NK agent compound binding to viral antigen and method of use

Abstract

The present disclosure describes a compound involved in NK cells and a method of using the compound. Generally, the compound comprises an NK engaging domain, a targeting domain that selectively binds to a target cell, and an NK activating domain that operably links the NK engaging domain and the targeting domain. In an exemplary embodiment, the targeting domain selectively binds to an HIV antigen.

Description

Technology Field

[0001] Cross-references regarding related applications

[0002] This application claims the benefit of priority to U.S. Serial No. 62 / 906,660 filed September 26, 2019, pursuant to 35 USC §119(e), the entire contents of which are incorporated herein by reference.

[0003] Government-supported technology

[0004] The present invention was made with government support pursuant to CA111412 and CA065493 awarded by the National Institutes of Health and CA036725, CA072669, CA077598 and CA197292 awarded by the National Cancer Institute. The government holds certain rights to the present invention.

[0005] Inclusion in sequence list

[0006] The data in the attached sequence list is incorporated by reference into this application. The attached sequence list text file, named GTBIO2130_1WO_Sequence_Listing.txt, was created on September 25, 2020, and is 96 kb. The file can be accessed using Microsoft Word on a computer running a Windows OS. Background Technology

[0007] 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 are involved in antibody-dependent cell-mediated cytotoxicity (ADCC). NK cells are regulated by IL-15. IL-15 can induce increased antigen-dependent cytotoxicity and lymphokine-activated killer activity, or mediate cytokine responses. NK cells can be activated to stimulate immune responses for the treatment of cancer and infectious diseases as NK-cell-based immunotherapy.

[0008] Although advancements in the efficacy and uses of antiretroviral drugs substantially improve the health and lifespan of HIV-infected individuals, these drugs are merely a stopgap measure to prevent progression to AIDS and limit further transmission of the virus. Despite the use of antivirals to suppress HIV replication, infected individuals retain a reservoir of latently HIV-infected cells capable of reactivating and re-establishing an active infection upon discontinuation of antiretroviral therapy. Therapeutic solutions require the reactivation and subsequent destruction of these latently infected cells. Antibody responses to HIV infection are generally ineffective due to the high mutation rate of the virus, which prevents the rapid elimination of epitopes recognized by the generated antibodies while they are present. However, various HIV-specific antibodies have recently been identified in infected individuals that possess strong neutralizing effects but lack the ability to induce antibody-dependent cell-mediated cytotoxicity (ADCC). (Patent Document 1) Korean Published Patent Application No. 10-2018-0057715

[0009] The present invention provides a compound that activates NK cells to stimulate an immune response for treating cancer and viral infections. The inventors have designed dual- and triple-specific killing agents (BiKE and TriKE™) composed of CD16 agents linked by IL-15 molecules. As used herein, the compound of the present invention may be referred to as 16 / 15 / X TriKE, where X represents a targeting domain. The X targeting domain may be indicated, for example, as a viral antigen, a cancer cell antigen, etc.

[0010] In certain embodiments, antibody constructs are engineered to utilize the broad specificity of these antibodies for target HIV by specifically re-inducing NK cell death to actively replicate infected cells through their recognition of membrane-expressed Env, and by triggering NK cell degranulation via the low-affinity Fc receptor, CD16. The addition of IL-15 as a linker further activates NK cells to enhance their response. IL-15 has also been identified as a potential reactivator of latently infected cells. Early studies indicate that NK cell cytokine production and the death of infected targets expressing HIV-Env are enhanced when incubated with the HIV-specific constructs of the present invention.

[0011] In one embodiment, the present invention provides a compound having an NK-involved domain comprising a moiety that selectively binds to CD16 or NKG2c; an NK-activating domain operably linked to the NK-involved domain comprising IL-15 or a functional fragment thereof; and a targeting domain that selectively binds to a viral antigen and operably linked to the NK-activating domain and the NK-involved domain. In some embodiments, 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-involved domain moiety comprises an antibody or a binding fragment thereof or a nanobody. In some embodiments, the antibody fragment comprises scFv, F(ab)2, or Fab. In some embodiments, the antibody or its binding fragment or nanobody is human. In some embodiments, the antibody or its binding fragment or nanobody is camelid. In some embodiments, 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 comprises a moiety antibody or a binding fragment thereof or a nanobody. In some embodiments, the antibody binding fragment comprises scFv, F(ab)2, or Fab. In some embodiments, the NK engaging domain comprises CD16, the NK activating 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 activating domain comprises IL-15, and the targeting domain selectively binds to a viral antigen derived from HIV.In some embodiments, the NK involvement 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 compound described herein comprises at least one side sequence adjacent to two of the domains. In some embodiments, the compound described herein further comprises a second side sequence connecting the two connected domains to a third domain. In some embodiments, the side sequence adjacent to the NK activation domain. In some embodiments, the first side sequence is C-terminus for the NK involvement domain, and the second side sequence is N-terminus for the antiviral targeting domain. In some embodiments, the compound described herein further comprises a second targeting domain. In some embodiments, the compound described herein further comprises a second NK involvement domain. In some embodiments, the compound described herein further comprises a second NK activation domain.

[0012] In addition, a composition comprising the compounds described in this specification and a pharmaceutically acceptable carrier is provided.

[0013] Additionally, in some embodiments, the method comprises the step of administering to a subject a compound described herein in an amount effective for inducing NK-mediated death of target cells. In some embodiments, the target cells are infected with a virus. In some embodiments, the virus is HIV, CMV, HPV, HCV, or adenovirus. In some embodiments, the virus is HIV.

[0014] In some embodiments, a method for stimulating the expansion of NK cells in vivo is provided, comprising the step of administering to a subject an amount of the compound described herein that is effective for stimulating 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 embodiment, the virus is HIV.

[0015] In some embodiments, the present specification provides a method for treating a viral infection in a subject, comprising the step of administering to the subject an amount of a compound described herein that is effective for treating 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.

[0016] In some embodiments, the present specification provides a compound comprising: a T cell engaging domain having a moiety that selectively binds to CD3; a T cell activating domain operably linked to the T cell engaging 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 operably linked to the T cell activating 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.

[0017] In some embodiments, the present specification provides a method comprising the step of administering to a subject a compound described herein in an amount effective for inducing T-cell-mediated death 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 the HIV Env protein (e.g., gp120).

[0018] In some embodiments, the present specification provides a method for stimulating the expansion of T cells in vivo, comprising the step of administering to a subject an amount of the compound described herein that is effective for stimulating the expansion of T 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 some embodiments, the virus is HIV.

[0019] In one embodiment, the present invention provides a method for treating mesothelioma comprising the step of administering to a subject a compound comprising the amino acid sequence of SEQ ID NO. 32 or 36, which includes the target domain of SEQ ID NO. 33, in an amount effective for inducing NK-mediated death of target cells expressing mesothelin.

[0020] In some embodiments, the present specification provides a method for preparing the compound described herein comprising (i) co-transfecting a first polynucleotide having a nucleotide sequence encoding an amino acid sequence containing an immunoglobulin heavy chain and a second polynucleotide having a nucleotide sequence encoding an amino acid sequence containing an immunoglobulin light chain within a mammalian cell, and (ii) collecting the 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 chain and heavy chain for the anti-HIV antibody comprise SEQ ID NOs 21 and 22; 31 and 30; 26 and 25; and 40 and 39, respectively.

[0021] In some embodiments, the present specification provides an isolated DNA sequence encoding the amino acid sequence described in the present specification.

[0022] The present invention also provides a pharmaceutical composition comprising the TriKE compounds described herein. For example, the present invention provides a pharmaceutical composition comprising SEQ ID NOs 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 NOs 5, 7, 24, 29, 32, 34, 36, or 37 in a pharmaceutically acceptable carrier.

[0023] In one embodiment, the present disclosure provides a method for preparing a compound of the present invention, comprising co-transfecting a first polynucleotide comprising a nucleotide sequence encoding an amino acid sequence containing 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 containing an immunoglobulin light chain of SEQ ID NO. 21, 26, 31, or 40, respectively, within a mammalian cell; and collecting a supernatant from the mammalian cell, wherein the obtained compound binds to a viral antigen. In one embodiment, the viral antigen is an HIV antigen.

[0024] In one embodiment, the present invention provides an isolated DNA sequence encoding the amino acid sequence of SEQ ID NOs 21, 22, 25, 26, 30, 31, 39, or 40. In another embodiment, the present invention provides a pharmaceutical composition comprising SEQ ID NOs 5, 7, 24, 29, 32, 34, 36, and 37 in a pharmaceutically acceptable carrier. In a further embodiment, the present invention provides a method for treating a subject comprising the step of 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. In a further embodiment, the present invention provides a method for treating a subject having AIDS or at risk of developing AIDS, comprising the step of administering to the subject a pharmaceutical composition comprising SEQ ID NOs 5, 7, 24, 29, and 37.

[0025] The above summary of the invention is not intended to describe every disclosed embodiment or every implementation of the invention. The following description illustrates exemplary embodiments more specifically. Throughout this application, guidance is provided in several places through a list of embodiments, which may be used in various combinations. In each case, the cited list serves only as a representative group and should not be interpreted as a standalone list. Brief explanation of the drawing

[0026] FIGS. 1a to 1c. The CD16 nanobody was derived from the disclosed llama nanobody (GeneBank sequence EF561291). The CD16 nanobody was conjugated to CD19 to test the ability of this CD16 agent to induce NK cell death. (a) The CD16 nanobody exhibited cytotoxic NK activity similar to rituximab-mediated death in a chromium release assay targeting CD19+Raji. (b) The CD16 CDR was cloned into a humanized camelid scaffold to generate the humanized CD16 agent HuEF91. The binding of HuEF91 was identical to that of CD16scFv, indicating that the humanized HuEF91 did not interfere with the specificity of the molecule. (c) The llama161533 TriKE (Sequence No. 3) can expand NK cells. Fig. 2. Map of CAM1615PGT121 (sequence numbers 6 and 7). Fig. 3. CAM1615PGT121 nucleotide sequence (sequence number 6). Lowercase and uppercase letters are used to represent the domain structure of the nucleic acid sequence, as further illustrated in Fig. 4. Fig. 4. CAM16, hma linker, IL15 WT, EASGGPE linker, PGT121, and stop sequence of the CAM1615PGT121 nucleotide sequence (Sequence No. 6). Lowercase and uppercase letters are used to indicate the domain structure of the nucleic acid sequence. Fig. 5. CAM1615PGT121 amino acid sequence (Sequence No. 7). PGT121 amino acid sequence (Sequence No. 8) is shown. Fig. 6. Two plasmids were co-transfected into mammalian cells to generate Fab-based antibodies. The amino acid sequence of the protein was generated from a plasmid containing humanized camelid anti-CD16 (SEQ ID NOs 16, 17). Fig. 7. Sequences of two proteins expressed from a single plasmid using 2A self-cleaving peptide (Sequence No. 19). Fig. 8. Amino acid sequence of CAM16_IL15_12A12scFv (HIV TriKE) (Sequence Nos. 37-40). Fig. 9. Amino acid sequence of CAM16_IL15_VLC01_scFV_TriKE (Sequence No. 24). Fig. 10. Amino acid sequence of CAM16_IL15_ 10E8_scFV_TriKE (Sequence No. 29). FIGS. 11a and 11b. Structure and function of HIV-specific BiKEs and TriKEs. Figure 11a illustrated is a schematic diagram illustrating the origin of the components for an initial bispecific HIV-targeting construct consisting of an anti-CD16 short-chain variable fragment linked to a Fab derived from the HIV broad-spectrum neutralizing antibody (bnAb) VRC01. Figure 11b is a schematic diagram of HIV bispecific and trispecific killing agents (BiKE and TriKE, respectively) and their proposed functions. FIGS. 12a to 12c. HIV-Env-specific BiKE binds to CD16-expressing NK cells and HIV-infected cell lines, and induces HIV-specific NK cell responses. FIG. 12a illustrates healthy donor-purified peripheral blood NK cells stained with biotinylated anti-His and fluorochrome-conjugated streptavidin for CD16, streptavidin control, or His-tagged BiKE. BiKE binds to NK cells reflecting CD16 expression. FIG. 12b illustrates uninfected CD4-expressing HeLa cells or HIV-infected HeLa-CD4s stained with HIV-Env BiKE. BiKE specifically binds to infected HeLa-CD4s but not to uninfected HeLa-CD4s, indicating the specificity of BiKE for cells expressing the HIV envelope. Figure 12c illustrates that purified healthy donor NK cells were incubated with infected or uninfected HeLa-CD4 cells that had or did not have HIV-Env BiKE. K562 cells and Raji cells with rituxin were used as controls. FIGS. 13a to 13c. HIV-Env BiKE specifically binds to primary infected T-cell lines and mediates NK cell death. FIG. 13a shows two HIV-infected T-cell lines, H9 HIV-IIIB and ACH-2, or their uninfected counterparts, H9 and CEM CD4, stained intracellularly for HIV capsid protein to confirm active HIV replication. FIG. 13b shows the same infected and uninfected T-cell lines stained with His-tagged HIV-Env BiKE and biotinylated anti-His+streptavidin or secondary alone. Although BiKE did not show binding to the uninfected T-cell lines, it showed binding to both infected clones, indicating specificity for actively infected T-cells. Figure 13c shows that purified NK cells from a healthy donor were co-cultured with uninfected or HIV-infected T-cell lines with or without HIV-Env BiKE and evaluated for NK degranulation (CD107a) and IFNγ production. FIGS. 14a to 14c. IL-15 containing HIV-TriKE activated immune subsets and induced viral transcription in latently infected primary and T-cell lines. FIG. 14a shows peripheral blood mononuclear cells incubated for 16 hours with equal molar amounts of rhIL-15 or IL-15 containing HIV-TriKE. NK and T-cell subsets were evaluated by flow cytometry for activation by CD69 expression. FIG. 14b shows latently infected human CD4+ T-cell line, ACH-2, incubated for 48 hours in the presence of 10 nM PMA, 10 ng / ml rhIL-15, or IL-15 containing equal molar amounts of TriKE. Subsequently, the cells were washed and intracellularly stained for HIV-gag (p24). IL-15 alone and both TriKE induced significant viral reactivation, as indicated by p24 expression. Figure 14c illustrates that purified CD4+ memory T-cells were isolated from antiretroviral-treated HIV-infected patients and cultured with rhIL-15, the IL-15 hyperagonist, Nant-803, or IL-15 containing TriKE. Each condition was incubated for 72 hours with or without the HDAC inhibitor, SAHA. Subsequently, cells were harvested, and nested PCR reactions were performed to identify HIV mRNA. Fig. 15. Targeting of solid tumors by second-generation TriKE molecules through multiple antigens. NCI-H460: Lung carcinoma (large cell lung cancer); NCI-H322: Bronchoalveolar carcinoma (cervical lymph node metastasis); CSPG4: Chondroitin sulfate proteoglycan 4; SS1: Mesothelin. FIGS. 16a to 16d. 51 Chromium release assays were performed with several different novel TriKEs to demonstrate that any targeting scFv can be prepared as a functional TriKE. (a) EpCAM+CD133+NG2+ non-small cell lung cancer NCI-H460 cells plus NK cells were incubated with 1615EPCAM133 TriKE or 1615NG2 TriKE (glial antigen 2 or CSPG4). Both 1615NG2 and 1615EpCAM133 have activity 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 No. 36) TriKE. Only 1615Meso is active. (c) Mesothelin+NG2+ ovarian cancer cells (Ovcar3 cells) were incubated with 1615NG2 TriKE or 1615SS1 TriKE. 1615Meso and 1615NG2 are active. (d) Raji cells were cultured with NK cells, and 51 It was studied in a Cr release assay. TriKE 16152219 (SEQ No. 12) simultaneously targets B cell markers CD19 and CD22. Only 16152219, 162219, and rituximab killed the CD22+CD19+ target. The control group did not. Fig. 17. TriKE sequence of sequence number 3 (Rama 161533 TriKE) (underlined part). Fig. 18. CAM1615SS1 (mesothelin) amino acid sequence (Sequence No. 32). The underlined part represents the TriKE sequence. The SS1 (mesothelin) scFV antibody fragment is shown (Sequence No. 33). FIG. 19. PGT121Fab was produced using a 2A self-cleaving peptide. In alternative embodiments, for example, a 2-plasmid system or an IRES may be used. FIGS. 20a to 20b. FIGS. 20a and 20b. Co-transfection of two plasmids in mammalian cells to generate Fab-based antibodies. Two separate plasmids (plasmid 1 and plasmid 2) were co-transfected in Expi-Cho-S cells for TriKE generation. The amino acid sequence containing the non-humanized camelid anti-CD16 is shown. Specific details for implementing the invention

[0027] Natural killer (NK) cells are cytotoxic lymphocytes of the innate immune system capable of immune surveillance. Like cytotoxic T cells, NK cells deliver membrane-permeable and apoptosis-inducing granzymes and stores of perforin granules. Unlike T cells, NK cells do not require antigen priming and do not recognize targets by engaging activating receptors in the absence of MHC recognition.

[0028] NK cells express CD16, an activating receptor that binds to the Fc portion of IgG antibodies, and are involved in antibody-dependent cell-mediated cytotoxicity (ADCC). NK cells are regulated by IL-15, which can induce antigen-dependent cytotoxicity and increased lymphokine-activated cytotoxic 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-activated functions contribute to improved cancer defense.

[0029] The present disclosure describes a multi-specific therapeutic compound that may be a trispecific killer agent compound (TriKE). A TriKE has three distinct binding domains: an NK cell engaging domain that binds to NK cells (e.g., CD16), an NK activating domain comprising a cytokine or a functional fragment thereof that binds to a receptor for such cytokines, and a targeting domain that binds to a marker present on a target cell (e.g., cancer cell). The design and production of TriKEs are extensively described, for example, in U.S. Patent Application Publication US2018 / 0282386, which is incorporated by reference in its entirety. TriKEs offer the advantage of combining an antibody-dependent cytotoxicity (ADCC)-promoting moiety and an expansion-related moiety (IL-15) on the same molecule.

[0030] Therapeutically, adoptive transfer of NK cells can induce remission in patients with refractory acute myeloid leukemia (AML) when stimulating NK cell survival and in vivo expansion in combination with lymphocyte-removal chemotherapy and IL-2. Such therapy may be limited by IL-2-mediated induction of regulatory T (Treg) cells and a lack of antigen specificity, which inhibit NK cell proliferation and function. The development of reagents that induce NK cell antigen specificity, expansion, and / or persistence while bypassing the negative effects of Treg inhibition can enhance NK-cell-based immunotherapy.

[0031] Although advancements in the efficacy and use of antiretroviral drugs have substantially improved the health and life expectancy of HIV-infected individuals, these drugs are merely a stopgap measure to prevent progression to AIDS and limit further transmission of the virus. Despite the use of antiretrovirals to suppress HIV replication, infected individuals retain a reservoir of latently HIV-infected cells capable of reactivating and re-establishing an active infection upon discontinuation of antiretroviral therapy. Therapeutic solutions require the reactivation and subsequent destruction of these latently infected cells. When antibody responses to HIV infection are present, they are generally ineffective due to the high mutation rate of the virus, which prevents the rapid elimination of epitopes recognized by the generated antibodies. However, recently, various HIV-specific antibodies have been identified in infected individuals that possess potent neutralizing effects but lack the ability to induce antibody-dependent cell-mediated cytotoxicity (ADCC).

[0032] Accordingly, the present invention addresses this problem by providing bi- and tri-specific natural killer cell agents (BiKE and TriKE) composed of short-chain variable fragments derived from broad-spectrum neutralizing antibodies (bnAb) against HIV-Env, and CD16 agents linked by IL-15 molecules. The purpose of these tri-specific antibody constructs is to utilize the broad specificity of these antibodies targeting HIV, specifically inducing NK cell death into 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, enhancing their response. IL-15 has also been identified as a potential reactivator of latently infected cells. Early studies from the inventors' laboratory demonstrated enhanced NK cell cytokine production and death of infected targets expressing HIV-Env when incubated with HIV-specific constructs. PBMCs from healthy donors incubated with TriKE exhibited a significant increase in immune cell activation in NK, CD4, and CD8 subsets, as well as the induction of NK cell proliferation. Additionally, IL-15, as a monomer or part of TriKE, demonstrates the ability to reactivate latently HIV-infected T-cells isolated from infected patients in vitro.

[0033] Recent trials of the IL-15 / IL-15Ra hyperagonist (Nant-803) in ART-treated HIV-infected patients also resulted in the detection of the virus in serum and immune activation. Together, these data indicate a role for HIV-bnAb-containing TriKEs in the reactivation and elimination of latently infected reservoirs by utilizing the ability of NK cells to mediate ADCC.

[0034] A dual-specific fusion was performed to introduce an anti-human anti-CD16 scFv derived from human phage display library technology (McCall et al., 1999. Mol Immunol. 36:433-445). NK cells mediate antibody-dependent cell-mediated cytotoxicity (ADCC) through the CD16 (FcγRIII) receptor. Signaling via the CD16 receptor induces calcium flux and phosphorylation of ITAM, triggering the release of cytokines such as interferon (IFNγ) and tumor necrosis factor (TNFα), as well as soluble granules. The dual-specific molecule was designed to trigger the CD16 receptor in conjunction with other targeting molecules (Gleason et al. Blood. 2014 (19):3016-26), so-called dual-specific killer agents (BiKE). When one scFv recognizes NK cells and a second scFv recognizes tumor antigens, BiKE can significantly enhance cytotoxic death in various human cancers. One exemplary BiKE targeted CD33 and enhanced NK cell responses in acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). MDS is a clonal xenogeneic stem cell disorder characterized by normal or hypercellular bone marrow (BM), accompanied by peripheral blood (PB) cytopenia and an increased risk of progression to AML.

[0035] NK cells respond to various cytokines, including IL-15, which are involved in NK cell homeostasis, proliferation, survival, activation, and / or development. IL-15 and IL-2 share several signaling components, including IL-2 / IL-15R□□ (CD122) and a common gamma chain (CD132). Unlike IL-2, IL-15 does not stimulate Tregs, thereby allowing NK cell activation while bypassing the Treg suppression of the immune response. In addition to promoting NK cell homeostasis and proliferation, IL-15 can rescue NK cell functional defects that may occur in the post-transplant environment. IL-15 also stimulates CD8+ T cell function, which can further enhance its immunotherapeutic potential. Furthermore, based on preclinical studies, the toxicity profile of IL-15 may be more favorable than that of IL-2 at low doses.

[0036] IL-15 plays a role in NK cell developmental homeostasis, proliferation, survival, and activation. IL-15 and IL-2 share several signaling components, including IL-2 / IL-15R□ (CD122) and a common gamma chain (CD132). IL-15 also activates NK cells and can restore functional defects in NK cell engraftment after hematopoietic stem cell transplantation (HSCT).

[0037] In one embodiment, the present disclosure describes a tri-specific killer (TriKE) molecule comprising, generally, one or more NK cell agent 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 cells that enhance cytokines, chemokines, and / or activating molecules), wherein each domain is operably linked to another domain. As used herein, the term “operably linked” refers to a direct or indirect covalent linkage. Accordingly, two operably linked domains may be directly covalently linked to each other. Conversely, two operably linked domains may be linked by mutual covalent linkages in an intervening moiety (e.g., a lateral sequence). Two domains may be considered operablely connected, for example, when separated by a third domain with or without one or more intervening side sequences. In an exemplary embodiment, the NK-involved domain is indicated by CD16, and the NK-activating domain is IL-15 or a functional fragment thereof.

[0038] The NK involvement domain may comprise any moiety that binds to and / or activates NK cells, and / or any moiety that blocks the inhibition of NK cells. In some embodiments, the NK involvement domain may comprise an antibody that selectively binds to a component on the surface of NK cells. In other embodiments, the NK involvement domain may comprise a ligand or small molecule that selectively binds to a component on the surface of NK cells. As used herein, the term “selectively binds” refers to an ability that differs between two or more alternatives, having differential affinity to any degree, for example, for a specific target. As used herein, the term “antibody” generally refers to an immunoglobulin or a fragment thereof, and accordingly includes 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. Accordingly, for the sake of brevity, references to antibodies that selectively bind to components on the surface of NK cells include any antibody fragment exhibiting the described binding characteristics. Similarly, references to ligands that selectively bind to components on the surface of NK cells include any fragment of a ligand exhibiting the described binding characteristics.

[0039] In some embodiments, the NK involvement domain may selectively bind to a receptor located at least partially on the surface of an NK cell. In certain embodiments, the NK involvement domain may bind to an NK cell and thereby provide the function of bringing the NK to spatial proximity to a target to which the targeting domain, described in more detail below, selectively binds. However, in certain embodiments, the NK involvement domain may activate the NK cell and, accordingly, also selectively bind to a receptor having an activating function. As described above, activation of the CD16 receptor can lead to antibody-dependent cell-mediated cytotoxicity. Accordingly, in certain embodiments, the NK involvement domain may include at least a portion of an effective anti-CD16 receptor antibody to selectively bind to the CD16 receptor. In other embodiments, the NK involvement cell domain may interfere with the mechanism of inhibiting the NK cell.

[0040] An NK-engaging domain can be designed to have a desired degree of NK selectivity and, accordingly, desired immune binding characteristics. For example, CD16 has been identified as the Fc receptors FcγRIIIa (CD16a) and FcγRIIIb (CD16b). These receptors subsequently bind to the Fc portion of IgG antibodies that 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. A TriKE embodiment comprising an NK-engaging domain containing an anti-CD16a antibody may bind to NK cells but not to neutrophils. Accordingly, the NK-engaging domain of TriKE can be designed to contain an anti-CD16a antibody in a manner that binds to NK cells but not to neutrophils.

[0041] Although the present specification describes the NK involvement domain in the context of various embodiments comprising anti-CD16 receptor scFv, the NK involvement domain may comprise any antibody or other ligand that selectively binds to the CD16 receptor. Additionally, the NK involvement domain may comprise an antibody or ligand 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.

[0042] The targeting domain may include any moiety that selectively binds to an intended target, such as, for example, tumor cells, targets in the cancer stroma, suppressor cells, for example, targets on CD33+ bone marrow-derived suppressor cells, or targets on virus-infected cells.

[0043] In other embodiments, the targeting domain may selectively bind to a target on a cell infected by a virus, such as, for example, adenovirus, HIV, CMV, and / or HPV. In an exemplary example of this specification, the targeting domain is an HIV epitope.

[0044] The NK activation domain may comprise an amino acid sequence that activates NK cells, promotes sustained NK cells, or otherwise enhances 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) sufficient to provide NK cell activation and / or sustained activity. In embodiments comprising more than one NK activation domain, the NK activation domains may be provided in sequence or in any other combination. Additionally, each cytokine-based NK activation domain may comprise the entire amino acid sequence of the cytokine, or may be an amino acid fragment, independent of the characteristics of other NK activation domains included in the TriKE molecule. Exemplary cytokines on which the NK activation domain may be based include, for example, IL-15, IL-18, IL-12, and IL-21. Accordingly, although the NK activation domain is described in detail in this specification in the context of an exemplary model embodiment derived from IL-15, TriKE can be designed using any suitable cytokine or an NK activation domain derived therefrom.

[0045] For the sake of brevity in this description, references to an NK activation domain by identifying the underlying cytokine include the entire amino acid sequence of the cytokine, any suitable amino acid fragment of the cytokine, and / or a modified form of the cytokine containing one or more amino acid substitutions. Accordingly, references to an "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, such as IL-15N72D or IL-15N72A.

[0046] The use of the IL-15 NK activation domain in TriKE can provide sustained NK cell activity, as demonstrated in a mouse model showing a rapid increase in human NK cells and reduction in tumors even after 3 weeks. In mice, NK cells are activated to produce a series of anticancer factors and cytokines. Furthermore, the IL-15 NK activation domain alters the chemistry of these molecules, and consequently, they can be more easily refolded and / or recovered in greater yield, making the TriKE molecule more suitable for clinical scale-up.

[0047] In some embodiments, the molecule may further comprise a side sequence capable of linking two of the aforementioned domains. In some embodiments, the presence of the side sequence may further increase NK cell activation. One exemplary side sequence comprises 20 amino acids of SEQ ID NO. 1 (see also US 2018 / 0282386). Another exemplary side sequence comprises 7 amino acids of SEQ ID NO. 2. Certain embodiments comprise more than one side sequence. As an example, SEQ ID NO. 1 comprises a side sequence of SEQ ID NO. 3 for linking an NK-activating domain (e.g., IL-15) to an NK-engaging domain (e.g., anti-CD16 receptor scFv). SEQ ID NO. 1 also comprises a side sequence of SEQ ID NO. 4 for linking an NK-activating domain to a targeting domain (e.g., anti-CD33 scFv).

[0048] 51Chromium release assays were performed with various different TriKEs, indicating that any scFv targeting cancer cells can be introduced into functional TriKEs. Non-small cell lung cancer (NCI-H460) cells were incubated with either 1615EPCAM133 TriKE or 1615NG2 TriKE. Both 1615NG2 and 1615EpCAM133 are active at various different E:T ratios (20:1, 10:1, and 5:1). Figure 19b illustrates melanoma cells incubated with 1615EPCAM133 TriKE. Mesothelin+EpCAM-NG2 MDA-435A melanoma cells were incubated with 1615EPCAM TriKE or 1615Meso TriKE (SEQ No. 32). Only 1615Meso is active. Ovarian cancer cells (Ovcar3 cells) were incubated with 1615NG2 TriKE or 1615Meso TriKE. Both TriKEs induced NK cytotoxic activity. Additionally, an anti-leukemia TriKE was prepared and recognized the leukemia markers CD19 and CD22. 16152219 TriKE was tested on CD22+CD19+ Raji cells and effectively killed them (as well as rituximab). Together, these data indicate that any scFv can be inserted into the generalized TriKE construct platform of 1615X, and that the resulting TriKE can induce NK cells to respond to and expand toward scFv targets. Additional exemplary TriKE molecules are listed in Table 1.

[0049]

[0050] In some embodiments, the NK cell agent may include the use of a humanized CD16 agent derived from an animal nanobody. Although the scFv has a heavy chain variable component and a light chain variable component linked by a linker, the nanobody consists of a single monomer variable chain, for example, a variable heavy chain or a variable light chain capable of specifically binding to a target. The nanobody may be derived from an antibody of any suitable animal, for example, a camelid (e.g., llama or camel) or a cartilaginous fish. The nanobody may provide superior physical stability, the ability to bind to deep grooves, and increased production yield compared to larger antibody fragments.

[0051] In one exemplary embodiment, the nanobody-based NK agent molecule may comprise a humanized CD16 nanobody derived from a disclosed llama nanobody called EF91 (GeneBank sequence EF561291; Behar et al., 2008. Protein Eng Des Sel. 21(1):1-10). The llama EF91 was initially engineered into BiKE-containing CD19 to test the ability of this CD16 agent to induce NK cell activation. It exhibited functionality similar to rituximab-mediated apoptosis in a chromium release assay targeting Raji (Fig. 1a). To confirm the functionality of the molecule, the CDR was cloned into a humanized camelid scaffold to humanize the CD16 agent now called HuEF91 (Vincke et al., 2009. J Biol Chem. 284(5):3273-3284). The binding of HuEF91 is identical to that observed using standard CD16 scFv, indicating that the introduction of the llama nanobody variable heavy chain into the humanized scaffold does not interfere with the specificity of the molecule. The use of HuEF91 as an NK agent in the TriKE molecule described herein can increase drug yield and / or increase stability and / or increase NK-cell-mediated ADCC efficacy.

[0052] In some embodiments, the immune agents described herein may be used to stimulate the patient's own immune system to eliminate tumor cells. Although studies have shown that T cells genetically modified to express chimeric antigen receptors (CARs) are potent clinical agents of anti-tumor activity, the production of T-CARs is expensive and complex. Other disadvantages include the risk of cytokine toxicity and the long-term persistence or neoplastic modification of T-CARs resulting in interactions with healthy tissues. The triple-specific killer agents described herein can serve as agents for ADCC and can expand NK cells without requiring in vitro genetic modification and gene therapy, thereby offering potential advantages over T-CAR systems. Because the immune agents are rapidly eliminated, the response cannot be sustained indefinitely, which can reduce the risk of cytokine toxicity of the immune agents compared to T-CARs.

[0053] In some embodiments, the triple-specific killer agent comprises a cytokine. In some embodiments, the triple-specific killer agent preferably comprises IL-15. IL-15 does not contain Treg, 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 proliferation signals in NK cells, thereby causing enhanced NK cell expansion and survival. These features may be beneficial during the use of the triple-specific killer agent in the treatment of cancer. In some embodiments, the inclusion of IL-15 in the triple-specific killer agent mediates the induced delivery of TriKE to NK / target cell synapses, potentially allowing IL-15 to accumulate more effectively at the tumor site compared to systemic IL-15.

[0054] In some embodiments, the immune agent increases the secretion of immune cell-mediated cytokines. In some embodiments, cytokine secretion is preferably antigen-specific. In some embodiments, these cytokines may include IFN-γ, GM-CSF, IL-6, IL-8, and / or TNF-α. In some embodiments, the production of these cytokines is preferably at physiological levels. In some embodiments, the production of these cytokines is at a level lower than that observed in IL-12 / IL-18 stimulated NK cells (Papadakis et al., 2004. J Immunol. 172:7002-7007). As shown in Example 2, the measurement of representative inflammatory cytokines, including GM-CSF, IL-6, IL-8, and TNF-α, using the cytokine Luminex assay, shows a statistically significant difference in GM-CSF secretion between BiKE and TriKE, but no difference in the secretion of other cytokines.

[0055] In some embodiments, the immune agent increases the proliferation of lymphocytes. Lymphocytes may include, for example, NK cells, γδ-T cells, and / or CD8 T cells. TetraKE or a larger molecule may be designed to include more than one NK cell agent domain and / or more than one NK activation domain.

[0056] 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 the subject in an amount effective for inducing NK-mediated death of the target cells. “Treat” or a variant thereof refers to reducing, limiting the progression, improving, or resolving symptoms or signs associated with a pathological condition to any degree. As used herein, “improve” refers to any reduction in the degree, severity, frequency, and / or likelihood of characteristic symptoms or clinical signs of a particular pathological condition; “symptom” refers to any subjective evidence of the disease or the patient’s pathological condition; and “sign” and “clinical sign” refer to objective physical findings associated with a particular pathological condition that may be observed by a person other than the patient.

[0057] “Treatment” may be therapeutic or prophylactic. “Therapeutic” and its variations refer to treatment that improves one or more existing symptoms or clinical signs associated with the condition. “Prophylactic” and its variations refer to treatment that limits, to some extent, the onset and / or appearance of symptoms or clinical signs of the condition. Generally, “therapeutic” treatment is initiated after the condition appears in the subject, and “prophylactic” treatment is initiated before the condition appears in the subject. Accordingly, in certain embodiments, the method may include 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. Accordingly, for example, a subject “at risk” of developing the specified condition is a subject who has the specified condition or possesses one or more signs of an increased risk of developing the specified 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 the condition. Exemplary indications of the condition may include, for example, genetic predisposition, family history, age, gender, geographical location, lifestyle, or medical history. Treatment may also continue even after symptoms have resolved, for example, to prevent or delay the recurrence of symptoms.

[0058] In the case of subjects infected with HIV, for example, "treatment" may include a reduction in viral load and / or improvement of symptoms.

[0059] In some cases, treatment may involve administering TriKE molecules to subjects so that the TriKE molecules can stimulate endogenous NK cells in vivo. When TriKE molecules are used as part of in vivo treatment, they can specifically target NK cell antigens with co-stimulation, survival enhancement, and expansion that may be antigen-specific. In other cases, TriKE can be used in vitro as an adjuvant for NK cell proton therapy.

[0060] Accordingly, TriKE molecules, whether NK-activated TriKE or T-cell-activated TriKE, may be administered before, during, or after the subject first exhibits symptoms or clinical signs of the pathology. Treatment initiated before the subject first exhibits symptoms or clinical signs related to the pathology may reduce the likelihood of the subject experiencing clinical evidence of the pathology compared to a subject not administered TriKE molecules, reduce the severity of symptoms and / or clinical signs of the pathology, or completely resolve the pathology. Treatment initiated after the subject first exhibits symptoms or clinical signs related to the pathology may reduce the severity of symptoms and / or clinical signs of the pathology or completely resolve the pathology compared to a subject not administered the composition.

[0061] The TriKE molecule may be any embodiment of the aforementioned TriKE molecule having a targeting domain that selectively binds to an appropriate target cell population. In some cases, the target cells may include virus-infected cells so that the method may include treating a viral infection. Accordingly, in some embodiments, the method may include improving at least one symptom or clinical sign of a viral infection.

[0062] In various embodiments, the TriKE targeting domain may include, for example, a polypeptide that selectively binds to mesothelin or viral antigens on HIV.

[0063] As used herein, "object" may be any animal, e.g., mammal (e.g., human, dog, cat, horse, cow, sheep, goat, monkey, etc.). In certain embodiments, the object may be a human.

[0064] The TriKE molecules 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. Such media and / or agents for pharmaceutically active substances are well known in the art. Any conventional media or agent is considered for use in therapeutic compositions except where it is used as an active ingredient. Additional active ingredients may also be introduced into the composition. As used herein, "pharmaceutically acceptable" refers to a substance that is not biologically or otherwise undesirable, that is, a substance that may be administered to an individual with the TriKE molecules without causing any undesirable effects or interacting in a harmful manner with any other component of the pharmaceutical composition containing said substance.

[0065] Accordingly, TriKE molecules can be formulated into pharmaceutical compositions. Pharmaceutical compositions can be formulated in various forms suitable for a preferred route of administration. Accordingly, 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., intranasal, intrapulmonary, intramural, vaginal, intrauterine, 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.

[0066] Accordingly, TriKE molecules may be provided in any suitable form, including but not limited to solutions, suspensions, emulsions, sprays, aerosols, or mixtures of any form. The composition may be delivered as a formulation with any pharmaceutically acceptable excipient, carrier, or excipient. For example, the formulation may be delivered in a conventional topical form, such as a cream, ointment, aerosol formulation, non-aerosol spray, gel, lotion, etc. The formulation may further include one or more additives, such as an adjuvant, a skin penetration enhancer, a colorant, a fragrance, a flavoring agent, a humectant, a thickener, etc.

[0067] The formulation can be conveniently presented in a unit dosage form and can be manufactured by methods widely known in the pharmaceutical field. A method for manufacturing a composition having a pharmaceutically acceptable carrier comprises the step of binding a TriKE molecule to a carrier constituting one or more auxiliary components. Generally, the formulation can be manufactured by uniformly and / or intimately binding the active molecule to a liquid carrier, a finely divided solid carrier, or both, and subsequently, if necessary, forming the product into a desired formulation.

[0068] The amount of TriKE molecules administered may vary depending on various factors, including, but not limited to, the specific TriKE molecule used, the subject's body weight, physical condition and / or age, and / or route of administration. Accordingly, the absolute weight of TriKE molecules contained in a provided 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. Consequently, it is not practical to generally present an amount constituting the effective amount of TriKE molecules for all possible applications. However, a person skilled in the art can easily determine an appropriate amount by taking these factors into full consideration.

[0069] In some embodiments, the method may comprise administering TriKE molecules sufficient to provide a dose of, for example, about 100 ng / kg to about 50 mg / kg to a subject, but in some embodiments, the method may be performed by administering TriKE molecules at a dose outside this range. In some of these embodiments, the method comprises administering TriKE molecules sufficient to provide a dose of about 10 µg / kg to about 5 mg / kg, for example, about 100 µg / kg to about 1 mg / kg to a subject.

[0070] Alternatively, the dosage can be calculated using the actual body weight obtained immediately before the start of the treatment course. For a dosage calculated in this way, the body surface area (m²) is calculated before the start of the treatment course using the Dubois method: m² = (body weight kg 0.425 × height cm 0.725) × 0.007184.

[0071] In some embodiments, the method may include administering sufficient TriKE molecules to provide, for example, a dose of about 0.01 mg / m2 to about 10 mg / m2.

[0072] In some embodiments, the TriKE molecule may be administered, for example, as a single dose or multiple doses per week, but in some embodiments, the method may be carried out by administering the TriKE molecule at a frequency outside this range. In certain embodiments, the TriKE molecule may be administered about once per month to about five times per week.

[0073] In some embodiments, the method further comprises administering one or more additional therapeutic agents. 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 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 the 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 administering the component (e.g., cytokine production, activation of a specific cell population, etc.) persists at the treatment site until one or more additional components are administered to the treatment site. Accordingly, co-administration combinations may include components that are not present in each other's chemical mixtures under certain circumstances. In other embodiments, TriKE molecules and additional therapeutic agents may be administered as part of a mixture or cocktail. In some embodiments, administration of TriKE molecules may enable the effect of other therapeutic modes at lower doses compared to administering other therapeutic agents or agents alone, thereby reducing the potential, severity, and / or degree of toxicity observed when higher doses of other therapeutic agents or agents are administered.

[0074] In some embodiments, the method may include administering a sufficient amount of the TriKE molecule described herein and administering at least one additional therapeutic agent, such administration exhibiting a therapeutic synergy. In some aspects of the method of the present invention, a measure of response to treatment observed after administering both the TriKE molecule described herein and the additional therapeutic agent is improved compared to the same measure of response to treatment observed after administering the TriKE molecule or the additional therapeutic agent alone. In some embodiments, the additional therapeutic agent may include additional agents targeting HIV, e.g., 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 may be combined with the TriKe composition before, during, or after the administration of TriKe.

[0075] In the foregoing description and the following claims, the term “and / or” means one or all of the listed elements or any combination of two or more of the listed elements; the terms “comprising,” “comprising,” and variations thereof shall be interpreted as open forms, that is, additional elements or steps are optional and may or may not be present; unless otherwise stated, the singular form and “at least one” are used interchangeably and mean one or more; and references to a numeric range by an endpoint include all numbers encompassed within such range (e.g., 1 to 5 include 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).

[0076] In the foregoing description, specific embodiments may be described separately for clarity. Unless otherwise clearly stated that features of a specific embodiment are incompatible with features of other embodiments, a specific embodiment may include a combination of compatible features described herein in relation to one or more embodiments.

[0077] For any method disclosed herein comprising separate steps, the steps may be performed in any feasible order. And, where appropriate, any combination of two or more steps may be performed simultaneously.

[0078] The present invention is illustrated by the following examples. It should be understood that specific examples, materials, quantities, and procedures should be broadly interpreted in accordance with the scope and spirit of the invention as described herein.

[0079] Example 1

[0080] 1615 Compilation of ANTIHIV

[0081] Since 1615x is a platform technology, it is also possible to use antiviral scFvs that are associated with or unrelated to cancer development. The synthesis and assembly of a hybrid polynucleotide encoding TriKE 1615 antiHIV (SEQ No. 5) were achieved using DNA shuffling and ligation techniques. The fully assembled polynucleotide has, from the 5' end to the 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 No. 1), modified IL-15, a 7-amino acid linker (EASGGPE SEQ No. 2), and the anti-HIV scFv; and finally, an XhoI restriction site.

[0082] Both HIV-envelope specific and IL-15-containing tri-specific killer agents (TriKEs) are reactivated and induce NK cell death in HIV-infected T-cells.

[0083] While advancements in the efficacy and use of antiretroviral drugs substantially improve the health and lifespan of HIV-infected individuals, these drugs serve only as a temporary measure to prevent progression to AIDS and limit further transmission of the virus. Despite the use of antiretrovirals to suppress HIV replication, infected individuals retain a reservoir of latently HIV-infected cells capable of reactivating and re-establishing an active infection upon discontinuation of antiretroviral therapy. Therapeutic solutions require the reactivation and subsequent destruction of these latently infected cells. When antibody responses to HIV infection are present, they are generally ineffective due to the rapid mutation rate of the virus, which can rapidly eliminate epitopes recognized by the generated antibodies. However, recently, various HIV-specific antibodies have been identified in infected individuals that possess potent neutralizing effects but lack the ability to induce antibody-dependent cell-mediated cytotoxicity (ADCC). Accordingly, the inventors designed bi- and tri-specific killing agents (BiKE and TriKE) composed of a short-chain variable fragment derived from a broad-spectrum neutralizing antibody (bnAb) against HIV-Env and a CD16 agent bound by an IL-15 molecule. The purpose of these tri-specific antibody constructs is to specifically re-induce NK cell death, thereby killing the newly expressed EnvThis utilizes the broad specificity of these antibodies against targeted HIV by actively replicating infected cells through recognition 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 to enhance their response. IL-15 has also been identified as a potential reactivator of latently infected cells. Early studies from the inventors' laboratory demonstrate enhanced NK cell cytokine production and apoptosis of infected targets expressing HIV-Env when incubated with HIV-specific constructs. PBMCs from healthy donors incubated with TriKE showed a significant increase in immune cell activation in NK, CD4, and CD8 subsets, as well as inducing NK cell proliferation. Furthermore, IL-15 demonstrates the ability to reactivate latently HIV-infected T-cells isolated from infected patients in vitro, either as a monomer or as part of TriKE. In a recent study of the IL-15 / IL-15Rα hyperagonist (Nant-803) in ART-treated HIV-infected patients, the virus was also detected in serum and immune activation. Together, these data indicate a potential role for HIV-bnAb-containing TriKEs in the reactivation and elimination of latently infected reservoirs by utilizing NK cell capabilities mediated by ADCC.

[0084] As an additional example, the broad-spectrum neutralizing antibody (bnAb) shown in Table 2 below, described herein as 16 / 15 / X TriKE, may be used in the composition and method. The antibody shown in Table 2 may be used, for example, in a TriKE composition comprising camCD16 / IL-15 / anti-HIV bnAb. Further examples of databases listing HIV blocking antibodies useful in the present invention may be publicly identified at the following sites, which are incorporated herein by reference in their entirety:

[0085] https: / / web.archive.org / web / 20131230231821 / http: / / bnaber.org / ;

[0086] https: / / www.hiv.lanl.gov / content / immunology / ab_search;

[0087] https: / / web.archive.org / web / 20131230231821 / http: / / bnaber.org /

[0088]

[0089]

[0090] Example 2

[0091] Triple-specific killer agents for mesothelin (TRIKE) target NK cells against lung cancer.

[0092] NK cells are important effectors in the treatment of hematological malignancies, but have been less effective in the treatment of solid tumors to date. Although lung cancer cells are generally refractory to NK cell death, the inventors sought to determine whether a small molecule that re-induced NK cell lysis against a common tumor antigen, mesothelin, could enhance NK cell death in the lung cancer environment. Mesothelin is a surface protein that is overexpressed in a number of cancers, including aggressive cancers of the lung wall, such as mesothelioma.

[0093] Comparison of peripheral blood NK cells from healthy donors with those of newly diagnosed cancer patients indicated that lung cancer patients maintained the expression of CD16 (Fc receptor) on the cell surface without differences in major subsets of NK cells. Accordingly, the inventors designed a trispecific killer agent (TriKE) consisting of a single-domain antibody against CD16 (sdAb) and a short-chain variable fragment against mesothelin (scFv). The sdAb and scFv were linked together by recombinant IL-15. When tested on peripheral blood NK cells from healthy donors, these agents were able to enhance NK cell proliferation in vitro. Furthermore, when peripheral blood NK cells were cultured with nine different lung cancer lineages, TriKE increased degranulation (in some cases > 60%) and IFNγ production (in some cases > 30%) in the cancer cells.

[0094] NK cells from the peripheral blood of lung cancer patients also proliferated in response to the drug alone. Additionally, when treated with TriKE, the patients' cells increased degranulation (> 60%) and IFNγ production (> 40%, which was significantly higher than the response of healthy donors) in response to lung cancer cells.

[0095] Checkpoint blocking antibodies are the current standard of care for lung cancer patients, and the inventors' future research focuses on the combination of these mesothelin-targeted TriKEs and checkpoint blocking in vitro and in vivo.

[0096] Example 3

[0097] Transient expression of anti-HIV / CAM16 BiKE and TriKE

[0098] Experimental design

[0099] For VRC01-b12CL / VRC07H G54H (see Table 4 for bnAb), the inventors first began by testing VRC Fab-based BiKE. bNAb takes years to evolve and accumulate three times more mutations than other antibodies.

[0100] Expression work

[0101] For expression in mammalian cell lines, VRC01-b12CL (light chain) was cloned into a suitable expression vector (pCoof40) along with a short linker (GGGGS2) and non-humanized CAM16. The cloning method used was Gibson assembly or the related HiFi assembly method (New England Biolabs). The humanized CAM16 biKE was also constructed in the same manner. The VRC07H G54H plasmid was not modified. Large-scale production of both plasmid DNAs was performed using the ZymoPURE Plasmid Maxiprep Kit (Zymo Research).

[0102] BiKE protein expression

[0103] The inventors used suspension-adapted CHO or HEK293 cell lines (ExpiCHO / Expi293F) for high levels of transient protein expression. A 1:1 ratio of heavy chain-large-light chain containing plasmids (1 uG DNA / mL ExpiCHO medium) was co-transfected using ExpiFectamine CHO or 293 Transfection Kits (Thermo Fisher) and incubated with shaking in a temperature and CO2-controlled incubator according to the manufacturer's instructions. After 4 to 5 days, or when the viability was measured as less than 75% of the cells, the supernatant was harvested by centrifugation at 2000 RPM on a benchtop centrifuge and subsequent filtration through a 0.22 micron filter. The filtered supernatant was subsequently stored at -80°C.

[0104] When ready for purification, the drug was thawed and isolated using TALON metal-affinity resin according to the manufacturer's instructions. The drug was subsequently quantified using the Bradford assay with BSA, which was used as a standard. Purity was assessed using densitometric analysis on a Bolt™ 4 to 12% Bis-Tris plus SDS-Page gel run in Bolt™ MES SDS run buffer and stained with Coomassie dye G-250.

[0105] Example 4

[0106] HIV-specific BiKE and TriKE

[0107] Figure 11 illustrates the structure and proposed function of HIV-specific BiKE and TriKE. Figure 11a is a schematic diagram illustrating the origin of components for an initial dual-specific HIV-targeting construct consisting of an anti-CD16 short-chain variable fragment linked to Fab derived from the HIV broad-spectrum neutralizing antibody (bnAb) VRC01. Figure 11b is a schematic diagram and proposed function of HIV dual- and tri-specific killing agents (BiKE and TriKE, respectively). BiKE binds to the HIV envelope expressed on infected cells via the bnAb component, while the anti-CD16 moiety binds to NK cells and signals via CD16 to elicit a functional response. Similar activity is proposed using TriKE with the addition of an IL-15 linker ability that activates NK cells, enhancing its function and inducing a proliferative response. IL-15 components can also activate latently infected T-cells, thereby facilitating recognition by HIV-Trike and the immune system as a whole.

[0108] Figure 12 illustrates that HIV-Env-specific BiKE binds to CD16-expressing NK cells and HIV-infected cell lines, and induces HIV-specific NK cell responses. Figure 12a shows purified peripheral blood NK cells from a healthy donor stained with biotinylated anti-His and fluorochrome-conjugated streptavidin for CD16, streptavidin control, or His-tagged BiKE. BiKE binds to NK cells reflecting CD16 expression. Figure 12b shows uninfected CD4-expressing HeLa cells or HIV-infected HeLa-CD4s stained with HIV-Env BiKE. BiKE specifically bound to infected HeLa-CD4s but not to uninfected HeLa-CD4s, indicating the specificity of BiKE for cells expressing the HIV envelope. Figure 12c illustrates the incubation of purified healthy donor NK cells with infected or uninfected HeLa-CD4 cells, either with or without HIV-Env BiKE. K562 cells and Raji containing rituxin were used as controls. HIV-Env BiKE elicited potent degranulation and cytokine responses only in infected HeLa cells, indicating the specific activity of BiKE. These responses were identical to those of the Raji + rituxin control group.

[0109] Figure 13 illustrates that HIV-Env BiKE specifically binds to primary infected T-cell lines and mediates NK cell apoptosis. Figure 13a illustrates the intracellular staining of two HIV-infected T-cell lines, H9 HIV-IIIB and ACH-2, or their uninfected counterparts, H9 and CEM CD4, for HIV capsid protein to confirm active HIV replication. Figure 13b illustrates the staining of the same infected and uninfected T-cell lines with His-tagged HIV-Env BiKE and biotinylated anti-His+streptavidin, or with the secondary agent alone. Although BiKE did not show binding to the uninfected T-cell line, it did show binding to both infected clones, indicating specificity for actively infected T-cells. Figure 13c illustrates the co-culture of purified NK cells from a healthy donor with and without HIV-Env BiKE with uninfected or HIV-infected T-cell lines and the evaluation of NK degranulation (CD107a) and IFNg production. HIV-BiKE enhanced both NK cell degranulation and cytokine production, specifically for infected T-cell lines, but not for uninfected T-cell lines.

[0110] Figure 14 illustrates that IL-15 containing HIV-TriKE activates immune subsets and induces viral transcription in latently infected primary and T-cell lines. Figure 14a illustrates the incubation of peripheral blood mononuclear cells for 16 hours with equal molar amounts of rhIL-15 or IL-15 containing HIV-TriKE. NK and T-cell subsets were evaluated by flow cytometry for activation via CD69 expression. Figure 14b illustrates the incubation of latently infected human CD4+ T-cell line, ACH-2, for 48 hours in the presence of 10 nM PMA, 10 ng / mL rhIL-15, or IL-15 containing equal molar amounts of TriKE. Subsequently, the cells were washed and intracellularly stained for HIV-gag (p24). IL-15 alone and both TriKE induced significant viral reactivation, as indicated by p24 expression. Figure 14c illustrates that purified CD4+ memory T-cells were isolated from antiretroviral-treated HIV-infected patients and cultured with rhIL-15, IL-15 hyperagonists, Nant-803, or IL-15 containing TriKE. Each condition was incubated for 72 hours with or without HDAC inhibitors and SAHA. Subsequently, cells were harvested, and HIV mRNA was identified by performing nested PCR reactions.

[0111] Both BiKE and TriKE molecules can be produced using the above method, for example, by co-transfection of two plasmids or polynucleotides encoding light and heavy chains separately. The two proteins can also be produced from a single plasmid or polynucleotide using, for example, a 2A self-cleaving peptide or IRES. Exemplary BiKE and TriKE molecules and amino acid sequences are shown in the drawings and sequences of this specification.

[0112] In addition to the various embodiments described in the above specification, the following additional embodiments are considered herein.

[0113] Embodiment 1.

[0114] NK-involved domain containing a moiety that selectively binds to CD16;

[0115] An NK activation domain operably linked to an NK involvement domain comprising IL-15 or a functional fragment thereof; and

[0116] A compound comprising a targeting domain that selectively binds to a viral antigen and is operably linked to an NK activation domain and an NK involvement domain.

[0117] Embodiment 2. The compound of claim 1, wherein CD16 comprises CD16a.

[0118] Embodiment 3. The compound of claim 1, wherein the viral antigen is present on a cell infected with the virus.

[0119] Example 4. The compound of claim 1, wherein the viral antigen is derived from HIV, CMV, HPV, HCV, or adenovirus.

[0120] Embodiment 5. The compound of claim 1, wherein the viral antigen is derived from HIV.

[0121] Embodiment 6. The compound of claim 1, wherein the NK-involved domain moiety comprises an antibody or a binding fragment thereof or a nanobody.

[0122] Example 7. The compound of claim 6, wherein the antibody fragment comprises scFv, F(ab)2, or Fab.

[0123] Embodiment 8. The compound of claim 6, wherein the antibody or its binding fragment or nanobody is human, humanized, or camelid.

[0124] Example 9. The compound of claim 6, wherein the antibody or its binding fragment or nanobody is a camelid.

[0125] Example 10. A compound according to claim 6, wherein IL-15 comprises the amino acid sequence of SEQ ID NO. 4 or a functional variant thereof.

[0126] Example 11. A compound according to claim 10, wherein the functional variant of IL-15 comprises an N72D or N72A amino acid substitution compared to SEQ ID NO. 4.

[0127] Embodiment 12. The compound of claim 1, wherein the targeting domain moiety comprises an antibody or a binding fragment thereof or a nanobody.

[0128] Example 13. The compound of claim 12, wherein the antibody-binding fragment comprises scFv, F(ab)2, or Fab.

[0129] Embodiment 14. The compound of claim 1, wherein the NK involvement domain comprises CD16, the NK activation domain comprises IL-15, and the targeting domain selectively binds to a viral antigen derived from HIV.

[0130] Embodiment 15. The compound of claim 1, wherein the NK involvement domain comprises CD16a, the NK activation domain comprises IL-15, and the targeting domain selectively binds to a viral antigen derived from HIV.

[0131] Embodiment 16. The compound of claim 1, wherein the NK involvement domain comprises NKG2c, the NK activation domain comprises IL-15, and the targeting domain selectively binds to a viral antigen derived from HIV.

[0132] Embodiment 17. The compound of claim 1, comprising at least one flanking sequence connecting two of the domains.

[0133] Embodiment 18. The compound of claim 17, further comprising a second lateral sequence connecting two connected domains together with a third domain.

[0134] Example 19. The compound of claim 18, wherein the adjacent sequence is adjacent to the NK activation domain.

[0135] Example 20. A compound according to claim 18, wherein the first side sequence is a C-terminus for the NK-involved domain and the second side sequence is an N-terminus for the anti-viral targeting domain.

[0136] Embodiment 21. A compound according to claim 1, further comprising a second targeting domain.

[0137] Embodiment 22. The compound of claim 1, further comprising a second NK-involved domain.

[0138] Embodiment 23. The compound of claim 1, further comprising a second NK activating domain.

[0139] Example 24. The compound of claim 1, wherein the compound is SEQ ID NO. 5, 7, 24, 29 or 37.

[0140] Embodiment 25. A composition comprising the following:

[0141] A compound of any one of claims 1 to 24; and

[0142] Pharmaceutically acceptable carrier.

[0143] Embodiment 26. As a method,

[0144] A method comprising the step of administering to a subject a compound of any one of claims 1 to 25 in an amount effective for inducing NK-mediated killing of target cells.

[0145] Embodiment 27. The method of claim 26, wherein the target cell is infected with a virus.

[0146] Embodiment 28. The method of claim 27, wherein the virus is HIV, CMV, HPV, HCV, or adenovirus.

[0147] Embodiment 29. The method of claim 28, wherein the virus is HIV.

[0148] Embodiment 30. A method for stimulating the expansion of NK cells in vivo,

[0149] A method comprising the step of administering to a subject an amount of a compound of any one of claims 1 to 25 that is effective in stimulating the expansion of NK cells in the subject.

[0150] Embodiment 31. The method of claim 30, wherein the subject is infected with a virus.

[0151] Embodiment 32. The method of claim 31, wherein the virus is HIV, CMV, HPV, HCV, or adenovirus.

[0152] Embodiment 33. The method of claim 32, wherein the virus is HIV.

[0153] Embodiment 34. A method for treating a viral infection in a subject,

[0154] A method comprising the step of administering to a subject an amount of a compound of any one of claims 1 to 25 that is effective for treating a viral infection.

[0155] Embodiment 35. The method of claim 34, wherein the subject is infected with HIV, CMV, HPV, HCV, or adenovirus.

[0156] Embodiment 36. The method of claim 35, wherein the subject is infected with HIV.

[0157] Example 37. Isolated nucleic acid sequence of SEQ ID NO. 6.

[0158] Example 38. Isolated amino acid sequence of SEQ ID NO. 7.

[0159] Example 39. An isolated amino acid sequence comprising the sequence of camCD16 / IL-15 / Sequence No. 8.

[0160] Example 40. Isolated amino acid sequence comprising SEQ ID NOs 9, 17, 27, 28, 13, 15, 16, 17, 18, 19, and 20.

[0161] Embodiment 41. The isolated amino acid of claim 40, further comprising the isolated amino acid sequence of SEQ ID NO. 10.

[0162] Example 42. An isolated amino acid sequence comprising SEQ ID NO. 18 operably linked to IL-15.

[0163] Example 43. Isolated amino acid sequences of SEQ ID NOs 20 to 26.

[0164] Embodiment 44. A method for preparing a compound of any one of claims 1 to 24, wherein

[0165] (i) co-transfecting, respectively, 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 within a mammalian cell;

[0166] (ii) A method comprising collecting a supernatant from mammalian cells, wherein the obtained compound binds to a viral antigen.

[0167] Embodiment 45. The method of claim 44, wherein the viral antigen is derived from HIV.

[0168] Embodiment 46. The method of claim 45, wherein the viral antigen is Env.

[0169] Example 47. Isolated DNA sequence encoding the amino acid sequence of SEQ ID NO. 21, 22, 25, 26, 30, 31, 39 or 40.

[0170] Example 48. A pharmaceutical composition comprising SEQ ID NOs 7, 24, 29, 32, 34, 36 and 37 in a pharmaceutically acceptable carrier.

[0171] Embodiment 49. A method for treating a subject, comprising the step of 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.

[0172] Embodiment 50. A method for treating a subject having AIDS or at risk of developing AIDS, comprising the step of administering to the subject a pharmaceutical composition comprising SEQ ID NOs 5, 7, 24, 29, and 37.

[0173] The full disclosures of all patents, patent applications, publications, and electronically available materials cited herein are incorporated by reference in their entirety. Although the invention has been described with reference to the embodiments above, modifications and variations will be understood to be included within the spirit and scope of the invention. Accordingly, the invention is limited only by the following claims.

[0174] order

[0175] Sequence No. 1 Linker

[0176]

[0177] Sequence No. 2 linker

[0178]

[0179] Sequence No. 3 Rama 161533

[0180]

[0181] Sequence No. 4 Il-15 (Human)

[0182]

[0183] Sequence No. 5 1615antiHIV

[0184]

[0185] Sequence No. 6 Cam1615PGT121

[0186]

[0187]

[0188] Sequence No. 7 Cam1615PGT121

[0189]

[0190] Sequence No. 8 PGT121

[0191]

[0192] Sequence number 9

[0193]

[0194] Sequence number 10

[0195]

[0196] Sequence number 11

[0197]

[0198] Sequence No. 12

[0199]

[0200] Sequence No. 13

[0201]

[0202]

[0203] Sequence No. 14

[0204]

[0205] Sequence number 15

[0206]

[0207] Sequence number 16

[0208]

[0209] Sequence number 17

[0210]

[0211] Sequence number 18

[0212]

[0213] Sequence No. 19

[0214]

[0215] Sequence number 20

[0216]

[0217] Sequence No. 21 (HIV antibody light chain)

[0218]

[0219] Sequence No. 22 (HIV antibody heavy chain)

[0220]

[0221] Sequence number 23

[0222]

[0223] Sequence No. 24

[0224]

[0225] Sequence number 25

[0226]

[0227] Sequence number 26

[0228]

[0229] Sequence number 27

[0230]

[0231] Sequence number 28

[0232]

[0233] Sequence number 29

[0234]

[0235]

[0236] Sequence number 30

[0237]

[0238] Sequence No. 31

[0239]

[0240] Sequence No. 32

[0241]

[0242] Sequence number 33

[0243]

[0244] Sequence No. 34

[0245]

[0246] Sequence number 35

[0247]

[0248]

[0249] Sequence number 36

[0250]

[0251] Sequence number 37

[0252]

[0253] Sequence number 38

[0254]

[0255] Sequence number 39

[0256]

[0257] Sequence number 40

[0258]

Claims

Claim 1 A compound comprising: an NK engaging domain having the amino acid sequence of SEQ ID NO. 18; an NK activating domain operably connected to the NK engaging domain, wherein the NK activating domain is IL-15; and a targeting domain that selectively binds to a viral antigen and operably connected to the NK activating domain and the NK engaging domain, wherein the viral antigen is an HIV antigen and the targeting domain has 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 the compound has the amino acid sequence of amino acids 19-534 of SEQ ID NO. 7 and SEQ ID NO. 24, amino acids 19-558 of SEQ ID NO. 29, or amino acids 19-545 of SEQ ID NO.

37. Claim 2 A compound according to claim 1, wherein the NK-involved domain comprises an antibody or an antigen-binding fragment thereof or a nanobody. Claim 3 In paragraph 2, a compound in which the antigen-binding fragment comprises scFv, F(ab')2, or Fab. Claim 4 In paragraph 2, a compound in which the antibody, antigen-binding fragment, or nanobody is human, humanized, or camelid. Claim 5 In paragraph 2, a compound in which the antibody, antigen-binding fragment, or nanobody is a camelid. Claim 6 A compound according to paragraph 2, wherein IL-15 has the amino acid sequence of SEQ ID NO.

4. Claim 7 A compound according to paragraph 2, wherein IL-15 has the amino acid sequence of SEQ ID NO. 4 and has an N72D or N72A amino acid substitution. Claim 8 A compound according to claim 1, wherein the targeting domain comprises an antibody, an antigen-binding fragment thereof, or a nanobody. Claim 9 In paragraph 8, a compound in which the antigen-binding fragment comprises scFv, F(ab')2, or Fab. Claim 10 A compound according to claim 1, further comprising a first side sequence that is a C-terminus for an NK-involved domain and a second side sequence that is an N-terminus for a targeting domain, wherein the first side sequence has the amino acid sequence of SEQ ID NO. 1 or SEQ ID NO. 2 and the second side sequence has the amino acid sequence of SEQ ID NO. 1 or SEQ ID NO.

2. Claim 11 A pharmaceutical composition for use in the manufacture of a drug for treating HIV infection, comprising: a compound of any one of claims 1 to 10; and a pharmaceutically acceptable carrier. Claim 12 A pharmaceutical composition for treating HIV infection in a subject, comprising (i) a compound of any one of claims 1 to 10, or (ii) a compound of any one of claims 1 to 10 and a pharmaceutically acceptable carrier. Claim 13 A method for preparing a compound of any one of claims 1 to 10, comprising: (i) a polynucleotide encoding the amino acid sequence of amino acid 19-534 of SEQ ID NO. 7, SEQ ID NO. 24, amino acid 19-558 of SEQ ID NO. 29, or amino acid 19-545 of SEQ ID NO. 37 in mammalian cells in vitro ( in vitro A method comprising: (ii) a step of transfecting in ); and (ii) a step of collecting a supernatant from said mammalian cell, wherein the obtained compound binds to a viral antigen. Claim 14 delete Claim 15 In paragraph 13, a method in which the viral antigen is HIV Env. Claim 16 A pharmaceutical composition for treating HIV infection in a subject, comprising, in a pharmaceutically acceptable carrier, a polypeptide having the amino acid sequence of amino acids 19-534 of SEQ ID NO. 7, SEQ ID NO. 24, amino acids 19-558 of SEQ ID NO. 29, or amino acids 19-545 of SEQ ID NO.

37. Claim 17 delete Claim 18 A pharmaceutical composition for treating a subject having AIDS or at risk of developing AIDS, comprising a polypeptide having the amino acid sequence of amino acids 19-534 of SEQ ID NO. 7, SEQ ID NO. 24, amino acids 19-558 of SEQ ID NO. 29, or amino acids 19-545 of SEQ ID NO.

37. 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 Claim 33 delete Claim 34 delete Claim 35 delete Claim 36 delete Claim 37 delete Claim 38 delete Claim 39 delete Claim 40 delete Claim 41 delete Claim 42 delete Claim 43 delete Claim 44 delete Claim 45 delete Claim 46 delete Claim 47 delete Claim 48 delete Claim 49 delete Claim 50 delete

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