NK engager compounds that bind to viral antigens and methods of use

Abstract

The present disclosure describes compounds that engage NK cells and methods of using the compounds. Generally, the compounds include an NK cell-engaging domain, a targeting domain that selectively binds to a target cell, and an NK activation domain that operably links the NK cell-engaging domain and the targeting domain. In an exemplary embodiment, the targeting domain selectively binds to an HIV antigen.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority under 35 U.S.C. §119(e) of U.S. Patent Application No. 62 / 906,660, filed September 26, 2019, the entire contents of which are incorporated herein by reference in their entirety.

[0002] Statement of government support This invention was made with government support under grants CA111412 and CA065493 awarded by the National Institutes of Health, and CA036725, CA072669, CA077598 and CA197292 awarded by the National Cancer Institute. The government has certain rights in this invention.

[0003] Incorporating a sequence listing The attached Sequence Listing material is incorporated by reference into this application. The attached Sequence Listing text file, named GTBIO2130_1WO_Sequence_Listing.txt, was created on September 25, 2020, and is 96kb. The file can be accessed using Microsoft Word on a computer using the Windows OS. [Background technology]

[0004] Background information Natural killer (NK) cells are cytotoxic lymphocytes of the innate immune system capable of immune surveillance. NK cells express CD16, an activating receptor that binds to the Fc portion of IgG antibodies and participates in antibody-dependent cell-mediated cytotoxicity (ADCC). NK cells are regulated by IL-15, which can induce antigen-dependent cytotoxicity, increased lymphokine-activated killer activity, and / or mediate cytokine responses. NK cells can be activated to stimulate immune responses to treat cancer and infectious diseases as NK cell-based immunotherapies.

[0005] Advances in the effectiveness and use of antiretroviral drugs have substantially improved the health and lifespan of HIV-infected individuals, but these drugs are only a temporary fix to prevent progression to AIDS and limit further transmission of the virus. Despite the use of antiviral agents to suppress HIV replication, infected individuals retain a reservoir of latently infected HIV cells, which can reactivate and re-establish active infection upon discontinuation of antiretroviral therapy. A curative solution requires the reactivation and subsequent destruction of these latently infected cells. While antibody responses to HIV infection exist, they are generally ineffective due to the high rate of viral mutation, which allows the virus to rapidly eliminate epitopes recognized by generated antibodies. However, in recent years, various HIV-specific antibodies have been identified in infected individuals that possess strong neutralizing activity but a reduced ability to induce antibody-dependent cell-mediated cytotoxicity (ADCC). Summary of the Invention

[0006] The present invention provides compounds for activating NK cells to stimulate immune responses to treat cancer and viral infections. The inventors have designed bispecific and trispecific killer engagers (BiKE and TriKE™) composed of a CD16 engager linked by an IL-15 molecule. As used herein, the compounds of the present invention may be referred to as 16 / 15 / X TriKE, where X represents a targeting domain. The X targeting domain may, for example, target a viral antigen, a cancer cell antigen, etc.

[0007] In certain embodiments, antibody constructs were constructed to target HIV using the broad specificity of these antibodies while simultaneously redirecting NK cell killing specifically to actively replicating infected cells through their recognition of membrane-expressed Env and triggering NK cell degranulation via the low-affinity Fc receptor, CD16. The addition of IL-15 as a linker further activates NK cells, thereby enhancing their response. IL-15 has also been identified as a potential reactivator of latently infected cells. Initial studies have shown that NK cell cytokine production and killing of infected targets expressing HIV-Env are enhanced when incubated with the HIV-specific constructs of the present invention.

[0008] In one embodiment, the present invention provides a compound comprising: an NK engaging domain comprising a portion that selectively binds to CD16 or NKG2c; an NK activation domain operably linked to the NK engaging domain, the NK activation domain comprising IL-15 or a functional fragment thereof; and a targeting domain that selectively binds to a viral antigen, the targeting domain operably linked to the NK activation domain and the NK engaging domain. In some embodiments, the CD16 is CD16a. In some embodiments, the viral antigen is present on an infected cell. In some embodiments, the viral antigen is derived from HIV, CMV, HPV, HCV, or adenovirus. In some embodiments, the viral antigen is derived from HIV. In some embodiments, the NK engaging domain portion comprises an antibody or binding fragment thereof, or a nanobody. In some embodiments, the antibody fragment comprises an scFv, F(ab)2, or Fab. In some embodiments, the antibody or binding fragment thereof, or nanobody is human. In some embodiments, the antibody or binding fragment thereof, or nanobody is camelid. In some embodiments, the IL-15 has the amino acid sequence of SEQ ID NO: 4, or a functional variant thereof. In some embodiments, the functional variant of IL-15 comprises an N72D or N72A amino acid substitution compared to SEQ ID NO:4. In some embodiments, the targeting domain portion comprises an antibody, or binding fragment thereof, or a nanobody. In some embodiments, the antibody binding fragment comprises an scFv, F(ab)2, or Fab. In some embodiments, the NK engaging domain comprises CD16, the NK activation domain comprises IL-15, and the targeting domain selectively binds to a viral antigen derived from HIV. In some embodiments, the NK engaging domain comprises CD16a, the NK activation domain comprises IL-15, and the targeting domain selectively binds to a viral antigen derived from HIV. In some embodiments, the NK engaging domain comprises NKG2c, the NK activation domain comprises IL-15, and the targeting domain selectively binds to a viral antigen derived from HIV.In some embodiments, the compounds described herein comprise at least one flanking sequence linking two of the domains. In some embodiments, the compounds described herein further comprise a second flanking sequence linking the two linked domains to a third domain. In some embodiments, the flanking sequence flanks the NK activation domain. In some embodiments, the first flanking sequence is C-terminal to the NK engaging domain and the second flanking sequence is N-terminal to the antiviral targeting domain. In some embodiments, the compounds described herein further comprise a second targeting domain. In some embodiments, the compounds described herein further comprise a second NK engaging domain. In some embodiments, the compounds described herein further comprise a second NK activation domain.

[0009] Compositions are also provided that include a compound described herein and a pharmaceutically acceptable carrier.

[0010] Furthermore, in some embodiments, a method is provided, comprising administering to a subject a compound described herein in an amount effective to induce NK-mediated killing of a target cell. In some embodiments, the target cell is infected with a virus. In some embodiments, the virus is HIV, CMV, HPV, HCV, or adenovirus. In some embodiments, the virus is HIV.

[0011] In some embodiments, a method for stimulating the expansion of NK cells in vivo is provided, the method comprising administering to a subject a compound described herein in an amount effective to stimulate the expansion of NK cells in the subject. In some embodiments, the subject is infected with a virus. In some embodiments, the virus is HIV, CMV, HPV, HCV, or adenovirus. In one aspect, the virus is HIV.

[0012] In some embodiments, provided herein is a method for treating a viral infection in a subject, the method comprising administering to the subject an amount of a compound described herein effective to treat the viral infection. In some embodiments, the subject is infected with HIV, CMV, HPV, HCV, or adenovirus. In one embodiment, the subject is infected with HIV.

[0013] In some embodiments, provided herein are compounds comprising: a T cell engaging domain having a portion that selectively binds to CD3; a T cell activation domain operably linked to the T cell engaging domain, the T cell activation domain comprising a cytokine of the IL-2 family or a functional fragment thereof; and a targeting domain that selectively binds to a viral antigen and is operably linked to the T cell activation domain and the T cell engaging domain. In some embodiments, the viral antigen is present on an infected cell. In some embodiments, the viral antigen is derived from HIV, CMV, HPV, HCV, or adenovirus. In some embodiments, the viral antigen is derived from HIV.

[0014] In some embodiments, provided herein are methods comprising administering to a subject a compound described herein in an amount effective to induce T cell-mediated killing of a target cell. In some embodiments, the target cell is infected with a virus. In some embodiments, the virus is HIV, CMV, HPV, HCV, or adenovirus. In one embodiment, the virus is HIV, and the target is an HIV Env protein (e.g., gp120).

[0015] In some embodiments, provided herein are methods for stimulating T cell expansion in vivo, the methods comprising administering to a subject a compound described herein in an amount effective to stimulate T cell expansion in the subject. In some embodiments, the subject is infected with a virus. In some embodiments, the virus is HIV, CMV, HPV, HCV, or adenovirus. In some embodiments, the virus is HIV.

[0016] In one embodiment, the present invention provides a method for treating mesothelioma, comprising administering to a subject a compound comprising the amino acid sequence of SEQ ID NO: 32 or 36, which contains the targeting domain of SEQ ID NO: 33, in an amount effective to induce NK-mediated killing of target cells expressing mesothelin.

[0017] In some embodiments, provided herein are methods for producing the compounds described herein, comprising: (i) co-transfecting a mammalian cell with a first polynucleotide having a nucleotide sequence encoding an amino acid sequence comprising an immunoglobulin heavy chain and a second polynucleotide having a nucleotide sequence encoding an amino acid sequence comprising an immunoglobulin light chain; and (ii) collecting a supernatant from the mammalian cell. In some embodiments, the viral antigen is derived from HIV, CMV, HPV, HCV, or adenovirus. In some embodiments, the viral antigen is derived from HIV. In some embodiments, the viral antigen is Env. For example, the light and heavy chains of anti-HIV antibodies comprise SEQ ID NOs: 21 and 22, 31 and 30, 26 and 25, and 40 and 39, respectively.

[0018] In some embodiments, provided herein are isolated DNA sequences that encode the amino acid sequences described herein.

[0019] The present invention also provides pharmaceutical compositions comprising the TriKE compounds described herein.For example, the present invention provides pharmaceutical compositions comprising SEQ ID NO: 5, 7, 24, 29, 32, 34, 36 or 37 in a pharmaceutically acceptable carrier.In one embodiment, the present invention provides a method for treating a subject by administering a pharmaceutical composition comprising SEQ ID NO: 5, 7, 24, 29, 32, 34, 36 or 37 in a pharmaceutically acceptable carrier.

[0020] In one embodiment, the disclosure provides a method of making a compound of the invention, the method comprising co-transfecting a mammalian cell with a first polynucleotide comprising a nucleotide sequence encoding an amino acid sequence comprising an immunoglobulin heavy chain of SEQ ID NO: 22, 25, 30, or 39 and a second polynucleotide comprising a nucleotide sequence encoding an amino acid sequence comprising an immunoglobulin light chain of SEQ ID NO: 21, 26, 31, or 40, and collecting a supernatant from the mammalian cell, wherein the resulting compound binds to a viral antigen. In one aspect, the viral antigen is an HIV antigen.

[0021] In one aspect, the present invention provides an isolated DNA sequence encoding the amino acid sequence of SEQ ID NO:21, 22, 25, 26, 30, 31, 39, or 40. In another aspect, the present invention provides a pharmaceutical composition comprising SEQ ID NO:5, 7, 24, 29, 32, 34, 36, and 37 in a pharmaceutically acceptable carrier. In a further aspect, the present invention provides a method of treating a subject comprising administering to the subject a pharmaceutical composition comprising SEQ ID NO:5, 7, 24, 29, 32, 34, 36, and 37 in a pharmaceutically acceptable carrier. In a further aspect, the present invention provides a method of treating a subject having or at risk of developing AIDS comprising administering to the subject a pharmaceutical composition comprising SEQ ID NO:5, 7, 24, 29, and 37.

[0022] The above summary of the present invention is not intended to describe each disclosed embodiment or every implementation of the present invention. The following description more particularly points out exemplary embodiments. In several places throughout the application, guidance is provided through lists of examples, which examples can be used in various combinations. In each instance, the recited list serves only as a representative group and should not be interpreted as an exclusive list. [Brief explanation of the drawings]

[0023] [Figure 1A]The CD16 nanobody was derived from a publicly available llama nanobody (GeneBank sequence EF561291). The CD16 nanobody was spliced ​​to CD19 to test the ability of this CD16 engager to drive NK cell killing. (A) The CD16 nanobody demonstrated cytolytic NK activity similar to rituximab-mediated killing in a chromium release assay using CD19+ Raji targets. [Figure 1B] The CD16 nanobody was derived from a publicly available llama nanobody (GeneBank sequence EF561291). The CD16 nanobody was spliced ​​into CD19 to test the ability of this CD16 engager to drive NK cell killing. (B) The CD16 CDRs were cloned into a humanized camelid scaffold to generate the humanized CD16 engager, HuEF91. HuEF91 binding was comparable to CD16 scFv binding, indicating that humanization of HuEF91 did not interfere with the specificity of the molecule. [Figure 1C] The CD16 nanobody was derived from a publicly available llama nanobody (GeneBank sequence EF561291). The CD16 nanobody was spliced ​​to CD19 to test the ability of this CD16 engager to drive NK cell killing. (C) Llama 161533 TriKE (SEQ ID NO: 3) can expand NK cells. [Figure 2] Map of CAM1615PGT121 (SEQ ID NOs: 6-7). [Figure 3] CAM1615PGT121 nucleotide sequence (SEQ ID NO: 6). As further shown in Figure 4, lowercase and uppercase letters are used to indicate the domain structure of the nucleic acid sequence. [Figure 4] CAM16, hma linker, IL15 WT, EASGGPE linker, PGT121, and termination sequence of the CAM1615PGT121 nucleotide sequence (SEQ ID NO: 6). Lowercase and uppercase letters are used to indicate the domain structure of the nucleic acid sequence. [Figure 5]CAM1615PGT121 amino acid sequence (SEQ ID NO: 7). PGT121 amino acid sequence (SEQ ID NO: 8) is shown. [Figure 6] The two plasmids were co-transfected into mammalian cells to produce Fab-based antibodies. Amino acid sequence of the protein produced from Plasmid 1 containing humanized camelid anti-CD16 (SEQ ID NOs: 16 and 17). [Figure 7] Sequence of two proteins expressed from a single plasmid using the 2A self-cleaving peptide. (SEQ ID NO: 19) [Figure 8] Amino acid sequence of CAM16_IL15_12A12scFv (HIV TriKE) (SEQ ID NOs: 37 to 40). [Figure 9] Amino acid sequence of CAM16_IL15_VLC01_scFV_TriKE (SEQ ID NO: 24). [Figure 10] Amino acid sequence of CAM16_IL15_10E8_scFV_TriKE (SEQ ID NO: 29). [Figure 11A] Structure and function of HIV-specific BiKE and TriKE. Figure 11(A) is a schematic diagram showing the origin of the components of the initial bispecific HIV-targeting construct composed of an anti-CD16 short variable fragment linked to a Fab derived from the HIV broadly neutralizing antibody (bnAb) VRC01. [Figure 11B] Structure and function of HIV-specific BiKE and TriKE. Figure 11(B) Schematic and proposed function of the HIV dual-specific and trispecific killer engagers (BiKE and TriKE, respectively). [Figure 12A] HIV-Env-specific BiKE binds to CD16-expressing NK cells and HIV-infected cell lines and induces HIV-specific NK cell responses. (Figure 12(A)) Purified peripheral blood NK cells from healthy donors were stained with biotinylated anti-His and fluorescent dye-conjugated streptavidin for CD16, streptavidin control, or His-tagged BiKE. BiKE binds to NK cells that reflect CD16 expression. [Figure 12B]HIV-Env-specific BiKE binds to CD16-expressing NK cells and HIV-infected cell lines and induces HIV-specific NK cell responses. Figure 12(B) Uninfected CD4-expressing HeLa cells or HIV-infected HeLa-CD4 were stained with HIV-Env BiKE. BiKE specifically bound to infected HeLa-CD4 but not to uninfected HeLa-CD4, demonstrating BiKE's specificity for cells expressing the HIV envelope. [Figure 12C] HIV-Env-specific BiKE binds to CD16-expressing NK cells, an HIV-infected cell line, and induces HIV-specific NK cell responses. (Figure 12(C)) Purified healthy donor NK cells were incubated with infected or uninfected HeLa-CD4 cells with or without HIV-Env BiKE. K562 cells with Rituxin and Raji were used as controls. [Figure 13A] HIV-Env BiKE specifically binds to primary infected T cell lines and mediates NK cell killing. Figure 13 (A) Two HIV-infected T cell lines, H9 HIV-IIIB and ACH-2, or their uninfected counterparts, H9 and CEM CD4, were intracellularly stained for HIV capsid proteins to confirm active HIV replication. [Figure 13B] HIV-Env BiKE specifically binds to primary infected T cell lines and mediates NK cell killing. (Figure 13(B)) The same infected and uninfected T cell lines were stained with His-tagged HIV-Env BiKE and biotinylated anti-His+ streptavidin, or with secondary alone. BiKE showed no binding to uninfected T cell lines but bound to both infected clones, demonstrating specificity for actively infected T cells. [Figure 13C] HIV-Env BiKE specifically binds to primary infected T cell lines and mediates NK cell killing. (Figure 13(C)) Purified NK cells from healthy donors were co-cultured with uninfected or HIV-infected T cell lines with or without HIV-Env BiKE and assessed for NK degranulation (CD107a) and IFNγ production. [Figure 14]Figures 14A-14C. IL-15-containing HIV-TriKE activates immune subsets and induces viral transcription in latently infected primary and T cell lines. (A) Peripheral blood mononuclear cells were incubated with equimolar rhIL-15 or IL-15-containing HIV-TriKE for 16 hours. NK and T cell subsets were assessed for activation by CD69 expression by flow cytometry. Figure 14(B) A latently infected human CD4+ T cell line, ACH-2, was incubated for 48 hours in the presence of 10 nM PMA, 10 ng / mL rhIL-15, or equimolar IL-15-containing TriKE. Cells were then washed and intracellularly stained for HIV-gag (p24). Both IL-15 alone and TriKE induced significant viral reactivation, as indicated by p24 expression. (C) Purified CD4+ memory T cells were isolated from antiretroviral-treated HIV-infected patients and cultured with rhIL-15, the IL-15 superagonist Nant-803, or IL-15-containing TriKE. Each condition was incubated with or without the HDAC inhibitor SAHA for 72 hours. Cells were then harvested and nested PCR reactions were performed to identify HIV mRNA. [Figure 15] Targeting solid tumors with second-generation TriKE molecules via several antigens: NCI-H460: lung cancer (large cell lung cancer), NCI-H322: bronchoalveolar carcinoma (cervical lymph node metastasis), CSPG4: chondroitin sulfate proteoglycan 4, SS1: mesothelin. [Figure 16]Figures 16A-16D. Chromium release assays were performed with several different novel TriKEs, demonstrating that any scFv targeting cancer cells can be made into functional TriKEs. (A) EpCAM+CD133+NG2+ non-small cell lung cancer NCI-H460 cells and NK cells were incubated with 1615EPCAM133 TriKE or 1615NG2 TriKE (neuron-glial antigen 2 or CSPG4). Both 1615NG2 and 1615EpCAM133 were active at several different E:T ratios (20:1, 10:1, and 5:1). (B) Mesothelin+EpCAM-CD133-NG2 MDA-435A melanoma cells were incubated with 1615EPCAM TriKE or 1615Meso TriKE (SEQ ID NO: 36). Only 1615Meso was active. (C) Mesothelin+NG2+ ovarian cancer cells (Ovcar3 cells) were incubated with 1615NG2 TriKE or 1615SS1 TriKE. 1615Meso and 1615NG2 were active. (D) Raji cells were cultured with NK cells and examined in a 51Cr release assay. TriKE 16152219 (SEQ ID NO: 12) simultaneously targets the B cell markers CD19 and CD22. Only 16152219, 162219, and rituximab killed CD22+CD19+ targets. The control did not kill. [Figure 17] TriKE sequence (underlined) in SEQ ID NO: 3 (Lama 161533 TriKE). [Figure 18] CAM1615SS1 (mesothelin) amino acid sequence (SEQ ID NO: 32). The underlined sequence indicates the TriKE sequence. SS1 (mesothelin) scFV antibody fragment is shown (SEQ ID NO: 33). [Figure 19] PGT121Fab was generated using the 2A self-cleaving peptide. In alternative embodiments, for example, a two plasmid system or an IRES can be used. [Figure 20]Figures 20A-20B. Co-transfection of two plasmids into mammalian cells to produce Fab-based antibodies. Two separate plasmids (Plasmid 1 and Plasmid 2) were co-transfected into Expi-Cho-S cells for TriKE production. The amino acid sequence containing non-humanized camelid anti-CD16 is shown. DETAILED DESCRIPTION OF THE INVENTION

[0024] Detailed Description of the Invention Natural killer (NK) cells are cytotoxic lymphocytes of the innate immune system capable of immune surveillance. Similar to cytotoxic T cells, NK cells deliver reservoirs of membrane-permeabilizing and apoptosis-inducing granzymes and perforin granules. Unlike T cells, NK cells do not require antigen priming and recognize targets by engaging activating receptors in the absence of MHC recognition.

[0025] NK cells express CD16, an activating receptor that binds to the Fc portion of IgG antibodies and is involved in antibody-dependent cell-mediated cytotoxicity (ADCC). NK cells are regulated by IL-15, which can induce increased antigen-dependent cytotoxicity, lymphokine-activated killer activity, and / or mediate interferon (IFN), tumor necrosis factor (TNF), and / or granulocyte-macrophage colony-stimulating factor (GM-CSF) responses. All of these IL-15-activating functions contribute to improved cancer defense.

[0026] The present disclosure describes a multispecific therapeutic compound that may be a trispecific killer engager compound (TriKE). TriKE has three distinct binding regions: an NK cell-engaging domain that binds to NK cells (e.g., CD16), an NK activation domain that contains a cytokine or a functional fragment thereof and binds to the cytokine's receptor, and a targeting domain that binds to a marker present on target cells (e.g., cancer cells). The design and production of TriKE is extensively described, for example, in U.S. Patent Application Publication No. 2018 / 0282386, the entire contents of which are incorporated by reference. TriKE offers the advantage of combining an antibody-dependent cellular cytotoxicity (ADCC)-promoting moiety and an expansion-associated moiety (IL-15) on the same molecule.

[0027] Therapeutically, adoptive transfer of NK cells, for example in combination with lymphodepleting chemotherapy and IL-2, can induce remission in patients with refractory acute myeloid leukemia (AML) when stimulating NK cell survival and in vivo expansion. This therapy can be limited by a lack of antigen specificity and the IL-2-mediated induction of regulatory T (Treg) cells, which suppress NK cell proliferation and function. Generating reagents that drive NK cell antigen specificity, expansion, and / or persistence while avoiding the negative effects of Treg inhibition could enhance NK cell-based immunotherapy.

[0028] Advances in the effectiveness and use of antiretroviral drugs have substantially improved the health and lifespan of HIV-infected individuals, but these drugs are only a temporary fix to prevent progression to AIDS and limit further transmission of the virus. Despite the use of antiretroviral agents to suppress HIV replication, infected individuals retain a reservoir of latently infected HIV cells, which can reactivate and re-establish active infection upon discontinuation of antiretroviral therapy. A curative solution requires the reactivation and subsequent destruction of these latently infected cells. While antibody responses to HIV infection exist, they are generally ineffective due to the high rate of viral mutation, which allows the virus to rapidly eliminate epitopes recognized by generated antibodies. However, in recent years, various HIV-specific antibodies have been identified in infected individuals that possess strong neutralizing activity but a reduced ability to induce antibody-dependent cell-mediated cytotoxicity (ADCC).

[0029] Therefore, the present invention addresses these issues by providing bispecific and trispecific natural killer cell engagers (BiKE and TriKE), which consist of a short-chain variable fragment derived from a broadly neutralizing antibody (bnAb) against HIV-Env linked by an IL-15 molecule and a CD16 engager. The purpose of this trispecific antibody construct is to utilize the broad specificity of these antibodies to target HIV while simultaneously redirecting NK cell killing specifically to actively replicating infected cells through its recognition of membrane-expressed Env and triggering NK cell degranulation via the low-affinity Fc receptor, CD16. The addition of IL-15 as a linker further activates NK cells, thereby enhancing their response. IL-15 has also been identified as a potential reactivator of latently infected cells. Initial studies from our laboratory have shown that NK cell cytokine production and killing of infected targets expressing HIV-Env were enhanced when incubated with HIV-specific constructs. PBMCs from healthy donors incubated with TriKE were shown to induce a significant increase in immune cell activation in NK, CD4, and CD8 subsets, as well as proliferation of NK cells. Furthermore, IL-15, either as a monomer or as part of TriKE, exhibits the ability to reactivate latently HIV-infected T cells isolated from infected patients in vitro.

[0030] A recent trial of an IL-15 / IL-15Ra superagonist (Nant-803) in ART-treated HIV-infected patients also resulted in serum virus detection and immune activation. Collectively, these data demonstrate a role for TriKE-containing HIV-bnAbs in reactivating and clearing the latently infected reservoir by exploiting the ability of NK cells to mediate ADCC.

[0031] Bispecific fusions incorporating anti-human anti-CD16 scFvs derived from human phage display library technology have been generated (McCall et al., 1999. Mol Immunol. 36:433-445). NK cells mediate antibody-dependent cell-mediated cytotoxicity (ADCC) via the CD16 (FcγRIII) receptor. Signaling through the CD16 receptor induces calcium flux and ITAM phosphorylation, leading to the formation of lytic granules and the release of cytokines such as interferon (IFNγ) and tumor necrosis factor (TNFα). Bispecific molecules have been designed to target the CD16 receptor in conjunction with other target molecules (Gleason et al. Blood. 2014(19):3016-26), so-called bispecific killer engagers (BiKEs). By using one scFv to recognize NK cells and a second scFv to recognize tumor antigens, BiKEs can significantly enhance cytotoxic killing in various human cancers. One exemplary BiKE targeted CD33 and enhanced NK cell responses against acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS), a clonal heterogeneous stem cell disorder characterized by normal or hypercellular bone marrow (BM) with peripheral blood (PB) cytopenias and a high risk of progression to AML.

[0032] NK cells are responsive to various cytokines, including IL-15, which is involved in NK cell homeostasis, proliferation, survival, activation, and / or development. IL-15 and IL-2 share several signaling components, including the IL-2 / IL-15R (CD122) and common gamma chain (CD132). Unlike IL-2, IL-15 does not stimulate Tregs, allowing NK cell activation while bypassing Treg inhibition of the immune response. In addition to promoting NK cell homeostasis and proliferation, IL-15 can rescue functional defects of NK cells that may arise in the post-transplant setting. IL-15 can also stimulate CD8+ T cell function, further enhancing its immunotherapeutic potential. Additionally, based on preclinical studies, the toxicity profile of IL-15 may be more favorable than that of IL-2 at low doses.

[0033] IL-15 plays a role in the developmental homeostasis, proliferation, survival, and activation of NK cells. IL-15 and IL-2 share several signaling components, including the IL-2 / IL-15R (CD122) and common gamma chain (CD132). IL-15 can also activate NK cells and reverse functional defects in NK cell engraftment after hematopoietic stem cell transplantation (HSCT).

[0034] In one aspect, the present disclosure describes trispecific killer engager (TriKE) molecules that generally include one or more NK cell engager domains (e.g., CD16, CD16+CD2, CD16+DNAM, CD16+NKp46), one or more targeting domains (e.g., targeting tumor cells or virus-infected cells), and one or more cytokine NK activation domains (e.g., IL-15, IL-12, IL-18, IL-21, or other NK cell-enhancing cytokines, chemokines, and / or activating molecules), each of which is operably linked to the other domains. As used herein, the term "operably linked" refers to a direct or indirect covalent bond. Thus, two operably linked domains can be directly covalently linked to each other. Conversely, two operably linked domains can be linked by a mutual covalent bond to an intervening moiety (e.g., and adjacent sequence). Two domains may be considered operably linked, for example, if they are separated by a third domain, with or without one or more intervening flanking sequences. In an exemplary embodiment, the NK engaging domain is directed against CD16 and the NK activating domain is IL-15 or a functional fragment thereof.

[0035] An NK engaging domain can include any moiety that binds to and / or activates NK cells and / or blocks inhibition of NK cells. In some embodiments, an NK engaging domain can include an antibody that selectively binds to a component on the surface of an NK cell. In other embodiments, an NK engaging domain can include a ligand or small molecule that selectively binds to a component on the surface of an NK cell. As used herein, the term "selectively binds" refers to the ability to distinguish between two or more alternatives, e.g., having any degree of differing affinity for a particular target. As used herein, "antibody" generally refers to an immunoglobulin or fragment thereof, and thus encompasses monoclonal antibodies, fragments thereof (e.g., scFv, Fab, F(ab')2, Fv, or other modified forms), combinations of monoclonal antibodies and / or fragments thereof, and / or combinations of polyclonal antibodies. Thus, for brevity, reference to an antibody that selectively binds to a component on the surface of an NK cell includes any antibody fragment that exhibits the described binding characteristics. Similarly, reference to a ligand that selectively binds to a component on the surface of an NK cell includes any fragment of the ligand that exhibits the described binding characteristics.

[0036] In some embodiments, the NK engaging domain can selectively bind to a receptor located at least partially on the surface of an NK cell. In certain embodiments, the NK engaging domain can function to bind NK cells, thereby bringing the NK into spatial proximity with a target to which the targeting domain, described in more detail below, selectively binds. However, in certain embodiments, the NK engaging domain can selectively bind to a receptor that activates NK cells and thus also has an activation function. As described above, activation of the CD16 receptor can induce antibody-dependent cell-mediated cytotoxicity. Thus, in certain embodiments, the NK engaging domain can comprise at least a portion of an anti-CD16 receptor antibody effective to selectively bind to the CD16 receptor. In other embodiments, the NK engager cell domain can interfere with mechanisms that inhibit NK cells.

[0037] The NK engaging domain can be designed to have the desired degree of NK selectivity and, therefore, the desired immune engagement properties. For example, CD16 has been identified as the Fc receptors FcγRIIIa (CD16a) and FcγRIIIb (CD16b). These receptors bind to the Fc portion of IgG antibodies and then activate NK cells for antibody-dependent cell-mediated cytotoxicity. Anti-CD16 antibodies selectively bind to NK cells but can also bind to neutrophils. Anti-CD16a antibodies selectively bind to NK cells but not neutrophils. TriKE embodiments that include an NK engaging domain containing an anti-CD16a antibody can bind to NK cells but not neutrophils. Therefore, in situations where it is desired to engage NK cells but not neutrophils, the NK engaging domain of TriKE can be designed to include an anti-CD16a antibody.

[0038] Although described herein with respect to various embodiments in which the NK engaging domain comprises an anti-CD16 receptor scFv, the NK engaging domain can comprise any antibody or other ligand that selectively binds to the CD16 receptor. Additionally, the NK engaging domain can comprise an antibody or ligand that selectively binds to any NK cell receptor, such as, for example, cytotoxicity receptor 2B4, the low affinity Fc receptor CD16, killer immunoglobulin-like receptor (KIR), CD2, NKG2A, TIGIT, NKG2C, LIR-1, and / or DNAM-1.

[0039] The targeting domain can include any moiety that selectively binds to an intended target, such as, for example, a tumor cell, a target in the cancer stroma, a target on inhibitory cells such as myeloid-derived suppressor cells that are CD33+, or a target on a virally infected cell.

[0040] In other embodiments, the targeting domain can selectively bind to a target on a cell infected by a virus, such as, for example, adenovirus, HIV, CMV, and / or HPV, In illustrative examples herein, the targeting domain is an HIV epitope.

[0041] The NK activation domain may include an amino acid sequence that activates NK cells, promotes NK cell persistence, or otherwise promotes NK cell activity. The NK activation domain may be or be derived from one or more cytokines capable of activating and / or maintaining NK cells. As used herein, the term "derived from" refers to an amino acid fragment of a cytokine (e.g., IL-15) sufficient to provide NK cell activation and / or maintenance of activity. In embodiments including two or more NK activation domains, the NK activation domains may be provided tandemly or in any other combination. In addition, each cytokine-based NK activation domain may comprise any of the cytokine's complete amino acid sequence or may be an amino acid fragment, regardless of the nature of the other NK activation domains included in the TriKE molecule. Exemplary cytokines on which the NK activation domain can be based include, for example, IL-15, IL-18, IL-12, and IL-21. Thus, although described in detail herein with respect to an exemplary model embodiment in which the NK activation domain is derived from IL-15, TriKE may be designed using an NK activation domain that is or is derived from any suitable cytokine.

[0042] For brevity of this description, reference to an NK activation domain by identifying the cytokine on which it may be based includes both the complete amino acid sequence of the cytokine, any suitable amino acid fragment of the cytokine, and / or modified versions of the cytokine containing one or more amino acid substitutions. Thus, reference to an "IL-15" NK activation domain includes an NK activation domain comprising the complete amino acid sequence of IL-15, an NK activation domain comprising a fragment of IL-15, or an NK activation domain that contains an amino acid substitution compared to the wild-type IL-15 amino acid sequence, such as, for example, IL-15N72D or IL-15N72A.

[0043] The use of the IL-15 NK activation domain in TriKE can provide sustained NK cell activity, as demonstrated in a mouse model showing dramatic increases in human NK cells and reductions in cancer even after three weeks. NK cells are activated in mice and produce numerous anti-cancer factors and cytokines. The IL-15 NK activation domain somehow alters the chemical properties of these molecules so that they are more easily refolded and / or can be recovered in higher yields, thus making TriKE molecules more suitable for clinical scale-up.

[0044] In some embodiments, the molecule can further comprise a flanking sequence that can link two of the above-mentioned domains. In some embodiments, the presence of a flanking sequence can further increase NK cell activation. One exemplary flanking sequence comprises the 20 amino acids of SEQ ID NO: 1 (see also U.S. Patent Application Publication No. 2018 / 0282386). Another exemplary flanking sequence comprises the 7 amino acids of SEQ ID NO: 2. Certain embodiments include two or more flanking sequences. As an example, SEQ ID NO: 1 includes the flanking sequence of SEQ ID NO: 3, which links the NK engaging domain (e.g., anti-CD16 receptor scFv) to the NK activation domain (e.g., IL-15). SEQ ID NO: 1 also includes the flanking sequence of SEQ ID NO: 4, which links the NK activation domain to the targeting domain (e.g., anti-CD33 scFv).

[0045] 51Chromium release assays were performed using several different TriKEs, demonstrating that any scFv targeting cancer cells can be incorporated into functional TriKEs. Non-small cell lung cancer (NCI-H460) cells were incubated with 1615EPCAM133 TriKE or 1615NG2 TriKE. Both 1615NG2 and 1615EpCAM133 were active at several different E:T ratios (20:1, 10:1, and 5:1). Figure 19B shows melanoma cells incubated with 1615EPCAM133 TriKE. Mesothelin + EpCAM-NG2 MDA-435A melanoma cells were incubated with 1615EPCAM TriKE or 1615Meso TriKE (SEQ ID NO: 32). Only 1615Meso had activity. Ovarian cancer (Ovcar3) cells were incubated with 1615NG2 TriKE or 1615Meso TriKE. Both TriKEs induced NK cell lytic activity. We also generated an anti-leukemia TriKE that recognizes the leukemia markers CD19 and CD22. 16152219 TriKE was tested on CD22+CD19+ Raji cells and killed them very effectively (similar to rituximab). Together, these data demonstrate that any scFv can be inserted into the 1615X generalized TriKE structural platform, and the resulting TriKE can direct NK cells to expand in response to the scFv target. Additional exemplary TriKE molecules are listed in Table 1.

[0046] Table 1. Exemplary TriKE molecules TIFF0007821723000001.tif110160 * >30% enhanced ADCC or cytotoxic activity by the TriKE platform ** Expansion: TriKE enhances NK cell expansion, but BiKE does not. *** Activation: TriKE enhances the production of various anti-cancer cytokines, including INFγ and TNFα.

[0047] In some embodiments, NK cell engagers can involve the use of humanized CD16 engagers derived from animal nanobodies. While scFvs have heavy and light variable chain components connected by a linker, nanobodies consist of a single monomeric variable chain, e.g., a variable heavy chain or a variable light chain, that can specifically engage a target. Nanobodies can be derived from antibodies of any suitable animal, such as, for example, camelids (e.g., llamas or camels) or cartilaginous fish. Nanobodies can offer superior physical stability, the ability to bind to deep grooves, and increased production yields compared to larger antibody fragments.

[0048] In one exemplary embodiment, the nanobody-based NK engager molecule can comprise a humanized CD16 nanobody derived from the published llama nanobody (GeneBank sequence EF561291, Behar et al., 2008. Protein Eng Des Sel. 21(1):1-10) and designated EF91. Llama EF91 was initially constructed on a CD19-containing BiKE to test the ability of this CD16 engager to drive NK cell activation. It demonstrated similar functionality to rituximab-mediated killing in a chromium release assay using the Raji target (Figure 1A). Once the functionality of the molecule was confirmed, the CDRs were cloned into a humanized camelid scaffold (Vincke et al., 2009. J Biol Chem. 284(5):3273-3284) to humanize the CD16 engager, designated HuEF91. The binding of HuEF91 was comparable to that observed using a standard CD16 scFv, indicating that incorporating the llama nanobody variable heavy chain into a humanized backbone does not interfere with the specificity of the molecule. The use of HuEF91 as an NK engager in the TriKE molecules described herein can increase drug yield, increase stability, and / or increase the efficacy of NK cell-mediated ADCC.

[0049] In some embodiments, the immune engagers described herein can be used to stimulate a patient's own immune system to eliminate tumor cells. Studies have shown that T cells engineered to express chimeric antigen receptors (CARs) are potent clinical mediators of antitumor activity, but T-CAR production is costly and complex. Other disadvantages include the risk of cytokine toxicity and the long-term persistence of T-CARs, which can lead to interactions with healthy tissue or neoplastic transformation. As described herein, trispecific killer engagers can function as mediators of ADCC and can expand NK cells without the need for ex vivo genetic modification and gene therapy, offering potential advantages over T-CAR systems. Because immune engagers are rapidly cleared, responses cannot be sustained indefinitely, potentially reducing the risk of cytokine toxicity of immune engagers compared to T-CARs.

[0050] In some embodiments, the trispecific killer engager comprises a cytokine. In some embodiments, the trispecific killer engager preferably comprises IL-15. IL-15 does not induce Tregs, and IL-15 is a regulator of NK cells. In addition to improving activation and cytotoxicity, IL-15 can regulate and initiate anti-apoptotic and proliferative signals on NK cells, resulting in enhanced NK cell expansion and survival. These characteristics may be beneficial during the use of trispecific killer engagers in cancer treatment. In some embodiments, the inclusion of IL-15 in the trispecific killer engager can mediate direct delivery of TriKE to the NK / target cell synapse, potentially allowing IL-15 to accumulate at tumor sites more effectively than systemic IL-15.

[0051] In some embodiments, the immune engager increases immune cell-mediated cytokine secretion. In some embodiments, cytokine secretion is preferably antigen-specific. In some embodiments, the cytokines can include IFN-γ, GM-CSF, IL-6, IL-8, and / or TNF-α. In some embodiments, the cytokine production is preferably at physiological levels. In some embodiments, the cytokine production is at levels lower than those observed in NK cells stimulated with IL-12 / IL-18 (Papadakis et al., 2004. J Immunol. 172:7002-7007). As shown in Example 2, cytokine Luminex analysis was used to measure characteristic inflammatory cytokines, including GM-CSF, IL-6, IL-8, and TNF-α, and showed a statistically significant difference in GM-CSF secretion between BiKE and TriKE, but no difference in the secretion of other cytokines.

[0052] In some embodiments, the immune engager increases lymphocyte proliferation. Lymphocytes can include, for example, NK cells, γδ T cells, and / or CD8 T cells. TetraKE, or larger molecules, can be designed to contain two or more NK cell engager domains and / or two or more NK activation domains.

[0053] In another aspect, the present disclosure describes a method for killing target cells in a subject. Generally, the method comprises administering a TriKE molecule to a subject in an amount effective to induce NK-mediated killing of target cells. "Treatment" or variations thereof refers to any degree of reducing, limiting the progression, improving, or eliminating symptoms or signs associated with a condition. As used herein, "ameliorating" refers to any reduction in the degree, severity, frequency, and / or likelihood of a symptom or clinical sign characteristic of a particular condition, "symptom" refers to any subjective evidence of a disease or patient's condition, and "sign" or "clinical sign" refers to an objective physical finding associated with a particular condition that can be detected by a person other than the patient.

[0054] "Treatment" can be either therapeutic or prophylactic. "Therapeutic" and its variants refer to treatment that improves one or more existing symptoms or clinical signs associated with a condition. "Prophylactic" and its variants refer to treatment that limits, to any extent, the onset and / or appearance of symptoms or clinical signs of a condition. Generally, "therapeutic" treatment is initiated after a condition manifests in a subject, while "prophylactic" treatment is initiated before the condition manifests in a subject. Thus, in certain embodiments, the method can include prophylactic treatment of a subject at risk of developing a condition. "At risk" refers to a subject who may or may not actually have the described risk. Thus, for example, a subject "at risk" for developing a particular condition is one who has one or more signs of increased risk of having or developing a particular condition compared to an individual lacking one or more indicia, regardless of whether the subject exhibits any symptoms or clinical signs of having or developing the condition. Exemplary signs of a condition can include, for example, genetic predisposition, ancestry, age, sex, geographic location, lifestyle, or medical history. Treatment may be continued after symptoms have resolved, for example to prevent or delay recurrence.

[0055] For example, in the case of a subject infected with HIV, "treatment" can include reducing viral load and / or ameliorating symptoms.

[0056] In some cases, the treatment can involve administering a TriKE molecule to a subject so that the TriKE molecule can stimulate endogenous NK cells in vivo. Using the TriKE molecule as part of an in vivo treatment can make the NK cells antigen-specific, resulting in simultaneous costimulation, improved survival, and expansion. In other cases, TriKE can be used in vitro as an adjuvant for NK cell adoptive transfer therapy.

[0057] Thus, whether NK-activating TriKE or T cell-activating TriKE, the TriKE molecule may be administered before, during, or after a subject first exhibits symptoms or clinical signs of a condition. Treatment initiated before a subject first exhibits symptoms or clinical signs associated with a condition may reduce the likelihood that the subject will experience clinical evidence of the condition, reduce the severity of the symptoms and / or clinical signs of the condition, and / or result in complete resolution of the condition, compared to subjects not administered the TriKE molecule. Treatment initiated after a subject first exhibits symptoms or clinical signs associated with a condition may reduce the severity of the symptoms and / or clinical signs of the condition, and / or result in complete resolution of the condition, compared to subjects not administered the composition.

[0058] The TriKE molecule can be any embodiment of the TriKE molecule described above, having a targeting domain that selectively binds to an appropriate target cell population. In some cases, the target cells can include virally infected cells, and the method can include treating the viral infection. Thus, in some embodiments, the method can include ameliorating at least one symptom or clinical sign of the viral infection.

[0059] In various embodiments, for example, a TriKE targeting domain can include a polypeptide that selectively binds to, for example, mesothelin or a viral antigen on HIV.

[0060] As used herein, a "subject" can be any animal, such as, for example, a mammal (e.g., a human, dog, cat, horse, cow, sheep, goat, monkey, etc.). In certain embodiments, the subject can be a human.

[0061] The TriKE molecules described herein can be formulated with a pharmaceutically acceptable carrier. As used herein, "carrier" includes any solvent, dispersion medium, vehicle, coating, diluent, antibacterial and / or antifungal agent, isotonic agent, absorption delaying agent, buffer, carrier solution, suspension, colloid, etc. The use of such media and / or agents for 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 therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the composition. As used herein, "pharmaceutically acceptable" refers to a material that is not biologically or otherwise undesirable, i.e., the material can be administered to an individual together with the TriKE molecule without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained.

[0062] Therefore, the TriKE molecule may be formulated into a pharmaceutical composition. Pharmaceutical compositions can be formulated in various forms adapted to the preferred route of administration. Thus, the composition can be administered via known routes, including, for example, oral, parenteral (e.g., intradermal, transdermal, subcutaneous, intramuscular, intravenous, intraperitoneal, etc.), or topical (e.g., intranasal, intrapulmonary, intramammary, intravaginal, intrauterine, intradermal, transdermal, rectal, etc.). Pharmaceutical compositions can be administered to mucosal surfaces, for example, by administration to the nasal or respiratory mucosa (e.g., by spray or aerosol). The composition can also be administered via sustained or delayed release.

[0063] Therefore, TriKE molecule can be provided in any suitable form, including but not limited to solution, suspension, emulsion, spray, aerosol, or any form of mixture.The composition can be delivered in a formulation containing any pharmaceutically acceptable excipient, carrier, or vehicle.For example, the formulation can be delivered in a conventional topical dosage form such as cream, ointment, aerosol formulation, non-aerosol spray, gel, lotion, etc.The formulation can further include one or more additives, including, for example, adjuvants, skin penetration enhancers, colorants, fragrances, flavorings, moisturizers, thickeners, etc.

[0064] Preparation can be conveniently presented in unit dosage form, and can be prepared by the method well known in the field of pharmacy.The method of preparing the composition that contains pharmaceutically acceptable carrier comprises the step of associating TriKE molecule with carrier that constitutes one or more accessory components.Generally, preparation can be prepared by associating active molecule with liquid carrier, finely divided solid carrier, or both uniformly and / or intimately, and then, if necessary, shape the product into desired preparation.

[0065] The amount of TriKE molecule administered can vary depending on various factors, including, but not limited to, the specific TriKE molecule used, the subject's weight, physical condition, and / or age, and / or the administration route. Thus, the absolute weight of the TriKE molecule contained in a given unit dosage form can vary widely, depending on factors such as the subject's species, age, weight, and physical condition, and / or the administration method. Therefore, it is not practical to generally provide an amount that constitutes an amount of TriKE molecule that is effective for all possible uses. However, those skilled in the art can easily determine the appropriate amount by appropriately considering such factors.

[0066] In some embodiments, the methods can include administering sufficient TriKE molecules to provide a subject with a dose of, for example, about 100 ng / kg to about 50 mg / kg, although in some embodiments, the methods can be practiced by administering TriKE molecules at doses outside this range. In some of these embodiments, the methods include administering sufficient TriKE molecules to provide a subject with a dose of, for example, about 10 μg / kg to about 5 mg / kg, e.g., about 100 μg / kg to about 1 mg / kg.

[0067] Alternatively, the dose may be calculated using actual body weight obtained immediately before the start of the treatment course. For doses calculated in this manner, body surface area (m2) is calculated before the start of the treatment course using the Dubois method: m2 = (weight in kg 0.425 × height in cm 0.725) × 0.007184.

[0068] In some embodiments, the methods can include administering sufficient TriKE molecule to provide a dose of, for example, about 0.01 mg / m 2 to about 10 mg / m 2 .

[0069] In some embodiments, the TriKE molecule can be administered, for example, in a single dose to multiple doses per week, although in some embodiments, the method can be practiced by administering the TriKE molecule at a frequency outside this range. In certain embodiments, the TriKE molecule can be administered from about once per month to about five times per week.

[0070] In some embodiments, the method further comprises administering one or more additional therapeutic agents. The one or more additional therapeutic agents may be administered before, after, and / or simultaneously with the administration of the TriKE molecule. The TriKE molecule and the additional therapeutic agent 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 either component administered alone. The two components may be co-administered simultaneously or sequentially. Simultaneously co-administered components may be provided in one or more pharmaceutical compositions. Sequential co-administration of two or more components includes when the components are administered such that each component is simultaneously present at the treatment site. Alternatively, sequential co-administration of two components may include when at least one component is removed from the treatment site, but at least one cellular effect (e.g., cytokine production, activation of a specific cell population, etc.) resulting from the administration of the component persists at the treatment site until one or more additional components are administered to the treatment site. Thus, a co-administered combination may, in certain circumstances, include components that are never present in a chemical mixture with each other. In other embodiments, the TriKE molecule and additional therapeutic agent may be administered as part of a mixture or cocktail. In some aspects, administration of a TriKE molecule may allow for the effectiveness of lower doses of other therapeutic modalities compared to administration of the other therapeutic agent or agent alone, thereby reducing the likelihood, severity, and / or extent of toxicity observed when higher doses of the other therapeutic agent or agent are administered.

[0071] In some embodiments, the methods can include administering a sufficient TriKE molecule as described herein and at least one additional therapeutic agent that exhibits therapeutic synergy. In some aspects of the methods of the present invention, a measure of response to treatment observed after administering both a TriKE molecule as described herein and the additional therapeutic agent is improved compared to the same measure of response to treatment observed after administering either the TriKE molecule or the additional therapeutic agent alone. In some embodiments, the additional therapeutic agent can include an additional agent targeting HIV, such as atazanavir (Reyataz), darunavir (Prezista), fosamprenavir (Lexiva), lopinavir; ritonavir (Norvir); tipranavir (Aptivus), or acyclovir. Other antiviral agents are known to those skilled in the art and can be combined with the TriKe composition before, during, or after TriKe administration.

[0072] In the foregoing description and in the claims that follow, the term "and / or" means one or all of the listed elements or a combination of any two or more of the listed elements; the terms "comprises," "comprising," and variations thereof are to be interpreted as open-ended, i.e., additional elements or steps are optional and may or may not be present; unless otherwise specified, "a," "an," "the," and "at least one" are used interchangeably and mean one or more; and the recitation of numerical ranges by endpoints includes all numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).

[0073] In the foregoing description, certain embodiments may be described in isolation for clarity. A particular embodiment may include any combination of compatible features described herein in connection with one or more embodiments, unless expressly specified otherwise that a feature of a particular embodiment is incompatible with a feature of another embodiment.

[0074] For any method disclosed herein that includes discrete steps, the steps can be performed in any feasible order, and, where appropriate, any combination of two or more steps can be performed simultaneously.

[0075] The present invention is illustrated by the following examples, it being understood that the particular examples, materials, amounts, and procedures are to be interpreted broadly in accordance with the scope and spirit of the invention described herein. [Example]

[0076] Example 1 Construction of 1615 anti-HIV Because 1615x is a platform technology, it is possible to use antiviral scFvs related to or unrelated to cancer development. Synthesis and assembly of a hybrid polynucleotide encoding TriKE 1615 anti-HIV (SEQ ID NO: 5) was achieved using DNA shuffling and ligation techniques. The fully assembled polynucleotide contains, from the 5' to 3' end, an NcoI restriction site, an ATG start codon, the VH and VL regions of the anti-CD16 scFv, a 20-amino acid segment (PSGQAGAAASESLFVSNHAY SEQ ID NO: 1), modified IL-15, a 7-amino acid linker (EASGGPE SEQ ID NO: 2), and the anti-HIV scFv. Finally, it contains an XhoI restriction site.

[0077] HIV-envelope-specific, IL-15-containing trispecific killer engager (TriKE) both reactivates and directs NK cell killing toward HIV-infected T cells Advances in the effectiveness and use of antiretroviral drugs have substantially improved the health and lifespan of HIV-infected individuals, but these drugs are only a temporary fix to prevent progression to AIDS and limit further transmission of the virus. Despite the use of antiviral agents to suppress HIV replication, infected individuals retain a reservoir of latently infected HIV cells, which can reactivate and re-establish active infection upon discontinuation of antiretroviral therapy. A curative solution requires the reactivation and subsequent destruction of these latently infected cells. While antibody responses to HIV infection exist, they are generally ineffective due to the high rate of viral mutation, which allows the virus to rapidly eliminate epitopes recognized by generated antibodies. However, in recent years, various HIV-specific antibodies have been identified in infected individuals that possess strong neutralizing activity but a reduced ability to induce antibody-dependent cell-mediated cytotoxicity (ADCC). Therefore, we designed bispecific and trispecific killer engagers (BiKE and TriKE) consisting of a short-chain variable fragment derived from a broadly neutralizing antibody (bnAb) against HIV-Env linked by an IL-15 molecule and a CD16 engager. The purpose of this trispecific antibody construct is to utilize the broad specificity of these antibodies to target HIV while simultaneously redirecting NK cell killing specifically to actively replicating infected cells through its recognition of membrane-expressed Env and triggering NK cell degranulation via the low-affinity Fc receptor, CD16. The addition of IL-15 as a linker should further activate NK cells, thereby enhancing their response. IL-15 has also been identified as a potential reactivator of latently infected cells. Initial studies from our laboratory have shown that NK cell cytokine production and killing of infected targets expressing HIV-Env were enhanced when incubated with the HIV-specific constructs of the present invention. PBMCs from healthy donors incubated with TriKE showed a significant increase in immune cell activation in the NK, CD4 and CD8 subsets, as well as induced proliferation of NK cells.Furthermore, IL-15, either as a monomer or as part of TriKE, demonstrates the ability to reactivate latently HIV-infected T cells isolated from infected patients in vitro. Recent testing of an IL-15 / IL-15Rα superagonist (Nant-803) in ART-treated HIV-infected patients also resulted in serum virus detection and immune activation. Collectively, these data suggest a potential role for TriKE-containing HIV-bnAbs in reactivating and clearing the latently infected reservoir by exploiting the ability of NK cells to mediate ADCC.

[0078] As a further example, the broadly neutralizing antibodies (bnAbs) set forth in Table 2 below can be used in the compositions and methods described herein for 16 / 15 / X TriKE. The antibodies set forth in Table 2 can be used, for example, in TriKE constructs comprising camCD16 / IL-15 / anti-HIV bnAbs. Further examples of databases listing HIV-blocking antibodies useful in the present invention are published at the following sites, which are incorporated herein by reference in their entirety: https: / / web.archive.org / web / 20131230231821 / http: / / bnaber.org / , https: / / www.hiv.lanl.gov / content / immunology / ab_search, https: / / web.archive.org / web / 20131230231821 / http: / / bnaber.org / .

[0079] (Table 2) Broadly neutralizing HIV antibodies TIFF0007821723000002.tif239170 # The disclosures of the above patents and applications, particularly with respect to bnAb sequences, are incorporated herein by reference.

[0080] Example 2 Trispecific killer engager for mesothelin (TRIKE) targets NK cells to lung cancer NK cells are important effectors in the treatment of hematologic malignancies but have so far been less effective in the treatment of solid tumors. Lung cancer cells are generally resistant to NK cell killing, so we sought to determine whether small molecules that redirect NK cell lysis against a common tumor antigen, mesothelin, could enhance NK cell killing in the lung cancer setting. Mesothelin is a surface protein overexpressed in several cancers, including mesothelioma, an aggressive cancer of the lung lining.

[0081] Comparison of peripheral blood NK cells from healthy donors and newly diagnosed cancer patients revealed that lung cancer patients maintained cell surface expression of CD16 (an Fc receptor) without differences in the major subsets of NK cells. Therefore, we designed a trispecific killer engager (TriKE) consisting of a single-domain antibody (sdAb) against CD16 and a single-chain variable fragment (scFv) against mesothelin. The sdAb and scFv were linked together by recombinant IL-15. When tested with peripheral blood NK cells from healthy donors, this drug was able to enhance NK cell proliferation in vitro. Additionally, when peripheral blood NK cells were cultured with nine different lung cancer lines, TriKE specifically increased degranulation (some >60%) and IFNγ production (some >30%) against cancer cells.

[0082] NK cells derived from the peripheral blood of lung cancer patients also proliferated in response to the drug alone. Furthermore, when treated with TriKE, the patient's cells increased degranulation (>60%) and IFNγ production (>40%, which was significantly greater than the healthy donor response) in response to lung cancer cells.

[0083] Checkpoint blockade antibodies are the current standard of care for lung cancer patients, and our further studies will focus on combining this mesothelin-targeted TriKE with checkpoint blockade both in vitro and in vivo.

[0084] Example 3 Transient expression of anti-HIV / CAM16 BIKE and TRIKE Experimental design For VRC01-b12CL / VRC07H G54H (see Table 4 for bnAbs), we started by testing the VRC Fab-based BiKE first. bNAbs take years to evolve and accumulate three times as many mutations as other antibodies.

[0085] Expression constructs For expression in mammalian cell lines, VRC01-b12CL (light chain) was cloned into a suitable expression vector (pCoof40) with a short linker (GGGGS2) and non-humanized CAM16. The cloning method used was Gibson assembly or the related method HiFi assembly (New England Biolabs). Humanized CAM16 BiKE was constructed similarly. The VRC07H G54H plasmid was unmodified. Large-scale preparations of both plasmid DNAs were made using the ZymoPURE Plasmid Maxiprep Kit (Zymo Research).

[0086] Expression of BiKE protein We used suspension-adapted CHO or HEK293 cell lines (ExpiCHO / Expi293F) for high-level transient protein expression. A 1:1 ratio of heavy chain to light chain-containing plasmids (1 μg DNA / mL ExpiCHO medium) was cotransfected using the ExpiFectamine CHO or 293 transfection kit (Thermo Fisher Scientific) and incubated with shaking in a temperature- and CO2-controlled incubator according to the manufacturer's instructions. After 4–5 days, or when viability was measured as less than 75% viable cells, the supernatant was harvested by centrifugation at 2000 RPM in a benchtop centrifuge followed by filtration through a 0.22 micrometer filter. The filtered supernatant was then stored at -80°C.

[0087] When ready for purification, the drug was thawed and isolated using TALON metal affinity resin according to the manufacturer's instructions. Drug was then quantified using a Bradford assay with BSA as the standard. Purity was assessed using densitometry on a Bolt™ 4-12% Bis-Tris+SDS-Page gel run in Bolt™ MES SDS running buffer and stained with Coomassie dye G-250.

[0088] Example 4 HIV-specific BIKE and TRIKE Figure 11 shows the structure and proposed function of HIV-specific BiKE and TriKE. Figure 11(A) is a schematic diagram showing the origin of the components of the initial bispecific HIV-targeting construct, composed of an anti-CD16 short-chain variable fragment linked to a Fab derived from the HIV broadly neutralizing antibody (bnAb) VRC01. Figure 11(B) is a schematic and proposed function of the HIV bispecific and trispecific killer engagers (BiKE and TriKE, respectively). BiKE binds to the HIV envelope expressed on infected cells via the bnAb component, while the anti-CD16 portion binds to NK cells, signals via CD16, and elicits a functional response. Similar activity is proposed using TriKE, with the added ability of an IL-15 linker to activate NK cells, enhance their function, and induce a proliferative response. The IL-15 component may also activate latently infected T cells, thereby making them susceptible to recognition by HIV-TriKE and the immune system as a whole.

[0089] Figure 12 shows that HIV-Env-specific BiKE binds to CD16-expressing NK cells and HIV-infected cell lines and induces HIV-specific NK cell responses. Figure 12(A) Purified peripheral blood NK cells from a healthy donor were stained with biotinylated anti-His and fluorescent dye-conjugated streptavidin for CD16, streptavidin control, or His-tagged BiKE. BiKE binds to NK cells that reflect CD16 expression. Figure 12(B) Uninfected CD4-expressing HeLa cells or HIV-infected HeLa-CD4 cells were stained with HIV-Env BiKE. BiKE specifically bound to infected HeLa-CD4 but not uninfected HeLa-CD4, demonstrating BiKE specificity for cells expressing the HIV envelope. Figure 12(C) Purified healthy donor NK cells were incubated with infected or uninfected HeLa-CD4 cells with or without HIV-Env BiKE. K562 cells with Rituxin and Raji were used as controls. HIV-Env BiKE induced strong degranulation and cytokine responses only in infected HeLa cells, demonstrating the specific activity of BiKE. These responses were comparable to those of the Raji + Rituxin control.

[0090] Figure 13 shows that HIV-Env BiKE specifically binds to primary infected T cell lines and mediates NK cell killing. Figure 13(A) Two HIV-infected T cell lines, H9 HIV-IIIB and ACH-2, or their uninfected counterparts, H9 and CEM CD4, were intracellularly stained for HIV capsid proteins to confirm active HIV replication. Figure 13(B) The same infected and uninfected T cell lines were stained with His-tagged HIV-Env BiKE and biotinylated anti-His+ streptavidin, or with secondary alone. BiKE showed no binding to uninfected T cell lines but bound to both infected clones, demonstrating specificity for active infected T cells. Figure 13(C) Purified NK cells from healthy donors were cocultured with uninfected or HIV-infected T cell lines with or without HIV-Env BiKE and assessed for NK degranulation (CD107a) and IFNg production. HIV-BiKE enhanced both NK cell degranulation and cytokine production specifically in infected, but not uninfected, T cell lines.

[0091] Figure 14 shows that IL-15-containing HIV-TriKE activates immune subsets and induces viral transcription in latently infected primary and T cell lines. (A) Peripheral blood mononuclear cells were incubated with equimolar rhIL-15 or IL-15-containing HIV-TriKE for 16 hours. NK and T cell subsets were assessed for activation by CD69 expression by flow cytometry. (B) The latently infected human CD4+ T cell line, ACH-2, was incubated for 48 hours in the presence of 10 nM PMA, 10 ng / mL rhIL-15, or equimolar IL-15-containing TriKE. Cells were then washed and intracellularly stained for HIV-gag (p24). Both IL-15 alone and TriKE induced significant viral reactivation, as indicated by p24 expression. (C) Purified CD4+ memory T cells were isolated from antiretroviral-treated HIV-infected patients and cultured with rhIL-15, the IL-15 superagonist Nant-803, or IL-15-containing TriKE. Each condition was incubated with or without the HDAC inhibitor SAHA for 72 hours. Cells were then harvested and nested PCR reactions were performed to identify HIV mRNA.

[0092] Both BiKE and TriKE molecules can be produced using the methods described above, including, for example, cotransfection of two plasmids or polynucleotides encoding the light and heavy chains separately. The two proteins can also be produced from a single plasmid or polynucleotide, for example, using a 2A self-cleaving peptide or an IRES. Exemplary BiKE and TriKE molecules and amino acid sequences are shown in the figures and sequences herein.

[0093] In addition to the various embodiments described in the specification above, the following additional embodiments are contemplated herein. Embodiment 1. an NK engaging domain comprising a portion that selectively binds to CD16; an NK activating domain operably linked to an NK engaging domain, comprising IL-15 or a functional fragment thereof; a targeting domain that selectively binds to a viral antigen and is operably linked to an NK activating domain and an NK engaging domain; A compound comprising: Embodiment 2. The compound of claim 1, wherein CD16 comprises CD16a. Embodiment 3. The compound of claim 1, wherein the viral antigen is present on an infected cell. Embodiment 4. The compound of claim 1, wherein the viral antigen is derived from HIV, CMV, HPV, HCV, or adenovirus. Embodiment 5. The compound of claim 1, wherein the viral antigen is derived from HIV. Embodiment 6. The compound of claim 1, wherein the NK engaging domain portion comprises an antibody or binding fragment thereof, or a nanobody. Embodiment 7. The compound of claim 6, wherein the antibody fragment comprises an scFv, F(ab)2, or Fab. Embodiment 8. The compound of claim 6, wherein the antibody or binding fragment thereof or nanobody is human, humanized, or camelid. Embodiment 9. The compound of claim 6, wherein the antibody or binding fragment thereof or nanobody is camelid. Embodiment 10. The compound of claim 6, wherein the IL-15 comprises the amino acid sequence of SEQ ID NO: 4 or a functional variant thereof. Embodiment 11. The compound of claim 10, wherein the functional variant of IL-15 comprises an N72D or N72A amino acid substitution compared to SEQ ID NO:4. Embodiment 12. The compound of claim 1, wherein the targeting domain portion comprises an antibody or binding fragment thereof, or a nanobody. Embodiment 13. The compound of claim 12, wherein the antibody-binding fragment comprises an scFv, F(ab)2, or Fab. Embodiment 14. The compound of claim 1, wherein the NK engaging domain comprises CD16, the NK activation domain comprises IL-15, and the targeting domain selectively binds to a viral antigen derived from HIV. Embodiment 15. The compound of claim 1, wherein the NK engaging domain comprises CD16a, the NK activation domain comprises IL-15, and the targeting domain selectively binds to a viral antigen derived from HIV. Embodiment 16. The compound of claim 1, wherein the NK engaging domain comprises NKG2c, the NK activation domain comprises IL-15, and the targeting domain selectively binds to a viral antigen derived from HIV. Embodiment 17. The compound of claim 1, comprising at least one flanking sequence connecting two of the domains. Embodiment 18. The compound of claim 17, further comprising a second flanking sequence connecting the two linked domains with a third domain. Embodiment 19. The compound of claim 18, wherein the flanking sequences flank the NK activation domain. Embodiment 20. The compound of claim 18, wherein the first flanking sequence is C-terminal to the NK engaging domain and the second flanking sequence is N-terminal to the antiviral targeting domain. Embodiment 21. The compound of claim 1, further comprising a second targeting domain. Embodiment 22. The compound of claim 1, further comprising a second NK-engaging domain. Embodiment 23. The compound of claim 1, further comprising a second NK activation domain. Embodiment 24. The compound of claim 1, which is SEQ ID NO: 5, 7, 24, 29, or 37. Embodiment 25. A compound according to any one of claims 1 to 24, a pharmaceutically acceptable carrier; A composition comprising: Embodiment 26. Administering to a subject a compound according to any one of claims 1 to 25 in an amount effective to induce NK-mediated killing of target cells. A method comprising: Embodiment 27. The method of claim 26, wherein the target cell is infected with a virus. Embodiment 28. The method of claim 27, wherein the virus is HIV, CMV, HPV, HCV, or adenovirus. Embodiment 29. The method of claim 28, wherein the virus is HIV. Embodiment 1. A method for stimulating the expansion of NK cells in vivo, comprising: A method comprising administering to a subject an amount of a compound according to any one of claims 1 to 25 effective to stimulate the expansion of NK cells in the subject. Embodiment 31. The method of claim 30, wherein the subject is infected with a virus. Embodiment 32. The method of claim 31, wherein the virus is HIV, CMV, HPV, HCV, or adenovirus. Embodiment 33. The method of claim 32, wherein the virus is HIV. Embodiment 34. 1. A method of treating a viral infection in a subject, comprising: A method comprising administering to a subject an amount of a compound according to any one of claims 1 to 25 effective to treat a viral infection. Embodiment 35. The method of claim 34, wherein the subject is infected with HIV, CMV, HPV, HCV, or adenovirus. Embodiment 36. The method of claim 35, wherein the subject is infected with HIV. Embodiment 37. An isolated nucleic acid sequence of SEQ ID NO:6. Embodiment 38. An isolated amino acid sequence of SEQ ID NO:7. Embodiment 39. An isolated amino acid sequence comprising the sequence camCD16 / IL-15 / SEQ ID NO:8. Embodiment 40. An isolated amino acid sequence comprising SEQ ID NOs: 9, 17, 27, 28, 13, 15, 16, 17, 18, 19, 20. Embodiment 41. The isolated amino acid of claim 40, further comprising the isolated amino acid sequence of SEQ ID NO: 10. Embodiment 42. An isolated amino acid sequence comprising SEQ ID NO: 18 operably linked to IL-15. Embodiment 43. An isolated amino acid sequence of SEQ ID NOs: 20-26. Embodiment 44. (i) co-transfecting a mammalian cell with a first polynucleotide comprising a nucleotide sequence encoding an amino acid sequence comprising an immunoglobulin heavy chain of SEQ ID NO: 22, 25, 30, or 39 and a second polynucleotide comprising a nucleotide sequence encoding an amino acid sequence comprising an immunoglobulin light chain of SEQ ID NO: 21, 26, 31, or 40, respectively; and (ii) harvesting the supernatant from the mammalian cells; 25. A method for producing the compound according to any one of claims 1 to 24, comprising: Embodiment 45. The method of claim 44, wherein the viral antigen is derived from HIV. Embodiment 46. The method of claim 45, wherein the viral antigen is Env. Embodiment 47. An isolated DNA sequence encoding the amino acid sequence of SEQ ID NO: 21, 22, 25, 26, 30, 31, 39, or 40. Embodiment 48. A pharmaceutical composition comprising SEQ ID NOs: 7, 24, 29, 32, 34, 36, and 37 in a pharmaceutically acceptable carrier. Embodiment 49. A method of treating a subject, comprising administering to the subject a pharmaceutical composition comprising SEQ ID NOs: 5, 7, 24, 29, 32, 34, 36, and 37 in a pharmaceutically acceptable carrier. Embodiment 50. A method of treating a subject having or at risk of developing AIDS, comprising administering to the subject a pharmaceutical composition comprising SEQ ID NOs: 5, 7, 24, 29, and 37.

[0094] The complete disclosures of all patents, patent applications, and publications cited herein, as well as electronically available materials, are incorporated by reference in their entirety. Although the present invention has been described with reference to the above examples, it will be understood that modifications and variations are encompassed within the spirit and scope of the invention. Accordingly, the present invention is limited only by the following claims.

[0095] array TIFF0007821723000003.tif244158TIFF0007821723000004.tif248158TIFF0007821723000005.tif243158 TIFF0007821723000006.tif249158TIFF0007821723000007.tif248158TIFF0007821723000008.tif188160

Claims

1. an NK engaging domain comprising amino acids 1-240 of SEQ ID NO:11, SEQ ID NO:18, or SEQ ID NO:5; a first flanking sequence C-terminal to the NK engaging domain; an NK activating domain operably linked to said NK engaging domain, said NK activating domain comprising IL-15; a second flanking sequence C-terminal to the NK activation domain; a targeting domain that selectively binds to a viral antigen, operably linked to said NK activation domain and said NK engaging domain, said targeting domain comprising the amino acid sequence of SEQ ID NO:8, SEQ ID NOs:25 and 26, SEQ ID NOs:30 and 31, or SEQ ID NOs:39 and 40, wherein said viral antigen is an HIV antigen; and comprising the amino acid sequence of SEQ ID NO: 7, 24, 29, or 37; compound.

2. The compound of claim 1 , wherein the viral antigen is present on an infected cell.

3. 2. The compound of claim 1, wherein the NK-engaging domain portion comprises an antibody or a binding fragment thereof or a nanobody.

4. The compound of claim 3 , wherein the antibody fragment comprises an scFv, F(ab)2, or Fab.

5. The compound of claim 3, wherein the antibody or binding fragment thereof or the nanobody is humanized or camelid.

6. The compound of claim 3, wherein the antibody or binding fragment thereof or the nanobody is from a camelid.

7. The compound of claim 3, wherein the IL-15 comprises the amino acid sequence of SEQ ID NO:

4.

8. The compound of claim 1 , wherein the targeting domain portion comprises an antibody or a binding fragment thereof or a nanobody.

9. The compound of claim 8 , wherein the antibody-binding fragment comprises an scFv, F(ab)2, or Fab.

10. The compound of claim 1 further comprising a second targeting domain.

11. 10. The compound of claim 1, further comprising a second NK-engaging domain.

12. The compound of claim 1 further comprising a second NK activation domain.

13. A compound according to any one of claims 1 to 12; a pharmaceutically acceptable carrier; A composition comprising:

14. A pharmaceutical composition comprising a compound according to any one of claims 1 to 12 in an amount effective to induce NK-mediated killing of a target cell.

15. 15. The pharmaceutical composition of claim 14, wherein the target cell is infected with HIV.

16. A pharmaceutical composition for stimulating the expansion of NK cells in a subject, comprising an amount of a compound according to any one of claims 1 to 12 effective to stimulate the expansion of NK cells.

17. 17. The pharmaceutical composition of claim 16, wherein the subject is infected with HIV.

18. A pharmaceutical composition for treating a viral infection in a subject, comprising a compound according to any one of claims 1 to 12 in an amount effective to treat the viral infection.

19. 19. The pharmaceutical composition of claim 18, wherein the subject is infected with HIV.

20. (i) transfecting a mammalian cell with a polynucleotide comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:8, SEQ ID NOs:25 and 26, SEQ ID NOs:30 and 31, or SEQ ID NOs:39 and 40; and (ii) collecting the supernatant from the mammalian cells. wherein the resulting compound binds to a viral antigen.

21. 21. The method of claim 20, wherein the viral antigen is derived from HIV.

22. 22. The method of claim 21, wherein the viral antigen is Env.

23. A pharmaceutical composition comprising a protein comprising the amino acid sequence of SEQ ID NO: 7, 24, 29, or 37 in a pharmaceutically acceptable carrier.

24. A pharmaceutical composition for treating a subject, comprising a protein comprising the amino acid sequence of SEQ ID NO: 7, 24, 29, or 37 in a pharmaceutically acceptable carrier.

25. A pharmaceutical composition for treating a subject having or at risk of developing AIDS, comprising a protein comprising the amino acid sequence of SEQ ID NO: 7, 24, 29, or 37.

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