Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2803—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
  • C07K16/2827—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/52—Cytokines; Lymphokines; Interferons
  • C07K14/54—Interleukins [IL]
  • C07K14/5443—IL-15
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2803—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
  • C07K16/283—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against Fc-receptors, e.g. CD16, CD32, CD64
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
  • C07K2317/22—Immunoglobulins specific features characterized by taxonomic origin from camelids, e.g. camel, llama or dromedary
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
  • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
  • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
  • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
  • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
  • C07K2317/622—Single chain antibody (scFv)
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
  • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/31—Fusion polypeptide fusions, other than Fc, for prolonged plasma life, e.g. albumin

Definitions

• the present invention relates generally to fusion proteins, and more specifically to B7-H3 targeting tri-specific killer engager molecules and their use to treat cancer.
• Immunotherapy is an individualized treatment that activates or suppresses the immune system to amplify or diminish an immune response and is developing rapidly for treating various forms of cancer.
• Immunotherapy for cancer such as chimeric antigen receptor (CAR)-T cells, CAR-natural killer (NK) cells, PD-1 and PD-L1 inhibitor, aims to help patients' immune system fight cancer.
• CAR chimeric antigen receptor
• NK CAR-natural killer
• PD-1 and PD-L1 inhibitor aims to help patients' immune system fight cancer.
• T cell depends on both the specific combination of T cell receptor (TCR) and peptide-bound major histocompatibility complex (MHC), and the interplay of co-stimulatory molecules of T cell with ligands on antigen presenting cells (APCs).
• TCR T cell receptor
• MHC major histocompatibility complex
• APCs antigen presenting cells
• B7-H3 could promote the activation of T cells and the production of IFN-y.
• Immune checkpoints such as PD-1, PD-L1, PD-L2, and CTLA4, are molecules holding many receptor-ligand interactions to evade the immune system and facilitate proliferation.
• mAbs monoclonal antibodies
• mAbs monoclonal antibodies
• the inhibitors targeting PD- 1 or CTLA4 were successfully used for treating patients with metastatic melanoma, with improved responses and prolonged survival.
• B7-H3 was found to be overexpressed among several kinds of human cancer cells and was correlated with disease deteriorations. B7-H3 was recognized as a co-stimulatory molecule for immune reactions such as T cell activation and IFN-y production. In the presence of anti-CD3 antibody mimicking the TCR signal, human B7-H3-Ig fusion protein increases the proliferation of both CD4+ and CD8+ T cells and enhances the cytotoxic T lymphocyte (CTL) activity in vitro. B7-H3 also has an antitumor effect on adenocarcinoma of the colon, which could also be regarded as a promising therapy for the treatment of colon cancers.
• CTL cytotoxic T lymphocyte
• B7-H3 was recognized as a co-stimulatory molecule that was not only abundantly expressed in pancreatic cancer but also associated with increased treatment efficacy. Although B7-H3 expression was detectable in most examined pancreatic cancer samples, and significantly upregulated in pancreatic cancer versus normal pancreas, patients with high tumor B7-H3 levels had a significantly better postoperative prognosis than patients with low tumor B7-H3 levels (Yang et al., ibid).
• NK cell cytotoxicity can occur by natural cytotoxicity, mediated via the natural cytotoxicity receptors (NCR), or by antibodies, such as rituximab, to trigger antibody-dependent cell-mediated cytotoxicity (ADCC) through CD 16, the activating low-affinity Fc-y receptor for immunoglobulin G (IgG) highly expressed by the CD56 dim subset of NK cells.
• NCR natural cytotoxicity receptors
• ADCC antibody-dependent cell-mediated cytotoxicity
• IgG immunoglobulin G
• CD 16/CD 19 BiKE and CD 16/CD 19/CD22 TriKE can trigger NK cell activation through direct signaling of CD 16 and induce directed secretion of lytic granules and target cell death. Furthermore, these reagents induce NK cell activation that leads to cytokine and chemokine production.
• the present invention is based on the development of B3-H7 targeting fusion proteins, and specifically B7-H3 targeting tri-specific killer engager molecules (TriKEs) and on methods of use thereof.
• TriKEs tri-specific killer engager molecules
• the present invention provides an isolated nucleic acid sequence as set forth in SEQ ID NO: 13 or 14 or a sequence having 90% identity thereto.
• the invention provides a protein encoded by a nucleic acid sequence as set forth in SEQ ID NO: 13 or 14 or a sequence having 90% identity thereto.
• amino acid sequence is selected from SEQ ID NO:6 or 7.
• the invention provides a fusion protein including the amino acid sequence set forth in SEQ ID NO:6 and 7, operably linked to each other in either orientation.
• the protein includes SEQ ID NO:6 and 7, in direct linkage between the C-terminus of SEQ ID NO:6 and the N-terminus of SEQ ID NO:7. In another aspect, the protein includes SEQ ID NO:7 and 6, in direct linkage between the C-terminus of SEQ ID NO:7 and the N-terminus of SEQ ID NO:6.
• the invention provides a fusion protein including the sequence set forth in SEQ ID NO: 1 and sequences having 90% or greater identity to SEQ ID NO: 1.
• the invention provides a fusion protein including in operably linkage, SEQ ID NO:2 or 19; 4, 17, or 18; 6 and 7, or 7 and 6.
• the fusion protein further includes a half-life extending (HLE) molecule.
• HLE half-life extending
• the HLE molecule is a Fc or a scFc antibody fragment including any one of SEQ ID NOs:21-25.
• SEQ ID NO:4 has anN72 substitution.
• the N72 mutation is N72A or N72D, set forth in SEQ ID NO: 17 and 18, respectively.
• sequence is SEQ ID NO:8.
• the invention provides a method of treating cancer in a subject including administering to the subject any of the fusion proteins described herein, thereby treating the cancer.
• the cancer is selected from non-small lung cancer, cutaneous squamous cell carcinoma, pancreatic cancer, primary hepatocellular carcinoma, colorectal carcinoma, clear cell renal carcinoma or breast cancer.
• the invention provides a fusion protein comprising in operable linkage, SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:6 and 7 in either orientation or SEQ ID NO: 19, SEQ ID NO: 17 or 18 and SEQ ID NO:6 and 7 in either orientation and nucleic acid sequences encoding such proteins.
• SEQ ID NO: 19 is operably linked to SEQ ID NO: 17 or 18 by a linker of SEQ ID NO:3 or 15.
• SEQ ID NO: 17 or 18 is operably linked to SEQ 6 and 7, in either orientation, by a linker of SEQ ID NO:5 or 16.
• the fusion protein further includes a half-life extending (HLE) molecule.
• HLE half-life extending
• the HLE molecule is a Fc or a scFc antibody fragment including any one of SEQ ID NOs:21-25.
• the invention provides a pharmaceutical composition including a therapeutically effective amount of a fusion protein including the amino acid sequence of SEQ ID NO: 1 or a sequence having 90% or greater identity to SEQ ID NO:1 and a pharmaceutically acceptable carrier.
• the invention provides a method of treating cancer in a subject including administering to the subject the pharmaceutical composition described herein.
• the invention provides a method of inducing natural killer (NK) cell activity against a cancer cell in a subject including administering to the subject a fusion protein including the sequence set forth in SEQ ID NO:1 and sequences having 90% or greater identity to SEQ ID NO: 1, thereby inducing NK cell activity against a cancer cell in the subject.
• inducing NK cell activity includes inducing NK cells degranulation, inducing NK cell production of interferon y, increasing a number of tumor infiltrating NK cells in the subject, and/or inducing or increasing NK cell proliferation.
• the invention provides a method of inhibiting tumor growth in a subject including administering to the subject a fusion protein including the sequence set forth in SEQ ID NO: 1 and sequences having 90% or greater identity to SEQ ID NO: 1, thereby inhibiting tumor growth in the subject.
• inhibiting tumor growth includes decreasing tumor cell survival.
• the invention provides a method of increasing survival of a subject having cancer including administering to the subject a fusion protein including the sequence set forth in SEQ ID NO: 1 and sequences having 90% or greater identity to SEQ ID NO: 1, thereby increasing survival of the subject.
• the invention provides a method of inducing natural killer (NK) mediated antibody-dependent cellular cytotoxicity against a cancer cell in a subject including administering to the subject a fusion protein including the sequence set forth in SEQ ID NO: 1 and sequences having 90% or greater identity to SEQ ID NO:1, thereby increasing survival of the subject.
• NK natural killer
• administering to a subject a fusion protein including the sequence set forth in SEQ ID NO: 1 and sequences having 90% or greater identity to SEQ ID NO: 1 further includes administering to the subject an anti-cancer treatment.
• the subject has cancer.
• the cancer is selected from the group consisting of lung cancer, prostate cancer, multiple myeloma, ovarian cancer and head and neck cancer.
• cancer cells are B7-H3 expressing cancer cells.
• the cancer is a treatment resistant cancer.
• FIGURES 1A-1D illustrate the construction and isolation of caml615B7-H3 trispecific killer engager (TriKE).
• FIGURE 1A is a schematic representation of the TriKE construct consisting of (left to right) camelid anti-CD16 VHH, Human IL- 15, and anti-B7-H3 scFv.
• FIGURE IB is a graph illustrating the chromatography trace from the first-step purification of caml615B7-H3 on an ion exchange (FFQ) column. The collection peak is indicated by the double-sided arrow.
• FIGURE 1C is a graph illustrating the chromatography trace from the second-step purification of caml615B7-H3 on a size exclusion chromatography (SEC) column.
• SEC size exclusion chromatography
• FIGURE ID is a photograph of a sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel indicating the purity of the final product after the two orthogonal column steps.
• SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis
• FIGURES 2A-2H illustrates that caml615B7-H3 TriKE induces potent, and specific, natural killer (NK) cell proliferation.
• Peripheral blood mononuclear cells PBMCs
• PBMCs Peripheral blood mononuclear cells
• FIGURE 2 A shows a representative histogram showing NK cell (CD56+ CD3-) proliferation measured as the dilution of the CellTrace Violet dye.
• FIGURE 2B is a graph illustrating pooled data showing the overall proportion of NK cells that proliferated.
• FIGURE 2C is a graph illustrating pooled data showing the proportion of NK cells that highly proliferated (measured as proliferation beyond three divisions).
• FIGURE 2E is a representative histogram showing T cell (CD56- CD3+) proliferation.
• FIGURE 2F is a graph illustrating pooled data showing the overall proportion of T cells proliferated.
• FIGURE 2G is a graph illustrating pooled data showing the proportion of T cells highly proliferated.
• FIGURES 3A-3B illustrate camB7-H3 TriKE binding specificity.
• FIGURE 3A is a schematic representation of the binding of the TriKE molecule to B7-H3 positive cancer cells.
• FIGURE 3B is a graph illustrating the binding specificity to WT B7-H3, BT-12 pediatric brain tumor lines highly express B7-H3 (WT) while B7-H3 KO BT-12 (red) cell line was produced using CRISPR.
• FIGURES 4A-4B illustrates the ADCC induction by the TriKE molecules.
• FIGURE 4A is a graph showing the ADCC induction of the WT TriKE.
• FIGURE 4B is a graph shoing the ADCC induction of the CD 16 TriKE.
• FIGURES 5A-5B illustrate functional assays conducted with hematologic malignancy cell lines with varying levels of B7-H3 expression from none to very high levels.
• FIGURES 6A-6B illustrates B7-H3 MFI on four myeloma cell lines by flow cytometry.
• FIGURE 6A is a graph showing the expression of B7-H3 on the myeloma lines RPMI-8226, U266, MM 1 S and H929 by flow cytometry.
• FIGURE 6B is a graph summarizing the data in FIGURE 6A.
• FIGURES 7A-7B illustrate the ability of peripheral blood NK cells with or without B7-H3-TriKE to kill myeloma cells in live imaging IncuCyte Zoom assays.
• FIGURE 7A is a graph showing the effects with effectortarget (E:T) ratios of 2:1 and 4:1 on MM IS cells.
• FIGURE 7B is a graph showing the effects with with effectortarget (E:T) ratios of 2:1 and 4:1 on U266 cells.
• FIGURES 8A-8D illustrate the ability of peripheral blood NK cells with or without B7-H3-TriKE to kill myeloma cells in live imaging IncuCyte Zoom assays.
• FIGURE 8A is a graph showing the effects with effectortarget (E:T) ratios of 2:1 and 4:1 on H929 cells.
• FIGURE 8B is a graph showing the effects with with effectortarget (E:T) ratios of 2:1 and 4: 1 on MM 1 S cells.
• FIGURE 8C is a graph showing the effects with with effectortarget (E:T) ratios of 2:1 and 4:1 on RPMI-8226 cells.
• FIGURE 8D is a graph showing the effects with with effectortarget (E:T) ratios of 2: 1 and 4: 1 on U266 cells.
• FIGURES 9A-9D illustrate the efficacy of B7-H3-TriKE with the proteasome inhibitor bortezomib (lOnM) and the immunomodulatory drug lenalidomide (5pM).
• FIGURE 9A is a graph showing the effect of the combination therapy on H929 cells after 48 hours.
• FIGURE 9B is a graph showing the effect of the combination therapy on RPMI-8226 cells after 48 hours.
• FIGURE 9C is a graph showing the effect of the combination therapy on MM 1 S cells after 48 hours.
• FIGURE 9D is a graph showing the effect of the combination therapy on U266 cells after 48 hours.
• FIGURES 10A-10D illustrate the effect B7-H3-TriKE on MDSC developed from CD33+ myeloid cells from healthy donors, when incubated with myeloma cells at 1 : 100 ratio.
• FIGURE 10A is a graph showing MDSC (CD14+CD1 lb+) expression of B7-H3.
• FIGURE 10B is a graph showing cell survival as measured by flow cytometry.
• FIGURE 10C is a plot of live, CD14+ cells
• FIGURE 10D is a graph illustrating H929 growth as measured over 48 hours by live cell imaging.
• FIGURES 11A-11B illustrate NK mediated killing of B7-H3 expressing MDSC.
• FIGURE 11A is a graph showing B7-H3 expression of MDSC.
• FIGURE 11B is a graph illustrating NK mediated killing of co-cultured MDSC with NK at E:T of 1:1 and compared killing with and without B7-H3 TriKE.
• FIGURES 12A-12D show that caml615B7-H3 TriKE enhances NK cell function against prostate tumor targets.
• FIGURE 12A is a graph illustrating pooled proportion of NK cells from healthy donors expressing CD107a+ (degranulation) against CREB5, 22RV1 and Enza-R targets.
• FIGURE 12B is a graph illustrating pooled proportion of NK cells from healthy donors expressing IFNy cytokine production against CREB5, 22RV1 and Enza-R targets.
• FIGURE 12C is a graph illustrating pooled proportion of NK cells from healthy donors expressing CD107a+ (degranulation).
• FIGURE 12D is a graph illustrating pooled proportion of NK cells from healthy donors expressing IFNy cytokine production.
• FIGURES 13A-13B show that camB7-H3 increase NK cell function against prostate cancer cells and NK proliferation as compared to IL - 15 Alone.
• FIGURE 13A is a graph showing percent of NK cells expressing IFNy against prostate cancer cell targets.
• FIGURE 13B is a graph illustrating the proportion of NK cells undergoing 3 or more rounds of division.
• FIGURES 14A-14L show that TriKEs can induce NK cell degranulation and inflammatory cytokine production against prostate cancer cell lines.
• FIGURE 14A is a graph showing pooled analysis of proportion of NK cells from healthy donors or prostate cancer patients expressing CD107a+ against CA-2 targets.
• FIGURE 14B is a graph showing pooled analysis of proportion of NK cells from healthy donors or prostate cancer patients expressing CD107a+ against PC-3 targets.
• FIGURE 14C is a graph showing pooled analysis of proportion of NK cells from healthy donors or prostate cancer patients expressing CD107a+ against DU- 145 targets.
• FIGURE 14D is a graph showing pooled analysis of proportion of NK cells from healthy donors or prostate cancer patients expressing IFNy against CA-2 targets.
• FIGURE 14E is a graph showing pooled analysis of proportion of NK cells from healthy donors or prostate cancer patients expressing IFNy against PC-3 targets.
• FIGURE 14F is a graph showing pooled analysis of proportion of NK cells from healthy donors or prostate cancer patients expressing IFNy against DU- 145 targets.
• FIGURE 14G is a graph showing pooled analysis of proportion of NK cells from healthy donors or prostate cancer patients expressing CD107a+ against LnCAP targets.
• FIGURE 14H is a graph showing pooled analysis of proportion of NK cells from healthy donors or prostate cancer patients expressing CD107a+ against VCAP targets.
• FIGURE 141 is a graph showing pooled analysis of proportion of NK cells from healthy donors or prostate cancer patients expressing CD107a+ against 22RV1 targets.
• FIGURE 14J is a graph showing pooled analysis of proportion of NK cells from healthy donors or prostate cancer patients expressing IFNy against LnCAP targets.
• FIGURE 14K is a graph showing pooled analysis of proportion of NK cells from healthy donors or prostate cancer patients expressing IFNy against VCAP targets.
• FIGURE 14L is a graph showing pooled analysis of proportion of NK cells from healthy donors or prostate cancer patients expressing IFNy against 22RV1 targets.
• FIGURES 15A-15D show that caml615B7-H3 TriKE enhances NK cell function of healthy donors (black bars) and prostate cancer patients (white bars) against prostate tumor targets.
• FIGURE 16 is a graph illustrating a PC3 standard IncuCyte Assay.
• FIGURE 17 shows photographs illustrating PC-3 spheroid under various treatment conditions.
• FIGURE 18 is a graph illustrating the size of PC-3 spheroids over time.
• FIGURE 19 is a graph illustrating the size of PC-3 spheroids over time.
• FIGURE 20 shows photographs illustrating the ability of B7-H3 TriKE and BiKE to mediate efficient killing of PC3 spheroids.
• FIGURE 21 is a graph illustrating cell index over time.
• FIGURES 22A-22F illustrate enzalutamide resistant prostate cancer cell line phenotyping for B7-H3.
• FIGURE 22A is a graph illustrating B7-H3 expression in CREB5+ cells.
• FIGURE 22B is a graph illustrating B7-H3 expression in C4-2 cells.
• FIGURE 22C is a graph illustrating B7-H3 expression in LN-CaP cells.
• FIGURE 22D is a graph illustrating B7- H3 expression in enzalutamide resistant LNCap cells.
• FIGURE 22E is a graph illustrating B7- H3 expression in PC3 cells.
• FIGURE 22F is a graph illustrating B7-H3 expression in 22RV1 cells.
• FIGURES 23A-23E illustrate how camB7-H3 TriKE induces activity against prostate cancer cells over a broader dynamic range than previous scFv version.
• FIGURE 23A is a graph illustrating percent CD107a+ NK cells in PBMCNK alone in the presence of 0.3nM TriKE.
• FIGURE 23B is a graph illustrating percent CD107a+ NK cells in PBMCNK alone in the presence of 3nM TriKE.
• FIGURE 23C is a graph illustrating percent CD107a+ NK cells in PBMCNK alone in the presence of 30nM TriKE.
• FIGURE 23D is a graph illustrating percent CD107a+ NK cells in PBMCNK and CA-2 cells in the presence of 0.3nM TriKE.
• FIGURE 23E is a graph illustrating percent CD107a+ NK cells in PBMCNK and CA-2 cells in the presence of 3nM TriKE.
• FIGURE 23F is a graph illustrating percent CD 107a+ NK cells in PBMCNK and CA-2 cells in the presence of 30nM TriKE.
• FIGURE 23G is a graph illustrating percent IFNy+ NK cells in PBMCNK alone in the presence of 0.3nM TriKE.
• FIGURE 23H is a graph illustrating percent IFNy+ NK cells in PBMCNK alone in the presence of 3nM TriKE.
• FIGURE 231 is a graph illustrating percent IFNy+ NK cells in PBMCNK alone in the presence of 30nM TriKE.
• FIGURE 23 J is a graph illustrating percent IFNy+ NK cells in PBMCNK and CA-2 cells in the presence of 0.3nM TriKE.
• FIGURE 23K is a graph illustrating percent IFNy+ NK cells in PBMCNK and CA-2 cells in the presence of 3nM TriKE.
• FIGURE 23E is a graph illustrating percent IFNy+ NK cells in PBMCNK and CA-2 cells in the presence of 30nM TriKE.
• FIGURES 24A-24F illustrate that B7-H3 TriKE enhances NK cell function against lung tumor targets.
• FIGURES 25A-25B illustrate the assessment of NK cell activity against HNSCC without treatment.
• FIGURE 25A is a graph illustrating percent CD 107a expression (as a marker for degranulation).
• FIGURE 25B is a graph illustrating percent intracellular IFN-y production.
• FIGURES 26A-26B illustrate the assessment of B7-H3 expression on tumor and immune cells.
• FIGURE 26A is a graph illustrating 5 HNSCC cell lines B7-H3 expression and binding affinity with B7-H3 single domain via flow cytometry.
• FIGURE 26B is a graph illustrating PBMCs from a healthy donor B7-H3 expression by flow cytometry.
• FIGURES 27A-27D illustrate the functional validation O1B7-H3 TriKE.
• FIGURE 27A is a graph illustrating CD 107a expression in PBMCs from healthy donors incubated with Cal27 trio.
• FIGURE 27B is a graph illustrating intracellular IFN-y production in PBMCs from healthy donors incubated with Cal27 trio.
• FIGURE 27C is a graph illustrating CD107a expression in PBMCs from healthy donors incubated with Cal33 trio.
• FIGURE 27D is a graph illustrating intracellular IFN-y production in PBMCs from healthy donors incubated with Cal33 trio.
• FIGURES 28A-28F illustrate real-time imaging assays.
• FIGURE 28A is a graph showing Nuclight red-labeled Cal27 survival after incubation with NK cells at an E:T of 5: 1 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.
• FIGURE 28B shows photographs illustrating spheroids ofNuclight red-labeled Cal27.
• FIGURE 28C is a graph illustrating the size of the spheroid in FIGURE 28B.
• FIGURE 28D is a graph showing Nuclight red-labeled Cal33 survival after incubation with NK cells at an E:T of 5:1 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.
• FIGURE 28E shows photographs illustrating spheroids of Nuclight red-labeled Cal33.
• FIGURE 28F is a graph illustrating the size of the spheroid in FIGURE 28E.
• FIGURES 29A-29B show that various doses of caml 615B7-H3 TriKE enhance NK cell function against ovarian tumor targets.
• FIGURE 29A is a graph illustrating pooled proportion of NK cells from healthy donors expressing CD107a+ (degranulation) against OVCAR8 and MA148 targets.
• FIGURE 29B is a graph illustrating pooled proportion of NK cells from healthy donors expressing IFNy cytokine production against OVCAR8 and MA 148 targets.
• FIGURES 30A-30I show that caml615B7-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 for 4hrs with 30 nM TriKE or control condition.
• FIGURE 301 is a graph illustrating that umor killing was evaluated using IncuCyte imaging assay. NuclightRed expressing OVCAR8 targets were incubated with enriched healthy donor NK over 48hrs, with Caspase 3/7 viability dye.
• the percentage of Live (Nuclight Red+Caspase 3/7-) tumor cells was quantified over a 48-h period and normalized to tumor alone. Readings were obtained every 15 min (representative of four separate experiments). * p ⁇ 0.05, ** p ⁇ 0.01, *** p ⁇ 0.001, and **** p ⁇ 0.0001.
• FIGURE 31 illustrates high dimensional analysis of caml615B7-H3 activated NK cells.
• FIGURES 32A-32F illustrate that caml615B7-H3 TriKE is efficacious in subduing ovarian tumor progression in vivo.
• FIGURE 32B is a graph showing bio luminescent imaging indicative of tumor progression, measured as Total flux radiance (p/s), over three weeks in the MA148 mouse model treated with enriched NK cells and noted treatments.
• FIGURE 32C is a graph illustrating bioluminescent imaging results on day 21 data point.
• FIGURE 32D is a photograph illustrating total flux radiance (p/s) on day 21.
• FIGURE 32E is a scatter plot of CD56+CD3- NK cell numbers from peritoneal lavages for the rhIL-15 and caml615B7-H3 TriKE treatment groups at D21.
• FIGURE 32F is a scatter plot of CD 16 media fluorescence intensity on NK cells from peritoneal lavages for the rhIL- 15 and caml615B7-H3 TriKE treatment groups at day 21. * p ⁇ 0.05, ** p ⁇ 0.01.
• the present invention is based on the development of B7-H3 targeting fusion proteins, and specifically B7-H3 targeting tri-specific killer engager molecules (TriKEs) and methods of use thereof.
• TriKEs tri-specific killer engager molecules
• the present invention provides an isolated nucleic acid sequence as set forth in SEQ ID NO: 13 or 14 or a sequence having 90% identity thereto.
• nucleic acid refers to polynucleotides 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, anti-sense DNA strands, shRNA, ribozymes, nucleic acids conjugated and oligonucleotides.
• a nucleic acid may be present as a single-stranded or double-stranded and linear or covalently circularly closed molecule.
• a nucleic acid can be isolated.
• isolated nucleic acid means, that the nucleic acid (i) was amplified in vitro, for example via polymerase chain reaction (PCR), (ii) was produced recombinantly by cloning, (iii) was purified, for example, by cleavage and separation by gel electrophoresis, (iv) was synthesized, for example, by chemical synthesis, or (vi) extracted from a sample.
• a nucleic might be employed for introduction into, i.e., transfection of, cells, in particular, in the form of RNA which can be prepared by in vitro transcription from a DNA template.
• the RNA can moreover be modified before application by stabilizing sequences, capping, and polyadenylation.
• amplified DNA or “PCR product” refers to an amplified fragment of DNA of defined size.
• PCR product detection methods include, but are not restricted to, gel electrophoresis using agarose or polyacrylamide gel and adding ethidium bromide staining (a DNA intercalant), labeled probes (radioactive or non-radioactive labels, southern blotting), labeled deoxyribonucleotides (for the direct incorporation of radioactive or non-radio active labels) or silver staining for the direct visualization of the amplified PCR products; restriction endonuclease digestion, that relies agarose or polyacrylamide gel or High-performance liquid chromatography (HPLC); dot blots, using the hybridization of the amplified DNA on specific labeled probes (radioactive or non-radioactive labels); high-pressure liquid chromatography using ultraviolet detection; electro-chemiluminescence coupled with
• nucleic acid 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 as described in U.S. Pat. 7,957,913; U.S. Pat. 7,776,616; U.S. Pat. 5,234,809; U.S. Pub. 2010/0285578; and U.S. Pub. 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 classic dideoxy sequencing reactions (Sanger method) using labeled terminators or primers and gel separation in slab or capillary, and next generation sequencing methods such as sequencing by synthesis using reversibly terminated labeled nucleotides, pyrosequencing, 454 sequencing, Illumina/Solexa sequencing, allele specific hybridization to a library of labeled oligonucleotide probes, sequencing by synthesis using allele specific hybridization to a library of labeled clones that is followed by ligation, real time monitoring of the incorporation of labeled nucleotides during a polymerization step, polony sequencing, and SOLiD sequencing.
• Separated molecules may be sequenced by sequential or single extension reactions using polymerases or ligases as well as by single or sequential differential hybridizations with libraries of probes.
• sequence identity or “percent identity” are used interchangeably herein.
• 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. When 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 the 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).
• the length of a 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 is at least 90% or 100%.
• the two sequences are the same length.
• Ranges of desired degrees of sequence identity are approximately 80% to 100% and integer values in between. Percent identities between a disclosed sequence and a claimed sequence 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, an exact 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 sequences provided herein retain the function of the original sequence (e.g., ability to bind B7-H3 or CD 16).
• 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 polypeptides and polynucleotides described herein are embodied within the disclosure.
• 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.
• Variants of the disclosed sequences also include peptides, or full-length protein, that contain substitutions, deletions, or insertions into the protein backbone, that would still leave at least about 70% homology to the original protein over the corresponding portion. A yet greater degree of departure from homology is allowed if like-amino acids, i.e., conservative amino acid substitutions, do not count as a change in the sequence. Examples of conservative substitutions involve amino acids that have the same or similar properties.
• Illustrative amino acid conservative substitutions include the changes of: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartate to glutamate; cysteine to serine; glutamine to asparagine; glutamate to aspartate; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine, glutamine, or glutamate; 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 to leucine.
• the invention provides a protein encoded by a nucleic acid sequence as set forth in SEQ ID NO: 13 or 14 or a sequence having 90% identity thereto.
• polypeptide refers to any chain of at least two amino acids, linked by a covalent chemical bound.
• polypeptide can refer to the complete amino acid sequence coding for an entire protein or to 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 is translated (in the case of mRNA) into a polypeptide in vitro or in vivo when placed under the control of appropriate regulatory sequences.
• a coding sequence can include, but is 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 will usually be located 3' to the coding sequence.
• the amino acid sequence is selected from SEQ ID NO:6 or 7.
• the nucleic acid sequences provided herein can encode for example a light chain or a heavy chain of an antibody, conferring to the encoded polypeptide a binding domain or targeting domain to a specific target. Such a polypeptide can be referred to as a targeting peptide.
• 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 an antigen.
• “Native antibodies” and “intact immunoglobulins”, or the like, are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains.
• the light chains from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (K) and lambda (X), based on the amino acid sequences of their constant domains.
• immunoglobulins can be assigned to different classes.
• immunoglobulins There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgGl, IgG2, IgG3, IgG4, IgA, and IgA2.
• the heavy-chain constant domains that correspond to the different classes of immunoglobulins are called a, 5, e, y, and p, respectively.
• the subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.
• each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies among the heavy chains of different immunoglobulin isotypes.
• Each heavy and light chain also has regularly spaced intrachain disulfide bridges.
• Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains.
• Each light chain has a variable domain at one end (VL) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light-chain variable domain is aligned with the variable domain of the heavy chain.
• Particular amino acid residues are believed to form an interface between the light- and heavy-chain variable domains.
• variable region includes three segments called complementarity-determining regions (CDRs) or hypervariable regions and a more highly conserved portions of variable domains are called the framework region (FR).
• the variable domains of heavy and light chains each includes four FR regions, largely adopting a [3-shcct configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of the [3-sheet structure.
• the CDRs in each chain are held together in close proximity by the FRs and, with the CDRs from the other chain, contribute to the formation of the antigen-binding domain or targeting domain of antibodies (see Kabat et al., NIH Publ. No. 91-3242, Vol. I, pages 647-669 [1991]).
• the constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody dependent cellular cytotoxicity.
• Antibodies can be cleaved experimentally with the proteolytic enzyme papain, which causes each of the heavy chains to break, producing three separate antibody fragments.
• the two units that consist of a light chain and a fragment of the heavy chain approximately equal in mass to the light chain are called the Fab fragments (i.e., the "antigen binding" fragments).
• the third unit, consisting of two equal segments of the heavy chain, is called the Fc fragment.
• the Fc fragment is typically not involved in antigen-antibody binding but is important in later processes involved in ridding the body of the antigen.
• antibody fragments include a portion of an intact antibody, preferably the antigen binding or variable region of the intact antibody.
• antibody fragments include Fab, Fab’ and F(ab’)2, Fc fragments or Fc-fusion products, single-chain Fvs (scFv), disulfide-linked Fvs (sdfv) and fragments including either a VL or VH domain; diabodies, tribodies and the like (Zapata et al. Protein Eng. 8(10): 1057-1062 [1995]).
• the Fab fragment contains 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 carboxy terminus of the heavy chain CHI domain including one or more cysteines from the antibody hinge region.
• Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear a free thiol group.
• F(ab') 2 antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.
• the Fc region of an antibody is the tail region of an antibody that interacts with cell surface receptors and some proteins of the complement system. This property allows antibodies to activate the immune system.
• the Fc region is composed of two identical protein fragments, derived from the second and third constant domains of the antibody's two heavy chains; IgM and IgE Fc regions contain three heavy chain constant domains (CH domains 2-4) in each polypeptide chain.
• the Fc regions of IgGs bear a highly conserved N-glycosylation site. Glycosylation of the Fc fragment is essential for Fc receptor-mediated activity.
• Fc-Fusion proteins also known as Fc chimeric fusion protein, Fc-Ig, Ig-based Chimeric Fusion protein and Fc-tag protein
• Fc-Fusion proteins have become valuable reagents for in vivo and in vitro research.
• the Fc-fused binding partner can range from a single peptide, a ligand that activates upon binding with a cell surface receptor, signaling molecules, the extracellular domain of a receptor that is activated upon dimerization or as a bait protein that is used to identify binding partners in a protein microarray.
• One of the most valuable features of the Fc domain in vivo is it can dramatically prolong the plasma half-life of the protein of interest, which for bio-therapeutic drugs, results in an improved therapeutic efficacy; an attribute that has made Fc-Fusion proteins attractive bio-therapeutic agents.
• the Fc fusion protein may be part of a pharmaceutical composition including an Fc fusion protein and a pharmaceutically acceptable carrier excipients or carrier.
• compositions 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 may include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium 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); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobin; and others.
• Fv is the minimum antibody fragment which contains a complete antigenrecognition and -binding site. This region consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association. It is in this configuration that 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 comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.
• Single-chain Fv or “sFv” antibody fragments comprise the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain.
• 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.
• 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 the skilled practitioner.
• the antibody of choice is a single chain Fv fragment (scFv). See WO 93/16185.
• nucleic acid sequences provided herein encode a light chain and a heavy chain that bind specifically to a B3-H7 protein.
• B7 Homolog 3 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 existing 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, B7-H3 protein has a very limited expression on normal tissues because of its post-transcriptional regulation by microRNAs. However, B7-H3 protein is expressed at high frequency on many different cancer types (60% of all cancers).
• B7-H3 In non-malignant tissues, B7-H3 has a predominantly 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 possesses non-immunological pro-tumorigenic functions such as promoting migration, invasion, angiogenesis, chemoresistance, epithelial-to-mesenchymal transition, and affecting tumor cell metabolism.
• B7-H3 Due to its selective expression on solid tumors and its pro-tumorigenic function, B7-H3 is the target of several anti-cancer agents including enoblituzumab, omburtamab, MGD009, MGC018, DS-7300a, and CAR-T cells.
• B7-H3 targeting peptide or “B7-H3 targeting protein” is meant to refer to any peptide or polypeptide (including protein and fusion protein) that can specifically bind to B7-H3.
• the B7-H3 targeting peptide can be an antibody, an antibody fragment, and the like, having specific binding to one or more target polypeptide, including B7-H3.
• the polypeptide encodes the light chain and the heavy chain of a B7- H3 targeting peptide.
• the nucleic acid sequence of SEQ ID NO: 13 can encode the light chain of a B7-H3 targeting peptide, having the amino acid sequence as set forth in SEQ ID NO:6.
• the nucleic acid sequence of SEQ ID NO: 14 can encode the heavy chain of a B7-H3 targeting peptide, having the amino acid sequence as set forth in SEQ ID NO:8.
• the invention provides a fusion protein including the amino acid sequence set forth in SEQ ID NO:6 and 7, operably linked to each other in either orientation.
• fusion molecule and “fusion protein” are used interchangeably and are meant to refer to a biologically active polypeptide, with or without a further effector molecule, usually a protein or peptide sequence covalently linked (z.e., fused) by recombinant, chemical or other suitable method.
• the fusion molecule can be used at one or several sites through a peptide linker sequence.
• the peptide linker may be used to assist in construction of the fusion molecule.
• preferred fusion molecules are fusion proteins.
• fusion molecule also can include conjugate molecules.
• operably linked to one another, it is meant that there is a direct or indirect covalent linking between the peptides composing the fusion protein.
• two domains that are operably linked may be directly covalently coupled to one another.
• the two operably linked domains may be connected by mutual covalent linking to an intervening moiety (e.g., and flanking sequence).
• Two domains may be considered operably linked if, for example, they are separated by the third domain, with or without one or more intervening flanking sequences.
• Methods for attaching two individual elements usually require the use of a linker.
• linker refers any bond, small molecule, or other vehicle which allows the substrate and the active agent to be targeted to the same area, tissue, or cell, for example by physically linking the individual portions of the conjugate.
• a linker can be any chemical moiety that is capable of linking a compound, usually a drug, to a cell-binding agent in a stable, covalent manner.
• the fusion proteins provided herein can for example include the amino acid sequences set forth in SEQ ID NOs:6 and 7, operably linked to each other in either orientation.
• the fusion protein can include the amino acid sequence set forth in SEQ ID NO:6 at a C-terminal of the fusion protein and the amino acid sequence set forth in SEQ ID NO: 7 at a N-terminal of the fusion protein; or the fusion protein can include the amino acid sequence set forth in SEQ ID NO:6 at a N-terminal of the fusion protein and the amino acid sequence set forth in SEQ ID NO:7 at a C-terminal of the fusion protein.
• the orientation of the amino acid sequences in the fusion protein do not alter the binding-specificity of the fusion protein to its target (i.e., B7-H3 targeting fusion protein).
• the light chain and the heavy chain of the B7-H3 targeting peptide can be operably linked to one another in either orientation without affecting the binding specificity or sensitivity of the targeting peptide.
• the protein includes SEQ ID NO:6 and 7, in direct linkage between the C-terminus of SEQ ID NO:6 and the N-terminus of SEQ ID NO:7.
• the protein includes SEQ ID NO:7 and 6, in direct linkage between the C- terminus of SEQ ID NO:7 and the N-terminus of SEQ ID NO:6.
• the fusion protein provided herein can include additional protein domain, such as additional targeting domain to provide the fusion protein with specific binding to one or more target polypeptide.
• the fusion protein can be a tri-specific killer engager (TriKE) molecule including the B7-H3 targeting peptide as the targeting domain.
• TriKE tri-specific killer engager
• NK cells are cytotoxic lymphocytes of the innate immune system capable of immune surveillance. Like cytotoxic T cells, NK cells deliver a store of membrane penetrating and apoptosis-inducing granzyme and perforin granules. Unlike T cells, NK cells do not require antigen priming and recognize targets by engaging activating receptors in the absence of MHC recognition. NK cells express CD 16, an activation receptor that binds to the Fc portion of IgG antibodies and is involved in antibody-dependent cell-mediated cytotoxicity (ADCC).
• ADCC antibody-dependent cell-mediated cytotoxicity
• NK cells are regulated by IL- 15, which can induce increased antigen-dependent cytotoxicity, lymphokine-activated killer activity, and/or mediate interferon (IFN), tumor-necrosis factor (TNF) and/or granulocyte-macrophage colony-stimulating factor (GM-CSF) responses. All of these IL- 15 -activated functions contribute to improved cancer defense.
• IFN mediate interferon
• TNF tumor-necrosis factor
• GM-CSF granulocyte-macrophage colony-stimulating factor
• NK cells can, for example, induce remission in patients with refractory acute myeloid leukemia (AML) when combined with lymphodepleting chemotherapy and IL-2 to stimulate survival and in vivo expansion of NK cells.
• AML refractory acute myeloid leukemia
• This therapy can be limited by lack of antigen specificity and IL-2-mediated induction of regulatory T (Treg) cells that suppress NK cell proliferation and function.
• Treg regulatory T
• Tri-specific killer engager molecule are targeting fusion protein including two domains capable of driving NK-cell-mediated killing of tumor cells (e.g., CD33+ tumor cells and/or EpCAM+ tumor cells) and an intramolecular NK activating domain capable of generating an NK cell self-sustaining signal 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.
• tumor cells e.g., CD33+ tumor cells and/or EpCAM+ tumor cells
• an intramolecular NK activating domain capable of generating an NK cell self-sustaining signal 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.
• NK cells are responsive to a variety of cytokines including, for example, IL- 15, which is involved in NK cell homeostasis, proliferation, survival, activation, and/or development.
• IL- 15 and IL-2 share several signaling components, including the IL-2/IL-15RP (CD 122) and the common gamma chain (CD 132).
• CD 122 the IL-2/IL-15RP
• CD 132 the common gamma chain
• IL- 15 does not stimulate Tregs, allowing for NK cell activation while bypassing Treg inhibition of the immune response.
• IL- 15 can rescue NK cell functional defects that can occur in the post-transplant setting.
• IL- 15 also can stimulate CD8+ T cell function, further enhancing its immunotherapeutic potential.
• IL- 15 plays a role in NK cell development homeostasis, proliferation, survival, and activation.
• IL- 15 and IL- 2 share several signaling components including the IL-2/IL-15RP (CD 122) and the common gamma chain (CD 132).
• IL- 15 also activates NK cells and can restore functional defects in engrafting NK cells after hematopoietic stem cell transplantation (HSCT).
• HSCT hematopoietic stem cell transplantation
• the fusion protein provided herein can be a TriKE molecule including one or more NK cell engager domains (e.g., CD 16, CD16+CD2, CD16+DNAM, CD16+NKp46), one or more targeting domains (that target, e.g., a tumor cell or virally-infected cell, such as the B7- H3 targeting peptide described herein), and one or more cytokine NK activating domains (e.g., IL-15, IL-12, IL-18, IL-21, or other NK cell enhancing cytokine, chemokine, and/or activating molecule), with each domain operably linked to the other domains.
• NK cell engager domains e.g., CD 16, CD16+CD2, CD16+DNAM, CD16+NKp46
• targeting domains that target, e.g., a tumor cell or virally-infected cell, such as the B7- H3 targeting peptide described herein
• the fusion protein described herein can be a TriKE molecule including a CD 16 NK cell engager domain, such as the CD 16 domain having the amino acid sequence set forth in SEQ ID NO:2 or 19; a B7-H3 targeting fusion protein domain, such as the B7-H3 fusion protein having the amino acid sequences set forth in SEQ ID NOs:6 and 7; and a IL-15 cytokine NK activating domain, such as the IL- 15 having the amino acid sequence set forth in SEQ ID NO:4, 17 or 18.
• a CD 16 NK cell engager domain such as the CD 16 domain having the amino acid sequence set forth in SEQ ID NO:2 or 19
• B7-H3 targeting fusion protein domain such as the B7-H3 fusion protein having the amino acid sequences set forth in SEQ ID NOs:6 and 7
• a IL-15 cytokine NK activating domain such as the IL- 15 having the amino acid sequence set forth in SEQ ID NO:4, 17 or 18.
• the different protein domains of the TriKE molecules can be in operable linkage with one another.
• linkers can be used to covalently attached the protein domains of the TriKE molecule to one another.
• the elements of a fusion protein can be in assembled operable linkage with one another using one or more linkers.
• Linkers can be susceptible to or be substantially resistant to acid-induced cleavage, light-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage and disulfide bond cleavage at conditions under which the compound or the antibody remains active.
• Linkers are classified upon their chemical motifs, well known in the art, including disulfide groups, hydrazine or peptides (cleavable), or thioester groups (non- cleavable).
• Linkers also include charged linkers, and hydrophilic forms thereof as known in the art.
• Suitable linker for the fusion of two or more protein or protein domains can include natural linkers, and empirical linkers. Natural linkers are derived from multi-domain proteins, which are naturally present between protein domains. Natural linkers can have several properties depending or their such as length, hydrophobicity, amino acid residues, and secondary structure, which can impact the fusion protein in different way.
• Empirical linkers can be classified in three types: flexible linkers, rigid linkers, and cleavable linkers.
• Flexible linkers can provide a certain degree of movement or interaction at the joined domains. They are generally composed of small, non-polar (e.g., Gly) or polar (e.g., Ser or Thr) amino acids, which provides flexibility, and allows for mobility of the connecting functional domains.
• Rigid linkers can successfully keep a fixed distance between the domains to maintain their independent functions, which can provide efficient separation of the protein domains or sufficient reduction of their interference with each other.
• Cleavable linkers can allow the release of functional domains in vivo. By taking advantage of unique in vivo processes, they can be cleaved under specific conditions such as the presence of reducing reagents or proteases. This type of linker can reduce steric hindrance, improve bioactivity, or achieve independent actions/metabolism of individual domains of recombinant fusion proteins after linker cleavage.
• linker include linkers having the amino acid sequences set forth in SEQ ID NOs: 3, 5, 10, 12, 15 and 16.
• SEQ ID NO:2 or 19, and 4, 17 or 18 are linked by SEQ ID NO:3 or SEQ ID NO:15.
• SEQ ID NO:4, 17 or 18 and 6 or 7 are linked by SEQ ID NO:5 or SEQ ID NO: 16.
• SEQ ID NO:6 and 7 are in operable linkage in either orientation.
• the invention provides a fusion protein including the sequence set forth in SEQ ID NO: 1 and sequences having 90% or greater identity to SEQ ID NO: 1.
• the invention provides a fusion protein including in operably linkage, SEQ ID NO:2 or 19; 4, 17, or 18; 6 and 7, or 7 and 6.
• the fusion protein described herein can include a wild-type (wt) IL- 15 or mutant IL- 15 cytokine NK activating domain.
• Mutant IL- 15 can for example include IL- 15 including a substitution of the N72 amino acid.
• Non-limiting examples of N72 substitutions include N72A and N72D mutations.
• SEQ ID NO:4 has an N72 substitution.
• the N72 mutation is N72A or N72D and the protein is set forth in SEQ ID NO: 17 or 18, respectively.
• the invention provides a fusion protein including SEQ ID NO: 19, SEQ ID NO: 17 or 18 and SEQ ID NO:6 and 7 in either orientation.
• SEQ ID NO: 19 is operably linked to SEQ ID NO: 17 or 18 by a linker of SEQ ID NO:3 or 15.
• SEQ ID NO: 17 or 18 is operably linked to SEQ 6 and 7, in either orientation by a linker of SEQ ID NO:5 or 16.
• the fusion protein can include in operable linkage a camelid or a human CD 16 NK cell engager domain (SEQ ID NO:2 or 19, respectively), a wt or a mutant IL-15 cytokine NK activating domain (SEQ ID NO:4, 17 or 18), and a light chain and a heavy chain of an of a B7- H3 targeting peptide (SEQ ID NO:6 and 7, respectively).
• the CD 16 NK cell engager domain can be linked to IL- 15 cytokine NK activating domain by a linker having an amino acid sequence set forth in SEQ ID NO:3 or 15.
• the IL-15 cytokine NK activating domain can be linked to the B7-H3 targeting peptide by a linker having an amino acid sequence set forth in SEQ ID NO:5 or 16.
• the IL-15 cytokine NK activating domain can 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).
• the fusion protein can include, in operable linkage, from anN-terminus to a 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.
• the fusion protein can include, in operable linkage, from a N-terminus to a 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.
• the fusion protein can include, in operable linkage, from a N- terminus to a 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, 3, 17, 5,
• the fusion protein further includes a half-life extending (HLE) molecule.
• HLE half-life extending
• the circulatory 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).
• the second general category of effector functions include those that operate after an immunoglobulin binds an antigen. In the case of IgG, these functions involve the participation of the complement cascade or Fc gamma receptor (FcyR)-bearing cells.
• Binding of the Fc region to an Fey R causes certain immune effects, for example, endocytosis of immune complexes, engulfinent and destruction of immunoglobulin- coated particles or microorganisms (also called antibodydependent 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, regulation of immune system cell activation, and regulation of immunoglobulin production.
• endocytosis of immune complexes also called antibodydependent phagocytosis, or ADCP
• ADCP antibodydependent phagocytosis
• ADCC antibody-dependent cell-mediated cytotoxicity
• Certain engineered binding polypeptides e.g., antibody variants (e.g., scFvs) or antibody fragments (e.g., Fab fragments)
• Fab fragments have short half- lives in vivo because they lack the Fc region that is required for FcRn binding and are rapidly filtered out of the blood by the kidneys owing to their small size.
• Engineered targeting polypeptides such as the fusion proteins described herein, can exhibit decreased binding to FcRn when compared to native binding polypeptides and, therefore, have decreased half-life in vivo.
• Fc variants with improved affinity for FcRn can have longer serum half-lives, and such molecules have useful applications in methods of treating mammals where long half-life of the administered polypeptide is desired, e.g., to treat a chronic disease or disorder.
• Fc variants with decreased FcRn binding affinity have shorter half-lives, and such molecules are also useful, for example, for administration to a mammal where a shortened circulation time may be advantageous, e.g., for in vivo diagnostic imaging or in situations where the starting polypeptide has toxic side effects when present in the circulation for prolonged periods.
• the fusion proteins described herein can include a half-life extending (HLE) molecule to extend their half-life in vivo upon administration to a subject.
• HLE half-life extending
• half-life refers to a biological half-life of a particular targeting polypeptide in vivo.
• Half-life may be represented by the time required for half the quantity administered to a subject to be cleared from the circulation and/or other tissues in the animal.
• the curve is usually biphasic with a rapid a-phase and longer [3-phase.
• the a-phase typically represents an equilibration of the administered targeting polypeptide between the intra- and extra-vascular space and is, in part, determined by the size of the polypeptide.
• the [3-phasc typically represents the catabolism of the targeting polypeptide in the intravascular space. Therefore, the term half-life as used herein preferably refers to the half-life of the targeting polypeptide in the [3- phase.
• the typical [3 phase half-life of a human antibody in humans is 21 days.
• An increased half-life is generally useful in in vivo applications of immunoglobulins, especially antibodies and most especially antibody fragments of small size.
• Approaches described in the art to achieve such effect comprise the fusion of the small bispecific antibody construct to larger proteins, which preferably do not interfere with the therapeutic effect of the protein construct.
• Examples for such further developments of bispecific T cell engagers are described in US 2017/0218078A1, which provides half-life extending formats (HLE formats) of bispecific T cell engaging molecules comprising a first domain binding to a target cell surface antigen, a second domain binding to an extracellular epitope of the human and/or the Macaca CD3e chain and a third domain, which is the specific Fc modality (the HLE molecule).
• half-life extending molecule As used herein, the terms “half-life extending 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.
• an HLE sequence generally includes a Fc region or scFc region of an immunoglobulin.
• Fc region refers to the portion of a native immunoglobulin formed by the respective Fc domains (or Fc moieties) of its two heavy chains.
• a native Fc region is homodimeric.
• scFc region single-chain Fc region
• scFc region refers to a synthetic Fc region comprised of Fc domains (or Fc moieties) genetically linked within a single polypeptide chain (i.e., encoded in a single contiguous genetic sequence). Accordingly, a genetically fused Fc region (i.e., a scFc region) is monomeric.
• Fc domain refers to the portion of a single immunoglobulin heavy chain beginning in the hinge region just upstream of the papain cleavage site (i.e., residue 216 in IgG, taking the first residue of heavy chain constant region to be 114) and ending at the C-terminus of the antibody. Accordingly, a complete Fc domain comprises at least a hinge domain, a CH2 domain, and a CH3 domain.
• the scFc region described herein includes at least two Fc domain which are genetically fused via a linker polypeptide (e.g., an Fc connecting peptide) interposed between said Fc moieties.
• the scFc region can include two identical Fc moieties or can include two non-identical Fc moieties.
• Non- limiting examples of Fc domain that can be used for the preparation of a HLE molecule (alone or in combination with another Fc domain through a linker polypeptide) that can be incorporated in any of the fusion proteins described herein include any of the polypeptides having an amino acid including any one of SEQ ID NOs:26-33.
• Non- limiting examples of linker polypeptide that can be used for the preparation of a scFc region that can be used for the preparation of a HLE molecule include any of the polypeptides having an amino acid including any one of SEQ ID NOs:34-35.
• the HLE molecules described herein can include a Fc domain having an amino acid including any one of SEQ ID NOs:26-33, or a scFc region including a first Fc domain having an amino acid comprising any one of SEQ ID NOs:26-33 fused to a second Fc domain having an amino acid comprising any one of SEQ ID NOs:26-33, through a linker having an amino acid including any one of SEQ ID NOs:34-35.
• the HLE molecule can include any one of SEQ ID NOs:21-25.
• the invention provides an isolated nucleic acid sequence encoding any of the fusion proteins described herein.
• the fusion proteins described herein such as the TriKE fusion proteins including a CD16 NK cell engager domain, such as the CD 16 domain having the amino acid sequence set forth in SEQ ID NO:2; a B7-H3 targeting fusion protein domain, such as the B7-H3 fusion protein having the amino acid sequences set forth in SEQ ID NOs:6 and 7; and a IL-15 cytokine NK activating domain, such as the IL- 15 having the amino acid sequence set forth in SEQ ID NO:4, in operable linkage, and as set forth in SEQ ID NO: 1
• the sequence is SEQ ID NO:8 or sequences having 90% or more sequence identity thereto.
• 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.
• subject refers to any individual or patient to which the subject methods are performed.
• the subject is human, although as will be appreciated by those in the art, the subject may be an animal.
• other animals including vertebrate such as rodents (including mice, rats, hamsters and guinea pigs), cats, dogs, rabbits, farm animals including cows, horses, goats, sheep, pigs, chickens, etc., and primates (including monkeys, chimpanzees, orangutans and gorillas) are included within the definition of subject.
• treatment is used interchangeably herein with the term “therapeutic method” and refers to both 1) therapeutic treatments or measures that cure, slow down, lessen symptoms of, and/or halt progression of a diagnosed pathologic conditions or disorder, and 2) and prophylactic/ preventative measures.
• Those in need of treatment may include individuals already having a particular medical disorder as well as those who may ultimately acquire the disorder (i.e., those needing preventive measures).
• terapéuticaally effective amount refers to that amount of the subject compound that will elicit the biological or medical response of a tissue, system, animal or human that is being sought by the researcher, veterinarian, medical doctor or other clinician. Generally, the response is either amelioration of symptoms in a patient or a desired biological outcome. Such amount should be sufficient to treat cancer.
• the effective amount can be determined as described herein.
• Administration routes can be enteral, topical or parenteral.
• administration routes include but are not limited to intracutaneous, subcutaneous, intravenous, intraperitoneal, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, transdermal, transtracheal, subcuticular, intraarticulare, subcapsular, subarachnoid, intraspinal and intrastemal, oral, sublingual buccal, rectal, vaginal, nasal ocular administrations, as well infusion, inhalation, and nebulization.
• parenteral administration and “administered parenterally” as used herein means modes of administration other than enteral and topical administration.
• the fusion proteins described herein can be formulated in pharmaceutical compositions comprising the fusion protein and a pharmaceutically acceptable carrier.
• pharmaceutically acceptable it is meant the carrier, diluent or excipient must be compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.
• carrier include, but are not limited to, liposome, nanoparticles, ointment, micelles, microsphere, microparticle, cream, emulsion, and gel.
• excipient examples include, but are not limited to, anti-adherents such as magnesium stearate, binders such as saccharides and their derivatives (sucrose, lactose, starches, cellulose, sugar alcohols and the like) protein like 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.
• diluent include, but are not limited to, water, alcohol, saline solution, glycol, mineral oil and dimethyl sulfoxide (DMSO).
• compositions can be administered in a variety of unit dosage forms depending upon 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.
• cancer refers to a group of diseases characterized by abnormal and uncontrolled cell proliferation starting at one site (primary site) with the potential to invade and to spread to other sites (secondary sites, metastases) which differentiates cancer (malignant tumor) from benign tumor. Virtually all the organs can be affected, leading to more than 100 types of cancer that can affect humans. Cancers can result from many causes including genetic predisposition, viral infection, exposure to ionizing radiation, exposure environmental pollutant, tobacco and/or alcohol use, obesity, poor diet, lack of physical activity or any combination thereof.
• neoplasm or “tumor” including grammatical variations thereof, means new and abnormal growth of tissue, which may be benign or cancerous.
• the neoplasm is indicative of a neoplastic disease or disorder, including but not limited, to various cancers.
• cancers can include prostate, pancreatic, biliary, colon, rectal, liver, kidney, lung, testicular, breast, ovarian, brain, and head and neck cancers, melanoma, sarcoma, multiple myeloma, leukemia, lymphoma, and the like.
• Exemplary cancers described by the national cancer institute include: Acute Lymphoblastic Leukemia, Adult; Acute Lymphoblastic Leukemia, Childhood; Acute Myeloid Leukemia, Adult; Adrenocortical Carcinoma; Adrenocortical Carcinoma, Childhood; AIDS- Related Lymphoma; AIDS-Related Malignancies; Anal Cancer; Astrocytoma, Childhood Cerebellar; Astrocytoma, Childhood Cerebral; Bile Duct Cancer, Extrahepatic; Bladder Cancer; Bladder Cancer, Childhood; Bone Cancer, Osteosarcoma/Malignant Fibrous Histiocytoma; Brain Stem Glioma, Childhood; Brain Tumor, Adult; Brain Tumor, Brain Stem Glioma, Childhood; Brain Tumor, Cerebellar Astrocytoma, Childhood; Brain Tumor, Cerebral Astrocytoma/Malignant Glioma, Childhood; Brain Tumor, Ependymo
• administration of the fusion proteins described herein can be in combination with one or more additional therapeutic agents.
• the phrases “combination therapy”, “combined with” and the like refer to the use of more than one medication or treatment simultaneously to increase the response.
• the fusion proteins of the present invention and the pharmaceutical composition thereof might for example be used in combination with other drugs or treatment in use to treat cancer.
• the administration of the fusion proteins to a subject can be in combination with a chemotherapeutic agent, surgery, radiotherapy, or a combination thereof.
• Such therapies can be administered prior to, simultaneously with, or following administration of the composition of the present invention.
• chemotherapeutic agent refers to any therapeutic agent used to treat cancer.
• 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, Tioguanine, Topotecan, Valrubicin, Vinblastine, Vincristine, Vindesine, Vinorelbine, panitumamab, Erbitux (cetuximab), matu
• immunotherapeutic agent examples include, but are not limited to, interleukins (11-2, 11-7, 11-12), cytokines (Interferons, G-CSF, imiquimod), chemokines (CCL3, CC126, CXCL7), immunomodulatory imide drugs (thalidomide and its analogues).
• the invention provides a pharmaceutical composition including a therapeutically effective amount of a fusion protein including the amino acid sequence of SEQ ID NO: 1 or a sequence having 90% or greater identity to SEQ ID NO:1 and a pharmaceutically acceptable carrier.
• the invention provides a method of treating cancer in a subject including administering to the subject the pharmaceutical composition described herein.
• Natural killer cells also known as NK cells or large granular lymphocytes (LGL) are a type of cytotoxic lymphocyte critical to the innate immune system that belong to the rapidly expanding family of known innate lymphoid cells (ILC) and represent 5-20% of all circulating lymphocytes in humans.
• the role of NK cells is analogous to that of cytotoxic T cells in the vertebrate adaptive immune response.
• NK cells provide rapid responses to virus- infected cell and other intracellular pathogens acting at around 3 days after infection and respond to tumor formation.
• immune cells detect the major histocompatibility complex (MHC) presented on infected cell surfaces, triggering cytokine release, causing the death of the infected cell by lysis or apoptosis.
• MHC major histocompatibility complex
• NK cells are unique, however, as they have the ability to recognize and kill stressed cells in the absence of antibodies and MHC, allowing for a much faster immune reaction. They were named "natural killers” because of the notion that they do not require activation to kill cells that are missing "self' markers of MHC class 1. This role is especially important because harmful cells that are missing MHC I markers cannot be detected and destroyed by other immune cells, such as T lymphocyte cells.
• NK cells 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 the sustaining of NK cell activity. NK cells also play a role in the adaptive immune response: numerous experiments have demonstrated their ability to readily adjust to the immediate environment and formulate antigen-specific immunological memory, fundamental for responding to secondary infections with the same antigen. The role of NK cells in both the innate and adaptive immune responses is becoming increasingly important in research using NK cell activity as a potential cancer therapy.
• the invention provides a method of inducing natural killer (NK) cell activity against a cancer cell in a subject including administering to the subject a fusion protein including the sequence set forth in SEQ ID NO:1 and sequences having 90% or greater identity to SEQ ID NO: 1, thereby inducing NK cell activity against a cancer cell in the subject.
• NK natural killer
• inducing NK cell activity includes inducing NK cells degranulation, inducing NK cell production of interferon y, increasing a number of tumor infiltrating NK cells in the subject, and/or inducing or increasing NK cell proliferation.
• Natural killer cells or large granular lymphocytes are a type of cytotoxic lymphocyte critical to the innate immune system that belong to the rapidly expanding family of known innate lymphoid cells (ILC) and represent 5-20% of all circulating lymphocytes in humans. They have different functions including: cytolytic granule mediated cell apoptosis, antibody-dependent cell-mediated cytotoxicity (ADCC) and cytokine-induced NK and cytotoxic T lymphocyte (CTL) activation.
• NK cells are cytotoxic; small granules in their cytoplasm contain proteins such as perforin and proteases known as granzymes. Upon release in close proximity to a cell slated for killing, perforin forms pores in the cell membrane of the target cell, creating an aqueous channel through which the granzymes and associated molecules can enter, inducing either apoptosis or osmotic cell lysis.
• apoptosis and cell lysis is important in immunology: lysing a virus-infected cell could potentially release the virions, whereas apoptosis leads to destruction of the virus inside, a-defensins, antimicrobial molecules, are also secreted by NK cells, and directly kill bacteria by disrupting their cell walls in a manner analogous to that of neutrophils.
• Infected cells are routinely opsonized with antibodies for detection by immune cells.
• Antibodies that bind to antigens can be recognized by FcyRIII (CD 16) receptors expressed on NK cells, resulting in NK activation, release of cytolytic granules and consequent cell apoptosis. This is a major killing mechanism of some monoclonal antibodies like rituximab (Rituxan), ofatumumab (Azzera), and others.
• Cytokines play a crucial role in NK cell activation. As these are stress molecules released by cells upon viral infection, they serve to signal to the NK cell the presence of viral pathogens in the affected area. 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 viral infections while the adaptive immune response generates antigen-specific cytotoxic T cells that can clear the infection. NK cells work to control viral infections by secreting IFNy and TNFa. IFNy activates macrophages for phagocytosis and lysis, and TNFa acts to promote direct NK tumor cell killing. Patients deficient in NK cells prove to be highly susceptible to early phases of herpes virus infection.
• NK cells have been reported to play a critical role in promoting drug-induced cell death in human triple-negative breast cancer. Since NK cells recognize target cells when they express non-self HLA antigens (but not self), autologous (patients' own) NK cell infusions have not shown any antitumor effects. Instead, investigators are working on using allogeneic cells from peripheral blood, which requires that all T cells be removed before infusion into the patients to remove the risk of graft versus host disease, which can be fatal. This can be achieved using an immunomagnetic column (CliniMACS). In addition, because of the limited number ofNK cells in blood (only 10% of lymphocytes are NK cells), their number needs to be expanded in culture. This can take a few weeks and the yield is donor dependent.
• the invention provides a method of inhibiting tumor growth in a subject including administering to the subject a fusion protein including the sequence set forth in SEQ ID NO: 1 and sequences having 90% or greater identity to SEQ ID NO: 1, thereby inhibiting tumor growth in the subject.
• inhibiting tumor growth includes decreasing tumor cell survival.
• the invention provides a method of increasing survival of a subject having cancer including administering to the subject a fusion protein including the sequence set forth in SEQ ID NO: 1 and sequences having 90% or greater identity to SEQ ID NO: 1, thereby increasing survival of the subject.
• survival of the subject is increased when the subject is administered the fusion protein of the invention, as compared to the survival in the absence of the administration, or upon administration of another treatment regimen that does not include the fusion protein of the invention.
• the invention provides a method of inducing natural killer (NK) mediated antibody-dependent cellular cytotoxicity against a cancer cell in a subject including administering to the subject a fusion protein including the sequence set forth in SEQ ID NO: 1 and sequences having 90% or greater identity to SEQ ID NO:1, thereby increasing survival of the subject.
• NK natural killer
• administering to a subject a fusion protein including the sequence set forth in SEQ ID NO: 1 and sequences having 90% or greater identity to SEQ ID NO: 1 further includes administering to the subject an anti-cancer treatment.
• the subject has cancer.
• the cancer is selected from the group consisting of lung cancer, prostate cancer, multiple myeloma, ovarian cancer and head and neck cancer.
• cancer cells are B7-H3 expressing cancer cells.
• the cancer is a treatment resistant cancer.
• Antigen-specific immunotherapies require overexpression of target antigens on tumor cells with minimal off-tumor expression on normal tissues. Ideally, the antigen displays high expression in a broad spectrum of cancers, making the immunotherapy applicable in a number of settings and basket clinical trials are becoming more popular if broad targets can be identified.
• B7-H3 a transmembrane costimulatory protein that is a member of the B7 family of checkpoint ligands, has gained interest as a target for immunotherapy. While it is involved both in the context of co-stimulation and inhibition by engaging receptors on T-cells, it has also been shown to contribute 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.
• a mouse model utilizing a B7-H3 -targeting CAR T construct that is reactive to mouse cells, has demonstrated anti-tumor responses in the absence of 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.
• Bispecific immune engagers such as blinatumomab have shown impressive clinical success.
• one of its single chain variable fragments (scFv) targets cancer cells and the other one targets CD3 on T cells. This creates an immune synapse between T cells and cancer cells, resulting in tumor killing.
• scFv single chain variable fragments
• activation and proliferation of T cells can result in cytokine release syndrome, disseminated intravascular coagulation, and nervous system events including encephalopathy and seizures.
• the present study aims at selectively engaging natural killer (NK) cells instead of T cells.
• NK cells are part of the innate immune system, play a major role in tumor surveillance, and have shown potential in a number of studies involving solid tumors and hematologic cancers. Due to these characteristics, a tri-specific killer engager (TriKE) platform consisting of a single chain variable fragment (scFv) targeting CD 16, the most potent activating receptor on NK cells, a scFv targeting a tumor associated antigen, and an IL- 15 moiety has been designed and described. The inventors have improved on this platform by adding a single domain antibody against CD 16, the result of which is better IL- 15 activity and overall function. IL- 15 is the most critical homeostatic cytokine for NK cell function.
• TriKE tri-specific killer engager
• NK cell expansion and survival it can amplify antibody-dependent cellular cytotoxicity (ADCC), it can induce lymphokine-activated killer activity, and it can enhance production of other co-stimulatory mediators like interferon gamma (IFNy) and tumor-necrosis factor alpha (TNFa).
• ADCC antibody-dependent cellular cytotoxicity
• IFNy interferon gamma
• TNFa tumor-necrosis factor alpha
• Described herein is a second-generation TriKE bioengineered with human IL- 15 as a modified crosslinker between a humanized camelid anti-CD16 VHH single domain antibody (sdAb) and an anti-B7-H3 scFv, termed caml615B7-H3.
• sdAb camelid anti-CD16 VHH single domain antibody
• caml615B7-H3 an anti-B7-H3 scFv
• Single-domain VHH antibodies derived from camelids are known to offer advantages over conventional VL-VH scFv fragments.
• the complementary determining regions (CDRs) from a camelid (llama) anti-CD16 were split into a universal, humanized, heavy chain scaffold. This humanized camelid sequence was used to manufacture caml615B7- H3.
• a hybrid gene encoding caml615B7-H3 was synthesized using DNA shuffling and DNA ligation techniques.
• the fully assembled gene (from 5' end to 3' end) encoded a Ncol restriction site; an ATG start codon; anti-human CD 16 VHH; a 20 amino acid (aa) segment, PSGQAGAAASESLFVSNHAY (SEQ ID NO:36); human wild-type IL- 15; the seven amino acid linker, EASGGPE (SEQ ID NO:37); anti-B7-H3 mAb 376.96 scFv; and aXhoI 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.
• IPTG isopropyl-D-thiogalactopyranoside
• Escherichia coli strain BL21 (DE3) (Novagen, Madison, WI, USA) was used for the expression of proteins after plasmid transfection. Bacterial expression resulted in the sequestering of target protein into inclusion bodies (IBs). Bacteria were cultured overnight in 800 mL Luria broth containing kanamycin (30 mg/mL). When absorbance reached 0.65 at 600 nm, gene expression was induced with Isopropyl [3-D-l-thiogalactopyranoside/IPTG (FischerBiotech, Fair Lawn, NJ, USA). Bacteria were harvested after 2 h.
• IBs inclusion bodies
• 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% glycerin, 50 rmnol/L Tris, 50 mmol/L NaCl, and 5 mmol/L EDTA (pH 8.0). The extract was washed 3 times.
• Removal of SLS was performed 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 HC1 (13.3 M) was added to the solution which was incubated at 37 °C for 2 to 3 h. The solution was diluted 20-fold with refolding buffer, 50 mM Tris, 0.5 M 1-arginine, 1 M Urea, 20% glycerol, 5 mM EDTA, pH 8.0.
• the mixture was refolded at 4 °C for two days and then dialyzed against five volumes of 20 mM Tris-HCl at pH 8.0 for 48 h at 4 °C, then eight volumes for 18 additional hours.
• the product was then purified over a fast flow Q ion exchange column and further purified by passage over a size exclusion column (Superdex 200, GE, Marlborough, MA, USA). Protein purity was determined with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) stained with Simply Blue Safe Stain (Invitrogen, Carlsbad, CA, USA).
• FIGURE 1A shows a schematic of the B7-H3 TriKE construct.
• B7-H3 TriKE includes single chain variable fragments from camelid nanobodies (cam) targeting CD 16 and B7-H3 joined by IL- 15 and two flexible linker regions to form a single peptide with molecular weight of ⁇ 46 kDa.
• FIGURE IB shows the absorbance tracing from the FFQ ion exchange column as the first phase of the purification with the eluant collected in 8-mL aliquots shown on the abscissa of the graph.
• the double-sided arrow shows the collection peak as drug exits the column.
• FIGURE 1C shows the absorbance tracing from the second purification phase, size exclusion chromatography (SEC).
• SEC size exclusion chromatography
• the wild-type IL-15 moiety in the caml615B7-H3 TriKE was designed to induce targeted delivery of a proliferative signal to NK cells.
• proliferation assays evaluating dilution of a CellTrace dye over a 7-day period were carried out on PBMCs treated with no treatment (NT), monomeric rhIL-15 (IL15), or the TriKE (caml615B7-H3). At the end of the seven days, cells were harvested and proliferation was evaluated by gating on CD56+CD3- cells.
• the camB7-H3 TriKE of the invention has demonstrated a strong binding specificity against WT B7-H3.
• BT- 12 pediatric brain tumor lines highly express B7-H3 (WT).
• a B7-H3 KO BT-12 cell line was produced using CRISPR (Theruvath et al). Similar specificity was noted using Raji (negative B7-H3) and prostate cancer cell lines C4-2 (positive B7-H3) and multiple other lines.
• B7-H3 BiKE had similar binding with positive and negative cell lines (Data not shown).
• NK-92 cells without or with CD 16 were incubated for 48 hours with dilutions of
• MA- 148 (established locally at the University of Minnesota) is a human epithelial high-grade serous ovarian carcinoma cell line.
• lines were transfected with a luciferase reporter construct using Invitrogen’s Lipofectamine Reagent and selective pressure applied with 10 pg/mL of blasticidin.
• Ovarian carcinoma 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).
• PBMCs Peripheral blood mononuclear cells
• IRB institutional review board
• Ovarian cancer specimens were collected in women diagnosed with advanced-stage ovarian or primary peritoneal carcinoma at time of primary debulking surgery.
• prostate cancer blood was obtained from two patients with metastatic castration resistant prostate cancer and one patient with metastatic hormone sensitive prostate cancer.
• lung cancer blood was obtained from seven unresectable lung cancer patients at the time of diagnosis, prior treatment. Cells were pelleted, lysed for red blood cells, cryopreserved in 10% DMSO/90% FBS, and stored in liquid nitrogen.
• PBMCs from healthy donors were labeled with CellTrace Violet Proliferation Dye (Invitrogen, Carlsbad, CA, USA) according to kit specifications. After staining, cells were cultured with TriKEs at noted concentrations, or equimolar concentrations of controls, and incubated in a humidified atmosphere containing 5% CO2 at 37 °C for seven days.
• CellTrace Violet Proliferation Dye Invitrogen, Carlsbad, CA, USA
• ADCC was measured in a flow cytometry assay by evaluating degranulation via CD 107a (lysosomal-associated membrane protein LAMP- 1) and intracellular IFNy production.
• CD 107a lysosomal-associated membrane protein LAMP- 1
• RPMI 1640 media supplemented with 10% fetal calf serum (RPML10).
• RPML10 fetal calf serum
• Fluorescein isothiocyate (FITC)-conjugated anti-human CD 107a monoclonal antibody (BD Biosciences, San Jose, CA, USA) was then added. Following an hour 37 °C incubation, GolgiStop (1 :1500, BD Biosciences) and GolgiPlug (1 :1000, BD Biosciences) were added for 3 h. After washing with phosphate buffered saline, the 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).
• FITC Fluorescein isothiocyate
• Tumor killing was evaluated in real-time using the IncuCyte platform. Magnetic- bead-enriched CD3-CD56+ NK effector cells were plated into 96-well flat clear-bottom polystyrene tissue-culture-treated microplates (Coming, Flintshire, UK) along with NuclightRed stably expressing OVCAR8 cells at a 2: 1 effectortarget ratio. Caspase-3/7 green dye (Sartorious, Ann Arbor, MI, USA) was added to pick up dying cells that have not yet lost NuclightRed fluorescence.
• PBMCs were incubated alone or with OVCAR8s at a 2: 1 ratio +/- caml615B7-H3 (30 nM) for 24 h. After harvesting samples, cells were counted, and viability was measured using trypan blue exclusion. Two hundred thousand cells from each donor were aliquoted into 5-mL polystyrene U-bottom tubes for barcoding and CyTOF staining. Cells were stained with Cisplatin (Fluidigm Product# 201064, San Francisco, CA, USA), followed by barcoding using the Cell-ID 20-Plex Pd Barcoding Kit (Fluidigm Product# 201060).
• MA- 148-Luc ovarian cancer cells were incorporated into a previously described NK cell xenogeneic mouse model system.
• PBMC peripheral blood mononuclear cell
• CD19 CD 19 depleted
• MA-148-luc cells are a subline of MA- 148 that have been transfected with a luciferase reporter gene, allowing for imaging of the mice each week to determine their bioluminescent activity and to monitor tumor progression. Briefly, mice were injected with 100 pL of 30 mg/mL luciferin substrate 10 min prior to imaging and then anesthetized via inhalation of isoflurane gas (25). Mice were then imaged using the Xenogen Ivis 100 imaging system and analyzed with Living Image 2.5 software (Xenogen Corporation, Alameda, CA, USA).
• GraphPad PRISM 8 (GraphPad Prism Software, Inc., San Diego, CA, USA) was used to create all statistical tests. For all in vitro studies, one-way ANOVA with repeated measures was used to calculate significance in comparisons to the caml615B7-H3 group. For mouse studies, two-way ANOVA was used to calculate significance in the longitudinal study, while one-way ANOVA was used to calculate the significance in differences in radiance at the day-21 timepoint. An unpaired t test was used to evaluate differences in cell counts and MFI. Bars represent mean ⁇ SEM. Statistical significance is displayed as * p ⁇ 0.05, ** p ⁇ 0.01, *** p ⁇ 0.001, and **** p ⁇ 0.0001.
• B7-H3 (CD276) expression on myeloma is associated with decreased progression free survival, it exhibits low expression on healthy tissue, and it is expressed on myeloid derived suppressor cells (MDSC), which promote myeloma growth.
• MDSC myeloid derived suppressor cells
• H929 cells were more potently killed in the presence of B7-H3-TriKE at E:T of 2:1 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).
• MDSC were developed from CD33+ myeloid cells from healthy donors using IL-6 and GM-CSF or by incubating them with myeloma cells at 1 : 100 ratio for seven days.
• MDSC CD14+CD1 lb+
• B7-H3 FIG. 10A
• MDSC were also isolated from the bone marrow aspirates of three newly diagnosed myeloma patients and exhibited 56-95 survival (aspirates were processed with lysis buffer and stained for CD 14, CDl lb, and B7-H3. Shown is a flow cytometry plot of live, CD14+ cells (FIGURE 10C).
• MDSC were incubated with myeloma cells and growth was measured over 48 hours by live cell imaging (FIGURE 10B). Addition of MDSC to cytotoxicity assays enhanced myeloma cell growth 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 relatively low B7-H3-e0xpressing H929 line. This also shows it can reverse MDSC-induced myeloma growth.
• caml615B7-H3 TriKE targets prostate cancer.
• the ability of the caml615B7-H3 TriKE to improve NK cell activity against prostate cancer was tested. All of the prostate cancer cell lines tested expressed B7-H3. For these tests, normal donor PBMCs and PBMCs obtained from metastatic prostate cancer patients were used.
• the data indicates that the caml615B7-H3 TriKE has promise in NK cell immunotherapy within the prostate cancer setting and shows that the NK cell function can be rescued on patients who require novel interventions due to poor outcomes with current therapeutic approaches.
• the signal induced by the TriKE with prostate cancer cells was stronger than that induced by a strong natural cytotoxicity signal and was specific to B7-H3.
• caml615B7-H3 TriKE were more potent at inducing NK function than IL- 15 alone and were also more potent at inducing NK cell proliferation, as compared to IL- 15 alone.
• tumor killing of PC-3 cells was evaluated in real-time using the IncuCyte platform, which emphasized the enhanced efficacy of the B7-H3 TriKE molecules to induce prostate cancer cell death.
• the B7-H3 TriKE molecules were also able to reduce PC-3 spheroid size over time.
• camB7-H3 TriKE were found to induce activity against prostate cancer cells over a broader dynamic range than previous scFv version, as evaluated by measuring the percent of CD107a+ and IFNy+ NK cells.
• caml615B7-H3 TriKE targets lung cancer.
• the ability of caml615B7-H3 TriKE to improve NK cell activity against B7-H3-expressing lung cancer was tested on normal donor PBMCs incubated with A549 and NCI-H322, two nonsmall cell lung cancer adenocarcinoma lines (FIGURES 24A-24D).
• FIGURES 24A-24D two nonsmall cell lung cancer adenocarcinoma lines
• the caml615B7-H3 TriKE significantly and robustly improved NK cell activation when compared to controls.
• Individual caml6 VHH or anti-B7-H3 scFv components were tested and showed no background NK cell activity against A549s.
• HNSCC Head and Neck Squamous Cell Carcinomas
• B7-H3 a checkpoint member of the B7 and CD28 families, is overexpressed on several solid tumors but is absent or not expressed on healthy tissues. It is a promising target for immunotherapy, and recent basket trials, particularly in prostate cancer, have demonstrated strong clinical signals.
• TriKE tri-specific killer engager
• This TriKE molecule includes an NK cell engaging domain containing a humanized camelid nanobody against CD 16, a camelid nanobody against B7-H3 and a wild type IL- 15 sequence between the two engagers.
• B7-H3 expression was assessed by flow cytometry on wild-type HNSCC cells and a paired version with a CRISPER KO of the FANCA gene and it was determined that the KO had no effect on B7-H3 expression.
• the TriKE activity against HNSCC should be present on both normal HNSCC and FA- HNSCC settings.
• NK cell responses against HNSCC lines in the presence of the B7-H3 TriKE were assessed through either flow cytometry based functional assays, to evaluate NK cell degranulation and cytokine secretion, or IncuCyte imaging assays, to directly assess target killing.
• NK cell degranulation and IFN-gamma production of B7-H3 TriKE-treated samples were higher compared to that of 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 the B7-H3 single domain or IL- 15 alone irrespective of the FANCA gene, both in 2D and 3D IncuCyte imaging assays.
• HNSCC cell lines UM-SCC-01, SFCI-SCC-07, JHU-SCC-FaDu, Cal27 and Cal33 to evaluate CD 107a expression (as a marker for degranulation) and intracellular IFN-y production.
• HNSCC cell lines did not induce NK cell cytolytic function without treatment.
• FIGURES 26A-26B 5 HNSCC cell lines were assessed for B7-H3 expression and binding affinity with B7-H3 single domain via flow cytometry and PBMCs from a healthy donor were assessed for B7-H3 expression by flow cytometry.
• B7-H3 is highly expressed on HNSCC but not on healthy immune cells.
• FIGURE 27A-27D B7-H3 TriKE induced NK cell activity against HNSCC.
• Error bars indicate standard error of mean, and statistical significance was determined as *p ⁇ .05, **p ⁇ .01, ***p ⁇ .001 and ****p ⁇ .0001
• B7-H3 TriKE induced NK cell killing against HNSCC in real-time imaging assays.
• caml 615B7-H3 TriKE exhibits Potent killing of ovarian cancer.
• the ability of caml615B7-H3 TriKE to mediate NK cell activity against ovarian cancer cells was evaluated.
• Ovarian cancer cells used displayed robust B7-H3 expression. Since B7-H3 has been shown to have a role in immune responses, the capacity of the caml615B7-H3 to induce activity against normal immune cells was evaluated in PBMCs.
• Flow cytometric assays allowing for gating on NK cells, determined that the caml615B7-H3 induced some background degranulation (CD107a) onNK cells in comparison to controls, but this activity was low.
• NK cell activity from normal donor PBMCs and ascites from the peritoneal cavity of ovarian cancer patients at the time of surgery, was assessed against MA- 148 cells, another high-grade serous ovarian adenocarcinoma cell line (FIGURES 29A-29B and 30G-30H).
• MA- 148 cells another high-grade serous ovarian adenocarcinoma cell line
• the caml615B7-H3 TriKE induced robust activity on normal donor NK cells. While NK cell activity from ovarian-cancer-derived ascites samples was decreased, as expected due to alterations in NK cell function driven by the tumor microenvironment and decreases in CD16 expression, the caml615B7-H3 TriKE induced significantly enhanced NK cell degranulation compared to controls.
• ovarian cancer tumor cells (OVCAR8s) was measured dynamically over a two-day period in the presence of enriched NK cells alone (No Treatment), NK cells and rhIL-15 (IL15), and NK cells and the caml615B7-H3 TriKE (FIGURE 301).
• 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 Caspase3/7 dye.
• the basic readout provided is the number of tumor cells alive (Red+Green-) normalized to tumor alone at the noted times.
• caml 615B7-H3 TriKE induced robust and rapid tumor killing when compared to controls.
• This data indicates that the caml615B7-H3 TriKE potently enhances activity against ovarian cancer cells in vitro.
• the caml615B7-H3 TriKE induced similar degranulation and stronger IFNy production against ovarian cancer when compared to a potent natural cytotoxicity signal, in the absence of TriKE, induced by K562 cells.
• FIGURE 31 high dimensional analysis of caml615B7-H3 TriKE activated cells was performed.
• a custom, 42 parameter, CyTOF (mass cytometry) NK cell targeted panel was used.
• PBMCs were left untreated, incubated with caml615B7-H3 TriKE for 24 h, incubated with tumor (OVCAR8s) for 24 h, or incubated with tumor and caml615B7- H3 TriKE for 24 h. Cells were then stained, fixed, and run on a CyTOF2.
• caml615B7-H3 TriKE mediates anti -tumor activity in vivo. Determination of in vivo activity is a critical step for translation. However, prior to evaluating the ability of the caml615B7-H3 TriKE to induce function against tumor the potential for toxicity was assessed. To do this NSG mice were irradiated, engrafted with 1 million NK cells, treated with nothing, IL- 15, or caml615B7-H3 for three weeks, and weights were tracked over the course of 90 days post initial treatment. Despite an initial drop in weight in all groups, likely due to the irradiation, no significant differences were seen in the TriKE treated group vs. the controls.
• B7-H3 displays these characteristics: it has high expression in a number of tumors and low expression in normal tissues.
• Targeted antibody-based therapies for B7-H3 are 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 thus far have been favorable. Radiolabeled antibodies targeting B7-H3 have been safely administered for at least 10 years.
• B7-H3 reportedly is expressed on vasculature and stroma fibroblasts, indicating that this antigen could be used to target the tumor vasculature and architecture.
• PDAC pancreatic ductal adenocarcinoma
• caml615B7-H3 TriKE delivers a specific IL- 15 signal to the NK cells, preventing off target toxi cities, and also mediates ADCC against a variety of adenocarcinoma cell lines in the ovarian, prostate, and lung cancer settings. This dual mechanism of action allows for enhanced NK cell proliferation, survival, and targeted activation.
• NK cell exhaustion either mediated by IL- 15 or through strong NK cell activation, could be operant.
• TriKEs are dependent on targeting CD 16 for activation and can be cleaved by the metalloproteinase ADAM 17.
• ADAM 17 metalloproteinase
• TriKEs overcome non-specific mechanisms of natural cytotoxicity by promoting an antigen-specific synapse 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 a number of solid tumor malignancies.
• B7-H3 is a robust target for TriKE molecules, selectively boosting NK-cell in vitro killing of ovarian cancer, prostate cancer, and lung cancer.
• the IL- 15 action is remarkably specific to NK-cell activity with little off-target effects on T cells. This provides the first in vivo xenograft data, supporting the notion that TriKEs can work against solid tumors and supports their future clinical development.

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