RU2812481C2 - Nk involving molecules and methods of their use - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: following is described: a group of inventions including a compound for activating NK cells, a composition for use in inducing proliferation of NK cells, a method of inducing NK-mediated destruction of a target cell in a subject, a method of stimulating the proliferation of NK cells in vivo, a method of inhibiting the growth of cancer cells in a subject, a method of stimulating secretion of inflammatory cytokines by NK cells in vivo, and a method of treating acute myeloid leukemia (AML) in a subject.

EFFECT: invention expands the arsenal of means for activating NK cells.

26 cl, 53 dwg, 1 tbl, 11 ex

Description

CROSS REFERENCE TO RELATED APPLICATIONS

[001] This application claims priority under 35 USC §119(e) No. 62/747983, filed October 19, 2018, the entire contents of which are incorporated herein by reference in their entirety.

GOVERNMENT FUNDING

[002] This invention was made with government support under CA111412 and CA65493 from the National Institutes of Health, under CA36725, Ca72669, Ca077598 and Ca197292 from the National Cancer Institute, and under CA150085 from the US Department of Defense. The government has certain rights in the invention.

LIST OF SEQUENCES

[003] The material in the attached sequence listing is hereby incorporated by reference to this application. The attached sequence listing text file, named GTBIO2090_1WO_SAXE_LISTING.TXT, was created on October 15, 2019 and is 23 KB in size. The file can be viewed using Microsoft Word on a computer that runs Windows.

BACKGROUND OF THE ART

TECHNICAL FIELD

[004] The invention relates generally to immunotherapy and more particularly to compositions useful in recruiting natural killer (NK) cells into an immune response.

GENERAL INFORMATION

[005] Natural killer (NK) cells are cytotoxic lymphocytes of the innate immune system capable of immune surveillance. Like cytotoxic T lymphocytes, NK cells supply a supply of membrane-permeable and apoptosis-inducing granules granzymes and perforins. Unlike T lymphocytes, NK cells do not require antigen induction and recognize targets through engagement by activating receptors in the absence of MHC recognition. NK cells express CD16, an activation receptor that binds to the Fc portion of IgG antibodies and is involved in antibody-dependent cellular cytotoxicity (ADCC). NK cells are regulated by IL-15, which can cause increased antigen-dependent cytotoxicity, lymphokine-activated killer activity, and/or interferon (IFN), tumor necrosis factor (TNF) and/or granulocyte-macrophage colony-stimulating factor (GM-) mediated responses. KSF). All of these functions, activated by IL-15, contribute to improved protection against cancer.

[006] Therapeutically beneficial adoptive transfer of NK cells can, for example, induce remission in patients with refractory acute myeloid leukemia (AML), in combination with lymphocyte-depleting chemotherapy and IL-2 to promote in vivo survival and expansion of NK cells. This therapy may be limited by the lack of antigen specificity and IL-2-mediated induction of regulatory T cells (Tregs), which suppress NK cell proliferation and function. Development of a reagent that manipulates the antigen specificity, proliferation, and/or survival of NK cells while avoiding the negative effects of Treg inhibition may improve NK cell-based immunotherapies.

SUMMARY OF THE INVENTION

[007] This 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 invention provides a compound comprising an NK domain engaging; an NK activating domain operably linked to an NK engaging domain; and a targeting domain that selectively binds to a target cell and is operably linked to an NK activating domain and an NK engaging domain, wherein the targeting domain selectively binds to CLEC12A.

[008] In some embodiments, the NK engaging domain includes a moiety that selectively binds CD16. In some embodiments, the NK engaging domain fragment includes 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 moiety includes a scFv, F(ab)2, or Fab. In some embodiments, the antibody or binding fragment, or nanobody, thereof is human or humanized. In some embodiments, the antibody or binding fragment, or nanobody, thereof is camelid.

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

[0010] In some embodiments, the targeting domain fragment includes an antibody or a binding fragment thereof, or a nanobody. In some embodiments, the antibody binding moiety includes a scFv, F(ab)2, or Fab.

[0011] In some embodiments, the NK engaging domain includes a moiety that selectively binds CD16, the NK activating domain includes IL-15, and the targeting domain selectively binds CLEC12A.

[0012] In some embodiments, the compounds and compositions described herein contain at least one flanking sequence connecting two domains. In some embodiments, the compounds and compositions described herein further comprise a second flanking sequence connecting the two linked domains to a third domain. In some embodiments, the flanking sequences flank the NK activating domain. In some embodiments, the first flanking sequence is C-terminal to the NK engagement domain and the second flanking sequence is N-terminal to the anti-CLEC12A targeting domain.

[0013] In some embodiments, provided herein is an isolated amino acid sequence comprising SEQ ID NO: 1. In some embodiments, provided herein is an isolated DNA sequence encoding the amino acid sequence of SEQ ID NO: 1.

[0014] In some embodiments, provided herein is an isolated amino acid sequence comprising SEQ ID NO: 2. In some embodiments, provided herein is an isolated DNA sequence encoding the amino acid sequence of SEQ ID NO: 2.

[0015] In some embodiments, provided herein is an isolated amino acid sequence comprising SEQ ID NO: 4. In some embodiments, provided herein is an isolated DNA sequence encoding the amino acid sequence of SEQ ID NO: 4.

[0016] In some embodiments, provided herein are compositions comprising the compounds described herein and a pharmaceutically acceptable carrier.

[0017] In some embodiments, methods provided herein include: administering to a subject a compound described herein in an amount effective to induce NK-mediated killing of a target cell. In some embodiments, the target cell is a cancer cell.

[0018] In some embodiments, provided herein are methods of stimulating in vivo division of NK cells, comprising: administering to a subject an amount of a compound described herein effective to stimulate the proliferation of NK cells in the subject.

[0019] In some embodiments, provided herein are methods of treating cancer in a subject, comprising: administering to the subject an amount of a compound described herein effective to treat cancer. In some embodiments, the cancer includes: 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 methods provided herein further include administering the compound before, concurrently with, or after chemotherapy, surgical resection of a tumor, or radiation therapy. In some embodiments, the chemotherapy includes altretamine, amsacrine, L-asparaginase, colaspase, bleomycin, busulfan, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, cyclophosphamide, cytophosphamide, cytarabine, dacarbazine, dactinomycin, 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, vincrestine, vindesine, and vinorelbine. In some embodiments, the hematopoietic cancer is AML.

BRIEF DESCRIPTION OF GRAPHIC MATERIALS

[0020] FIG. 1A-1C illustrate NK cell proliferation (Figure 1A), NK cell killing (Figure 1B), and functional analysis (Figure 1C) upon treatment with CLEC12A TriKE.

[0021] FIG. Figure 2 illustrates the percentage of CD33 and Clec12A surface expression on primary AML samples from 10 patients.

[0022] FIG. 3A-3B illustrate CD16-IL15-CLEC12A TriKE (Fig. 3A) and mechanism of action (Fig. 3B).

[0023] FIG. 4 illustrates the binding of CD16-IL15-CLEC12A TriKE to targets that express CLEC12A.

[0024] FIG. 5A-5B illustrate CD16-IL15-CLEC12A TriKE promotion of NK cell proliferation.

[0025] FIG. 6A-6C illustrate CD16-IL15-CLEC12A TriKE induction of degranulation (Figure 6A) and cytokine production (Figure 6B-C) against targeted AML cells.

[0026] FIG. 7A-7B illustrate CD16-IL15-CLEC12A TriKE induction of killing of targeted AML cells.

[0027] FIG. 8A-8D illustrate CD16-IL15-CLEC12A TriKE-induced killing of AML primary targets in vitro.

[0028] FIG. 9A-9C illustrate CD16-IL15-CLEC12A TriKE induction of NK cell proliferation.

[0029] FIG. 10A-10D illustrate validation of CD16-IL15-CLEC12A TriKE function.

[0030] FIG. 11A-11C illustrate CD16-IL15-CLEC12A TriKE induction of target cell killing in a real-time imaging assay. Tumor targets of THP-1 are shown.

[0031] FIG. 12A-12G illustrate CD16-IL15-CLEC12A TriKE-induced killing of primary AML blast cells.

[0032] FIG. 13A-13G illustrate that CD16-IL15-CLEC12A limits TriKE tumor growth in vivo.

[0033] FIG. 14A-14C illustrate validation of CD16-IL15-CLEC12A TriKE binding.

[0034] FIG. 15A-15B illustrate CD16-IL15-CLEC12A TriKE induction of target cell killing in a real-time imaging assay. Tumor targets of HL-60 are shown.

[0035] FIG. 16A-16B illustrate CD16-IL15-CLEC12A TriKE-mediated target killing. The target gating strategy (Figure 16A) and target cell killing (Figure 16B) are shown. AML blast cell targets are shown.

[0036] FIG. 17 illustrates a gating strategy for identifying cancer stem cells in bone marrow samples from AML patients.

[0037] FIG. 18a-18B illustrate the expression of CLEC12A and CD33 within the CD34pos progenitor compartment in bone marrow. Shown are cell populations from two representative donors (Figure 18A) and cell colonies following treatment with the indicated TriKE (Figure 18B).

[0038] FIG. 19 illustrates a gating strategy for identifying different subpopulations of CD34pos precursors in bone marrow samples from healthy subjects.

DETAILED DESCRIPTION OF THE INVENTION

[0039] Natural killer (NK) cells are cytotoxic lymphocytes of the innate immune system capable of immune surveillance. Like cytotoxic T lymphocytes, NK cells supply a supply of membrane-permeable and apoptosis-inducing granules granzymes and perforins. Unlike T lymphocytes, NK cells do not require antigen induction and recognize targets through engagement by activating receptors in the absence of MHC recognition. NK cells express CD16, an activation receptor that binds to the Fc portion of IgG antibodies and is involved in antibody-dependent cellular cytotoxicity (ADCC). NK cells are regulated by IL-15, which can cause increased antigen-dependent cytotoxicity, lymphokine-activated killer activity, and/or interferon (IFN), tumor necrosis factor (TNF) and/or granulocyte-macrophage colony-stimulating factor (GM-) mediated responses. KSF). All of these functions, activated by IL-15, contribute to improved protection against cancer.

[0040] Therapeutically beneficial adoptive transfer of NK cells can, for example, induce remission in patients with refractory acute myeloid leukemia (AML), in combination with lymphocyte-depleting chemotherapy and IL-2 to promote in vivo survival and expansion of NK cells. This therapy may be limited by the lack of antigen specificity and IL-2-mediated induction of regulatory T cells (Tregs), which suppress NK cell proliferation and function. Development of a reagent that manipulates the antigen specificity, proliferation, and/or survival of NK cells while avoiding the negative effects of Treg inhibition may improve NK cell-based immunotherapies.

[0041] This disclosure describes the generation of a trispecific molecule that includes two domains capable of directing NK cell-mediated killing of tumor cells (e.g., CD33+ and/or CD33- tumor cells) and an intramolecular NK activating domain capable of generating a self-sustaining signal NK cells. The trispecific molecule can promote NK cell proliferation and/or enhance NK cell-mediated cytotoxicity against, for example, HL-60 targets, cancer cells, or cancer cell-derived cell lines.

[0042] The invention is based on the development of trispecific CD16/IL-15/CD33 killer cell-engaging molecules (TriKE) for the destruction of acute myeloid leukemia (AML) cells by natural killer cells. This molecule contains an anti-CD16 camel nanobody to activate NK cells, an anti-CD33 single chain variable fragment (scFv) to engage cancer targets, and an IL15 molecule that drives NK cell priming, proliferation, and survival. Using an earlier version of this molecule, CD33 TriKE was shown to be effective in activating NK cells against AML targets in vitro and in vivo. These preclinical data led to the launch of a clinical trial in refractory AML patients at the University of Minnesota, with the launch of Q3 2018. Although these previous studies have tested the use of TriKE as an effective strategy for targeting NK cells in cancer immunotherapy, CD33 has limitations as a target antigen.

[0043] The high mortality and low five-year survival rates (26%) for patients with AML may be associated with chemotherapy resistance and disease relapse. Most of the chemotherapy-resistant leukemia stem cells (LSCs) predicted to promote relapse do not express CD33. In addition, all hematopoietic stem cells and normal myeloid cells express CD33, thus targeting this antigen can lead to severe hematopoietic defects and target/non-tumor toxicity. To address these limitations, this paper describes the development of TriKE that targets CLEC12A or C-type lectin-like molecule 1 (CLL1). CLEC12A is highly expressed on AML cells and more than 70% of CD33-negative cells express CLEC12A. It has been identified as a stem cell marker in AML that is selectively overexpressed in LSCs. CLEC12A is expressed by CD34+/CD38− HSCs, but not by conventional CD34+/CD38 hematopoietic stem cells in regenerating bone marrow, thereby minimizing off-target effects. Member A of family 12 with a C-type lectin domain is a protein that in humans is 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 have characteristic protein folding and have diverse functions such as cell adhesion, cell-cell signaling, glycoprotein handling, and roles in inflammation and immune response. The protein encoded by this gene is a negative regulator of granulocyte and monocyte function. Several transcript variants of this gene have been described as the result of alternative splicing, but the origin of some full-length transcripts from these variants has not been determined. This gene is closely related to other members of the CTL/CTLD superfamily in the natural killer gene complex region on chromosome 12p13.

[0044] BiKE and TriKE connections

[0045] Bispecific hybrids have been produced that contain anti-human anti-CD16 scFv obtained through human phage display library technology (McCall et al., 1999. Mol Immunol. 36:433-445). NK cells mediate antibody-dependent cellular cytotoxicity (ADCC) through the CD16 receptor (FcγRIII). Signaling through the CD16 receptor induces calcium fluxes and ITAM phosphorylation, causing the release of lytic granules and cytokines such as interferon (IFNγ) and tumor necrosis factor (TNFα). A bispecific molecule has been designed to target the CD16 receptor in combination with other target molecules (Gleason et al. Blood. 2014 (19):3016-26), as a so-called bispecific killer-engaging (BiKE) molecule. . With one scFv recognizing NK cells and a second scFv recognizing tumor antigen, BiKE can markedly enhance cytotoxic killing in a variety of human cancers. One exemplary BiKE targets CD33 and enhanced NK cell responses against acute myeloid leukemia (AML) and myelodiplastic syndrome (MDS). MDS is a clonal heterogeneous stem cell disorder characterized by normal or hypercellular bone marrow (BM) with peripheral blood cytopenias (PB) and an increased risk of progression to AML.

[0046] NK cells respond to various 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 in engrafted NK cells after hematopoietic stem cell transplantation (HSCT). IL-15 and IL-2 share several signaling components, including IL-2/IL-15Rβ (CD122) and a common gamma chain (CD132). Unlike IL-2, IL-15 does not stimulate Tregs, allowing the activation of NK cells, bypassing the suppression of the immune response by Treg cells. In addition to promoting NK cell homeostasis and proliferation, IL-15 can restore functional defects in NK cells that may occur in the post-transplant setting. IL-15 can also stimulate the function of CD8+ T cells, further enhancing their immunotherapeutic potential. Additionally, based on preclinical studies, the toxicity profiles of IL-15 may be superior to IL-2 at low doses. In accordance with some embodiments, the compositions described herein can be used to activate NK cells and control the priming, proliferation and survival of NK cells.

[0047] This disclosure describes, in one aspect, trispecific killer-engaging (TriKE) molecules, which typically include one, or one or more targeting domains (which target, for example, a tumor cell or a virus-infected cell ), and one or more NK cytokine activation domains (e.g., IL-15, IL-12, IL-18, IL-21, or other cytokines, chemokines and/or activating molecules that enhance NK cell function), each domain functionally linked to other domains. As used herein, the term "operably linked" refers to direct or indirect covalent linkage. Thus, two functionally related domains can be directly covalently linked to each other. Alternatively, two operably linked domains may be joined by covalent linkage to an intervening moiety (eg, a flanking sequence). Two domains may be considered functionally related if, for example, they are separated by a third domain, with or without one or more intervening flanking sequences.

[0048] Exemplary BiKE and TriKE molecules or compounds are described in WO2017062604, the disclosure of which is incorporated herein by reference in its entirety.

[0049] This disclosure describes, in some embodiments, compounds that include an NK domain engaging; an NK activating domain operably linked to the NK engaging domain; and a targeting domain that selectively binds to a target cell and is operably linked to an NK activating domain and an NK engaging domain, wherein the targeting domain selectively binds to a target molecule. The target molecule may be expressed, for example, on the surface of the target cell. The target cell can be, for example, a tumor cell. In some embodiments, the target domain selectively binds to CLEC12A.

[0050] As used herein, the terms "selectively binding" or "selectively binds" as applied to the interaction of a binding molecule or domain described herein, for example, an antibody or engagement domain, activation domain or targeting domain, and its binding partner, for example, an antigen or a receptor, means that the interaction depends on the presence of a particular structure, such as an antigenic determinant, or an epitope, or an amino acid sequence, on the binding partner. In other words, a binding molecule or domain preferentially binds or recognizes a binding partner, even when the binding partner is present in a mixture of other molecules. Binding may 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.

[0051] The compounds described herein may or may not have a His tag. The His tag makes protein purification possible and can be useful in research development, for example. The His tag may be placed at the C-terminal or N-terminal end of the compounds or molecules described herein and may include a spacer N-terminal or C-terminal to the His tag. For example, a His tag that is placed at the C-terminus of a compound or molecule described herein may include a spacer N-terminal to the His tag. As another example, a His tag that is placed at the N-terminus of a compound or molecule described herein may include a spacer C-terminal to the His tag. An exemplary His tag with spacer is SEQ ID NO: 3. SEQ ID NO: 3 may be placed at the C-terminus of the compounds described herein. One skilled in the art will appreciate that any number of His repeats may constitute a His tag, and that any spacer sequence of any length may or may not be used.

[0052] In some embodiments, the TriKE compound or molecule that selectively binds to CLEC12A includes the amino acid sequence of SEQ ID NO: 1. In some embodiments, the TriKE compound or molecule that selectively binds to CLEC12A includes the amino acid sequence SEQ ID NO: 2. In some embodiments, the targeting domain of compounds described herein that selectively binds CLEC12A includes the selected amino acid sequence SEQ ID NO: 4.

[0053] Also disclosed herein are nucleic acid sequences that encode SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 4. For example, SEQ ID NO: 1 may be encoded by SEQ ID NO: 5, SEQ ID NO: 2 may be encoded by SEQ ID NO: 6, and SEQ ID NO: 4 may be encoded by SEQ ID NO: 7. One skilled in the art will appreciate that any functional variants of nucleic acid molecules proposed herein are contemplated herein disclosure of the invention. Functional variants are nucleic acid sequences that can be translated to provide an amino acid sequence homologous or identical to that of the translated parent molecule.

[0054] NK-involving domain

[0055] The NK engaging domain may include any moiety that binds to and/or activates an NK cell, and/or any moiety that blocks NK cell inhibition. Exemplary NK cell engagement domains include a moiety that binds, for example, CD16, CD16+CD2, CD16+DNAM, or CD16+NKp46. In some embodiments, the engagement domain includes a moiety that selectively binds CD16. In some embodiments, the NK engaging domain activates an NK cell. In some embodiments, the NK engaging domain blocks NK cell inhibition.

[0056] In some embodiments, the NK engaging domain may include an antibody that selectively binds to a component of the NK cell surface. In other embodiments, the NK engaging domain may include a ligand or small molecule that selectively binds to an NK cell surface component. As used herein, the term "selectively binds" refers to the ability to discriminate between two or more options, such as, for example, having a differing affinity, to any degree, for a particular target. As used herein, "antibody" generally refers to an immunoglobulin or a fragment thereof, and thus includes a monoclonal antibody, a fragment thereof (e.g., scFv, Fab, F(ab')2, Fv, or other modified forms), a combination monoclonal antibodies and/or fragments thereof, and/or a combination of polyclonal antibodies. Thus, for brevity, reference to an antibody that selectively binds to an NK cell surface component includes any antibody fragment that exhibits the described binding pattern. Likewise, reference to a ligand that selectively binds to an NK cell surface component includes any moiety of the ligand that exhibits the described binding pattern.

[0057] In some embodiments, the NK engaging domain may selectively bind to a receptor at least partially located on the surface of an NK cell. In some embodiments, the NK engaging domain may provide an NK cell binding function, and thereby bring the NK into spatial proximity to a target to which the targeting domain—described in more detail below—selectively binds. However, in some embodiments, the NK engaging domain may selectively bind to a receptor that activates the NK cell and therefore also has an activating function. As described above, activation of the CD16 receptor can cause antibody-dependent cellular cytotoxicity. Thus, in some embodiments, the NK engaging domain may include at least a portion of an anti-CD16 receptor antibody effective to selectively bind to the CD16 receptor. In other embodiments, the NK cell-engaging domain may interfere with mechanisms that inhibit NK cells. In such embodiments, the NK engaging domain may include, for example, anti-PD1/PDL1, anti-NKG2A, anti-TIGIT, anti-killer immunoglobulin receptor (KIR), and/or any other inhibitory blocking domain.

[0058] The NK engagement domain can be engineered to obtain the desired degree of NK selectivity and therefore the desired immune pattern of engagement. For example, CD16 has been defined as the Fc receptors: FcγRIIIa (CD16a) and FcγRIIIb (CD16b). These receptors bind to the Fc portion of IgG antibodies, which then activates the NK cell for antibody-dependent cellular 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. An embodiment of TriKE that includes an NK engaging domain that includes an anti-CD16a antibody can bind to NK cells but does not bind to neutrophils. Thus, in circumstances where it is desired to recruit NK cells but not neutrophils, it is possible to design an NK recruiting domain for TriKE to enable an anti-CD16a antibody.

[0059] Although described herein in the context of various embodiments in which the NK engaging domain comprises an anti-CD16 receptor scFv, the NK engaging domain may include any antibody or other ligand that selectively binds to the CD16 receptor. Moreover, the NK engaging domain may include an antibody or ligand that selectively binds to any NK cell receptor, such as, for example, cellular cytotoxicity receptor 2B4, low affinity Fc receptor CD16, killer cell immunoglobulin-like receptors (KIR), CD2 , NKG2A, TIGIT, NKG2C, LIR-1, and/or DNAM-1. In one embodiment, the composition of the invention is a construct in the NKG2C/IL-15/CD33 functional relationship. It should be understood that the placement of fragments may be changed based on activity assays (eg, CD33/IL-15/NKG2C).

[0060] In some embodiments, the NK-engaging domain includes an antibody or a binding fragment or nanobody thereof. The binding antibody fragment may be a scFv, F(ab)2, or Fab. In some embodiments, the NK engaging domain includes a nanobody. In some embodiments, the NK cell engagement molecule may include the use of a humanized CD16 engagement molecule derived from an animal nanobody. While scFv has a heavy chain variable region component and a light chain variable region component connected by a linker, the nanobody consists of one monomeric chain variable region, that is, a heavy chain variable region or a light chain variable region, which is capable of specifically engaging a target. The nanobody can be derived from an antibody from any suitable animal, such as, for example, a camelid (eg, llama or camel), or a cartilaginous fish. The nanobody can provide superior physical stability, the ability to bind into deep grooves, and increased product yield compared to larger antibody fragments.

[0061] In one illustrative embodiment, the nanobody-based NK-engaging molecule may include a humanized CD16 nanobody derived from a published llama nanobody (GeneBank sequence EF561291; Behar et al., 2008. Protein Eng Des Sel. 21(1): 1-10), designated as EF91. Llama EF91 was initially inserted into BiKE containing CD19 to test the ability of this CD16 engagement molecule to lead to NK cell activation. It showed similar functionality to rituximab-mediated killing in the chromium release assay with Raji targets. After confirming the functionality of the molecule, the CDRs were cloned into a humanized camel scaffold (Vincke et al., 2009. J Biol Chem. 284(5):3273–3284) to humanize the molecule involving CD16, now designated HuEF91. HuEF91 binding is equivalent to that observed using a standard CD16 scFv, indicating that insertion of the llama nanobody heavy chain variable region into the humanized scaffold does not interfere with the specificity of the molecule. The use of HUEF91 as an NK engagement molecule in the TriKE engagement molecules described herein may increase drug yield, increase persistence, and/or increase the efficiency of NK cell-mediated ADCC.

[0062] Thus, in some embodiments, the antibody or binding fragment, or nanobody, thereof is human or humanized. In some embodiments, the antibody or binding fragment, or nanobody, thereof is camelid.

[0063] NK activating domain

[0064] An NK activating domain may include an amino acid sequence that activates NK cells, promotes the maintenance of NK cells, or otherwise promotes NK cell activity. The NK activating domain may be, or may 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 (eg, IL-15) that is sufficient to provide NK cell activating and/or maintaining activity. In embodiments that include more than one NK activating domain, the NK activating domains may be provided sequentially or in any other combination. Additionally, each cytokine-based NK activating domain may include either the entire amino acid sequence of the cytokine or may be an amino acid fragment, regardless of the nature of the other NK activating domains included in the TriKE molecule. Exemplary cytokines on which the NK activating domain may 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 illustrative model embodiment in which the NK activating domain is derived from IL-15, TriKE can be designed using an NK activating domain that is, or is derived from, any suitable (its) cytokine(s).

[0065] For brevity, in this specification, reference to an NK activating domain by defining the cytokine on which it is based includes the complete amino acid sequence of the cytokine, any suitable amino acid fragment of the cytokine, and also a modified version of the cytokine that includes one or more amino acid substitutions. Thus, reference to the NK activating domain of “IL-15” includes an NK activating domain that includes the complete amino acid sequence of IL-15, an NK activating domain that includes a fragment of IL-15, or an NK activating domain such such as IL-15N72D or IL-15N72A, which includes an amino acid substitution compared to the amino acid sequence of wild type IL-15.

[0066] Use of the NK activating domain of IL-15 in TriKE can provide sustained NK cell activity - as demonstrated in a mouse model showing that human NK cells increase dramatically and cancer cells decrease, even after three weeks. NK cells are activated in mice to produce a suite of anticancer factors and cytokines. In addition, the NK activating domain of IL-15 can alter the chemistry of these molecules so that they are more easily refolded and/or released in greater yield, making TriKE molecules more suitable for clinical scale-up.

[0067] Thus, in some embodiments, the NK activating domain includes a cytokine or a functional fragment thereof. In some embodiments, the activating domain includes IL-15 or a functional fragment thereof. In some embodiments, the IL-15 is wild-type IL-15. In some embodiments, the IL-15 is human. In some embodiments, the 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, the functional variant of IL-15 contains amino acid substitutions N72D or N72A as compared to SEQ ID NO: 9.

[0068] As used herein, the term "functional variant" refers to a molecule, including a binding molecule, for example, containing a nucleotide and/or amino acid sequence that is changed by one or more nucleotides and/or amino acids compared to the nucleotide and/or amino acids amino acid sequences of the original molecule. For a binding molecule, the functional variant is still able to compete for binding with the binding partner of the original binding molecule. In other words, modifications to the amino acid and/or nucleotide sequence of the original binding molecule do not significantly affect or change the binding characteristics of the binding molecule encoded by the nucleotide sequence or containing the amino acid sequence, i.e., the binding molecule is still able to recognize and bind its target. A functional variant may have conservative sequence modifications, including nucleotide and amino acid substitutions, insertions and deletions. These modifications can be made using standard methods known in the art, such as site-directed mutagenesis and PCR-mediated random mutagenesis.

[0069] Functional variants may also include, but are not limited to, derivatives that are substantially similar in primary structural sequence but that contain, for example, in vitro or in vivo chemical and/or biochemical modifications that are not found in the original binding molecule. Such modifications include, but are not limited to: acetylation, acylation, ADP-ribosylation, amidation, flavin covalent addition, heme moiety covalent addition, nucleotide or nucleotide derivative covalent addition, lipid or lipid derivative covalent addition, phosphotidylinositol covalent addition, cross-linking, cyclization , disulfide bond formation, demethylation, covalent cross-link formation, cysteine formation, pyroglutamate formation, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, PEGylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer RNA-mediated addition of amino acids to proteins such as arginylation, ubiquitination, etc.

[0070] Targeting domain and targets

[0071] The targeting domain may include any moiety that selectively binds to an intended target, such as, for example, a tumor cell, a target in cancer stroma, a target on an inhibitory cell, such as myeloid-derived suppressor cells that are CD33+, or target on a virus-infected cell. Thus, the targeting domain may include, for example, an antitumor antibody such as: rituximab (anti-CD20), afutuzumab (anti-CD20), trastuzumab (anti-HER2/neu), pertuzumab (anti-HER2/neu), labetuzumab (anti-CEA), adecatumumab (anti-EpCAM), cituzumab bogatox (anti-EpCAM), edrecolomab (anti-EpCAM), arkitumomab (anti-CEA), bevacizumab (anti-VEGF-A), cetuximab (anti-EGFR ), nimotuzumab (anti-EGFR), panitumumab (anti-EGFR), zalutumumab (anti-EGFR), gemtuzumab ozogamicin (anti-CD33), lintuzumab (anti-CD33), etaracizumab (anti-integrin α _v β ₃ ), intetumumab (anti-CD51), ipilimumab (anti-CD152), oregovomab (anti-CA-125), votumumab (anti-tumor antigen CTAA16.88) or pemtumumab (anti-MUC1), anti-CD19, anti-CD22, anti-CD133, anti-CD38, antimesothelin, anti-ROR1, CSPG4, SS1 or IGFR1. Any tumor marker can be a target. In some embodiments, the targeting domain, or tumor-associated target antigen, may include: CD133, CD20, HER2, CEA, EpCAM, VEGF-A, EGFR, CD33, αVβ3 integrin, CD51, CD152, CD125, CTAA16 .88, MUC1, CD19, CD22, CD38, mesothelin, ROR1, CSPG4, SS1 or IGFR1, NKG2 family members including but not limited to 2A, 2B, 2C, etc., BCMA, APRIL, B7H3 and PSMA, in as an example.

[0072] 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 malignant 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 may selectively bind to a target on a cell infected with a virus, such as, for example, EBV, HBV, HCV and/or HPV. In some embodiments, the viral target is a tumor marker or tumor antigen. The target can be any viral tumor marker or non-viral tumor marker.

[0073] The targeting domain fragment may include an antibody or antibody binding fragment, or a nanobody, as described above. The antibody binding fragment may contain scFv, F(ab)2, or Fab.

[0074] In some specific embodiments, the targeting domain may include an anti-CLEC12A antibody. In other specific embodiments, a second targeting domain may be embedded. The second targeting domain may include a moiety that can bind to any of the targets described above. In some embodiments, the second targeting domain may selectively bind CD33.

[0075] In some embodiments, the compounds described herein include an NK engagement domain having a moiety that selectively binds CD16, an activating domain having IL-15, and a targeting domain that selectively binds CLEC12A. The terms "CLEC12A Trike", "1615CLEC12A TriKe" and "CD16-IL15-CLEC12A TriKE" may be used interchangeably to refer to a TriKE that targets CLEC12A unless the context clearly indicates otherwise.

[0076] Flanking sequences

[0077] In some embodiments, the compounds described herein may further include a flanking sequence or linker sequence that may connect two of the above-described domains. The terms "flanking sequence" and "linker sequence" may be used interchangeably unless the context clearly indicates otherwise. In some embodiments, the presence of a flanking sequence may further increase NK cell activation. Any amino acid sequence may be a flanking sequence or a linker sequence. One exemplary flanking sequence includes the 20 amino acids of SEQ ID NO: 13. Another exemplary flanking sequence includes the seven amino acids of SEQ ID NO: 14. Still other exemplary flanking sequences include SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 15. As another example, any number of repeats of an amino acid sequence may be a flanking sequence or a linker sequence. For example, any number of repeats of SEQ ID NO: 15 may be a flanking sequence or a linker sequence. Sequence repeats may be complete or partial, and complete or partial repeats may be at the beginning, i.e., at the N-terminus, or at the end, i.e., at the C-terminus, of a flanking sequence or linker sequence. The flanking sequences can be in any orientation.

[0078] Some embodiments (eg, 1615CLEC12A TriKE without His tag, SEQ ID NO: 1 or 1615CLEC12A TriKE with His tag, SEQ ID NO: 2) may include more than one flanking sequence. As one example, SEQ ID NO: 1 and/or SEQ ID NO: 2 includes the flanking sequence of SEQ ID NO: 11 for linking an NK engagement domain (e.g., CD16 anti-receptor scFv) to an 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 for linking the NK activating domain to the targeting domain (eg, anti-CLEC12A scFv). The flanking sequences that connect the domains of a molecule may be the same or different. For example, the same or different flanking sequences may connect an NK engaging domain (eg, an anti-CD16 scFv) to an NK activating domain (eg, IL-15), and an NK activating domain to a targeting domain (eg, an anti-CLEC12A scFv). In some embodiments, constructs that lack a flanking sequence exhibit reduced activity compared to constructs that possess a flanking sequence.

[0079] In some embodiments, the compounds described herein include at least one flanking sequence connecting two domains. In some embodiments, the compounds described herein further include a second flanking sequence connecting the two joined domains to a third domain. In some embodiments, the flanking sequences are the same. In some embodiments, the flanking sequences are different.

[0080] In some embodiments, the flanking sequences flank the NK activating domain. In some embodiments, the first flanking sequence is C-terminal to the NK engagement domain. In some embodiments, the second flanking sequence is N-terminal to the anti-CLEC12A targeting domain. In some embodiments, the first flanking sequence is C-terminal to the NK engagement domain and the second flanking sequence is N-terminal to the anti-CLEC12A targeting domain.

[0081] Ready-made forms

[0082] The compounds described herein can be formulated with a pharmaceutically acceptable carrier. As used herein, the term "carrier" includes any solvent, dispersion medium, carrier, coating, diluent, antibacterial and/or antifungal agent, isotonic agent, absorption retarding agent, buffer, carrier solution, suspension, colloid, etc. etc. The use of such media and/or agents for pharmaceutically active substances is well known in the art. Unless any conventional vehicle or agent is incompatible with the active ingredient, it is intended for use in therapeutic compositions. Additional active ingredients may also be included in the compositions. As used herein, the term "pharmaceutically acceptable" refers to a material that is not biologically or otherwise undesirable, i.e., the material can be administered to an individual along with the TriKE molecule without causing any undesirable biological effects or interacting in a harmful manner with any of the other components of the pharmaceutical composition in which it is contained.

[0083] Thus, the TriKE molecule can be formulated as a pharmaceutical composition. The pharmaceutical composition can be prepared in various forms adapted to the preferred route of administration. Thus, the composition may be administered through known routes, including, for example, oral, parenteral (e.g., intradermal, transdermal, subcutaneous, intramuscular, intravenous, intraperitoneal, etc.) or topical (e.g., intranasal, intrapulmonary, intramammary, intravaginal , intrauterine, intradermal, transdermal, rectal, etc.). The pharmaceutical composition can be applied to the surface of the mucosa, for example, by application, for example, to the mucous membrane of the nose or respiratory tract (for example, using a spray or aerosol). The composition can also be administered by sustained or delayed release.

[0084] Thus, the TriKE molecule may be provided in any suitable form, including, without limitation, a solution, suspension, emulsion, spray, aerosol, or any mixture form. The composition may be supplied in formulated form with any pharmaceutically acceptable excipient, carrier or vehicle. For example, the drug may be delivered in a conventional topical dosage form such as, for example, a cream, ointment, aerosol formulation, non-aerosol spray, gel, lotion, etc. The formulation may further include one or more additives including, for example, an adjuvant, a skin penetration enhancer, a colorant, a flavoring agent, a fragrance, a humectant, a thickener, and the like.

[0085] The composition may be conveniently presented in a dosage unit form and may be prepared by methods well known in the pharmaceutical art. Methods of preparing a composition with a pharmaceutically acceptable carrier include the step of combining the TriKE molecule with a carrier that contains one or more accessory ingredients. In general, the formulation can be prepared by uniformly and/or thoroughly combining 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 forms.

[0086] Methods of treatment

[0087] In some embodiments, methods provided herein include administering to a subject a compound or molecule described herein in an amount effective to induce NK-mediated killing of a target cell. Any cell can be a target cell. In some embodiments, the target cell is a cancer cell. The methods described herein may include administering to a subject a TriKE molecule in an amount effective to induce NK-mediated killing of target cells in the subject. In some embodiments, TriKE molecules are administered to treat a disease or pathology in a subject.

[0088] As used herein, the term “treat” or variations thereof refers to reducing, limiting the progression, alleviating or eliminating, to any extent, symptoms or signs associated with a pathology. As used herein, the term “relief” refers to any reduction in the extent, severity, frequency, and/or likelihood of a symptom or clinical sign characteristic of a particular pathology; "symptom" refers to any subjective evidence of disease or pathology in a patient; and "sign" or "clinical sign" refers to an objective physical finding related to a specific pathology that can be detected by someone other than the subject or patient.

[0089] As used herein, the term “subject” refers to any person or patient on whom the methods disclosed herein are performed. The term "subject" may be used interchangeably with the term "individual" or "patient". The "subject" may be any animal, such as, for example, a mammal (eg, dog, cat, horse, cow, sheep, goat, monkey, etc.). In some embodiments, the subject may be a human.

[0090] “Treatment” may be therapeutic or prophylactic. “Therapeutic” and its variants refer to treatment that alleviates one or more existing symptoms or clinical signs associated with a pathology. “Prophylactic” and its variants refer to treatment that limits to any extent the development and/or occurrence of a symptom, or clinical sign, of a pathology. In general, "therapeutic" treatment is initiated after pathology is manifested in the subject, whereas "prophylactic" treatment is initiated before pathology is manifested in the subject. Thus, in some embodiments, the method may include prophylactically treating a subject at risk of developing a pathology. “At risk” refers to an entity that may or may not actually have the risk described. Thus, for example, a subject “at risk” of developing a specified pathology is a subject who has one or more signs of increased risk of acquiring or developing a specified pathology compared to people who lack one or more signs, regardless of whether the subject exhibits any symptoms or clinical signs of acquiring or developing a pathology. Exemplary features of pathology may include, for example, genetic predisposition, ancestry, age, gender, geographic location, lifestyle, or clinical information. Treatment may also be continued after symptoms have disappeared, for example to prevent or delay their recurrence.

[0091] In other embodiments, provided herein are methods of promoting the proliferation of NK cells in vivo, which include administering to a subject a compound or molecule described herein in an amount effective to stimulate the proliferation of NK cells in the subject. In some embodiments, TriKE molecules are administered to treat a disease or pathology in a subject. Using the TriKE molecule as part of an in vivo treatment can render NK cells antigen-specific, with simultaneous costimulation, increased survival, and proliferation, which may be antigen-specific. In other cases, TriKE can be used in vitro as an adjuvant for NK cell adoptive transfer therapy.

[0092] In still other embodiments, provided herein are methods of treating cancer that include administering to a subject a compound or molecule described herein effective for treating 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, 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 a lymphoma. In some embodiments, the hematopoietic cancer is a leukemia. In some embodiments, the hematopoietic cancer is acute myeloid leukemia (AML).

[0093] As used herein, the term “myeloid leukemia” refers to a leukemia characterized by proliferation of myeloid tissue and an abnormal increase in the number of granulocytes, myelocytes and myeloblasts in the circulating blood. This term is synonymous with the terms myelocytic leukemia, myelogenous leukemia, myeloma leukemia and granulocytic leukemia. The term “myeloid leukemia” may refer to, but is not limited to, acute and chronic myeloid leukemia (AML and CML), acute promyelocytic leukemia (APL), chronic myelomonocytic leukemia (“CMML”), myelodysplastic syndrome, and juvenile myelomonocytic leukemia, which involves myeloid elements bone marrow (eg, leukocytes, erythrocytes and megakaryocytes), and includes all subtypes that are defined by morphological, histochemical and immunological methods well known to those skilled in the art. AML subtypes include, according to the FAB classification: FAB-M0, FAB-M1, FAB-M2, FAB-M3, FAB-M4, FAB-M5, FAB-M6 and FAB-M7.

[0094] As used herein, the term “myelodysplastic syndrome” covers a heterogeneous group of closely related clonal hematopoietic disorders that arise in the early hematopoietic cells of the bone marrow. All disorders are characterized by impaired morphology and maturation of bone marrow cells (dysmyelopoiesis), and peripheral blood cytopenia resulting from ineffective blood cell production. In other words, maturing blood cells often die in the bone marrow before reaching full maturity and entering the blood, which explains the low concentrations of blood cells. Patients suffering from myelodysplastic syndrome may also have a collection of very immature bone marrow cells called leukemic blast cells.

[0095] The amount of TriKE molecule administered may vary depending on various factors, including, but not limited to, the specific TriKE molecule used, the weight, physical condition, and/or age of the subject, and/or the route of administration. Thus, the absolute weight of the TriKE molecule included in a given unit dosage form may vary widely and depends on factors such as the species, age, weight and physical condition of the subject, and/or route of administration. Accordingly, it is not practical to establish, in general, an amount that constitutes an amount of a TriKE molecule that is effective for all possible uses. However, those skilled in the art can readily determine the appropriate amount with due consideration of such factors.

[0096] In some embodiments, the method may include administering to the subject sufficient amounts of the TriKE molecule to provide a dose, for example, from about 100 ng/kg to about 50 mg/kg, although in some embodiments, the methods may be accomplished by administering TriKE molecules at a dose outside this range. In some of these embodiments, the method includes administering to the subject sufficient amounts of the TriKE molecule to provide a dose of about 10 μg/kg to about 5 mg/kg, such as a dose of about 100 μg/kg to about 1 mg/kg.

[0097] Alternatively, the dose may be calculated using actual body weight obtained immediately before the start of treatment. For doses calculated in this way, the body surface area (m2) is calculated before the start of treatment using the Dubois method: m2 = (weight kg 0.425 × height cm 0.725) × 0.007184.

[0098] In some embodiments, the method may include administering sufficient amounts of the TriKE molecule to provide a dose, for example, from about 0.01 mg/m2 to about 10 mg/m2.

[0099] In some embodiments, the TriKE molecule may be administered in, for example, one dose to multiple doses per week, although in some embodiments the method may be performed by administering the TriKE molecule at a frequency outside this range. In some embodiments, the TriKE molecule can be administered from about once a month to about five times a week.

[00100] In some embodiments, the method further includes administering one or more additional therapeutic agents. One or more additional therapeutic agents may be administered before, after, and/or simultaneously with administration of the TriKE molecule. The TriKE molecule and additional therapeutic agents may be co-administered. As used herein, the term “co-administered” refers to two or more components of a combination that are administered such that the therapeutic or prophylactic effects of the combination may be greater than the therapeutic or prophylactic effects of each component administered separately. The two components may be administered together or sequentially. At the same time, the co-administered components may be provided in one or more pharmaceutical compositions. Sequential co-administration of two or more components includes instances in which the components are administered such that each component can be present at the treatment site simultaneously. Alternatively, sequential coadministration of two components may include cases in which at least one component has been removed from the treatment site, but at least one cellular effect of the component administration (e.g., cytokine production, activation of a specific cell population, etc.) d) is maintained at the treatment site until one or more additional components are introduced into the treatment site. Thus, a co-administered combination may, in certain circumstances, include components that would never exist in a chemical mixture with each other. In other embodiments, the TriKE molecule and the additional therapeutic agent may be administered as part of a mixture or cocktail. In some aspects, administration of a TriKE molecule may provide the effectiveness of a lower dose of other therapeutic modalities compared to administration of another therapeutic agent or agents themselves, thereby reducing the likelihood, severity and/or extent of toxicity observed with administration of a higher dose of another therapeutic agent. or agents.

[00101] Illustrative additional therapeutic agents include: altretamine, amsacrine, L-asparaginase, colaspase, bleomycin, busulfan, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, cyclophosphamide, cytophosphamide, cytarabine, dacarbazine, dactinomycin, 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, vincrestine, vindesine, and vinorelbine.

[00102] Accordingly, in some embodiments, methods of treating cancer provided herein further include administering a compound, molecule, composition, or formulation described herein before, concurrently with, or after chemotherapy, surgical resection of a tumor, or radiation therapy. Chemotherapy includes, for example: altretamine, amsacrine, L-asparaginase, colaspase, bleomycin, busulfan, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, cyclophosphamide, cytophosphamide, cytarabine, dacarbazine, dactinomycin, 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, vincrestine, vindesine, and vinorelbine.

[00103] In some embodiments, the methods provided herein may include administering a sufficient amount of TriKE molecules as described herein and administering at least one additional therapeutic agent, with the administration of TriKE molecules and at least one additional a therapeutic agent demonstrating therapeutic synergy. In some aspects of the methods of this invention, the outcome measurement of treatment response observed after administration of both the TriKE molecule as described herein and an additional therapeutic agent is improved compared to the same outcome measurement of treatment response observed after administration alone. either a TriKE molecule or an additional therapeutic agent. In some embodiments, the additional therapeutic agent may include an additional agent targeting EpCAM, including, for example, a monoclonal antibody specific for EpCAM, such as, for example, catumaxomab, a monoclonal hybrid antibody targeting EpCAM and CD3.

[00104] As used herein, the term “and/or” means one or all of the listed elements, or a combination of any two or more of the listed elements; The terms “comprises,” “comprising,” and variations thereof are to be construed as open-ended, i.e., additional elements or steps are optional and may or may not be present.

[00105] As used herein, singular forms include plural forms unless the context clearly indicates otherwise. Unless otherwise noted, the singular forms and “at least one” may be used interchangeably, and may mean one or more than one. Thus, for example, references to “method” include one or more methods and/or steps of the type described herein that will become apparent to those skilled in the art upon reading this disclosure, etc.

[00106] As used herein, listing numeric ranges by endpoint includes all numbers included in that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3, 80, 4, 5, etc.).

[00107] "About" as used herein, when the term refers to a measurable quantity such as quantity, duration, etc., is meant to cover variations of ±20%, or ±10%, or ±5%, or even ± 1% of the specified value, since such deviations are suitable for the disclosed methods or for performing the disclosed methods.

[00108] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art to which this invention belongs.

[00109] As used herein, the term “protein” refers to any polymer chain of amino acids. The terms "peptide" and "polypeptide" are used interchangeably with the term "protein" and also refer to the polymer chain of amino acids. The term “protein” includes native or artificial proteins, protein fragments, and protein sequence polypeptide analogs. A protein can be monomeric or polymeric. The term "protein" covers fragments and variants thereof (including fragments of variants) unless otherwise inconsistent with the context.

[00110] As used herein, the term “nucleic acid” refers to any deoxyribonucleic acid (DNA) molecule, ribonucleic acid (RNA) molecule, or nucleic acid analogs. A DNA or RNA molecule can be double-stranded or single-stranded and can be of any size. Examples of nucleic acids include, but are not limited to: chromosomal DNA, plasmid DNA, cDNA, cell-free DNA (cfDNA), mRNA, tRNA, rRNA, miRNA, microRNA (microRNA or miR), hnRNA. Exemplary nucleic acid analogs include peptide nucleic acids, morpholino and locked nucleic acids, glycolic nucleic acids, and threose nucleic acids.

[00111] In the foregoing description, for the sake of clarity, certain embodiments may be described separately. Unless specifically indicated that features of a particular embodiment are incompatible with features of another embodiment, certain embodiments may include a combination of compatible features described herein in connection with one or more embodiments.

[00112] For any method disclosed herein that includes discrete steps, the steps may be performed in any feasible order. Additionally, if necessary, any combination of two or more steps can be performed simultaneously.

[00113] The present invention is illustrated by the following examples. It should be understood that the specific examples, materials, quantities and procedures are to be interpreted broadly within the scope and spirit of the invention as set forth herein.

EXAMPLES

EXAMPLE 1

[00114] This example describes the development of CD16-IL15-CLEC12A TriKE.

[00115] TriKE 1615CLEC12A was developed in a mammalian cell system to ensure the presence of appropriate post-translational modifications. TriKE was confirmed to specifically bind to target HL60 and THP1 cells that express CLEC12A compared to Raji cells that do not express CLEC12A. Treatment of peripheral blood mononuclear cells (PBMCs) with 1615CLEC12A TriKE resulted in a significant increase in NK cell specific proliferation over 7 days, as measured by CellTrace dilution, compared to treatment with CLEC12A scFv or IL15 alone (69.7 ± 6. 7% versus 11.9 ± 2.5% versus 38.4 ± 7.3%) (Figure 1A). An IncuCyte Zoom assay was performed to measure NK cell killing. In it, target HL-60 cells were labeled with a caspase 3/7 reagent, the color change of which indicates the death of the target cell. 1615CLEC12A TriKE was able to induce greater target cell killing than scFv or IL-15 CLEC12A, as measured by the number of live target cells at the end of the 48-hour assay (53.9 ± 1.9% vs. 103.3 ± 3.4 % compared to 71.1 ± 1.4%). 1615CLEC12A TriKE induced an increase in NK cell degranulation, as measured by CD107a expression, against HL60 AML tumor targets in a 4-hour functional assay, compared with scFv or IL15 CLEC12A treatment alone (62.3 ± 1.1% vs. 19.4 ± 3.8% compared to 27.5 ± 4.9%). In this analysis there was also an increase in cytokine production as measured by IFNg and TNFa, respectively (16.7 ± 4.2% versus 2.3 ± 1.5% versus 4.7 ± 1.9% and 18.0 ±5.1% vs. 2.5±1.7% vs. 4.6±2.5%) (Figure 1B). A similar enhanced functional response was observed with THP1 AML tumor targets. In these functional assays, treatment with 1615CLEC12A TriKE caused less background activation compared with CD33 TriKE, indicating less off-target effects on PBMCs. To confirm the clinical relevance of this molecule, the efficacy of 1615CLEC12A TriKE against primary AML targets was tested. AML blast cells were identified as cells with low SB (side light scatter), intermediate levels of CD45 and high levels of CD34. Of the 9 AML samples tested, 7 expressed high levels of CD33 (70.4 ± 6.3%) and CLEC12A (78.1 ± 5.2%). In functional assays with these samples, 1615CLEC12A TriKE was able to induce greater expression of CD107a and IFNg, as well as enhance killing of tumor targets, as measured by live/dead staining, compared with CLEC12A scFv or IL15 ( Fig. 1C ). In these assays, the efficacy of 1615CLEC12A TriKE was comparable to that of CD33 TriKE. These data demonstrate that 1615CLEC12A TriKE stimulates specific NK cell proliferation, degranulation, cytokine secretion, and killing of tumor targets in vitro. In addition to AML, CLEC12A is expressed on cancer cells and LSCs from patients with myelodysplastic syndromes (MDS). These results highlight the clinical potential of 1615CLEC12A TriKE alone or in combination with CD33 TriKE for the treatment of MDS and AML.

EXAMPLE 2

[00116] This example describes the expression of CD33 and CLEC12A on AML cells.

[00117] The majority of all deaths from hematopoietic malignancies are caused by acute myeloid leukemia (AML), which has a five-year survival rate of only 26%, highlighting the need for new treatments. The most common antigen used to target AML cells is CD33. However, there are many limitations in developing anti-CD33 drugs. For example, not all cancer cells express CD33, including cancer cells from 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 lymphocytes, express CD33, leading to off-target toxicity. In addition, cancer stem cells, which are thought to promote relapse, do not express CD33.

[00118] A new antigen called C-type lectin-like molecule 1 (CLL-1) or CLEC12A has been targeted to address the above limitations.

[00119] FIG. Figure 2 shows the percentage of surface expression of CD33 and CLEC12A measured by flow cytometry analysis of primary AML samples from 10 patients. CLEC12A is expressed at high levels on AML cells. About 70% of CD33-negative cells expressed CLEC12A. CLEC12A expression was restricted to a subset of myeloid cells, limiting off-target toxicity. CLEC12A is present on leukemia stem cells but not on hematopoietic stem cells.

[00120] These data provide evidence that CLEC12A is a surface marker on both primary AML cells that express CD33 and those that do not express CD33. Thus, in some embodiments, CLEC12A can be targeted by a trispecific killer cell-engaging molecule (TriKE) to AML and other cells that express or lack CD33.

EXAMPLE 3

[00121] This example describes a trispecific killer cell-engaging molecule (TriKE) targeting CLEC12A.

[00122] To target cancer cells using natural killer (NK) cells, a trispecific killer cell-engaging molecule (TriKE) was developed containing an anti-CD16 heavy chain antibody that activates NK cells, an IL-15 molecule that drives priming , NK cell proliferation and survival, and anti-CLEC12A single chain variable fragment (scFv), which involves cancer targets. The CD16-IL15-CLEC12A TriKE design and mechanism of action are shown in Fig. 3A-B.

[00123] TriKE contains an anti-CD16 heavy chain antibody constructed by incorporating the anti-CD16 V _HH llama CDR into a humanized V _HH cakras (Figure 3A). It is coupled to a wild-type IL-15 molecule, which is coupled to a scFv from the anti-CLEC12A antibody. TriKE (SEQ ID NO: 1) was produced in a mammalian system with Expi-293 cells and contained a His tag which was used to purify the molecule. In some embodiments, the TriKE molecule may lack a His tag. A TriKE molecule that lacks a His tag may be suitable for use in clinical applications, although a TriKE molecule containing a His tag can also be used. TriKE creates an immunological link between the CLEC12A+ tumor cell and the NK cell, promoting the release of cytotoxic granules and secretion of cytokines that kill the target cell (Figure 3B).

[00124] In addition to the scFv from the anti-CLEC12A antibody described above as an illustrative example (SEQ ID NO: 4; corresponding to SC02-357 of US Pat. No. 7,741,443), the TriKE targeting domain may contain any sequence capable of targeting or binding to CLEC12A , for example, 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 US 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.

EXAMPLE 4

[00125] This example describes confirmation of binding of CD16-IL15-CLEC12A TriKE to target cells.

[00126] CLEC12A+ HL-60 and CLEC12A- Raji targets were incubated with 1615CLEC12A TriKE or scFv at equimolar concentrations. Binding was assessed using an anti-His antibody that binds to the His tag on TriKE or scFv. A streptavidin secondary antibody was used and detected by flow cytometry. Data in Fig. 4 show that the 1615CLEC12A TriKE is associated with HL-60 targets, but not with Raji targets.

[00127] The binding of various 1615CLEC12A TriKE components was tested by ELISA against CD16 (Figure 14A), IL15 receptor alpha (Figure 14B) and CLEC12A extracellular domain (ED; Figure 14C). 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 in subsequent experiments unless otherwise noted.

[00128] These data indicate that each 1615CLEC12A TriKE component binds to its corresponding target molecule and that CD16-IL15-CLEC12A TriKE specifically binds to CLEC12A-expressing targets.

EXAMPLE 5

[00129] This example describes NK cell proliferation induced by CD16-IL15-CLEC12A TriKE.

[00130] PBMCs were labeled with Cell Trace and incubated with equimolar concentrations of IL-15, CLEC12A scFv, or CLEC12A TriKE for 7 days (Figure 5A). The NK cell population was assessed by assessing Cell Trace dye dilution in the CD56+ CD3− population using flow cytometry (Figure 5B). The percentage of NK cells that divided was calculated using FlowJo Analyzer. Statistics reflect significant differences between groups calculated by one-way ANOVA, * P < 0.05, ** P < 0.005, N = 10. A higher percentage of dividing NK cells was observed in the presence of CLEC12A TriKE compared to IL-15 or CLEC12A scFv.

[00131] In other experiments, PBMCs were isolated from fresh healthy donor samples (n=6), labeled with CellTrace Violet, and incubated for 7 days with 1615CLEC12A TriKE or control agents at 30 nM as described above. After the incubation period, cells were harvested and NK cell proliferation (CD3−, CD56+) was assessed by flow cytometry.

[00132] Pooled data (Figure 9A) and representative histograms (Figure 9B) show NK cell proliferation (via CellTrace dilution) for different treatment groups. Fig. 9C shows pooled NK cell counts (45 seconds at constant speed) during product collection. One-way analysis of variance (ANOVA) with repeated measures was used to calculate differences compared to the 1615CLEC12A group. Error bars indicate +/- rms error of the mean. Statistical significance is defined as ** P < 0.005, **** P < 0.0001. A significantly higher percentage of dividing NK cells was observed with CLEC12A TriKE treatment compared with no treatment, IL-15 treatment, or CLEC12A scFv treatment.

[00133] These data show that 1615CLEC12A TriKE induced strong proliferation of NK cells.

EXAMPLE 6

[00134] This example describes the functional verification of CD16-IL15-CLEC12A TriKE.

[00135] PBMCs were incubated with CLEC12A+ HL-60 and THP1 cells at a 2:1 effector to target ratio in the presence of IL-15, CLEC12A scFv or CLEC12A TriKE at equimolar concentrations. Surface CD107a, to assess degranulation (Fig. 6A), intracellular IFNg (Fig. 6B), and TNFa, to assess inflammatory cytokine production (Fig. 6C), were analyzed on CD56 ⁺ CD3 ^- NK cells by flow cytometry. Statistical comparison of CLEC12A TriKE treatment with control IL-15 or CLEC12A scFv treatment reflected significant differences between groups calculated by one-way ANOVA, ** P < 0.005, N=6. Higher percentages of surface CD107a staining and higher percentages of intracellular IFNg and TNFa staining on NK cells were observed in the presence of CLEC12A TriKE compared to no treatment, IL-15 treatment, or CLEC12A scFv treatment of target HL60 and THP1 cells.

[00136] In other experiments, frozen PBMCs from healthy donors (n=6) were incubated with the indicated treatment agents (30 nM) to assess CD107a expression as a marker of degranulation (Figure 10A), intracellular IFNγ production (Figure 10B), or intracellular expression TNFa in NK cells (CD3-, CD56+; Figure 10C) in a 4-hour assay. Cells were assessed with PBMCs alone or in the presence of THP1 and HL-60 targets at an effector/target ratio of 2:1. NK cell activation (CD3−, CD56+) in PBMCs was assessed using CD69 expression in a 4-hour assay with PBMCs alone or in the presence of targets THP1 and HL-60 at an effector/target ratio of 2:1 (Figure 10D). One-way analysis of variance (ANOVA) with repeated measures was used to calculate differences compared to the 1615CLEC12A group. Error bars indicate +/- rms error of the mean. Statistical significance is defined as * P, .05, ** P, .01, *** P, .001, and **** P, .0001.

[00137] There was greater activation of NK cells, as demonstrated by increased staining for CD107a, IFNg, TNFa, and CD69 when incubated with CLEC12A TriKE compared to no treatment, IL-15 treatment, or scFv treatment for both THP1 and target cells. HL60 cells (Fig. 10A-D).

[00138] These data show that 1615CLEC12A TriKE induced degranulation and cytokine production against AML cell targets, including targets THP1 and HL-60.

EXAMPLE 7

[00139] This example describes CD16-IL15-CLEC12A TriKE-induced killing of AML targets.

[00140] Enriched NK cells were incubated with CLEC12A+ targets HL-60 (Figure 7A) and THP1 (Figure 7B) at an effector to target ratio of 2:1 in the presence of IL-15, CLEC12A scFv or CLEC12A TriKE at equimolar concentrations. Target cells were labeled with CellTrace Far Red and Caspase 3/7 green apoptosis assay reagent (Essen Biosciences). Killing was assessed using an Incucyte Zoom machine and analyzed by normalizing the cell count to the original target cell count. Graphs in Fig. 7A-B display the following (starting from top): (i) no processing (first from top); (ii) CLEC12A scFv treatment (second from top); (iii) IL-15 treatment (third from top/second from bottom); (iv) CLEC12A TriKE treatment (fourth from top/bottom). The percentage of live target cells was lowest with CLEC12A TriKE treatment.

[00141] 1615CLEC12A TriKE-mediated induction of target cell killing was assessed in a real-time imaging assay. Enriched NK cells (CD3-, CD56+) were incubated with CellTrace Far Red-labeled THP-1 cells at a 2:1 effector to target ratio with the indicated treatment agents (30 nM) for 48 hours in an IncuCyte S3 imager. The number of dead THP-1 cells was measured using Caspase 3/7 reagent. Fig. 11A shows a quantification of the percentage of live THP-1 (CellTrace Far Red/Caspase 3/7) tumor targets normalized to only the targets at time 0 hours. Data readings were performed every 30 minutes for 48 hours. Representation of 3 separate experiments. The first from the top corresponds to no treatment, the second from the top corresponds to the CLEC12A scFv treatment, the third from the top corresponds to the IL-15 treatment, and the fourth from the top corresponds to the CLEC12A TriKE treatment. The lowest percentage of live THP-1 cells was seen in the presence of CLEC12A TriKE.

[00142] FIG. 11B are representative images (original magnification 34: 2.82 mm/pixel) at 0, 18 and 36 hours showing THP-1 cells (larger cells) and NK cells (smaller cells). At 0 hours there were few dead cells for all treatment conditions indicated. At 18 and 36 hours, several clusters of dead THP-1 cells were observed for the no-treatment and CLEC12A scFv conditions, with some clusters of dying THP-1 cells present for the IL-15 treatment, and many more clusters of dying cells present for the treatment with CLEC12A TriKE.

[00143] FIG. 11C shows quantification of the percentage of live THP-1 tumor targets at various effector to target ratios (1:1, 2:1 and 5:1). The first, second and third starting from the top correspond to NK only, the fourth starting from the top corresponds to 1:1 NK+CLEC12A TriKE, the fifth starting from the top corresponds to 2:1 NK+CLEC12A TriKE, the sixth starting from the top corresponds to 5:1 NK+CLEC12A TriKE. Lower percentages of live THP-1 cells were observed for all effector to target ratios compared to NK cells only, with an effector to target ratio of 5:1 producing the lowest percentage of live THP-1 cells, but this reduced THP-1 cell viability was greatest when CLEC12A TriKE treatment was present.

[00144] In other experiments, enriched NK cells were incubated with CellTrace Far Red-labeled HL-60 cells at a 2:1 effector to target ratio with the indicated treatment agents (30 nM each) for 48 hours in an IncuCyte s3 imager. The number of dead HL-60 cells was measured using Caspase 3/7 reagent.

[00145] FIG. 15A shows quantification of the percentage of live HL-60 (Celltracace Dar Red/Caspase 3/7) tumor targets normalized to targets only and the 0 hour time point. Data readings were performed every 30 minutes for 48 hours. Representation of 3 separate experiments. The first from the top corresponds to no treatment, the second from the top corresponds to the CLEC12A scFv treatment, the third from the top corresponds to the IL-15 treatment, and the fourth from the top corresponds to the CLEC12A TriKE treatment. The lowest percentage of live HL-60 cells was observed in the presence of CLEC12A TriKE.

[00146] FIG. 15B is representative images at 0, 18 and 36 hours showing target HL-60 cells (larger cells) and NK cells (smaller cells). At 0 hours there were few dead cells for all treatment conditions indicated. At 18 and 36 hours, several clusters of dead HL-60 cells were observed for the no-treatment and CLEC12A scFv conditions, with some clusters of dying HL-60 cells present for the IL-15 treatment, and many more clusters of dying cells present for the treatment with CLEC12A TriKE.

[00147] These data demonstrate CD16-IL15-CLEC12A TriKE-induced killing of AML targets, including THP-1 and HL-60 target cells.

EXAMPLE 8

[00148] This example illustrates CD16-IL15-CLEC12A TriKE-induced killing of primary targeted AML blast cells in vitro.

[00149] Enriched NK cells were incubated with primary AML blast cells at a 2:1 effector to target ratio in the presence of IL-15, CLEC12A scFv, CLEC12A TriKE, or CD33 TriKE at equimolar concentrations. Fig. 8A shows a gating scheme for detecting AML blast cells using the FlowJo Analyzer. Fig. 8B shows the percentage of AML blast cell killing determined by the live/dead marker after blast cell gating after 48 hours. Surface expression of CD107a to assess degranulation (Fig. 8C), and intracellular IFNg to assess inflammatory cytokine production (Fig. 8D) was determined on CD56 ⁺ CD3 ^- NK cells by flow cytometry after 4 hours. Statistical analysis shows significant differences between groups calculated using one-way analysis of variance, * P < 0.05 ** P < 0.005, N = 10. The percentage of dead AML blast cells and staining percentages for CD107A and IFNγ were significantly higher when incubated with CLEC12A TriKE compared to incubation with IL-15 or incubation with CLEC12A scFv (Figure 8B-D).

[00150] In other experiments, primary AML blast cells (BS-B Low, CD45int, CD117+, CD14-, CD34+) were assessed for CD33 and CLEC12A expression using flow cytometry (Figure 12A). The cells expressed CD33, CLEC12A, or both CD33 and CLEC12A as demonstrated.

[00151] Enriched NK cells (CD56+, CD3-) from healthy donors (n=10) were incubated with primary AML blast cells with the indicated treatment agents (30 nM) to assess CD107a expression as a marker of degranulation (Figure 12B) and intracellular IFNg production (Figure 12C) in a 4-hour assay at an effector to target ratio of 2:1. CLEC12A TriKE and CD33 TriKE induced degranulation and IFNg production as shown. Target cell killing was also assessed using flow cytometry and a live/dead marker at 48 hours (Figure 12D). Treatment with IL-15, CLEC12A TriKE, and CD33 TriKE resulted in killing of AML blast cells as shown, with greater CLEC12A TriKE-mediated killing.

[00152] The ratio of different groups of AML blast cells (based on CD33 and CLEC12A expression) was monitored after 48 hours to assess the specificity of 1615CLEC12A TriKE compared to 1615CD33 TriKE (Figure 12E). The bars show the % of live AML blast cells corresponding to the following (starting at 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 CLEC12A scFv, CLEC12A+CD33+, CLEC12A+CD33-, CLEC12A-CD33+, and CLEC12A-CD33-; (iv) for CLEC12A TriKE, CLEC12A+CD33+, CLEC12A-CD33+, and CLEC12A-CD33-; (v) for CD33 TriKE, CLEC12A+CD33+ and CLEC12A+CD33-. These data support the specificity of 1615CLEC12A TriKE compared to 1615CD33 TriKE.

[00153] 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 agents (30 nM) to assess the killing of cancer stem cells (CS- In Low, CD45int, CD34+, CD38-) in the 4-hour assay at an effector/target ratio of 2:1 (Figure 12F). Typical cytometry plots show killing of CLEC12A and CD33 positive cancer stem cells.

[00154] FIG. 12G shows combined data from a cancer stem cell killing assay showing the percentage of cancer stem cells present at the end of the assay. One-way analysis of variance (ANOVA) with repeated measures was used to calculate differences compared to the 1615CLEC12A group. Error bars indicate +/- rms error of the mean. Statistical significance is defined as * P, .05, ** P, .01, *** P, .001, and **** P, .0001. Treatment with CLEC12A resulted in a significant decrease in the percentage of LSCs compared to no treatment.

[00155] To test primary AML blast cells as target cells for CLEC12A TriKE-mediated killing, a gating strategy was used. The gating strategy for detecting primary AML blast cells is demonstrated in FIG. 16a. Primary AML blast cells (n=5) were then incubated with the indicated treatment agents (30 nM) to assess target cell killing using flow cytometry and a live/dead marker at 48 hours (Figure 16B). The percentage of dead AML blast cells at 48 hours is shown.

[00156] These data indicate that 1615CLEC12A TriKE induced a small increase in the number of primary AML blast cells killed in patient samples during blast crisis when NK cells are limited.

EXAMPLE 9

[00157] This example describes the restriction of tumor growth in vivo mediated by 1615CLEC12A TriKE.

[00158] FIG. 13A shows the design of experiments with HL-60luc mice. The model was established by conditioning mice with NSG (225 cGy) and then intravenously injecting HL-60luc cells (7.5 × 10 ⁵ cells/mouse). Three days later, 1 × ¹⁰⁶ human NK cells from a healthy donor (calculated from magnetically depleted CD3/CD19 product) activated overnight with 10 ng/ml IL-15 were infused. 1615CLEC12A TriKE or 161533 TriKE (20 μg) was administered PnTSrTTPT for the next 3 weeks of the study (15 doses total), and the control group received HL-60luc cells only.

[00159] Quantification of luminescence of the four treated groups on days 7, 14 and 21 after NK infusion is shown in Figure 13B. Each dot represents a different mouse and the bars represent the mean +/- standard deviation. One-way analysis of variance (ANOVA) without comparison comparisons was used to calculate differences compared to the 1615CLEC12A group. Statistical significance was defined as * P , 0.05, *** P , 0.001, and **** P , 0.0001. Fig. 13C shows individual mouse photoluminescence (dark areas) after 2 min exposure on days 7, 14 and 21. Treatment of CLEC12A with TriKE resulted in decreased tumor burden at all time points examined compared to the HL60 control. Additionally, at day 21, there was a significant reduction in tumor burden with CLEC12A TriKE treatment compared to control NK cell treatment.

[00160] FIG. 13D shows the experimental design of pdx mice. The model was established by preparing NSG mice with SGM3 (125 cGy) and then intravenously injecting HL-60luc cells (7.5 × ¹⁰⁵ cells/mouse). Tumors were allowed to grow until at least 1% AML blast cells appeared in the blood. Subsequently, 1 × ¹⁰⁶ human NK cells from a healthy donor (calculated from magnetically depleted CD3/CD19 product) activated overnight with 10 ng/ml IL-15 were infused. 1615CLEC12A TriKE or 161533 TriKE (20 μg) was administered PnWtSrTtPt for the next 3 weeks of the study (15 doses total), and the control group received NK cells only without treatment. Mice were sacrificed on day 21, and the percentage of AML blast cells (CD45int, CD33+) in femur bone marrow was counted by flow cytometry (Figure 13E). Each dot represents an individual mouse. The percentage of NK cells (CD3-, CD56+) in bone marrow (Figure 13F) and peripheral blood (Figure 13G) samples was calculated using flow cytometry. Manifestations of activity were recorded for 60 seconds and the number of acts of manifestations of human NK cell activity was counted. Representative dot plots are shown showing the number of NK cell (CD56+ CD3-) activity events within the CD45+ gating). One-way analysis of variance (ANOVA) without comparison comparisons was used to calculate differences compared to the 1615CLEC12A group. Error bars indicate mean +/- standard deviation. Statistical significance was defined as * P , 0.05, *** P , 0.001, and **** P , 0.0001. The results show that NK+CLEC12A TriKE treatment significantly reduced the percentage of primary AML blast cells compared to tumor alone (Figure 13E). Additionally, treatment of NK+CLEC12A TriKE resulted in 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.

[00161] Taken together, these results demonstrate that 1615CLEC12A TriKE limits tumor growth in vivo.

EXAMPLE 10

[00162] This example describes the analysis of stem and progenitor cells.

[00163] Cancer stem cells were identified in bone marrow samples using the gating strategy shown in FIG. 17. A gating strategy to identify different subpopulations of CD34 ^pos precursors in healthy bone marrow samples is shown in FIG. 19. The gating strategy shown in FIG. 19 was used to analyze the cell populations shown in FIG. 18A.

[00164] FIG. 18A shows expression of CLEC12A and CD33 in the CD34 ^pos progenitor compartment in bone marrow from two representative healthy donors. HSC: hematopoietic stem cells, CPP: pluripotent progenitor, LPPP: lymphoid pluripotent progenitor, CLM: common lymphoid progenitor, CMP: common myeloid progenitor, GMP: granulocyte-macrophage progenitor, MEP: megakaryocyte-erythroid progenitor. CLEC12A expression was observed in the AMP, GMP, and MEP populations, but it was relatively lower in the HSC, PPP, OLP, and DILI populations. Additionally, with the exception of the OAB population, CLEC12A was detected at lower levels than CD33. Colonies of erythroid burst forming units (E-BFU) and erythroid colony forming units (E-CFU) were counted after treatment with 1615CLEC12A TriKE or 161533 TriKE (Figure 18B). More E-PFU and GM-CFU colonies were observed with 1615CLEC12A TriKE treatment compared to 161533 TriKE treatment.

[00165] Data show that CLEC12A was differentially expressed in normal donor stem and progenitor cell populations, and that treatment with 161533 TriKE reduced stem cell formation and/or differentiation compared to CLEC12A TriKE treatment. Without being limited by theory, this indicates that although CLEC12A can be used to target leukemia stem cells, it should ensure normal resumption of hematopoiesis, whereas targeting CD33 is more likely to affect resumption. In other words, targeting CLEC12A should have fewer off-target effects in terms of normal myeloid regeneration.

[00166] Thus, taken together, the above data indicate that CD16-IL15-CLEC12A TriKE specifically binds to target cells expressing CLEC12A, promoted NK cell proliferation, enhanced NK cell function, promoted clearance of AML cell lines in Incucyte zoom assays, and induced the destruction of primary AML and MDS blast cells.

EXAMPLE 11

[00167] This example illustrates the creation of TetraKE targeting CLEC12A and a second target or tumor antigen.

[00168] It is possible to design a TetraKE molecule (tetramer) that contains more than one targeting domain. As an example, TetraKE may contain an NK engaging domain, an NK activating domain, and two targeting domains. Any of the NK engaging domains and NK activating domains described herein may be used. Targeting domains can be directed, for example, to various targets or tumor antigens. Any combination of targets or tumor antigens can be included in TetraKE. For example, the first targeting domain may bind to CLEC12A, while the second targeting domain may bind to another target or tumor antigen.

[00169] Any of the targets or tumor antigens described herein can be included in TetraKE with a first targeting domain that binds CLEC12A, including, for example, a second targeting domain that binds CD133, CD20, HER2, CEA, EpCAM , VEGF-A, EGFR, CD33, αVβ3 integrin, CD51, CD152, CD125, CTAA16.88, MUC1, CD19, CD22, CD38, mesothelin, ROR1, CSPG4, SS1, or IGFR1, NKG2C, BCMA, APRIL, B7H3, and PSMA, or viral antigen derived from EBV, HBV, HCV and/or HPV. Additionally, TetraKE domains can be operably linked to each other using flanking or linker sequences, as described herein. An exemplary TetraKE includes a compound that has a moiety that selectively binds CD16, an NK activating domain that contains IL-15, a first targeting domain that selectively binds CLEC12A, and a second targeting domain that selectively binds CD33.

SEQ ID NO: 1

Mkwvtfisllflfssaysqvqlvesggglvqpggslrlscaasgltfssynmgwfrqapgqgleavasitwsgrdtfyadsvkgrftisrdnskntlylqmnslraedtavyycaanpwpvaaprsgtywgqgtlvtvsssggggsggggsggggsggggsgnwvnvisdlkkiedliqsmhid atlytesdvhpsckvtamkcfllelqvislesgdasihdtvenliilannslssngnvtesgckeceeleeknikeflqsfvhivqmfintsgstsgsgkpgsgegstkgqvqlqesgpglvkpsetlsltcvvsggsisssnwwswvrqppgkglewigeiyhsgspdynpslksrvtisvdksrnqfs lklssvtaadtavyycakvstggffdywgqgtlvtvssggggsggggsggggseieltqspsslsasvgdrvtitcrasqsissylnwyqqkpgkapklliyaasslqsgvpsrfsgsgsgtdftltisslqpedfatyycqqsystpptfgpgtkveik

SEQ ID NO: 2

Mkwvtfisllflfssaysqvqlvesggglvqpggslrlscaasgltfssynmgwfrqapgqgleavasitwsgrdtfyadsvkgrftisrdnskntlylqmnslraedtavyycaanpwpvaaprsgtywgqgtlvtvsssggggsggggsggggsggggsgnwvnvisdlkkiedliqsmhid atlytesdvhpsckvtamkcfllelqvislesgdasihdtvenliilannslssngnvtesgckeceeleeknikeflqsfvhivqmfintsgstsgsgkpgsgegstkgqvqlqesgpglvkpsetlsltcvvsggsisssnwwswvrqppgkglewigeiyhsgspdynpslksrvtisvdksrnqfs lklssvtaadtavyycakvstggffdywgqgtlvtvssggggsggggsggggseieltqspsslsasvgdrvtitcrasqsissylnwyqqkpgkapklliyaasslqsgvpsrfsgsgsgtdftltisslqpedfatyycqqsystpptfgpgtkveikvdehhhhhhhhhh

SEQ ID NO: 3

vdehhhhhhhhhh

SEQ ID NO: 4

qvqlqesgpglvkpsetlsltcvvsggsisssnwwswvrqppgkglewigeiyhsgspdynpslksrvtisvdksrnqfslklssvtaadtavyycakvstggffdywgqgtlvtvssggggsggggsggggseieltqspsslsasvgdrvtitcrasqsissylnwyqqkpgkap klliyaasslqsgvpsrfsgsgsgtdftltisslqpedfatyycqqsystpptfgpgtkveik

SEQ ID NO: 5

atgaagtgggtaacctttatttcccttctttttctctttagctcggcttattcccaggtgcagctggtggagtctgggggaggcttggtgcagcctgggggctctctgagactctctcctgtgcagcctctggcctcaccttcagtagctataacatgggctggttccgccaggctccagggcaaggccttgaggctgcatctattacctggag tggtcgggacacattctatgcagactccgtgaagggccgattcaccatctccagagacaactccaagaacactctctatctgcaaatgaacagcctgcgcgcggaggacacggccgtttattattgtgctgcaaacccctggccagtggcggcgccacgtagtggcacctactggggccaagggaccctggtcaccgtctcctcatctggcggcggcggtt ctggtggaggaggtagtggggggggaggaagcggagggggtggctcagggaactgggtgaatgtaataagtgatttgaaaaaaattgaagatcttattcaatctatgcatattgatgctactttatatacggaaagtgatgttcaccccagttgcaaagtaacagcaatgaagtgctttctcttggagttacaagttatttcacttgagtccggaga tgcaagtattcatgatacagtagaaaatctgatcatcctagcaaacaacagtttgtcttctaatgggaatgtaacagaatctggatgcaaagaatgtgaggaactggaggaaaaaaatattaaagaatttttgcagagttttgtacatattgtccaaatgttcatcaacacttctggcagtaccagcgggtcagggaaacctggcagtgggg aaggttccacaaaaggtcaagtacaactccaggagtccgggccagggttggtcaagccatccgagacgcttagtttgacctgtgttgtcagcggaggctctatatcatcttcaaactggtggtcttgggtacggcaaccaccgggcaaggggctcgaatggatcggggaaatctaccactccggaagccccgactataatccgtcactgaagagc agagtcactatatccgtggacaagagcagaaaccaattttctcttaagctctcctcagtgacagcagcagatacagcggtctattattgtgccaaggtatcaacaggcggattctcttcgattattggggacagggcactttggttacggtttcttctggaggcgggggaagtggtggaggggggtctgggggaggtggctcagaaatcgaacttac gcagtcaccctcctccctctcagcatccgtaggtgacagagttacgataacctgtagagcaagtcaatccatttctagctaccttaactggtatcagcaaaaacctgggaaagcccccaagctgcttatctatgcggcatcctccctccaaagtggagttcccagtcggttcagtggttccggctcagggactgactttaccctcacaatcagctcat tgcaaccagaggactttgcaacgtattactgtcagcaaagctactcaacgccgcctacgttcggtcccggaaccaaagttgagattaaagtagacgaacaccatcatcatcatcaccatcaccaccattga

SEQ ID NO: 6

atgaagtgggtaacctttatttcccttctttttctctttagctcggcttattcccaggtgcagctggtggagtctgggggaggcttggtgcagcctgggggctctctgagactctctcctgtgcagcctctggcctcaccttcagtagctataacatgggctggttccgccaggctccagggcaaggccttgaggctgcatctattacctggag tggtcgggacacattctatgcagactccgtgaagggccgattcaccatctccagagacaactccaagaacactctctatctgcaaatgaacagcctgcgcgcggaggacacggccgtttattattgtgctgcaaacccctggccagtggcggcgccacgtagtggcacctactggggccaagggaccctggtcaccgtctcctcatctggcggcggcggtt ctggtggaggaggtagtggggggggaggaagcggagggggtggctcagggaactgggtgaatgtaataagtgatttgaaaaaaattgaagatcttattcaatctatgcatattgatgctactttatatacggaaagtgatgttcaccccagttgcaaagtaacagcaatgaagtgctttctcttggagttacaagttatttcacttgagtccggaga tgcaagtattcatgatacagtagaaaatctgatcatcctagcaaacaacagtttgtcttctaatgggaatgtaacagaatctggatgcaaagaatgtgaggaactggaggaaaaaaatattaaagaatttttgcagagttttgtacatattgtccaaatgttcatcaacacttctggcagtaccagcgggtcagggaaacctggcagtgggg aaggttccacaaaaggtcaagtacaactccaggagtccgggccagggttggtcaagccatccgagacgcttagtttgacctgtgttgtcagcggaggctctatatcatcttcaaactggtggtcttgggtacggcaaccaccgggcaaggggctcgaatggatcggggaaatctaccactccggaagccccgactataatccgtcactgaagagc agagtcactatatccgtggacaagagcagaaaccaattttctcttaagctctcctcagtgacagcagcagatacagcggtctattattgtgccaaggtatcaacaggcggattctcttcgattattggggacagggcactttggttacggtttcttctggaggcgggggaagtggtggaggggggtctgggggaggtggctcagaaatcgaacttac gcagtcaccctcctccctctcagcatccgtaggtgacagagttacgataacctgtagagcaagtcaatccatttctagctaccttaactggtatcagcaaaaacctgggaaagcccccaagctgcttatctatgcggcatcctccctccaaagtggagttcccagtcggttcagtggttccggctcagggactgactttaccctcacaatcagctcat tgcaaccagaggactttgcaacgtattactgtcagcaaagctactcaacgccgcctacgttcggtcccggaaccaaagttgagattaaatga

SEQ ID NO: 7

caagtacaactccaggagtccgggccagggttggtcaagccatccgagacgcttagtttgacctgtgttgtcagcggaggctctatatcatcttcaaactggtggtcttgggtacggcaaccaccgggcaaggggctcgaatggatcggggaaatctaccactccggaagccccgactataatccgtcactgaagagcagagtcactatatccgtgg acaagagcagaaaccaattttctcttaagctctcctcagtgacagcagcagatacagcggtctattattgtgccaaggtatcaacaggcggattcttcgattattggggacagggcactttggttacggtttcttctggaggcgggggaagtggtggaggggggtctgggggaggtggctcagaaatcgaacttacgcagtcaccctcctccct ctcagcatccgtaggtgacagagttacgataacctgtagagcaagtcaatccatttctagctaccttaactggtatcagcaaaaacctgggaaagcccccaagctgcttatctatgcggcatcctccctccaaagtggagttcccagtcggttcagtggttccggctcagggactgactttaccctcacaatcagctcattgcaaccagaggactttgca acgtattactgtcagcaaagctactcaacgccgcctacgttcggtcccggaaccaaagttgagattaaa

SEQ ID NO: 8

QVQLVESGGGLVQPGGSLRLSCAASGLTFSSYNMGWFRQAPGQGLEAVASITWSGRDTFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAANPWPVAAPRSGTYWGQGTLVTVSS

SEQ ID NO: 9

NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

SEQ ID NO: 10

Mkwvtfisllflfssays

SEQ ID NO: 11

sggggsggggsggggsggggsg

SEQ ID NO: 12

gstsgsgkpgsgegstkg

SEQ ID NO: 13

PSGQAGAAASESLFVSNHAY

SEQ ID NO: 14

EASGGPE

SEQ ID NO: 15

GGGGSGGGGS

SEQ ID NO: 16

MGWSCIILFLVATATGVHSS

SEQ ID NO: 17

MGWSCIILFLVATATGVHS

SEQ ID NO: 18

EVQLVESGGELVQAGGSLRLSCAASGLTFSSYNMGWFRRAPGKEREFVASITWSGRDTFYADSVKGRFTISRDNAKNTVYLQMSSLKPEDTAVYYCAANPWPVAAPRSGTYWGQGTQVTVSSVDE

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 the 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 Inhumanized Cam16

[00170] Any and all references and citations to other documents, such as patents, patent applications, patent publications, journals, books, articles, web content, that have been made in this disclosure are hereby incorporated herein by reference in fully for all purposes.

[00171] Although the present invention has been described with reference to specific details of certain embodiments in the above examples, it should be understood that modifications and variations are within the spirit and scope of the invention. Accordingly, the present invention is limited only by the following claims.

--->

LIST OF SEQUENCES

<110> REGENTS OF THE UNIVERSITY OF MINNESOTA

MILLER, Jeffrey S.

FELICES, Martin

VALLERA, Daniel A.

LENVIK, Todd R.

<120> NK INVOLVING MOLECULES AND METHODS OF THEIR APPLICATION

<130>GTBIO2090-1WO

<150> US 62/747983

<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

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

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 ggctccagg 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 aggggtggc 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 aatccattc 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 ggctccagg 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 aggggtggc 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 aatccattc 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

15

<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

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His Ser Ser

20

<210> 17

<211> 19

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 17

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His Ser

<210> 18

<211> 125

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 18

Glu Val Gln Leu Val Glu Ser Gly Gly Glu Leu Val Gln Ala 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 Arg Ala Pro Gly Lys Glu Arg Glu Phe 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 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 (36)

1. A compound for activating NK cells containing: a natural killer (NK) cell-engaging domain containing a moiety that selectively binds CD16; an NK activating domain operably linked to said NK engaging domain, containing an antibody or a binding fragment thereof, or a nanobody; And a targeting domain that selectively binds to a target cell and is operably linked to said NK activating domain and an NK engaging domain, wherein said targeting domain selectively binds to CLEC12A. 2. The compound according to claim 1, characterized in that said binding fragment of the antibody contains scFv, F(ab)2 or Fab. 3. The compound according to claim 1, characterized in that the antibody or its binding fragment or nanobody is human or humanized. 4. The compound according to claim 1, characterized in that the antibody or its binding fragment or nanobody is camel. 5. The compound according to claim 1, characterized in that the NK activating domain contains IL-15 or a functional fragment thereof. 6. The compound according to claim 5, characterized in that said IL-15 contains the amino acid sequence SEQ ID NO: 9 or a functional variant thereof. 7. The compound according to claim 1, characterized in that the targeting domain fragment contains an antibody or a binding fragment thereof, or a nanobody. 8. The compound according to claim 7, characterized in that the binding fragment of the antibody contains scFv, F(ab)2 or Fab. 9. The compound of claim 1, wherein the NK engaging domain contains a moiety that selectively binds CD16, the NK activating domain contains IL-15, and the targeting domain selectively binds CLEC12A. 10. A compound according to claim 1, containing at least one flanking sequence connecting two of said domains. 11. The compound of claim 10, further comprising a second flanking sequence connecting the two joined domains to the third domain. 12. The compound according to claim 11, characterized in that said flanking sequences flank the NK activating domain. 13. The compound of claim 12, wherein the first flanking sequence is C-terminal to the NK engagement domain and the second flanking sequence is N-terminal to the anti-CLEC12A targeting domain. 14. The compound according to claim 1, characterized in that the CLEC12A domain is presented in SEQ ID NO: 4. 15. Composition for use in inducing proliferation of NK cells, containing: connection according to claim 1; And pharmaceutically acceptable carrier. 16. A method of inducing NK-mediated killing of a target cell in a subject, comprising: administering to the subject a compound of claim 1 in an amount effective to induce NK-mediated killing of the target cell. 17. The method according to claim 16, characterized in that said target cell is a cancer cell. 18. A method for stimulating the proliferation of NK cells in vivo , including: administering to a subject an amount of a compound of claim 1 effective to stimulate the proliferation of NK cells in said subject. 19. A method of inhibiting the growth of cancer cells in a subject, comprising: administering to said subject an amount of a compound of claim 1 effective to inhibit the growth of cancer cells. 20. The method according to claim 19, characterized in that the cancer includes prostate cancer, lung cancer, colon cancer, rectal cancer, bladder cancer, melanoma, kidney cancer, renal cancer, oral cancer, pharyngeal cancer, pancreatic cancer gland cancer, uterine cancer, thyroid cancer, skin cancer, head and neck cancer, cervical cancer, ovarian cancer or hematopoietic cancer. 21. The method of claim 20, further comprising administering the compound before, simultaneously with, or after chemotherapy, surgical resection of the tumor, or radiation therapy. 22. The method of claim 21, characterized in that chemotherapy includes altretamine, amsacrine, L-asparaginase, colaspase, bleomycin, busulfan, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, cyclophosphamide, cytophosphamide, cytarabine, dacarbazine, dactinomycin, 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, vincrestine, vindesine, and vinorelbine. 23. The method according to claim 19, characterized in that the hematopoietic cancer is acute myeloid leukemia (AML). 24. A method for stimulating the secretion of inflammatory cytokines by NK cells in vivo , where the method includes: administering to the subject an effective amount of a compound of claim 1 to stimulate the secretion of inflammatory cytokines. 25. The method of claim 24, wherein the inflammatory cytokines include IFNγ and TNFα. 26. A method of treating acute myeloid leukemia (AML) in a subject, where the method includes: administering to the subject an effective amount of a compound of claim 1 for the treatment of AML.