Immunotherapy Compounds and Methods

Abstract

The immunotherapy compound comprises an NK cell binding domain, an NK activation domain, and a targeting domain. The targeting domain selectively binds to HER2, HER3, or HER2 / HER3 heterodimer complexes and operably links to the NK activation domain and the NK cell binding domain. The compound may be administered to a subject for the induction of NK-mediated apoptosis of cancer cells, stimulation of NK cell expansion in the subject, and / or cancer treatment.

Description

Technology Field

[0001] Cross-reference regarding related applications

[0002] This application claims priority to U.S. provisional patent application No. 62 / 901,198, filed September 16, 2019, the entirety of which is incorporated herein by reference.

[0003] Government support

[0004] This invention was made with government support under CA197292 granted by the National Institutes of Health. The government holds specific rights to this invention.

[0005] Sequence list

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

[65535] US 2018 / 0282386 A1US 2014 / 0072581 A1

[0007] outline

[0008] In one aspect, the present disclosure describes a multispecific immunotherapeutic compound comprising an NK cell binding domain, an NK activation 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 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 comprises the amino acid sequence of SEQ ID NO: 2.

[0010] In some embodiments, the NK cell binding domain moiety may comprise an antibody or its binding fragment. In some of these embodiments, the antibody or its binding fragment may be human, humanized, or camel.

[0011] 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.

[0012] In some embodiments, the targeting domain comprises an antibody or a binding fragment thereof. In some of these embodiments, the antibody binding fragment may comprise a 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.

[0013] In some embodiments, the immunotherapy compound may include a second targeting domain.

[0014] In some embodiments, the immunotherapy compound may include a second NK cell binding domain.

[0015] In some embodiments, the immunotherapy compound may include a second NK activation domain.

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

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

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

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

[0020] In another aspect, the present disclosure describes a method for treating cancer in a subject. Generally, the method comprises administering an effective amount of any embodiment of the composition or compound summarized above to the subject for treating cancer.

[0021] In some embodiments, the composition or compound is administered before, simultaneously with, or after chemotherapy, surgical resection of a tumor, or radiation therapy. In some of these embodiments, the chemotherapy includes altretamine, amsacrin, L-asparaginase, colaspase, bleomycin, busulfan, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, cyclophosphamide, cytophosphan, cytarabine, dacarbazine, dactinomycin, daunorubicin, docetaxel, doxorubicin, epirubicin, etoposide, fluorouracil, fludarabine, potemustine, ganciclovir, gemcitabine, hydroxyurea, idarubicin, ifosfamide, irinotecan, lomustine, melphalan, mercaptopurine, methotrexate, mitoxantrone, mitomycin C, nimustine, oxaliplatin, paclitaxel, pemetrexed, It may include procarbazine, raltitrexed, temozolomide, tenifoside, thioguanine, thiotepa, topotecan, vinblastine, vincristine, vindecin, or vinorelbine.

[0022] The foregoing overview is not intended to describe all embodiments of the invention or each disclosed embodiment. The following description more particularly illustrates exemplary embodiments. Throughout this application, guidance is provided through a list of examples that may be used in various combinations. In each case, the mentioned list serves merely as a representative group and should not be interpreted as an exclusive list. Brief explanation of the drawing

[0023] The patent or application file contains at least one drawing in color. A copy of the patent or patent application publication containing the color drawing will be provided by the Patent Office upon request and payment of the necessary fees. Fig. 1. Design, production, and purification of cam1615HER2. (A) Schematic diagram of an exemplary expression vector having an array of coding regions encoding components of cam1615HER2 (from left to right): Camelian anti-CD16 VHH, human IL-15, anti-HER2 scFV. (B) Gene map of an exemplary expression vector encoding cam1615HER2, including target gene sites and restriction sites on the pET28c vector. Fig. 2. Production and purification of cam1615HER2. (A) SDS-PAGE gel stained with Coomassie blue dye showing the size and purity of the final product after two orthogonal column steps. MWS: Molecular weight standard; NR: Unreduced; R: Reduced. Concentration measurements were performed to determine the final purity. (B) Chromatographic trace generated from the first-step purification of cam1615HER2 on an ion exchange (FFQ) column. The collected peak is indicated by a double arrow. (C) Chromatographic trace generated from the second-step purification of cam1615HER2 on a size exclusion column. The collected peak is indicated by a double arrow. Fig. 3. Effect of cam1615HER2 on NK cell expansion and CD56 undergoing proliferation measured by flow cytometry + CD3 -Absolute numbers showing the effect of TriKE treatment on the percentage of NK cells. PBMCs from six different normal donors were analyzed individually. In addition to cam1615HER2, IL-15 was used as a control. (A) Cam1615HER2 treatment shows that the percentage of highly proliferating NK cells was significantly different compared to the control. (B) Cam1615HER2 treatment shows that the percentage of total NK cells was significantly different compared to the control. (C) Cam1615HER2 treatment shows that the number of original NK counts was significantly different compared to the NT control. (D) Cam1615HER2 treatment shows highly proliferating CD3 + CD56 - It did not increase the percentage of T cells. (E) cam1615HER2 treatment total CD3 + CD56 - It did not increase the percentage of T cells. (F) cam1615HER2 treatment was original CD3 + CD56 - It did not increase the number of T cells. Fig. 4. cam1615HER2 TriKE binding to target cell lines. (A) FITC-directly labeled cam1615HER2 binding to SKOV-3 cell line. (B) FITC-directly labeled cam1615HER2 binding to SK-BR-3 cell line. (C) FITC-directly labeled cam1615HER2 binding to UMSCC-11B cell line. BAC3 is a CD3-binding molecule and functions as a negative control for binding. Fig. 5. Functional activity correlated with the binding activity of cam1615HER2 TriKE. (A) CD107a functional activity was elevated in cultures of cam1615HER2-treated PBMC plus SKOV3 cells compared to IL-15 and untreated controls. PBMCs from 10 different normal donors were analyzed individually. (B) Enhanced IFN-γ activity in the same PBMC / SKOV3 cultures. (C) CD107a functional activity was elevated in cultures of PBMC plus the breast cancer cell line SK-BR-3 (7 different donors were analyzed). (D) Enhanced IFN-γ activity in the same PBMC / SK-BR-3 cultures. (E) CD107a was not elevated when the UMSCC-11B head and neck cancer cell line was tested in the same analysis (4 different donors were analyzed). Fig. 6. Test of the ability of TriKE to enhance apoptosis in drug (tamoxifen)-resistant MCF-7L-TamR breast carcinoma cells. (A) CD56 cells without cancer cells + CD3 - The background cytotoxic activity of 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) CD56 cells without cancer cells + CD3 -Background intracellular IFN-γ activity of NK cells. (F) Intracellular IFN-γ activity using PBMCs incubated with parental MCF-7L cell lines. (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 was correlated with CD107a activity. Fig. 7. INCUCYTE (Essen Bioscience, Inc., Ann Arbor, Michigan) data measuring real-time apoptosis of SKOV3 ovarian cancer cells in the presence of PBMCs, demonstrating CD107a cytotoxicity data. (A) Globule size; (B) Globule intensity. cam1615HER2 induces a rapid decline in target cells measured over a 120-hour period compared to lower activity in untreated, anti-cam16 alone (CAM16), and IL-15 alone (IL-15) controls. N=7 donors / group. Fig. 8. Visual evidence that cam1615HER2 induces a rapid time-dependent decline in target cells (right column) measured over a 72-hour period compared to lower activity in untreated (left column), anti-cam16 alone, and IL-15 alone controls. N=7 donors / group. Fig. 9. Ascites fluid test from ovarian cancer patients as a source of effector cells. (A) CD107a background activity when cells from patient ascites fluid are incubated without MA-148 ovarian cancer cells. (B) CD107a activity when ascites fluid cells are incubated with MA-148 ovarian cancer cells. (C) CD107a background activity when cells from a normal donor are incubated without MA-148 ovarian cancer cells. (D) CD107a activity when normal donor cells are incubated with MA-148 ovarian cancer cells. (E) IFN-γ background activity when cells from patient ascites fluid are incubated without MA-148 ovarian cancer cells. (F) IFN-γ activity when ascites fluid cells are incubated with MA-148 ovarian cancer cells. (G) IFN-γ background activity when cells from a normal donor are 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 untreated. In each case, 9–13 different donors were analyzed independently, and the data were averaged. Fig. 10. In vivo efficacy of cam1615HER2 in a xenograft model. Cells were stably transfected with firefly luciferase for the purpose of real-time bioluminescent imaging. (A) Bioluminescent imaging of a group of 6 NSG mice provided with SKOV3 and NK cells via the peritoneal cavity. The image shows the total flux for each animal and indicates that 5 out of 6 animals in the untreated group had advanced tumors. Tumor development began in a single animal in the untreated group that showed minimal activity. (B) Day 38 imaging of a group of 6 mice also provided with SKOV3 and NK cells but treated with cam1615HER2 TriKE. Fig. 11. (A) Despite multiple TriKE injections, there was minimal change in animal body weight, indicating that the treatment was not toxic compared to the untreated control group. (B) Scatter plot of data from the same experiment on day 46 post-tumor inoculation. Data are expressed as total flux radiance (p / s). Treated mice were compared to untreated mice. The difference was significant as determined by Student's T-test (p=0.0216). (C) Line plot of time (days) indicating that the treatment group began to relapse. (D) Survival plot of data over an extended time interval. The difference between the treatment and untreated groups is significant. Fig. 12. Test of the ability of cam1615HER2 TriKE to enhance apoptosis in other HER2-expressing ovarian cancer cell lines. (A) CD107a activity when PBMC NK cells are incubated with OVCAR3 ovarian cancer cells. (B) CD107a activity when PBMC NK cells are incubated with OVCAR5 ovarian cancer cells. (C) CD107a activity when PBMC NK cells are incubated with SKOV3 ovarian cancer cells. (D) IFN-γ activity when PBMC NK cells are incubated with OVCAR3 ovarian cancer cells. (E) IFN-γ activity when PBMC NK cells are incubated with OVCAR5 ovarian cancer cells. (F) IFN-γ activity when PBMC NK cells are incubated with SKOV3 ovarian cancer cells. SKOV3 data were performed with the following negative controls: untreated (NT), anti-cam16 alone (CAM16), IL-15 (IL15), and anti-HER2 antibody alone (e23). Specific details for implementing the invention

[0024] Detailed description of exemplary embodiments

[0025] The present disclosure describes therapeutic compounds that target tumor cells expressing human epidermal growth factor receptor-2 (HER-2) and / or human epidermal growth factor receptor-3 (HER-3), which are members of the epidermal growth factor receptor (EGFR) family of transmembrane receptor tyrosine kinases. HER2 has a direct association with cancer because its overexpression is associated with a poor prognosis in breast cancer and triggers intracellular signaling pathways related to cell proliferation, differentiation, and survival. HER2 and HER3 can form a heterodimeric complex.

[0026] In many embodiments, the immunotherapy compound may be a trispecific killer binder compound (TriKE). TriKE has three distinct binding domains: an NK cell binding domain that binds to NK cells (e.g., CD16), an NK activation domain comprising its functional fragment that binds to a cytokine or a receptor for that cytokine, and a targeting domain that binds to a marker present on a target cell (e.g., cancer cell). The design and manufacture of TriKE are extensively described, for example, in U.S. Patent Application Publication No. US 2018 / 0282386 A1. TriKE provides the advantage of combining an antibody-dependent cytotoxicity (ADCC)-promoting moiety and an extension-related moiety (IL-15) on the same molecule.

[0027] One or more binding regions or domains within the immunotherapy compound may comprise antibodies. As used herein, the term "antibody" generally refers to an immunoglobulin or a fragment thereof, and thus encompasses monoclonal antibodies and their fragments. 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 their fragments. For example, camels produce functional antibodies without light chains. These single-domain antibody fragments (VHH or NANOBODIES) (Ablynx NV, Gent, Belgium) have several advantages for biotechnological applications. They are well expressed in microorganisms and possess high stability and solubility. In certain embodiments of the TriKE compounds described herein, the NK cell binding domain is a camel single-domain antibody fragment.

[0028] Although the immunotherapy compound is 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 immunotherapy compound described herein may comprise any other suitable HER2-targeting and / or HER3-targeting moiety. Accordingly, in various embodiments, the targeting domain may recognize HER2, HER3, and / or HER2 / HER3 heterodimers. HER2 / HER3 heterodimers are found in many breast cancers and many HER2 + It is detected in tumors. HER2 / HER3 dimers are associated with proliferation, distant metastasis, and / or poor patient treatment outcomes.

[0029] Exemplary alternative targeting moiety comprises antibodies and antibody fragments that specifically bind to HER2, HER3 and / or HER2 / HER3 heterodimers. Exemplary antibody fragments include e23 or its functional fragment (e.g., sequence identification number: 15), trastuzumab or its functional fragment (e.g., sequence identification number: 16 and European Patent No. EP 3457139 A1), sequence identification number: 17, sequence identification number: 18, rumletuzumab or its functional fragment (RG7116; (Liu et al., 2019, Biol Proced Online 21:5); for example, sequence identification number: 19, sequence identification number: 20), cerivantumab or its functional fragment (MM-121; (Liu et al., 2019, Biol Proced Online 21:5); for example, sequence identification number: 21 or sequence identification number: 22), KTN3379 / CDX-3379 or its functional fragment ((Liu et al., 2019, Biol Proced Online 21:5); e.g., sequence identification number: 23), patritumab or its functional fragment (U3-1287; (Liu et al., 2019, Biol Proced Online 21:5); for example, sequence identification number: 24, sequence identification number: 25, sequence identification number: 26 or sequence identification number: 27), elgemtumab (LJM716, (Liu et al., 2019, Biol Proced Online 21:5)) or its functional fragment, U3-1402(Liu et al., 2019, Biol Proced Online 21:5) or its functional fragment, AV-203 (Liu et al., 2019, Biol Proced Online 21:5) or its functional fragment, GSK2849330(Liu et al., 2019, Biol Proced Online 21:5) or its functional fragment, MM-111 (Liu et al., 2019, Biol Proced Online 21:5) or its functional fragment, MCLA-128 (Liu et al., 2019, Biol Proced Online21:5) or its functional fragment, istiratumab (MM-141; (Liu et al., 2019, Biol Proced Online 21:5)) or its functional fragment, duligotumab (MEHD7945A; (Liu et al., 2019, Biol Proced Online 21:5)) or its functional fragment or pertuzumab or its functional variant, but is not limited thereto.

[0030] Although described herein in the context of exemplary embodiments comprising a single-domain antibody (sdAb) in which the NK cell binding domain binds to CD16, the immunotherapeutic compound may comprise any other suitable NK binding moiety. Exemplary alternative NK binding moiety comprises, but is not limited to, any amino acid sequence capable of selectively binding to a receptor located at least partially on the surface of NK cells. Thus, by performing the function of binding to NK cells, the NK cell binding domain can bring the NK to spatial proximity to the target to which the targeting domain selectively binds. In certain embodiments, the NK cell binding domain may selectively bind to a receptor that activates NK cells and thus also possesses an activating function. For example, activation of the CD16 receptor can induce antibody-dependent cell-mediated cytotoxicity. Thus, the NK cell binding domain of the exemplary cam1615HER2 compound possesses NK activating activity. In another embodiment, the NK cell binding domain may interfere with the mechanism of inhibiting NK cells. In this embodiment, 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 inhibition blocking domain.

[0031] The NK cell binding domain may include a ligand or antibody that selectively binds to any NK cell receptor, e.g., cytotoxic receptor 2B4, low-affinity Fc receptor CD16, killer immunoglobulin-like receptor (KIR), CD2, NKG2A, TIGIT, NKG2C, LIR-1 and / or DNAM-1.

[0032] An NK cell binding domain can be designed to possess a desired degree of NK selectivity and, consequently, desired immune binding characteristics. For example, CD16 is identified as the Fc receptors FcγRⅢa (CD16a) and FcγRⅢb (CD16b). These receptors bind to the Fc portion of IgG antibodies and subsequently activate NK cells against antibody-dependent cell-mediated cytotoxicity. Anti-CD16 antibodies selectively bind to NK cells but can also bind to neutrophils. Anti-CD16a antibodies selectively bind to NK cells but do not bind to neutrophils. Immunotherapy compounds containing an NK cell binding domain with an anti-CD16a antibody can bind to NK cells but cannot bind to neutrophils. Therefore, in situations where it is desired to bind NK cells but not neutrophils, the NK cell binding domain of an immunotherapy compound can be designed to include an anti-CD16a antibody.

[0033] Although the NK activation domain has been described herein in the context of exemplary embodiments comprising a fragment of human IL-15, the NK activation domain may comprise any amino acid sequence that activates NK cells, promotes the sustainment of NK cells, or otherwise promotes NK cell activity. The NK activation domain may be one or more cytokines capable of activating and / or sustaining NK cells, or may be derived therefrom. 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, that is, has sequence similarity or sequence identity with the referenced cytokine sufficient to provide NK cell activation and / or sustainment activity. Exemplary cytokines on which the NK activation domain may be based include, for example, IL-15, IL-18, IL-12, and IL-21. Therefore, the exemplary cam1615HER2 compound contains an NK activation domain derived from IL-15, but the HER2-targeting compound can be any suitable cytokine or designed to have an NK activation domain derived therefrom.

[0034] For the sake of brevity in this description, references to the NK activation domain may refer to a functional variant of the cytokine or the entire amino acid sequence of the cytokine by identifying the underlying cytokine. A functional variant of the cytokine may include a modified version of the cytokine containing one or more amino acid deletions, additions, and / or substitutions, and / or any suitable amino acid fragment of the cytokine. Thus, references to the "IL-15" NK activation domain include an NK activation domain containing the entire amino acid sequence of IL-15, an NK activation domain containing a fragment of IL-15, or an NK activation domain containing amino acid substitutions compared to the wild-type IL-15 amino acid sequence, e.g., IL-15N72D or IL-15N72A.

[0035] Although described above in the context of exemplary embodiments where the immunotherapy compound is a trispecific killer binder compound (i.e., TriKE), the compositions and methods described herein may involve the use of immunotherapy compounds modified to include additional domains. For example, the immunotherapy compound may be designed to be a larger molecule having more than one targeting domain, more than one NK cell binding domain, and / or more than one NK activation domain. In embodiments comprising more than one NK activation domain, the NK activation domains may be provided in series or in any other combination. Any cytokine-based NK activation domain may comprise the entire amino acid sequence of the cytokine, may be an amino acid fragment, or may be a modified version of the cytokine, independently of the nature of other NK activation domains included in the immunotherapy compound.

[0036] Exemplary additional targeting domains include, but are not limited to, any moiety that selectively binds to an intended target, e.g., a tumor cell, a target in the tumor stroma, a suppressor cell, e.g. a CD33+ myeloid-derived suppressor cell, or a target on a virus-infected cell. Therefore, the targeting domain is, for example, anti-tumor antibodies, such as rituximab (anti-CD20), aputuzumab (anti-CD20), pertuzumab (anti-HER2 / neu), rabetuzumab (anti-CEA), adecatumumab (anti-EpCAM), citatuzumab vogatox (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), etalacizumab (anti-integrin α vβ3), intertumumab (anti-CD51), ipilimumab (anti-CD152), oregovomab (anti-CA-125), botumumab (anti-tumor antigen CTAA16.88) or femtumumab (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, It may include any one or more of the sequence number 1-119 of Anti-PD-L1, European Patent No. EP 3457139 A1, or a functional variant of any of the above.

[0037] An amino acid sequence is a "functional variant" of a reference amino acid sequence if it possesses a specified amount of sequence identity or sequence specificity compared to a reference amino acid sequence. An amino acid sequence is a "functional fragment" of a reference amino acid sequence if it contains an amino acid sequence shorter than the full length of the reference amino acid sequence. A "functional fragment" may additionally possess a specified amount of sequence identity or sequence specificity compared to a reference amino acid sequence.

[0038] 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 the length of their sequences; gaps within either or both sequences are allowed to make the alignment to optimize the number of identical amino acids, but nevertheless, the amino acids within each sequence must be maintained in their proper order.

[0039] Pairwise comparative analysis of amino acid sequences can be performed using the BESTFIT algorithm in the GCG package (version 10.2, Madison, Wisconsin). Alternatively, polypeptides are [Tatiana et al., ( FEMS Microbiology Letter As described in , 174, 247-250 (1999)), the Blastp program of the BLAST 2 search algorithm can be compared and is available on the National Center for Biotechnology Information (NCBI) website. Matrix = BLOSUM62; default values ​​for all BLAST 2 search parameters, including open gap penalty = 11, extended gap penalty = 1, gap x_dropoff = 50, expectation = 10, word size = 3, and filter on, can be used.

[0040] In the comparison of two amino acid sequences, structural similarity may be referred to as 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 substitutions for amino acid residues within an amino acid sequence may 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. Cationally charged (basic) amino acids include arginine, lysine, and histidine. Anionally charged (acidic) amino acids include aspartic acid and glutamic acid. Therefore, conservative substitutions include, for example, Lys for Arg to retain a positive charge and vice versa; Glu for Asp to retain a negative charge and vice versa; Ser for Thr to retain a free -OH; and Gln for Asn to retain a free -NH2.

[0041] Accordingly, amino acids belonging to a group of amino acids having specific sizes or characteristics (e.g., charge, hydrophobicity, or hydrophilicity) can be substituted with other amino acids, particularly in regions of the protein not directly associated with biological activity, without altering the protein's activity. Regions within the amino acid sequence not directly associated with biological activity can be inferred from alignment analysis, which identifies regions where variability (e.g., addition, deletion, or non-conservative substitution) 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.

[0042] The NK binding domain, NK activation domain, or targeting domain may 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 with a reference amino acid sequence (e.g., a reference antibody fragment, a cytokine, or a cytokine fragment).

[0043] The NK binding domain, NK activation domain, or targeting domain may 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 identity with a reference amino acid sequence.

[0044] Immunotherapy compounds as described herein may also be designed to provide additional sequences, such as the addition of an added C-terminal or N-terminal amino acid, which can facilitate purification, for example, by capture on a column or by the use of an antibody. Such tags include, for example, histidine-rich tags that enable the purification of polypeptides on a nickel column. Such genetic modification techniques and suitable additional sequences are widely known in the field of molecular biology.

[0045] The present disclosure also provides polynucleotides encoding any of the immunotherapy compounds described herein and complements to the sequences of such polynucleotides. When the amino acid sequence of any one of the immunotherapy compound polypeptides described herein (or one or more component fragments of the immunotherapy compound) is presented, a person skilled in the art can determine the full range of polynucleotides encoding the amino acid sequence using ordinary, routine methods.

[0046] Fig. 1A shows an exemplary construction of an exemplary embodiment of a second-generation TriKE, referred herein as cam1615HER2 (Sequence Identification No.: 1), capable of both antibody-dependent cytotoxicity (ADCC) and NK cell expansion. The cam1615HER2 TriKE contains anti-CD16 VHH as an NK cell binding domain. Anti-CD16 VHH is a variable region of the heavy chain of a camel antibody. Fig. 1B is a plasmid map showing the arrangement of the TriKE coding sequence in a pET expression vector. Fig. 2B shows the absorbance trace of the target protein and bacterial fractions as they pass through an FFQ ion exchange column as the first step of purification from the inclusion body. The eluent was collected in an 8 ml aliquot. Fig. 2C shows the absorbance trace from the second purification step, size exclusion chromatography (SEC). The double arrows indicate the target peak collected as cam1615HER2 exits the column. Figure 2A shows the final product (fractions C2-D4) analyzed using SDS-PAGE with Coomassie blue staining, which provides evidence of a homogeneous product, as mostly single bands. The final product was more than 90% pure and had a molecular weight of approximately 55 kDa.

[0047] However, cam1615HER2 TriKE can be constructed in a different way. It is possible to design multiple constructs, each encoding and directing the synthesis of a portion of a complete immunotherapy compound. For example, an anti-HER2 light chain (e.g., SEQ ID: 17) can be encoded on one plasmid, and an anti-HER2 heavy chain (e.g., SEQ ID: 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 the anti-HER2 heavy chain can dimerize to form an anti-HER2 Fab as a targeting moiety of the immunotherapy compound. cam1615HER2 TriKE can be constructed in this manner. For example, Sequence ID No. 31 provides an amino acid sequence expressed from an exemplary first plasmid, which is an amino acid sequence containing a signal peptide, an anti-HER2 light chain, a linker, an IL-15 amino acid sequence, a second linker, and a camel anti-CD16 single-domain antibody fragment. Sequence ID No. 32 provides an amino acid sequence expressed from an exemplary second plasmid, which is an amino acid sequence containing a signal peptide and an anti-HER2 heavy chain.

[0048] As another example, a single plasmid construct may contain all components of a complete immunotherapy compound. For example, Sequence ID No. 33 provides an amino acid sequence expressed from an exemplary single-plasmid construct, which is an amino acid sequence comprising a signal sequence, an anti-HER2 heavy chain fragment, a T2A self-cleavage peptide, a second signal sequence, an anti-HER2 light chain fragment, a linker, an IL-15 amino acid sequence, a second linker, and a camel anti-CD16 single-domain antibody fragment. When expressed, the T2A peptide may self-cleave to separate the anti-HER2 heavy chain fragment from the remainder of the immunotherapy compound and dimerize it with the anti-HER2 light chain fragment.

[0049] In the exemplary cam1615HER2 TriKE, the human IL-15 TriKE moiety possesses the ability to expand the molecule. Therefore, the ability of the IL-15 moiety within cam1615HER2 to influence NK expansion was measured. Figures 3A and 3B show highly proliferated CD56 after 7 days of incubation with 50 nM cam1615HER2 TriKE compared to the untreated control (NT) and IL-15 control. + CD3 - It shows the percentage of NK cells and the total percentage of NK cells. Both significantly increased after incubation with cam1615HER2. Similarly, exposure to cam1615HER2 also significantly improved the total NK count compared to the NT control group (Fig. 3C). Figs. 3D-F show CD3 compared to the untreated control group. + CD56 - It shows that the percentage and number of T cells were not increased. Taken together, these studies indicate that HER2 TriKE stimulates the expansion of NK cells but not T cells. The data also show that the IL-15 moiety within TriKE is in functional and viable morphological alignment.

[0050] Cytotoxicity is a characteristic of NK immunotherapy. To establish antibody-dependent cytotoxicity (ADCC) efficacy, various cell lines were analyzed for CD107a expression, an accepted measure of NK cell cytotoxicity. cam1615HER2 TriKE was tested on SKOV3 and SK-BR-3 because it is known that cases of breast cancer and some ovarian cancers overexpress ERBB2, making it a desirable target for antibody-designated targeting. The UMSCC-11B cell line was tested as a negative control because it exhibits minimal HER2 expression. First, various agents were FITC-labeled, and binding was measured by testing their direct binding to their targets using flow cytometry. SKOV3 (Fig. 4A) and SK-BR-3 (Fig. 4B) showed the highest levels of HER2 TriKE binding. UMSCC-11B showed lower levels of binding (Fig. 4C).

[0051] When ADCC activity was measured, cam1615HER2 TriKE showed highly elevated CD107a activity in SKOV3 and SK-BR-3 cell lines compared to IL-15 and untreated controls (Figs. 5A, 5C). When ADCC was tested on UMSCC-11B cell lines, cam1615HER2 TriKE had almost no effect (Fig. 5E). Additionally, SKOV-3 (Fig. 5B) and SK-BR-3 (Fig. 5D) cam1615HER2 showed elevated IFN-γ activity when treated with cam1615HER2 TriKE.

[0052] cam1615HER2 TriKE was further tested on the breast cancer cell line MCF-7L. Figure 6A shows the CD107a effector cell background without added target cells. Figure 6B shows CD107a activity when the target was added. The highest level of apoptosis was observed with cam1615HER2 TriKE compared to the control. Cam16 itself possessed activity, but it was not high. The MCF-7L sub-line (MCF-7L-TamR) is tamoxifen-resistant and showed a similar pattern of CD107a activity when treated with cam1615HER2 TriKE (Figure 6C). It is known that NK cells, when activated to induce apoptosis, also secrete anticancer cytokines, such as IFN-γ. Figure 6E shows that IFN-γ levels quantified by intracellular staining in the same samples were elevated with cam1615HER2 TriKE and minimally affected in the control, indicating NK cell activation. This was also true for MCF-7L-TamR (Fig. 6F). Fig. 6D demonstrates cam1615HER2 TriKE-induced apoptosis in SK-BR-3 cells and indicates that it induces apoptosis similar to trastuzumab. Fig. 6H shows that cam1615HER2 TriKE has much greater IFN-γ enhancing activity than trastuzumab. Taken together, these data demonstrate that HER2 is an effective target for immune binders on human breast cancer cells and that innate immunotherapy is highly effective in vitro against drug-resistant breast cancer cell lines.

[0053] Figure 12 provides data showing the activity of cam1615HER2 TriKE on two additional HER2-expressing cell lines, OVCAR-3 and OVCAR-5. Again, cam1615HER2 TriKE shows elevated CD107a activity (Figures 12A, 12B) and IFN-γ activity (Figures 12D, 12E) compared to the control. Repeat experiments evaluating SKOV3 include anti-HER2 scFv alone (e23), cam16 VHH alone, IL-15 alone, and a wider number of negative controls, including untreated controls. Again, the drug shows elevated CD107a activity (Figure 12C) and elevated IFN-γ activity (Figure 12F) compared to the control.

[0054] Apoptosis was further evaluated in real-time over 2 days using the INCUCYTE ZOOM platform (Essen Biosciences, Inc., Ann Arbor, Michigan). SKOV3 grown in culture as spheroids was studied. SKOV3 stably transduced with NUCLIGHT RED (Essen Biosciences, Inc., Ann Arbor, Michigan) was incubated with enriched NK cells and cam1615HER2 TriKE, free IL-15, and cam16 VHH alone or without treatment. Caspase 3 / 7 green reagent was added to detect apoptosis. Dying cells turn green, and dying NUCLIGHT RED Raji cells turn yellow, allowing for the tracking of remaining living cells. When the data were compiled, cam1615HER2 TriKE induced an impressive decline in SKOV3 globule size (Fig. 7A) and globule intensity (Fig. 7B) over 72 hours of continuous measurement compared to IL-15, cam16 VHH alone, and untreated controls. Fig. 8 provides images showing that cam1615HER2 induces a rapid time-dependent decline in target cells measured over a 72-hour period. The findings in this direct-death assay correlated with the findings in the CD107a assay.

[0055] To determine whether NK cells from consenting cancer patients would be effective in the analysis, 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 with the effector without cancer cells compared to cam1615HER2 TriKE or the control group. Figure 9B shows a significant increase in CD107a expression at the effector plus tumor target upon treatment with cam1615HER2 TriKE compared to the untreated control group. Regarding IFN-γ activity, Figure 9E shows low activity in the effector without a target upon treatment with cam1615HER2 TriKE. When effector cells were added, cam1615HER2 TriKE showed enhanced IFN-γ activity (Figure 9F). For comparison, Fig. 9C shows the CD107a background of normal donor effector cells, and Fig. 9D shows the CD107a activity of normal donor cells with MA148 cancer cells. Fig. 9G shows the IFN-γ background of normal donor effector cells, and Fig. 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, but the activity of normal cells was slightly higher. Nevertheless, effector cells from patients can stimulate TriKE.

[0056] 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 of the untreated group (Figure 10A) and treated group (Figure 10B) of mice approximately 6 weeks after intraperitoneal inoculation of SKOV3 tumor cells. Advanced tumor progression was evident in the untreated mouse group (Figure 10A) and was clearly reduced in the treated mouse group (Figure 10B).

[0057] Figure 11A shows the minimum change in animal body weight over a 46-day period, 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 and shows that tumor growth is significantly inhibited in the treatment group versus the untreated group, even on day 46 after tumor administration. Figure 11C shows the overall progression of tumor growth (bioluminescence) over time. Over the 46-day period, tumor growth was significantly inhibited in the treatment group, but tumor growth began to recur on day 39. Figure 11D shows longer-term results in the survival plot. By day 52, 5 out of 6 animals in the untreated group had died, and by day 60, all animals in the untreated group had died. In contrast, 50% of the animals in the cam1615HER2-treated group were still alive by day 72. The remaining animals in the cam1615HER2-TriKE-treated group still exhibited tumor load; therefore, the treatment was inhibitory, not curative. Taken together, these data demonstrate that cam1615HER2 TriKE is effective in inhibiting the growth of human ovarian carcinomas in vivo.

[0058] Accordingly, the data presented herein demonstrate that HER2 acts as an immunotherapeutic target for ovarian and breast cancer when targeted by immunotherapeutic compounds, such as trispecific killer binders (TriKEs). Exemplary immunotherapeutic compounds are effective against tamoxifen-refractory cells and can therefore readily kill cancer cells resistant to tamoxifen or other chemotherapy agents. The present disclosure further demonstrates that HER2-targeted NK-binding TriKEs, which are exemplary immunotherapeutic compounds, can suppress cancer in vivo in an intraperitoneal ovarian cancer xenograft model, which involves engrafting both human NK cells and cancer cells into xenogeneic mice. The data presented herein further provide a basis for immunotherapeutic compounds targeting HER3 and / or HER2 / HER3 heterodimers.

[0059] The immunotherapy compounds described herein may be formulated with pharmaceutically acceptable carriers. As used herein, "carriers" include any solvent, dispersion medium, vehicle, coating, diluent, antibacterial and / or antifungal agent, isotonic agent, absorption retardant, buffer, carrier solution, suspension, colloid, etc. The use of such media and / or agents for pharmaceutical active substances is widely known in the art. Any conventional media or agent is considered for use in therapeutic compositions except where it is incompatible with the active ingredient. An auxiliary active ingredient may also be incorporated into the composition. As used herein, "pharmaceutically acceptable" refers to a substance that is not biologically or otherwise undesirable; that is, the substance may be administered to an individual with the immunotherapy compound without interacting in a harmful manner with any other component of the pharmaceutical composition in which it is contained or causing any undesirable biological effects.

[0060] Accordingly, immunotherapy compounds can be formulated into pharmaceutical compositions. Pharmaceutical compositions can be formulated in various forms adapted to a preferred route of administration. Thus, the composition may be administered via known routes, including, for example, oral, parenteral (e.g., intradermal, transdermal, subcutaneous, intramuscular, intravenous, intraperitoneal, etc.), or topical (e.g., nasal, intrapulmonary, intramammary, vaginal, intrauterine, intradermal, transdermal, rectal, etc.). The pharmaceutical composition may be administered to mucosal surfaces, for example, by administration to the nasal or respiratory mucosa (e.g., by spray or aerosol). The composition may also be administered via sustained or delayed release. In certain embodiments, the composition is administered intraperitoneally, intravenously, or subcutaneously.

[0061] Accordingly, the immunotherapy compound may be provided in any suitable form, including but not limited to a solution, suspension, emulsion, spray, aerosol, or any mixture of any form. The composition may be delivered as a formulation with any pharmaceutically acceptable excipient, carrier, or vehicle. For example, the formulation may be delivered in a conventional topical form of administration, e.g., a cream, ointment, aerosol formulation, non-aerosol spray, gel, lotion, etc. The formulation may further include one or more additives, e.g., adjuvants, skin penetration enhancers, colorants, fragrances, flavorings, moisturizers, thickeners, etc. In certain embodiments, the composition may be formulated as a solution or suspension.

[0062] The formulation may be conveniently provided in the form of a unit dose and may be manufactured by methods widely known in the field of pharmaceutical technology. A method for manufacturing a composition having a pharmaceutically acceptable carrier comprises the step of binding an immunotherapy compound to a carrier constituting one or more auxiliary components. Generally, the formulation may be manufactured by uniformly and / or densely binding an active molecule to a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, forming the product into a desired formulation.

[0063] Accordingly, in another aspect, the present disclosure describes a method for treating cancer in a subject. Generally, the method comprises administering an effective amount of an immunotherapy compound to treat cancer to the subject. “Treat” or a variation thereof refers to reducing, limiting progression, alleviating, or resolving symptoms or signs associated with a condition to any degree. As used herein, “alleviate” refers to any reduction in the degree, severity, frequency, and / or likelihood of characteristic clinical signs or symptoms of a particular condition; “symptom” refers to any subjective evidence of the disease or the patient’s condition; and “sign” or “clinical sign” refers to objective physical findings associated with a particular condition that may be observed by something other than the patient.

[0064] “Treatment” may be therapeutic or prophylactic. “Therapeutic” and variations thereof refer to treatment that alleviates one or more existing symptoms or clinical signs associated with the condition. “Prophylactic” and variations thereof refer to treatment that limits the occurrence and / or appearance of symptoms or clinical signs of the condition to any degree. Generally, “therapeutic” treatment is initiated after the condition appears in the subject, whereas “prophylactic” treatment is initiated before the condition appears in the subject. Accordingly, in certain embodiments, the method may involve prophylactic treatment of a subject at risk of developing the condition. “At risk” refers to a subject who may or may not actually possess the described risk. Thus, for example, a subject “at risk” of developing a specific condition is a subject who possesses one or more signs of an increased risk of having or developing the specific condition compared to an individual lacking one or more signs, regardless of whether the subject has the condition or exhibits any symptoms or clinical signs of developing it. Exemplary signs of the condition may include, for example, genetic predisposition, ancestry, age, gender, geographical location, lifestyle, or medical history. Treatment may also continue after symptoms have resolved, for example, to prevent or delay recurrence.

[0065] Therefore, immunotherapy compounds may be administered to the 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 of the subject experiencing clinical evidence of the condition, reduce the severity of symptoms and / or clinical signs of the condition, and / or completely resolve the condition compared to a subject not administered the immunotherapy compound. Treatment initiated after the subject first exhibits symptoms or clinical signs associated with the condition may reduce the severity of symptoms and / or clinical signs of the condition and / or completely resolve the condition compared to a subject not administered the immunotherapy compound.

[0066] The amount of immunotherapy compound administered may vary depending on various factors, including but not limited to the specific immunotherapy compound administered, the subject's body weight, physical condition and / or age, and / or route of administration. Consequently, the absolute weight of the immunotherapy compound contained in a given unit dosage form can vary widely and depends on factors, such as the subject's species, age, body weight and physical condition, and / or method of administration. Therefore, it is not practical to generally present an amount that constitutes the quantity of the immunotherapy compound effective for all possible applications. However, a person skilled in the art can easily determine an appropriate amount by taking these factors into account.

[0067] In some embodiments, the method may involve administering an immunotherapy compound sufficient to provide a subject with, for example, a dose of about 100 ng / kg / day to about 10 mg / kg / day, but in some embodiments, the method may be performed by administering the immunotherapy compound at a dose outside this range.

[0068] In some embodiments, the method may comprise administering an immunotherapy compound sufficient to provide a minimum dose of at least 100 ng / kg / day, e.g., 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.

[0069] In some embodiments, the method is 10 mg / kg / day or less, e.g., 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, 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 It includes administering an immunotherapy compound sufficient to provide a maximum dose of μg / kg / day or less, 20 μg / kg / day or less, or 10 μg / kg / day or less. Where the immunotherapy compound is not absent but is present in the specified amount and up to the specified amount, the immunotherapy compound provides a dose of “less than” the specified amount.

[0070] In some embodiments, the method comprises administering an immunotherapy compound sufficient to provide a dose characterized by a range having an endpoint defined by any identified minimum dose and any maximum dose greater than the selected minimum dose. For example, in some embodiments, the method may comprise administering an immunotherapy compound sufficient to provide a subject 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.

[0071] In certain embodiments, the method comprises administering an immunotherapy compound sufficient to provide a dose equal to any minimum dose or any maximum dose listed above. Accordingly, for example, in certain embodiments, the method may comprise administering an immunotherapy compound sufficient to provide a dose such as 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.

[0072] In some embodiments, the immunotherapy compound may be administered, for example, as a single dose to multiple doses per week, but in some embodiments, the method may be performed by administering the immunotherapy compound at a frequency outside this range. In certain embodiments, the immunotherapy compound may be administered about once a month to about five times a week. In some embodiments, the doses described above, in terms of the amount of immunotherapy compound administered over a 24-hour period, are administered in a 7-day cycle of 4 days of treatment and 3 days of rest.

[0073] In some embodiments, the immunotherapy compound may be administered, for example, in a single dose or multiple cycles of treatment, but in some embodiments, the method may be performed by administering the immunotherapy compound for a period outside these ranges. In some embodiments, the immunotherapy compound may be administered for three weeks. In these embodiments, each week may be a treatment cycle, for example, the exemplary treatment cycle described in the paragraph above. In other embodiments, the immunotherapy compound may be administered for a greater number of 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 may be one week or more, one month or more, or one year or more.

[0074] In some embodiments, the method further comprises administering one or more additional therapeutic agents. One or more additional therapeutic agents (e.g., chemotherapy agents) may be administered before, after, and / or simultaneously with the administration of the immunotherapy compound. The immunotherapy compound and the additional therapeutic agents may 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 any single component administered alone. Two components may be co-administered simultaneously or sequentially. Components co-administered simultaneously may be provided as one or more pharmaceutical compositions. Sequential co-administration of two or more components includes cases where the components are administered such that each component is simultaneously present at the treatment site. Alternatively, sequential co-administration of two components may include cases where at least one component is removed from the treatment site, but at least one cellular effect of the component administration (e.g., cytokine production, activation of a specific cell population, etc.) persists at the treatment site until one or more additional components are administered to the treatment site. Thus, the co-administered combination may, under certain circumstances, include components that never exist as a chemical mixture with each other. In other embodiments, the immunotherapy compound and additional therapeutic agents may be administered as part of a mixture or cocktail. In some aspects, the administration of the immunotherapy compound may allow for the efficacy of other modes of treatment at lower doses compared to the administration of other therapeutic agents or therapeutic agents alone, and accordingly, may reduce the likelihood, severity, and / or degree of toxicity observed when higher doses of other therapeutic agents or therapeutic agents are administered.

[0075] Exemplary additional therapeutic agents include altretamine, amsacrine, L-asparaginase, colaspase, bleomycin, busulfan, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, cyclophosphamide, cytophosphan, cytarabine, dacarbazine, dactinomycin, daunorubicin, docetaxel, doxorubicin, epirubicin, etoposide, fluorouracil, fludarabine, potemustine, ganciclovir, gemcitabine, hydroxyurea, idarubicin, ifosfamide, irinotecan, lomustine, melphalan, mercaptopurine, methotrexate, mitoxantrone, mitomycin C, nimustine, oxaliplatin, paclitaxel, pemetrexed, procarbazine, Raltitrexed, temozolomide, tenifoside, thioguanine, thiotepa, topotecan, vinblastine, vincristine, vindesin, and vinorelbine, anti-HER2 antibody therapy, anti-HER3 antibody therapy (e.g., (Liu et al., 2019, Biol Proced Online (See 21:5) or anti-HER2 / HER3 heterodimer complex antibody therapy (e.g., (Liu et al., 2019, Biol Proced Online Includes (see 21:5).

[0076] In some embodiments, the method may comprise administering a sufficient amount of the immunotherapy compound as described herein and administering at least one additional therapeutic agent demonstrating a therapeutic synergy. In some aspects of the method of the present invention, a measure of response to treatment observed after administering both the immunotherapy compound as described herein and the additional therapeutic agent is improved compared to the same measure of response to treatment observed after administering the immunotherapy compound or the additional therapeutic agent alone. In some embodiments, the additional therapeutic agent may comprise an additional agent targeting EpCAM, for example, an EpCAM-specific monoclonal antibody, such as catumaxomab, a monoclonal hybrid antibody targeting EpCAM and CD3.

[0077] In some embodiments, administering an immunotherapy compound to a subject may stimulate endogenous NK cells in vivo. Using an immunotherapy compound as part of an in vivo method may make NK cells antigen-specific, along with co-stimulation, enhancement of survival, and expansion. In other cases, the immunotherapy compound may be used in vitro as an adjuvant for NK cell adoptive delivery therapy.

[0078] In the foregoing description and the following claims, the term “and / or” means one or all of the enumerated elements or a combination of any two or more of the enumerated elements; the terms “comprising,” “comprising,” and variations thereof are to be interpreted as open-ended—i.e., additional elements or steps are optional and may or may not exist; unless otherwise specified, expressions in the singular form and “at least one” are used interchangeably and mean one or more than one; references to a numeric range by an endpoint include all numbers contained within the range (e.g., 1 to 5 include 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).

[0079] In the foregoing description, specific embodiments may be described separately for clarity. Unless otherwise clearly stated that a feature of a specific embodiment is incompatible with a feature of another embodiment, a specific embodiment may include a combination of the compatibility features described herein in relation to one or more embodiments.

[0080] For any method disclosed herein comprising separate steps, the steps may be performed in any feasible order. Additionally, where appropriate, any combination of two or more steps may be performed simultaneously.

[0081] The present invention is illustrated by the following examples. Specific examples, materials, quantities, and procedures should be understood to be broadly interpreted in accordance with the scope and spirit of the invention as set forth herein.

[0082] Examples

[0083] Build of cam1615HER TriKE

[0084] DNA fragment encoding the CDR region from camelization anti-CD16 (Vincke et al., 2009, J. Biol. Chem (. 284(5):3273-3284) was spliced ​​into a universal humanized nanobody scaffold previously shown to allow grafts of antigen-binding loops with the transfer of antigen specificity and affinity (Behar et al., 2008, Protein Engineering, Design & Selection 21(1):1-10). cam1615HER2 (sequence identification number: 1) was constructed using this new sequence. cam1615HER2 fully assembled hybrid gene encoding TriKE cam1615HER2 is NcoI restriction site; ATG start codon; anti-human CD16 VHH; 20-amino acid (aa) fragment of PSGQAGAAASESLFVSNHAY (Sequence ID No.: 3); human IL-15; 7-amino acid linker of EASGGPE (Sequence ID No.: 5); anti-HER2 scFv (Batra et al., 1992, Proc Natl Acad SciUSA 89(13):5867-5871) and codes for the XhoI restriction site (from the 5' end to the 3' end). The resulting hybrid gene (sequence identification number: 7) was spliced ​​into the pET28c expression vector under the control of the isopropyl-D-thiogalactopyranoside (IPTG) inducible T7 promoter. The DNA target gene encoding cam1615HER2 was 1,517 base pairs long. Wild-type human IL-15 was used, and mutated forms of the cytokine were not used. The Biomedical Genomics Center at the University of Minnesota in St. Paul, Minnesota, USA, verified the in-frame accuracy of the gene sequence and target gene.

[0085] Protein purification from inclusion bodies

[0086] For protein expression after plasmid transfection 에스케리키아 콜라이 Strain BL21(DE3) (Novagen, Madison, Wisconsin) was used. Bacteria were cultured overnight in 800-ml 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 the inclusion body. After expression, the bacteria were harvested, then homogenized in a buffer (50 mM Tris, 50 mM NaCl, and 5 mM EDTA pH 8.0), the pellet was sonicated, and centrifuged. To extract protein from the pellet, a solution of 0.3% sodium deoxycholate, 5% Triton X-100, 10% glycerin, 50 mmol / L Tris, 50 mmol / L NaCl, and 5 mmol / L EDTA (pH 8.0) was used, and the extract was washed three times.

[0087] Proteins from inclusion bodies require refolding. Therefore, a sodium N-laurosyl-sarcosine (SLS) air oxidation method modified from a previously described method was used (Vallera et al., 2005, Leuk Res29(3):331-341). Briefly, the inclusions were dissolved in 100 mM Tris and 2.5% SLS (Sigma-Aldrich, St. Louis, Missouri). The pellet was removed by centrifugation. 50 μM CuSO4 was added to the solution, followed by incubation at room temperature for 20 hours with rapid stirring for air oxidation of the -SH groups. Removal of SLS was performed by adding 6 M urea and 10% AG 1-X8 resin (200-400 mesh, chloride form) (Bio-Rad Laboratories, Inc., Hercules, California) to the surfactant-solubilized protein solution. The next step involved adding 13.3 M guanidine-HCl to the protein solution and incubating at 37 °C for 2 to 3 hours. 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 at pH 8.0 for 48 hours at 4 °C, followed by dialyzing against 8 volumes for an additional 18 hours. Subsequently, the product was purified by first by high-speed flow Q ion exchange chromatography, followed by passing through a size exclusion column (SUPERDEX 200, Cytiva, Marlborough, Massachusetts). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed using Simply Blue Life Stain (Invitrogen, Carlsbad, California) to evaluate protein size and purity.

[0088] cancer cell lines

[0089] The following cell lines were obtained from the American Type Culture Collection (Manassas, Virginia): MCF-7L (tubular breast carcinoma), MCF-7L-TamR (tamoxifen-resistant sub-strain of MCF-7L), SKOV3 (ovarian ascites), SK-BR-3 (breast carcinoma derived from a metastatic site), and 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 SKOV3-luc. For in vivo experiments, SKOV3 was transfected with a luciferase reporter construct using LIPOFECTAMINE (Invitrogen, Carlsbad, California), and SKOV3-luc was prepared by applying a selective pressure with 10 μg / ml blastocidin. UMSCC-11B was certified by STR testing performed by the Fragment Analysis Facility at Johns Hopkins University. MA148 (regionally established at the University of Minnesota) is a human epithelial ovarian carcinoma cell line. Cell lines were maintained at 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 atmosphere containing 5% CO2. When adherent cells had grown to over 90%, they were overcultured using trypsin-EDTA for isolation. Standard hemocytoplasmic counting was used for cell counting. Only cells with a viability > 95%, as determined by trypan blue exclusion, were used in the experiment.

[0090] Evaluation of cytotoxicity and NK cell activation

[0091] Antibody-dependent cytotoxicity (ADCC) was measured using CD107a (lysosomal-associated membrane protein LAMP-1) flow cytometry analysis. For effector cells, PBMCs were obtained from healthy volunteers after obtaining donor consent and Institutional Review Board (IRB) 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 IRB approval. All specimens were collected from women diagnosed with advanced-stage ovarian or primary peritoneal carcinoma at the time of primary volume reduction surgery. Cells were pelleted, lysed to remove erythrocytes, cryopreserved in 10% DMSO / 90% FBS, and stored in liquid nitrogen.

[0092] PBMCs were incubated overnight in RPMI 1640 medium supplemented with 10% fetal bovine serum (RPMI-10) (37°C, 5% CO2), washed three times with RPMI-10, and then suspended in medium or tumor target cells. Subsequently, cells were incubated with TriKE or a control at 37°C for 10 minutes. Then, fluorescein isothiocyanate (FITC)-conjugated anti-human CD107a monoclonal antibody (BD Biosciences, San Jose, California) was added and incubated for 1 hour. After incubation, GolgiStop (1:1,500, BD Biosciences, San Jose, California) and GolgiPlug (1:1,000, BD Biosciences, San Jose, California) 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-CD16 mAb, and PE-CF594-conjugated anti-CD3 mAb (BioLegend, San Diego, California). Cells were incubated at 4 °C for 15 minutes, washed, and fixed with 2% paraformaldehyde.

[0093] Intracellular IFN-γ was measured as an indicator of NK cell activation. Briefly, cells were exposed to permeability buffer (BD Biosciences, San Jose, California) and incubated with Pacific Blue-conjugated anti-human IFN-γ (BioLegend, San Diego, California) for 20 minutes. Finally, cells were washed and CD56 + CD3 - It was evaluated by fluorescence-activated cell sorting analysis using LSRII flow cytometry (BD Biosciences, San Jose, California) gating on the cells.

[0094] Cytotoxic efficacy was additionally measured in real time. Magnetic bead-enriched CD56+ CD3 - NK effector cells were plated with red-labeled tumor cells on 96-well flat clear-bottom polystyrene tissue-culture-treated microplates (Corning, Flintshire, UK) and transferred to an IncuCyte Zoom platform (Essen Biosciences, Inc., Ann Arbor, Michigan) housed inside a cell incubator at 37°C / 5% CO2. Images were taken from three technical replicates at 15-minute intervals for 48 hours using a 4× objective lens and then analyzed using IncuCyte Basic Software (Essen Biosciences, Inc., Ann Arbor, Michigan).

[0095] NK cell expansion via IL-15 stimulation

[0096] To measure the efficacy of TriKE dependent on its functional IL-15 moiety, PBMCs or enriched NK cells from healthy donors were labeled with CELLTRACE Violet Growth Dye (Invitrogen, Carlsbad, California) according to the kit instructions. After staining, effector cells were cultured with 50 nM TriKE or a control and incubated for 7 days at 37°C under a humidified atmosphere containing 5% CO2. Cells were harvested, stained for viability with Live / Dead reagent (Invitrogen, Carlsbad, California), and surface-stained for viable CD3-16 PE / Cy7 (BioLegend, San Diego, California) and anti-CD3 PE-CF594 (BD Biosciences, Franklin Lakes, NJ). - CD56 + The NK cell population was gated. Data analysis was performed using FlowJo software (Flowjo enterprise LCC, version 7.6.5, Atsweland, Oregon).

[0097] In vivo mouse research and imaging

[0098] Although the efficacy of HER2 TriKE has been previously reported (Vallera et al., 2016, Clinical Cancer Research 22(14):3440-3450), the growth of the human ovarian cancer cell line SCOV3 was tested in a modified scid / hu mouse model. The cell line was transfected with a luciferase reporter gene to allow real-time monitoring of tumor progression via bioluminescent imaging. NSG mice (NOD.Cg-Prkdc scid I12rg tm1Wjl / SzJ, n = 5 / group) 2 × 10 5 SCOV3 cells were injected intraperitoneally, followed by low-dose whole-body radiation (275 cGy) 3–5 days later. The following day, all groups were provided with highly enriched NK cells (PBMCs depleted of CD3 and CD19) and treatment with cam1615HER2 TriKE was initiated. A single course of treatment consisted of 50 μg of the drug delivered intraperitoneally five times a week (MTWThF) for two weeks, followed by maintenance therapy three times a week (MWF) until day 60. Live mice were imaged weekly. In each imaging session, 100 μl of 30 mg / ml luciferin substrate was injected into the mice over 10 minutes, followed by imaging under isoflurane gas sedation. Imaging data were collected using a Xenogen Ivis 100 imaging system equipped with Living Image 2.5 software (Xenogen Corporation, Hopkinton, Massachusetts). Mice were weighed weekly whenever possible.

[0099] The full disclosures of all patents, patent applications, publications, and electronically available materials cited herein (e.g., nucleotide sequence submissions in GenBank and RefSeq, amino acid sequence submissions in SwissProt, PIR, PRF, and PDB, and translations from annotated coding regions in GenBank and RefSeq) are incorporated by reference in their entirety. In the event of any discrepancy between the disclosure(s) of the present application and the disclosure(s) of any document incorporated by reference herein, the disclosure(s) of the present application shall prevail. The above detailed description and examples are provided merely for clarity. It is understood that there are no unnecessary limitations therefrom. Because variations obvious to a person skilled in the art will be included within the invention as defined by the claims, the invention is not limited to the exact details shown and described.

[0100] Unless otherwise indicated, all numerical values ​​expressing the amount, molecular weight, etc. of a component used in the specification and claims should be understood as being modified by the term "approximately" in all cases. Accordingly, unless otherwise indicated, numerical parameters presented in the specification and claims are approximations that may vary depending on the desired characteristics to be obtained by the present invention. At least, without attempting to limit the scope of the claims, each numerical parameter should be interpreted in light of the number of reported significant figures and by applying ordinary rounding techniques.

[0101] The numerical ranges and parameters presenting the broad scope of the present invention are approximations, but the numerical values ​​presented in specific embodiments are reported as accurately as possible. However, all numerical values ​​inherently contain a range inevitably generated from the standard deviation found in their respective test measurements.

[0102] All headings are for the convenience of the reader and, unless otherwise specified, should not be used to limit the meaning of the text following the heading.

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Claims

Claim 1 A compound comprising: an NK cell binding domain comprising a moiety that selectively binds to CD16, as presented in the amino acid sequence of SEQ ID NO: 2 or amino acids 1 to 124 of SEQ ID NO: 1; a first flanking sequence present at the C-terminus of the NK cell binding domain; an NK activation domain operably linked to the NK cell binding domain, comprising IL-15 as presented in the amino acid sequence of SEQ ID NO: 4 or a functional variant of IL-15 presented in an amino acid sequence different from SEQ ID NO: 4 and N72D or N72A amino acid substitutions; a second flanking sequence present at the C-terminus of the NK activation domain; and a targeting domain that selectively binds to HER2, as presented in the amino acid sequence of SEQ ID NO:

6. Claim 2 In paragraph 1, CD16 is a compound comprising CD16a. Claim 3 In claim 1, a compound consisting of the amino acid sequence of sequence identification number:

1. Claim 4 A pharmaceutical composition for use in cancer treatment comprising a compound of any one of claims 1 to 3; and a pharmaceutically acceptable carrier. Claim 5 A pharmaceutical composition in which the cancer is breast cancer or ovarian cancer, as described in paragraph 4. Claim 6 In claim 4, the composition is administered before, simultaneously with, or after chemotherapy, immunotherapy, surgical resection of a tumor, or radiation therapy, wherein chemotherapy comprises altretamine, amsacrin, L-asparaginase, colaspase, bleomycin, busulfan, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, cyclophosphamide, cytophosphan, cytarabine, dacarbazine, dactinomycin, daunorubicin, docetaxel, doxorubicin, epirubicin, etoposide, fluorouracil, fludarabine, potemustine, ganciclovir, gemcitabine, hydroxyurea, idarubicin, ifosfamide, irinotecan, lomustine, melphalan, mercaptopurine, methotrexate, mitoxantrone, A pharmaceutical composition comprising mitomycin C, nimustine, oxaliplatin, paclitaxel, pemetrexed, procarbazine, raltitrexed, temozolomide, tenifoside, thioguanine, thiotepa, topotecan, vinblastine, vincristine, vindecin, or vinorelbine. Claim 7 A composition used in the manufacture of a medicine for treating breast cancer or ovarian cancer in a subject, comprising a compound of any one of claims 1 to 3. Claim 8 In claim 7, treating breast or ovarian cancer in a subject further comprises administering a compound before, simultaneously with, or after chemotherapy, surgical resection of a tumor, or radiation therapy, wherein chemotherapy comprises altretamine, amsacrin, L-asparaginase, colaspase, bleomycin, busulfan, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, cyclophosphamide, cytophosphan, cytarabine, dacarbazine, dactinomycin, daunorubicin, docetaxel, doxorubicin, epirubicin, etoposide, fluorouracil, fludarabine, potemustine, ganciclovir, gemcitabine, hydroxyurea, idarubicin, ifosfamide, irinotecan, lomustine, melphalan, mercaptopurine, A composition comprising methotrexate, mitoxantrone, mitomycin C, nimustine, oxaliplatin, paclitaxel, pemetrexed, procarbazine, raltitrexed, temozolomide, tenifoside, thioguanine, thiotepa, topotecan, vinblastine, vincristine, vindecin, or vinorelbine. Claim 9 In paragraph 4, a pharmaceutical composition used in the manufacture of a medicine for treating breast cancer or ovarian cancer in a subject. Claim 10 delete Claim 11 delete Claim 12 delete Claim 13 delete Claim 14 delete Claim 15 delete Claim 16 delete Claim 17 delete Claim 18 delete Claim 19 delete Claim 20 delete Claim 21 delete Claim 22 delete Claim 23 delete Claim 24 delete Claim 25 delete Claim 26 delete Claim 27 delete Claim 28 delete Claim 29 delete Claim 30 delete Claim 31 delete Claim 32 delete

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