Immunotherapeutic compounds and methods

Abstract

The immunotherapeutic compound includes an NK cell engaging domain, an NK activating domain, and a targeting domain. The targeting domain selectively binds to a HER2, HER3, or HER2 / HER3 heterodimer complex and is operably linked to the NK activating domain and the NK cell engaging domain. The compound can be administered to a subject to induce NK-mediated killing of cancer cells, stimulate expansion of NK cells in the subject, and / or for treating cancer.

Description

[0001] Cross-reference of related applications

[0002] 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.

[0003] Government funding

[0004] This invention was developed with government support under license number CA197292 granted by the National Institutes of Health (NIH). The government holds certain rights to this invention.

[0005] sequence list

[0006] This application contains a sequence list submitted electronically to the United States Patent and Trademark Office via EFS-Web. The sequence list is an ASCII text file titled “Seq_Listing-0110-000632_ST25.txt”, 70 kilobytes in size, created on September 15, 2020. Information contained in this sequence list is incorporated herein by reference.

[0007] Overview

[0008] In one aspect, this disclosure describes multispecific immunotherapeutic compounds comprising an NK cell-binding domain, an NK activation domain, and a targeting domain. The targeting domain selectively binds to HER2, HER3, or a HER2 / HER3 heterodimer complex and is operatively linked to the NK activation domain and the NK cell-binding domain.

[0009] 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 includes the amino acid sequence of SEQ ID NO:2.

[0010] In some embodiments, the NK cell binding domain may include an antibody or a binding fragment thereof. In some of these embodiments, the antibody or its binding fragment may be human, humanized, or camelid-derived.

[0011] In some embodiments, the NK activating 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 as compared to SEQ ID NO: 4.

[0012] In some embodiments, the targeting domain comprises an antibody or binding fragment thereof. In some of these embodiments, the antibody binding fragment can comprise an scFv, F(ab)2, Fab, or a 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.

[0013] In some embodiments, the immunotherapeutic compound can comprise a second targeting domain.

[0014] In some embodiments, the immunotherapeutic compound can comprise a second NK cell engaging domain.

[0015] In some embodiments, the immunotherapeutic compound can comprise a second NK activating domain.

[0016] In another aspect, the present disclosure describes a composition comprising any of the embodiments of the therapeutic compound outlined above and a pharmaceutically acceptable carrier.

[0017] In some embodiments, the composition can further comprise an additional therapeutic agent. In some of these embodiments, the additional therapeutic agent can comprise a chemotherapeutic agent. In some embodiments, the additional therapeutic agent can comprise a therapeutic agent that targets HER2, HER3, or a HER2 / HER3 heterodimeric complex.

[0018] In another aspect, the present disclosure describes a method comprising administering to a subject any of the embodiments of the compound or composition outlined above in an amount effective to induce NK-mediated killing of cancer cells.

[0019] In another aspect, the present disclosure describes a method for stimulating expansion of NK cells in vivo. Generally, the method comprises administering to a subject any of the embodiments of the compound or composition outlined above in an amount effective to stimulate expansion of NK cells in the subject.

[0020] In another aspect, the present disclosure describes methods of treating cancer in a subject. Generally, the methods include administering to the subject an amount of any of the embodiments of the compounds or compositions outlined above effective to treat the cancer.

[0021] In some embodiments, the compound or composition is administered prior to, concurrently with, or following chemotherapy, surgical resection of a tumor, or radiation therapy. In some of these embodiments, the chemotherapy can include hexamethylmelamine, amsacrine, L-asparaginase, colaspase, bleomycin, busulfan, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladrabine, 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.

[0022] The above summary is not intended to describe each disclosed embodiment or every implementation of the present application. The following description more particularly exemplifies illustrative embodiments. Throughout this application, guidance is provided by way of example in several places by use of lists of examples. The list of examples is merely representative, and should not be interpreted as an exclusive list. BRIEF DESCRIPTION OF DRAWINGS

[0024] This patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0025] Figure 1. Design, production, and purification of cam1615HER2. (A) Schematic of an exemplary expression vector containing a coding region arrangement that encodes the following cam1615HER2 components (from left to right): a camelid anti-CD16 VHH, human IL-15, an anti-HER2 scFV. (B) Gene map of an exemplary expression vector encoding cam1615HER2, including restriction sites and target gene locations on the pET28c vector.

[0026] Figure 2. Production and purification of cam1615HER2. (A) SDS-PAGE gel stained with Coomassie blue dye showing purity and size of final product after two orthogonal column steps. MWS: molecular weight standard; NR: non-reduced; R: reduced. Densitometry was performed to obtain final purity. (B) Chromatogram resulting from the first step of purification of cam1615HER2 on an ion exchange (FFQ) column. Peak collection is indicated by double headed arrow. (C) Chromatogram resulting from the second step of purification of cam1615HER2 on a size exclusion column. Peak collection is indicated by double headed arrow.

[0027] Figure 3 . Effect of cam1615HER2 on NK cell expansion and absolute numbers showing the effect of TriKE treatment on C56 + CD3 - NK cell percentage as measured by flow cytometry. PBMC from 6 different normal donors were assayed separately. IL-15 was used as a control in addition to cam1615HER2. (A) cam1615HER2 treatment showed a significant difference in the percentage of highly proliferating NK cells when compared to control. (B) cam1615HER2 treatment showed a significant difference in the percentage of total NK cells when compared to control. (C) cam1615HER2 treatment showed a significant difference in the number of naive NK counts when compared to 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 naive CD3 + CD56 - T cells.

[0028] 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 was used as a negative control for binding.

[0029] Figure 5. Functional activity correlates with binding activity of cam1615 HER2 TriKE. (A) CD 107a functional activity is elevated in PBMC plus SKOV3 cell cultures treated with cam1615 HER2 compared to IL-15 and no treatment controls. PBMC from 10 different normal donors were assayed individually. (B) Enhanced IFN-γ activity in the same PBMC / SKOV3 cultures. (C) CD 107a functional activity is elevated in PBMC plus breast cancer cell line SK-BR-3 cultures (7 different donors assayed). (D) Enhanced IFN-γ activity in the same PBMC / SK-BR-3 cultures. (E) CD 107a is not elevated when UMSCC-11B head and neck cancer cell line is tested in the same assay (4 different donors assayed).

[0030] Figure 6. Testing the ability of TriKE to enhance killing of tamoxifen resistant MCF-7L-TamR breast cancer cells by a cytotoxic drug (tamoxifen). (A) CD 56 + CD3 - NK cells in the absence of cancer cells. (B) CD 107a activity using PBMC incubated with the parental MCF-7L cell line. (C) CD 107a activity using PBMC incubated with tamoxifen resistant MCF-7L-TamR cells. (D) CD 107a activity using PBMC incubated with SKBR-3 breast cancer cells. (E) CD 56 + CD3 - NK cells in the absence of cancer cells. (B) CD 107a activity using PBMC incubated with the parental MCF-7L cell line. (C) CD 107a activity using PBMC incubated with tamoxifen resistant MCF-7L-TamR cells. (D) CD 107a activity using PBMC incubated with SKBR-3 breast cancer cells. (E) CD 56

[0031] Figure 7. INCUCYTE (Essen Bioscience, Inc., Ann Arbor, MI) data measuring real-time killing of SKOV3 ovarian cancer cells in the presence of PBMCs, confirming cytotoxicity data for CD 107a. (A) Spheroid size; (B) Spheroid intensity. Cam 1615HER2 resulted in a sharp decline in target cells measured over a 120 hour time period compared to lower activity of no treatment, anti-cam 16 alone (CAM 16), and IL-15 alone (IL 15) controls. N = 7 donors / group.

[0032] Figure 8 . Visual evidence that cam 1615HER2 resulted in a sharp, time-dependent decline in target cells measured over a 72 hour time period compared to lower activity of no treatment (left column), anti-cam 16 alone, and IL-15 alone controls (right column). N = 7 donors / group.

[0033] Figure 9. Testing ascites from ovarian cancer patients as effector cell source. (A) CD 107a background activity when cells from patient ascites fluid were incubated without MA-148 ovarian cancer cells. (B) CD 107a activity when ascites cells were incubated with MA-148 cell ovarian cancer cells. (C) CD 107a background activity when cells from normal donors were incubated without MA-148 ovarian cancer cells. (D) CD 107a activity when normal donor cells were incubated with MA-148 cell ovarian cancer cells. (E) IFN-γ background activity when cells from patient ascites fluid were incubated without MA-148 ovarian cancer cells. (F) IFN-γ activity when ascites cells were incubated with MA-148 cell ovarian cancer cells. (G) IFN-γ background activity when cells from normal donors were incubated without MA-148 ovarian cancer cells. (H) IFN-γ activity when normal donor cells were incubated with MA-148 cell ovarian cancer cells. Controls are IL-15 and no treatment. In each case, independent determinations were made on 9-13 different donors and data averaged.

[0034] Figure 10. In vivo efficacy of cam1615HER2 in xenograft model. Cells were stably transfected with firefly luciferase for the purpose of real-time bioluminescence imaging. (A) Bioluminescence imaging of a group of 6 NSG mice given SKOV3 and NK cells intraperitoneally. The images show the total flux for each animal and indicate that 5 of the 6 animals in the no treatment group had advanced tumors. The single animal in the no treatment group that showed minimal activity went on to develop a tumor. (B) A group of 6 mice given SKOV3 and NK cells but treated with cam1615HER2 TriKE imaged on day 38.

[0035] Figure 11. (A) Minimal change in animal weight despite multiple TriKE injections, indicating that the treatment was not toxic compared to the no treatment control. (B) Scatter plot of data from the same experiment on day 46 after tumor inoculation. Data is expressed as total flux radiation (p / s). Treated mice were compared to untreated mice. The difference was significant (p = 0.0216) as determined by Student's T test. (C) Time (days) line graph indicating that the treatment group has begun to relapse. (D) Survival plot of data over an extended time interval. The difference between the treated versus the untreated group was significant.

[0036] Figure 12 . cam1615HER2 TriKE was tested for its ability to enhance killing of other HER2 expressing ovarian cancer cell lines. (A) CD 107a activity when PBMC NK cells were incubated with OVCAR3 ovarian cancer cells. (B) CD 107a activity when PBMC NK cells were incubated with OVCAR5 ovarian cancer cells. (C) CD 107a activity when PBMC NK cells were incubated with SKOV3 ovarian cancer cells. (D) IFN-γ activity when PBMC NK cells were incubated with OVCAR3 ovarian cancer cells. (E) IFN-γ activity when PBMC NK cells were incubated with OVCAR5 ovarian cancer cells. (F) IFN-γ activity when PBMC NK cells were incubated with SKOV3 ovarian cancer cells. SKOV3 data was performed with the following negative controls: no treatment (NT), anti-cam16 alone (CAM16), IL-15 (IL15), and anti-HER2 antibody alone (e23).

[0037] DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0038] The present 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), which are members of the epidermal growth factor receptor (EGFR) family of transmembrane receptor tyrosine kinases. HER2 is directly associated with cancer because its overexpression is associated with poor prognosis in breast cancer and it triggers intracellular signaling pathways associated with cell proliferation, differentiation, and survival. Her2 and HER3 can form heterodimeric complexes.

[0039] In many embodiments, the immunotherapeutic compound can be a trispecific killer engager compound (TriKE). TriKEs have 3 separate binding regions: an NK cell engaging domain that binds to an NK cell (e.g., CD16), an NK activating domain comprising a cytokine or functional fragment thereof that binds to a receptor for the cytokine, and a targeting domain that binds to a marker present on a target cell (e.g., a cancer cell). The design and production of TriKEs are described extensively in, e.g., U.S. Patent Application Publication No. US 2018 / 0282386 Al. TriKEs have the advantage of combining antibody-dependent cellular cytotoxicity (ADCC) promoting moieties and expansion-associated moieties (IL-15) on the same molecule.

[0040] One or more of the binding regions or domains in the immunotherapeutic compound can include an antibody. As used herein, the term “antibody” generally refers to an immunoglobulin or fragment thereof, and thus includes a monoclonal antibody, fragments thereof. Exemplary antibody fragments include, but are not limited to, scFv, Fab, F(ab’)2, 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 without light chains. These single domain antibody fragments (VHH or NANOBODIES (Ablynx N.V., Ghent, Belgium)) have multiple advantages for biotechnological 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 engaging domain is a camelid single domain antibody fragment.

[0041] While described herein in the context of exemplary embodiments in which the immunotherapeutic compound has a targeting domain comprising a scFv targeting HER2 having the amino acid sequence of SEQ ID NO: 6, the immunotherapeutic compounds described herein can include any other suitable HER2- and / or HER3-targeting moiety. Thus, in various embodiments, the targeting domain can recognize HER2, HER3, and / or HER2 / HER3 heterodimers. HER2 / HER3 heterodimers are detected in many breast cancers and many HER2 + HER2 / HER3 heterodimers are associated with proliferation, distant metastasis, and / or poor patient prognosis.

[0042] Exemplary alternative targeting moieties include antibodies and antibody fragments that specifically bind to HER2, HER3, and / or HER2 / HER3 heterodimers. Exemplary antibody fragments include, but are not limited to, 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. EP3457139 Al), 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 Online21 :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, duligotumab (MEHD7945A; Liu et al., 2019, Biol Proced Online 21 :5) or a functional fragment thereof, or pertuzumab or a functional variant thereof.

[0043] While described herein in the context of exemplary embodiments in which the NK cell engaging domain comprises a single domain antibody (sdAb) that binds to CD16, the immunotherapeutic compound can comprise any other suitable NK engaging moiety. Exemplary alternative NK engaging moieties include, but are not limited to, any amino acid sequence that can selectively bind to a receptor that is at least partially located on the surface of an NK cell. Thus, the NK cell engaging domain can function to bind an NK cell and thereby bring the NK cell in spatial proximity to a target to which the targeting domain selectively binds. In certain embodiments, the NK cell engaging domain can selectively bind to a receptor that activates an NK cell and thus also has an activating function. For example, activation of the CD16 receptor can trigger antibody-dependent cell-mediated cytotoxicity. Thus, the NK cell engaging domain of an exemplary cam1615 HER2 compound has an NK activating activity. In other embodiments, the NK cell engaging domain can interrupt a mechanism that inhibits an NK cell. In such embodiments, the NK cell engaging domain can comprise, for example, an anti-PD-1 / PD-L1, an anti-NKG2A, an anti-TIGIT, an anti-killer cell immunoglobulin receptor (KIR), and / or any other inhibitory blocking domain.

[0044] The NK cell engaging domain can comprise an antibody or ligand that selectively binds to any NK cell receptor, such as the cytotoxicity receptor 2B4, the low affinity Fc receptor CD16, the killer cell immunoglobulin-like receptor (KIR), CD2, NKG2A, TIGIT, NKG2C, LIR-1, and / or DNAM-1.

[0045] NK cell engaging domains can be designed to have a desired degree of NK selectivity, and thus a desired immunological engaging property. For example, CD16 has been identified as Fc receptor FcyRIIIa (CD16a) and FcyRIIIb (CD16b). These receptors bind to the Fc portion of IgG antibodies and then activate NK cells to produce antibody-dependent cell-mediated cytotoxicity. Anti-CD16 antibodies bind to NK cells selectively, but can also bind to neutrophils. Anti-CD16a antibodies bind to NK cells selectively, but not to neutrophils. Immunotherapeutic compounds comprising NK cell engaging domains with anti-CD16a antibodies can bind to NK cells but not to neutrophils. Thus, in situations where it can be desirable to engage NK cells but not neutrophils, the NK cell engaging domains of immunotherapeutic compounds can be designed to include anti-CD16a antibodies.

[0046] While described herein in the context of exemplary embodiments in which the NK activating domain comprises a human IL-15 fragment, the NK activating domain can comprise any amino acid sequence that activates NK cells, promotes maintenance of NK cells, or otherwise promotes NK cell activity. The NK activating domain can be or can be derived from one or more cytokines that can activate and / or maintain NK cells. As used herein, the term "derived from" refers to an amino acid fragment of a cytokine (e.g., IL-15) that is a functional variant of the cytokine in question - i.e., has sufficient sequence similarity or sequence identity to the cytokine in question to provide NK cell activation and / or maintenance activity. Exemplary cytokines upon which the NK activating domain can be based include, for example, IL-15, IL-18, IL-12, and IL-21. Thus, while exemplary cam1615 HER2 compounds include an NK activating domain derived from IL-15, a compound targeting HER2 can be designed to have an NK activating domain that is or is derived from any suitable cytokine.

[0047] For brevity in this specification, reference to an NK activating domain by identifying the cytokine upon which the NK activating domain is based can refer to the full amino acid sequence of the cytokine or a functional variant of the cytokine. The functional variant of the cytokine can include any suitable amino acid fragment of the cytokine and / or a modified version of the cytokine that includes one or more amino acid deletions, additions, and / or substitutions. Thus, reference to an "IL-15" NK activating domain includes an NK activating domain comprising the full amino acid sequence of IL-15, an NK activating domain comprising a fragment of IL-15, or an NK activating domain comprising amino acid substitutions compared to the wild-type IL-15 amino acid sequence (such as IL-15N72D or IL-15N72A).

[0048] While described above in the context of exemplary embodiments in which the immunotherapeutic compound is a trispecific killer cell engager compound (i.e., a TriKE), the compositions and methods described herein can involve the use of immunotherapeutic compounds modified to include additional domains. For example, the immunotherapeutic compound can be designed as a larger molecule with more than one targeting domain, more than one NK cell engaging domain, and / or more than one NK activating domain. In embodiments that include more than one NK activating domain, the NK activating domains can be provided in series or in any other combination. Any cytokine-based NK activating domain can include the complete amino acid sequence of the cytokine, can be an amino acid fragment, or can be a modified version of the cytokine, regardless of the nature of the other NK activating domains included in the immunotherapeutic compound.

[0049] Exemplary additional targeting domains include, but are not limited to, any moiety that selectively binds to an intended target, such as a tumor cell, a target in the cancer stroma, a target on an inhibitory cell (such as a myeloid-derived suppressor cell that is CD33+), or a target on a virally infected cell. Thus, the targeting domain can include, for example, an anti-tumor antibody, such as rituximab (anti-CD20), afutuzumab (anti-CD20), pertuzumab (anti-HER2 / neu), labetuzumab (anti-CEA), adecatumumab (anti-EpCAM), citatuzumab 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 alpha V), ipilimumab (anti-CTLA-4), ipilimumab (anti-CTLA-4), tremelimumab (anti-CTLA-4), and the like. vβ3), intetumumab (anti-CD51), ipilimumab (anti-CD152), oregovomab (anti-CA-125), votumumab (anti-tumor antigen CTAA16.88), or pemtumumab (anti-MUCl), 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-RORl, anti-CSPG4, anti-SSl, or anti-HSPG2, anti-IGF-l, anti-ROR-l, anti-uPAR, anti-VEGFR, anti-LIV-l, anti-SGN-CD70A, anti-IL-3, anti-IL-4R, anti-epithelial mesenchymal transition (EMT), anti-TRAIL, anti-PD-Ll, any one or more of SEQ ID NOs: 1-119 of European Patent No. EP 3457139 Al, or a functional variant of any of the foregoing.

[0050] An amino acid sequence is a "functional variant" of a reference amino acid sequence if it has a specified 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 it contains less than the full-length amino acid sequence of the reference amino acid sequence. A "functional fragment" can further have a specified amount of sequence identity or sequence specificity compared to the reference amino acid sequence.

[0051] The 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 their sequence length; although the amino acids in each sequence must retain their proper order, gaps in one or both sequences are allowed when making the alignment to optimize the number of identical amino acids.

[0052] A 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 can be compared using the Blastp program of the BLAST 2 search algorithm, e.g., Tatiana et al. (2005) Nucleic Acids Res. 33:W104-10, the contents of which are incorporated herein by reference. FEMS Microbiol LettBLAST 2.0 (Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997); Zhang et al., Genome Res. 7:649-656 (1997); Wootton et al., Comput. Chem. 17: 149-163 (1993); Altschul et al., J. Mol. Biol. 21 1 : 501- 510 (1993); Altschul, Methods Cell Biol. 48: 109- 159 (1994); Madden et al., Methods Enzymol. 266: 131-141 (1996); Tatusova 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 BLAST 2 search parameters can be used, including Matrix = BLOSUM62; Open Gap Penalty = 11, Extend Gap Penalty = 1, Gap x_dropoff = 50, Expect = 10, Word Size = 3, and Filter On.

[0053] In comparisons of two amino acid sequences, structural similarity can be referred to in terms of percent "identity" or can be referred to in terms of percent "similarity." "Identity" refers to the presence of identical amino acids. "Similarity" refers to the presence of not only identical amino acids, but also to the allowance for conservative substitutions. A conservative substitution of an amino acid residue in an amino acid sequence can be selected from other members of the class to which the amino acid residue belongs. For example, non-polar (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, 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 Gin for Asn to maintain a free -NH2.

[0054] Thus, amino acids belonging to a group of amino acids having a particular size or characteristic (e.g., charge, hydrophobicity, or hydrophilicity) can be substituted for another amino acid without changing the activity of the protein, particularly in regions of the protein not directly related to the biological activity. Regions of an amino acid sequence not directly related to the biological activity can be inferred from an alignment analysis, which identifies regions where variability (e.g., additions, deletions, or non-conservative substitutions) exist when comparing related amino acid sequences. Alignment analysis can be performed using the amino acid sequences provided herein and / or readily available in databases.

[0055] The NK engaging domain, the NK activating domain, or the targeting domain can include 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 similarity to a reference amino acid sequence (e.g., a reference antibody fragment, a cytokine, or a cytokine fragment).

[0056] The NK engaging domain, NK activating domain, or targeting domain can 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.

[0057] The immunotherapeutic compounds as described herein can also be designed to provide additional sequences, such as the addition of extra C-terminal or N-terminal amino acids, which can facilitate purification, for example, by capture on a column or using antibodies. Such tags include, for example, a histidine-rich tag that allows for purification of the polypeptide on a nickel column. Such genetic modification techniques and suitable additional sequences are well known in the art of molecular biology.

[0058] 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 ordinary skill in the art can determine the entire scope of polynucleotides that encode that amino acid sequence using routine and routine methods.

[0059] Figure 1A An exemplary construct of an exemplary embodiment of a second generation TriKE, referred to herein as cam1615HER2 (SEQ ID NO: 1), is shown, which is capable of both antibody-dependent cellular cytotoxicity (ADCC) and NK cell expansion. The cam1615HER2 TriKE includes an anti-CD16 VHH as the NK cell engaging domain. The anti-CD16 VHH is a variable region of a camelid antibody heavy chain. Figure 1B A plasmid map showing the location of the TriKE coding sequence in the pET expression vector. FIG. 2B shows an absorbance trace of the fractions of bacteria and target protein as they pass through a FFQ ion exchange column as the first stage of purification from inclusion bodies. Eluate was collected in 8 ml aliquots. Figure 2C An absorbance trace from the second stage of purification, size exclusion chromatography (SEC), is shown. The double-headed arrow shows the peak of interest that was collected as the cam1615HER2 exited the column. Figure 2A The final product (fractions C2-D4) is shown to be primarily a single band when analyzed using SDS-PAGE with Coomassie blue staining, which provides evidence of a homogenous product. The final product is greater than 90% pure and has a molecular weight of about 55 kDa.

[0060] However, the cam1615HER2 TriKE can be constructed in other ways. Multiple constructs can be designed, each of which encodes a portion of the complete immunotherapeutic compound and directs its synthesis. For example, the anti-HER2 light chain (e.g., SEQ ID NO: 17) can be encoded based on one plasmid, while the anti-HER2 heavy chain (e.g., SEQ ID NO: 18) can be encoded based on a second plasmid. When both plasmids are introduced into a host cell and expressed, the anti-HER2 light chain and the anti-HER2 heavy chain can dimerize to form an anti-HER2 Fab as the targeting moiety of the immunotherapeutic compound. The cam1615HER2 TriKE can be constructed in this way. For example, SEQ ID NO: 31 provides an amino acid sequence expressed from an exemplary first plasmid that contains a signal peptide, an anti-HER2 light chain, a linker, an IL-15 amino acid sequence, a second linker, and a camelid CD 16 single domain antibody fragment. SEQ ID NO: 32 provides an amino acid sequence expressed from an exemplary second plasmid that contains a signal peptide and an anti-HER2 heavy chain.

[0061] As another example, a single plasmid construct can include all components of the complete immunotherapeutic compound. For example, SEQ ID NO: 33 provides an amino acid sequence expressed from an exemplary single plasmid construct in which the amino acid sequence includes 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-CD 16 single domain antibody fragment. When expressed, the T2A peptide self-cleaves, separating the anti-HER2 heavy chain fragment from the rest of the immunotherapeutic compound, enabling it to dimerize with the anti-HER2 light chain fragment.

[0062] In the exemplary cam1615HER2 TriKE, the human IL-15 TriKE portion has the capacity to expand the molecule. Thus, the ability of the IL-15 portion in cam1615HER2 to affect NK expansion was determined. Figure 3 A and Figure 3 B shows that highly proliferative CD56 + CD3 - NK cells and the total percentage of NK cells. Both were significantly elevated after incubation with cam1615HER2. Likewise, exposure to cam1615HER2 also significantly increased the total NK count Figure 3 C). Figure 3 D-F show that CD3 + CD56- The percentage and number of T cells did not increase. In summary, these studies indicate that HER2 TriKE stimulates the expansion of NK cells rather than T cells. The data also suggest that the IL-15 portion of TriKE is functional and in a viable conformational arrangement.

[0063] Cytotoxicity is a hallmark of NK cell immunotherapy. To determine the efficacy of antibody-dependent cytotoxicity (ADCC), CD107a expression in various cell lines was analyzed, a recognized measure of NK cell cytotoxicity. cam1615HER2 TriKE was tested against SKOV3 and SK-BR-3 because ERBB2 is known to be overexpressed in breast cancer and some ovarian cancer cases, making it a desirable target for antibody-directed targeting. The UMSCC-11B cell line was used as a negative control because it expressed minimal HER2. Binding was measured by first labeling various reagents with FITC and then testing their direct binding to the targets by flow cytometry. SKOV3 (Figure 4A) and SK-BR-3 (Figure 4A) were tested. Figure 4B The highest level of HER2 TriKE binding was observed. UMSCC-11B showed a lower level of binding. Figure 4C ).

[0064] When ADCC activity was measured, cam1615HER2 TriKE showed highly elevated CD107a activity against SKOV3 and SK-BR-3 cell lines compared to IL-15 and untreated controls (Figs. 5A, 5C). When ADCC was tested on the UMSCC-11B cell line, cam1615HER2 TriKE showed almost no effect. Figure 5E Furthermore, when treated with cam1615HER2 TriKE, SKOV-3 (Fig. 5B) and SK-BR-3 (Fig. 5D) cam1615HER2 showed increased IFN-γ activation.

[0065] The cam1615HER2 TriKE assay was further tested against the breast cancer cell line MCF-7L. Figure 6A shows the background of CD107a effector cells without the addition of target cells. Figure 6BThe image shows CD107a activity upon addition of the target. The highest level of killing was observed with cam1615HER2 TriKE compared to the control. Cam16 itself does exhibit activity, but not at such a high level. The MCF-7L subline (MCF-7L-TamR) is tamoxifen-resistant and showed a similar pattern of CD107a activity upon treatment with cam1615HER2 TriKE (Fig. 6C). NK cells are known to secrete anti-cancer cancer factors, such as IFN-γ, when activated for killing. Fig. 6E shows that cam1615HER2 TriKE increased IFN-γ levels quantified by intracellular staining in the same sample, and that it was minimally affected in the control, indicating NK cell activation. For MCF-7L-TamR ( Figure 6F The same applies. Figure 6D The study confirmed that cam1615HER2 TriKE killed SK-BR-3 cells and showed that it had the same killing power as trastuzumab. Figure 6H The results showed that cam1615HER2 TriKE exhibited even greater IFN-γ enhancing activity than trastuzumab. In summary, these data demonstrate that HER2 is an effective target for immune binding agents on human breast cancer cells, and that innate immunotherapy is highly effective against drug-resistant breast cancer cell lines in vitro.

[0066] Figure 12 Data are provided showing the activity of cam1615HER2 TriKE against OVCAR-3 and OVCAR-5 (two other cell lines expressing HER2). Similarly, compared to the control, cam1615HER2 TriKE showed increased CD107a activity (…). Figure 12 A, 12B) and IFN-γ activity ( Figure 12 D, 12E). Repeated experiments evaluating SKOV3 included a wider number of negative controls, including anti-HER2 scFv alone (e23), cam16 VHH alone, IL-15 alone, and no treatment control. Similarly, compared to controls, the drug showed elevated CD107a activity ( Figure 12 C) and increased IFN-γ activity ( Figure 12 F).

[0067] Further real-time assessment of killing was performed over two days using the INCUCYTE ZOOM platform (Essen Bioscience, Inc., Ann Arbor, MI). SKOV3, grown as spheroids in culture, were investigated. SKOV3 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 no treatment. Caspase 3 / 7 green reagent was added to detect cell death. Dead cells turn green and dead NUCLIGHT RED Raji cells turn yellow, which allows tracking of remaining live cells. When compiling data, cam1615HER2 TriKE induced a significant decrease in SKOV3 spheroid size Figure 7A ) and spheroid intensity Figure 7B ) over 72 hours of continuous measurement compared to IL-15, cam16 VHH alone, and untreated controls. Figure 8 The provided images show that cam1615HER2 TriKE causes a sharp time-dependent decrease in target cells measured over a 72 hour time period. The results of this direct killing assay correlate with the results of the CD 107a assay.

[0068] To determine if NK cells from consenting cancer patients can function in the assay, NK cells from 6 consenting ovarian cancer patients, but not normal volunteers, were obtained and tested against MA-148 ovarian cancer cells. Figure 9A shows that the CD 107a background after treating effectors in the absence of cancer cells was lower than with cam1615HER2 TriKE or controls. Figure 9B Figure 9A shows that the CD 107a background after treating effectors in the absence of cancer cells was lower than with cam1615HER2 TriKE or controls. Figure 9B shows that CD 107a expression in effectors plus tumor targets was significantly elevated after treatment with cam1615HER2 TriKE compared to untreated controls. Regarding IFN-γ activity, Figure 9E shows that activity in effectors in the absence of targets was low when treated with cam1615HER2 TriKE. When effector cells were added, cam1615HER2 TriKE showed enhanced IFN-γ activity Figure 9F ) in effectors plus tumor targets. Figure 9C shows the CD 107a background and Figure 9D Figure 9G shows the IFN-γ background and Figure 9HIFN-γ activity of normal donor cells with MA148 cancer cells is shown. Although the trend is similar when normal or patient NK cells are mixed with cancer cells, the activity of normal cells is slightly higher. Nonetheless, effector cells from patients are still able to perform TriKE stimulation.

[0069] Figure 10 Data showing in vivo efficacy of cam1615 HER2 TriKE using the SCID / hu / NK xenograft model is shown. Figure 10 A and 10B show visual imaging data of untreated (A) and treated (B) groups of mice approximately 6 weeks after intraperitoneal inoculation of SKOV3 tumor cells. Advanced tumor progression is evident in the untreated group of mice (A) and is significantly reduced in the treated group of mice (B). Figure 10 A) and treated (B) groups of mice. Advanced tumor progression is evident in the untreated group of mice (A) and is significantly reduced in the treated group of mice (B). Figure 10 A) and treated (B) groups of mice. Advanced tumor progression is evident in the untreated group of mice (A) and is significantly reduced in the treated group of mice (B). Figure 10 A) and treated (B) groups of mice. Advanced tumor progression is evident in the untreated group of mice (A) and is significantly reduced in the treated group of mice (B). Figure 10 A) and treated (B) groups of mice. Advanced tumor progression is evident in the untreated group of mice (A) and is significantly reduced in the treated group of mice (B). A) and treated (B) groups of mice. Advanced tumor progression is evident in the untreated group of mice (A) and is significantly reduced in the treated group of mice (B).

[0070] A) and treated (B) groups of mice. Advanced tumor progression is evident in the untreated group of mice (A) and is significantly reduced in the treated group of mice (B). Figure 11A A) and treated (B) groups of mice. Advanced tumor progression is evident in the untreated group of mice (A) and is significantly reduced in the treated group of mice (B). Figure 11B A) and treated (B) groups of mice. Advanced tumor progression is evident in the untreated group of mice (A) and is significantly reduced in the treated group of mice (B). Figure 11C A) and treated (B) groups of mice. Advanced tumor progression is evident in the untreated group of mice (A) and is significantly reduced in the treated group of mice (B). Figure 11D A) and treated (B) groups of mice. Advanced tumor progression is evident in the untreated group of mice (A) and is significantly reduced in the treated group of mice (B). A) and treated (B) groups of mice. Advanced tumor progression is evident in the untreated group of mice (A) and is significantly reduced in the treated group of mice (B).

[0071] Accordingly, the data presented herein demonstrate that HER2 can serve as an immunotherapeutic target for ovarian and breast cancer when targeted by an immunotherapeutic compound, such as a TriKE compound. Exemplary immunotherapeutic compounds are effective against tamoxifen refractory cells and thus can readily kill cancer cells that are resistant to tamoxifen or other chemotherapeutic agents. The present disclosure further demonstrates that exemplary immunotherapeutic compounds (HER2-targeted NK engaging TriKE) can inhibit cancer in vivo in an intraperitoneal ovarian cancer xenograft model, a model that requires both human NK cells and cancer cells to be transplanted in a xenomouse. The data presented herein further provide a basis for immunotherapeutic compounds that target HER3 and / or HER2 / HER3 heterodimers.

[0072] The immunotherapeutic compounds described herein can be formulated with a pharmaceutically acceptable carrier. As used herein, "carrier" includes any solvent, dispersion medium, vehicle, coating, diluent, antibacterial and / or antifungal agent, isotonic agent, absorption delaying agent, buffer, carrier solution, suspension, colloid, and the like. The use of such media and / or agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions. As used herein, "pharmaceutically acceptable" means that the material is not biologically or otherwise undesirable, i.e., the material can be administered to an individual along with the immunotherapeutic compound without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained.

[0073] The immunotherapeutic compounds can thus be formulated into pharmaceutical compositions. Pharmaceutical compositions can be formulated in a wide variety of dosage forms. Thus, the compositions can be given by known routes, including, e.g., orally, parenterally (e.g., intradermally, transdermally, subcutaneously, intramuscularly, intravenously, intraperitoneally, etc.) or topically (e.g., intranasally, intrapulmonarily, intramastoidally, intravaginally, intrauterinely, intradermally, transdermally, rectally, etc.). Pharmaceutical compositions can be administered to mucosal surfaces, such as by administration to, e.g., 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.

[0074] Thus, the immunotherapeutic compound can be provided in any suitable form, including but not limited to solutions, suspensions, emulsions, sprays, aerosols, or any form of admixture. The composition can be delivered in a formulation with any pharmaceutically acceptable excipient, carrier, or vehicle. For example, the formulation can be delivered in a conventional topical dosage form, such as a cream, ointment, aerosol formulation, non-aerosol spray, gel, lotion, and the like. The formulation can further include one or more additives, including, for example, adjuvants, skin penetration enhancers, colorants, fragrances, flavorings, humectants, thickening agents, and the like. In certain embodiments, the composition can be formulated as a solution or suspension.

[0075] The formulations can conveniently be presented in unit dosage form and can be prepared by methods well known in the art of pharmacy. Methods of preparing compositions with pharmaceutically acceptable carriers include bringing into association the immunotherapeutic compound with the carrier and / or one or more accessory ingredients. In general, the formulation can be prepared by uniformly and / or intimately bringing into association the active molecule with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product into the desired formulation.

[0076] Thus, in another aspect, the present disclosure describes methods of treating cancer in a subject. Generally, the methods include administering to the subject an immunotherapeutic compound in an amount effective to treat the cancer. "Treating" or variants thereof mean reducing to any extent the symptoms or signs associated with a disorder, limiting progression of it, ameliorating or effecting an improvement in the symptoms or signs associated with a disorder. As used herein, "ameliorating" means any decrease in the extent, severity, frequency, and / or likelihood of symptoms or clinical signs characteristic of a particular disorder; "symptom" means any subjective evidence of disease or patient condition; and "sign" or "clinical sign" means an objective physical finding associated with a particular condition that can be discovered by someone other than the patient.

[0077] "Treatment" can be therapeutic or prophylactic. "Therapeutic," and variants thereof, refers to a treatment that ameliorates one or more existing symptoms or clinical signs associated with a disorder. "Prophylactic," and variants thereof, refers to a treatment that limits the development and / or onset of symptoms or clinical signs of a disorder to any extent. Generally, a "therapeutic" treatment is initiated after a subject exhibits a disorder, while a "prophylactic" treatment is initiated before a subject exhibits a disorder. Thus, in certain embodiments, the methods can involve prophylactic treatment of a subject at risk for developing a disorder. "At risk" refers to a subject who actually can or can not have the risk. Thus, for example, a subject "at risk" of developing a particular disorder is a subject who has one or more indicators of an increased risk of having or developing the particular disorder (as compared to an individual lacking the indicator(s)), regardless of whether the subject exhibits any symptoms or clinical signs of having or developing the disorder. Exemplary indicators of a disorder can include, for example, genetic predisposition, lineage, age, sex, geographic location, lifestyle, or medical history. Treatment can also continue after symptoms have resolved, e.g., to prevent or delay their recurrence.

[0078] Thus, an immunotherapeutic compound can be administered to a subject before, during, or after the subject first exhibits a symptom or clinical sign of a disorder. Treatment initiated before a subject first exhibits a symptom or clinical sign associated with a disorder can result in the subject experiencing a reduced likelihood of developing clinical evidence of the disorder, reducing the severity of symptoms and / or clinical signs of the disorder, and / or completely resolving the disorder, as compared to a subject not administered the immunotherapeutic compound. Treatment initiated after a subject first exhibits a symptom or clinical sign associated with a disorder can result in reducing the severity of symptoms and / or clinical signs of the disorder, and / or completely resolving the disorder, as compared to a subject not administered the immunotherapeutic compound.

[0079] The amount of an immunotherapeutic compound administered can vary according to a variety of factors, including, but not limited to, the particular immunotherapeutic compound being administered, the subject's body weight, physical condition and / or age, and / or the route of administration. Thus, the absolute weight of immunotherapeutic compound included in a given unit dose can vary widely, depending on such factors as the species, age, body weight and physical condition of the subject, and / or the method of administration. It is understood, therefore, that the amount of an immunotherapeutic compound constituting an effective amount of the compound for any particular application will not always be the same. However, the appropriate amount can be readily determined by one of ordinary skill in the art, given the teachings provided herein.

[0080] In some embodiments, the methods can comprise administering sufficient immunotherapeutic compound to provide a dose of, e.g., about 100 ng / kg / day to about 10 mg / kg / day to the subject, although in some embodiments, the methods can be carried out by administering an immunotherapeutic compound at a dose outside this range.

[0081] In some embodiments, the methods can comprise administering sufficient immunotherapeutic compound to provide a minimum dose of at least 100 ng / kg / day, such as 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.

[0082] In some embodiments, the methods comprise administering sufficient immunotherapeutic compound to provide a maximum dose of no more than 10 mg / kg / day, such as no more than 5 mg / kg / day, no more than 4 mg / kg / day, no more than 3 mg / kg / day, no more than 2 mg / kg / day, no more than 1 mg / kg / day, no more than 900 μg / kg / day, no more than 800 μg / kg / day, no more than 700 μg / kg / day, no more than 600 μg / kg / day, no more than 500 μg / kg / day, no more than 400 μg / kg / day, no more than 300 μg / kg / day, no more than 200 μg / kg / day, no more than 100 μg / kg / day, no more than 90 μg / kg / day, no more than 80 μg / kg / day, no more than 70 μg / kg / day, no more than 60 μg / kg / day, no more than 50 μg / kg / day, no more than 40 μg / kg / day, no more than 30 μg / kg / day, no more than 20 μg / kg / day, or no more than 10 μg / kg / day. An immunotherapeutic compound provides a dose of "no more than" a specified amount when the immunotherapeutic compound is present in an amount up to and including the specified amount.

[0083] In some embodiments, the methods comprise administering sufficient immunotherapeutic compound to provide a dose characterized by a range having endpoints defined by any of the minimum doses and any of the maximum doses greater than the selected minimum dose determined above. For example, in some embodiments, the methods can comprise administering sufficient immunotherapeutic compound to provide 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 5 μg / kg / day to 100 μg / kg / day, etc. to the subject.

[0084] In certain embodiments, the methods comprise administering sufficient immunotherapeutic compound to provide a dosage equal to any of the minimum dosages or any of the maximum dosages listed above. Thus, for example, in certain embodiments, the methods can comprise administering sufficient immunotherapeutic compound to provide a dosage 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.

[0085] In some embodiments, the immunotherapeutic compound can be administered, for example, in a single dose to multiple doses per week, although in some embodiments, the methods 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 above dosages, described in terms of the amount of immunotherapeutic compound administered over a 24 hour period, are administered in 7 day cycles of 4 days of treatment and 3 days of rest.

[0086] In some embodiments, the immunotherapeutic compound can be administered, for example, in a single dose to multiple cycles of treatment, although in some embodiments, the methods can be carried out by administering the immunotherapeutic compound for a duration outside this range. In some embodiments, the immunotherapeutic compound can be administered for 3 weeks. In such embodiments, each week can be a treatment cycle, such as the exemplary treatment cycles described in the preceding paragraph. In other embodiments, the immunotherapeutic compound can be administered for a greater number of treatment cycles, with no interval between one set of treatment cycles and a subsequent set of treatment cycles. The interval between one set of treatment cycles and a subsequent set of treatment cycles can be an interval of one or more weeks, one or more months, or one or more years.

[0087] In some embodiments, the methods further comprise administering one or more additional therapeutic agents. The one or more additional therapeutic agents (e.g., chemotherapeutic agents) can be administered prior to, after, and / or concurrently with the immunotherapeutic compound. The immunotherapeutic compound and additional therapeutic agent(s) can be co-administered. As used herein, “co-administered” means that the two or more components of the combination are administered such that the therapeutic or prophylactic effect of the combination can be greater than the therapeutic or prophylactic effect of either component administered alone. The two components can be co-administered simultaneously or sequentially. The components that are co-administered simultaneously can be provided in one or more pharmaceutical compositions. Sequential co-administration of two or more components includes situations in which the components are administered such that each component can be present at the treatment site at the same time. Alternatively, sequential co-administration of two components can include situations in which at least one component has been cleared from the treatment site but at least one cellular effect of the administration of the components (e.g., cytokine production, activation of a certain cell population, etc.) persists at the treatment site until one or more additional components are administered to the treatment site. Thus, in some cases, a co-administered combination can include components that are never present together in a chemical mixture. In other embodiments, the immunotherapeutic compound and additional therapeutic agent(s) can be administered as part of a mixture or cocktail. In some aspects, administration of the immunotherapeutic compound can allow for a lower dosage of the other therapeutic modality to be effective when compared to administration of the one or more other therapeutic agents alone, thereby reducing the likelihood, severity, and / or extent of toxicity observed when higher dosages of the one or more other therapeutic agents are administered.

[0088] Exemplary additional therapeutic agents include hexamethylmelamine, amsacrine, L- asparaginase, L-colaspase, 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, and vinorelbine, anti-HER2 antibody therapy (see, e.g., Liu et al., 2019, Biol Proced Online 21:5) or anti-HER2 / HER3 heterodimeric complex antibody therapy (see, e.g., Liu et al., 2019, BiolProced Online 21:5).

[0089] In some embodiments, the methods can include administration of an immunotherapeutic compound as described herein and administration of at least one additional therapeutic agent to show therapeutic synergy. In some aspects of the methods of the application, a measure of response to treatment observed after administration of both an immunotherapeutic compound as described herein and an additional therapeutic agent is improved over the same measure of response to treatment observed after administration of either the immunotherapeutic compound or the additional therapeutic agent alone. In some embodiments, the additional therapeutic agent can include an additional drug targeting EpCAM, including, for example, EpCAM-specific monoclonal antibodies, such as the monoclonal hybrid antibody catumaxomab that targets EpCAM and CD3.

[0090] In some embodiments, administration of an immunotherapeutic compound to a subject can stimulate endogenous NK cells in vivo. Use of an immunotherapeutic compound as part of an in vivo method can serve to simultaneously co-stimulate NK cells to be antigen-specific, to enhance survival and expansion of NK cells. In other cases, an immunotherapeutic compound can be used in vitro as an adjuvant for NK cell adoptive transfer therapy.

[0091] In the foregoing description and in the following claims, the term "and / or" means one or all of the listed elements or combination of any two or more of the listed elements; the term "comprising," including variations thereof, should be interpreted as encompassing the elements or steps that follow it as options that can or can not be present, unless otherwise specified; "a," "an," and "the" are used interchangeably and mean one or more, unless otherwise specified; and recitation of numerical ranges by endpoints includes all numbers subsumed

[0092] In the foregoing description, for the purposes of clarity, specific embodiments are described. Certain embodiments can include combinations of compatible features described herein in connection with one or more embodiments, unless the features are incompatible with each other as explicitly specified otherwise.

[0093] For any method disclosed herein that includes discrete steps, the steps can be conducted in any feasible order. And, depending on the context, any combination of two or more steps can be conducted simultaneously.

[0094] The application is illustrated by the following examples. It is to be understood that the particular examples, materials, amounts, and procedures are to be interpreted broadly and are meant only to illustrate the scope and spirit of the application as described herein. Example

[0095] Construction of cam1615HER TriKE

[0096] DNA fragments encoding CDR regions from a camelized anti-CD16 (Vincke et al., 2009, J. Biol. Chem. 284(5):3273-3284) were spliced into a generic, humanized nanobody scaffold that was previously shown to allow grafting of antigen binding loops and transfer of antigen specificity and affinity (Behar et al., 2008, Protein Engineering, Design & Selection 21(1): 1-10). This new sequence was used to make cam1615HER2 (SEQ ID NO: 1). The fully assembled hybrid gene encoding cam1615HER2 TriKE cam1615HER2 encoding (from 5' end to 3' end) a Ncol restriction site, an ATG start codon, an anti-human CD16 VHH, a 20 amino acid (aa) fragment 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 a Xhol restriction site. The resulting hybrid gene (SEQ ID NO: 7) was spliced into a 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. Wild-type human IL-15 was used instead of a mutant form of the cytokine. The gene sequence and in-frame accuracy of the target gene was verified by the Biomedical Genomics Center, University of Minnesota, St. Paul, MN.

[0097] Purification of protein from inclusion bodies

[0098] E. coli strain BL21 (DE3) (Novagen, Madison, WI) was used for protein expression after plasmid transfection. Bacteria were grown overnight in 800-ml Luria broth containing 50 μg / ml kanamycin. When the culture reached an absorbance of 0.65 at 600 nm, expression was induced by the addition of IPTG (Thermo Fisher Scientific, Inc., FairLawn, NJ). Bacterial expression resulted in the target protein being packaged within inclusion bodies. After expression, bacteria were harvested and then homogenized in a buffer (50 mM Tris, 50 mM NaCl and 5 mM EDTA pH 8.0) and the pellet was sonicated and centrifuged. To extract the protein from the pellet, 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) was used and the extract was washed 3 times.

[0099] Proteins from inclusion bodies require refolding. Therefore, a modified version of a previously described method (Vallera et al., 2005, Leuk Res29(3):331-341). Briefly, inclusion bodies were solubilized in 100 mM Tris, 2.5% SLS (Sigma-Aldrich, St. Louis, MO). The precipitate was removed by centrifugation. To the solution, 50 mM CuSO4 was added and then incubated at room temperature for 20 hours with rapid stirring for 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. Next, 13.3 M guanidine-HCl was added to the protein solution, which was then incubated at 37 °C for 2-3 hours. The solution was diluted 20-fold with a 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 two days. To remove the buffer, the sample was dialyzed against 5 volumes of 20 mM Tris-HCl at pH 8.0 at 4 °C for 48 hours and then against 8 volumes for an additional 18 hours. The product was then purified first by fast flow Q ion exchange chromatography and then by 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 protein size and purity.

[0100] Cancer cell lines

[0101] The following cell lines were obtained from the American Type Culture Collection (Manassas, VA): MCF-7L (breast ductal carcinoma), MCF-7L-TamR (tamoxifen-resistant subline of MCF-7), SKOV3 (ovarian ascites), SK-BR-3 (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 Surg132:668-677), HL-60 (acute promyelocytic leukemia), MA-148 (ovarian cancer), and SKOV3-luc. For in vivo experiments, SKOV3-luc was made by transfecting SKOV3 with a luciferase reporter construct using Lipofectamine (Invitrogen, Carlsbad, CA) and selective pressure with 10 pg / ml blastocidin. UMSCC-11B was authenticated by STR testing by the Fragment Analysis Facility, John Hopkins University. MA148 (established locally at the University of Minnesota) is a human epithelial ovarian cancer cell line. Cell lines were maintained in RPMI 1640 RPMI supplemented with 10-20% fetal bovine serum (FBS) and 2 mmol / L L-glutamine. Cell lines were incubated at a constant 37 °C in a humidified environment containing 5% CO2. When adherent cells were greater than 90% confluent, they were passaged using trypsin-EDTA for detachment. For cell counts, a standard hemocytometer was used. Only those cells with a viability > 95% as determined by trypan blue exclusion were used for experiments.

[0102] Evaluation of cytotoxicity and NK cell activation

[0103] Antibody-dependent cellular cytotoxicity (ADCC) was measured using CD 107a (lysosome-associated membrane protein LAMP-1) flow cytometry assay. For effector cells, PBMC were obtained from normal volunteers after written informed consent and Institutional Review Board approval. Cancer target cells were grown 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 after Institutional Review Board approval. All samples were collected from women diagnosed with advanced ovarian cancer or primary peritoneal cancer at the time of initial debulking surgery. Cell pellets were lysed to remove red blood cells, cryopreserved in 10% DMSO / 90% FBS and stored in liquid nitrogen.

[0104] PBMCs were incubated overnight (37°C, 5% CO2) in RPMI 1640 medium supplemented with 10% fetal bovine serum (RPMI-10) and suspended with tumor target cells or medium after washing three times with RPMI-10. Cells were then incubated with TriKE or controls for 10 minutes at 37°C. Then fluorescein isothiocyanate (FITC)-conjugated anti-human CD 107a monoclonal antibody (BD Biosciences, San Jose, CA) was added and incubated for 1 hour. After 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 (37°C, 5% CO2). After washing with phosphate-buffered saline, cells were stained with PE / Cy7-conjugated anti-CD56 mAb, APC / Cy7-conjugated anti-CD 16 mAb, and PE-CF594-conjugated anti-CD3 mAb (BioLegend, San Diego, CA). Cells were incubated for 15 minutes at 4°C, washed, and fixed with 2% paraformaldehyde.

[0105] 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 minutes. Cells were finally washed and evaluated for CD56 + CD3 - Cells were gated and evaluated by fluorescence-activated cell sorting analysis.

[0106] Cytotoxicity potency was additionally measured in real time. CD56 + CD3 -NK effector cells were plated with red labeled tumor cells into 96-well clear bottom polystyrene tissue-cultured treated microplates (Corning, Flintshire, UK) and the plate was transferred to an INCUCYTE ZOOM platform (Essen Bioscience, Inc., Ann Arbor, MI) that was housed in a cell incubator at 37°C / 5% CO2. Images from 3 technical replicates were taken every 15 minutes using a 4x objective for 48 hours and then analyzed using IncuCyte Basic Software (Essen Bioscience, Inc., Ann Arbor, MI).

[0107] Expansion of NK cells upon IL-15 stimulation

[0108] To measure TriKE efficacy dependent on its functional IL-15 portion, PBMCs or enriched NK cells from healthy donors were labeled with CELLTRACE violet proliferation dye (Invitrogen, Carlsbad, CA) according to the kit instructions. After staining, effector cells were cultured with 50 nM TriKE or control and incubated at 37°C in a humidified atmosphere containing 5% CO2for 7 days. Cells were harvested, stained for viability with Live / Dead reagent (Invitrogen, Carlsbad, CA, USA) and surface stained for anti-CD56 PE / Cy7 (Biolegend, San Diego, CA) and anti-CD3 PE-CF594 (BD Biosciences, Franklin Lakes, NJ) to gate live CD3 - CD56 + NK cell population. Data analysis was performed using FlowJo software (Flowjo enterprise LCC, version 7.6.5, Ashland, OR).

[0109] In vivo studies and imaging in mice

[0110] Efficacy of HER2 TriKE was tested in a scid / hu mouse model previously reported (Vallera et al., 2016, Clin Cancer Res 22(14):3440-3450) but modified for growth of human ovarian carcinoma cell line SCOV3. Cell line was transfected with luciferase reporter to allow real-time monitoring of tumor progression by bioluminescent imaging. NSG mice (NOD.Cg-Prkdc scid Il2rgtm1Wjl SzJ, n = 5 / group) were injected intraperitoneally with 2 x 10 5 SzJ, n = 5 / group) were injected intraperitoneally with 2 x 10

[0111] The complete disclosure of all patents, patent applications, and publications cited herein are incorporated by reference for all purposes as if each had been individually incorporated by reference. To the extent there is any inconsistency between the disclosure of the present application and the disclosure of any document incorporated by reference herein, the disclosure of the present application shall prevail. The foregoing detailed description and examples have been given for clarity of understanding only. No unnecessary limitations are to be understood therefrom. The application is not limited to the exact details shown and described, for variations obvious to one skilled in the art will be included within the application defined by the claims.

[0112] Unless otherwise indicated, all numbers expressing quantities of components, molecular weight, etc. in the specification and claims are to be understood as being modified in all instances by the term "about." Accordingly, unless otherwise indicated, the numerical parameters listed in the specification and claims are approximations. At the very least, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Furthermore, unless otherwise indicated, all measurements are understood to be made at 25 °C and at an atmosphere of one atmosphere pressure.

[0113] Notwithstanding that the numerical ranges and parameters setting forth the broadest scope of the application are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0114] All headings are for the convenience of the reader and are not to be used in the interpretation of the meaning of the text under the headings.

[0115] SEQUENCE LIST FREE TEXT

[0116] SEQ ID NO: 1 - cam1615HER2 amino acid sequence:

[0117]

[0118] SEQ ID NO: 2 - cam16 (amino acids 3-124 of SEQ ID NO: 1)

[0119]

[0120] SEQ ID NO: 3 - hma linker (amino acids 125-144 of SEQ ID NO: 1)

[0121] PSGQAGAAAS ESLFVSNHAY

[0122] SEQ ID NO: 4 - human IL-15 (amino acids 145-258 of SEQ ID NO: 1)

[0123]

[0124] SEQ ID NO: 5 - linker (amino acids 259-265 of SEQ ID NO: 1)

[0125] EASGGPE

[0126] SEQ ID NO: 6 - anti-HER2 (amino acids 266-501 of SEQ ID NO: 1)

[0127]

[0128] SEQ ID NO: 7 - cam1615HER2 DNA sequence

[0129]

[0130] SEQ ID NO: 8 - Ncol restriction site and start codon (1-8 of SEQ ID NO: 7)

[0131] CCATGGAG

[0132] SEQ ID NO: 9 - cam16 (9-374 of SEQ ID NO: 7)

[0133]

[0134] SEQ ID NO: 10 - hma linker (375-434 of SEQ ID NO: 7)

[0135] ccgtctggtc aggctggtgc tgctgctagc gaatctctgt tcgtttctaa ccacgcttac

[0136] SEQ ID NO: 11 - human IL-15 (435-776 of SEQ ID NO: 7)

[0137]

[0138] SEQ ID NO: 12 - linker (777-797 of SEQ ID NO: 7)

[0139] gaagcttccg gaggtcccga g

[0140] SEQ ID NO: 13 - anti-HER2 (798-1505 of SEQ ID NO: 7)

[0141]

[0142] SEQ ID NO: 14 - two stop codons and Xhol restriction site (1506-1517 of SEQ ID NO: 7)

[0143] taatagctcg aga

[0144] SEQ ID NO: 15 - e23 anti-HER2 amino acid sequence

[0145]

[0146] SEQ ID NO: 16 - trastuzumab-based scFv

[0147]

[0148] SEQ ID NO: 17 - anti-HER2 light chain

[0149]

[0150] SEQ ID NO: 18 - Anti-HER2 heavy chain

[0151]

[0152] SEQ ID NO: 19 - Margetuximab heavy chain

[0153]

[0154] SEQ ID NO: 20 - Margetuximab light chain

[0155]

[0156] SEQ ID NO: 21 - Seribantumab heavy chain

[0157]

[0158] SEQ ID NO: 22 - Seribantumab light chain

[0159]

[0160] SEQ ID NO: 23 - KTN3379 light chain

[0161]

[0162] SEQ ID NO: 24 - Pertuzumab subunit 1

[0163]

[0164] SEQ ID NO: 25 - Pertuzumab subunit 2

[0165]

[0166] SEQ ID NO: 26 - Pertuzumab subunit 3

[0167]

[0168] SEQ ID NO: 27 - Pertuzumab subunit 4

[0169]

[0170] SEQ ID NO: 28 - Trastuzumab-based TriKE amino acid sequence

[0171]

[0172] SEQ ID NO: 29 - TriKE DNA sequence based on trastuzumab - human codon optimized

[0173]

[0174] SEQ ID NO: 30 - TriKE DNA sequence based on trastuzumab - E. coli codon optimized

[0175]

[0176] SEQ ID NO: 31 - Signal peptide / anti-HER2 light chain / linker / wtIL15 / linker / cam16

[0177]

[0178] SEQ ID NO: 32 - Signal peptide / anti-HER2 heavy chain

[0179]

[0180] SEQ ID NO: 33 - Signal peptide / anti-HER2 heavy chain Fab / T2A / signal peptide / anti- HER2 light chain / linker / wtIL15 / linker / cam16

[0181]

[0182] SEQ ID NO: 34 - Linker

[0183] SGGGGSGGGG SGGGGSGGGG SG

[0184] SEQ ID NO: 35 - Linker

[0185] GSTSGSGKPG SGEGSTKG

[0186] SEQ ID NO: 36 - Signal peptide

[0187] MGWSCIILFL VATATGVHS

[0188] SEQ ID NO: 37 - T2A self-cleaving peptide

[0189] EGRGSLLTCG DVEENPGP.

Claims

1. A compound consisting of: a moiety that selectively binds to CD 16 and an NK cell engaging domain as set forth in the amino acid sequence of SEQ ID NO: 2 or amino acids 1-124 of SEQ ID NO: 1; a first flanking sequence located C-terminal to the NK cell engaging domain; an NK activating domain operably linked to the NK cell engaging domain that is IL-15 or a functional variant of IL-15, wherein the IL-15 is as set forth in the amino acid sequence of SEQ ID NO: 4 and the functional variant of IL-15 is as set forth in an amino acid sequence that differs from SEQ ID NO: 4 by an N72D or N72A amino acid substitution; a second flanking sequence located C-terminal to the NK activating domain; and a targeting domain that selectively binds to HER2 and is as set forth in the amino acid sequence of SEQ ID NO:

6.

2. The compound of claim 1, wherein the CD 16 comprises CD16a.

3. The compound of claim 1, wherein the compound consists of the amino acid sequence of SEQ ID NO:

1.

4. The compound of claim 1, wherein the NK activating domain is a functional variant of IL-15, which is as set forth in an amino acid sequence that differs from SEQ ID NO: 4 by an N72D or N72A amino acid substitution.

5. A composition comprising: the compound of any one of claims 1-4; and a pharmaceutically acceptable carrier.

6. The composition of claim 5, further comprising an additional therapeutic agent.

7. The composition of claim 6, wherein the additional therapeutic agent comprises a therapeutic agent that targets HER2.

8. Use of the compound of any one of claims 1-4 in the manufacture of a medicament for treating breast cancer or ovarian cancer in a subject.

9. The use of claim 8, the treatment further comprising administering the compound prior to, concurrently with, or following chemotherapy, surgical resection of a tumor, or radiotherapy.

10. The use of claim 9, wherein the chemotherapy comprises hexamethylmelamine, amsacrine, L-asparaginase, L-asparaginase, bleomycin, busulfan, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladrabine, cyclophosphamide, cyclophosphamide, 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.

11. Use of the composition of any one of claims 5-7 in the manufacture of a medicament for treating breast cancer or ovarian cancer in a subject.

12. The use of claim 11, further comprising administering the composition prior to, concurrent with, or subsequent to chemotherapy, immunotherapy, surgical resection of a tumor, or radiation therapy.

13. The use of claim 12, wherein the chemotherapy comprises hexamethylmelamine, amsacrine, L-asparaginase, L-asparaginase, bleomycin, busulfan, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladrabine, cyclophosphamide, cyclophosphamide, 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.

14. The use of claim 12, wherein the immunotherapy targets HER2.

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