B7-H3 TARGETED FUSION PROTEINS AND METHODS OF USING SAME

Abstract

The present invention provides B7-H3 targeted fusion proteins and methods of use thereof. The targeted fusion proteins comprise a B7-H3 targeted trispecific killer engager molecule comprising a B7-H3 targeted binding protein, a CD16 targeted binding protein, and an interleukin-15 protein. The methods of use include methods of treating cancer, inducing natural killer (NK) cell activity against cancer cells, inhibiting tumor growth, increasing survival of a subject with cancer, and inducing NK-mediated antibody-dependent cellular cytotoxicity against cancer cells in a subject.

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. Provisional Application No. 63 / 245,132, filed September 16, 2021. The disclosure of the prior application is deemed to be a part of this specification and is incorporated by reference in its entirety into the disclosure of this application.

[0002] Incorporating sequence tables The material in the attached sequence listing is incorporated by reference into this application. The attached sequence listing xml file G1421US00_GTBIO2180-1WO.xml was created on September 13, 2022 and is 55kb in size.

[0003] FIELD OF THEINVENTION The present invention relates generally to fusion proteins, and more specifically to B7-H3 targeted trispecific killer engager molecules and their use to treat cancer. [Background technology]

[0004] Background information Immunotherapy, a personalized treatment that activates or suppresses the immune system to amplify or reduce immune responses, is being rapidly developed to treat various types of cancer. Immunotherapies for cancer, such as chimeric antigen receptor (CAR)-T cells, CAR-natural killer (NK) cells, and PD-1 and PD-L1 inhibitors, aim to help the patient's immune system fight cancer. T cell activation depends on both the specific combination of the T cell receptor (TCR) with peptide-binding major histocompatibility complex (MHC) and the interaction of T cell costimulatory molecules with ligands in antigen-presenting cells (APCs). The B7 family, a peripheral membrane protein in activated APCs, has been shown to be involved in regulating T cell responses. Recent studies have shown that upregulation of inhibitory B7 molecules in the cancer microenvironment is highly associated with tumor immune escape. As a newly identified member of the B7 family, B7-H3 can promote T cell activation and IFN-γ production.

[0005] Different B7 molecules have either positive or negative costimulatory signals while regulating immune cell responses. Immune checkpoints such as PD-1, PD-L1, PD-L2, and CTLA4 are molecules with many receptor-ligand interactions to evade the immune system and promote proliferation. Several monoclonal antibodies (mAbs) that block these proteins have been developed to downregulate inhibitory immune responses and promote the cytotoxicity of T cells to eliminate tumor cells. Among immune checkpoint blockade drugs, inhibitors targeting PD-1 or CTLA4 have been successfully used to treat patients with metastatic melanoma, improving response and prolonging lifespan. This success has led to the development of such agents to treat a wide range of malignancies, including renal cell carcinoma (RCC), NSCLC, and acute myeloid leukemia (AML), which have further improved response rates and extended patient survival compared to conventional treatments (Yang et al., Int J Biol Sci 2020;16(11):1767-1773 (Non-Patent Document 1)).

[0006] B7-H3 was found to be overexpressed in several types of human cancer cells and correlated with disease progression. B7-H3 was recognized as a costimulatory molecule for immune responses such as T cell activation and IFN-γ production. In the presence of anti-CD3 antibodies that mimic TCR signals, human B7-H3-Ig fusion protein increases the proliferation of both CD4+ and CD8+ T cells and enhances cytotoxic T lymphocyte (CTL) activity in vitro. B7-H3 also has an antitumor effect against adenocarcinoma of the colon, which may also be considered as a promising therapy for the treatment of colon cancer. In a study of human pancreatic cancer patients, B7-H3 was recognized as a costimulatory molecule that was not only abundantly expressed in pancreatic cancer but also associated with increased treatment efficacy. B7-H3 expression was detectable in most pancreatic cancer samples examined and was significantly upregulated in pancreatic cancer compared to normal pancreas, whereas patients with high tumor B7-H3 levels had a significantly better postoperative prognosis than those with low tumor B7-H3 levels (Yang et al., ibid.).

[0007] Despite some success, there are limitations that reduce the overall efficiency of mAb therapy. The development of CD16-directed bispecific and trispecific single-chain variable fragment (BiKE and TriKE) recombinant molecules, which lack the Fc portion of the whole antibody and have target specificity for CD16, circumvents most of these undesirable limitations while inducing high effector function (Gleason et al., Mol Cancer Ther; 11(12); 2674-84, 2012). As a result, recombinant reagents are attractive for clinical use in enhancing natural killer (NK) cell immunotherapy.

[0008] The ability of NK cells to recognize and kill targets is regulated by a complex repertoire of inhibitory and activating cell surface receptors. NK cell cytotoxicity can occur through innate cytotoxicity mediated by natural cytotoxicity receptors (NCRs) or by targeting the CD56 receptor of NK cells with antibodies such as rituximab. dimThis can occur by triggering antibody-dependent cellular cytotoxicity (ADCC) via CD16, an activating low-affinity Fc-gamma receptor for immunoglobulin G (IgG) that is highly expressed by a subset of CD16. CD16 / CD19 BiKE and CD16 / CD19 / CD22 TriKE can trigger NK cell activation through direct CD16 signaling, inducing directional secretion of lytic granules and target cell death. Furthermore, these reagents induce NK cell activation leading to cytokine and chemokine production. [Prior art documents] [Non-patent literature]

[0009] [Non-Patent Document 1] Yang et al.,Int J Biol Sci 2020;16(11):1767-1773 [Non-Patent Document 2] Gleason et al.,Mol Cancer Ther;11(12); 2674-84,2012 Summary of the Invention

[0010] The present invention is based on the development of a B3-H7 targeted fusion protein, specifically a B7-H3 targeted trispecific killer engager molecule (TriKE), and methods of using same.

[0011] In one embodiment, the invention provides an isolated nucleic acid sequence as set forth in SEQ ID NO: 13 or 14, or a sequence having 90% identity thereto.

[0012] In another embodiment, the present invention provides a protein encoded by the nucleic acid sequence set forth in SEQ ID NO: 13 or 14, or a sequence having 90% identity thereto.

[0013] In one aspect, the amino acid sequence is selected from SEQ ID NO:6 or SEQ ID NO:7.

[0014] In an additional embodiment, the invention provides a fusion protein comprising the amino acid sequences set forth in SEQ ID NOs: 6 and 7 operably linked to each other in either orientation.

[0015] In one embodiment, the protein comprises SEQ ID NOs:6 and 7 directly linked between the C-terminus of SEQ ID NO:6 and the N-terminus of SEQ ID NO:7. In another embodiment, the protein comprises SEQ ID NOs:7 and 6 directly linked between the C-terminus of SEQ ID NO:7 and the N-terminus of SEQ ID NO:6.

[0016] In a further embodiment, the present invention provides a fusion protein comprising a sequence as shown in SEQ ID NO:1 and a sequence having 90% or greater identity to SEQ ID NO:1.

[0017] In one embodiment, the invention provides a fusion protein comprising, operably linked, SEQ ID NO: 2 or 19; SEQ ID NO: 4, 17 or 18; SEQ ID NO: 6 and 7, or SEQ ID NO: 7 and 6.

[0018] In one embodiment, SEQ ID NO:2 or 19 and SEQ ID NO:4, 17 or 18 are linked by SEQ ID NO:3 or SEQ ID NO:15. In another embodiment, SEQ ID NO:4, 17 or 18 and SEQ ID NO:6 or 7 are linked by SEQ ID NO:5 or SEQ ID NO:16. In other embodiments, SEQ ID NO:6 and 7 are operably linked in either orientation. In some embodiments, the fusion protein further comprises a half-life extension (HLE) molecule. In one embodiment, the HLE molecule is an Fc or scFc antibody fragment comprising any one of SEQ ID NOs:21-25. In some embodiments, SEQ ID NO:4 has an N72 substitution. In various embodiments, the N72 mutation is N72A or N72D as shown in SEQ ID NOs:17 and 18, respectively.

[0019] In additional embodiments, the invention provides an isolated nucleic acid sequence encoding any of the fusion proteins described herein.

[0020] In one embodiment, the sequence is SEQ ID NO:8.

[0021] In another embodiment, the invention provides a method of treating cancer in a subject comprising administering to the subject any of the fusion proteins described herein, thereby treating the cancer.

[0022] In one aspect, the cancer is selected from non-small cell carcinoma, cutaneous squamous cell carcinoma, pancreatic cancer, primary hepatocellular carcinoma, colorectal cancer, clear cell renal cell carcinoma, or breast cancer.

[0023] In additional embodiments, the invention provides a fusion protein comprising, operably linked, SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:6 and 7, in either orientation, or a fusion protein comprising SEQ ID NO:19, SEQ ID NO:17 or SEQ ID NO:18, and SEQ ID NO:6 and 7, in either orientation, and nucleic acid sequences encoding such proteins.

[0024] In one aspect, SEQ ID NO:19 is operably linked to SEQ ID NO:17 or 18 by a linker of SEQ ID NO:3 or 15. In another aspect, SEQ ID NO:17 or 18 is operably linked to SEQ ID NOs:6 and 7, in either orientation, by a linker of SEQ ID NO:5 or 16. In some aspects, the fusion protein further comprises a half-life extension (HLE) molecule. In one aspect, the HLE molecule is an Fc or scFc antibody fragment comprising any one of SEQ ID NOs:21-25.

[0025] In one embodiment, the invention provides a pharmaceutical composition comprising a therapeutically effective amount of a fusion protein comprising the amino acid sequence of SEQ ID NO:1, or a sequence having 90% or greater identity to SEQ ID NO:1, and a pharma- ceutically acceptable carrier.

[0026] In another embodiment, the present invention provides a method of treating cancer in a subject, comprising administering to the subject a pharmaceutical composition described herein.

[0027] In an additional embodiment, the present invention provides a method of inducing natural killer (NK) cell activity against cancer cells in a subject, comprising administering to the subject a fusion protein comprising a sequence as set forth in SEQ ID NO:1 and a sequence having 90% or greater identity to SEQ ID NO:1, thereby inducing NK cell activity against cancer cells in the subject.

[0028] In one aspect, inducing NK cell activity includes inducing NK cell degranulation, inducing NK cell production of interferon-gamma, increasing the number of tumor-infiltrating NK cells in a subject, and / or inducing or increasing NK cell proliferation.

[0029] In one embodiment, the present invention provides a method of inhibiting tumor growth in a subject, comprising administering to the subject a fusion protein comprising a sequence as set forth in SEQ ID NO:1 and a sequence having 90% or greater identity to SEQ ID NO:1, thereby inhibiting tumor growth in the subject.

[0030] In one aspect, inhibiting tumor growth comprises reducing tumor cell survival.

[0031] In another embodiment, the present invention provides a method of increasing survival of a subject having cancer, comprising administering to the subject a fusion protein comprising a sequence as set forth in SEQ ID NO:1 and a sequence having 90% or greater identity to SEQ ID NO:1, thereby increasing survival of the subject.

[0032] In an additional embodiment, the present invention provides a method for inducing natural killer (NK)-mediated antibody-dependent cytotoxicity against cancer cells in a subject, comprising administering to the subject a fusion protein comprising a sequence as set forth in SEQ ID NO:1 and a sequence having 90% or greater identity to SEQ ID NO:1, thereby increasing survival of the subject.

[0033] In one aspect, administering to a subject a fusion protein comprising a sequence as set forth in SEQ ID NO:1 and a sequence having 90% or greater identity to SEQ ID NO:1 further comprises administering to the subject an anti-cancer treatment.

[0034] In another embodiment, the subject has cancer. In some embodiments, the cancer is selected from the group consisting of lung cancer, prostate cancer, multiple myeloma, ovarian cancer, and head and neck cancer. In other embodiments, the cancer cells are B7-H3 expressing cancer cells. In some embodiments, the cancer is a treatment resistant cancer. [Brief description of the drawings]

[0035] [Figure 1A] 1A-1D show the construction and isolation of cam1615B7-H3 trispecific killer engager (TriKE). FIG. 1A is a schematic representation of the TriKE construct, consisting of (from left to right) camelid anti-CD16 VHH, human IL-15, and anti-B7-H3 scFv. FIG. 1B is a graph illustrating a chromatographic trace from a first-stage purification of cam1615B7-H3 on an ion exchange (FFQ) column. The collected peak is indicated by a double-headed arrow. FIG. 1C is a graph illustrating a chromatographic trace from a second-stage purification of cam1615B7-H3 on a size-exclusion chromatography (SEC) column. The collected peak is indicated by a double-headed arrow. FIG. 1D is a photograph of a sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel showing the purity of the final product after two orthogonal column steps. The gel lanes represent molecular markers, non-reduced (NR) products, and reduced (R) products. [Figure 1B] See legend to Figure 1A. [Figure 1C] See legend to Figure 1A. [Figure 1D] See legend to Figure 1A. [Figure 2A]Figures 2A-2H show that cam1615B7-H3 TriKE induces potent and specific natural killer (NK) cell proliferation. Peripheral blood mononuclear cells (PBMCs) were labeled with CellTrace Violet and incubated with equimolar concentrations of 5 nM rhIL-15 or cam1615B7-H3 TriKE for 7 days. On day 7, cells were harvested and stained for flow cytometric evaluation. Figure 2A shows a representative histogram showing NK cell (CD56+CD3-) proliferation, measured as dilution of CellTrace Violet dye. Figure 2B is a graph illustrating pooled data showing the overall percentage of expanded NK cells. Figure 2C is a graph illustrating pooled data showing the percentage of highly expanded (measured as proliferation of more than 3 divisions) NK cells. Figure 2D is a graph illustrating pooled data showing NK cell counts in cultures (N=9). Figure 2E is a representative histogram showing T cell (CD56-CD3+) proliferation. Figure 2F is a graph illustrating pooled data showing the overall percentage of expanded T cells. Figure 2G is a graph illustrating pooled data showing the percentage of highly expanded T cells. Figure 2H is a graph illustrating pooled data showing T cell counts in cultures (N=9). *p<0.05, **p<0.01, ***p<0.001. [Figure 2B] See legend to Figure 2A. [Figure 2C] See legend to Figure 2A. [Figure 2D] See legend to Figure 2A. [Figure 2E] See legend to Figure 2A. [Figure 2F] See legend to Figure 2A. [Figure 2G] See legend to Figure 2A. [Figure 2H] See legend to Figure 2A. [Figure 3A]Figures 3A-3B illustrate camB7-H3 TriKE binding specificity. Figure 3A is a schematic representation of the binding of TriKE molecules to B7-H3 positive cancer cells. Figure 3B illustrates the binding specificity to WT B7-H3, where the BT-12 pediatric brain tumor line highly expresses B7-H3 (WT) and CRISPR was used to generate the B7-H3 KO BT-12 (red) cell line. [Figure 3B] See legend to Figure 3A. [Figure 4A] Figures 4A-4B illustrate ADCC induction by TriKE molecules: Figure 4A is a graph showing ADCC induction of WT TriKE, and Figure 4B is a graph showing ADCC induction of CD16 TriKE. [Figure 4B] See legend to Figure 4A. [Figure 5A] Figures 5A-5B illustrate functional assays performed on hematological malignancy cell lines with different levels of B7-H3 expression, ranging from none to very high levels. Figure 5A is a graph illustrating NK cell activation measured using CD107a measured by flow cytometry (n=3), IncuCyte, or xCelligence assay. Figure 5B is a graph illustrating NK cell activation measured using IFN-gamma measured by flow cytometry (n=3), IncuCyte, or xCelligence assay. [Figure 5B] See legend to Figure 5A. [Figure 6A] Figures 6A-6B illustrate B7-H3 MFI in four myeloma cell lines by flow cytometry. Figure 6A is a graph illustrating expression of B7-H3 in myeloma lines RPMI-8226, U266, MM1S, and H929 by flow cytometry. Figure 6B is a graph summarizing the data in Figure 6A. [Figure 6B] See legend to Figure 6A. [Figure 7]Figures 7A-7B illustrate the ability of peripheral blood NK cells to kill myeloma cells with or without B7-H3-TriKE in a live imaging IncuCyte Zoom assay. Figure 7A is a graph showing the effect of effector:target (E:T) ratios of 2:1 and 4:1 on MM1S cells. Figure 7B is a graph showing the effect of effector:target (E:T) ratios of 2:1 and 4:1 on U266 cells. [Figure 8] 8A-8D illustrate the ability of peripheral blood NK cells to kill myeloma cells with or without B7-H3-TriKE in a live imaging IncuCyte Zoom assay. FIG. 8A is a graph showing the effect of 2:1 and 4:1 effector:target (E:T) ratios on H929 cells. FIG. 8B is a graph showing the effect of 2:1 and 4:1 effector:target (E:T) ratios on MM1S cells. FIG. 8C is a graph showing the effect of 2:1 and 4:1 effector:target (E:T) ratios on RPMI-8226 cells. FIG. 8D is a graph showing the effect of 2:1 and 4:1 effector:target (E:T) ratios on U266 cells. [Figure 9] 9A-9D illustrate the efficacy of B7-H3-TriKE with the proteasome inhibitor bortezomib (10 nM) and the immunomodulatory drug lenalidomide (5 μM). FIG. 9A is a graph showing the effect of combination therapy on H929 cells after 48 hours. FIG. 9B is a graph showing the effect of combination therapy on RPMI-8226 cells after 48 hours. FIG. 9C is a graph showing the effect of combination therapy on MM1S cells after 48 hours. FIG. 9D is a graph showing the effect of combination therapy on U266 cells after 48 hours. [Figure 10A]Figures 10A-10D illustrate the effect of B7-H3-TriKE on MDSCs expressed from CD33+ bone marrow cells from healthy donors when incubated with myeloma cells at a ratio of 1:100. Figure 10A is a graph showing MDSC (CD14+CD11b+) expression of B7-H3. Figure 10B is a graph showing cell survival measured by flow cytometry. Figure 10C is a plot of viable CD14+ cells. Figure 10D is a graph illustrating H929 proliferation measured over 48 hours by live cell imaging. [Figure 10B] See legend to Figure 10A. [Figure 10C] See legend to Figure 10A. [Figure 10D] See legend to Figure 10A. [Figure 11A] Figures 11A-11B illustrate NK-mediated killing of B7-H3 expressing MDSC. Figure 11A is a graph showing B7-H3 expression on MDSC. Figure 11B is a graph illustrating NK-mediated killing of MDSC co-cultured with NK at 1:1 E:T and comparing killing with or without B7-H3 TriKE. [Figure 11B] See legend to Figure 11A. [Figure 12A] 12A-12D show that cam1615B7-H3 TriKE enhances NK cell function against prostate tumor targets. FIG. 12A is a graph illustrating the pooled percentage of NK cells from healthy donors expressing CD107a+ (degranulation) against CREB5, 22RV1, and Enza-R targets. FIG. 12B is a graph illustrating the pooled percentage of NK cells from healthy donors expressing IFNγ cytokine production against CREB5, 22RV1, and Enza-R targets. FIG. 12C is a graph illustrating the pooled percentage of NK cells from healthy donors expressing CD107a+ (degranulation). FIG. 12D is a graph illustrating the pooled percentage of NK cells from healthy donors expressing IFNγ cytokine production. [Figure 12B] See legend to Figure 12A. [Figure 12C] See legend to Figure 12A. [Figure 12D] See legend to Figure 12A. [Figure 13A] Figures 13A-13B show that camB7-H3 increases NK cell function and NK proliferation against prostate cancer cells compared to IL-15 alone. Figure 13A is a graph showing the percentage of NK cells expressing IFNγ against prostate cancer cell targets. Figure 13B is a graph illustrating the proportion of NK cells that divide 3 or more times. [Figure 13B] See legend to Figure 13A. [Figure 14A]14A-14L show that TriKE can induce NK cell degranulation and inflammatory cytokine production against prostate cancer cell lines. FIG. 14A is a graph showing a pooled analysis of the percentage of NK cells from healthy donors or prostate cancer patients expressing CD107a+ against CA-2 targets. FIG. 14B is a graph showing a pooled analysis of the percentage of NK cells from healthy donors or prostate cancer patients expressing CD107a+ against PC-3 targets. FIG. 14C is a graph showing a pooled analysis of the percentage of NK cells from healthy donors or prostate cancer patients expressing CD107a+ against DU-145 targets. FIG. 14D is a graph showing a pooled analysis of the percentage of NK cells from healthy donors or prostate cancer patients expressing IFNγ against CA-2 targets. FIG. 14E is a graph showing a pooled analysis of the percentage of NK cells from healthy donors or prostate cancer patients expressing IFNγ against PC-3 targets. FIG. 14F is a graph showing a pooled analysis of the percentage of NK cells from healthy donors or prostate cancer patients expressing IFNγ against the DU-145 target. FIG. 14G is a graph showing a pooled analysis of the percentage of NK cells from healthy donors or prostate cancer patients expressing CD107a+ against the LnCAP target. FIG. 14H is a graph showing a pooled analysis of the percentage of NK cells from healthy donors or prostate cancer patients expressing CD107a+ against the VCAP target. FIG. 14I is a graph showing a pooled analysis of the percentage of NK cells from healthy donors or prostate cancer patients expressing CD107a+ against the 22RV1 target. FIG. 14J is a graph showing a pooled analysis of the percentage of NK cells from healthy donors or prostate cancer patients expressing IFNγ against the LnCAP target. FIG. 14K is a graph showing a pooled analysis of the percentage of NK cells from healthy donors or prostate cancer patients expressing IFNγ against the VCAP target. FIG. 14L is a graph showing a pooled analysis of the percentage of NK cells from healthy donors or prostate cancer patients expressing IFNγ against the 22RV1 target. [Figure 14B] See legend to Figure 14A. [Figure 14C] See legend to Figure 14A. [Figure 14D] See legend to Figure 14A. [Figure 14E] See legend to Figure 14A. [Figure 14F] See legend to Figure 14A. [Figure 14G] See legend to Figure 14A. [Figure 14H] See legend to Figure 14A. [Figure 14I] See legend to Figure 14A. [Figure 14J] See legend to Figure 14A. [Figure 14K] See legend to Figure 14A. [Figure 14L] See legend to Figure 14A. [Figure 15A] Figures 15A-15D show that cam1615B7-H3 TriKE enhances the function of NK cells from healthy donors (black bars) and prostate cancer patients (white bars) against prostate tumor targets. Figure 15A shows a pooled analysis of the percentage of NK cells from healthy donors or prostate cancer patients expressing CD107a+ (top) or IFNγ (bottom) against C4-2 targets (N=9 for healthy donors, N=3 for patients). Figure 15B shows a pooled analysis of the percentage of NK cells from healthy donors or prostate cancer patients expressing CD107a+ (top) or IFNγ (bottom) against DU145 targets (N=9 for healthy donors, N=3 for patients). Figure 15C is a graph showing a pooled analysis of the percentage of NK cells from healthy donors or prostate cancer patients expressing CD107a+ (top) or IFNγ (bottom) against LNCaP targets (N=9 for healthy donors, N=3 for patients). Figure 15D is a graph showing a pooled analysis of the percentage of NK cells from healthy donors or prostate cancer patients expressing CD107a+ (top) or IFNγ (bottom) against PC3 targets (N=9 for healthy donors, N=3 for patients). *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. [Figure 15B] See legend to Figure 15A. [Figure 15C]See legend to Figure 15A. [Figure 15D] See legend to Figure 15A. [Figure 16] Graph illustrating PC3 standard IncuCyte assay. [Figure 17] Photographs illustrating PC-3 spheroids under various treatment conditions. [Figure 18] 1 is a graph illustrating the size of PC-3 spheroids over time. [Figure 19] 1 is a graph illustrating the size of PC-3 spheroids over time. [Figure 20] Photographs illustrating the ability of B7-H3 TriKE and BiKE to mediate efficient killing of PC3 spheroids are shown. [Figure 21] 1 is a graph illustrating Cell Index over time. [Figure 22] 22A-22F illustrate the enzalutamide-resistant prostate cancer cell line phenotype of B7-H3. FIG. 22A is a graph illustrating B7-H3 expression in CREB5+ cells. FIG. 22B is a graph illustrating B7-H3 expression in C4-2 cells. FIG. 22C is a graph illustrating B7-H3 expression in LN-CaP cells. FIG. 22D is a graph illustrating B7-H3 expression in enzalutamide-resistant LNCap cells. FIG. 22E is a graph illustrating B7-H3 expression in PC3 cells. FIG. 22F is a graph illustrating B7-H3 expression in 22RV1 cells. [Figure 23A]Figures 23A-23L illustrate how camB7-H3 TriKE induces activity against prostate cancer cells over a wider dynamic range than previous scFv versions. Figure 23A is a graph illustrating the percentage of CD107a+ NK cells in PBMCNK alone in the presence of 0.3 nM TriKE. Figure 23B is a graph illustrating the percentage of CD107a+ NK cells in PBMCNK alone in the presence of 3 nM TriKE. Figure 23C is a graph illustrating the percentage of CD107a+ NK cells in PBMCNK alone in the presence of 30 nM TriKE. Figure 23D is a graph illustrating the percentage of CD107a+ NK cells in PBMCNK and CA-2 cells in the presence of 0.3 nM TriKE. Figure 23E is a graph illustrating the percentage of CD107a+ NK cells in PBMCNK and CA-2 cells in the presence of 3 nM TriKE. Figure 23F is a graph illustrating the percentage of CD107a+ NK cells in PBMCNK and CA-2 cells in the presence of 30 nM TriKE. Figure 23G is a graph illustrating the percentage of IFNγ+ NK cells in PBMCNK alone in the presence of 0.3 nM TriKE. Figure 23H is a graph illustrating the percentage of IFNγ+ NK cells in PBMCNK alone in the presence of 3 nM TriKE. Figure 23I is a graph illustrating the percentage of IFNγ+ NK cells in PBMCNK alone in the presence of 30 nM TriKE. Figure 23J is a graph illustrating the percentage of IFNγ+ NK cells in PBMCNK and CA-2 cells in the presence of 0.3 nM TriKE. Figure 23K is a graph illustrating the percentage of IFNγ+ NK cells in PBMCNK and CA-2 cells in the presence of 3 nM TriKE. FIG. 23L is a graph illustrating the percentage of IFNγ+ NK cells in PBMCNK and CA-2 cells in the presence of 30 nM TriKE. [Figure 23B] See legend to Figure 23A. [Figure 23C] See legend to Figure 23A. [Figure 23D] See legend to Figure 23A. [Figure 23E] See legend to Figure 23A. [Figure 23F] See legend to Figure 23A. [Figure 23G] See legend to Figure 23A. [Figure 23H] See legend to Figure 23A. [Figure 23I] See legend to Figure 23A. [Figure 23J] See legend to Figure 23A. [Figure 23K] See legend to Figure 23A. [Figure 23L] See legend to Figure 23A. [Figure 24A] Figures 24A-24F show that B7-H3 TriKE enhances NK cell function against lung tumor targets. Figure 24A is a graph showing a pooled analysis of the percentage of NK cells from healthy donors expressing CD107a+ against A549 tumor targets (N=6). Figure 24B is a graph showing a pooled analysis of the percentage of NK cells from healthy donors expressing IFNγ against A549 tumor targets (N=6). Figure 24C is a graph showing a pooled analysis of the percentage of NK cells from healthy donors expressing CD107a+ against NCI-H322 tumor targets (N=5). Figure 24D is a graph showing a pooled analysis of the percentage of NK cells from healthy donors expressing IFNγ against NCI-H322 tumor targets (N=5). Figure 24E is a graph showing the pooled percentage of NK cells from healthy donors (black bars) or lung cancer patients (white bars) expressing CD107a+ against NCI-H460 (N=7). Figure 24F is a graph showing the pooled percentage of NK cells from healthy donors (black bars) or lung cancer patients (white bars) expressing IFNγ against NCI-H460 (N=7). **p<0.01, ***p<0.001, and ****p<0.0001. [Figure 24B] See legend to Figure 24A. [Figure 24C] See legend to Figure 24A. [Figure 24D] See legend to Figure 24A. [Figure 24E] See legend to Figure 24A. [Figure 24F] See legend to Figure 24A. [Figure 25A] Figures 25A-25B illustrate the assessment of NK cell activity against HNSCC without treatment. Figure 25A is a graph illustrating the percentage of CD107a expression (as a marker for degranulation). Figure 25B is a graph illustrating the percentage of intracellular IFN-γ production. [Figure 25B] See legend to Figure 25A. [Figure 26A] Figures 26A-26B illustrate the assessment of B7-H3 expression in tumor and immune cells. Figure 26A is a graph illustrating B7-H3 expression in five HNSCC cell lines by flow cytometry and binding affinity to B7-H3 single domains. Figure 26B is a graph illustrating B7-H3 expression in PBMCs from healthy donors by flow cytometry. [Figure 26B] See legend to Figure 26A. [Figure 27] Figures 27A-27D illustrate functional validation of B7-H3 TriKE. Figure 27A is a graph illustrating CD107a expression in PBMCs from healthy donors incubated with Cal27 trios. Figure 27B is a graph illustrating IFN-y production in PBMCs from healthy donors incubated with Cal27 trios. Figure 27C is a graph illustrating CD107a expression in PBMCs from healthy donors incubated with Cal33 trios. Figure 27D is a graph illustrating IFN-y production in PBMCs from healthy donors incubated with Cal33 trios. [Figure 28A]Figures 28A-F illustrate real-time imaging assays. Figure 28A is a graph showing the survival of Nuclight red-labeled Cal27 after 48 hours of incubation in an IncuCyte Zoom imager with NK cells at 5:1 E:T in different conditions: no treatment or 3 nM IL-15, B7-H3 SD, and B7-H3 TriKE. Figure 28B shows a photograph illustrating Nuclight red-labeled Cal27 spheroids. Figure 28C is a graph illustrating the size of the spheroids in Figure 28B. Figure 28D is a graph showing the survival of Nuclight red-labeled Cal33 after 48 hours of incubation in an IncuCyte Zoom imager with NK cells at 5:1 E:T in different conditions: no treatment or 3 nM IL-15, B7-H3 SD, and B7-H3 TriKE. Figure 28E shows a photograph illustrating Nuclight red-labeled Cal33 spheroids, and Figure 28F is a graph illustrating the size of the spheroids in Figure 28E. [Figure 28B] See legend to Figure 28A. [Figure 28C] See legend to Figure 28A. [Figure 28D] See legend to Figure 28A. [Figure 28E] See legend to Figure 28A. [Figure 28F] See legend to Figure 28A. [Figure 29A] Figures 29A-29B show that various doses of cam1615B7-H3 TriKE enhance NK cell function against ovarian tumor targets. Figure 29A is a graph showing the pooled percentage of NK cells from healthy donors expressing CD107a+ (degranulation) against OVCAR8 and MA148 targets. Figure 29B is a graph showing the pooled percentage of NK cells from healthy donors expressing IFNγ cytokine production against OVCAR8 and MA148 targets. [Figure 29B] See legend to Figure 29A. [Figure 30A]Figures 30A-30I show that cam1615B7-H3 TriKE enhances NK cell function against ovarian tumor targets. Healthy donor or ovarian cancer PBMC cells were incubated with ovarian tumor targets at 2:1 E:T ratio with 30 nM TriKE or control condition for 4 hours. Figure 30A is a graph showing the pooled percentage of NK cells from healthy donors expressing CD107a+ (degranulation) against OVCAR8 targets (N=8). Figure 30B is a graph showing the pooled percentage of NK cells from healthy donors expressing IFNγ cytokine production against OVCAR8 targets (N=8). Figure 30C is a graph showing the pooled percentage of NK cells from healthy donors expressing CD107a+ (degranulation) against OVCAR3 targets (N=4). Figure 30D is a graph showing the pooled percentage of NK cells from healthy donors expressing IFNγ cytokine production against OVCAR3 targets (N=4). FIG. 30E is a graph showing the pooled percentage of NK cells from healthy donors expressing CD107a+ (degranulation) against OVCAR5 targets (N=7). FIG. 30F is a graph showing the pooled percentage of NK cells from healthy donors expressing IFNγ cytokine production against OVCAR5 targets (N=7). FIG. 30G is a graph illustrating a pooled analysis of the percentage of NK cells from healthy donors (black bars) or ovarian cancer patients (white bars) expressing CD107a+ (degranulation) against MA-148 targets (N=9). FIG. 30H is a graph illustrating a pooled analysis of the percentage of NK cells from healthy donors (black bars) or ovarian cancer patients (white bars) expressing IFNγ against MA-148 targets (N=6). FIG. 30I is a graph illustrating that tumor killing was assessed using an IncuCyte imaging assay. NuclightRed expressing OVCAR8 targets were incubated with enriched healthy donor NKs with caspase 3 / 7 viability dye for 48 hours. The percentage of viable (Nuclight Red+Caspase 3 / 7-) tumor cells was quantified over 48 hours and normalized to tumor alone. Readings were obtained every 15 minutes (representative of 4 separate experiments).*p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. [Figure 30B] See legend to Figure 30A. [Figure 30C] See legend to Figure 30A. [Figure 30D] See legend to Figure 30A. [Figure 30E] See legend to Figure 30A. [Figure 30F] See legend to Figure 30A. [Figure 30G] See legend to Figure 30A. [Figure 30H] See legend to Figure 30A. [Figure 30I] See legend to Figure 30A. [Figure 31-1] High-dimensional analysis of cam1615B7-H3 activated NK cells. Concatenated analysis of PBMCs from three donors incubated alone, with 30 nM cam1615B7-H3 TriKE, with OVCAR8 tumor (2:1 E:T), or with 30 nM cam1615B7-H3 TriKE and OVCAR8 tumor. Data were visualized with viSNE (Cytobank) and gated on CD45+CD56+CD3- cells. [Figure 31-2] See description of Figure 31-1. [Figure 32A]32A-32F illustrate that cam1615B7-H3 TriKE is effective in suppressing ovarian tumor progression in vivo. FIG. 32A shows a xenogeneic ovarian cancer MA-148-Luc model in female NSG mice (N=5 / treatment group). FIG. 32B is a graph showing bioluminescence imaging showing tumor progression measured as total flux radiance (p / s) over 3 weeks in the MA148 mouse model treated with enriched NK cells and the indicated treatments. FIG. 32C is a graph illustrating the bioluminescence imaging results at the day 21 data point. FIG. 32D is a photograph illustrating the total flux radiance (p / s) at day 21. FIG. 32E is a scatter plot of CD56+CD3- NK cell counts from peritoneal lavage fluid of rhIL-15 and cam1615B7-H3 TriKE treatment groups at day 21. Figure 32F is a scatter plot of CD16 medium fluorescence intensity in NK cells from peritoneal lavage fluids of rhIL-15 and cam1615B7-H3 TriKE treatment groups on day 21. *p<0.05, **p<0.01. [Figure 32B] See legend to Figure 32A. [Figure 32C] See legend to Figure 32A. [Fig. 32D] See legend to Figure 32A. [Figure 32E] See legend to Figure 32A. [Fig. 32F] See legend to Figure 32A. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0036] Detailed Description of the Invention The present invention is based on the development of B7-H3 targeted fusion proteins, and specifically, the B7-H3 targeted trispecific killer engager molecule (TriKE), and methods of using same.

[0037] Before the compositions and methods of the present invention are described, it is to be understood that the invention is not limited to the particular compositions, methods, and experimental conditions described, as such compositions, methods, and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the invention will be limited only by the appended claims.

[0038] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to "a method" includes one or more methods, and / or steps, of the type described herein that will be apparent to those skilled in the art after reading this disclosure and so forth.

[0039] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0040] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, but it is understood that modifications and variations are within the spirit and scope of this disclosure. Preferred methods and materials are described herein.

[0041] In one embodiment, the invention provides an isolated nucleic acid sequence as set forth in SEQ ID NO: 13 or 14, or a sequence having 90% identity thereto.

[0042] As used herein, the term "nucleic acid" or "oligonucleotide" refers to a polynucleotide, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). Nucleic acids include, but are not limited to, genomic DNA, cDNA, mRNA, iRNA, miRNA, tRNA, ncRNA, rRNA, and recombinantly produced and chemically synthesized molecules, such as aptamers, plasmids, antisense DNA strands, shRNA, ribozymes, conjugated nucleic acids, and oligonucleotides. According to the present invention, nucleic acids can exist as single-stranded or double-stranded, and linear or covalently closed circular molecules. Nucleic acids can be isolated. The term "isolated nucleic acid" means that the nucleic acid is (i) amplified in vitro, for example by polymerase chain reaction (PCR), (ii) produced recombinantly by cloning, (iii) purified, for example by cleavage and separation by gel electrophoresis, (iv) synthesized, for example by chemical synthesis, or (vi) extracted from a sample. The nucleic acid may be used for introduction into cells, i.e. for transfection of cells, in particular in the form of RNA, which may be prepared by in vitro transcription from a DNA template. Furthermore, the RNA may be modified before application by stabilizing sequences, capping and polyadenylation.

[0043] As used herein, "amplified DNA" or "PCR product" refers to an amplified fragment of DNA of a defined size. A variety of techniques for detecting PCR products are available and well known in the art. Methods for detection of PCR products include, but are not limited to, gel electrophoresis using agarose or polyacrylamide gels and adding ethidium bromide staining (DNA intercalant), labeled probes (radioactive or non-radioactively labeled, Southern blotting), labeled deoxyribonucleotides (for direct incorporation of radioactive or non-radioactive labels), or silver staining for direct visualization of the amplified PCR products; restriction endonuclease digestion relying on agarose or polyacrylamide gels or high performance liquid chromatography (HPLC); dot blots using hybridization of amplified DNA to specific labeled probes (radioactive or non-radioactively labeled); high pressure liquid chromatography with ultraviolet detection; electrochemiluminescence coupled with potential-initiated chemical reactions / photon detection; and direct sequencing using radioactive or fluorescently labeled deoxyribonucleotides to determine the exact order of nucleotides comprising the DNA fragment of interest, oligo ligation assay (OLA), PCR, qPCR, DNA sequencing, fluorescence, gel electrophoresis, magnetic beads, allele-specific primer extension (ASPE) and / or direct hybridization.

[0044] In general, nucleic acids can be extracted, isolated, amplified, or analyzed by a variety of techniques, such as those described by Green and Sambrook, Molecular Cloning: A Laboratory Manual (Fourth Edition), Cold Spring Harbor Laboratory Press, Woodbury, NY 2,028 pages (2012); or those described in U.S. Patent No. 7,957,913; U.S. Patent No. 7,776,616, U.S. Patent No. 5,234,809, U.S. Patent Application Publication No. 2010 / 0285578, and U.S. Patent Application Publication No. 2002 / 0190663. Examples of nucleic acid analysis include, but are not limited to, sequencing and DNA-protein interaction. Sequencing may be by any method known in the art. DNA sequencing techniques include classical dideoxy sequencing reactions (Sanger method) using labeled terminators or primers and in-slab or in-capillary gel separation, as well as next-generation sequencing methods such as sequencing by synthesis using reversibly terminated labeled nucleotides, pyrosequencing, 454 sequencing, Illumina / Solexa sequencing, allele-specific hybridization with a library of labeled oligonucleotide probes, allele-specific hybridization with a library of labeled clones followed by ligation, real-time monitoring of the incorporation of labeled nucleotides during the polymerization step, polony sequencing, and SOLiD sequencing. The separated molecules may be sequenced by sequential or single extension reactions using polymerases or ligases, as well as by single or sequential differential hybridization with a library of probes.

[0045] The terms "sequence identity" or "percent identity" are used interchangeably herein. To determine the percent identity of two polypeptide molecules or two polynucleotide sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first polypeptide or polynucleotide for optimal alignment with a second polypeptide or polynucleotide sequence). The amino acids or nucleotides at corresponding amino acid or nucleotide positions are then compared. If a position in the first sequence is occupied by the same amino acid or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions / total number of positions (i.e., overlapping positions) x 100). In some embodiments, the length of the reference sequence (e.g., SEQ ID NO: 13 or 14) aligned for comparison purposes is at least 80% of the length of the comparison sequence, and in some embodiments, at least 90% or 100%. In one embodiment, the two sequences are the same length.

[0046] The desired degree of sequence identity ranges from about 80% to 100%, and integer values ​​therebetween. The percent identity between the disclosed and claimed sequences can be at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9%. In general, a perfect match indicates 100% identity over the length of the reference sequence (e.g., SEQ ID NO: 13 or 14). Preferably, sequences that are not 100% identical to the sequences provided herein retain the function of the original sequence (e.g., the ability to bind to B7-H3 or CD16).

[0047] Polypeptides and polynucleotides that are about 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5% or more identical to the polypeptides and polynucleotides described herein are embodied within the present disclosure. For example, a polypeptide can have 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO:13 or 14.

[0048] Variants of the disclosed sequences also include peptides or full-length proteins that contain substitutions, deletions, or insertions into the protein backbone that still retain at least about 70% identity to the original protein over the corresponding portions. Larger deviations from homology are permitted where similar amino acids, i.e., conservative amino acid substitutions, are not counted as sequence changes. Examples of conservative substitutions include amino acids with the same or similar properties. Exemplary conservative amino acid substitutions include alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartic acid to glutamic acid; cysteine ​​to serine; glutamine to asparagine; glutamic acid to aspartic acid; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine, glutamine, or glutamic acid; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine, or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; valine to isoleucine, and then to leucine.

[0049] In another embodiment, the invention provides a protein encoded by the nucleic acid sequence set forth in SEQ ID NO: 13 or 14, or a sequence having 90% identity thereto.

[0050] The terms "peptide", "polypeptide" and "protein" are used interchangeably herein and refer to any chain of at least two amino acids linked by a covalent chemical bond. As used herein, a polypeptide may refer to a complete amino acid sequence that encodes an entire protein or a portion thereof. A "protein coding sequence" or a sequence that "encodes" a particular polypeptide or peptide is a nucleic acid sequence that is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vitro or in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxyl) terminus. Coding sequences include, but are not limited to, cDNA from prokaryotic or eukaryotic mRNA, genomic DNA sequences from prokaryotic or eukaryotic DNA, and even synthetic DNA sequences. A transcription termination sequence is typically located 3' to the coding sequence.

[0051] In one aspect, the amino acid sequence is selected from SEQ ID NO:6 or SEQ ID NO:7.

[0052] The nucleic acid sequences provided herein can, for example, encode a light or heavy chain of an antibody, and confer upon the encoded polypeptide a binding domain or a targeting domain to a particular target. Such polypeptides can be referred to as targeting peptides.

[0053] The term "antibody" generally refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen-binding site that immunospecifically binds to an antigen. "Native antibodies" and "intact immunoglobulins" and the like are usually heterotetrameric glycoproteins of about 150,000 daltons, consisting of two identical light (L) chains and two identical heavy (H) chains. Light chains from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (κ) and lambda (λ), based on the amino acid sequence of their constant domains. Depending on the amino acid sequence of the constant domains of their heavy chains, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy-chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

[0054] In a typical antibody molecule, each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide bonds varies between the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced interchain disulfide bridges. Each heavy chain has a variable domain (VH) at one end followed by several constant domains. Each light chain has a variable domain (VL) at one end and a constant domain at the other end, with the constant domain of the light chain aligning with the first constant domain of the heavy chain and the variable domain of the light chain aligning with the variable domain of the heavy chain. Certain amino acid residues are believed to form an interface between the light chain variable domain and the heavy chain variable domain. Each variable region contains three segments called complementarity determining regions (CDRs) or hypervariable regions, and the more highly conserved portions of the variable domains are called framework regions (FRs). The variable domains of the heavy and light chains each contain four FR regions that are primarily in a β-sheet configuration, connected by three CDRs, which form loops that connect and sometimes form part of the β-sheet structure. The CDRs in each chain are held tightly together by the FRs and, together with the CDRs from the other chain, contribute to the formation of the antigen-binding or targeting domain of the antibody (see Kabat et al., NIH Puhl. No. 91-3242, Vol. I, pages 647-669

[1991] ). The constant domains are not directly involved in binding the antibody to an antigen, but exhibit various effector functions, such as participating in antibody-dependent cellular cytotoxicity.

[0055] Antibodies can be experimentally cleaved with the proteolytic enzyme papain, which cleaves each of the heavy chains and produces three separate antibody fragments. Two units, consisting of a light chain and a fragment of the heavy chain of approximately equal mass to the light chain, are called Fab fragments (i.e., "antigen-binding" fragments). The third unit, consisting of two equal segments of the heavy chain, is called the Fc fragment. The Fc fragment typically does not participate in antigen-antibody binding, but is important in subsequent processes involved in removing antigens from the body. As used herein, "antibody fragment" includes a portion of an intact antibody, preferably the antigen-binding or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab' and F(ab')2, Fc fragments or Fc fusion products, single chain Fv (scFv), disulfide-linked Fv (sdfv), as well as fragments containing either the VL or VH domains, diabodies, tribodies, and the like (Zapata et al. Protein Eng. 8(10):1057-1062

[1995] ).

[0056] Fab fragments contain the constant domain of the light chain and the first constant domain (CHI) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxyl terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine ​​residues of the constant domains bear a free thiol group. F(ab')2 antibody fragments were originally produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0057] The Fc region of an antibody is the tail region of the antibody that interacts with cell surface receptors and several proteins of the complement system. This property allows the antibody to activate the immune system. In IgG, IgA and IgD antibody isotypes, the Fc region consists of two identical protein fragments derived from the second and third constant domains of the two heavy chains of the antibody, whereas the Fc region of IgM and IgE contains three heavy chain constant domains (CH domains 2-4) in each polypeptide chain. The Fc region of IgG has a highly conserved N-glycosylation site. Glycosylation of the Fc fragment is essential for Fc receptor-mediated activity. The N-glycans attached to this site are mainly complex type, core-fucosylated, biantennary structures. In addition, a small proportion of these N-glycans also have bisected GlcNAc and α-2,6-linked sialic acid residues.

[0058] Fc fusion proteins (also known as Fc chimeric fusion proteins, Fc-Ig, Ig-based chimeric fusion proteins, and Fc-tag proteins) consist of the Fc domain of IgG genetically linked to a peptide or protein of interest. Fc-fusion proteins have become valuable reagents for in vivo and in vitro research. Fc fusion binding partners can range from single peptides to ligands that activate upon binding to cell surface receptors, signaling molecules, extracellular domains of receptors that are activated upon dimerization, or as bait proteins used to identify binding partners in protein microarrays. One of the most valuable in vivo features of the Fc domain is that it can dramatically extend the plasma half-life of a protein of interest, an attribute that provides biotherapeutics with improved therapeutic efficacy, making Fc fusion proteins attractive biotherapeutics. Fc fusion proteins can be part of a pharmaceutical composition that includes the Fc fusion protein and a pharma- ceutically acceptable carrier excipient or carrier. Pharmaceutically acceptable carriers, excipients, or stabilizers are well known in the art (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. Ed. (1980)).Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed and include the following: buffers such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol); polypeptides of low molecular weight (less than about 10 residues); serum albumin, gelatin, etc. hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as TWEEN™, PLURONICS™, or polyethylene glycol (PEG).

[0059] "Fv" is the minimum antibody fragment that contains a complete antigen recognition and binding site. This region consists of a dimer of one heavy chain variable domain and one light chain variable domain in tight but non-covalent association. In this configuration, the three CDRs of each variable domain interact to define an antigen binding site on the surface of the VH-VL dimer. Collectively, the six CDRs confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three CDRs specific for an antigen) has the ability to recognize and bind to an antigen, although with a lower affinity than the entire binding site.

[0060] "Single-chain Fv" or "sFv" antibody fragments comprise the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Preferably, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the sFv to form the desired structure for antigen binding. For a review of sFvs, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0061] Various techniques have been developed for the production of antibody fragments. Traditionally, these fragments were obtained via proteolytic digestion of intact antibodies (see, e.g., Morimoto et al., Journal of Biochemical and Biophysical Methods 24:107-117 (1992), and Brennan et al., Science, 229:81 (1985)). However, these fragments can now be produced directly by recombinant host cells. For example, antibody fragments can be isolated from the antibody phage libraries mentioned above. Alternatively, Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab')2 fragments (Carter et al., Bio / Technology 10:163-167 (1992)). According to another approach, F(ab')2 fragments can be isolated directly from recombinant host cell culture. Other techniques for the production of antibody fragments will be apparent to those skilled in the art. In other embodiments, the antibody of choice is a single chain Fv fragment (scFv). See WO93 / 16185.

[0062] In various embodiments, the nucleic acid sequences provided herein encode light and heavy chains that specifically bind to the B3-H7 protein.

[0063] B7 homolog 3 (B7-H3), also known as cluster of differentiation 276 (CD276), is a human protein encoded by the CD276 gene. The B7-H3 protein is a 316 amino acid long type I transmembrane protein that exists in two isoforms determined by its extracellular domain. In mice, the extracellular domain consists of a single pair of immunoglobulin variable (IgV)-like and immunoglobulin constant (IgC)-like domains, whereas in humans it consists of one pair (2Ig-B7-H3) or two identical pairs (4Ig-B7-H3) due to exon duplication. B7-H3 mRNA is expressed in most normal tissues. In contrast, the B7-H3 protein has very limited expression in normal tissues due to its post-transcriptional regulation by microRNAs. However, the B7-H3 protein is expressed at high frequency in many different cancer types (60% of all cancers).

[0064] In non-malignant tissues, B7-H3 has a primarily inhibitory role in adaptive immunity, suppressing T cell activation and proliferation. In malignant tissues, B7-H3 is an immune checkpoint molecule that inhibits tumor antigen-specific immune responses. B7-H3 also has non-immune tumor-promoting functions, such as promoting migration, invasion, angiogenesis, chemoresistance, epithelial to mesenchymal transition, and influencing tumor cell metabolism. Due to its selective expression in solid tumors and its tumor-promoting functions, B7-H3 is the target of several anti-cancer drugs, including enoblituzumab, ombultamab, MGD009, MGC018, DS-7300a, and CAR-T cells.

[0065] As used herein, the term "B7-H3 targeting peptide" or "B7-H3 targeting protein" is meant to refer to any peptide or polypeptide (including proteins and fusion proteins) that can specifically bind to B7-H3. A B7-H3 targeting peptide can be an antibody, antibody fragment, etc. that specifically binds to one or more target polypeptides, including B7-H3. In some embodiments, the polypeptide encodes a light chain and a heavy chain of a B7-H3 targeting peptide. In one embodiment, the nucleic acid sequence of SEQ ID NO: 13 can encode a light chain of a B7-H3 targeting peptide having an amino acid sequence set forth in SEQ ID NO: 6. In another embodiment, the nucleic acid sequence of SEQ ID NO: 14 can encode a heavy chain of a B7-H3 targeting peptide having an amino acid sequence set forth in SEQ ID NO: 8.

[0066] In an additional embodiment, the invention provides a fusion protein comprising the amino acid sequences set forth in SEQ ID NOs: 6 and 7 operably linked to each other in either orientation.

[0067] The terms "fusion molecule" and "fusion protein" are used interchangeably and are meant to refer to a biologically active polypeptide with or without an additional effector molecule, usually a protein or peptide sequence, covalently linked (i.e., fused) by recombinant, chemical, or other suitable methods. If necessary, the fusion molecule can be fused at one or several sites via a peptide linker sequence. Alternatively, a peptide linker may be used to aid in the construction of the fusion molecule. In particular, the preferred fusion molecule is a fusion protein. In general, the fusion molecule may also include a conjugate molecule.

[0068] "Operably linked" to each other means that there is a direct or indirect covalent bond between the peptides that make up the fusion protein. Thus, two operably linked domains can be directly covalently linked to each other. Conversely, two operably linked domains can be linked by mutual covalent bonds to an intervening moiety (e.g., a flanking sequence). Two domains can be considered to be operably linked, for example, when they are separated by a third domain, with or without one or more intervening flanking sequences.

[0069] The method for linking two individual elements usually requires the use of a linker. As used herein, the term "linker" refers to any bond, small molecule, or other vehicle that allows the substrate and active agent to target the same region, tissue, or cell, for example by physically linking the individual parts of the conjugate. A linker can be any chemical moiety that can link a compound, usually a drug, to a cell-binding agent in a stable covalent manner.

[0070] Fusion proteins provided herein can include, for example, the amino acid sequences set forth in SEQ ID NOs: 6 and 7 operably linked to each other in either orientation. For example, a fusion protein can include the amino acid sequence set forth in SEQ ID NO: 6 at the C-terminus of the fusion protein and the amino acid sequence set forth in SEQ ID NO: 7 at the N-terminus of the fusion protein, or the fusion protein can include the amino acid sequence set forth in SEQ ID NO: 6 at the N-terminus of the fusion protein and the amino acid sequence set forth in SEQ ID NO: 7 at the C-terminus of the fusion protein. The orientation of the amino acid sequences within the fusion protein does not alter the binding specificity of the fusion protein (i.e., the B7-H3 targeting fusion protein) for its target.

[0071] The light and heavy chains of the B7-H3 targeting peptide can be operably linked to each other in either orientation without affecting the binding specificity or sensitivity of the targeting peptide. In one aspect, the protein comprises SEQ ID NOs:6 and 7 directly linked between the C-terminus of SEQ ID NO:6 and the N-terminus of SEQ ID NO:7. In another aspect, the protein comprises SEQ ID NOs:7 and 6 directly linked between the C-terminus of SEQ ID NO:7 and the N-terminus of SEQ ID NO:6.

[0072] The fusion proteins provided herein can include additional protein domains, such as additional targeting domains to provide the fusion protein with specific binding to one or more target polypeptides. For example, the fusion protein can be a trispecific killer engager (TriKE) molecule that includes a B7-H3 targeting peptide as a targeting domain.

[0073] NK cells are cytotoxic lymphocytes of the innate immune system capable of immune surveillance. Similar to cytotoxic T cells, NK cells deliver stored granzymes and perforin granules that are membrane permeable and apoptosis-inducing. Unlike T cells, NK cells do not require antigen priming and recognize targets by engaging activating receptors in the absence of MHC recognition. 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 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.

[0074] Therapeutically, adoptive transfer of NK cells can induce remission in patients with refractory acute myeloid leukemia (AML), for example, when combined with lymphodepleting chemotherapy and IL-2 to stimulate NK cell survival and in vivo expansion. This therapy can be limited by the lack of antigen specificity and IL-2-mediated induction of regulatory T (Treg) cells that suppress NK cell proliferation and function. Generating reagents that drive NK cell antigen specificity, proliferation and / or persistence while bypassing the negative effects of Treg inhibition can enhance NK cell-based immunotherapy.

[0075] Trispecific killer engager molecules are targeting fusion proteins that contain two domains capable of driving NK cell-mediated killing of tumor cells (e.g., CD33+ tumor cells and / or EpCAM+ tumor cells) and an intracellular NK activation domain capable of generating an NK cell self-sustaining signal that can drive NK cell proliferation and / or enhance NK cell-driven cytotoxicity against, for example, HL-60 targets, cancer cells, or cancer cell-derived cell lines.

[0076] NK cells are responsive to various cytokines, including IL-15, which are involved in, for example, NK cell homeostasis, proliferation, survival, activation, and / or development. IL-15 and IL-2 share several signaling components, including IL-2 / IL-15Rβ (CD122) and common gamma chain (CD132). Unlike IL-2, IL-15 does not stimulate Tregs, allowing NK cell activation while bypassing Treg inhibition of immune responses. Besides promoting NK cell homeostasis and proliferation, IL-15 may rescue functional defects of NK cells that may occur in post-transplant situations. IL-15 may also stimulate the function of CD8+ T cells, further enhancing their immunotherapeutic potential. In addition, based on preclinical studies, the toxicity profile of IL-15 may be more favorable than IL-2 at low doses. 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-15Rβ (CD122) and the common gamma chain (CD132). IL-15 can also activate NK cells and restore functional defects in NK cell engraftment after hematopoietic stem cell transplantation (HSCT).

[0077] The fusion proteins provided herein can be TriKE molecules that include one or more NK cell engager domains (e.g., CD16, CD16+CD2, CD16+DNAM, CD16+NKp46), one or more targeting domains (e.g., that target tumor cells or virally infected cells, such as the B7-H3 targeting peptides described herein), 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 operably linked to the other domains.

[0078] For example, a fusion protein described herein can be a TriKE molecule comprising a CD16 NK cell engager domain, such as a CD16 domain having the amino acid sequence set forth in SEQ ID NO:2 or 19, a B7-H3 targeting fusion protein domain, such as a B7-H3 fusion protein having the amino acid sequence set forth in SEQ ID NO:6 and 7, and an IL-15 cytokine NK activation domain, such as IL-15 having the amino acid sequence set forth in SEQ ID NO:4, 17 or 18.

[0079] The different protein domains of a TriKE molecule can be operably linked to one another, for example, linkers can be used to covalently link the protein domains of a TriKE molecule to one another.

[0080] The elements of the fusion protein can be operably linked and assembled together using one or more linkers. The linker can be sensitive or substantially resistant to acid-induced cleavage, photoinduced cleavage, peptidase-induced cleavage, esterase-induced cleavage, and disulfide bond cleavage under conditions under which the compound or antibody remains active. Linkers are classified based on chemical motifs known in the art, including disulfide groups, hydrazine or peptide (cleavable), or thioether groups (non-cleavable). Linkers also include charged linkers and hydrophilic forms thereof known in the art.

[0081] Linkers suitable for fusing two or more proteins or protein domains include natural linkers and empirical linkers.Natural linkers are derived from multi-domain proteins that naturally occur between protein domains.Natural linkers can have several properties depending on their length, hydrophobicity, amino acid residues, and secondary structure, etc., which can affect fusion proteins in different ways.

[0082] The study of linkers in natural multidomain proteins has led to the creation of many empirical linkers with various sequences and conformations for the construction of recombinant fusion proteins. Empirical linkers can be classified into three types: flexible linkers, rigid linkers, and cleavable linkers. Flexible linkers can provide some degree of movement or interaction to the linked domains. They are generally composed of small, non-polar amino acids (e.g., Gly) or polar amino acids (e.g., Ser or Thr), which provide flexibility and lead to mobility of the functional domains connected. Rigid linkers can successfully maintain a fixed distance between the domains to maintain their independent functions, which can provide efficient separation of protein domains or sufficient reduction of their interference with each other. Cleavable linkers can allow the release of functional domains in vivo. By utilizing inherent in vivo processes, cleavable linkers can be cleaved under certain conditions, such as the presence of reducing agents or proteases. This type of linker can reduce steric hindrance, improve biological activity, or achieve independent action / metabolism of individual domains of the recombinant fusion protein after linker cleavage.

[0083] Non-limiting examples of linkers include linkers having the amino acid sequences set forth in SEQ ID NOs: 3, 5, 10, 12, 15, and 16.

[0084] In one aspect, SEQ ID NO:2 or 19 and SEQ ID NO:4, 17 or 18 are linked by SEQ ID NO:3 or SEQ ID NO:15. In another aspect, SEQ ID NO:4, 17 or 18 and SEQ ID NO:6 or 7 are linked by SEQ ID NO:5 or SEQ ID NO:16. In other aspects, SEQ ID NOs:6 and 7 are operably linked in either orientation.

[0085] In a further embodiment, the present invention provides a fusion protein comprising a sequence as shown in SEQ ID NO:1 and a sequence having 90% or greater identity to SEQ ID NO:1.

[0086] In one embodiment, the invention provides a fusion protein comprising, operably linked, SEQ ID NO: 2 or 19; SEQ ID NO: 4, 17 or 18; SEQ ID NO: 6 and 7, or SEQ ID NO: 7 and 6.

[0087] The fusion proteins described herein may comprise a wild-type (wt) IL-15 or a mutant IL-15 cytokine NK activation domain. The mutant IL-15 may include, for example, a mutant IL-15 comprising a substitution of the N72 amino acid. Non-limiting examples of N72 substitutions include N72A and N72D mutations.

[0088] In some embodiments, SEQ ID NO: 4 has an N72 substitution. In various embodiments, the N72 mutation is N72A or N72D, and the protein is set forth in SEQ ID NO: 17 or 18, respectively.

[0089] In yet another embodiment, the invention provides a fusion protein comprising SEQ ID NO:19, SEQ ID NO:17 or 18, and SEQ ID NOs:6 and 7, in either orientation.

[0090] In one aspect, SEQ ID NO:19 is operably linked to SEQ ID NO:17 or 18 by a linker of SEQ ID NO:3 or 15. In another aspect, SEQ ID NO:17 or 18 is operably linked to SEQ ID NOs:6 and 7, in either orientation, by a linker of SEQ ID NO:5 or 16.

[0091] The fusion protein may comprise, in operable linkage, a camelid or human CD16 NK cell engager domain (SEQ ID NO: 2 or 19, respectively), a wt or mutant IL-15 cytokine NK activation domain (SEQ ID NO: 4, 17 or 18), and a light chain and a heavy chain of a B7-H3 targeting peptide (SEQ ID NO: 6 and 7, respectively). The CD16 NK cell engager domain may be linked to the IL-15 cytokine NK activation domain by a linker having the amino acid sequence set forth in SEQ ID NO: 3 or 15. The IL-15 cytokine NK activation domain may be linked to the B7-H3 targeting peptide by a linker having the amino acid sequence set forth in SEQ ID NO: 5 or 16. The IL-15 cytokine NK activation domain may be linked to the heavy chain of the B7-H3 targeting peptide (linked to the light chain) or to the light chain of the B7-H3 targeting peptide (linked to the heavy chain).

[0092] For example, a fusion protein can comprise, operably linked from the N-terminus to the C-terminus: SEQ ID NOs: 2, 4, 6, and 7; SEQ ID NOs: 2, 4, 7, and 6; SEQ ID NOs: 19, 17, 6, and 7; SEQ ID NOs: 19, 17, 7, and 6; SEQ ID NOs: 19, 18, 6, and 7, or SEQ ID NOs: 19, 18, 7, and 6 may include.

[0093] Specifically, the fusion protein comprises, operably linked from the N-terminus to the C-terminus: SEQ ID NOs: 2, 3, 4, 5, 6, and 7; SEQ ID NOs: 2, 3, 4, 16, 6, and 7; SEQ ID NOs: 2, 15, 4, 5, 6, and 7; SEQ ID NOs: 2, 15, 4, 16, 6, and 7; SEQ ID NOs: 2, 3, 4, 5, 7, and 6; SEQ ID NOs: 2, 3, 4, 16, 7, and 6; SEQ ID NOs: 2, 15, 4, 5, 7, and 6, or SEQ ID NOs: 2, 15, 4, 16, 7, and 6 may include.

[0094] In other embodiments, the fusion protein comprises, operably linked from the N-terminus to the C-terminus: SEQ ID NOs: 19, 3, 17, 5, 6, and 7; SEQ ID NOs: 19, 3, 17, 16, 6, and 7; SEQ ID NOs: 19, 15, 17, 5, 6, and 7; SEQ ID NOs: 19, 15, 17, 16, 6, and 7; SEQ ID NOs: 19, 3, 17, 5, 7, and 6; SEQ ID NOs: 19, 3, 17, 16, 7, and 6; SEQ ID NOs: 19, 15, 17, 5, 7, and 6; SEQ ID NOs: 19, 15, 17, 16, 7, and 6; SEQ ID NOs: 19, 3, 18, 5, 6, and 7; SEQ ID NOs: 19, 3, 18, 16, 6, and 7; SEQ ID NOs: 19, 15, 18, 5, 6, and 7; SEQ ID NOs: 19, 15, 18, 16, 6, and 7; SEQ ID NOs: 19, 3, 18, 5, 7, and 6; SEQ ID NOs: 19, 3, 18, 16, 7, and 6; SEQ ID NOs: 19, 15, 18, 5, 7, and 6, or SEQ ID NOs: 19, 15, 18, 16, 7, and 6 may include.

[0095] In some embodiments, the fusion protein further comprises a half-life extension (HLE) molecule.

[0096] The circulating half-life of targeting proteins such as IgG immunoglobulins can be regulated by the affinity of the Fc region for the neonatal Fc receptor (FcRn). A second general category of effector functions includes those that function after the immunoglobulin binds to an antigen. In the case of IgG, these functions involve the participation of the complement cascade or cells that bear Fc gamma receptors (FcγR). Binding of the Fc region to FcγR triggers certain immune effects, such as endocytosis of immune complexes, phagocytosis and destruction of immunoglobulin-coated particles or microorganisms (also called antibody-dependent phagocytosis, or ADCP), clearance of immune complexes, lysis of immunoglobulin-coated target cells by killer cells (called antibody-dependent cell-mediated cytotoxicity, or ADCC), release of inflammatory mediators, modulation of immune system cell activation, and modulation of immunoglobulin production. While certain engineered binding polypeptides (e.g., antibody variants (e.g., scFv) or antibody fragments (e.g., Fab fragments)) benefit from their smaller molecular size and / or monovalency, they suffer from several disadvantages due to the absence of a functional Fc region. For example, Fab fragments lack the Fc region necessary for FcRn binding and, due to their small size, are rapidly filtered from the blood by the kidneys and therefore have a short half-life in vivo.

[0097] Engineered targeting polypeptides such as fusion proteins described herein may exhibit reduced binding to FcRn when compared to native binding polypeptides, and therefore have reduced half-life in vivo. Fc variants with improved affinity to FcRn may have longer serum half-life, and such molecules have useful applications in methods of treating mammals where a long half-life of the administered polypeptide is desired, for example, to treat chronic diseases or disorders. In contrast, Fc variants with reduced FcRn binding affinity have shorter half-life, and such molecules are also beneficial, for example, for administration to mammals where a shortened circulation time may be advantageous, for example, in vivo diagnostic imaging, or in situations where the starting polypeptide has toxic side effects if present in the circulation for a long period of time.

[0098] The fusion proteins described herein can include a half-life extension (HLE) molecule to extend their half-life in vivo upon administration to a subject.

[0099] As used herein, the term "half-life" refers to the biological half-life of a particular targeting polypeptide in vivo. Half-life can be represented by the time required for half of the amount administered to a subject to be removed from the circulation and / or other tissues in an animal. When a clearance curve of a targeting polypeptide is constructed as a function of time, the curve is usually biphasic with a rapid α-phase and a longer β-phase. The α-phase typically represents the equilibration of the administered targeting polypeptide between the intravascular and extravascular spaces and is determined, in part, by the size of the polypeptide. The β-phase typically represents the catabolism of the targeting polypeptide in the intravascular space. Thus, the term half-life as used herein preferably refers to the half-life of a targeting polypeptide in the β-phase. The typical β-phase half-life of a human antibody in humans is 21 days.

[0100] Increased half-life is generally useful in in vivo applications of immunoglobulins, particularly antibodies, and most particularly antibody fragments of small size. Approaches described in the art to achieve such an effect include fusing small bispecific antibody constructs to larger proteins, which preferably do not interfere with the therapeutic effect of the protein construct. An example of such further development of bispecific T cell engagers is described in US2017 / 0218078A1, which provides a half-life extended format (HLE format) of bispecific T cell engagement molecules, comprising a first domain that binds to a target cell surface antigen, a second domain that binds to an extracellular epitope of the human and / or macaque CD3 epsilon chain, and a third domain that is a specific Fc modality (HLE molecule).

[0101] As used herein, the terms "half-life extension molecule," "HLE sequence," and the like, are meant to refer to any molecule, such as a protein or polypeptide, that can be linked or fused to a polypeptide of interest to increase or extend its half-life in vivo. Specifically, an HLE sequence generally comprises the Fc or scFc region of an immunoglobulin.

[0102] As used herein, the term "Fc region" refers to the portion of a native immunoglobulin formed by the respective Fc domains (or Fc portions) of its two heavy chains. A native Fc region is a homodimer. In contrast, the term "genetically fused Fc region" or "single-chain Fc region" (scFc region) as used herein refers to a synthetic Fc region consisting of Fc domains (or Fc portions) genetically linked (i.e., encoded in a single contiguous gene sequence) within a single polypeptide chain. Thus, a genetically fused Fc region (i.e., scFc region) is monomeric.

[0103] As used herein, the term "Fc domain" refers to that portion of a single immunoglobulin heavy chain beginning at the hinge region just upstream of the papain cleavage site (i.e., residue 216 in IgG, where the first residue of the heavy chain constant region is 114) and ending at the C-terminus of the antibody. Thus, a complete Fc domain includes at least the hinge, CH2, and CH3 domains.

[0104] The scFc region described herein comprises at least two Fc domains genetically fused via a linker polypeptide (e.g., an Fc connecting peptide) interposed between the Fc moieties. The scFc region can comprise two identical Fc moieties or can comprise two non-identical Fc moieties.

[0105] Non-limiting examples of Fc domains that can be used to prepare HLE molecules that can be incorporated into any of the fusion proteins described herein (either alone or in combination with another Fc domain via a linker polypeptide) include any of the polypeptides having an amino acid sequence comprising any one of SEQ ID NOs: 26-33.

[0106] Non-limiting examples of linker polypeptides that can be used in the preparation of HLE molecules and that can be used in the preparation of scFc regions include any of the polypeptides having an amino acid sequence comprising any one of SEQ ID NOs: 34 to 35.

[0107] The HLE molecules described herein can comprise an Fc domain having amino acids comprising any one of SEQ ID NOs: 26-33, or an scFc region comprising a first Fc domain having amino acids comprising any one of SEQ ID NOs: 26-33 fused to a second Fc domain having amino acids comprising any one of SEQ ID NOs: 26-33 via a linker having amino acids comprising any one of SEQ ID NOs: 34-35. For example, the HLE molecule can comprise any one of SEQ ID NOs: 21-25.

[0108] In additional embodiments, the invention provides an isolated nucleic acid sequence encoding any of the fusion proteins described herein.

[0109] A fusion protein as described herein can be encoded by a nucleic acid sequence, e.g., a TriKE fusion protein as set forth in SEQ ID NO: 1, comprising in operative linkage a CD16 NK cell engager domain, such as a CD16 domain having the amino acid sequence set forth in SEQ ID NO: 2, a B7-H3 targeting fusion protein domain, such as a B7-H3 fusion protein having the amino acid sequence set forth in SEQ ID NO: 6 and 7, and an IL-15 cytokine NK activation domain, such as an IL-15 having the amino acid sequence set forth in SEQ ID NO: 4. In one aspect, the sequence is SEQ ID NO: 8, or a sequence having 90% or greater sequence identity thereto.

[0110] In another embodiment, the invention provides a method of treating cancer in a subject comprising administering to the subject any of the fusion proteins described herein, thereby treating the cancer.

[0111] The term "subject" as used herein refers to any individual or patient on whom the method of the present invention is carried out. Generally, the subject is a human being, but as will be understood by those skilled in the art, the subject may also be an animal. Thus, other animals, including rodents (including mice, rats, hamsters, and guinea pigs), cats, dogs, rabbits, domestic animals such as cows, horses, goats, sheep, pigs, chickens, and primates (including monkeys, chimpanzees, orangutans, and gorillas), are included within the definition of a subject.

[0112] The term "treatment" is used interchangeably herein with the term "therapy" and refers to both 1) therapeutic procedures or measures that cure, slow, alleviate symptoms, and / or halt progression of a diagnosed pathological condition or disorder, and 2) as well as prophylactic / preventative measures. Those in need of treatment can include individuals who already have a particular medical disorder, as well as those who may eventually reach the disorder (i.e., those in need of preventative measures).

[0113] The terms "therapeutically effective amount", "effective dose", "therapeutically effective dose", "effective amount" and the like refer to that amount of the compound that elicits the biological or medical response in a tissue, system, animal or human that is being sought by a researcher, veterinarian, physician or other clinician. Generally, the response is either an improvement in symptoms or a desired biological outcome in a patient. Such an amount should be sufficient to treat cancer. An effective amount can be determined as described herein.

[0114] The terms "administration of" and / or "administering" should be understood to mean providing a therapeutically effective amount of a pharmaceutical composition to a subject in need of treatment. The route of administration can be enteral, topical, or parenteral. Thus, routes of administration include, but are not limited to, intradermal, subcutaneous, intravenous, intraperitoneal, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, transdermal, transtracheal, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal, oral, sublingual buccal, rectal, vaginal, nasal intraocular administration, as well as injection, inhalation, and spray. As used herein, the phrases "parenteral administration" and "administered parenterally" refer to modes of administration other than enteral and topical administration.

[0115] The fusion proteins described herein can be formulated into pharmaceutical compositions comprising the fusion protein and a pharma- ceutically acceptable carrier. By "pharmaceutically acceptable" it is meant that the carrier, diluent or excipient is compatible with the other ingredients of the formulation and not deleterious to the recipient thereof. Examples of carriers include, but are not limited to, liposomes, nanoparticles, ointments, micelles, microspheres, microparticles, creams, emulsions, and gels. Examples of excipients include, but are not limited to, anti-adhesives such as magnesium stearate, binders such as sugars and their derivatives (such as sucrose, lactose, starch, cellulose, sugar alcohols, etc.) proteins, gelatin and synthetic polymers, lubricants such as talc and silica, and preservatives such as antioxidants, vitamin A, vitamin E, vitamin C, retinyl palmitate, selenium, cysteine, methionine, citric acid, sodium sulfate, and parabens. Examples of diluents include, but are not limited to, water, alcohol, saline solution, glycol, mineral oil, and dimethyl sulfoxide (DMSO).

[0116] Pharmaceutical compositions can be administered in various unit dosage forms depending on the method of administration. Suitable unit dosage forms include, but are not limited to, powders, tablets, pills, capsules, lozenges, suppositories, patches, nasal sprays, injectables, implantable sustained release formulations, lipid complexes, etc.

[0117] The methods described herein are directed to the treatment of cancer. The term "cancer" refers to a group of diseases characterized by abnormal and uncontrolled cell growth beginning at one site (primary site) with the potential to invade and metastasize to other sites (secondary sites, metastases), distinguishing cancer (malignant tumors) from benign tumors. Virtually every organ can be affected, leading to over 100 types of cancer that can affect humans. Cancer can result from many causes, including genetic predisposition, viral infection, exposure to ionizing radiation, exposure to environmental pollutants, tobacco and / or alcohol use, obesity, poor diet, lack of physical activity, or any combination thereof. As used herein, "tumor" or "tumor," including grammatical variations thereof, refers to a new and abnormal growth of tissue that may be benign or cancerous. In related aspects, tumor refers to a neoplastic disease or disorder, including, but not limited to, various cancers. For example, such cancers may include prostate cancer, pancreatic cancer, biliary tract cancer, colon cancer, rectal cancer, liver cancer, kidney cancer, lung cancer, testicular cancer, breast cancer, ovarian cancer, brain cancer, head and neck cancer, melanoma, sarcoma, multiple myeloma, leukemia, lymphoma, and the like.

[0118] Exemplary cancers described by the National Cancer Institute include acute lymphoblastic leukemia, adult; acute lymphoblastic leukemia, pediatric; adult acute myeloid leukemia, adult; adrenal cortical carcinoma; adrenal cortical carcinoma, pediatric; AIDS-related lymphoma; AIDS-related malignancies; anal cancer; astrocytoma, pediatric cerebellum; astrocytoma, pediatric cerebral; cholangiocarcinoma, extrahepatic; bladder cancer; bladder cancer, pediatric; bone cancer, osteosarcoma / malignant fibrous histiocytoma; brain stem glioma, pediatric; brain tumor, adult; brain tumor, brain stem glioma, pediatric; brain tumor, cerebellar astrocytoma, pediatric; brain tumor, cerebral astrocytoma / malignant glioma, pediatric; brain tumor, ependymoma, pediatric ;Brain tumor, medulloblastoma, children;Brain tumor, supratentorial primitive neuroectodermal tumor, children;Brain tumor, visual pathway and hypothalamic glioma, children;Brain tumor, children (other);Breast cancer;Breast cancer and pregnancy;Breast cancer, children;Breast cancer, male;Bronchial adenoma / carcinoid, children:Carcinoid tumor, children;Carcinoid tumor, gastrointestinal;Carcinoma, adrenal cortex;Carcinoma, pancreatic islet cell;Carcinoma of unknown primary;Central nervous system lymphoma, primary;Cerebellar astrocytoma, children;Cerebral astrocytoma / malignant glioma, children;Cervical carcinoma;Childhood cancer;Chronic lymphocytic leukemia;Chronic myelogenous leukemia;Chronic myeloproliferative disorder;Clear cell sarcoma of tendon sheath;Colon Cancer;Colorectal cancer, children;Cutaneous T-cell lymphoma;Uterine cancer;Ependymoma, children;Epithelial carcinoma, ovary;Esophageal cancer;Esophageal cancer, children;Ewing's tumor;Extracranial germ cell tumor, children;Extragonadal germ cell tumor;Extrahepatic bile duct cancer;Eye cancer, intraocular melanoma;Eye cancer, retinal germ cell tumor;Gallbladder cancer;Gastric (Stomach) cancer;Gastric cancer, children;Gastrointestinal carcinoid tumor;Germ cell tumor, extracranial, children;Germ cell tumor, extragonadal;Germ cell tumor, ovary;Gestational trophoblastic tumor;Glioma, children's brain stem;Glioma, children's visual pathway and hypothalamus;Hairy cell leukemia;Head and neck cancer;Hepatocellular (liver) carcinoma, adults (primary);hepatocellular (liver) cancer, childhood (primary);Hodgkin's lymphoma, adult;Hodgkin's lymphoma, childhood;Hodgkin's lymphoma, gestational age;hypopharyngeal cancer;hypothalamic and visual pathway glioma, childhood;intraocular melanoma;islet cell carcinoma (endocrine pancreas);Kaposi's sarcoma;kidney cancer;laryngeal cancer;laryngeal cancer, childhood;leukemia, acute lymphoblastic, adult;leukemia, acute lymphoblastic, childhood;leukemia, acute myeloid, adult;leukemia, acute myeloid, childhood;leukemia, chronic lymphocytic;leukemia, chronic myeloid;leukemia, hairy cell;lip and oral cavity cancer;liver cancer, adult (primary);liver cancer, childhood (primary);Lung cancer, non-small cell;Lung cancer, small cell;Lymphoblastic leukemia, adult, acute;Lymphoblastic leukemia, childhood, acute;Lymphocytic leukemia, chronic;Lymphoma, AIDS-related;Lymphoma, central nervous system (primary);Lymphoma, cutaneous T cell;Lymphoma, Hodgkin, adult;Lymphoma, Hodgkin, childhood;Lymphoma, Hodgkin, gestational age;Lymphoma, non-Hodgkin, adult;Lymphoma, non-Hodgkin, childhood;Lymphoma, non-Hodgkin, gestational age;Lymphoma, primary central nervous system;Macroglobulinemia, Waldenström;Male breast cancer;Malignant mesothelioma, adult;Malignant mesothelioma, childhood;Malignant thymoma;medulloblastoma, childhood;melanoma;melanoma, intraocular;Merkel cell carcinoma;malignant mesothelioma;metastatic squamous cell carcinoma of the neck of unknown primary;multiple endocrine neoplasia syndrome, childhood;multiple myeloma / plasma cell neoplasm;mycosis fungoides;myelodysplastic syndrome;myeloid leukemia, chronic;myeloid leukemia, childhood acute;myeloma, multiple;myeloproliferative disorder, chronic;nasal cavity and paranasal sinus cancer;nasopharyngeal cancer;nasopharyngeal cancer, childhood;neuroblastoma;non-Hodgkin's lymphoma, adult;non-Hodgkin's lymphoma, childhood;non-Hodgkin's lymphoma, gestational age;non-small cell lung cancer;oral cancer, childhood;oral cavity and lip cancer;oropharyngeal cancer; Osteosarcoma / malignant fibrous histiocytoma of bone;Ovarian cancer, children;Ovarian epithelial cancer;Ovarian germ cell tumors;Ovarian low malignant potential tumors;Pancreatic cancer;Pancreatic cancer, children;Pancreatic cancer, islet cell;Sino-nasal and nasal cancer;Parathyroid cancer;Penile cancer;Pheochromocytoma;Pineal and supratentorial primitive neuroectodermal tumors, children;Pituitary tumors;Plasma cell neoplasm / multiple myeloma;Pleuropulmonary blastoma;Pregnancy and breast cancer;Pregnancy and Hodgkin's lymphoma;Pregnancy and non-Hodgkin's lymphoma;Primary central nervous system lymphoma;Primary liver cancer, adults;Primary liver cancer, children;Prostate cancer;Rectal cancer;Renal cell (kidney) cancer;Renal cell carcinoma, Pediatric;Kidney and ureter, transitional cell carcinoma;Retinal germ cell tumor;Rhabdomyosarcoma, pediatric;Salivary gland cancer;Salivary gland cancer, pediatric;Sarcoma, Ewing's tumor;Sarcoma, Kaposi;Sarcoma (osteosarcoma) / malignant fibrous histiocytoma of bone;Sarcoma, rhabdomyosarcoma, pediatric;Sarcoma, soft tissue, adult;Sarcoma, soft tissue, pediatric;Sezary syndrome;Skin cancer;Skin cancer, pediatric;Epidermal layer carcinoma (melanoma);Skin carcinoma, Merkel cell;Small cell lung cancer;Small intestine cancer;Soft tissue sarcoma, adult;Soft tissue sarcoma, pediatric;Neck squamous cell carcinoma of unknown primary, metastatic;Stomach (Gastric) cancer;Gastric cancer, pediatric;These include: supratentorial primitive neuroectodermal tumor, childhood;cutaneous T-cell lymphoma;testicular cancer;thymoma, childhood;malignant thymoma;thyroid cancer;thyroid cancer, childhood;transitional cell carcinoma of the kidney and ureter;trophoblastic tumor, gestational;carcinoma of unknown primary site, childhood;abnormal cancer of childhood;ureter and kidney, transitional cell carcinoma;urethral cancer;uterine sarcoma;vaginal cancer;optic pathway and hypothalamic glioma, childhood;vulvar cancer;Waldenstrom's macroglobulinemia;and Wilms' tumor.

[0119] In one aspect, the cancer is selected from non-small cell lung cancer, cutaneous squamous cell carcinoma, pancreatic cancer, primary hepatocellular carcinoma, colorectal cancer, clear cell renal cell carcinoma, or breast cancer.

[0120] In some embodiments, administration of the fusion proteins described herein may be combined with one or more additional therapeutic agents. The phrases "combination therapy," "in combination," and the like refer to the simultaneous use of one or more therapeutic agents or treatments to increase response. The fusion proteins of the present invention and pharmaceutical compositions thereof may be used in combination with other drugs or treatments used to treat, for example, cancer. In particular, administration of the fusion proteins to a subject may be combined with chemotherapy, surgery, radiation therapy, or a combination thereof. Such therapy may be administered prior to, simultaneously with, or after administration of the compositions of the present invention.

[0121] As used herein, the term "chemotherapeutic agent" refers to any therapeutic agent used to treat cancer. Examples of chemotherapeutic agents include, but are not limited to, actinomycin, azacitidine, azathioprine, bleomycin, bortezomib, carboplatin, capecitabine, cisplatin, chlorambucil, cyclophosphamide, cytarabine, daunorubicin, docetaxel, doxifluridine, doxorubicin, epirubicin, epothilone, etoposide, fluorouracil, gemcitabine, hydroxyurea, Idarubicin, imatinib, irinotecan, mechlorethamine, mercaptopurine, methotrexate, mitoxantrone, oxaliplatin, paclitaxel, pemetrexed, teniposide, thioguanine, topotecan, valrubicin, vinblastine, vincristine, vindesine, vinorelbine, panitumamab, erbitux (cetuximab), matuzumab, IMC-IIF8, TheraCIMhR3, denosumab, Avastin (bevacizumab), Humira (adalimumab), Herceptin (trastuzumab), Remicade (infliximab), rituximab, Synagis (palivizumab), Milotarg (gemtuzumab oxogamicin), Sarcrisa (isatuximab), Raptiva (efalizumab), Tysabri (natalizumab), Zenapax (dacliximab), NeutroSpec (Technetium (99mTc) fanolesomab), tocilizumab, ProstaScint (indium -yl-labeled capromab pendetide), Bexar (tositumomab), Zevalin (ibritumomab tiucetan conjugated to yttrium-90 (IDEC-Y2B8), Xolair (omalizumab), MabThera (rituximab), LeoPro (buciximab), MabCampus (alemtuzumab), Simulect (basiliximab), Leukoscan (sulesomab), CEAscan (arcitumomab), Verluma (nofetumomab), Panorex (edrecolomab), alemtuzumab, CDP870 , natalizumab, Giotrif (afatinib), Lynparza (olaparib), Perjeta (pertuzumab), Otodivo (nivolumab), Boslif (bosutinib), Cabometyx (cabozantinib), Ogivri (trastuzumab-dkst), Sutent (sunitinib malate), Adcetris (brentuximab vedotin), Alecensa (alectinib), Calquence (acalabrutinib), Yescarta (ciloleucel), Verzenio (abemaciclib), Keytruda (pembrolizumab), A Examples of immunotherapeutic agents include, but are not limited to, interleukins (Il-2, Il-7, Il-12), cytokines (interferon, G-CSF, imiquimod), chemokines (CCL3, CCl26, CXCL7), immunomodulatory imide drugs (thalidomide and its analogs).

[0122] In one embodiment, the invention provides a pharmaceutical composition comprising a therapeutically effective amount of a fusion protein comprising the amino acid sequence of SEQ ID NO:1, or a sequence having 90% or greater identity to SEQ ID NO:1, and a pharma- ceutically acceptable carrier.

[0123] In another embodiment, the present invention provides a method of treating cancer in a subject, comprising administering to the subject a pharmaceutical composition described herein.

[0124] Natural killer cells, also known as NK cells or large granular lymphocytes (LGLs), are a type of cytotoxic lymphocyte important to the innate immune system. They belong to a rapidly expanding family of known innate lymphoid cells (ILCs) and account for 5-20% of all circulating lymphocytes in humans. The role of NK cells is similar to that of cytotoxic T cells in the adaptive immune response of vertebrates. NK cells provide a rapid response against virus-infected cells and other intracellular pathogens acting approximately 3 days after infection and respond to tumor formation. Typically, immune cells detect major histocompatibility complexes (MHC) present on the infected cell surface, which triggers cytokine release and causes the death of the infected cell by lysis or apoptosis. However, NK cells are unique and allow for a much faster immune response, as they have the ability to recognize and kill stressed cells even in the absence of antibodies and MHC. They were named "natural killers" due to the idea that they do not require activation to kill cells that lack the MHC class 1 "self" marker. This role is particularly important because harmful cells that lack the MHC I marker cannot be detected and destroyed by other immune cells, such as T lymphocytes.

[0125] In addition to natural killer cells being effectors of innate immunity, both activating and inhibitory NK cell receptors play important functional roles including self-tolerance and maintenance of NK cell activity. NK cells also play a role in adaptive immune responses, and many experiments have demonstrated the ability of NK cells to rapidly adapt to their immediate environment and create antigen-specific immune memory, essential for responding to secondary infections with the same antigen. The role of NK cells in innate and adaptive immune responses is becoming increasingly important in studies using NK cell activity as a potential cancer therapy.

[0126] In an additional embodiment, the present invention provides a method of inducing natural killer (NK) cell activity against cancer cells in a subject, comprising administering to the subject a fusion protein comprising a sequence as set forth in SEQ ID NO:1 and a sequence having 90% or greater identity to SEQ ID NO:1, thereby inducing NK cell activity against cancer cells in the subject.

[0127] In one aspect, inducing NK cell activity includes inducing NK cell degranulation, inducing NK cell production of interferon-gamma, increasing the number of tumor-infiltrating NK cells in a subject, and / or inducing or increasing NK cell proliferation.

[0128] Natural killer cells or large granular lymphocytes (LKL) are a type of cytotoxic lymphocyte important to the innate immune system, belonging to a rapidly expanding family of known innate lymphoid cells (ILCs) and accounting for 5-20% of all circulating lymphocytes in humans. They have diverse functions, including cytolytic granule-mediated cell apoptosis, antibody-dependent cell-mediated cytotoxicity (ADCC), and cytokine-induced NK and cytotoxic T lymphocyte (CTL) activation.

[0129] NK cells are cytotoxic, and small granules in their cytoplasm contain proteins such as perforin and proteases known as granzymes. When released in close proximity to a cell to be killed, perforin forms pores in the target cell's plasma membrane, creating aqueous channels through which granzymes and related molecules can enter, inducing either apoptosis or osmotic cytolysis. The distinction between apoptosis and cytolysis is important in immunology, as lysing a virus-infected cell can potentially release virions, whereas apoptosis results in the destruction of the virus inside. α-defensin, an antibacterial molecule, is also secreted by NK cells and kills bacteria directly by destroying their cell walls in a manner similar to neutrophils.

[0130] Infected cells are usually opsonized with antibodies for detection by immune cells. Antibodies that bind to antigens can be recognized by the FcγRIII (CD16) receptor expressed on NK cells, leading to NK activation, release of lytic granules, and eventual apoptosis of the target cell. This is the primary killing mechanism of some monoclonal antibodies, such as rituximab (Rituxan), ofatumumab (Azzera), and others.

[0131] Cytokines play an important role in NK cell activation. These are stress molecules released by cells upon viral infection and therefore serve to signal the presence of viral pathogens in the affected area to NK cells. Cytokines involved in NK activation include IL-12, IL-15, IL-18, IL-2, and CCL5. NK cells are activated in response to interferons or macrophage-derived cytokines. They serve to contain the viral infection while the adaptive immune response generates antigen-specific cytotoxic T cells that can clear the infection. NK cells function to control viral infections by secreting IFNγ and TNFα. IFNγ activates macrophages for phagocytosis and lysis, while TNFα acts to promote direct NK tumor cell killing. Patients with NK cell deficiencies have proven highly susceptible to the early stages of herpes virus infection.

[0132] Tumor-infiltrating NK cells have been reported to play a key role in promoting drug-induced cell death in human triple-negative breast cancer. Because NK cells recognize target cells when they express non-self HLA antigens (not self), autologous (patient's own) NK cell infusions have not shown antitumor effects. Instead, researchers are working on using allogeneic cells from peripheral blood, which require the removal of all T cells before infusion into the patient to eliminate the risk of graft-versus-host disease, which can be fatal. This can be achieved using immunomagnetic columns (CliniMACS). Also, because there are a limited number of NK cells in the blood (only 10% of lymphocytes are NK cells), the number needs to be expanded in culture. This can take several weeks, and the yield is donor-dependent.

[0133] In one embodiment, the present invention provides a method of inhibiting tumor growth in a subject, comprising administering to the subject a fusion protein comprising a sequence as set forth in SEQ ID NO:1 and a sequence having 90% or greater identity to SEQ ID NO:1, thereby inhibiting tumor growth in the subject.

[0134] In one aspect, inhibiting tumor growth comprises reducing tumor cell survival.

[0135] In another embodiment, the present invention provides a method of increasing survival of a subject having cancer, comprising administering to the subject a fusion protein comprising a sequence set forth in SEQ ID NO:1 and a sequence having 90% or greater identity to SEQ ID NO:1, thereby increasing survival of the subject.

[0136] By increasing survival it is meant that the survival of a subject is increased when the subject is administered the fusion protein of the invention as compared to survival in the absence of the fusion protein, or as compared to administration of another treatment regimen that does not include the fusion protein of the invention.

[0137] In an additional embodiment, the present invention provides a method for inducing natural killer (NK)-mediated antibody-dependent cytotoxicity against cancer cells in a subject, comprising administering to the subject a fusion protein comprising a sequence as set forth in SEQ ID NO:1 and a sequence having 90% or greater identity to SEQ ID NO:1, thereby increasing survival of the subject.

[0138] In one aspect, administering to a subject a fusion protein comprising a sequence as set forth in SEQ ID NO:1 and a sequence having 90% or greater identity to SEQ ID NO:1 further comprises administering to the subject an anti-cancer treatment.

[0139] In another embodiment, the subject has cancer. In some embodiments, the cancer is selected from the group consisting of lung cancer, prostate cancer, multiple myeloma, ovarian cancer, and head and neck cancer. In other embodiments, the cancer cells are B7-H3 expressing cancer cells. In some embodiments, the cancer is a treatment resistant cancer.

[0140] Below are presented examples illustrating the development, characterization, and evaluation of the effectiveness of the B7-H3 TriKE molecule contemplated for the described uses. The following examples are provided to further illustrate embodiments of the present invention, but are not intended to limit the scope of the invention. While they are exemplary of examples that may be used, other procedures, methodologies, or techniques known to those skilled in the art may alternatively be used. EXAMPLES

[0141] Example 1 Trispecific development and characterization Antigen-specific immunotherapy requires that the target antigen be overexpressed in tumor cells and have minimal extratumoral expression in normal tissues. Ideally, the antigen would show high expression in a wide range of cancers, making immunotherapy applicable in several settings, and basket clinical trials would be more common if broad targets could be identified. B7-H3, a transmembrane costimulatory protein that is a member of the B7 family of checkpoint ligands, has gained interest as an immunotherapy target. It has been shown to participate in both costimulatory and inhibitory settings by associating with receptors on T cells, while also contributing to immune evasion through expression on antigen-presenting cells such as macrophages and tumor cells within the tumor microenvironment. B7-H3 expression is high in many types of cancer, but very low in normal tissues. Mouse models utilizing B7-H3-targeted CAR T constructs reactive against mouse cells have demonstrated antitumor responses without toxicity, further highlighting the safety profile of B7-H3 as a target. Ninety-three percent of ovarian tumors express B7-H3, and expression is associated with advanced stage, high recurrence, and poor survival. Similar findings exist for other types of carcinomas, including colon, prostate, pancreatic, non-small cell lung, and gastric cancer, indicating that B7-H3 may be a useful marker in cancer biology, progression, and treatment across a range of different cancers. Due to these characteristics, there are currently several ongoing clinical trials targeting this antigen in forms ranging from Fc-optimized antibodies (NCT02982941) to CAR T cells (NCT04077866).

[0142] Bispecific immune engagers such as blinatumomab have shown excellent clinical success. As a single engineered molecule, one of its single-chain variable fragments (scFv) targets cancer cells and the other targets CD3 in T cells. This creates an immune synapse between T cells and cancer cells, resulting in tumor killing. However, T cell activation and proliferation can lead to cytokine release syndrome, disseminated intravascular coagulation, and neurological events including encephalopathy and seizures. Therefore, this study aims to selectively engage natural killer (NK) cells instead of T cells. NK cells are part of the innate immune system and play a major role in tumor surveillance, showing potential in many studies involving solid tumors and hematological cancers. Due to these features, a trispecific killer engager (TriKE) platform has been designed and described, consisting of a single-chain variable fragment (scFv) targeting CD16, the most potent activating receptor in NK cells, an scFv targeting a tumor-associated antigen, and an IL-15 moiety. We improved this platform by adding a single domain antibody against CD16, the result being better IL-15 activity and overall function. IL-15 is the most important homeostatic cytokine for NK cell function. It is necessary for NK cell proliferation and survival, and can amplify antibody-dependent cellular cytotoxicity (ADCC), induce lymphokine-activated killer activity, and enhance the production of other costimulatory mediators such as interferon gamma (IFNγ) and tumor necrosis factor alpha (TNFα).

[0143] Herein, we describe a second generation TriKE bioengineered with human IL-15 as a modified cross-linker between a humanized camelid anti-CD16 VHH single domain antibody (sdAb) and an anti-B7-H3 scFv, designated cam1615B7-H3. Thus, in a single molecule, two important therapeutic properties are combined: the ability to specifically enhance NK cell proliferation accompanied by the ability to enhance ADCC. While cam1615B7-H3 demonstrated potent and specific induction of NK cell activity against a variety of solid tumors in vitro, it also showed potent activity in a xenogeneic ovarian cancer model. Thus, targeting B7-H3 with TriKE may have a high therapeutic effect in NK cell-based immunotherapy of several solid cancers.

[0144] Construction of cam1615B7-H3 TriKE It is known that single domain VHH antibodies derived from camelids offer advantages over conventional VL-VH scFv fragments. The complementarity determining regions (CDRs) from camelid (llama) anti-CD16 were split into a universal, humanized heavy chain scaffold. This humanized camelid sequence was used to generate cam1615B7-H3. A hybrid gene encoding cam1615B7-H3 was synthesized using DNA shuffling and DNA ligation techniques. The fully assembled gene encoded (from the 5' to 3' end): an NcoI restriction site; an ATG start codon; an anti-human CD16 VHH; a 20 amino acid (aa) segment, PSGQAGAAASESLFVSNHAY (SEQ ID NO: 36); human wild-type IL-15; a 7 amino acid linker, EASGGPE (SEQ ID NO: 37); an anti-B7-H3 mAb 376.96 scFv; and a XhoI restriction site. The resulting hybrid gene was spliced ​​into the pET28c expression vector under the control of an isopropyl-D-thiogalactopyranoside (IPTG)-inducible T7 promoter. The DNA target gene encoding cam1615B7-H3 was 1527 base pairs. The Biomedical Genomics Center, University of Minnesota (St. Paul, MN, USA) verified the accuracy of the gene sequence and in-frame of the construct.

[0145] Purification of proteins from inclusion bodies Escherichia coli strain BL21(DE3) (Novagen, Madison, WI, USA) was used for expression of proteins after plasmid transfection. Bacterial expression resulted in sequestration of the target protein into inclusion bodies (IBs). Bacteria were grown overnight in 800 mL of Luria Broth containing kanamycin (30 mg / mL). Expression was induced with isopropyl β-D-1-thiogalactopyranoside / IPTG (FischerBiotech, Fair Lawn, NJ, USA) when the absorbance at 600 nm reached 0.65. Bacteria were harvested after 2 hours. After a homogenization step in a buffer solution (50 mM Tris, 50 mM NaCl, and 5 mM EDTA pH 8.0), the pellet was sonicated and centrifuged. Proteins were extracted from the pellet using a solution of 0.3% sodium deoxycholate, 5% Triton X-100, 10% glycerol, 50 mmol / L Tris, 50 mmol / L NaCl, and 5 mmol / L EDTA (pH 8.0). The extract was washed three times.

[0146] Bacterial expression in inclusion bodies requires refolding. Therefore, the protein was refolded using the sodium N-lauroylsarcosine (SLS) air oxidation method (20). IB was dissolved in 100 mM Tris, 2.5% SLS (Sigma, St. Louis, MO USA) and clarified by centrifugation. Then, 50 μM CuSO4 was added to the solution, which was then incubated at room temperature for 20 h with rapid stirring for air oxidation of -SH groups. SLS was removed by adding 6 M urea and 10% AG 1-X8 resin (200-400 mesh, chloride form) (Bio-Rad Laboratories, Hercules, CA, USA) to the detergent-solubilized protein solution. Guanidine HCl (13.3 M) was added to the solution and incubated at 37 °C for 2-3 h. The solution was diluted 20-fold with refolding buffer: 50 mM Tris, 0.5 M l-arginine, 1 M urea, 20% glycerol, 5 mM EDTA (pH 8.0). The mixture was refolded for 2 days at 4°C and then dialyzed against 5 volumes of 20 mM Tris-HCl, pH 8.0 for 48 h at 4°C, then against 8 volumes for another 18 h. The product was then purified on a fast-flow Q ion exchange column and further purified by passing through a size exclusion column (Superdex 200, GE, Marlborough, MA, USA). Protein purity was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) stained with Simply Blue Safe Stain (Invitrogen, Carlsbad, CA, USA).

[0147] Generation and purification of B7-H3-targeted TriKE To construct a second generation TriKE capable of both ADCC and NK cell expansion, we modified the existing TriKE platform. A wild-type human IL-15 crosslinker with two modified flanking regions was inserted between two antibody fragments, a VHH humanized camelid anti-CD16 fragment at the N-terminus and an anti-B7-H3 fragment at the C-terminus, to generate cam1615B7-H3. Figure 1A shows a schematic diagram of the B7-H3 TriKE construct. B7-H3 TriKE contains a single chain variable fragment from camelid nanobody (cam) targeting IL-15 and CD16 and B7-H3 linked by two flexible linker regions to form a single peptide with a molecular weight of approximately 46 kDa. This differs from BiKE, which consists of camCD16 and camB7-H3 with a single flexible linker region, forming a single peptide of approximately 35 kDa. NK cell-mediated target lysis is directed to B7-H3-expressing tumor cells via the formation of a direct physical linkage with TriKE or BiKE of B7-H3. IL-15 then stimulates the NK cells. Figure 1B shows an absorbance trace from a FFQ ion exchange column as the first stage of purification with the eluate collected in 8 mL aliquots shown on the horizontal axis of the graph. The double-headed arrow indicates the collected peak as the drug exits the column. Figure 1C shows an absorbance trace from the second purification stage, size exclusion chromatography (SEC). The first peak was collected from the column, and the various drug-containing fractions were pooled and analyzed using SDS-PAGE with Coomassie blue staining for the presence of a homogenous product (Figure 1D). The final product was >90% pure with a molecular weight of approximately 55 kDa, with a predicted molecular weight of 54.58 kDA. Similar to other TriKE molecules, this TriKE has a rapid clearance profile due to its size, with an expected EC50 in the range of hours.

[0148] cam1615B7-H3 TriKE induces potent and specific NK cell proliferation The wild-type IL-15 portion of cam1615B7-H3 TriKE was designed to induce targeted delivery of proliferation signals to NK cells. To test this, we performed a proliferation assay evaluating the dilution of CellTrace dye over 7 days on PBMCs that were untreated (NT), treated with monomeric rhIL-15 (IL15), or TriKE (cam1615B7-H3). At the end of the 7 days, cells were harvested and proliferation was assessed by gating on CD56+CD3- cells. While untreated (NT) resulted in low proliferation with low NK cell numbers, cam1615B7-H3 induced an overall increase in proliferation similar to that induced by rhIL-15 (Figures 2A-2D), with no significant difference between these two groups. Because IL-15 acts on both NK and T cells, we next assessed specificity by gating on T cells (CD56-CD3+). In contrast to rhIL-15, which induced robust proliferation of T cells (Figures 2E and 2F), minimal T cell proliferation was seen in the TriKE-treated group, clearly indicating that cam1615B7-H3 TriKE IL-15 delivery is more restricted to NK cells. This difference was particularly striking for the robustly proliferating population (through three divisions), where cam1615B7-H3 TriKE showed significantly less proliferation than the untreated group (Figure 2G), whereas T cell numbers did not differ between the untreated and TriKE-treated groups (Figure 2H). This data indicates that the cam16 engager in cam1615B7-H3 TriKE specifically delivers IL-15 to NK cells, but not to T cells.

[0149] As shown in Figures 3A and 3B, our camB7-H3 TriKE demonstrated strong binding specificity to WT B7-H3. The BT-12 pediatric brain tumor line highly expresses B7-H3 (WT). Using CRISPR, we generated a B7-H3 KO BT-12 cell line (Theruvath et al.). Similar specificity was observed using Raji (negative B7-H3) and prostate cancer cell line C4-2 (positive B7-H3) as well as several other lines. B7-H3 BiKE had similar binding to positive and negative cell lines (data not shown).

[0150] NK-92 cells, with or without CD16, were incubated with dilutions of NCI IL-15 and GTB-5550 for 48 hours. Metabolic activity was then measured using resazurin (n=4). As shown in Figures 4A-4B, in CD16+NK-92, the TriKE molecule was found to be twice as potent as NCI IL-15.

[0151] Example 2 General Materials and Methods Cancer cell lines and antibodies MA-148 (established locally at the University of Minnesota) is a human epithelial high-grade serous ovarian cancer cell line. For in vivo experiments, luciferase reporter constructs were transfected using Invitrogen's Lipofectamine reagent and selective pressure was applied with 10 μg / mL blasticidin. The ovarian cancer cell lines OVCAR5 and OVCAR8 were obtained from the DTP, DCTD Tumor Repository, sponsored by the Biological Testing Branch, Developmental Therapeutics Program, National Cancer Institute (NCI), National Institutes of Health (NIH, Frederick, MD, USA). Other cell lines, including OVCAR3 (ovarian), C4-2 (prostate), DU145 (prostate), LNCaP (prostate), PC-3 (prostate), A549 (lung), NCI-H322 (lung), NCI-H460 (lung), and Raji cells (Burkitt's lymphoma), were obtained from the American Type Culture Collection. All lines express high levels of B7-H3, except for Raji cells, which were used as a negative control. Lines were maintained in RPMI1640 medium supplemented with 10–20% fetal bovine serum (FBS) and 2 mmol / L L-glutamine. Lines were incubated at a constant 37°C in a humidified atmosphere containing 5% CO2. When adherent cells were >90% confluent, they were passaged using trypsin-EDTA for detachment. A standard hemocytometer was used for cell counting. Only cells with viability >95% were used for the experiments. The sequence of the monoclonal antibody scFv fragment 376.96 was obtained by Dr. Ferrone and used to construct TriKE.

[0152] cell products Peripheral blood mononuclear cells (PBMCs) were obtained from normal volunteers or subjects after consent and Institutional Review Board (IRB) approval (9709M00134 and 1607M91103) according to the guidelines by the Committee on the Use of Human Subjects in Research and in accordance with the Declaration of Helsinki. For in vivo studies, fresh PBMCs were magnetically depleted (i.e., passed three times through a magnet) for CD3 and CD19 positive cells three times according to the manufacturer's recommendations (STEMCELL Technologies, Cambridge, MA, USA) to generate NK cell enriched products. Ovarian cancer specimens (ascites) were collected from women diagnosed with advanced stage ovarian or primary peritoneal cancer at the time of primary debulking surgery. For prostate cancer, blood was collected from two patients with metastatic castration-resistant prostate cancer and one patient with metastatic hormone-sensitive prostate cancer. For lung cancer, blood was collected from seven patients with pretreatment unresectable lung cancer at diagnosis, cells were pelleted, red blood cells were lysed, and cryopreserved in 10% DMSO / 90% FBS and stored in liquid nitrogen.

[0153] IL-15-stimulated NK cell proliferation To measure the ability of TriKE to specifically induce NK cell proliferation via the IL-15 moiety, PBMCs from healthy donors were labeled with CellTrace Violet proliferation dye (Invitrogen, Carlsbad, CA, USA) according to the kit specifications. After staining, cells were cultured with the indicated concentrations of TriKE or equimolar controls and incubated for 7 days at 37 °C in a humidified atmosphere containing 5% CO2. Cells were harvested, stained for viability with Live / Dead reagent (Invitrogen, Carlsbad, CA, USA), and surface stained with anti-CD56 PE / Cy7 (Biolegend, San Diego, CA, USA) and anti-CD3 PE-CF594 (BD Biosciences, Franklin Lakes, NJ, USA) to gate on viable CD56+CD3- NK cell or CD56-CD3+ T cell populations. Data analysis was performed using FlowJo software (FlowJo LCC, version 7.6.5, Ashland, OR, USA).

[0154] Assessment of cytotoxicity and NK cell activation ADCC was measured in a flow cytometry assay by assessing CD107a (lysosome-associated membrane protein LAMP-1)-mediated degranulation and intracellular IFNγ production. Upon thawing, PBMCs or ascites cells from normal donors and patients were placed overnight (37°C, 5% CO2) in RPMI1640 medium supplemented with 10% fetal bovine serum (RPMI-10). The next morning, after washing twice with RPMI-10, they were suspended with tumor target cells or in medium. Cells were then incubated with TriKE or control for 10 min at 37°C. Fluorescein isothiocyanate (FITC)-conjugated anti-human CD107a monoclonal antibody (BD Biosciences, San Jose, CA, USA) was then added. After 1 h incubation at 37°C, GolgiStop (1:1500, BD Biosciences) and GolgiPlug (1:1000, BD Biosciences) were added for 3 h. After washing with phosphate-buffered saline, cells were stained with PE / Cy 7-conjugated anti-CD56 mAb, APC / Cy 7-conjugated anti-CD16 mAb, and PE-CF594-conjugated anti-CD3 mAb (BioLegend, San Diego, CA, USA). Cells were incubated for 15 min at 4°C, washed, and fixed with 2% paraformaldehyde. Cells were then permeabilized using intracellular perm buffer (BioLegend) and IFNγ production was assessed by detection via aBV650-conjugated anti-human IFNγ antibody (BioLegend). Samples were washed and evaluated on an LSRII flow cytometer (BD Biosciences, San Jose, Calif., USA).

[0155] Real-time tumor killing assay Tumor killing was assessed in real time using the IncuCyte platform. Magnetic bead-enriched CD3-CD56+ NK effector cells were plated in 96-well clear flat-bottom polystyrene tissue culture treated microplates (Corning, Flintshire, UK) with OVCAR8 cells stably expressing NuclightRed at an effector:target ratio of 2:1. Caspase-3 / 7 green dye (Sartorious, Ann Arbor, MI, USA) was added to pick up stained cells that had not yet lost NuclightRed fluorescence. The described treatments were then added at a concentration of 30 nM, and the plates were placed in the IncuCyte ZOOM® platform housed in a cell incubator at 37°C / 5% CO2. Images from three technical replicates were taken every 15 minutes for 48 hours using a 4X objective and then analyzed using IncuCyte™ Basic Software v2018A (Sartorious). The graphed readout represents the percentage of viable OVCAR8 targets (NuclightRed+Caspase-3 / 7-) normalized to viable targets alone at the starting (0 hour) time point.

[0156] Mass Cytometry (CyTOF) For mass cytometry (CyTOF) studies, PBMCs were incubated with + / -cam1615B7-H3 (30 nM) for 24 hours, either alone or with OVCAR8 at a 2:1 ratio. After samples were harvested, cells were counted and viability was measured using trypan blue exclusion. 200,000 cells from each donor were aliquoted into 5 mL polystyrene U-bottom tubes for barcoding and CyTOF staining. Cells were barcoded using the Cell-ID 20-Plex Pd Barcoding Kit (Fluidigm product number 201060) after staining with cisplatin (Fluidigm product number 201064, San Francisco, CA, USA). After barcoding, all cells were combined in a single 5 mL polystyrene U-bottom tube and incubated in surface marker antibody cocktail for 30 minutes at 4°C.

[0157] After surface staining, cells were then fixed using 2% PFA. For intracellular staining, cells were permeabilized by incubation with Triton-X 0.1% for 5 minutes at room temperature, followed by incubation with intracellular antibody cocktail for 30 minutes at 4°C. Stained cells were then incubated overnight with Cell-ID Intercalator (Fluidigm product number 201192A). The following morning, cells were washed and subjected to the CyTOF 2 instrument. Washing steps were completed using either Maxpar PBS (Fluidigm product number 201058), Maxpar Cell Staining Buffer (Fluidigm product number 201068), or Millipur Water at 1600 RPM for 4 minutes. For custom tagged antibodies: conjugation of heavy metals to specific ScFvs is performed using Maxpar Antibody Labeling Kit (Fluidigm). This protocol involves partial antibody reduction using 0.5M TCEP: Pierce Bond-Breaker TCEP solution (Thermo Scientific Product No. 77720, Waltham, MA, USA), and comprehensive buffer exchange using centrifugal filter units of both 3 kDa and 50 kDa size (Millipore Product No. UFC500396, UFC505096, Burlington, MA, USA). After antibody conjugation, the yield is measured and the final reagent is stored in antibody stabilizer (Boca Scientific Product No. 131 000, Westwood, MA, USA). The reagent is then titrated and validated for known flow cytometry antibodies. Data from three donors were combined. Concatenation of FCS files was completed in combination with Cytobank and Flowjo. All Visne analysis was performed in Cytobank.

[0158] In vivo mouse studies and imaging MA-148-Luc ovarian cancer cells were incorporated into the NK cell xenogeneic mouse model system described previously. NSG mice (NOD.Cg-Prkdcscid Il2rgtm1 Wjl / SzJ, n=5 / group) were IP injected with 2.0×105 MA-148-luc cells and then conditioned 3 days later with low-dose total body irradiation (225cGy). The following day, all groups received highly enriched NK cells (magnetically CD3 and CD19 depleted PBMCs) equivalent to 1 million NK cells / mouse and began the drug regimen. One course of treatment consisted of IP injections of 30 μg TriKE or 5 μg rhIL-15 administered daily (Monday-Friday) for 3 weeks. MA-148-luc cells are a subline of MA-148 transfected with a luciferase reporter gene, allowing imaging of mice each week to determine their bioluminescence activity and monitor tumor progression. Briefly, 10 min before imaging, mice were injected with 100 μL of 30 mg / mL luciferin substrate and then anesthetized by inhalation of isoflurane gas (25). Mice were then imaged using a Xenogen Ivis 100 imaging system and analyzed by Living Image 2.5 software (Xenogen Corporation, Alameda, CA, USA). At the end of the experiment (day 21), all animals were sacrificed and postmortem peritoneal lavage fluids were analyzed for human NK cell content by flow cytometry. Animal imaging and analysis were performed at the University of Minnesota Imaging Center. Mouse studies were performed following approval from and in accordance with the guidelines of the Institutional Animal Care and Use Committee (IACUC) of the University of Minnesota (protocol 1908-37330A).

[0159] statistical analysis All statistical tests were performed using GraphPad PRISM8 (GraphPad Prism Software, Inc., San Diego, CA, USA). For all in vitro studies, one-way ANOVA with repeated measures was used to calculate significance in comparisons with the cam1615B7-H3 group. In mouse studies, two-way ANOVA was used to calculate significance in longitudinal studies, and one-way ANOVA was used to calculate significance in differences in radiation dose at the 21 day time point. Unpaired t-tests were used to evaluate differences in cell counts and MFI. Bars represent mean ± SEM. Statistical significance is indicated as *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001.

[0160] Example 3 Blood Cancer Cells The efficacy of the H7-B3 TriKE molecule of the present invention was evaluated in several hematological cancer cell lines. Unless otherwise stated, the methods used were as described in Example 2.

[0161] Functional assays were performed using hematological malignancy cell lines with different levels of B7-H3 expression, ranging from zero to very high levels, as illustrated in Figures 5A-5B. NK cell activation was measured using CD107a and IFN-gamma measured by flow cytometry (n=3), IncuCyte, or xCelligence assays.

[0162] We next focused on multiple myeloma, where expression of B7-H3 (CD276) is associated with decreased progression-free survival, is low in healthy tissues, and is expressed on myeloid-derived suppressor cells (MDSCs) that promote myeloma growth.

[0163] As shown in Figures 6A-6B, high expression of B7-H3 was found by flow cytometry in the myeloma lines RPMI-8226, U266, and MM1S, with relatively low expression in H929.

[0164] In a live imaging IncuCyte Zoom assay, the ability of peripheral blood NK cells to kill myeloma cells with or without B7-H3-TriKE was compared with increasing doses of TriKE. Maximal killing occurred at a concentration of 3 nM. A statistically significant increase was found for NK cell-mediated killing of all four myeloma lines with the addition of 3 nM B7-H3-TriKE. Against U266 and MM1S, B7-H3-TriKE significantly enhanced killing at effector:target (E:T) ratios of 2:1 and 4:1. RPMI-8226 was relatively resistant to NK cell cytotoxicity, whereas B7-H3-TriKE enhanced killing at an E:T of 4:1. H929 cells were killed more potently at E:T 2:1 in the presence of B7-H3-TriKE, but there was no difference in killing at E:T 4:1, likely due to high natural cytotoxicity in both groups (see Figures 7A-7B and 8A-8D).

[0165] The efficacy of B7-H3-TriKE with the proteasome inhibitor bortezomib (10 nM) and the immunomodulatory drug lenalidomide (5 μM) was also tested. Cytotoxicity curves were performed in triplicate and compared by repeated measures ANOVA. Combination treatment with B7-H3-TriKE, NK cells, and lenalidomide demonstrated synergistic killing of H929 cells after 48 hours of live cell imaging (p=0.047), but combination with bortezomib did not further enhance killing compared to NK cells and TriKE alone (Figure 9A). Both lenalidomide and bortezomib showed a trend toward improved killing against MM1S when administered with NK cells and B7-H3 TriKE, but did not reach statistical significance (Figure 9B). Combination treatment with B7-H3-TriKE, NK cells, and lenalidomide or bortezomib demonstrated synergistic killing of RPMI-8226 cells after 48 hours of live cell imaging (p<0.001 and 0.015, respectively) (Figure 9C). Bortezomib in combination with B7-H3-TriKE and NK cells enhanced killing in U266 cells (p=0.037) (Figure 9D).

[0166] MDSCs were generated from CD33+ myeloid cells from healthy donors using IL-6 and GM-CSF or by incubation with myeloma cells at a ratio of 1:100 for 7 days. MDSCs (CD14+CD11b+) displayed high expression of B7-H3 (Figure 10A). MDSCs were also isolated from bone marrow aspirates of three newly diagnosed myeloma patients and showed survival of 56–95% (aspirates were treated with lysis buffer and stained for CD14, CD11b, and B7-H3. Flow cytometry plots of viable CD14+ cells are shown (Figure 10C). MDSCs were incubated with myeloma cells and proliferation was measured over 48 h by viable cell imaging (Figure 10B). Addition of MDSCs to the cytotoxicity assay enhanced myeloma cell proliferation, but was overcome by B7-H3 TriKE and NK cells (Figure 10D). B7-H3-TriKE significantly enhanced NK cell-mediated killing of myeloma cells, even in the H929 line with relatively low B7-H3 expression. This also indicates that MDSC-induced myeloma proliferation can be reversed.

[0167] After MDSCs expressed B7-H3, MDSCs were co-cultured with NK at 1:1 E:T and killing with or without B7-H3 TriKE was compared (see Figures 11A and 11B).

[0168] Example 4 Efficacy of H7-B3 TriKE in prostate cancer The efficacy of the H7-B3 TriKE molecule of the present invention was evaluated in several prostate cancer cell lines. Unless otherwise stated, the method used is as described in Example 2.

[0169] As shown in Figures 12A-12D, 14A-14L, and 15A-15D, cam1615B7-H3 TriKE targets prostate cancer. The ability of cam1615B7-H3 TriKE to improve NK cell activity against prostate cancer was tested. All prostate cancer cell lines tested expressed B7-H3. These studies used normal donor PBMCs and PBMCs obtained from metastatic prostate cancer patients. Although metastatic prostate cancer patients showed a slight decrease in NK cell activity when compared to normal donors, cam1615B7-H3 TriKE enhanced degranulation and IFNγ production in both normal donor and patient NK cells against C4-2, DU145, LNCaP, and PC3 prostate cancer adenocarcinoma cell lines when compared to controls (Figures 14A-14G, 14J, and 15A-15D). Individual cam16 VHH or anti-B7-H3 scFv components did not induce increased NK cell activation against C4-2. Thus, this data indicates that cam1615B7-H3 TriKE holds promise for NK cell immunotherapy in the prostate cancer setting and can rescue NK cell function in patients who require novel interventions due to poor outcomes with current therapeutic approaches. The signal induced in prostate cancer cells by TriKE was stronger than that induced by potent natural cytotoxic signals and was specific for B7-H3.

[0170] As shown in Figures 13A-13B, cam1615B7-H3 TriKE was more potent at inducing NK function than IL-15 alone, and was also more potent at inducing NK cell proliferation compared to IL-15 alone.

[0171] As illustrated in FIG. 16, tumor killing of PC-3 cells was assessed in real time using the IncuCyte platform, which highlighted the enhanced efficacy of the B7-H3 TriKE molecule in inducing prostate cancer cell death.

[0172] As shown in Figures 17-20, the B7-H3 TriKE molecule was also able to reduce PC-3 spheroid size over time.

[0173] As shown in FIG. 21, it was demonstrated that killing of prostate cancer cells by the B7-H3 TriKE molecule occurred rapidly (within hours) after initiation of treatment.

[0174] Various enzalutamide-resistant prostate cancer cells were phenotyped for expression of B7-H3. As illustrated in Figures 22A-22F, all cell lines tested expressed B7-H3.

[0175] As shown in Figures 23A-23L, camB7-H3 TriKE was found to induce activity against prostate cancer cells over a wider dynamic range than previous scFv versions, as assessed by measuring the percentage of CD107a+ and IFNγ+ NK cells.

[0176] Example 5 Efficacy of H7-B3 TriKE in lung cancer The efficacy of the H7-B3 TriKE molecule of the present invention was evaluated in several lung cancer cell lines. Unless otherwise stated, the methods used were as described in Example 2.

[0177] As illustrated in Figures 24A-24F, cam1615B7-H3 TriKE targets lung cancer. The ability of cam1615B7-H3 TriKE to improve NK cell activity against B7-H3-expressing lung cancer was tested in normal donor PBMCs incubated with two non-small cell lung cancer adenocarcinoma lines, A549 and NCI-H322 (Figures 24A-24D). In both cases, cam1615B7-H3 TriKE significantly and robustly improved NK cell activation when compared to controls. Individual cam16 VHH or anti-B7-H3 scFv components were tested and showed no background NK cell activity against A549. Normal donor PBMCs and PBMCs prior to any therapy from a patient with newly diagnosed unresectable lung cancer were incubated with NCI-H460 cells, a large cell lung cancer cell line. As the data clearly show, cam1615B7-H3 treatment strongly increased NK cell function against large cell lung cancer in both normal donor and patient samples when compared to controls (Figures 24E-24F). TriKE-mediated NK cell degranulation and IFNγ production against lung cancer cells was higher than that seen when NK cells were incubated with K562 targets alone. Activation against lung cancer cell lines was specific for B7-H3 expression, as it was higher than activation by B7-H3-Raji cells. Thus, the data indicate that cam1615B7-H3 TriKE has broad B7-H3-specific activity against many solid tumor targets.

[0178] Example 6 Efficacy of H7-B3 TriKE in head and neck cancer The efficacy of the H7-B3 TriKE molecule of the present invention was evaluated in several head and neck cancer cell lines. Unless otherwise stated, the methods used were as described in Example 2.

[0179] Worldwide, head and neck squamous cell carcinoma (HNSCC) accounts for approximately 900,000 cases and 400,000 deaths. In some situations, such as Fanconi anemia (FA), patients undergo curative treatment (allogeneic stem cell transplantation) but develop HNSCC in early adulthood at a high incidence. Current treatment strategies for non-FA HNSCC patients include surgery, chemotherapy, and radiation therapy. However, these are not viable treatment options for FA HNSCC patients due to poor tolerance to high toxicity levels of chemotherapy and radiation. Thus, there is a significant need for novel and targeted therapeutic interventions for the treatment of FA HNSCC patients.

[0180] B7-H3, a checkpoint member of the B7 and CD28 families, is overexpressed in several solid tumors but is absent or not expressed in healthy tissues. It is a promising target for immunotherapy, and recent basket trials, particularly in prostate cancer, have demonstrated strong clinical signals. Here, we developed the ability of a trispecific killer engager (TriKE) containing a B7-H3 targeting component to direct NK cell killing to B7-H3-expressing head and neck cancer targets. The TriKE molecule contains an NK cell engaging domain, including a humanized camelid nanobody against CD16; a camelid nanobody against B7-H3; and a wild-type IL-15 sequence between these two engagers. Expression of B7-H3 was assessed by flow cytometry in wild-type HNSCC cells and in a paired version in which the FANCA gene was KO'd by CRISPER, and it was determined that KO did not affect B7-H3 expression. Therefore, TriKE activity against HNSCC should be present in both normal HNSCC and FA-HNSCC settings.

[0181] NK cell responses against HNSCC cell lines in the presence of B7-H3 TriKE were assessed by either flow cytometry-based functional assays to assess NK cell degranulation and cytokine secretion, or IncuCyte imaging assays to directly assess target killing. NK cell degranulation and IFN-gamma production in B7-H3 TriKE-treated samples was higher compared to control samples treated with B7-H3 single domain or IL-15 alone. B7-H3 TriKE also induced more HNSCC target cell killing by NK cells compared to treatment with B7-H3 single domain or IL-15 alone, regardless of the FANCA gene, in both 2D and 3D IncuCyte imaging assays. Ongoing experiments will evaluate the functionality and efficacy of B7-H3 TriKE in vivo. Taken together, this data shows that B7-H3 TriKE can drive NK cell activity against the camelid nanobody B7-H3-CD16 against B7-H3-expressing HNSCC cells, indicating that B7-H3-targeted TriKE may be used clinically to treat HNSCC or FA-HNSCC patients.

[0182] As shown in Figures 25A-25B, frozen PBMCs (N=3) from healthy donors were incubated with five HNSCC cell lines: UM-SCC-01, SFCI-SCC-07, JHU-SCC-FaDu, Cal27 and Cal33 for 5 hours to assess CD107a expression (as a marker for degranulation) and intracellular IFN-y production. HNSCC cell lines did not induce NK cell cytolytic function without treatment.

[0183] As shown in Figures 26A-26B, five HNSCC cell lines were assessed for B7-H3 expression via flow cytometry to assess binding affinity with the B7-H3 single domain, and PBMCs from healthy donors were assessed for B7-H3 expression by flow cytometry. B7-H3 is highly expressed in HNSCC, but not in healthy immune cells.

[0184] As shown in Figures 27A-27D, B7-H3 TriKE induced NK cell activity against HNSCC. Frozen PBMCs (N=3) from healthy donors were incubated with (A-B) Cal27 trio and (B-C) Cal33 trio (each trio consisting of two clones of HNSCC WT and HNSCC FANCA KO lines) for 5 hours with different treatments: no treatment or 3 nM IL-15, MOPC, B7-H3 SD, and B7-H3 TriKE to assess CD107a expression (as a marker of degranulation) and intracellular IFN-y production. Error bars indicate standard error of the mean and statistical significance was determined as *p<.05, **p<.01, ***p<.001, and ****p<.0001.

[0185] As shown in Figures 28A-28F, B7-H3 TriKE induced NK cell killing against HNSCC in a real-time imaging assay. Enriched NK cells (N=4) were incubated with Nuclight Red-labeled Cal27 in 5:1 E:T in different conditions: no treatment or 3 nM IL-15, B7-H3 SD, and B7-H3 TriKE for 48 hours in an IncuCyte Zoom imager. Percentage quantification of viable cells was performed by normalizing hourly counts of red blood cells to target alone at t=0. Spheroids of Nuclight red-labeled Cal27 were formed for 72 hours and then incubated with enriched NK cells (N=4) in 5:1 E:T in different conditions: no treatment or 3 nM IL-15, B7-H3 SD, and B7-H3 TriKE for 96 hours in an IncuCyte S3 imager. Representative images showing spheroids over time. Percent quantification of mean red object area (viable cells) was performed by normalizing the mean red object area counts over time to target alone at t=0. The same set of assays was performed in Cal33.

[0186] There is a significant need for targeted therapies that can effectively eliminate HNSCC cells while sparing healthy cells. Herein, we describe a preclinical study of TriKE molecules against the B7-H3 ligand expressed in HNSCC. We found that treatment with B7-H3 TriKE effectively induced NK cell degranulation and cytokine production against HNSCC, as well as promoted targeted killing of HNSCC in vitro. Ongoing experiments will evaluate the functionality and efficacy of B7-H3 TriKE in vivo. Future studies will include investigating the HNSCC tumor microenvironment and evaluating the efficacy of B7-H3 TriKE in the HNSCC tumor microenvironment, as well as evaluating whether the HPV status of HNSCC has any implications on the efficacy of TriKE in the HNSCC tumor microenvironment, since previous studies have reported differential NK cell activity in the HPV+ / - HNSCC tumor microenvironment.

[0187] Example 7 Efficacy of H7-B3 TriKE in ovarian cancer The efficacy of the H7-B3 TriKE molecule of the present invention was evaluated in several ovarian cancer cell lines. Unless otherwise stated, the methods used were as described in Example 2.

[0188] As shown in Figures 29A-29B and 30A-30I, cam1615B7-H3 TriKE shows potent killing of ovarian cancer. The ability of cam1615B7-H3 TriKE to mediate NK cell activity against ovarian cancer cells was evaluated. The ovarian cancer cells used showed robust B7-H3 expression. Because B7-H3 has been shown to have a role in immune responses, the ability of cam1615B7-H3 to induce activity against normal immune cells was evaluated in PBMCs. Flow cytometry assays allowed gating of NK cells and determined that cam1615B7-H3 induced some background degranulation (CD107a) in NK cells compared to controls, but this activity was low. No background noise was seen with IFNγ. In contrast, when PBMCs were incubated with various high-grade serous ovarian cancer cell lines, including OVCAR8, OVCAR3, and OVCAR5, robust NK cell degranulation and intracellular IFNγ production was observed compared to no treatment and rhIL-15 alone (Figures 29A-29B and 30A-30F). To determine whether individual components of TriKE can induce NK cell activity by themselves, individual cam16 VHH, IL-15, or anti-B7-H3 scFv components were incubated with PBMCs and OVCAR8 cells and activity was determined. The data clearly show that individual components do not enhance NK cell activity against OVCAR8 cells. NK cell activity from normal donor PBMCs and ascites from the peritoneal cavity of an ovarian cancer patient at the time of surgery was evaluated against MA-148 cells, another high-grade serous ovarian adenocarcinoma cell line (Figures 29A-29B and 30G-30H). Compared to controls, cam1615B7-H3 TriKE induced robust activity against normal donor NK cells. NK cell activity from ovarian cancer-derived ascites samples was reduced as expected due to altered NK cell function and reduced CD16 expression driven by the tumor microenvironment, but cam1615B7-H3 TriKE induced significantly enhanced NK cell degranulation compared to controls.Finally, killing of ovarian cancer tumor cells (OVCAR8) was measured dynamically over two days in the presence of enriched NK cells alone (no treatment), NK cells and rhIL-15 (IL15), and NK cells and cam1615B7-H3 TriKE (Figure 30I). In this assay, tumor cells can be tracked with a stably expressed fluorescent protein (NucLight Red), and detection of early apoptosis, used to exclude recent cell death, is mediated by a green fluorescent caspase 3 / 7 dye. The basic readout provided is the number of viable tumor cells (red + green -), normalized to tumor alone at the stated time point. As shown, cam1615B7-H3 TriKE induced robust and rapid tumor killing when compared to the control. This data indicates that cam1615B7-H3 TriKE potently enhances activity against ovarian cancer cells in vitro. Notably, cam1615B7-H3 TriKE induced similar degranulation and stronger IFNγ production compared to the strong natural cytotoxic signal induced by K562 cells in the absence of TriKE. The fold NK cell activation against all ovarian cancer cell lines, calculated as activation in PBMC+tumor+TriKE divided by activation in PBMC+TriKE alone, was higher than that by the B7-H3-negative Raji line, indicating the B7-H3 specificity of TriKE.

[0189] High-dimensional analysis of cam1615B7-H3 TriKE-activated cells was performed as shown in Figure 31. A custom 42-parameter, CyTOF (mass cytometry) NK cell target panel was used to broadly evaluate the phenotypic and functional effects of TriKE activation on NK cells. PBMCs were left untreated, incubated with cam1615B7-H3 TriKE for 24 hours, incubated with tumor (OVCAR8) for 24 hours, or incubated with tumor and cam1615B7-H3 TriKE for 24 hours. Cells were then stained, fixed, and subjected to CyTOF2. Samples (three biological replicates per condition) were concatenated and data were visualized with viSNE, which shows the localization of individual cells in a 2D plot using all expression information to explore multidimensional data (Figure 31). Data were analyzed using CD56 bright and CD56 dim The distribution of NK cells in effectors and tumors was not altered. Both activation markers CD25 and CD69, as well as the chemokine receptor CXCR3, were induced by TriKE treatment. Granzyme B, involved in the cytolytic activity of NK cells, was primed in effectors + TriKE, whereas in the presence of tumor targets (effectors + tumors + TriKE), these granzyme B-high cells disappeared, likely due to ADCC-driven specific degranulation. Interestingly, when effectors were exposed to tumors in the presence of TriKE, the expression of both inhibitory KIRs (KIR2DL1, KIR2DL3, and KIR3DL1) and activating KIRs (KIR2DS1 and KIR2DS4) was reduced. This also seemed to be true for NKG2D, whereas the natural cytotoxicity receptors (NCRs: NKp30, NKp44, and NKp46) were less affected. Finally, the inhibitory receptor TIGIT did not appear to be affected either. Taken together, the data demonstrate a dynamic change in NK cell phenotype following TriKE-mediated activation.

[0190] As illustrated in Figures 32A-32F, cam1615B7-H3 TriKE mediates antitumor activity in vivo. Determination of in vivo activity is a critical step for translation. However, before evaluating the ability of cam1615B7-H3 TriKE to induce function against tumors, potential toxicity was evaluated. To do this, NSG mice were irradiated and engrafted with 1 million NK cells treated with no treatment, IL-15, or cam1615B7-H3 for 3 weeks, and body weight was tracked over 90 days after initial treatment. Although there was an initial loss of body weight in all groups, likely due to irradiation, no significant differences were observed between TriKE-treated groups and controls. This is not surprising given the low toxicity profile of IL-15 and the safety profile of B7-H3. Although in vitro data indicates that cam1615B7-H3 TriKE can potently activate NK cells against various tumors, to evaluate whether this TriKE has efficacy in a preclinical model, we used the xenogeneic mouse model of ovarian cancer described previously (Figure 32A). In this model, human NK cells and human high-grade serous MA-148-luc cells are injected into the peritoneal cavity of NSG mice. Longitudinal analysis of tumor progression showed that cam1615B7-H3-treated mice exhibited the lowest tumor progression when compared to IL-15-treated or tumor-only mice (Figure 32B). At the time of harvest (day 21), cam1615B7-H3-treated mice had significantly lower tumor burden than the tumor-only group (Figures 32C-32D). Peritoneal lavage fluid at this time point showed similar human NK cell numbers in the rhIL-15 and cam1615B7-H3 treated groups, indicating that the difference in tumor control was not solely due to differences in NK cell numbers (Figure 32E). In relation to the mechanism of action of cam1615B7-H3 TriKE, TriKE-treated mice had NK cells with higher levels of CD16 expression than IL-15-treated mice (Figure 32F). PD-1 expression, which is often associated with immune cell exhaustion, also had a trend toward lower (but not significant) expression in TriKE-treated mice compared to IL-15-treated mice.

[0191] Example 8 Conclusion and Discussion An ideal targeted immunotherapeutic intervention for solid tumors would have broad recognition across a variety of carcinomas with limited or no on-target off-tumor toxicity. B7-H3 exhibits these characteristics, with high expression in many tumors and low expression in normal tissues. Targeted antibody-based therapy against B7-H3 is currently being explored in the clinic (NCT04185038, NCT02982941, NCT03406949, NCT03729596, NCT04077866, and NCT02475213). Both the safety profile and efficacy of anti-B7-H3 antibodies in clinical trials to date have been good. Radiolabeled antibodies targeting B7-H3 have been safely administered for at least 10 years. The drug is considered safe enough to be used in the pediatric brain. Interestingly, B7-H3 has been reported to be expressed in the vasculature and stromal fibroblasts, indicating that this antigen can be used to target tumor vasculature and architecture. A clear correlation exists between high B7-H3 expression and various tumor growth parameters, including fewer tumor-infiltrating lymphocytes, faster cancer progression, and poor clinical outcomes in several cancers, such as pancreatic ductal adenocarcinoma (PDAC), prostate cancer, ovarian cancer, lung cancer, and clear cell renal cell carcinoma. Furthermore, natural cytotoxicity against most cancers is usually not sufficient for endogenous NK cells to suppress cancer progression, as highlighted by the low natural cytotoxicity against most tumor lines tested in this study. Taken together, these studies make a very compelling case for targeting B7-H3.

[0192] However, none of the previous therapeutic approaches combines cytokine signaling and ADCC, two important components for optimal NK cell immunotherapy. The cam1615B7-H3 protein described herein employs that optimal combination. Our data show that cam1615B7-H3 TriKE delivers specific IL-15 signals to NK cells, prevents off-target toxicity, and also mediates ADCC against various adenocarcinoma cell lines in the context of ovarian, prostate, and lung cancer. This dual mechanism of action allows for enhanced NK cell proliferation, survival, and target activation. Our previous studies comparing TriKE to a bispecific killer engager (BiKE) lacking IL-15 showed that the IL-15 portion of TriKE induces increased proliferation, survival, STAT5 signaling, and enhanced priming of NK cells. However, it should be noted that our in vitro studies show some induction of overall T cell proliferation by TriKE, although minimal in nature, compared to treatment with equimolar concentrations of IL-15. This indicates that TriKE induces more specificity than monomeric IL-15, but still triggers T cell proliferation at low levels. Interestingly, while overall T cell proliferation is increased by TriKE compared to no treatment, proliferation beyond three divisions is actually reduced, and there is no difference in T cell numbers at the end of culture when comparing these two groups. Exploration in more complex models and patients is required to fully outline the specificity of cam1615B7-H3 TriKE and evaluate its impact on T cells and more importantly T cell toxicity.

[0193] Although the results of the preclinical ovarian cancer mouse model were promising, and the treated animals had stable conditions, the treatment was not curative in this model. This may be due to a variety of factors. Human NK cell donors are variable, a problem that may be solved by breakthroughs in NK cell products such as induced pluripotent stem cell-derived NK cells (iNK). Also, the TriKE molecule is small, less than 65 kDa in size, and is rapidly cleared by the kidney, resulting in suboptimal dosing. Different donors may clear at different rates. Alternatively, NK cell depletion, either mediated by IL-15 or by strong NK cell activation, may be operant. TriKE relies on targeting CD16 for activation, but can be cleaved by the metalloproteinase ADAM17. We and others have previously described low levels of CD16 in NK cells derived from the ascites of women with ovarian cancer, and that the MA-148 xenogeneic mouse model mimics this phenomenon. CD16 cleavage can be mediated either by the tumor itself or by overactivation of NK cells by the inflammatory tumor microenvironment, as it can be triggered by both ADAM17 activation and cytokine receptors. This is not unique to ovarian cancer, as reduced CD16 expression on NK cells has been described in other tumor settings. Although CD16 downregulation may not be seen in all tumor settings, our ascites data indicate that in the setting of low CD16 expression, TriKE can still mediate tumor killing, albeit in a reduced manner. However, many years have passed since the discovery of the NK cell-specific cleavage inhibitor TriKE. Combination with clinically tested ADAM17 inhibitors or a recently described and currently clinically tested cellular product bearing a non-cleavable CD16 receptor (NCT04023071) should significantly improve the activity of TriKE in situations where CD16 is downregulated.

[0194] While the majority of immunotherapy modalities focus on checkpoint blockade and T cells, natural killer cells have many characteristics that make them ideal candidates for cell-based therapy against solid tumors. These studies focus on a unique biological platform technology that incorporates IL-15 as a bispecific antibody cross-linker to promote NK cell-mediated targeting for a broad range of cancers. TriKE overcomes the nonspecific mechanisms of natural cytotoxicity by promoting antigen-specific synapses intended to enhance functional NK cell-mediated killing, activation, and proliferation. The TriKE molecule described in this study targets B7-H3, a member of the B7 costimulatory family of Ig proteins that is overexpressed in several solid tumor malignancies. B7-H3 was found to be a robust target for the TriKE molecule, selectively promoting NK cell in vitro killing of ovarian, prostate, and lung cancers. IL-15 action is remarkably specific for NK cell activity with little off-target effects on T cells. This provides the first in vivo xenograft data supporting the notion that TriKE can act against solid tumors and support their future clinical development.

[0195] array: TIFF2024536791000002.tif213152TIFF2024536791000003.tif223153TIFF2024536791000004.tif223156 TIFF2024536791000005.tif220152TIFF2024536791000006.tif223151TIFF2024536791000007.tif103150

[0196] Although the 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 invention is limited only by the scope of the following claims.

Claims

1. An isolated nucleic acid sequence set forth in SEQ ID NO: 13 or 14, or a sequence having 90% identity thereto.

2. A protein encoded by the nucleic acid sequence of claim 1.

3. The protein of claim 2, wherein the amino acid sequence is selected from SEQ ID NO: 6 or 7.

4. The amino acid sequences set forth in SEQ ID NOs: 6 and 7 are operably linked to each other in either orientation. A fusion protein comprising:

5. the protein a) comprises SEQ ID NOs: 6 and 7 directly linked between the C-terminus of SEQ ID NO: 6 and the N-terminus of SEQ ID NO: 7, or b) comprises SEQ ID NOs: 7 and 6 directly linked between the C-terminus of SEQ ID NO: 7 and the N-terminus of SEQ ID NO: 6, The fusion protein of claim 4.

6. A fusion protein comprising the sequence shown in SEQ ID NO: 1 and a sequence having 90% or more identity to SEQ ID NO:

1.

7. A fusion protein comprising, operably linked, SEQ ID NO: 2 or 19; SEQ ID NO: 4, 17 or 18; SEQ ID NO: 6 and 7, or SEQ ID NO: 7 and 6.

8. a) SEQ ID NO: 2 or 19 and SEQ ID NO: 4, 17 or 18 are linked by SEQ ID NO: 3 or SEQ ID NO: 15; b) SEQ ID NO: 4, 17 or 18 and SEQ ID NO: 6 or 7 are linked by SEQ ID NO: 5 or SEQ ID NO: 16, and / or c) SEQ ID NOs: 6 and 7 are operably linked in either orientation; The fusion protein of claim 7.

9. further comprising a half-life extension (HLE) molecule; 9. The fusion protein of claim 8, wherein the HLE molecule is an Fc or scFc antibody fragment comprising any one of SEQ ID NOs: 21-25.

10. SEQ ID NO:4 has an N72 substitution; The fusion protein of claim 8, wherein the N72 mutation is N72A or N72D.

11. The fusion protein of claim 10, wherein the protein is represented by SEQ ID NO: 17 or 18.

12. An isolated nucleic acid sequence encoding the fusion protein of claim 6.

13. 13. The isolated nucleic acid sequence of claim 12, which is SEQ ID NO:

8.

14. 10. The fusion protein of claim 6 for use in a method for treating cancer in a subject, the method comprising administering the fusion protein to the subject, thereby treating the cancer.

15. 15. The fusion protein of claim 14, wherein the cancer is selected from non-small cell lung cancer, cutaneous squamous cell carcinoma, pancreatic cancer, primary hepatocellular carcinoma, colorectal cancer, clear cell renal cell carcinoma, or breast cancer.

16. A fusion protein comprising SEQ ID NO: 19, SEQ ID NO: 17 or 18, and SEQ ID NO: 6 and 7 in either orientation.

17. a) SEQ ID NO: 19 is operably linked to SEQ ID NO: 17 or 18 by a linker of SEQ ID NO: 3 or 15; b) SEQ ID NO: 17 or 18 is operably linked to SEQ ID NO: 6 and 7 in either orientation by a linker of SEQ ID NO: 5 or 16; and / or c) further comprising a half-life extension (HLE) molecule; The fusion protein of claim 16.

18. 18. The fusion protein of claim 17, wherein the HLE molecule is an Fc or scFc antibody fragment comprising any one of SEQ ID NOs: 21-25.

19. A pharmaceutical composition comprising a therapeutically effective amount of a fusion protein comprising the amino acid sequence of SEQ ID NO: 1 or a sequence having 90% or more identity to SEQ ID NO: 1, and a pharmaceutically acceptable carrier.

20. 20. The pharmaceutical composition of claim 19, for use in a method of treating cancer in a subject comprising administering the pharmaceutical composition to the subject.

21. A fusion protein comprising the sequence shown in SEQ ID NO: 1 and a sequence having 90% or more identity to SEQ ID NO: 1 for use in the following methods: a) a method of inducing natural killer (NK) cell activity against cancer cells in a subject, the method comprising administering to the subject the fusion protein, thereby inducing NK cell activity against cancer cells in the subject; wherein inducing NK cell activity comprises inducing NK cell degranulation, inducing NK cell production of interferon-γ, increasing the number of tumor-infiltrating NK cells in the subject, and / or inducing or increasing NK cell proliferation. b) a method of inhibiting tumor growth in a subject, comprising administering to the subject the fusion protein, thereby inhibiting tumor growth in the subject; wherein inhibiting tumor growth comprises reducing tumor cell survival. c) a method of increasing the survival of a subject having cancer, comprising administering to said subject said fusion protein, thereby increasing the survival of said subject; or d) A method for inducing natural killer (NK)-mediated antibody-dependent cellular cytotoxicity against cancer cells in a subject, comprising administering to the subject the fusion protein, thereby increasing the survival of the subject.

22. a) the method further comprises administering an anti-cancer treatment to the subject; and / or b) the subject has cancer; i) the cancer is selected from the group consisting of lung cancer, prostate cancer, multiple myeloma, ovarian cancer, and head and neck cancer; ii) the cancer cells are B7-H3-expressing cancer cells, and / or iii) the cancer is a treatment-resistant cancer; 21. The pharmaceutical composition of claim 20.

23. a) the method further comprises administering an anti-cancer treatment to the subject; and / or b) the subject has cancer; i) the cancer is selected from the group consisting of lung cancer, prostate cancer, multiple myeloma, ovarian cancer, and head and neck cancer; ii) the cancer cells are B7-H3-expressing cancer cells, and / or iii) the cancer is a treatment-resistant cancer; 22. The fusion protein of claim 21.

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