Immunotherapeutic compounds and methods

Abstract

To provide methods and compounds for inducing NK-mediated killing of cancer cells, stimulating proliferation of NK cells in a subject, and / or treating cancer.SOLUTION: The immunotherapeutic compound comprises an NK cell binding domain, an NK activating domain, and a targeting domain. The targeting domain selectively binds to HER2, HER3, or HER2 / HER3 heterodimer complexes and is operably linked to an NK activating domain and an NK cell binding domain. The compound can be administered to a subject to induce NK-mediated killing of cancer cells, stimulate proliferation of NK cells in the subject, and / or treat cancer.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 901,198, filed September 16, 2019, which is incorporated herein by reference in its entirety.

[0002] government funding This invention was made with government support under CA197292 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0003] Sequence Listing This application contains a Sequence Listing that has been submitted electronically to the U.S. Patent and Trademark Office via EFS-Web as an ASCII text file titled "Seq_Listing-0110-000632_ST25.txt" and 70 kilobytes in size, created on September 15, 2020. The information contained in the Sequence Listing is incorporated herein by reference. Summary of the Invention

[0004] In one aspect, the present disclosure describes multispecific immunotherapeutic compounds comprising an NK cell binding domain, an NK activation domain, and a targeting domain that selectively binds to HER2, HER3, or the HER2 / HER3 heterodimer complex and is operably linked to the NK activation domain and the NK cell binding domain.

[0005] In some embodiments, the NK cell binding domain specifically binds to CD16. In these embodiments, CD16 may be CD16a or CD16b. In some of these embodiments, the NK cell binding domain comprises the amino acid sequence of SEQ ID NO:2.

[0006] In some embodiments, the portion of the NK cell binding domain may comprise an antibody or binding fragment thereof. In some of these embodiments, the antibody or binding fragment thereof may be human, humanized, or camelid.

[0007] In some embodiments, the NK activation domain comprises an IL-15 component. In some of these embodiments, the IL-15 component comprises the amino acid sequence of SEQ ID NO: 4 or a functional variant thereof. In some of these embodiments, the functional variant of IL-15 comprises an N72D or N72A amino acid substitution compared to SEQ ID NO: 4.

[0008] In some embodiments, the targeting domain comprises an antibody or binding fragment thereof. In some of these embodiments, the antibody binding fragment may comprise an scFv, F(ab)2, Fab, or single-domain antibody fragment. In some of these embodiments, the targeting domain comprises the amino acid sequence of SEQ ID NO:6, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, or SEQ ID NO:27.

[0009] In some embodiments, the immunotherapeutic compound may include a second targeting domain.

[0010] In some embodiments, the immunotherapeutic compound may comprise a second NK-activating domain.

[0011] In another aspect, the present disclosure describes compositions comprising any embodiment of the therapeutic compounds summarized above and a pharmaceutically acceptable carrier.

[0012] In some embodiments, the composition may further comprise an additional therapeutic agent. In some of these embodiments, the additional therapeutic agent may comprise a chemotherapeutic agent. In some embodiments, the additional therapeutic agent may comprise a therapeutic agent that targets HER2, HER3, or the HER2 / HER3 heterodimer complex.

[0013] In another aspect, the present disclosure describes a method comprising administering to a subject any embodiment of the compounds or compositions summarized above in an amount effective to induce NK-mediated killing of cancer cells.

[0014] In another aspect, the present disclosure describes a method for stimulating NK cell proliferation in vivo. Generally, the method includes administering to a subject an amount of any embodiment of the compounds or compositions summarized above effective to stimulate NK cell proliferation in the subject.

[0015] In another aspect, the present disclosure describes a method of treating cancer in a subject. Generally, the method includes administering to the subject an amount of any embodiment of the compound or composition summarized above effective to treat the cancer.

[0016] In some embodiments, the compound or composition is administered before, concurrently with, or after chemotherapy, surgical removal of tumors, or radiation therapy. In some of these embodiments, the chemotherapy is selected from the group consisting of altretamine, amsacrine, L-asparaginase, collaspase, bleomycin, busulfan, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, cyclophosphamide, cytophosphane, cytarabine, dacarbazine, dactinomycin, daunorubicin, docetaxel, doxorubicin, epirubicin, etoposide, fluorouracil, fludarabine, These may include fotemustine, ganciclovir, gemcitabine, hydroxyurea, idarubicin, ifosfamide, irinotecan, lomustine, melphalan, mercaptopurine, methotrexate, mitoxantrone, mitomycin C, nimustine, oxaliplatin, paclitaxel, pemetrexed, procarbazine, raltitrexed, temozolomide, teniposide, thioguanine, thiotepa, topotecan, vinblastine, vincristine, vindesine, or vinorelbine.

[0017] The above summary is not intended to describe each disclosed embodiment or every implementation of the present invention. The following description more particularly exemplifies exemplary embodiments. In several places throughout the application, guidance is provided through lists of examples that can be used in various combinations. In each instance, the recited list serves only as a representative group and should not be interpreted as an exclusive list.

[0018] The patent or application file contains at least one drawing executed in color. Copies of the patent or patent application publication with such color drawing(s) will be provided by the Office upon request and payment of the necessary fee. [Brief explanation of the drawings]

[0019] [Figure 1] Design, production, and purification of cam1615HER2. (A) Schematic diagram of an exemplary expression vector with the arrangement of coding regions encoding the components of cam1615HER2 (from left to right): camelid anti-CD16 VHH, human IL-15, anti-HER2 scFV. (B) Genetic map of an exemplary expression vector encoding cam1615HER2, including restriction enzyme sites and target gene location on the pET28c vector. [Figure 2] Production and purification of cam1615HER2. (A) SDS-PAGE gel stained with Coomassie blue dye showing the purity and size of the final product after two orthogonal column steps. MWS: molecular weight standard; NR: non-reduced; R: reduced. Concentration measurements were performed to derive the final purity. (B) Chromatography trace obtained from the first-step purification of cam1615HER2 on an ion-exchange (FFQ) column. The collected peak is indicated by a double-headed arrow. (C) Chromatography trace obtained from the second-step purification of cam1615HER2 on a size-exclusion column. The collected peak is indicated by a double-headed arrow. [Figure 3]Absolute numbers showing the effect of cam1615HER2 on NK cell proliferation and the effect of TriKE treatment on the percentage of proliferating C56+CD3- NK cells measured by flow cytometry. PBMCs from six different normal donors were assayed separately. In addition to cam1615HER2, IL-15 was used as a control. (A) cam1615HER2 treatment resulted in a significantly different percentage of highly proliferating NK cells compared to the control. (B) cam1615HER2 treatment resulted in a significantly different percentage of total NK cells compared to the control. (C) cam1615HER2 treatment resulted in a significantly different number of raw NK counts compared to the NT control. (D) cam1615HER2 treatment did not increase the percentage of highly proliferating CD3+CD56- T cells. (E) cam1615HER2 treatment did not increase the percentage of total CD3+CD56- T cells. (F) cam1615HER2 treatment did not increase the number of live CD3+CD56- T cells. [Figure 4] cam1615HER2 TriKE binding to target cell lines. (A) cam1615HER2 directly labeled with FITC binding to SKOV-3 cell line. (B)) cam1615HER2 directly labeled with FITC binding to SK-BR-3 cell line. (C)) cam1615HER2 directly labeled with FITC binding to UMSCC-11B cell line. BAC3 is a CD3-binding molecule and serves as a negative control for binding. [Figure 5]Functional activity correlates with the binding activity of cam1615HER2 TriKE. (A) CD107a functional activity was elevated in cultures of PBMCs and SKOV3 cells treated with cam1615HER2 compared to IL-15 and untreated controls. PBMCs from 10 different normal donors were assayed separately. (B) Enhanced IFN-γ activity in the same PBMC / SKOV3 culture. (C) CD107a functional activity was elevated in cultures of PBMCs and the breast cancer cell line SK-BR-3 (seven different donors assayed). (D) Enhanced IFN-γ activity in the same PBMC / SK-BR-3 culture. (E) CD107a was not elevated when the UMSCC-11B head and neck cancer cell line was tested in the same assay (four different donors assayed). [Figure 6] Testing the ability of TriKE to enhance killing of drug (tamoxifen)-resistant MCF-7L-TamR breast cancer cells. (A) Background cytotoxic activity of cancer cell-free CD56+CD3- NK cells was tested using CD107a flow cytometry. (B) CD107a activity using PBMCs incubated with the parental MCF-7L cell line. (C) CD107a activity using PBMCs incubated with tamoxifen-resistant MCF-7L-TamR cells. (D) CD107a activity using PBMCs incubated with SKBR-3 breast cancer cells. (E) Background intracellular IFN-γ activity of cancer cell-free CD56+CD3- NK cells. (F) Intracellular IFN-γ activity using PBMCs incubated with the parental MCF-7L cell line. (G) Intracellular IFN-γ activity using PBMCs incubated with tamoxifen-resistant MCF-7L-TamR cells. (H) Intracellular IFN-γ activity using PBMCs incubated with SKBR-3 breast cancer cells. Intracellular IFN-γ activity correlated with CD107a activity. [Figure 7]INCUCYTE (ESSEN BIOSCIENCE, INC., Ann Arbor, MI) data measuring SKOV3 ovarian cancer cell killing in real time in the presence of PBMCs and confirming CD107a cytotoxicity data. (A) Spheroid size; (B) Spheroid strength. Cam1615HER2 causes a rapid decrease in target cells measured over 120 hours compared to low activity in untreated, anti-cam16 alone (CAM16), and IL-15 alone (IL15) controls. N=7 donors / group. [Figure 8] Visual evidence that cam1615HER2 causes a rapid, time-dependent decrease in target cells measured over 72 hours (right column) compared to the decrease in activity in no treatment (left column), anti-cam16 alone, and IL-15 alone controls. N=7 donors / group. [Figure 9] Testing of ascites from ovarian cancer patients as a source of effector cells. (A) CD107a background activity when cells from patient ascites were incubated without MA-148 ovarian cancer cells. (B) CD107a activity when ascites cells were incubated with MA-148 ovarian cancer cells. (C) CD107a background activity when cells from a normal donor were incubated without MA-148 ovarian cancer cells. (D) CD107a activity when normal donor cells were incubated with MA-148 ovarian cancer cells. (E) IFN-γ background activity when cells from patient ascites were incubated without MA-148 ovarian cancer cells. (F) IFN-γ activity when ascites cells were incubated with MA-148 ovarian cancer cells. (G) IFN-γ background activity when cells from a normal donor were incubated without MA-148 ovarian cancer cells. (H) IFN-γ activity when normal donor cells were incubated with MA-148 ovarian cancer cells. Controls were IL-15 and no treatment. In each case, 9–13 different donors were analyzed independently and the data were averaged. [Figure 10]In vivo efficacy of cam1615HER2 in a xenograft model. Cells were stably transfected with firefly luciferase for real-time bioluminescence imaging. (A) Bioluminescence imaging of a group of six NSG mice intraperitoneally injected with SKOV3 and NK cells. The image shows the total flux for each animal, indicating that five of the six animals in the untreated group had progressive tumors. The one animal in the untreated group that showed minimal activity continued to develop a tumor. (B) Day 38 imaging of a group of six mice that also received SKOV3 and NK cells but were treated with cam1615HER2 TriKE. [Figure 11] (A) Despite multiple TriKE injections, animals experienced minimal changes in body weight, indicating that treatment was not toxic compared to untreated controls. (B) Scatter plot of data from the same experiment 46 days after tumor inoculation. Data are expressed as total flux radiance (p / s). Treated mice are compared to untreated mice. The difference is significant as determined by Student's t-test (p=0.0216). (C) Line graph of time (days) showing when the treated group began to relapse. (d) Survival plot of data over an extended time interval. The difference between the treated and untreated groups is significant. [Figure 12]Testing the ability of cam1615HER2TriKE to enhance killing of other HER2-expressing ovarian cancer cell lines. (A) CD107a activity when PBMCNK cells were incubated with OVCAR3 ovarian cancer cells. (B) CD107a activity when PBMCNK cells were incubated with OVCAR5 ovarian cancer cells. (C) CD107a activity when PBMCNK cells were incubated with SKOV3 ovarian cancer cells. (D) IFN-γ activity when PBMCNK cells were incubated with OVCAR3 ovarian cancer cells. (E) IFN-γ activity when PBMCNK cells were incubated with OVCAR5 ovarian cancer cells. (F) IFN-γ activity when PBMCNK cells were incubated with SKOV3 ovarian cancer cells. SKOV3 data were performed using the following negative controls: no treatment (NT), anti-cam16 alone (CAM16), IL-15 (IL15), and anti-HER2 antibody alone (e23). DETAILED DESCRIPTION OF THE INVENTION

[0020] This disclosure generally describes therapeutic compounds that target tumor cells expressing human epidermal growth factor receptor-2 (HER2) and / or human epidermal growth factor receptor-3 (HER3), members of the epidermal growth factor receptor (EGFR) family of transmembrane receptor tyrosine kinases. HER2 is directly linked to cancer because its overexpression is associated with poor prognosis in breast cancer and triggers intracellular signaling pathways related to cell proliferation, differentiation, and survival. HER2 and HER3 can form heterodimeric complexes.

[0021] In many embodiments, the immunotherapeutic compound may be a trispecific killer engager compound (TriKE). TriKE has three distinct binding regions: an NK cell binding domain (e.g., CD16) that binds to NK cells, an NK activation domain containing a cytokine or a functional fragment thereof that binds to the cytokine's receptor, and a targeting domain that binds to a marker present on a target cell (e.g., a cancer cell). The design and production of TriKE is extensively described, for example, in U.S. Patent Application Publication No. US2018 / 0282386 A1. TriKE offers the advantage of combining an antibody-dependent cellular cytotoxicity (ADCC) facilitating moiety and an expansion-related moiety (IL-15) on the same molecule.

[0022] One or more binding regions or domains of an immunotherapeutic compound may comprise an antibody. As used herein, the term "antibody" generally refers to an immunoglobulin or fragment thereof, and thus encompasses monoclonal antibodies and fragments thereof. Exemplary antibody fragments include, but are not limited to, scFv, Fab, F(ab'), Fv, single-domain Ab (sdAb), or other modified forms (e.g., humanized), and / or combinations of monoclonal antibodies and / or fragments thereof. For example, camelids produce functional antibodies lacking light chains. These single-domain antibody fragments (VHH or NANOBODIES (Ablynx NV, Ghent, Belgium)) have several advantages for biotechnology applications. They are well expressed in microorganisms and have high stability and solubility. In certain embodiments of the TriKE compounds described herein, the NK cell-binding domain is a camelid single-domain antibody fragment.

[0023] Although immunotherapeutic compounds are described herein in the context of an exemplary embodiment having a targeting domain comprising a HER2-targeting scFv having the amino acid sequence of SEQ ID NO: 6, the immunotherapeutic compounds described herein may also comprise any other suitable HER2 and / or HER3 targeting moieties. Thus, in various embodiments, the targeting domain can recognize HER2, HER3, and / or HER2 / HER3 heterodimers. HER2 / HER3 heterodimers are used to treat many breast cancers and many HER2 + Detected in tumors, HER2 / HER3 dimers are associated with proliferation, distant metastasis, and / or poor patient outcomes.

[0024] Exemplary alternative targeting moieties include antibodies and antibody fragments that specifically bind to HER2, HER3, and / or HER2 / HER3 heterodimers. Exemplary antibody fragments include e23 or a functional fragment thereof (e.g., SEQ ID NO: 15), trastuzumab or a functional fragment thereof (e.g., SEQ ID NO: 16 and European Patent No. EP 3457139 A1), SEQ ID NO: 17, SEQ ID NO: 18, lumretuzumab or a functional fragment thereof (RG7116; Liu et al., 2019, Biol Proced Online 21:5; e.g., SEQ ID NO: 19, SEQ ID NO: 20), seribantumab or a functional fragment thereof (MM-121; Liu et al., 2019, Biol Proced Online 21:5; e.g., SEQ ID NO: 21, or SEQ ID NO: 22), KTN3379 / CDX-3379 or a functional fragment thereof (Liu et al., 2019, Biol Proced Online 21:5; e.g., SEQ ID NO: 23), patritumab or a functional fragment thereof (U3-1287; Liu et al., 2019, Biol Proced Online 21:5; e.g., SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, or SEQ ID NO: 27), elgemtumab (LJM716, Liu et al., 2019, Biol Proced Online 21:5) or a functional fragment thereof, U3-1402 (Liu et al., 2019, Biol Proced Online 21:5) or a functional fragment thereof, AV-203 (Liu et al., 2019, Biol Proced Online 21:5) or a functional fragment thereof, GSK2849330 (Liu et al., 2019, Biol Proced Online 21:5) or a functional fragment thereof, MM-111 (Liu et al., 2019, Biol Proced Online 21:5) or a functional fragment thereof, MCLA-128 (Liu et al., 2019, Biol Proced Online 21:5) or a functional fragment thereof, istiratumab (MM-141; Liu et al., 2019, Biol Proced Online 21:5) or a functional fragment thereof, duligotuzumab (MEHD7945A; Liu et al., 2019, Biol Proced Online 21:5) or a functional fragment thereof, or pertuzumab, or a functional variant thereof.

[0025] Although described herein in the context of an exemplary embodiment in which the NK cell binding domain comprises a single-domain antibody (sdAb) that binds to CD16, the immunotherapeutic compound may comprise any other suitable NK-binding moiety. Exemplary alternative NK-binding moieties include, but are not limited to, any amino acid sequence capable of selectively binding to a receptor located at least in part on the surface of an NK cell. Thus, the NK cell binding domain functions to bind NK cells, thereby bringing the NK into spatial proximity with the target to which the targeting domain selectively binds. In certain embodiments, the NK cell binding domain can selectively bind to a receptor that activates NK cells and thus also possesses activation function. For example, activation of the CD16 receptor can induce antibody-dependent cellular cytotoxicity. Thus, the NK cell binding domain of the exemplary cam1615HER2 compound possesses NK-activating activity. In other embodiments, the NK cell binding domain can interfere with mechanisms that inhibit NK cells. In such embodiments, the NK cell binding domain may include, for example, anti-PD-1 / PD-L1, anti-NKG2A, anti-TIGIT, anti-killer immunoglobulin receptor (KIR), and / or any other inhibitory blocking domain.

[0026] The NK cell binding domain may comprise, for example, an antibody or ligand that selectively binds to any NK cell receptor, such as the cytotoxicity receptor 2B4, the low affinity Fc receptor CD16, killer immunoglobulin-like receptor (KIR), CD2, NKG2A, TIGIT, NKG2C, LIR-1, and / or DNAM-1.

[0027] The NK cell-binding domain can be designed to have the desired degree of NK selectivity and, therefore, the desired immune binding properties. For example, CD16 has been identified as the Fc receptors FcγRIIIa (CD16a) and FcγRIIIb (CD16b). These receptors bind to the Fc portion of IgG antibodies and activate NK cells 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 not neutrophils. Immunotherapeutic compounds containing an NK cell-binding domain with an anti-CD16a antibody can bind to NK cells but not neutrophils. Therefore, in situations where it is desired to bind NK cells but not neutrophils, the NK cell-binding domain of an immunotherapeutic compound can be designed to include an anti-CD16a antibody.

[0028] Although described herein in the context of an exemplary embodiment in which the NK activation domain comprises a fragment of human IL-15, the NK activation domain can comprise any amino acid sequence that activates NK cells, promotes NK cell persistence, or otherwise promotes NK cell activity. The NK activation domain may be or be derived from one or more cytokines capable of activating and / or maintaining NK cells. As used herein, the term "derived from" refers to an amino acid fragment of a cytokine (e.g., IL-15) that is a functional variant of the referenced cytokine, i.e., has sufficient sequence similarity or sequence identity to the referenced cytokine, to provide NK cell activation and / or persistence. Exemplary cytokines on which the NK activation domain can be based include, for example, IL-15, IL-18, IL-12, and IL-21. Thus, although the exemplary cam1615HER2 compound comprises an NK activation domain derived from IL-15, a HER2-targeting compound can be designed to have an NK activation domain that is or is derived from any suitable cytokine.

[0029] For brevity of description in this specification, reference to an NK activation domain by identifying the cytokine on which it is based can refer to either the complete amino acid sequence of the cytokine or a functional variant of the cytokine. A functional variant of a cytokine may include any suitable amino acid fragment of the cytokine and / or a modified version of the cytokine containing one or more amino acid deletions, additions, and / or substitutions. Thus, reference to an "IL-15" NK activation domain includes an NK activation domain comprising the complete amino acid sequence of IL-15, an NK activation domain comprising a fragment of IL-15, or an NK activation domain such as IL-15N72D or IL-15N72A, which contain amino acid substitutions compared to the wild-type IL-15 amino acid sequence.

[0030] Although described above in the context of an exemplary embodiment in which the immunotherapeutic compound is a trispecific killer-engaging compound (i.e., TriKE), the compositions and methods described herein may also include the use of immunotherapeutic compounds modified to include additional domains. For example, immunotherapeutic compounds can be larger molecules designed to have multiple targeting domains, multiple NK cell-binding domains, and / or multiple NK activation domains. In embodiments containing multiple NK activation domains, the NK activation domains may be provided in tandem or in any other combination. The cytokine-based NK activation domain may comprise the complete amino acid sequence of a cytokine, be an amino acid fragment, or be a modified version of a cytokine, regardless of the nature of the other NK activation domains included in the immunotherapeutic compound.

[0031] Exemplary additional targeting domains include, but are not limited to, any moiety that selectively binds to an intended target, such as, for example, a tumor cell, a target in the cancer stroma, a target on an inhibitory cell such as a CD33+ myeloid-derived suppressor cell, or a target on a cell infected with a virus. Thus, the targeting domain may be, for example, rituximab (anti-CD20), afutuzumab (anti-CD20), pertuzumab (anti-HER2 / neu), labetuzumab (anti-CEA), adecatumumab (anti-EpCAM), sitatuzumab bogatox (anti-EpCAM), edrecolomab (anti-EpCAM), arcitumomab (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 β3), intetumumab (anti-CD51), ipilimumab (anti-CD152), oregovomab (anti-CA-125), votumumab (anti-tumor antigen CTAA16.88), or pemtumumab (anti-MUC1), anti-CD19, anti-CD20, anti-CD22, anti-CD23, anti-CD30, anti-CD38, anti-CD45, anti-CD52, anti-CD70, anti-CD74, anti-CD133, anti-mesothelin, anti-ROR1, anti-CSPG4, anti-SS1, or anti-HSPG2, anti-IGF-1, anti-ROR-1, anti-uPAR, anti-VEGFR, anti-LIV-1, anti-SGN-CD70A, anti-IL-3, anti-IL-4R, anti-epithelial-mesenchymal transition (EMT), anti-TRAIL, anti-PD-L1, European Patent No. EP 3457139 A1, or a functional variant of any of the foregoing.

[0032] An amino acid sequence is a "functional variant" of a reference amino acid sequence if the "functional variant" amino acid sequence has a certain amount of sequence identity or sequence specificity compared to the reference amino acid sequence. An amino acid sequence is a "functional fragment" of a reference amino acid sequence if the "functional fragment" amino acid sequence is less than the full-length amino acid sequence of the reference amino acid sequence. A "functional fragment" may further have a certain amount of sequence identity or sequence specificity compared to the reference amino acid sequence.

[0033] Sequence similarity and / or sequence identity of two amino acid sequences can be determined by aligning the residues of the amino acid sequences to optimize the number of identical amino acids along the length of the sequences; to optimize the number of identical amino acids, gaps in either or both sequences are allowed in creating the alignment, but the amino acids in each sequence must still remain in proper order.

[0034] Pairwise comparison analysis of amino acid sequences can be performed using the BESTFIT algorithm in the GCG package (version 10.2, Madison, WI). Alternatively, polypeptides are described by Tatiana et al. (FEMS Microbiol Lett, 174, 247-250 (1999)) and are available on the National Center for Biotechnology Information (NCBI) website. Default values ​​for all BLAST2 search parameters can be used, including matrix = BLOSUM62; open gap penalty = 11; extension gap penalty = 1; gap x_dropoff = 50; expect = 10; wordsize = 3; and filter on.

[0035] In comparing two amino acid sequences, structural similarity can be expressed in terms of percent "identity" or percent "similarity." "Identity" refers to the presence of identical amino acids. "Similarity" refers not only to the presence of identical amino acids but also allows for the presence of conservative substitutions. Conservative substitutes for amino acid residues within an amino acid sequence can be selected from other members of the class to which the amino acid residue belongs. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and tyrosine. Polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Positively charged (basic) amino acids include arginine, lysine, and histidine. Negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Thus, conservative substitutions include, for example, substitution of Lys for Arg and vice versa to maintain a positive charge; Glu for Asp and vice versa to maintain a negative charge; Ser for Thr to maintain a free -OH; and Gln for Asn to maintain a free -NH2.

[0036] Thus, an amino acid belonging to a group of amino acids having a particular size or property (e.g., charge, hydrophobicity, or hydrophilicity) can be substituted with another amino acid without altering the activity of the protein, particularly in regions of the protein that are not directly related to biological activity. Regions within an amino acid sequence that are not directly related to biological activity can be inferred from alignment analysis, which identifies regions where variability (e.g., additions, deletions, or non-conservative substitutions) exists when comparing related amino acid sequences. Alignment analysis can be performed using the amino acid sequences provided herein and / or amino acid sequences readily available in databases.

[0037] The NK binding domain, NK activation domain, or targeting domain may comprise an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a reference amino acid sequence (e.g., a reference antibody fragment, cytokine, or cytokine fragment).

[0038] The NK binding domain, NK activation domain, or targeting domain may comprise an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a reference amino acid sequence.

[0039] The immunotherapeutic compounds described herein can also be designed to provide additional sequences, such as the addition of additional C- or N-terminal amino acids, which can facilitate purification, for example, by column capture or the use of antibodies. Such tags include, for example, histidine-rich tags that allow for purification of the polypeptide on a nickel column. Such genetic modification techniques and appropriate additional sequences are well known in the field of molecular biology.

[0040] The present disclosure also provides polynucleotides encoding any of the immunotherapeutic compounds described herein, as well as the complements of such polynucleotide sequences. Given the amino acid sequence of any one of the immunotherapeutic compound polypeptides described herein (or one or more component fragments of the immunotherapeutic compound), one of skill in the art can determine the full range of polypeptides encoded by that amino acid sequence using conventional, routine methods.

[0041] Figure 1A shows an exemplary construction of an exemplary embodiment of a second-generation TriKE, herein designated cam1615HER2 (SEQ ID NO: 1), capable of both antibody-dependent cellular cytotoxicity (ADCC) and NK cell proliferation. cam1615HER2 TriKE contains an anti-CD16 VHH as the NK cell-binding domain. The anti-CD16 VHH is the variable region of the heavy chain of a camelid antibody. Figure 1B is a plasmid map showing the placement of the TriKE coding sequence in a pET expression vector. Figure 2B shows an absorbance trace of bacterial and target protein fractions as they pass through an FFQ ion-exchange column as the first step of purification from inclusion bodies. The eluate was collected in 8 ml aliquots. Figure 2C shows an absorbance trace from the second purification step, size-exclusion chromatography (SEC). The double-headed arrow indicates the target peak collected as cam1615HER2 exited the column. Figure 2A shows the final product (fractions C2–D4) as a nearly single band when analyzed using SDS-PAGE with Coomassie blue staining, providing evidence of a homogeneous product. The final product was over 90% pure and had a molecular weight of approximately 55 kDa.

[0042] However, cam1615HER2 TriKE can be constructed in other ways. It is possible to design multiple constructs, each encoding and directing the synthesis of a portion of a complete immunotherapeutic compound. For example, an anti-HER2 light chain (e.g., SEQ ID NO: 17) can be encoded on one plasmid, and an anti-HER2 heavy chain (e.g., SEQ ID NO: 18) can be encoded on a second plasmid. When both plasmids are introduced into a host cell and expressed, the anti-HER2 light chain and anti-HER2 heavy chain can dimerize to form an anti-HER2 Fab as the targeting portion of the immunotherapeutic compound. cam1615HER2 TriKE can be constructed in this manner. For example, SEQ ID NO: 31 provides the amino acid sequence expressed from an exemplary first plasmid, including the signal peptide, anti-HER2 light chain, linker, IL-15 amino acid sequence, second linker, and camelid anti-CD16 single-domain antibody fragment. SEQ ID NO: 32 provides the amino acid sequence expressed from an exemplary second plasmid, including the signal peptide and anti-HER2 heavy chain.

[0043] As another example, a single plasmid construct may contain all components of a complete immunotherapeutic compound. For example, SEQ ID NO: 33 provides the amino acid sequence expressed from an exemplary single plasmid construct, where the amino acid sequence provides a signal sequence, an anti-HER2 heavy chain fragment, a T2A self-cleaving peptide, a second signal sequence, an anti-HER2 light chain fragment, a linker, an IL-15 amino acid sequence, a second linker, and a camelid anti-CD16 single domain antibody fragment. Upon expression, the T2A peptide self-cleaves, separating the anti-HER2 heavy chain fragment from the remainder of the immunotherapeutic compound so that it can dimerize with the anti-HER2 light chain fragment.

[0044] In the exemplary cam1615HER2 TriKE, the human IL-15 TriKE portion provides the molecular expansion capability. Thus, the ability of the IL-15 portion of cam1615HER2 to induce NK expansion was identified. Figures 3A and 3B show the highly expanded CD56 cells after 7 days of incubation with 50 nM cam1615HER2 TriKE compared to untreated controls (NT) and IL-15 controls. + CD3 - The percentage of NK cells and the total percentage of NK cells are shown. Both were significantly elevated after incubation with cam1615HER2. Similarly, exposure to cam1615HER2 also significantly enhanced the total NK count compared to the NT control (Figure 3C). Figures 3D-F show the CD3 + CD56 - The results show that the percentage and raw numbers of T cells are not elevated compared to untreated controls. Taken together, these studies demonstrate that HER2 TriKE stimulates the proliferation of NK cells but not T cells. The data also demonstrate that the IL-15 moiety within TriKE is functional and in a viable conformational arrangement.

[0045] Cytotoxicity is a hallmark of NK immunotherapy. To establish the efficacy of antibody-dependent cellular cytotoxicity (ADCC), various cell lines were analyzed for expression of CD107a, which is recognized as an indicator of NK cell cytotoxicity. cam1615HER2 TriKE was tested against SKOV3 and SK-BR-3 cells. This is because breast cancer and some ovarian cancer cases are known to overexpress ERBB2, making them desirable targets for antibody targeting. The UMSCC-11B cell line was tested as a negative control because it has minimal expression of HER2. Binding was first measured by labeling various agents with FITC and then testing direct binding to the target by flow cytometry. SKOV3 (Figure 4A) and SK-BR-3 (Figure 4B) showed the highest levels of HER2 TriKE binding. UMSCC-11B showed lower levels of binding (Figure 4C).

[0046] When measuring ADCC activity, cam1615HER2 TriKE showed highly elevated CD107a activity against SKOV3 and SK-BR-3 cell lines compared to IL-15 and untreated controls (Figures 5A and 5C). When ADCC was tested on the UMSCC-11B cell line, cam1615HER2 TriKE had little effect (Figure 5E). Furthermore, SKOV-3 (Figure 5B) and SK-BR-3 (Figure 5D) cam1615HER2 cells showed elevated IFN-γ activation when treated with cam1615HER2 TriKE.

[0047] cam1615HER2 TriKE was further tested against the breast cancer cell line MCF-7L. Figure 6A shows the background of CD107a effector cells without added target cells. Figure 6B shows CD107a activity when target cells were added. The highest level of killing was observed with cam1615HER2 TriKE compared to the control. Cam16 itself had activity, but not as much. A subline of MCF-7L (MCF-7L-TamR) is tamoxifen-resistant and showed a similar CD107a activity pattern when treated with cam1615HER2 TriKE (Figure 6C). NK cells are also known to secrete anti-cancer cytokines, such as IFN-γ, when activated and killed. Figure 6E shows that IFN-γ levels, quantified in the same samples by intracellular staining, were elevated with cam1615HER2 TriKE and minimally affected in the control, indicating NK cell activation. The same was true for MCF-7L-TamR (Figure 6F). Figure 6D confirms the killing of SK-BR-3 cells by cam1615HER2 TriKE, demonstrating its efficacy as well as trastuzumab. Figure 6H shows that cam1615HER2 TriKE has even greater IFN-γ enhancing activity than trastuzumab. Taken together, these data demonstrate that HER2 is a valid target for immune engagement of human breast cancer cells and that innate immune therapy is highly effective against drug-resistant breast cancer cell lines in vitro.

[0048] Figure 12 provides data showing the activity of cam1615HER2 TriKE against two additional HER2-expressing cell lines, OVCAR-3 and OVCAR-5. Again, cam1615HER2 TriKE demonstrates elevated CD107a activity (Figures 12A and 12B) and IFN-γ activity (Figures 12D and 12E) compared to controls. Repeat experiments evaluating SKOV3 included a broader number of negative controls, including anti-HER2 scFv alone (e23), cam16 VHH alone, IL-15 alone, and a no treatment control. Again, the drug demonstrated elevated CD107a activity (Figure 12C) and IFN-γ activity (Figure 12F) compared to controls.

[0049] Death was further assessed in real time over two days using the INCUCYTE ZOOM platform (Essen Bioscience, Inc., Ann Arbor, MI). SKOV3 cells grown in culture as spheroids were studied. SKOV3 cells stably transduced with NUCLIGHT RED (Essen Bioscience, Inc., Ann Arbor, MI) were incubated with enriched NK cells and cam1615HER2 TriKE, free IL-15, cam16 VHH alone, or untreated. To detect cell death, caspase 3 / 7 green reagent was added. Dying cells turned green, while dying NUCLIGHT RED Raji cells turned yellow, allowing tracking of remaining viable cells. When data were collected, cam1615HER2 TriKE caused a striking reduction in SKOV3 spheroid size (Figure 7A) and spheroid intensity (Figure 7B) compared to IL-15, cam16 VHH alone, and untreated controls over 72 hours of continuous measurement. Figure 8 provides images showing that cam1615HER2 causes a rapid, time-dependent reduction in target cells measured over 72 hours. The results of this direct kill assay correlated with those of the CD107a assay.

[0050] To determine whether NK cells from consenting cancer patients would function in the assay, NK cells were obtained from six consenting ovarian cancer patients instead of normal volunteers and tested against MA-148 ovarian cancer cells. Figure 9A shows a lower CD107a background upon treatment of effectors without cancer cells than with cam1615HER2 TriKE or control. Figure 9B shows a significant increase in CD107a expression on effectors and tumor targets upon treatment with cam1615HER2 TriKE compared to untreated controls. Regarding IFN-γ activity, Figure 9E shows lower activity on effectors without targets when treated with cam1615HER2 TriKE. Upon addition of effector cells, cam1615HER2 TriKE exhibits enhanced IFN-γ activity (Figure 9F). For comparison, Figure 9C shows the CD107a background of normal donor effector cells, and Figure 9D shows CD107a activity of normal donor cells with MA148 cancer cells. Figure 9G shows the IFN-γ background of normal donor effector cells, and Figure 9H shows the IFN-γ activity of normal donor cells with MA148 cancer cells. When normal or patient NK cells were mixed with cancer cells, the trends were similar, although the activity of normal cells was slightly higher. Nevertheless, patient effector cells were capable of TriKE stimulation.

[0051] Figure 10 shows data demonstrating the in vivo efficacy of cam1615HER2 TriKE using a SCID / hu / NK xenograft model. Figures 10A and 10B show visual imaging data for untreated (Figure 10A) and treated (Figure 10B) groups of mice approximately 6 weeks after intraperitoneal inoculation of SKOV3 tumor cells. Advanced tumor progression was evident in the untreated group of mice (Figure 10A) and was clearly reduced in the treated group of mice (Figure 10B).

[0052] Figure 11A shows minimal changes in animal weight over 46 days, indicating that cam1615HER2 TriKE is not toxic despite daily administration. Figure 11B is a comparative snapshot of total tumor bioluminescence (total flux) from two independent experiments, demonstrating that tumor growth is significantly inhibited in the treated versus untreated groups even 46 days after tumor administration. Figure 11C shows the overall progression of tumor growth (bioluminescence) over time. Over 46 days, tumor growth is significantly inhibited in the treated group, but tumor growth begins to recur on day 39. Figure 11D shows longer-term results in a survival plot. Five of six animals in the untreated group die by day 52, and all untreated animals die by day 60. In contrast, here, the cam1615HER2-treated group still has a 50% survival rate by day 72. The remaining animals in the cam1615HER2-TriKE-treated group still exhibited tumor burden, indicating that treatment was suppressive, not curative. Taken together, these data confirm that cam1615HER2 TriKE is effective in inhibiting human ovarian cancer growth in vivo.

[0053] Thus, the data presented herein demonstrate that HER2, when targeted by immunotherapeutic compounds such as trispecific killer-engaging (TriKE) compounds, can serve as an immunotherapeutic target for ovarian and breast cancer. The exemplary immunotherapeutic compounds are effective against tamoxifen-refractory cells and can therefore readily kill cancer cells resistant to tamoxifen or other chemotherapeutic agents. The present disclosure further demonstrates that the exemplary immunotherapeutic compound, HER2-targeting NK-binding TriKE, can inhibit cancer in vivo in an intraperitoneal ovarian cancer xenograft model (a model involving the transplantation of both human NK cells and cancer cells in xenogeneic mice). The data presented herein further provide a basis for immunotherapeutic compounds targeting HER3 and / or the HER2 / HER3 heterodimer.

[0054] The immunotherapeutic compounds described herein can be formulated with a pharmaceutically acceptable carrier. As used herein, "carrier" includes any solvent, dispersion medium, vehicle, coating agent, diluent, antibacterial and / or antifungal agent, isotonic agent, absorption delaying agent, buffer, carrier solution, suspending agent, colloid, etc. The use of such media and / or agents for pharmaceutical active substances is well known in the art. Except insofar as a conventional media or agent is incompatible with the active ingredient, its use in therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the composition. As used herein, "pharmaceutically acceptable" refers to a material that is not biologically or otherwise undesirable. That is, the material can be administered to an individual together with the immunotherapeutic compound without causing undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is included.

[0055] Thus, immunotherapeutic compounds can be formulated into pharmaceutical compositions. Pharmaceutical compositions can be formulated in a variety of forms compatible with the preferred route of administration. Thus, compositions can be, for example, oral, parenteral (e.g., intradermal, transdermal, subcutaneous, intramuscular, intravenous, intraperitoneal, etc.), or topical (e.g., intranasal, intrapulmonary, intramammary, intravaginal, intrauterine, intradermal, subcutaneous, rectal, etc.) administration. Pharmaceutical compositions can be administered to mucosal surfaces, such as by administration to the nasal or respiratory mucosa (e.g., by spray or aerosol). Compositions can also be administered via sustained or delayed release. In certain embodiments, compositions are administered intraperitoneally, intravenously, or subcutaneously.

[0056] Thus, the immunotherapeutic compound can be provided in any suitable form, including, but not limited to, a solution, suspension, emulsion, spray, aerosol, or any form of mixture. The composition can be delivered in a formulation containing any pharmaceutically acceptable excipient, carrier, or vehicle. For example, the formulation can 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 fragrance, a flavoring, a moisturizer, a thickener, etc. In certain embodiments, the composition can be formulated into a solution or suspension.

[0057] Preparations can be conveniently presented in unit dosage form and can be prepared by methods well known in the field of pharmacy.The method of preparing the composition containing pharmaceutically acceptable carrier comprises combining immunotherapeutic compounds with carrier that constitutes one or more auxiliary ingredients.Generally, preparations can be prepared by uniformly and / or intimately combining active molecule with liquid carrier, finely divided solid carrier, or both, and then, if necessary, shaping the product into desired preparation.

[0058] Thus, in another aspect, the present disclosure describes a method for treating cancer in a subject. Generally, the method includes administering to the subject an amount of an immunotherapeutic compound effective to treat the cancer. "Treatment" or variations thereof refers to alleviating, inhibiting progression, improving, or reversing to some extent the symptoms or signs associated with a condition. As used herein, "ameliorating" refers to a decrease in the degree, severity, frequency, and / or likelihood of a symptom or clinical sign characteristic of a particular condition; "symptom" refers to subjective evidence of a disease or patient's condition; "sign" or "clinical sign" refers to an objective physical finding associated with a particular condition that can be detected by someone other than the patient.

[0059] "Treatment" may be therapeutic or prophylactic. "Therapeutic" and variations thereof refer to treatment that improves one or more existing symptoms or clinical signs associated with a condition. "Prophylactic" and variations thereof refer to treatment that limits, to some extent, the onset and / or appearance of symptoms or clinical signs of a condition. Generally, "therapeutic" treatment is initiated after a condition manifests in a subject, while "prophylactic" treatment is initiated before a condition manifests in a subject. Thus, in certain embodiments, a method may include prophylactic treatment of a subject at risk of developing a condition. "At risk" refers to a subject who may or may not actually have the described risk. Thus, for example, a subject "at risk" of developing a particular condition is one who has one or more signs that puts them at increased risk of having or developing a particular condition compared to an individual lacking one or more signs, regardless of whether the subject exhibits any symptoms or clinical signs that they have or are developing the condition. Exemplary signs of a condition may include, for example, genetic predisposition, ancestry, age, sex, geographic location, lifestyle, or medical history. Treatment may also be continued after symptoms have resolved, for example, to prevent or delay recurrence.

[0060] Thus, the immunotherapeutic compound can be administered to a subject before, during, or after the subject first exhibits symptoms or clinical signs of the condition. Treatment initiated before the subject first exhibits symptoms or clinical signs associated with the condition may reduce the likelihood that the subject will experience clinical evidence of the condition, reduce the severity of the symptoms and / or clinical signs, and / or completely reverse the condition, compared to subjects not administered the immunotherapeutic compound. Treatment initiated after the subject first exhibits symptoms or clinical signs associated with the condition may reduce the severity of the symptoms and / or clinical signs and / or completely reverse the condition, compared to subjects not administered the immunotherapeutic compound.

[0061] The amount of immunotherapeutic compound to be administered can vary depending on various factors, including but not limited to the specific immunotherapeutic compound to be administered, body weight, physical condition, and / or age of subject, and / or administration route.Therefore, the absolute weight of immunotherapeutic compound contained in a given unit dosage form can vary greatly, and depends on factors such as the species, age, body weight and physical condition of subject, and / or administration method.Therefore, it is not practical to generally indicate the amount that constitutes the amount of immunotherapeutic compound that is effective for all possible uses.However, those skilled in the art can easily determine the appropriate amount by fully considering such factors.

[0062] In some embodiments, the method may include administering sufficient immunotherapeutic compound to provide a dose of, for example, about 100 ng / kg / day to about 10 mg / kg / day to the subject, although in some embodiments, the method can be practiced by administering the immunotherapeutic compound at a dose outside this range.

[0063] In some embodiments, the method may include administering sufficient immunotherapeutic compound to provide a minimum dose of at least 100 ng / kg / day, such as, for example, at least 1 μg / kg / day, at least 5 μg / kg / day, at least 10 μg / kg / day, at least 25 μg / kg / day, at least 50 μg / kg / day, at least 100 μg / kg / day, at least 200 μg / kg / day, at least 300 μg / kg / day, at least 400 μg / kg / day, at least 500 μg / kg / day, at least 600 μg / kg / day, at least 700 μg / kg / day, at least 800 μg / kg / day, at least 900 μg / kg / day, or at least 1 mg / kg / day.

[0064] In some embodiments, the method comprises administering a dose of 0.5 mg / kg / day or less, e.g., 5 mg / kg / day or less, 4 mg / kg / day or less, 3 mg / kg / day or less, 2 mg / kg / day or less, 1 mg / kg / day or less, 900 μg / kg / day or less, 800 μg / kg / day or less, 700 μg / kg / day or less, 600 μg / kg / day or less, 500 μg / kg / day or less, 400 μg / kg / day or less, 300 μg / kg / day or less, 200 μg / kg / day or less, The method includes administering sufficient immunotherapeutic compound to provide a maximum dose of 10 mg / kg / day or less, such as 100 μg / kg / day or less, 90 μg / kg / day or less, 80 μg / kg / day or less, 70 μg / kg / day or less, 60 μg / kg / day or less, 50 μg / kg / day or less, 40 μg / kg / day or less, 30 μg / kg / day or less, 20 μg / kg / day or less, or 10 μg / kg / day or less. The immunotherapeutic compound provides a dose "not exceeding" the specified amount, provided that the immunotherapeutic compound is not absent, but is present in an amount less than the specified amount.

[0065] In some embodiments, the method includes administering sufficient immunotherapeutic compound to provide a dose characterized by a range having endpoints defined by any minimum dose identified above and any maximum dose greater than the selected minimum dose. For example, in some embodiments, the method may include administering sufficient immunotherapeutic compound to provide a subject with a dose of about 10 μg / kg / day to about 10 mg / kg / day, a dose of about 100 μg / kg / day to about 1 mg / kg / day, a dose of about 5 μg / kg / day to about 100 μg / kg / day, etc.

[0066] In certain embodiments, the method comprises administering sufficient immunotherapeutic compound to provide a dose equal to any minimum dose or any maximum dose described above. Thus, for example, in certain embodiments, the method may comprise administering sufficient immunotherapeutic compound to provide a dose of 1 μg / kg / day, 5 μg / kg / day, 10 μg / kg / day, 25 μg / kg / day, 50 μg / kg / day, 100 μg / kg / day, 200 μg / kg / day, 500 μg / kg / day, 1 mg / kg / day, 5 mg / kg / day, etc.

[0067] In some embodiments, the immunotherapeutic compound can be administered, for example, from a single dose to multiple doses per week, although in some embodiments, the method can be carried out by administering the immunotherapeutic compound at a frequency outside this range. In certain embodiments, the immunotherapeutic compound can be administered from about once a month to about five times a week. In some embodiments, the doses described above, relative to the amount of immunotherapeutic compound administered over a 24-hour period, are administered in a 7-day cycle of 4 days on and 3 days off.

[0068] In some embodiments, the immunotherapeutic compound can be administered, for example, from a single dose to multiple treatment cycles, although in some embodiments, the method can be practiced by administering the immunotherapeutic compound for a period outside this range. In some embodiments, the immunotherapeutic compound can be administered for three weeks. In such embodiments, each week can be a treatment cycle, such as the exemplary treatment cycle described in the previous paragraph. In other embodiments, the immunotherapeutic compound can be administered for more treatment cycles without a gap between one set of treatment cycles and subsequent sets of treatment cycles. The gap between one set of treatment cycles and subsequent sets of treatment cycles can be a gap of one week or more, one month or more, or one year or more.

[0069] In some embodiments, the method further comprises administering one or more additional therapeutic agents. The one or more additional therapeutic agents (e.g., chemotherapeutic agents) can be administered before, after, and / or simultaneously with the administration of the immunotherapeutic compound. The immunotherapeutic compound and the additional therapeutic agent can be co-administered. As used herein, "co-administered" refers to two or more components of a combination administered such that the therapeutic or prophylactic effect of the combination may be greater than the therapeutic or prophylactic effect of either component administered alone. The two components can be co-administered simultaneously or sequentially. Components co-administered simultaneously may be provided in one or more pharmaceutical compositions. Sequential co-administration of two or more components includes when the components are administered such that each component is present at the treatment site at the same time. Alternatively, sequential co-administration of two components may include when at least one component is removed from the treatment site, but at least one cellular effect of administering the component (e.g., cytokine production, activation of a specific cell population, etc.) persists at the treatment site until one or more additional components are administered to the treatment site. Thus, a co-administered combination can, in certain circumstances, include components that are never present in a chemical mixture with each other. In other embodiments, the immunotherapeutic compound and additional therapeutic agent can be administered as part of a mixture or cocktail. In some aspects, administration of the immunotherapeutic compound allows for the effectiveness of lower dosages of the other therapy when compared to administration of one or more other therapeutic agents alone, thereby reducing the likelihood, severity, and / or extent of toxicity observed when higher doses of the other therapeutic agent are administered.

[0070] Exemplary additional therapeutic agents include altretamine, amsacrine, L-asparaginase, colspase, bleomycin, busulfan, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, cyclophosphamide, cytophosphane, 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, vincristine, vindesine, or vinorelbine, anti-HER2 antibody therapy, anti-HER3 antibody therapy (see, e.g., Liu et al., 2019, Biol Proced Online 21:5), or anti-HER2 / HER3 heterodimer complex antibody therapy (see, e.g., Liu et al., 2019, Biol Proced Online 21:5).

[0071] In some embodiments, the methods may include administering sufficient immunotherapeutic compounds described herein and at least one additional therapeutic agent that demonstrates therapeutic synergy. In some aspects of the methods of the invention, the measured response to treatment observed after administering both the immunotherapeutic compounds described herein and the additional therapeutic agent is improved compared to the same measured response to treatment observed after administering either the immunotherapeutic compound or the additional therapeutic agent alone. In some embodiments, the additional therapeutic agent may include an additional agent that targets EpCAM, including, for example, an EpCAM-specific monoclonal antibody such as catumaxomab, or a monoclonal hybrid antibody that targets EpCAM and CD3.

[0072] In some embodiments, immunotherapeutic compounds can be administered to a subject to stimulate endogenous NK cells in vivo. Use of immunotherapeutic compounds as part of an in vivo method can render NK cells antigen-specific, with simultaneous costimulation, enhanced survival, and proliferation. In other cases, immunotherapeutic compounds can be used in vitro as adjuvants for NK cell adoptive transfer therapy.

[0073] In the foregoing description and in the claims that follow, 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. Unless otherwise specified, "a," "an," "the," and "at least one" are used interchangeably to mean one or more; the recitation of numerical ranges by endpoints includes all numbers within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).

[0074] In the foregoing description, for clarity, certain embodiments may be described in isolation. A particular embodiment may include any combination of compatible features described herein in connection with one or more embodiments, unless expressly specified that a feature of a particular embodiment is incompatible with a feature of another embodiment.

[0075] For any method disclosed herein that includes discrete steps, the steps can be performed in any practicable order, and, where appropriate, any combination of two or more steps can be performed simultaneously.

[0076] The present invention is illustrated by the following examples, it being understood that the particular examples, materials, amounts, and procedures are to be interpreted broadly in accordance with the scope and spirit of the invention described herein. [Example]

[0077] Building the cam1615HER TriKE DNA fragments encoding the CDR regions from camelized anti-CD16 (Vincke et al., 2009, J. Biol. Chem. 284(5):3273-3284) were spliced ​​into a universal humanized nanobody scaffold previously shown to allow grafting of antigen-binding loops with transfer of antigen specificity and affinity (Behar et al., 2008, Protein Engineering, Design & Selection 21(1):1-10). This novel sequence was used to generate cam1615HER2 (SEQ ID NO: 1). The fully assembled hybrid gene, cam1615HER2, encoding cam1615HER2 TriKE encodes (from the 5' to 3' end): an NcoI restriction site; an ATG start codon; an anti-human CD16 VHH; a 20-amino acid (aa) segment, PSGQAGAAASESLFVSNHAY (SEQ ID NO: 3); human IL-15; a 7-amino acid linker, EASGGPE (SEQ ID NO: 5); an anti-HER2 scFv (Batra et al., 1992, Proc Natl Acad Sci USA 89(13):5867-5871); and an XhoI restriction site. The resulting hybrid gene (SEQ ID NO: 7) was spliced ​​into the pET28c expression vector under the control of an isopropyl-D-thiogalactopyranoside (IPTG)-inducible T7 promoter. The DNA target gene encoding cam1615HER2 was 1517 base pairs long. Wild-type human IL-15 was used, but not a mutant form of the cytokine. The Center for Biomedical Genomics at the University of Minnesota, St. Paul, Minnesota, verified the gene sequence and in-frame accuracy of the target genes.

[0078] Purification of proteins from inclusion bodies Escherichia coli strain BL21(DE3) (Novagen, Madison, WI) was used for protein expression after plasmid transfection. Bacteria were grown overnight in 800 ml of Luria broth containing 50 μg / ml kanamycin. Expression was induced by adding IPTG (Thermo Fisher Scientific, Inc., Fair Lawn, NJ) when the medium reached an absorbance of 0.65 at 600 nm. Bacterial expression resulted in the packaging of the target protein into inclusion bodies. After expression, bacteria were harvested and homogenized in buffer (50 mM Tris, 50 mM NaCl, and 5 mM EDTA pH 8.0), and the pellet was sonicated and centrifuged. Proteins were extracted from the pellet using a solution of 0.3% sodium deoxycholate, 5% Triton X-100, 10% glycerol, 50 mmol / L Tris, 50 mmol / L NaCl, and 5 mmol / L EDTA (pH 8.0). The extract was washed three times.

[0079] Proteins from inclusion bodies require refolding. Therefore, we used a sodium N-lauroyl-sarcosine (SLS) air oxidation method, modified from a previously described method (Vallera et al., 2005, Leuk Res 29(3):331-341). Briefly, inclusion bodies were dissolved in 100 mM Tris, 2.5% SLS (Sigma-Aldrich, St. Louis, MO). The pellet was removed by centrifugation. 50 μM CuSO4 was added to the solution, and the mixture was incubated at room temperature for 20 hours with rapid stirring to allow air oxidation of -SH groups. SLS removal was performed by adding 6 M urea and 10% AG 1-X8 resin (200-400 mesh, chloride form) (Bio-Rad Laboratories, Inc., Hercules, CA) to the detergent-solubilized protein solution. In the next step, 13.3 M guanidine-HCl was added to the protein solution and incubated at 37 °C for 2–3 h. The solution was diluted 20-fold with refolding buffer (50 mM Tris, 0.5 M L-arginine, 1 M urea, 20% glycerol, 5 mM EDTA, pH 8.0). The mixture was incubated at 4 °C for 2 days. To remove the buffer, the sample was dialyzed against 5 volumes of 20 mM Tris-HCl, pH 8.0, at 4 °C for 48 h, then against 8 volumes for an additional 18 h. The product was then purified first by fast-flow Q ion exchange chromatography and then by passing it through a size-exclusion column (SUPERDEX 200, Cytiva, Marlborough, MA). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed using Simply Blue Life Stain (Invitrogen, Carlsbad, CA) to assess the size and purity of the protein.

[0080] Cancer cell lines The following cell lines were obtained from the American Type Culture Collection (Manassas, VA): MCF-7L (ductal carcinoma), MCF-7L-TamR (a tamoxifen-resistant subline of MCF-7), SKOV3 (ovarian ascites), SK-BR-3 (a breast carcinoma derived from a metastatic site), UMSCC-11B squamous cell carcinoma derived from a laryngeal tumor (Worsham et al., 2006, Arch Otolaryngol Head Neck Surg 132:668-677), HL-60 (acute promyelocytic leukemia), MA-148 (ovarian carcinoma), and oyobiSKOV3-luc. For in vivo experiments, SKOV3-luc was generated by transfecting SKOV3 with a luciferase reporter construct using LIPOFECTAMINE (Invitrogen, Carlsbad, CA) under selection pressure with 10 μg / ml blastocidin. UMSCC-11B was authenticated by STR testing performed by the Johns Hopkins University Fragment Analysis Facility. MA148 (established locally at the University of Minnesota) is a human epithelial ovarian cancer cell line. The line was maintained in RPMI 1640 medium supplemented with 10–20% fetal bovine serum (FBS) and 2 mmol / L-glutamine. The line was cultured at a constant 37°C in a humidified atmosphere containing 5% CO2. When adherent cells reached >90% confluence, they were passaged using trypsin-EDTA for detachment. A standard hemocytometer was used for cell counts. Only cells with >95% viability, as determined by trypan blue exclusion, were used for experiments.

[0081] Assessment of cytotoxicity and NK cell activation Antibody-dependent cellular cytotoxicity (ADCC) was measured using a CD107a (lysosome-associated membrane protein LAMP-1) flow cytometry assay. For effector cells, PBMCs were obtained from healthy volunteers after obtaining donor consent and institutional review board approval. Cancer target cells were propagated from cell lines as described above. For experiments using patient effector cells, the University of Minnesota Cancer Center Tissue Procurement Facility obtained high-grade serous ascites samples from patients diagnosed with ovarian cancer, with institutional review board approval. All specimens were collected from women diagnosed with advanced-stage ovarian or primary peritoneal cancer at the time of primary debulking surgery. Cells were pelleted, lysed to remove red blood cells, and cryopreserved in 10% DMSO / 90% FBS and stored in liquid nitrogen.

[0082] PBMCs were incubated overnight at 37°C, 5% CO2 in RPMI 1640 medium supplemented with 10% fetal bovine serum (RPMI-10), washed three times with RPMI-10, and then suspended in tumor target cells or medium. Cells were then incubated with TriKE or control for 10 minutes at 37°C. Fluorescein isothiocyanate (FITC)-conjugated anti-human CD107a monoclonal antibody (BD Biosciences, San Jose, CA) was then added and incubated for 1 hour. Following incubation, GolgiStop (1:1,500, BD Biosciences, San Jose, CA) and GolgiPlug (1:1,000, BD Biosciences, San Jose, CA) were added for 3 hours at 37°C, 5% CO2. After washing with phosphate-buffered saline, cells were stained with PE / Cy7-conjugated anti-CD56 mAb, APC / Cy7-conjugated anti-CD16 mAb, and PE-CF594-conjugated anti-CD3 mAb (BioLegend, San Diego, CA). Cells were incubated at 4°C for 15 min, washed, and fixed with 2% paraformaldehyde.

[0083] Intracellular IFN-γ was measured as an indicator of NK cell activation. Briefly, cells were exposed to permeabilization buffer (BD Biosciences, San Jose, CA) and incubated with Pacific Blue-conjugated anti-human IFN-γ (BioLegend, San Diego, CA) for 20 min. Finally, cells were washed and analyzed by CD56 immunoblotting. + CD3 - Cells were assessed by fluorescence-activated cell sorting analysis using an LSRII flow cytometer (BD Biosciences, San Jose, CA) with gating.

[0084] Cytotoxicity potency was further measured in real time. + CD3 - NK effector cells were plated in 96-well flat-bottom polystyrene tissue culture-treated microplates (Corning, Flintshire, UK) along with red-labeled tumor cells, and the plates were transferred to an INCUCYTE ZOOM platform (Essen Bioscience, Inc., Ann Arbor, MI) housed in a cell incubator at 37°C / 5% CO Images from three technical replicates were taken every 15 min for 48 h using a 4x objective and analyzed using INCUCYTE Basic Software (Essen Bioscience, Inc., Ann Arbor, MI).

[0085] IL-15-stimulated NK cell proliferation To measure the efficacy of TriKE, which depends on the functional IL-15 moiety, PBMCs or enriched NK cells from healthy donors were labeled with CELLTRACE violet proliferation dye (Invitrogen, Carlsbad, CA) according to the kit specifications. After staining, effector cells were cultured with 50 nM TriKE or control and incubated at 37°C in a humidified atmosphere containing 5% CO for 7 days. Cells were harvested and stained for viability with Live / Dead reagent (Invitrogen, Carlsbad, CA), and surface stained with anti-CD56 PE / Cy7 (Biolegend, San Diego, CA) and anti-CD3 PE-CF594 (BD Biosciences, Franklin Lakes, NJ) to identify viable CD3 - CD56 + The NK cell population was gated. Data analysis was performed using FlowJo software (Flowjo Enterprises LCC, version 7.6.5, Ashland, OR).

[0086] In vivo mouse studies and imaging The efficacy of HER2 TriKE was tested in a previously reported scid / hu mouse model (Vallera et al., 2016, Clin Cancer Res 22(14):3440-3450), but modified for growth in the human ovarian cancer cell line SCOV3. This line was transfected with a luciferase reporter gene to allow real-time monitoring of tumor progression via bioluminescence imaging. NSG mice (NOD.Cg-Prkd) were used. cscid Il2rg tm1Wjl / SzJ, n = 5 / group) 5SCOV3 cells were injected intraperitoneally, followed by low-dose total-body irradiation (275 cGy) 3–5 days later. The following day, all groups received highly enriched NK cells (magnetically CD3- and CD19-depleted PBMCs) and initiated treatment with cam1615HER2 TriKE. A single treatment course consisted of 50 μg of the drug administered intraperitoneally five times weekly (MTWThF) for 2 weeks, followed by maintenance therapy three times weekly (MWF) until day 60. For each imaging session, mice were injected with 100 μl of 30 mg / ml luciferin substrate for 10 min and imaged under isoflurane gas sedation. Imaging data were collected using a Xenogen Ivis 100 imaging system with Living Image 2.5 software (Xenogen Corporation, Hopkinton, MA). Mice were weighed weekly when possible.

[0087] The complete disclosures of all patents, patent applications, and publications, and electronically available materials (e.g., nucleotide sequence submissions in GenBank and RefSeq, including amino acid sequence submissions in, e.g., SwissProt, PIR, PRF, PDB, and translations from the annotated coding regions of GenBank and RefSeq) are incorporated herein by reference in their entirety. In the event of a discrepancy between the disclosure of this application and the disclosure of a document incorporated herein by reference, the disclosure of this application shall control. The foregoing detailed description and examples are provided for clarity of understanding only. No unnecessary limitations should be understood therefrom. The invention is not limited to the exact details shown and described. Variations obvious to those skilled in the art will be encompassed within the invention as defined by the claims.

[0088] Unless otherwise indicated, all numbers expressing quantities of ingredients, molecular weights, and the like used in the specification and claims should be understood in all instances to be modified by the term "about." Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the scope of the claims to the doctrine of equivalents, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. [Sequence List Free Text]

[0089] SEQ ID NO:1 - cam1615HER2 Amino Acid Sequence: MEQVQLVESG GGLVQPGGSL RLSCAASGLT FSSYNMGWFR QAPGQGLEAV ASITWSGRDT FYADSVKGRF TISRDNSKNT LYLQMNSLRA EDTAVYYCAA NPWPVAAPRS GTYWGQGTLV TVSSPSGQAG AAASESLFVS NHAYNWVNVI SDLKKIEDLI QSMHIDATLY TESDVHPSCK VTAMKCFLLE LQVISLESGD ASIHDTVENL IILANNSLSS NGNVTESGCK ECEELEEKNI KEFLQSFVHI VQMFINTSEA SGGPEDVQLT QSPAILSASP GEKVTMTCRA TPSVSYMHWY QQKPGSSPKP WIYTTSNLAS GVPARFSGGG SGTSYSLTVS RVEAEDAATY YCQQWSRSPP TFGGGSKLEI KGSTSGSGKS SEGKGVQLQE SGPEVVKPGG SMKISCKTSG YSFTGHTMNW VKQSHGKNLE WIGLINPYNG DTNYNQKFKG KATFTVDKSS STAYMELLSL TSEDSAVYYC ARRVTDWYFD VWGAGTTVTV S SEQ ID NO:2 - cam16 (amino acids 3-124 of SEQ ID NO:1) QVQLVESGGG LVQPGGSLRL SCAASGLTFS SYNMGWFRQA PGQGLEAVAS ITWSGRDTFY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAANP WPVAAPRSGT YWGQGTLVTV SS SEQ ID NO:3 - hma linker (amino acids 125-144 of SEQ ID NO:1) PSGQAGAAAS ESLFVSNHAY SEQ ID NO:4 - human IL-15 (amino acids 145-258 of SEQ ID NO:1) NWVNVISDLK KIEDLIQSMH IDATLYTESD VHPSCKVTAM KCFLLELQVI SLESGDASIH DTVENLIILA NNSLSSNGNV TESGCKECEE LEEKNIKEFL QSFVHIVQMF INTS SEQ ID NO:5 - linker (amino acids 259-265 of SEQ ID NO:1) EASGGPE SEQ ID NO:6 - anti-HER2 (amino acids 266-501 of SEQ ID NO:1) DVQLTQSPAI LSASPGEKVT MTCRATPSVS YMHWYQQKPG SSPKPWIYTT SNLASGVPAR FSGGGSGTSY SLTVSRVEAE DAATYYCQQW SRSPPTFGGG SKLEIKGSTS GSGKSSEGKG VQLQESGPEV VKPGGSMKIS CKTSGYSFTG HTMNWVKQSH GKNLEWIGLI NPYNGDTNYN QKFKGKATFT VDKSSSTAYM ELLSLTSEDS AVYYCARRVT DWYFDVWGAG TTVTVS SEQ ID NO:7 - cam1615HER2 DNA sequence CCATGGAGca ggtgcagctg gtggagtctg ggggaggctt ggtgcagcct gggggctctc tgagactctc ctgtgcagcc tctggcctca ccttcagtag ctataacatg ggctggttcc gccaggctcc agggcaaggc cttgaggctg tagcatctat tacctggagt ggtcgggaca cattctatgc agactccgtg aagggccgat tcaccatctc cagagacaac tccaagaaca ctctctatct gcaaatgaac agcctgcgcg cggaggacac ggccgtttat tattgtgctg caaacccctg gccagtggcg gcgccacgta gtggcaccta ctggggccaa gggaccctgg tcaccgtctc ctcaccgtct ggtcaggctg gtgctgctgc tagcgaatct ctgttcgttt ctaaccacgc ttacAACTGG GTGAATGTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA ATACGGAAAG TGATGTTCAC CCCAGTTGCA AAGTAACAGC AATGAAGTGC TTTCTCTTGG AGTTACAAGT TATTTCACTT GAGTCCGGAG ATGCAAGTAT TCATGATACA GTAGAAAAATC TGATCATCCT AGCAAACAAC AGTTTGTCTT CTAATGGGAA TGTAACAGAA TCTGGATGGATGCA AAGAATGTGA GGAACTGGAG GAAAAAATA TTAAAGAATT TTTGCAGAGT TTTGTACATA TTGTCCAAAT GTTCATCAAC ACTTCTgaag cttccggagg tcccgagGAC GTCCAGCTGA CCCAGTCTCC AGCAATCCTG TCTGCATCTC CAGGGGGAGAA GGTCACAATG ACTTGCAGGG CCACCCCAAG TGTAAGTTAC ATGCACTGGT ATCAGCAGAAGCCAGGATCC TCCCCCAAAC CTTGGATTTA TACCACATCC AACCTGGCTT CTGGAGTCCC TGCTCGCTTC AGTGGCGGTG GGTCTGGGAC CTCTTACTCT CTCACAGTCA GCAGAGTGGA GGCTGAAGAT GCTGCCACTT ATTACTGCCA GCAGTGGAGT CGTAGCCCAC CCACGTTCGG AGGGGGGTCC AAGCTGGAAA TAAAAGGTTC TACCTCTGGT TCTGGTAAAT CTTCTGAAGG TAAAGGTGTG CAGCTGCAGG AGTCAGGACC TGAGGTGGTG AAGCCTGGAG GTTCAATGAA GATATCCTGC AAGACTTCTG GTTACTCATT CACTGGCCAC ACCATGAACT GGGTGAAGCA GAGCCATGGA AAGAACCTTG AGTGGATTGG ACTTATTAAT CCTTACAATG GTGATACTAA CTACAACCAG AAGTTCAAGG GCAAGGCCAC ATTTACTGTA GACAAGTCGT CCAGCACAGC CTACATGGAG CTCCTCAGTC TGACATCTGA GGACTCTGCA GTCTATTACT GTGCAAGGAG GGTTACGGAC TGGTACTTCG ATGTCTGGGG CGCAGGGACC ACGGTCACCG TCTCCtaata gctcgag SEQ ID NO:8 - NcoI restriction site and start codon (1-8 of SEQ ID NO:7) CCATGGAG SEQ ID NO:9 - cam16 (9-374 of SEQ ID NO:7) caggtgcagc tggtggagtc tgggggaggc ttggtgcagc ctgggggctc tctgagactc tcctgtgcag cctctggcct caccttcagt agctataaca tgggctggtt ccgccaggct ccagggcaag gccttgaggc tgtagcatct attacctgga gtggcctcctc tgaagggccg attcaccatc tccagagaca actccaagaa cactctctat ctgcaaatga acagcctgcg cgcggaggac acggccgttt attattgtgc tgcaaacccc tggccagtgg cggcgccacg tagtggcacc tactggggcc aagggaccct ggtcaccgtc tcccat SEQ ID NO:10 - hma linker (375-434 of SEQ ID NO:7) ccgtctggtc aggctggtgc tgctgctagc gaatctctgt tcgtttctaa ccacgcttac SEQ ID NO:11 - human IL-15 (435-776 of SEQ ID NO:7) AACTGGGTGA ATGTAATAAG TGATTTGAAA AAAATTGAAG ATCTTATTCA ATCTATGCAT ATTGATGCTA CTTTATATAC GGAAAGTGAT GTTCACCCCA GTTGCAAAGT AACAGCAATG AAGTGCTTTC TCTTGGAGTT ACAAGTTATT TCACTTGAGT CCGGAGATGC AAGTATTCAT GATACAGTAG AAAATCTGAT CATCCTAGCA AACAACAGTT TGTCTTCTAA TGGGAATGTA ACAGAATCTG GATGCAAAGA ATGTGAGGAA CTGGAGGAAA AAAATTTAA AGAATTTTTG CAGAGTTTTG TACATATTGT CCAAATGTTC ATCAACACTT CT SEQ ID NO:12 - Linkers (777-797 of SEQ ID NO:7) gaagcttccg gaggtccg g SEQ ID NO:13 - Anti-HER2 (798-1505 of SEQ ID NO:7) GACGTCCAGC TGACCCAGTC TCCAGCAATC CTGTCTGCAT CTCCAGGGGA GAAGGTCACA ATGACTTGCA GGGCCACCCC AAGTGTAAGT TACATGCACT GGTATCAGCA GAAGCCAGGA TCCTCCCCCA AACCTTGGAT TTATACCACA TCCAACCTGG CTTCTGGAGT CCCTGCTCGC TTCAGTGGCG GTGGGTCTGG GACCTCTTAC TCTCTCACAG TCAGCAGAGT GGAGGCTGAA GATGCTGCCA CTTATTACTG CCAGCAGTGG AGTCGTAGCC CACCCACGTT CGGAGGGGGG TCCAAGCTGG AAATAAAAGG TTCTACCTCT GGTTCTGGTA AATCTTCTGA AGGTAAAGGT GTGCAGCTGC AGGAGTCAGG ACCTGAGGTG GTGAAGCCTG GAGGTTCAAT GAAGATATCC TGCAAGACTT CTGGTTACTC ATTCACTGGC CACACCATGA ACTGGGTGAA GCAGAGCCAT GGAAAGAACC TTGAGTGGAT TGGACTTATT AATCCTTACA ATGGTGATAC TAACTACAAC CAGAAGTTCA AGGGCAAGGC CACATTTACT GTAGACAAGT CGTCCAGCAC AGCCTACATG GAGCTCCTCA GTCTGACATC TGAGGACTCT GCAGTCTATT ACTGTGCAAG GAGGGTTACG GACTGGTACT TCGATGTCTG GGGCGCAGGG ACCACGGTCA CCGTCTCC SEQ ID NO:14 - Two stop codons and XhoI restriction site (1506-1517 of SEQ ID NO:7) taatagctcg aga SEQ ID NO:15 - e23 anti-HER2 amino acid sequence DVQLTQSPAI LSASPGEKVT MTCRATPSVS YMHWYQQKPG SSPKPWIYTT SNLASGVPAR FSGGGSGTSY SLTVSRVEAE DAATYYCQQW SRSPPTFGGG SKLEIKGSTS GSGKSSEGKG VQLQESGPEV VKPGGSMKIS CKTSGYSFTG HTMNWVKQSH GKNLEWIGLI NPYNGDTNYN QKFKGKATFT VDKSSSTAYM ELLSLTSEDS AVYYCARRVT DWYFDVWGAG TTVTVS SEQ ID NO:16 - Trastuzumab-based scFv EVQLVESGGG LVQPGGSLRL SCAASGFNIK DTYIHWVRQA PGKGLEWVAR IYPTNGYTRY ADSVKGRFTI SADTSKNTAY LQMNSLRAED TAVYYCSRWG GDGFYAMDYW GQGTLVTVSS GSTSGSGKPG SGEGSTKGDI QMTQSPSSLS ASVGDRVTIT CRASQDVNTA VAWYQQKPGK APKLLIYSAS FLYSGVPSRF SGSRSGTDFT LTISSLQPED FATYYCQQHY TTPPTFGQGT KVEIKRTV SEQ ID NO:17 - Anti-HER2 light chain DIQMTQSPSS LSASVGDRVT ITCRASQDVN TAVAWYQQKP GKAPKLLIYS ASFLYSGVPS RFSGSRSGTD FTLTISSLQP EDFATYYCQQ HYTTPPTFGQ GTKVEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC SEQ ID NO:18 - Anti-HER2 heavy chain EVQLVESGGG LVQPGGSLRL SCAASGFNIK DTYIHWVRQA PGKGLEWVAR IYPTNGYTRY ADSVKGRFTI SADTSKNTAY LQMNSLRAED TAVYYCSRWG GDGFYAMDYW GQGTLVTVSS ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKKVEP KSCDK SEQ ID NO:19 - Lumretuzumab heavy chain QVQLVQSGAE VKKPGASVKV SCKASGYTFR SSYISWVRQA PGQGLEWMGW IYAGTGSPSY NQKLQGRVTM TTDTSTSTAY MELRSLRSDD TAVYYCARHR DYYSNSLTYW GQGTLVTVSS ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKKVEP KSCDKTHTCP PCPAPELLGG PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSRDE LTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPG SEQ ID NO:20 - Lumretuzumab light chain DIVMTQSPDS LAVSLGERAT INCKSSQSVL NSGNQKNYLT WYQQKPGQPP KLLIYWASTR ESGVPDRFSG SGSGTDFTLT ISSLQAEDVA VYYCQSDYSY PYTFGQGTKL EIKRTVAAPS VFIFPPSDEQ LKSGTASVVC LLNNFYPREA KVQWKVDNAL QSGNSQESVT EQDSKDSTYS LSSTLTLSKA DYEKHKVYAC EVTHQGLSSP VTKSFNRGEC SEQ ID NO:21 - Seribantumab heavy chain EVQLLESGGG LVQPGGSLRL SCAASGFTFS HYVMAWVRQA PGKGLEWVSS ISSSGGWTLY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCTRGL KMATIFDYWG QGTLVTVSSA STKGPSVFPL APCSRSTSES TAALGCLVKD YFPEPVTVSW NSGALTSGVH TFPAVLQSSG LYSLSSVVTV PSSNFGTQTY TCNVDHKPSN TKVDKTVERK CCVECPPCPA PPVAGPSVFL FPPKPKDTLM ISRTPEVTCV VVDVSHEDPE VQFNWYVDGV EVHNAKTKPR EEQFNSTFRV VSVLTVVHQD WLNGKEYKCK VSNKGLPAPI EKTISKTKGQ PREPQVYTLP PSREEMTKNQ VSLTCLVKGF YPSDIAVEWE SNGQPENNYK TTPPMLDSDG SFFLYSKLTV DKSRWQQGNV FSCSVMHEAL HNHYTQKSLS LSPGK SEQ ID NO:22 - Seribantumab light chain QSALTQPASV SGSPGQSITI SCTGTSSDVG SYNVVSWYQQ HPGKAPKLII YEVSQRPSGV SNRFSGSKSG NTASLTISGL QTEDEADYYC CSYAGSSIFV IFGGGTKVTV LGQPKAAPSV TLFPPSSEEL QANKATLVCL VSDFYPGAVT VAWKADGSPV KVGVETTKPS KQSNNKYAAS SYLSLTPEQW KSHRSYSCRV THEGSTVEKT VAPAECS SEQ ID NO:23 - KTN3379 light chain QSVLTQPPSA SGTPGQRVTI SCSGSLSNIG LNYVSWYQQL PGTAPKLLIS RNNQRPSGVP DRFSGSKSGT SASLAISGLR SEDEADYYCA AWDDSPPGEA FGGGTKLTVL GQPKAAPSVT LFPPSSEELQ ANKATLVCLI SDFYPGAVTV AWKADSSPVK AGVETTKPSK QSNNKYAASS YLSLTPEQWK SHRSYSCQVT HEGSTVEKTV APTECS SEQ ID NO:24 - Patritumab Subunit 1 QVQLQQWGAG LLKPSETLSL TCAVYGGSFS GYYWSWIRQP PGKGLEWIGE INHSGSTNYN PSLKSRVTIS VETSKNQFSL KLSSVTAADT AVYYCARDKW TWYFDLWGRG TLVTVSSAST KGPSVFPLAP SSKSTSGGTA ALGCLVKDYF PEPVTVSWNS GALTSGVHTF PAVLQSSGLY SLSSVVTVPS SSLGTQTYIC NVNHKPSNTK VDKRVEPKSC DKTHTCPPCP APELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPGK SEQ ID NO:25 - Patritumab Subunit 2 QVQLQQWGAG LLKPSETLSL TCAVYGGSFS GYYWSWIRQP PGKGLEWIGE INHSGSTNYN PSLKSRVTIS VETSKNQFSL KLSSVTAADT AVYYCARDKW TWYFDLWGRG TLVTVSSAST KGPSVFPLAP SSKSTSGGTA ALGCLVKDYF PEPVTVSWNS GALTSGVHTF PAVLQSSGLY SLSSVVTVPS SSLGTQTYIC NVNHKPSNTK VDKRVEPKSC DKTHTCPPCP APELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPGK SEQ ID NO:26 - Patritumab Subunit 3 DIEMTQSPDS LAVSLGERAT INCRSSQSVL YSSSNRNYLA WYQQNPGQPP KLLIYWASTR ESGVPDRFSG SGSGTDFTLT ISSLQAEDVA VYYCQQYYST PRTFGQGTKV EIKRTVAAPS VFIFFPSDEQ LKSGTASVVC LLNNFYPREA KVQWKVDNAL QSGNSQESVT EQDSKDSTYS LSSTLTLSKA DYEKHKVYAC EVTHQGLSSP VTKSFNRGEC SEQ ID NO:27 - Patritumab Subunit 4 DIEMTQSPDS LAVSLGERAT INCRSSQSVL YSSSNRNYLA WYQQNPGQPP KLLIYWASTR ESGVPDRFSG SGSGTDFTLT ISSLQAEDVA VYYCQQYYST PRTFGQGTKV EIKRTVAAPS VFIFPPSDEQ LKSGTASVVC LLNNFYPREA KVQWKVDNAL QSGNSQESVT EQDSKDSTYS LSSTLTLSKA DYEKHKVYAC EVTHQGLSSP VTKSFNRGEC SEQ ID NO:28 - Trastuzumab-based TriKE amino acid sequence QVQLVESGGG LVQPGGSLRL SCAASGLTFS SYNMGWFRQA PGQGLEAVAS ITWSGRDTFY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAANP WPVAAPRSGT YWGQGTLVTV SSSGGGGSGG GGSGGGGSGG GGSGNWVNVI SDLKKIEDLI QSMHIDATLY TESDVHPSCK VTAMKCFLLE LQVISLESGD ASIHDTVENL IILANNSLSS NGNVTESGCK ECEELEEKNI KEFLQSFVHI VQMFINTSGS TSGSGKPGSG EGSTKGEVQL VESGGGLVQP GGSLRLSCAA SGFNIKDTYI HWVRQAPGKG LEWVARIYPT NGYTRYADSV KGRFTISADT SKNTAYLQMN SLRAEDTAVY YCSRWGGDGF YAMDYWGQGT LVTVSSGSTS GSGKPGSGEG STKGDIQMTQ SPSSLSASVG DRVTITCRAS QDVNTAVAWY QQKPGKAPKL LIYSASFLYS GVPSRFSGSR SGTDFTLTIS SLQPEDFATY YCQQHYTTPP TFGQGTKVEI KRTV SEQ ID NO:29 - Trastuzumab-based TriKE DNA sequence - Human codon optimized CAGGTGCAGC TGGTGGAGTC TGGGGGAGGC TTGGTGCAGC CTGGGGGCTC TCTGAGACTC TCCTGTGCAG CCTCTGGCCT CACCTTCAGT AGCTATAACA TGGGCTGGTT CCGCCAGGCT CCAGGGCAAG GCCTTGAGGC TGTAGCATGCTGA AGTTTCAGA GCAGACTCCG TGAAGGGCCG ATTCACCATC TCCAGAGACA ACTCCAAGAA CACTCTCTAT CTGCAAATGA ACAGCCTGCG CGCGGAGGAC ACGGCCGTTT ATTATTGTGC TGCAAACCCC TGGCCAGTGG CGGCGCCACG TAGTGGCACC TACTGGGCCGCCGTCGGTCGGTC GCGGCGGCGG TTCTGGTGGA GGAGGTAGTG GGGGGGGAGG AAGCGGAGGG GGTGGCTCAG GGAACTGGGT GAATGTAATA AGTGATTTGA AAAAAATTGA AGATCTTATT CAATCTATGC ATATTGATGC TACTTTATAT ACGGAAAGTGCC CATTCCAGTCA GTACTTATT TGAAGTGCTT TCTCTTGGAG TTACAAGTTA TTTCACTTGA GTCCGGAGAT GCAAGTATTC ATGATACAGT AGAAAATCTG ATCATCCTAG CAAACAACAG TTTGTCTTCT AATGGGAATG TAACAGAATC TGGATGCAAA GAATGTGAGG AACTGAAGAATTATTAG AGAATTATTAG TGTACATATT GTCCAAATGT TCATCAACAC TTCTGGCAGT ACCAGCGGGT CAGGGAAACC TGGCAGTGGG GAAGGTTCCA CAAAAGGTGA GGTTCAGCTC GTGGAATCCG GCGGCGGGCT GGTCCAACCA GGTGGGAGTC TCCCTGTC ATGTCGGAGCCAATATAAAAGA TACATATATA CATTGGGTAA GACAGGCCCC CGGTAAGGGT CTGGAGTGGG TTGCCAGAAT TTATCCCACT AATGGATACA CTCGTTACGC CGATTCTGTG AAAGGCCGGT TTACCATCTC TGCTGACACC TCAAAGAACA CCAGGCGACTGACCGACCGACCGCCG GGCTGTCTAT TATTGCTCTC GCTGGGGCGG CGACGGGTTT TATGCCATGG ATTACTGGGG TCAGGGGACC CTAGTTACAG TGAGCAGCGG TAGTACTTCT GGGAGCGGCA AGCCTGGCTC CGGAGAAGGA TCCACCAAAG GGGATATCCA GATGAGATCCGCATTCATTCATT CAGTGTGGGC GACCGGGTCA CGATCACCTG TAGGGCATCT CAGGACGTAA ACACAGCGGT GGCATGGTAC CAGCAAAAAC CTGGAAAGGC CCCAAAACTT TTGATCTACA GCGCTAGCTT CTTATACTCC GGCGTCCCCT CACGATTCTC CGGCTCCAGA AGTGGATTCACCTT GTCTTCATT CCGAGGATTT TGCTACCTAT TATTGCCAGC AACACTACAC AACCCCTCCG ACTTTCGGAC AAGGGACAAA GGTGGAAATT AAGAGGACTG TG SEQ ID NO:30 - Trastuzumab-based TriKE DNA sequence - E. coli codon optimized CAGGTGCAGC TGGTGGAGTC TGGTGGCGGC TTGGTGCAGC CTGGTGGCTC TCTGCGCCTG TCCTGTGCGG CCTCTGGCCT CACCTTCAGC AGCTATAACA TGGGCTGGTT CCGCCAGGCT CCAGGACAAG GCCTTGAGGC TGTGGCGTGA GCCTTGA GCCTTG GCGGACTCCG TGAAAGGCCG TTTCACCATC TCGCGTGACA ATTCCAAGAA CACGCTGTAT CTGCAAATGA ACAGCCTGCG CGCGGAGGAC ACGGCCGTTT ATTATTGTGC GGCAAACCCC TGGCCGGTTG CGGCGCCGCG TAGTGGCACC TACTGGGCCGCCGGTCGTCGTC GCGGCGGCGG TAGCGGTGGC GGAGGTAGCG GGGGGGGTGG AAGCGGTGGT GGTGGCTCAG GGAACTGGGT GAATGTAATA AGTGATTTGA AAAAAATTGA AGATCTGATT CAGAGCATGC ATATTGATGC GACGTTATAT ACGGAATCGAG ATGTTCCACCAAGCGATTGATTGATT TTTGTTAGAG TTACAAGTTA TTTCACTTGA GTCGGGAGAT GCAAGTATTC ATGATACTGT AGAAAATCTG ATCATCCTGG CAAACAACAG CTTGTCGTCG AATGGGAATG TAACAGAATC TGGATGCAAA GAATGTGAAG AACTGGAAGA AAATAAATCATTATT AGAATTAGATTTTTT GTCCAAATGT TCATCAACAC TTCTGGCAGT ACCAGCGGTT CAGGTAAACC GGGCAGCGGG GAAGGTTCCA CAAAAGGTGA AGTTCAGCTC GTGGAAAGCG GCGGCGGTCT GGTCCAGCCA GGTGGGAGTC TCCGCCTGTC ATGTTCCGCGGTGCCGTATATCAAAGA TACATATATA CATTGGGTAA GACAGGCCCC GGGTAAGGGT CTGGAGTGGG TTGCGCGTAT TTATCCGACG AATGGATACA CTCGTTACGC CGATTCTGTG AAAGGCCGCT TTACCATCTC AGCCGACACC TCAAAGAACA CCGCGTACTT ACAGATGAAC TCCCTGCGCG CAGAAGACAC GGCTGTCTAT TATTGCTCGC GCTGGGGCGG CGACGGTTTT TATGCCATGG ATTACTGGGG TCAGGGGACC CTAGTTACTG TGAGCAGCGG TAGTACTTCT GGGAGCGGCA AACCTGGCTC CGGAGAAGGT TCGACCAAAG GGGATATCCA GATGACGCAG AGCCCGTCAT CACTGTCGGC CAGTGTGGGC GATCGGGTCA CGATCACCTG CCGTGCATCG CAGGATGTAA ATACAGCGGT GGCATGGTAC CAGCAAAAAC CTGGAAAGGC CCCAAAACTT CTGATCTACA GCGCTAGCTT CTTATACTCC GGCGTCCCGT CACGATTTTC CGGCTCCCGT AGTGGAACGG ACTTTACTCT GACAATTTCT TCGCTTCAGC CCGAGGATTT TGCTACCTAT TATTGCCAGC AACACTACAC CACCCCGCCG ACTTTCGGCC AAGGGACGAA AGTGGAAATT AAGAGGACGG TG SEQ ID NO:31 - Signal peptide / Anti-HER2 light chain / linker / wtIL15 / linker / cam16 MGWSCIILFL VATATGVHSD IQMTQSPSSL SASVGDRVTI TCRASQDVNT AVAWYQQKPG KAPKLLIYSA SFLYSGVPSR FSGSRSGTDF TLTISSLQPE DFATYYCQQH YTTPPTFGQG TKVEIKRTVA APSVFIFPPS DEQLKSGTAS VVCLLNNFYP REAKVQWKVD NALQSGNSQE SVTEQDSKDS TYSLSSTLTL SKADYEKHKV YACEVTHQGL SSPVTKSFNR GECGGGGSGG GGSNWVNVIS DLKKIEDLIQ SMHIDATLYT ESDVHPSCKV TAMKCFLLEL QVISLESGDA SIHDTVENLI ILANNSLSSN GNVTESGCKE CEELEEKNIK EFLQSFVHIV QMFINTSGST SGSGKPGSGE GSTKGQVQLV ESGGGLVQPG GSLRLSCAAS GLTFSSYNMG WFRQAPGQGL EAVASITWSG RDTFYADSVK GRFTISRDNS KNTLYLQMNS LRAEDTAVYY CAANPWPVAA PRSGTYWGQG TLVTVSS SEQ ID NO:32 - Signal peptide / Anti-HER2 heavy chain MGWSCIILFL VATATGVHSE VQLVESGGGL VQPGGSLRLS CAASGFNIKD TYIHWVRQAP GKGLEWVARI YPTNGYTRYA DSVKGRFTIS ADTSKNTAYL QMNSLRAEDT AVYYCSRWGG DGFYAMDYWG QGTLVTVSSA STKGPSVFPL APSSKSTSGG TAALGCLVKD YFPEPVTVSW NSGALTSGVH TFPAVLQSSG LYSLSSVVTV PSSSLGTQTY ICNVNHKPSN TKVDKKVEPK SCDK SEQ ID NO:33 - Signal peptide / Anti-HER2 heavy chain Fab / T2A / signal peptide / Anti-HER2 light chain / linker / wtIL15 / linker / cam16 MGWSCIILFL VATATGVHSE VQLVESGGGL VQPGGSLRLS CAASGFNIKD TYIHWVRQAP GKGLEWVARI YPTNGYTRYA DSVKGRFTIS ADTSKNTAYL QMNSLRAEDT AVYYCSRWGG DGFYAMDYWG QGTLVTVSSA STKGPSVFPL APSSKSTSGG TAALGCLVKD YFPEPVTVSW NSGALTSGVH TFPAVLQSSG LYSLSSVVTV PSSSLGTQTY ICNVNHKPSN TKVDKKVEPK SCDKEGRGSL LTCGDVEENP GPMGWSCIIL FLVATATGVH SDIQMTQSPS SLSASVGDRV TITCRASQDV NTAVAWYQQK PGKAPKLLIY SASFLYSGVP SRFSGSRSGT DFTLTISSLQ PEDFATYYCQ QHYTTPPTFG QGTKVEIKRT VAAPSVFIFP PSDEQLKSGT ASVVCLLNNF YPREAKVQWK VDNALQSGNS QESVTEQDSK DSTYSLSSTL TLSKADYEKH KVYACEVTHQ GLSSPVTKSF NRGECGGGGS GGGGSNWVNV ISDLKKIEDL IQSMHIDATL YTESDVHPSC KVTAMKCFLL ELQVISLESG DASIHDTVEN LIILANNSLS SNGNVTESGC KECEELEEKN IKEFLQSFVH IVQMFINTSG STSGSGKPGS GEGSTKGQVQ LVESGGGLVQ PGGSLRLSCA ASGLTFSSYN MGWFRQAPGQ GLEAVASITW SGRDTFYADS VKGRFTISRD NSKNTLYLQM NSLRAEDTAV YYCAANPWPV AAPRSGTYWG QGTLVTVSS SEQ ID NO:34 - Linker SGGGGSGGGG SGGGGSGGGG SG SEQ ID NO:35 - Linker GSTSGSGKPG SGEGSTKG SEQ ID NO:36 - Signal peptide MGWSCIILFL VATATGVHS SEQ ID NO:37 - T2A self-cleaving peptide EGRGSLLTCG DVEENPGP

Claims

1. an NK cell-binding domain comprising an antibody or binding fragment thereof that selectively binds to CD16; a first flanking sequence at the C-terminus of the NK cell binding domain; an NK activation domain operably linked to an NK cell binding domain comprising IL-15; a second flanking sequence at the C-terminus of the NK activation domain; and in turn comprising a targeting domain that selectively binds to HER2; wherein the targeting domain comprises the amino acid sequence of SEQ ID NO:6; SEQ ID NO:15; SEQ ID NO:16; or SEQ ID NO:17 and SEQ ID NO:

18. compound.

2. The compound of claim 1, wherein the CD16 comprises CD16a.

3. The compound of claim 1 , wherein the NK cell binding domain comprises the amino acid sequence of SEQ ID NO:

2.

4. The compound of claim 1 , wherein the portion of the NK cell binding domain comprises an antibody or a binding fragment thereof.

5. 5. The compound of claim 4, wherein the antibody or binding fragment thereof is a human, humanized, or camelid antibody or binding fragment thereof.

6. The compound of claim 1, wherein the IL-15 comprises the amino acid sequence of SEQ ID NO:

4.

7. 2. The compound of claim 1, wherein the IL-15 comprises an N72D or N72A amino acid substitution compared to SEQ ID NO:

4.

8. The compound of any one of claims 1 to 7, wherein the targeting domain comprises an antibody or a binding fragment thereof.

9. The compound of claim 8 , wherein the antibody-binding fragment comprises an scFv.

10. 9. The compound of claim 8, wherein the targeting domain comprises trastuzumab or e23.

11. The compound of claim 1 , wherein the first flanking sequence connects two domains.

12. The compound of claim 11 , wherein the second flanking sequence connects the two linked domains to a third domain.

13. The compound of claim 12 , wherein the second flanking sequence is adjacent to the NK activation domain.

14. 13. The compound of claim 12, wherein the first flanking sequence is C-terminal to the NK cell binding domain and the second flanking sequence is N-terminal to the targeting domain.

15. The compound of any one of claims 1 to 14, further comprising a second targeting domain.

16. The compound of any one of claims 1 to 15, further comprising a second NK cell binding domain.

17. The compound of any one of claims 1 to 16, further comprising a second NK activation domain.

18. A compound according to any one of claims 1 to 17; and Pharmaceutically acceptable carrier A composition comprising:

19. 20. The composition of claim 18, further comprising an additional therapeutic agent.

20. 20. The composition of claim 19, wherein the additional therapeutic agent comprises a therapeutic agent that targets HER2, HER3, or the HER2 / HER3 heterodimer complex.

21. A pharmaceutical composition comprising an amount of a compound according to any one of claims 1 to 17 effective to induce NK-mediated cancer cell killing.

22. A pharmaceutical composition for stimulating NK cell proliferation in vivo, comprising a compound according to any one of claims 1 to 17 in an amount effective to stimulate NK cell proliferation in a subject.

23. 20. A pharmaceutical composition for treating cancer in a subject, comprising a compound according to any one of claims 1 to 17 in an amount effective to treat the cancer.

24. 24. The pharmaceutical composition of claim 23, wherein the compound is administered before, simultaneously with, or after chemotherapy, surgical resection of a tumor, or radiation therapy.

25. The chemotherapy may be altretamine, amsacrine, L-asparaginase, colspase, bleomycin, busulfan, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, cyclophosphamide, cytophosphan, cytarabine, dacarbazine, dactinomycin, daunorubicin, docetaxel, doxorubicin, epirubicin, etoposide, fluorouracil, fludarabine, fotemustine, gancyclo[alpha]pyrrolidone, cyclosporine ...

23. The pharmaceutical composition of claim 22, comprising riboflavin, gemcitabine, hydroxyurea, idarubicin, ifosfamide, irinotecan, lomustine, melphalan, mercaptopurine, methotrexate, mitoxantrone, mitomycin C, nimustine, oxaliplatin, paclitaxel, pemetrexed, procarbazine, raltitrexed, temozolomide, teniposide, thioguanine, thiotepa, topotecan, vinblastine, vincristine, vindesine, or vinorelbine.

26. A pharmaceutical composition comprising the composition of any one of claims 18 to 20 in an amount effective to induce NK-mediated cancer cell killing.

27. A pharmaceutical composition for stimulating NK cell proliferation in vivo, comprising the composition of any one of claims 18 to 20 in an amount effective to stimulate NK cell proliferation in a subject.

28. A pharmaceutical composition for treating cancer in a subject, comprising the composition of any one of claims 18 to 20 in an amount effective to treat cancer.

29. 29. The pharmaceutical composition of claim 28, wherein the composition is administered before, simultaneously with, or after chemotherapy, immunotherapy, surgical resection of a tumor, or radiation therapy.

30. The chemotherapy may be altretamine, amsacrine, L-asparaginase, colspase, bleomycin, busulfan, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, cyclophosphamide, cytophosphan, cytarabine, dacarbazine, dactinomycin, daunorubicin, docetaxel, doxorubicin, epirubicin, etoposide, fluorouracil, fludarabine, fotemustine, gancyclo[alpha]pyrrolidone, cyclosporine ...

30. The pharmaceutical composition of claim 29, comprising riboflavin, gemcitabine, hydroxyurea, idarubicin, ifosfamide, irinotecan, lomustine, melphalan, mercaptopurine, methotrexate, mitoxantrone, mitomycin C, nimustine, oxaliplatin, paclitaxel, pemetrexed, procarbazine, raltitrexed, temozolomide, teniposide, thioguanine, thiotepa, topotecan, vinblastine, vincristine, vindesine, or vinorelbine.

31. 30. The pharmaceutical composition of claim 29, wherein the immunotherapy targets HER2, HER3, or the HER2 / HER3 heterodimer complex.

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