of intracellular staining for IFNg and TNFa were observed on NK cells in the presence of CLEC12A TriKE compared to no treatment, treatment with IL-15, or treatment with CLEC12A scFv of HL60 and THP1 target cells.

[0147] In other experiments, cryopreserved PBMC from healthy donors (n = 6) were incubated with the indicated treatments (30 nM) to estimate CD107a expression as a marker of degranulation ( Figure 10A ), intracellular IFNg production ( Figure 10B ), or intracellular TNFa expression in NK cells (CD3-, CD56+; Figure 10C ) in 4-hour assays. Cells were estimated alone or in the presence of THP1 and HL-60 targets at an effector / target ratio of 2:1. Activation of NK cells (CD3-, CD56+) in PBMC was estimated using CD69 expression in 4-hour assays alone or in the presence of THP1 and HL-60 targets at an effector / target ratio of 2:1 ( Figure 10D ). One-way analysis of variance (ANOVA) with repeated measures was used to calculate differences for the 1615 CLEC12A group. Error bars represent + / - standard error of the mean. Statistical significance was determined as *P,.05, **P,.01, ***P,.001, and ****P,.0001.

[0148] Greater NK cell activation was observed when incubated with CLEC12A TriKE compared to no treatment, treatment with IL-15, or treatment with scFv of THP1 target cells and HL60 target cells, as demonstrated by increased staining for CD107a, IFNg, TNFa, and CD69 ( Figure 10A -D).

[0149] These data indicate that 1615 CLEC12A TriKE induced degranulation and cytokine production against AML target cells (including THP1 and HL-60 targets).

[0150] Example 7

[0151] This example describes the killing of AML targets induced by CD16-IL15-CLEC12A TriKE.

[0152] Enriched NK cells were incubated with CLEC12A+HL-60 ( Figure 7A ) and THP1 ( Figure 7B ) targets at an effector-to-target ratio of 2:1 in the presence of equimolar concentrations of IL-15, CLEC12A scFv, or CLEC12A TriKE. Target cells were labeled with CellTrace Far Red dye and caspase 3 / 7 green apoptosis assay reagent (Essen Biosciences). Killing was evaluated using an Incucyte Zoom instrument and analyzed by normalizing the cell number to the initial number of target cells. Figure 7A The graph in -B depicts the following (from the top): (i) no treatment (the first from the top); (ii) treatment with CLEC12A scFv (the second from the top); (iii) treatment with IL-15 (the third from the top / second from the bottom); (iv) treatment with CLEC12A TriKE (the fourth from the top / bottom). The percentage of live target cells was lowest after treatment with CLEC12A TriKE.

[0153] The induction of 1615 CLEC12A TriKE-mediated target cell killing was estimated in a real-time imaging assay. Enriched NK cells (CD3-, CD56+) were incubated with CellTrace Far Red-labeled THP-1 cells at an effector-to-target ratio of 2:1 in an IncuCyte S3 imager with the indicated treatment (30 nM) for 48 hours. Dead THP-1 cells were measured using a caspase 3 / 7 reagent. Figure 11AQuantification of the percentage of live THP-1 tumor targets (CellTrace FarRed / caspase 3 / 7) normalized to individual targets at the 0-hour time point. Readings were taken every 30 minutes over 48 hours. Representative of 3 separate experiments. The first from the top corresponds to no treatment, the second from the top corresponds to treatment with CLEC12AscFv, the third from the top corresponds to treatment with IL-15, and the fourth from the top corresponds to treatment with CLEC12ATriKE. The lowest percentage of live THP-1 cells was observed in the presence of CLEC12A TriKE.

[0154] Figure 11B Representative images (original magnification 34: 2.82 mm / pixel) after 0, 18, and 36 hours, which show THP-1 cells (larger cells) and NK cells (smaller cells). After 0 hours, for all indicated treatment conditions, few dead cells were present. After 18 and 36 hours, few clusters of dead THP-1 cells appeared under the no-treatment and CLEC12A scFv conditions throughout, where for treatment with IL-15, some dying THP-1 cell clusters were present, and more dying cell clusters were present in the case of treatment with CLEC12ATriKE.

[0155] Figure 11C Quantification of the percentage of live THP-1 tumor targets at different effector-to-target ratios (1:1, 2:1, and 5:1). The first, second, and third from the top correspond to NK alone, the fourth from the top corresponds to 1:1 NK+CLEC12A TriKE, the fifth from the top corresponds to 2:1 NK+CLEC12A TriKE, and the sixth from the top corresponds to 5:1 NK+CLEC12A TriKE. A lower percentage of live THP-1 cells was observed for all effector-to-target ratios compared to NK cells alone, where the 5:1 effector-to-target ratio resulted in the lowest percentage of live THP-1 cells, but this decrease in THP-1 cell viability was greatest in the presence of CLEC12A TriKE treatment.

[0156] In other experiments, enriched NK cells were incubated with CellTrace Far Red-labeled HL-60 cells at a 2:1 effector-to-target ratio in an IncuCyte S3 imager with the indicated treatments (30 nM each) for 48 hours. Dead HL-60 cells were assayed using a caspase 3 / 7 reagent.

[0157] Figure 15AShows the quantification of the percentage of viable HL-60 tumor targets (CellTrace Far Red / caspase 3 / 7) normalized to the individual target and the 0-hour time point. Readings were taken every 30 minutes over 48 hours. Representative of 3 individual experiments. The first from the top corresponds to no treatment, the second from the top corresponds to treatment with CLEC12AscFv, the third from the top corresponds to treatment with IL-15, and the fourth from the top corresponds to treatment with CLEC12A TriKE. The lowest percentage of viable HL-60 cells was observed in the presence of CLEC12A TriKE.

[0158] Figure 15B Are representative images after 0, 18, and 36 hours, which show HL-60 target cells (larger cells) and NK cells (smaller cells). After 0 hours, for all indicated treatment conditions, few dead cells were present. After 18 and 36 hours, few clusters of dead HL-60 cells appeared under the no-treatment and CLEC12A scFv conditions throughout, where for treatment with IL-15, some dying HL-60 cell clusters were present, and more dying cell clusters were present in the case of treatment with CLEC12A TriKE.

[0159] These data indicate that CD16-IL15-CLEC12A TriKE induced the killing of AML targets (including THP-1 and HL-60 target cells).

[0160] Example 8

[0161] This example illustrates the killing of primary AML blasts targets induced by CD16-IL15-CLEC12A TriKE in vitro.

[0162] Enriched NK cells were incubated with primary AML blasts at an effector-to-target ratio of 2:1 in the presence of equimolar concentrations of IL-15, CLEC12A scFv, CLEC12A TriKE, or CD33 TriKE. Figure 8A Shows the gating protocol for identifying AML blasts using FlowJo Analyzer. Figure 8B Shows the percentage of killing of AML blasts as evaluated by a live / dead marker after gating the blasts at 48 hours. After 4 hours, surface CD107a expression was evaluated on CD56 + CD3 - NK cells to estimate degranulation ( Figure 8C ) and intracellular IFNg to estimate inflammatory cytokine production ( Figure 8D)。Statistical data reflected significant differences between groups calculated by one-way ANOVA, *P<0.05, **P<0.005, N = 10. The percentage of dead AML blasts and the percentages of CD107a and IFNγ staining were significantly higher when incubated with CLEC12A TriKE compared to incubation with IL-15 or CLEC12A scFv( Figure 8B -D).

[0163] In other experiments, the expression of CD33 and CLEC12A on primary AML blasts (SSCh low, CD45 intermediate, CD117+, CD14-, CD34+) was evaluated using flow cytometry( Figure 12A ). Cells expressed CD33, CLEC12A, or both CD33 and CLEC12A, as shown.

[0164] Enriched NK cells (CD56+, CD3-) from healthy donors (n = 10) were incubated with primary AML blasts with the indicated treatments (30 nM) to estimate CD107a expression as degranulation( Figure 12B ) and intracellular IFNγ production( Figure 12C ) as markers in a 4-hour assay at an effector / target ratio of 2:1. CLEC12A TriKE and CD33 TriKE induced degranulation and IFNγ production, as shown. Target cell killing was also estimated using flow cytometry and a live / dead marker within 48 hours( Figure 12D ). Treatment with IL-15, CLEC12A TriKE, and CD33 TriKE led to killing of AML blasts, as shown, with greater killing mediated by CLEC12A TriKE

[0165] The proportion of AML blasts in different groups (based on CD33 and CLEC12A expression) was tracked over 48 hours to evaluate the specificity of 1615CLEC12A TriKE compared to 1615CD33 TriKE Figure 12E)。The bars showing the percentage of AML blasts surviving correspond to the following (from the top of each bar): (i) for no treatment, CLEC12A+CD33+ and CLEC12a+CD33-; (ii) for IL-15, CLEC12A+CD33+, CLEC12A+CD33- and CLEC12a-CD33-; (iii) for CLEC12AscFv, CLEC12A+CD33+, CLEC12A+CD33-, CLEC12A-CD33+ and CLEC12A-CD33-; (iv) for CLEC12ATriKE, CLEC12A+CD33+, CLEC12A-CD33+ and CLEC12A-CD33-; (v) for CD33 TriKE, CLEC12A+CD33+ and CLEC12A+CD33-. These data confirm the specificity of 1615CLEC12A TriKE compared to 1615CD33 TriKE.

[0166] In other experiments, enriched NK cells (CD56+, CD3-) from healthy donors (n = 5) were incubated with bone marrow samples from AML patients with the indicated treatment (30 nM) to assess killing of cancer stem cells (SSCh low, CD45 intermediate, CD34+, CD38-) in a 4-hour assay at an effector / target ratio of 2:1 ( Figure 12F ). Representative flow cytometry plots show killing of CLEC12A and CD33 positive cancer stem cells.

[0167] Figure 12G Pooled data from the cancer stem cell killing assay are shown, showing the percentage of cancer stem cells present at the end of the assay. One-way ANOVA with repeated measures was used to calculate differences for the 1615CLEC12A group. Error bars represent + / - standard error of the mean. Statistical significance was determined as * P,.05, ** P,.01, *** P,.001 and **** P,.0001. Treatment with CLEC12A TriKE resulted in a significant decrease in the percentage of LSCs compared to no treatment.

[0168] To examine primary AML blasts as target cells for killing mediated by CLEC12A TriKE, a gating strategy was employed. The gating strategy for identifying primary AML blasts is shown in Figure 16A . Primary AML blasts (n = 5) were then incubated with the indicated treatment (30 nM) for 48 hours to estimate target cell killing using flow cytometry and a live / dead marker ( Figure 16B)。The percentage of dead AMP primitive cells after 48 hours is shown.

[0169] These data indicate that 1615 CLEC12A TriKE induced a slight increase in the killing of primary AML primitive cells in patient samples during primitive cell crisis with NK cell restriction.

[0170] Example 9

[0171] This example describes the restriction of 1615 CLEC12A TriKE-mediated tumor growth in vivo.

[0172] Figure 13A A schematic diagram of the HL-60luc mouse experiment is shown. The model was established by conditioning NSG mice (225 cGy) and then intravenously injecting HL-60luc cells (7.5 × 105 cells / mouse). Three days later, 1 × 10 6 normal human donor NK cells activated overnight with 10 ng / ml of IL-15 (calculated based on the CD3 / CD19 product of magnetic depletion) were infused. During the next 3-week study, 1615 CLEC12A TriKE or 161533 TriKE (20 μg) was administered MTWThF (a total of 15 doses), and the control group received only HL-60luc cells.

[0173] The quantification of luminescence from four treatment groups at 7 days, 14 days, and 21 days after NK infusion is shown in Figure 13B . Each point represents a different mouse, and the bars represent the mean ± standard deviation. One-way analysis of variance (ANOVA) without a matched comparison was used to calculate the differences for the 1615 CLEC12A group. Statistical significance was determined as *P,.05, ***P,.001, and ****P,.0001. Figure 13C The photoluminescence (dark area) of individual mice after 2 minutes of exposure at 7 days, 14 days, and 21 days is shown. Compared with the HL60 control, CLEC12A TriKE treatment resulted in a reduced tumor burden at all examined time points. In addition, at 21 days, a significant reduction in the tumor burden after CLEC12A TriKE treatment was observed compared to the NK cell control treatment.

[0174] Figure 13D A schematic diagram of the pdx mouse experiment is shown. The model was established by conditioning NSG SGM3 mice (125 cGy) and then intravenously injecting HL-60luc cells (7.5 × 10 5 cells / mouse). Tumor growth was allowed until at least 1% of AML primitive cells were present in the blood. Then, 1 × 10 of NK cells activated overnight with 10 ng / ml of IL-15 were infused.6 Normal human donor NK cells (calculated based on CD3 / CD19 depletion). During the next 3-week study, 1615CLEC12A TriKE or 161533 TriKE (20 μg) was administered MTWThF (15 doses total), and the control group received NK cells but no treatment. Mice were sacrificed on day 21, and the percentage of AML blasts (CD45 intermediate, CD33+) in the bone marrow from the femur was calculated by flow cytometry ( Figure 13E ). Each point represents a different mouse. The percentage of NK cells (CD3−, CD56+) in bone marrow samples ( Figure 13F ) and peripheral blood ( Figure 13G ) was calculated by flow cytometry. Events were collected within 60 seconds, and the number of human NK cell events was counted. Representative dot plots showing the number of NK (CD56+CD3−) cell events within the CD45+ gate are shown. One-way ANOVA with no matching comparison was used to calculate the differences for the 1615CLEC12A group. Error bars represent mean ± standard deviation. Statistical significance was determined as *P, .05, ***P, .001, and ****P, .0001. Results showed that NK+CLEC12A TriKE treatment significantly reduced the percentage of primary AML blasts ( Figure 13E ) compared to tumor alone. In addition, NK+CLEC12A TriKE treatment led to a significant increase in the percentage of NK cells in the bone marrow ( Figure 13F ) and peripheral blood ( Figure 13G ) compared to tumor alone or tumor+NK treatment.

[0175] In summary, these results indicate that 1615CLEC12A TriKE limits tumor growth in vivo.

[0176] Example 10

[0177] This example describes the analysis of stem cells and progenitor cells.

[0178] Using Figure 17 The gating strategy shown to identify cancer stem cells in bone marrow samples. The gating strategy for determining different CD34 Figure 19 progenitor cell subsets in healthy bone marrow samples is shown in pos . Figure 19 The gating strategy shown is used to analyze the Figure 18A cell population shown.

[0179] Figure 18A CD34 in the bone marrow from two representative healthy donors is shown posExpression of CLEC12A and CD33 in the progenitor cell compartment. HSC: hematopoietic stem cell, MPP: multipotent progenitor, LMPP: lymphoid-primed multipotent progenitor, CLP: common lymphoid progenitor, CMP: common myeloid progenitor, GMP: granulocyte-macrophage progenitor, MEP: megakaryocyte-erythroid progenitor. CLEC12A expression was observed in the CMP, GMP, and MEP populations, but was relatively low in the HSC, MPP, CLP, and LMPP populations. In addition, CLEC12A levels were found to be lower than CD33 except in the GMP population. After treatment with 1615CLEC12A TriKE or 161533TriKE, burst-forming unit-erythroid (BFU-E) colonies and colony-forming unit-erythroid (CFU-E) colonies were counted ( Figure 18B ). Larger numbers of BFU-E and CFU-GM colonies were observed after treatment with 1615CLEC12A TriKE compared to treatment with 161533 TriKE.

[0180] Data show that CLEC12A is differentially expressed in normal donor stem and progenitor cell populations and that treatment with 161533 TriKE reduces stem cell formation and / or differentiation compared to treatment with CLEC12A TriKE. Without being limited by theory, this suggests that while CLEC12A can be used to target leukemic stem cells, normal hematopoiesis should be permitted, whereas CD33 targeting is more likely to affect reconstitution. In other words, CLEC12A targeting should have fewer off-target effects with respect to normal bone marrow reconstitution.

[0181] In summary, the above data show that CD16-IL15-CLEC12A TriKE specifically binds to target cells expressing CLEC12A, promotes NK cell proliferation, enhances NK cell function, promotes killing of AML cell lines in the Incucyte Zoom assay, and induces killing of primary AML and MDS blasts.

[0182] Example 11

[0183] This example illustrates the generation of a TetraKE targeting CLEC12A and a second target or tumor antigen.

[0184] TetraKE (tetramer) molecules can be designed to contain more than one targeting domain. As an example, a TetraKE can contain an NK engagement domain, an NK activation domain, and two targeting domains. Any NK engagement domain and NK activation domain described herein can be used. For example, the targeting domains can target different targets or tumor antigens. Any combination of targets or tumor antigens can be included in the TetraKE. For example, the first targeting domain can bind to CLEC12A, while the second targeting domain can bind to another target or tumor antigen.

[0185] Any target or tumor antigen described herein can be included in a TetraKE having a first targeting domain that binds to CLEC12A, including, for example, a second targeting domain that binds to CD133, CD20, HER2, CEA, EpCAM, VEGF-A, EGFR, CD33, integrin αVβ3, CD51, CD152, CD125, CTAA16.88, MUC1, CD19, CD22, CD38, mesothelin, ROR1, CSPG4, SS1 or IGFR1, NKG2C, BCMA, APRIL, B7H3, and PSMA, or a viral antigen derived from EBV, HBV, HCV, and / or HPV. In addition, the TetraKE domains can be operably linked to each other using flanking or linker sequences as described herein. An exemplary TetraKE contains a compound having a moiety that selectively binds to CD16, an NK activation domain that includes IL-15, a first targeting domain that selectively binds to CLEC12A, and a second targeting domain that selectively binds to CD33.

[0186] SEQ ID NO.:1

[0187] MKWVTFISLLFLFSSAYSQVQLVESGGGLVQPGGSLRLSCAASGLTFSSYNMGWFRQAPGQGLEAVASITWSGRDTFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAANPWPVAAPRSGTYWGQGTLVTVSSSGGGGSGGGGSGGGGSGGGGSGNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSGSTSGSGKPGSGEGSTKGQVQLQESGPGLVKPSETLSLTCVVSGGSISSSNWWSWVRQPPGKGLEWIGEIYHSGSPDYNPSLKSRVTISVDKSRNQFSLKLSSVTAADTAVYYCAKVSTGGFFDYWGQGTLVTVSSGGGGSGGGGSGGGGSEIELTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPTFGPGTKVEIK

[0188] SEQ ID NO.:2

[0189] MKWVTFISLLFLFSSAYSQVQLVESGGGLVQPGGSLRLSCAASGLTFSSYNMGWFRQAPGQGLEAVASITWSGRDTFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAANPWPVAAPRSGTYWGQGTLVTVSSSGGGGSGGGGSGGGGSGGGGSGNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSGSTSGSGKPGSGEGSTKGQVQLQESGPGLVKPSETLSLTCVVSGGSISSSNWWSWVRQPPGKGLEWIGEIYHSGSPDYNPSLKSRVTISVDKSRNQFSLKLSSVTAADTAVYYCAKVSTGGFFDYWGQGTLVTVSSGGGGSGGGGSGGGGSEIELTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPTFGPGTKVEIKVDEHHHHHHHHHH

[0190] SEQ ID NO.:3

[0191] VDEHHHHHHHHHH

[0192] SEQ ID NO.:4

[0193] QVQLQESGPGLVKPSETLSLTCVVSGGSISSSNWWSWVRQPPGKGLEWIGEIYHSGSPDYNPSLKSRVTISVDKSRNQFSLKLSSVTAADTAVYYCAKVSTGGFFDYWGQGTLVTVSSGGGGSGGGGSGGGGSEIELTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPTFGPGTKVEIK

[0194] SEQ ID NO.:5

[0195]

[0196] SEQ ID NO.:6

[0197]

[0198] SEQ ID NO.:7

[0199] caagtacaactccaggagtccgggccagggttggtcaagccatccgagacgcttagtttgacctgtgttgtcagcggaggctctatatcatcttcaaactggtggtcttgggtacggcaaccaccgggcaaggggctcgaatggatcggggaaatctaccactccggaagccccgactataatccgtcactgaagagcagagtcactatatccgtggacaagagcagaaaccaattttctcttaagctctcctcagtgacagcagcagatacagcggtctattattgtgccaaggtatcaacaggcggattcttcgattattggggacagggcactttggttacggtttcttctggaggcgggggaagtggtggaggggggtctgggggaggtggctcagaaatcgaacttacgcagtcaccctcctccctctcagcatccgtaggtgacagagttacgataacctgtagagcaagtcaatccatttctagctaccttaactggtatcagcaaaaacctgggaaagcccccaagctgcttatctatgcggcatcctccctccaaagtggagttcccagtcggttcagtggttccggctcagggactgactttaccctcacaatcagctcattgcaaccagaggactttgcaacgtattactgtcagcaaagctactcaacgccgcctacgttcggtcccggaaccaaagttgagattaaa

[0200] SEQ ID NO.:8

[0201] QVQLVESGGGLVQPGGSLRLSCAASGLTFSSYNMGWFRQAPGQGLEAVASITWSGRDTFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAANPWPVAAPRSGTYWGQGTLVTVSS

[0202] SEQ ID NO.:9

[0203] NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

[0204] SEQ ID NO.:10

[0205] MKWVTFISLLFLFSSAYS

[0206] SEQ ID NO.:11

[0207] SGGGGSGGGGSGGGGSGGGGSG

[0208] SEQ ID NO.:12

[0209] GSTSGSGKPGSGEGSTKG

[0210] SEQ ID NO.:13

[0211] PSGQAGAAASESLFVSNHAY

[0212] SEQ ID NO.:14

[0213] EASGGPE

[0214] SEQ ID NO.:15

[0215] GGGGSGGGGS

[0216] SEQ ID NO.:16

[0217] MGWSCIILFLVATATGVHSS

[0218] SEQ ID NO.:17

[0219] MGWSCIILFLVATATGVHS

[0220] SEQ ID NO.:18

[0221] EVQLVESGGELVQAGGSLRLSCAASGLTFSSYNMGWFRRAPGKEREFVASITWSGRDTFYADSVKGRFTISRDNAKNTVYLQMSSLKPEDTAVYYCAANPWPVAAPRSGTYWGQGTQVTVSSVDE

[0222] SEQ ID NO. Description SEQ ID NO.: 1 CLEC12A TriKE SEQ ID NO.: 2 CLEC12A TriKE with His - Tag and Spacer SEQ ID NO.: 3 His - Tag and Spacer SEQ ID NO.: 4 CLEC12A Targeting Domain SEQ ID NO.: 5 DNA Encoding CLEC12A TriKE with His - Tag and Spacer SEQ ID NO.: 6 DNA Encoding CLEC12A TriKE SEQ ID NO.: 7 DNA Encoding CLEC12A Targeting Domain SEQ ID NO.: 8 Humanized Cam16 SEQ ID NO.: 9 Wild - type IL - 15 SEQ ID NO.: 10 Signal Peptide SEQ ID NO.: 11 Linker SEQ ID NO.: 12 Linker SEQ ID NO.: 13 Linker SEQ ID NO.: 14 Linker SEQ ID NO.: 15 Linker SEQ ID NO.: 16 Signal Peptide SEQ ID NO.: 17 Signal Peptide SEQ ID NO.: 18 Non - Humanized Cam16

[0223] Any and all references and citations to other documents, such as patents, patent applications, patent publications, journals, books, papers, web content, throughout this disclosure are hereby incorporated by reference in their entirety for all purposes.

[0224] Although the invention has been described in connection with specific details of certain embodiments of the invention in the foregoing examples, it is to be understood that modifications and variations are included within the spirit and scope of the invention. Accordingly, the invention is limited only by the appended claims. Sequence Listing <110> The Regents of the University of Minnesota J•S•Miller M·Felices D·A·Valera T·R·Renwick <120> NK Conjugate Molecules and Methods of Use Thereof <130> GTBIO2090-1WO <150> US 62 / 747,983 <151> 2018-10-19 <160> 18 <170> PatentIn version 3.5 <210> 1 <211> 534 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 1 Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala 1 5 10 15 Tyr Ser Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro 20 25 30 Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Leu Thr Phe Ser 35 40 45 Ser Tyr Asn Met Gly Trp Phe Arg Gln Ala Pro Gly Gln Gly Leu Glu 50 55 60 Ala Val Ala Ser Ile Thr Trp Ser Gly Arg Asp Thr Phe Tyr Ala Asp 65 70 75 80 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr 85 90 95 Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 100 105 110 Tyr Cys Ala Ala Asn Pro Trp Pro Val Ala Ala Pro Arg Ser Gly Thr 115 120 125 Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ser Gly Gly Gly 130 135 140 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 145 150 155 160 Ser Gly Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp 165 170 175 Leu Ile Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp 180 185 190 Val His Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu 195 200 205 Leu Gln Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr 210 215 220 Val Glu Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly 225 230 235 240 Asn Val Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys 245 250 255 Asn Ile Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe 260 265 270 Ile Asn Thr Ser Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly 275 280 285 Glu Gly Ser Thr Lys Gly Gln Val Gln Leu Gln Glu Ser Gly Pro Gly 290 295 300 Leu Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Val Val Ser Gly 305 310 315 320 Gly Ser Ile Ser Ser Ser Asn Trp Trp Ser Trp Val Arg Gln Pro Pro 325 330 335 Gly Lys Gly Leu Glu Trp Ile Gly Glu Ile Tyr His Ser Gly Ser Pro 340 345 350 Asp Tyr Asn Pro Ser Leu Lys Ser Arg Val Thr Ile Ser Val Asp Lys 355 360 365 Ser Arg Asn Gln Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp 370 375 380 Thr Ala Val Tyr Tyr Cys Ala Lys Val Ser Thr Gly Gly Phe Phe Asp 385 390 395 400 Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly 405 410 415 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Glu Leu Thr 420 425 430 Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile 435 440 445 Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu Asn Trp Tyr Gln 450 455 460 Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Ser 465 470 475 480 Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr 485 490 495 Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr 500 505 510 Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Pro Thr Phe Gly Pro Gly 515 520 525 Thr Lys Val Glu Ile Lys 530 <210> 2 <211> 547 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 2 Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala 1 5 10 15 Tyr Ser Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro 20 25 30 Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Leu Thr Phe Ser 35 40 45 Ser Tyr Asn Met Gly Trp Phe Arg Gln Ala Pro Gly Gln Gly Leu Glu 50 55 60 Ala Val Ala Ser Ile Thr Trp Ser Gly Arg Asp Thr Phe Tyr Ala Asp 65 70 75 80 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr 85 90 95 Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 100 105 110 Tyr Cys Ala Ala Asn Pro Trp Pro Val Ala Ala Pro Arg Ser Gly Thr 115 120 125 Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ser Gly Gly Gly 130 135 140 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 145 150 155 160 Ser Gly Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp 165 170 175 Leu Ile Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp 180 185 190 Val His Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu 195 200 205 Leu Gln Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr 210 215 220 Val Glu Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly 225 230 235 240 Asn Val Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys 245 250 255 Asn Ile Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe 260 265 270 Ile Asn Thr Ser Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly 275 280 285 Glu Gly Ser Thr Lys Gly Gln Val Gln Leu Gln Glu Ser Gly Pro Gly 290 295 300 Leu Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Val Val Ser Gly 305 310 315 320 Gly Ser Ile Ser Ser Ser Asn Trp Trp Ser Trp Val Arg Gln Pro Pro 325 330 335 Gly Lys Gly Leu Glu Trp Ile Gly Glu Ile Tyr His Ser Gly Ser Pro 340 345 350 Asp Tyr Asn Pro Ser Leu Lys Ser Arg Val Thr Ile Ser Val Asp Lys 355 360 365 Ser Arg Asn Gln Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp 370 375 380 Thr Ala Val Tyr Tyr Cys Ala Lys Val Ser Thr Gly Gly Phe Phe Asp 385 390 395 400 Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly 405 410 415 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Glu Leu Thr 420 425 430 Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile 435 440 445 Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu Asn Trp Tyr Gln 450 455 460 Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Ser 465 470 475 480 Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr 485 490 495 Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr 500 505 510 Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Pro Thr Phe Gly Pro Gly 515 520 525 Thr Lys Val Glu Ile Lys Val Asp Glu His His His His His His His 530 535 540 His His His 545 <210> 3 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 3 Val Asp Glu His His His His His His His His His His 1 5 10 <210> 4 <211> 240 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 4 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Val Val Ser Gly Gly Ser Ile Ser Ser Ser 20 25 30 Asn Trp Trp Ser Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp 35 40 45 Ile Gly Glu Ile Tyr His Ser Gly Ser Pro Asp Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Val Thr Ile Ser Val Asp Lys Ser Arg Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Val Ser Thr Gly Gly Phe Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 115 120 125 Gly Gly Gly Gly Ser Glu Ile Glu Leu Thr Gln Ser Pro Ser Ser Leu 130 135 140 Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln 145 150 155 160 Ser Ile Ser Ser Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala 165 170 175 Pro Lys Leu Leu Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro 180 185 190 Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 195 200 205 Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser 210 215 220 Tyr Ser Thr Pro Pro Thr Phe Gly Pro Gly Thr Lys Val Glu Ile Lys 225 230 235 240 <210> 5 <211> 1644 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 5 atgaagtggg taacctttat ttcccttctt tttctcttta gctcggctta ttcccaggtg 60 cagctggtgg agtctggggg aggcttggtg cagcctgggg gctctctgag actctcctgt 120 gcagcctctg gcctcacctt cagtagctat aacatgggct ggttccgcca ggctccaggg 180 caaggccttg aggctgtagc atctattacc tggagtggtc gggacacatt ctatgcagac 240 tccgtgaagg gccgattcac catctccaga gacaactcca agaacactct ctatctgcaa 300 atgaacagcc tgcgcgcgga ggacacggcc gtttattatt gtgctgcaaa cccctggcca 360 gtggcggcgc cacgtagtgg cacctactgg ggccaaggga ccctggtcac cgtctcctca 420 tctggcggcg gcggttctgg tggaggaggt agtggggggg gaggaagcgg agggggtggc 480 tcagggaact gggtgaatgt aataagtgat ttgaaaaaaa ttgaagatct tattcaatct 540 atgcatattg atgctacttt atatacggaa agtgatgttc accccagttg caaagtaaca 600 gcaatgaagt gctttctctt ggagttacaa gttatttcac ttgagtccgg agatgcaagt 660 attcatgata cagtagaaaa tctgatcatc ctagcaaaca acagtttgtc ttctaatggg 720 aatgtaacag aatctggatg caaagaatgt gaggaactgg aggaaaaaaa tattaaagaa 780 tttttgcaga gttttgtaca tattgtccaa atgttcatca acacttctgg cagtaccagc 840 gggtcaggga aacctggcag tggggaaggt tccacaaaag gtcaagtaca actccaggag 900 tccgggccag ggttggtcaa gccatccgag acgcttagtt tgacctgtgt tgtcagcgga 960 ggctctatat catcttcaaa ctggtggtct tgggtacggc aaccaccggg caaggggctc 1020 gaatggatcg gggaaatcta ccactccgga agccccgact ataatccgtc actgaagagc 1080 agagtcacta tatccgtgga caagagcaga aaccaatttt ctcttaagct ctcctcagtg 1140 acagcagcag atacagcggt ctattattgt gccaaggtat caacaggcgg attcttcgat 1200 tattggggac agggcacttt ggttacggtt tcttctggag gcgggggaag tggtggaggg 1260 gggtctgggg gaggtggctc agaaatcgaa cttacgcagt caccctcctc cctctcagca 1320 tccgtaggtg acagagttac gataacctgt agagcaagtc aatccatttc tagctacctt 1380 aactggtatc agcaaaaacc tgggaaagcc cccaagctgc ttatctatgc ggcatcctcc 1440 ctccaaagtg gagttcccag tcggttcagt ggttccggct cagggactga ctttaccctc 1500 acaatcagct cattgcaacc agaggacttt gcaacgtatt actgtcagca aagctactca 1560 acgccgccta cgttcggtcc cggaaccaaa gttgagatta aagtagacga acaccatcat 1620 catcatcacc atcaccacca ttga 1644 <210> 6 <211> 1605 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 6 atgaagtggg taacctttat ttcccttctt tttctcttta gctcggctta ttcccaggtg 60 cagctggtgg agtctggggg aggcttggtg cagcctgggg gctctctgag actctcctgt 120 gcagcctctg gcctcacctt cagtagctat aacatgggct ggttccgcca ggctccaggg 180 caaggccttg aggctgtagc atctattacc tggagtggtc gggacacatt ctatgcagac 240 tccgtgaagg gccgattcac catctccaga gacaactcca agaacactct ctatctgcaa 300 atgaacagcc tgcgcgcgga ggacacggcc gtttattatt gtgctgcaaa cccctggcca 360 gtggcggcgc cacgtagtgg cacctactgg ggccaaggga ccctggtcac cgtctcctca 420 tctggcggcg gcggttctgg tggaggaggt agtggggggg gaggaagcgg agggggtggc 480 tcagggaact gggtgaatgt aataagtgat ttgaaaaaaa ttgaagatct tattcaatct 540 atgcatattg atgctacttt atatacggaa agtgatgttc accccagttg caaagtaaca 600 gcaatgaagt gctttctctt ggagttacaa gttatttcac ttgagtccgg agatgcaagt 660 gcaatgaagt gctttctctt ggagttacaa gttatttcac ttgagtccgg agatgcaagt 660 attcatgata cagtagaaaa tctgatcatc ctagcaaaca acagtttgtc ttctaatggg 720 attcatgata cagtagaaaa tctgatcatc ctagcaaaca acagtttgtc ttctaatggg 720 aatgtaacag aatctggatg caaagaatgt gaggaactgg aggaaaaaaa tattaaagaa 780 aatgtaacag aatctggatg caaagaatgt gaggaactgg aggaaaaaaa tattaaagaa 780 tttttgcaga gttttgtaca tattgtccaa atgttcatca acacttctgg cagtaccagc 840 tttttgcaga gttttgtaca tattgtccaa atgttcatca acacttctgg cagtaccagc 840 gggtcaggga aacctggcag tggggaaggt tccacaaaag gtcaagtaca actccaggag 900 gggtcaggga aacctggcag tggggaaggt tccacaaaag gtcaagtaca actccaggag 900 tccgggccag ggttggtcaa gccatccgag acgcttagtt tgacctgtgt tgtcagcgga 960 tccgggccag ggttggtcaa gccatccgag acgcttagtt tgacctgtgt tgtcagcgga 960 ggctctatat catcttcaaa ctggtggtct tgggtacggc aaccaccggg caaggggctc 1020 ggctctatat catcttcaaa ctggtggtct tgggtacggc aaccaccggg caaggggctc 1020 gaatggatcg gggaaatcta ccactccgga agccccgact ataatccgtc actgaagagc 1080 gaatggatcg gggaaatcta ccactccgga agccccgact ataatccgtc actgaagagc 1080 agagtcacta tatccgtgga caagagcaga aaccaatttt ctcttaagct ctcctcagtg 1140 agagtcacta tatccgtgga caagagcaga aaccaatttt ctcttaagct ctcctcagtg 1140 acagcagcag atacagcggt ctattattgt gccaaggtat caacaggcgg attcttcgat 1200 acagcagcag atacagcggt ctattattgt gccaaggtat caacaggcgg attcttcgat 1200 tattggggac agggcacttt ggttacggtt tcttctggag gcgggggaag tggtggaggg 1260 tattggggac agggcacttt ggttacggtt tcttctggag gcgggggaag tggtggaggg 1260 gggtctgggg gaggtggctc agaaatcgaa cttacgcagt caccctcctc cctctcagca 1320 gggtctgggg gaggtggctc agaaatcgaa cttacgcagt caccctcctc cctctcagca 1320 tccgtaggtg acagagttac gataacctgt agagcaagtc aatccatttc tagctacctt 1380 aactggtatc agcaaaaacc tgggaaagcc cccaagctgc ttatctatgc ggcatcctcc 1440 ctccaaagtg gagttcccag tcggttcagt ggttccggct cagggactga ctttaccctc 1500 acaatcagct cattgcaacc agaggacttt gcaacgtatt actgtcagca aagctactca 1560 acgccgccta cgttcggtcc cggaaccaaa gttgagatta aatga 1605 <210> 7 <211> 720 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 7 caagtacaac tccaggagtc cgggccaggg ttggtcaagc catccgagac gcttagtttg 60 acctgtgttg tcagcggagg ctctatatca tcttcaaact ggtggtcttg ggtacggcaa 120 ccaccgggca aggggctcga atggatcggg gaaatctacc actccggaag ccccgactat 180 aatccgtcac tgaagagcag agtcactata tccgtggaca agagcagaaa ccaattttct 240 cttaagctct cctcagtgac agcagcagat acagcggtct attattgtgc caaggtatca 300 acaggcggat tcttcgatta ttggggacag ggcactttgg ttacggtttc ttctggaggc 360 gggggaagtg gtggaggggg gtctggggga ggtggctcag aaatcgaact tacgcagtca 420 ccctcctccc tctcagcatc cgtaggtgac agagttacga taacctgtag agcaagtcaa 480 tccatttcta gctaccttaa ctggtatcag caaaaacctg ggaaagcccc caagctgctt 540 atctatgcgg catcctccct ccaaagtgga gttcccagtc ggttcagtgg ttccggctca 600 gggactgact ttaccctcac aatcagctca ttgcaaccag aggactttgc aacgtattac 660 tgtcagcaaa gctactcaac gccgcctacg ttcggtcccg gaaccaaagt tgagattaaa 720 <210> 8 <211> 122 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 8 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Leu Thr Phe Ser Ser Tyr 20 25 30 Asn Met Gly Trp Phe Arg Gln Ala Pro Gly Gln Gly Leu Glu Ala Val 35 40 45 Ala Ser Ile Thr Trp Ser Gly Arg Asp Thr Phe Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Asn Pro Trp Pro Val Ala Ala Pro Arg Ser Gly Thr Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 9 <211> 114 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 9 Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile 1 5 10 15 Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His 20 25 30 Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln 35 40 45 Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu 50 55 60 Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val 65 70 75 80 Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile 85 90 95 Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn 100 105 110 Thr Ser <210> 10 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 10 Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala 1 5 10 15 Tyr Ser <210> 11 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 11 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 Gly Gly Gly Gly Ser Gly 20 <210> 12 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 12 Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr 1 5 10 15 Lys Gly <210> 13 <211> 20 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 13 Pro Ser Gly Gln Ala Gly Ala Ala Ala Ser Glu Ser Leu Phe Val Ser 1 5 10 15 Asn His Ala Tyr 20 <210> 14 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 14 Glu Ala Ser Gly Gly Pro Glu 1 5 <210> 15 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 15 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 <210> 16 <211> 20 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 16 Methionine Glycine Tryptophan Serine Cysteine Isoleucine Isoleucine Leucine Phenylalanine Leucine Valine Alanine Threonine Alanine Threonine Glycine 1 5 10 15 Valine Histidine Serine Serine 20 <210> 17 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 17 Methionine Glycine Tryptophan Serine Cysteine Isoleucine Isoleucine Leucine Phenylalanine Leucine Valine Alanine Threonine Alanine Threonine Glycine 1 5 10 15 Valine Histidine Serine <210> 18 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 18 Glutamic Acid Valine Glutamine Leucine Valine Glutamic Acid Serine Glycine Glycine Glutamic Acid Leucine Valine Glutamine Alanine Glycine Glycine 1 5 10 15 Serine Leucine Arginine Leucine Serine Cysteine Alanine Alanine Serine Glycine Leucine Threonine Phenylalanine Serine Serine Tyrosine 20 25 30 Asparagine Methionine Glycine Tryptophan Phenylalanine Arginine Arginine Alanine Proline Glycine Lysine Glutamic Acid Arginine Glutamic Acid Phenylalanine Valine 35 40 45 Alanine Serine Isoleucine Threonine Tryptophan Serine Glycine Arginine Aspartic Acid Threonine Phenylalanine Tyrosine Alanine Aspartic Acid Serine Valine 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 Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Asn Pro Trp Pro Val Ala Ala Pro Arg Ser Gly Thr Tyr Trp 100 105 110 Gly Gln Gly Thr Gln Val Thr Val Ser Ser Val Asp Glu 115 120 125

Claims

1. A compound, comprising: an NK engagement domain comprising a portion that binds CD16; an NK activation domain operably linked to the NK engagement domain; and a targeting domain that binds to a target cell and is operably linked to the NK activation domain and the NK engagement domain, wherein the targeting domain binds to CLEC12A, and wherein the amino acid sequence of the compound is represented by SEQ ID NO.:1 or SEQ ID NO.:

2.

2. A composition, comprising: the compound according to claim 1; and a pharmaceutically acceptable carrier.

3. Use of the compound according to claim 1 in the manufacture of a medicament for the treatment of acute myeloid leukemia (AML) in a subject.

Images