B7H3 target protein and method of use thereof

Abstract

To provide multispecific B7H3 target compounds and methods for using them. [Solution] A multispecific B7H3 target compound and a method of using the same are provided. The multispecific compound includes a bispecific target protein comprising a B7H3 target domain and an immune cell binding domain; and a triplicate target protein comprising a B7H3 target domain, an immune cell binding domain and an immune cell activation domain. The method of use includes a method of inducing NK-mediated cell death, a method of inducing NK expansion in vivo, and a method of treating cancer.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefit of priority under 35 U.S. SC § 119(e) of U.S. Provisional Patent Application No. 63 / 033,989, filed on 3 June 2020 (which is incorporated by reference in its entirety).

[0002] Sequence List This application includes a sequence listing submitted electronically via EFS-Web to the United States Patent and Trademark Office on June 2, 2021, as an ASCII text file named "0110-000661WO01_ST25.txt" with a size of 40 kilobytes. The information contained in the sequence listing is incorporated herein by reference. [Overview of the project] [Means for solving the problem]

[0003] In one embodiment, the present disclosure describes an anti-B7H3 protein comprising at least one of SEQ ID NOs: 4, 5, or 6, or a functional variant thereof.

[0004] In another embodiment, the disclosure describes anti-B7H3 proteins comprising SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, the CDR region of SEQ ID NO: 1, the CDR region of SEQ ID NO: 2, the CDR region of SEQ ID NO: 3, or functional variants thereof.

[0005] In another aspect, this disclosure describes multispecific compounds. Generally, multispecific compounds include a target domain and an immune cell binding domain operably linked to the target domain. The target domain includes an anti-B7H3 protein.

[0006] In some embodiments, the immune cells are T cells or natural killer (NK) cells. In embodiments where the immune cells are NK cells, the immune cell-binding domain may include a ligand or antibody that specifically binds to CD16. In embodiments where the immune cell-binding domain includes an antibody that specifically binds to CD16, the antibody may be scFv, F(ab)2, Fab, or a single-domain antibody (sdAb).

[0007] In some embodiments, the immune cell binding domain may include SEQ ID NO: 19. In some embodiments, the target domain may include SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. In some embodiments, the immune cell binding domain may include SEQ ID NO: 19, and the target domain may include SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

[0008] In some embodiments, the immune cell binding domain and the target domain are linked by a linker having one of the amino acid sequences of SEQ ID NOs: 12-18. In some of these embodiments, the immune cell binding domain and the target domain are linked by a linker having the amino acid sequence of SEQ ID NO: 14.

[0009] In some embodiments, the anti-B7H3 multispecific compound comprises amino acids 19-294 of SEQ ID NO: 20 or amino acids 19-284 of SEQ ID NO: 21.

[0010] In some embodiments, the anti-B7H3 multispecific compound may be a triplespecific compound further comprising an immune cell activation domain. In embodiments where the immune cells are NK cells, the immune cell activation domain comprises a cytokine or a functional portion thereof. In some of these embodiments, the cytokine is IL-15 or a functional variant thereof.

[0011] In some embodiments, the triple-specific compound may comprise the amino acids of SEQ ID NO: 19 as an immune cell binding domain, the amino acids of SEQ ID NO: 19 as an immune cell activation domain, and one of the amino acids of SEQ ID NOs from 1 to 3 as a target domain. The functional domain may be linked by one or a combination of two or more linkers shown in SEQ ID NOs from 12 to 18. In some of these embodiments, the immune cell binding domain may be linked to the immune cell activation domain by a linker having the amino acid sequence of SEQ ID NO: 14. In some embodiments, the immune cell activation domain and the target domain may be linked by a linker having the amino acid sequence of SEQ ID NO: 15.

[0012] In some embodiments, the anti-B7H3 trispecific compound may contain the amino acid sequence of SEQ ID NO: 22 or SEQ ID NO: 23.

[0013] In some embodiments, functional variants of IL-15 include the amino acid substitution N72D or N72A compared to SEQ ID NO: 11.

[0014] In another aspect, this disclosure describes isolated nucleic acid sequences encoding any embodiment of the anti-B7H3 multispecific compounds described herein.

[0015] In another embodiment, the disclosure describes a host cell comprising any embodiment of the isolated nucleic acid outlined above. In some embodiments, the host cell is a T cell, an NK cell, or a macrophage.

[0016] In another embodiment, the disclosure describes a pharmaceutical composition comprising an anti-B7H3 multispecific compound and a pharmaceutically acceptable carrier.

[0017] In another aspect, the present disclosure generally describes a method comprising administering to a subject an effective amount of an anti-B7H3 multispecific compound to induce natural killer (NK)-mediated killing of cells. The anti-B7H3 multispecific compound comprises a target domain comprising an anti-B7H3 protein and an NK-binding domain operably linked to the target domain.

[0018] In another aspect, the present disclosure describes a method for stimulating the expansion of natural killer (NK) cells in vivo. Generally, the method comprises administering to a subject an effective amount of an anti-B7H3 multispecific compound. The anti-B7H3 multispecific compound comprises a target domain comprising an anti-B7H3 protein and an NK-binding domain operably linked to the target domain.

[0019] In another aspect, the present disclosure describes a method of treating a subject having or at risk of having cancer. Generally, the method comprises administering to a subject an effective amount of an anti-B7H3 multispecific compound. The anti-B7H3 multispecific compound comprises a target domain comprising an anti-B7H3 polypeptide and an NK-binding domain operably linked to the target domain. In some embodiments, the cancer cells express B7H3.

[0020] In another aspect, the present disclosure describes a chimeric antigen receptor compound comprising an anti-B7H3 polypeptide.

[0021] In another aspect, the present disclosure describes a targeted therapy compound comprising a target domain and a therapeutic domain linked to the target domain. The target domain comprises an anti-B7H3 polypeptide. In some embodiments, the targeted therapeutic agent provides a targeted immunotherapy. In some embodiments, the therapeutic domain comprises an agent, a therapeutic radioisotope, a toxin, a cytokine, or a chemokine.

[0022] In another aspect, the present disclosure describes a targeted imaging compound comprising a target domain and an imaging domain linked to the target domain. The target domain comprises an anti-B7H3 polypeptide. In some embodiments, the imaging domain comprises a colorimetric label, a fluorescent label, a radioactive label, a magnetic label, or an enzyme label.

[0023] In another aspect, the present disclosure describes a capture assay device comprising an anti-B7H3 polypeptide immobilized on a substrate.

[0024] The patent or application file contains at least one drawing created in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fees.

Brief Description of the Drawings

[0025] [Figure 1A] A schematic diagram of a trispecific killer engager incorporating an exemplary embodiment of an anti-B7H3 protein is illustrated. Figure 1A illustrates a schematic diagram of an exemplary anti-B7H3 protein sequence (e.g., sdAb) incorporated into a trispecific killer engager backbone that also includes a recombinant human IL-15 and an anti-CD16 single domain antibody (sdAb) arm, all linked via short linkers. Figure 1B illustrates the amino acid sequence of the novel anti-B7H3 protein. [Figure 1B] A schematic diagram of a trispecific killer engager incorporating an exemplary embodiment of an anti-B7H3 protein is illustrated. Figure 1A illustrates a schematic diagram of an exemplary anti-B7H3 protein sequence (e.g., sdAb) incorporated into a trispecific killer engager backbone that also includes a recombinant human IL-15 and an anti-CD16 single domain antibody (sdAb) arm, all linked via short linkers. Figure 1B illustrates the amino acid sequence of the novel anti-B7H3 protein. [Figure 2A-2B]The degranulation of NK cells and the production of interferon-gamma (IFN-γ) as measured by flow cytometry are illustrated. Figure 2A is a bar graph illustrating NK cell degranulation (%CD107a+) when a triple-specific killer promoter (30 nM) was incubated with peripheral blood mononuclear cells (PBMCs) alone. Figure 2B is a bar graph illustrating NK cell degranulation when a triple-specific killer promoter (30 nM) was co-cultured with PBMCs and prostate cancer cells (PC3). Figure 2C is a bar graph illustrating NK cell degranulation when a triple-specific killer promoter (30 nM) was co-cultured with PBMCs and prostate cancer cells (DU145). Figure 2D is a bar graph illustrating IFN-γ production when a triple-specific killer promoter (30 nM) was incubated with PBMs alone. Figure 2E is a bar graph illustrating IFN-γ production when a triple-specific killer engager (30 nM) is co-cultured with PBMCs and PC3 cells. Figure 2F is a bar graph illustrating IFN-γ production when a triple-specific killer engager (30 nM) is co-cultured with PBMCs and DU145 cells. [Figure 2C-2D]The degranulation of NK cells and the production of interferon-gamma (IFN-γ) as measured by flow cytometry are illustrated. Figure 2A is a bar graph illustrating NK cell degranulation (%CD107a+) when a triple-specific killer promoter (30 nM) was incubated with peripheral blood mononuclear cells (PBMCs) alone. Figure 2B is a bar graph illustrating NK cell degranulation when a triple-specific killer promoter (30 nM) was co-cultured with PBMCs and prostate cancer cells (PC3). Figure 2C is a bar graph illustrating NK cell degranulation when a triple-specific killer promoter (30 nM) was co-cultured with PBMCs and prostate cancer cells (DU145). Figure 2D is a bar graph illustrating IFN-γ production when a triple-specific killer promoter (30 nM) was incubated with PBMs alone. Figure 2E is a bar graph illustrating IFN-γ production when a triple-specific killer engager (30 nM) is co-cultured with PBMCs and PC3 cells. Figure 2F is a bar graph illustrating IFN-γ production when a triple-specific killer engager (30 nM) is co-cultured with PBMCs and DU145 cells. [Figure 2E-2F]The degranulation of NK cells and the production of interferon-gamma (IFN-γ) as measured by flow cytometry are illustrated. Figure 2A is a bar graph illustrating NK cell degranulation (%CD107a+) when a triple-specific killer promoter (30 nM) was incubated with peripheral blood mononuclear cells (PBMCs) alone. Figure 2B is a bar graph illustrating NK cell degranulation when a triple-specific killer promoter (30 nM) was co-cultured with PBMCs and prostate cancer cells (PC3). Figure 2C is a bar graph illustrating NK cell degranulation when a triple-specific killer promoter (30 nM) was co-cultured with PBMCs and prostate cancer cells (DU145). Figure 2D is a bar graph illustrating IFN-γ production when a triple-specific killer promoter (30 nM) was incubated with PBMs alone. Figure 2E is a bar graph illustrating IFN-γ production when a triple-specific killer engager (30 nM) is co-cultured with PBMCs and PC3 cells. Figure 2F is a bar graph illustrating IFN-γ production when a triple-specific killer engager (30 nM) is co-cultured with PBMCs and DU145 cells. [Figure 3A-3B] The degranulation of NK cells and the production of IFN-γ, as measured by flow cytometry, are illustrated. Figure 3A is a bar graph illustrating the degranulation (%CD107a+) of NK cells when a triple-specific killer promoter (30 nM) was co-cultured with PBMCs and prostate cancer cells (LnCAP). Figure 3B is a bar graph illustrating the degranulation of NK cells when a triple-specific killer promoter (30 nM) was co-cultured with PBMCs and prostate cancer cells (C4-2). Figure 3C is a bar graph illustrating the production of IFN-γ when a triple-specific killer promoter (30 nM) was co-cultured with PBMCs and LnCAP cells. Figure 3D is a bar graph illustrating the production of IFN-γ when a triple-specific killer promoter (30 nM) was co-cultured with PBMCs and C4-2 cells. [Figure 3C-3D]The degranulation of NK cells and the production of IFN-γ, as measured by flow cytometry, are illustrated. Figure 3A is a bar graph illustrating the degranulation (%CD107a+) of NK cells when a triple-specific killer promoter (30 nM) was co-cultured with PBMCs and prostate cancer cells (LnCAP). Figure 3B is a bar graph illustrating the degranulation of NK cells when a triple-specific killer promoter (30 nM) was co-cultured with PBMCs and prostate cancer cells (C4-2). Figure 3C is a bar graph illustrating the production of IFN-γ when a triple-specific killer promoter (30 nM) was co-cultured with PBMCs and LnCAP cells. Figure 3D is a bar graph illustrating the production of IFN-γ when a triple-specific killer promoter (30 nM) was co-cultured with PBMCs and C4-2 cells. [Figure 4A-4B] The degranulation of NK cells and the production of IFN-γ, as measured by flow cytometry, are illustrated. Figure 4A is a bar graph illustrating the degranulation of NK cells when the triple-specific killer engager (30 nM) was incubated with PBMCs alone. Figure 4B is a bar graph illustrating the degranulation of NK cells when the triple-specific killer engager (30 nM) was co-cultured with PBMCs and C4-2 cells. Figure 4C is a bar graph illustrating the degranulation of NK cells when the triple-specific killer engager (30 nM) was co-cultured with PBMCs and lung cancer cells (A549). Figure 4D is a bar graph illustrating the production of IFN-γ when the triple-specific killer engager (30 nM) was incubated with PBMCs alone. Figure 4E is a bar graph illustrating IFN-γ production when a triple-specific killer engager (30 nM) is co-cultured with PBMCs and C4-2 cells. Figure 4F is a bar graph illustrating IFN-γ production when a triple-specific killer engager (30 nM) is co-cultured with PBMCs and A549 cells. [Figure 4C-4D]The degranulation of NK cells and the production of IFN-γ, as measured by flow cytometry, are illustrated. Figure 4A is a bar graph illustrating the degranulation of NK cells when the triple-specific killer engager (30 nM) was incubated with PBMCs alone. Figure 4B is a bar graph illustrating the degranulation of NK cells when the triple-specific killer engager (30 nM) was co-cultured with PBMCs and C4-2 cells. Figure 4C is a bar graph illustrating the degranulation of NK cells when the triple-specific killer engager (30 nM) was co-cultured with PBMCs and lung cancer cells (A549). Figure 4D is a bar graph illustrating the production of IFN-γ when the triple-specific killer engager (30 nM) was incubated with PBMCs alone. Figure 4E is a bar graph illustrating IFN-γ production when a triple-specific killer engager (30 nM) is co-cultured with PBMCs and C4-2 cells. Figure 4F is a bar graph illustrating IFN-γ production when a triple-specific killer engager (30 nM) is co-cultured with PBMCs and A549 cells. [Figure 4E-4F] The degranulation of NK cells and the production of IFN-γ, as measured by flow cytometry, are illustrated. Figure 4A is a bar graph illustrating the degranulation of NK cells when the triple-specific killer engager (30 nM) was incubated with PBMCs alone. Figure 4B is a bar graph illustrating the degranulation of NK cells when the triple-specific killer engager (30 nM) was co-cultured with PBMCs and C4-2 cells. Figure 4C is a bar graph illustrating the degranulation of NK cells when the triple-specific killer engager (30 nM) was co-cultured with PBMCs and lung cancer cells (A549). Figure 4D is a bar graph illustrating the production of IFN-γ when the triple-specific killer engager (30 nM) was incubated with PBMCs alone. Figure 4E is a bar graph illustrating IFN-γ production when a triple-specific killer engager (30 nM) is co-cultured with PBMCs and C4-2 cells. Figure 4F is a bar graph illustrating IFN-γ production when a triple-specific killer engager (30 nM) is co-cultured with PBMCs and A549 cells. [Figure 5A-5B] The degranulation of NK cells and the production of IFN-γ, as measured by flow cytometry, are illustrated. Figure 5A is a bar graph illustrating the degranulation of NK cells when a triple-specific killer promoter (30 nM) was co-cultured with PBMCs and lung cancer cells (NCI-H460s). Figure 5B is a bar graph illustrating the degranulation of NK cells when a triple-specific killer promoter (30 nM) was co-cultured with PBMCs and ovarian cancer cells (OVCAR8). Figure 5C is a bar graph illustrating the degranulation of NK cells when a triple-specific killer promoter (30 nM) was co-cultured with PBMCs and ovarian cancer cells (MA148). Figure 5D is a bar graph illustrating the production of IFN-γ when a triple-specific killer promoter (30 nM) was co-cultured with PBMCs and NCI-H460s cells. Figure 5E is a bar graph illustrating IFN-γ production when the triple-specific killer engager (30 nM) is co-cultured with PBMC and OVCAR8 cells. Figure 5F is a bar graph illustrating IFN-γ production when the triple-specific killer engager (30 nM) is co-cultured with PBMC and MA148 cells. [Figure 5C-5D]The degranulation of NK cells and the production of IFN-γ, as measured by flow cytometry, are illustrated. Figure 5A is a bar graph illustrating the degranulation of NK cells when a triple-specific killer promoter (30 nM) was co-cultured with PBMCs and lung cancer cells (NCI-H460s). Figure 5B is a bar graph illustrating the degranulation of NK cells when a triple-specific killer promoter (30 nM) was co-cultured with PBMCs and ovarian cancer cells (OVCAR8). Figure 5C is a bar graph illustrating the degranulation of NK cells when a triple-specific killer promoter (30 nM) was co-cultured with PBMCs and ovarian cancer cells (MA148). Figure 5D is a bar graph illustrating the production of IFN-γ when a triple-specific killer promoter (30 nM) was co-cultured with PBMCs and NCI-H460s cells. Figure 5E is a bar graph illustrating IFN-γ production when the triple-specific killer engager (30 nM) is co-cultured with PBMC and OVCAR8 cells. Figure 5F is a bar graph illustrating IFN-γ production when the triple-specific killer engager (30 nM) is co-cultured with PBMC and MA148 cells. [Figure 5E-5F]The degranulation of NK cells and the production of IFN-γ, as measured by flow cytometry, are illustrated. Figure 5A is a bar graph illustrating the degranulation of NK cells when a triple-specific killer promoter (30 nM) was co-cultured with PBMCs and lung cancer cells (NCI-H460s). Figure 5B is a bar graph illustrating the degranulation of NK cells when a triple-specific killer promoter (30 nM) was co-cultured with PBMCs and ovarian cancer cells (OVCAR8). Figure 5C is a bar graph illustrating the degranulation of NK cells when a triple-specific killer promoter (30 nM) was co-cultured with PBMCs and ovarian cancer cells (MA148). Figure 5D is a bar graph illustrating the production of IFN-γ when a triple-specific killer promoter (30 nM) was co-cultured with PBMCs and NCI-H460s cells. Figure 5E is a bar graph illustrating IFN-γ production when the triple-specific killer engager (30 nM) is co-cultured with PBMC and OVCAR8 cells. Figure 5F is a bar graph illustrating IFN-γ production when the triple-specific killer engager (30 nM) is co-cultured with PBMC and MA148 cells. [Figure 6A-6B] The degranulation of NK cells, as measured by flow cytometry, is illustrated in the following bar graphs. Figure 6A is a bar graph illustrating the degranulation of NK cells when a triple-specific killer promoter (0.3 nM) was incubated with PBMCs alone. Figure 6B is a bar graph illustrating the degranulation of NK cells when a triple-specific killer promoter (3 nM) was incubated with PBMCs alone. Figure 6C is a bar graph illustrating the degranulation of NK cells when a triple-specific killer promoter (30 nM) was incubated with PBMCs alone. Figure 6D is a bar graph illustrating the degranulation of NK cells when a triple-specific killer promoter (0.3 nM) was co-cultured with PBMCs and C4-2 cells. Figure 6E is a bar graph illustrating the degranulation of NK cells when a triple-specific killer promoter (3 nM) was co-cultured with PBMCs and C4-2 cells. Figure 6F is a bar graph illustrating NK cell degranulation when a triple-specific killer engager (30 nM) was co-cultured with PBMCs and C4-2 cells. [Figure 6C-6D] The degranulation of NK cells, as measured by flow cytometry, is illustrated in the following bar graphs. Figure 6A is a bar graph illustrating the degranulation of NK cells when a triple-specific killer promoter (0.3 nM) was incubated with PBMCs alone. Figure 6B is a bar graph illustrating the degranulation of NK cells when a triple-specific killer promoter (3 nM) was incubated with PBMCs alone. Figure 6C is a bar graph illustrating the degranulation of NK cells when a triple-specific killer promoter (30 nM) was incubated with PBMCs alone. Figure 6D is a bar graph illustrating the degranulation of NK cells when a triple-specific killer promoter (0.3 nM) was co-cultured with PBMCs and C4-2 cells. Figure 6E is a bar graph illustrating the degranulation of NK cells when a triple-specific killer promoter (3 nM) was co-cultured with PBMCs and C4-2 cells. Figure 6F is a bar graph illustrating NK cell degranulation when a triple-specific killer engager (30 nM) was co-cultured with PBMCs and C4-2 cells. [Figure 6E-6F] The degranulation of NK cells, as measured by flow cytometry, is illustrated in the following bar graphs. Figure 6A is a bar graph illustrating the degranulation of NK cells when a triple-specific killer promoter (0.3 nM) was incubated with PBMCs alone. Figure 6B is a bar graph illustrating the degranulation of NK cells when a triple-specific killer promoter (3 nM) was incubated with PBMCs alone. Figure 6C is a bar graph illustrating the degranulation of NK cells when a triple-specific killer promoter (30 nM) was incubated with PBMCs alone. Figure 6D is a bar graph illustrating the degranulation of NK cells when a triple-specific killer promoter (0.3 nM) was co-cultured with PBMCs and C4-2 cells. Figure 6E is a bar graph illustrating the degranulation of NK cells when a triple-specific killer promoter (3 nM) was co-cultured with PBMCs and C4-2 cells. Figure 6F is a bar graph illustrating NK cell degranulation when a triple-specific killer engager (30 nM) was co-cultured with PBMCs and C4-2 cells. [Figures 7A-7B]The production of IFN-γ, as measured by flow cytometry, is illustrated in the following bar graphs. Figure 7A is a bar graph illustrating the production of IFN-γ when the triple-specific killer engager (0.3 nM) was incubated with PBMCs alone. Figure 7B is a bar graph illustrating the production of IFN-γ when the triple-specific killer engager (3 nM) was incubated with PBMCs alone. Figure 7C is a bar graph illustrating the production of IFN-γ when the triple-specific killer engager (30 nM) was incubated with PBMCs alone. Figure 7D is a bar graph illustrating the production of IFN-γ when the triple-specific killer engager (0.3 nM) was co-cultured with PBMCs and C4-2 cells. Figure 7E is a bar graph illustrating the production of IFN-γ when the triple-specific killer engager (3 nM) was co-cultured with PBMCs and C4-2 cells. Figure 7F is a bar graph illustrating IFN-γ production when a triple-specific killer engager (30 nM) was co-cultured with PBMCs and C4-2 cells. [Figure 7C-7D] The production of IFN-γ, as measured by flow cytometry, is illustrated in the following bar graphs. Figure 7A is a bar graph illustrating the production of IFN-γ when the triple-specific killer engager (0.3 nM) was incubated with PBMCs alone. Figure 7B is a bar graph illustrating the production of IFN-γ when the triple-specific killer engager (3 nM) was incubated with PBMCs alone. Figure 7C is a bar graph illustrating the production of IFN-γ when the triple-specific killer engager (30 nM) was incubated with PBMCs alone. Figure 7D is a bar graph illustrating the production of IFN-γ when the triple-specific killer engager (0.3 nM) was co-cultured with PBMCs and C4-2 cells. Figure 7E is a bar graph illustrating the production of IFN-γ when the triple-specific killer engager (3 nM) was co-cultured with PBMCs and C4-2 cells. Figure 7F is a bar graph illustrating IFN-γ production when a triple-specific killer engager (30 nM) was co-cultured with PBMCs and C4-2 cells. [Figures 7E-7F]The production of IFN-γ, as measured by flow cytometry, is illustrated in the following bar graphs. Figure 7A is a bar graph illustrating the production of IFN-γ when the triple-specific killer engager (0.3 nM) was incubated with PBMCs alone. Figure 7B is a bar graph illustrating the production of IFN-γ when the triple-specific killer engager (3 nM) was incubated with PBMCs alone. Figure 7C is a bar graph illustrating the production of IFN-γ when the triple-specific killer engager (30 nM) was incubated with PBMCs alone. Figure 7D is a bar graph illustrating the production of IFN-γ when the triple-specific killer engager (0.3 nM) was co-cultured with PBMCs and C4-2 cells. Figure 7E is a bar graph illustrating the production of IFN-γ when the triple-specific killer engager (3 nM) was co-cultured with PBMCs and C4-2 cells. Figure 7F is a bar graph illustrating IFN-γ production when a triple-specific killer engager (30 nM) was co-cultured with PBMCs and C4-2 cells. [Figure 8] The image illustrates the enhancement of cytolytic activity against ovarian cancer spheroids by a tripspecific killer engager incorporating an exemplary anti-B7H3 protein. [Figure 9] This graph illustrates the quantification of the enhancement of cytolytic activity against ovarian cancer spheroids by a tripspecific killer enhancer incorporating an exemplary anti-B7H3 protein. [Modes for carrying out the invention]

[0026] This disclosure describes anti-B7H3 polypeptides, compounds and devices comprising anti-B7H3 polypeptides, and methods for suing such compounds and devices. Exemplary platforms in which anti-B7H3 polypeptides may be used include, but are not limited to, chimeric antigen receptor therapies (e.g., CAR-NK therapy, CAR-T therapy, CAR-macrophage therapy, etc.), multispecific immune cell engager technologies (e.g., bispecific killer engagers, triplicate killer engagers, bispecific T cell engagers, triplicate T cell engagers, etc.), targeted immunotherapy (e.g., targeted ADAM17 blocker (TAB) therapy), therapeutic drug delivery (e.g., antibody-drug conjugates, therapeutic radioisotope delivery, toxin delivery, cytokine delivery, chemokine delivery), imaging technologies (labeled constructs and / or labeled radioisotopes delivery), and cell and / or ligand capture technologies (e.g., ELISA, etc.).

[0027] B7 homolog 3 (B7H3), also known as surface antigen classification 276 (CD276), is a human protein encoded by the CD276 gene. The B7H3 protein is a 316-amino acid type I transmembrane protein that exists in two isoforms, determined by its extracellular domain. In mice, the extracellular domain consists of a single pair of immunoglobulin variable (IgV)-like and immunoglobulin constant (IgC)-like domains, whereas in humans, it consists of either one pair (2Ig-B7H3) or two identical pairs (4Ig-B7H3) resulting from exon duplication. B7H3 mRNA is expressed in most normal tissues. In contrast, the B7H3 protein has very limited expression on normal tissues due to its post-transcriptional regulation by microRNA. In normal tissues, B7H3 plays a primarily inhibitory role in adaptive immunity, suppressing T cell activation and proliferation.

[0028] B7H3 is overexpressed in several types of human cancer cells. In malignant tissues, B7H3 is an immune checkpoint molecule that inhibits tumor antigen-specific immune responses. B7H3 also has non-immunological pre-tumorogenic functions such as promoting migration, invasion, angiogenesis, chemotherapy resistance, epithelial-mesenchymal transition, and effects on tumor cell metabolism. B7H3 is recognized as a co-stimulatory molecule in immune responses, including, but not limited to, T cell activation and IFN-γ production. In the presence of anti-CD3 antibodies that mimic TCR signaling, the human B7H3-Ig fusion protein acts on CD4 + and CD8 + B7H3 increases the proliferation of both T cells and enhances cytotoxic T lymphocyte (CTL) activity in vitro. B7H3 also has antitumor effects against colonic adenocarcinoma. B7H3 is also expressed in pancreatic cancer and is associated with enhanced therapeutic efficacy. Pancreatic cancer patients with high tumor B7H3 levels have a significantly better postoperative prognosis than those with low tumor B7H3 levels (Yang et al., Int J Biol Sci 2020;16(11):1767-1773). Due to its selective expression on solid tumors and its pre-tumorogenic function, B7H3 is a target for several anticancer drugs, including enobirituzumab, ombartamab, MGD009, MGC018, DS-7300a, and CAR-T cells.

[0029] Therefore, in one embodiment, anti-B7H3 polypeptides may be incorporated into immunotherapy compounds. Immunotherapy compounds can provide personalized therapies that activate or suppress the immune system and amplify or reduce the immune response, and are being rapidly developed to treat various forms of cancer. Immunotherapy for cancer, such as chimeric antigen receptor (CAR)-T cells, CAR-natural killer (NK) cells, and PD-1 and PD-L1 inhibitors, aims to assist the target immune system in fighting cancer. T cell activation depends on both a specific combination of major histocompatibility complex (MHC) bound to the T cell receptor (TCR) and peptides, and the interaction of T cell costimulatory molecules with ligands on antigen-presenting cells (APCs). The B7 family of superficial membrane proteins on activated APCs has been shown to be involved in regulating the T cell response. Recent studies have shown that upregulation of inhibitory B7 molecules within the tumor microenvironment is highly associated with tumor immune evasion. B7H3, a newly identified member of the B7 family, may promote T cell activation and IFN-γ production.

[0030] In one embodiment, the disclosure describes an anti-B7H3 polypeptide comprising at least one complementarity-determining region (CDR), such as those shown in SEQ ID NOs: 4, 5, and 6. Exemplary anti-B7H3 polypeptides comprising each of these CDRs are shown by the amino acid sequences of SEQ ID NOs: 1, 2, and 3. As used herein, the terms “peptide,” “polypeptide,” and “protein” are interchangeable and refer to any chain of at least two amino acids linked by a covalent chemical bond. Therefore, as used herein, the term “polypeptide” may refer to a complete amino acid sequence or a portion thereof that codes for an entire protein. As used herein, the terms “anti-B7H3 peptide,” “anti-B7H3 protein,” “B7H3-binding peptide,” and “B7H3-target peptide” generally refer to any peptide or polypeptide (including proteins or fusion proteins) that can specifically bind to B7H3.

[0031] In some embodiments, the anti-B7H3 polypeptide may be an antibody or antibody fragment containing a CDR region. As used herein, “CDR region” refers to one or more of the three complementarity-determining regions (CDRs) of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3. The CDRs are identified separately as SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6. In some embodiments, the polypeptide encodes the light and heavy chains of the B7H3 target protein.

[0032] A "protein-coding sequence," or a sequence that "codes" a particular polypeptide, is a nucleic acid sequence that, when controlled by an appropriate regulatory sequence, is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide, either in vitro or in vivo. The boundaries of a coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxyl) terminus. Coding sequences may include, but are not limited to, cDNA from prokaryotic or eukaryotic mRNA, genomic DNA sequences from prokaryotic or eukaryotic DNA, and even synthetic DNA sequences. The transcription termination sequence is usually located 3' to the coding sequence.

[0033] The term "antibody" refers to a molecule that contains at least one antigen-binding site that binds immunospecifically to a specific antigen target of interest. Therefore, the term "antibody" is not limited to, but includes, full-length antibodies and / or their variants, fragments thereof, peptide bodies and their variants, monoclonal antibodies (including full-length monoclonal antibodies), multispecific antibodies formed from at least two intact antibodies (e.g., bispecific antibodies), human antibodies, humanized antibodies, and antibody mimics that mimic the structure and / or function of an antibody or a specific fragment or part thereof, such as single-chain antibodies and their fragments. Therefore, as used herein, the term “antibody” encompasses, but is not limited to, Fab, Fab' and F(ab')2, pFc', Fd, single-domain antibodies (sdAb), variable fragments (Fv), single-chain variable fragments (scFv), or disulfide-bonded Fv(sdFv); bispecific antibodies or bivalent bispecific antibodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments (e.g., tribodies), including antibody fragments capable of binding to or to a part thereof a biomolecule (such as an antigen or receptor). Antibodies may belong to any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass.

[0034] The anti-B7H3 proteins described herein may be any protein that selectively binds to B7H3. Exemplary anti-B7H3 proteins include SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and their functional variants. As used herein, a protein is a "functional variant" of a reference protein if its amino acid sequence has a specific amount of identity with the reference protein and retains the activity of the reference protein. Structural similarity between two proteins can be determined by aligning the residues of the two proteins (e.g., a candidate protein and, for example, the protein of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3) and optimizing the number of identical amino acids along the length of their sequences; gaps in one or both sequences are acceptable when performing alignment to optimize the number of identical amino acids, but nevertheless, the amino acids within each sequence must be maintained in their proper order. A candidate protein is a protein being compared to a reference protein (e.g., SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3). Candidate proteins can, for example, be isolated from animals, produced using recombinant techniques, or synthesized chemically or enzymatically.

[0035] Pairwise comparative analysis of amino acid sequences can be performed, for example, using the best-fit algorithm in the GCG package (version 10.2, Madison WI). Alternatively, proteins may be compared using the Blasp program of the BLAST2 search algorithm, as described by Tatiana et al. (FEMS Microbiol Lett, 174, 247-250 (1999)) and available on the National Center for Biotechnology Information (NCBI) website. Default values ​​for all BLAST2 search parameters may be used, including matrix=BLOSUM62; open gap penalty=11, extension gap penalty=1, gap x_dropoff=50, expected value=10, word size=3, and filter-on.

[0036] In comparing two amino acid sequences, structural similarity may be referred to by percentage "identity" or percentage "similarity." "Identity" refers to the presence of identical amino acids. "Similarity" refers not only to the presence of identical amino acids but also to the presence of conserved substitutions. Conservative substitutions in amino acids in anti-B7H3 proteins may be selected from other members of the class to which the amino acid belongs. For example, it is well known in the field of protein biochemistry that amino acids belonging to a classification of amino acids with specific sizes or properties (such as charge, hydrophobicity, and hydrophilicity) can be substituted for other amino acids without altering protein activity, particularly in regions of the protein not directly related to biological activity. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and tyrosine. Polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Positively charged (basic) amino acids include arginine, lysine, and histidine. Negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Conservative substitutions include, for example, Lys for Arg and its inverse to maintain a positive charge; Glu for Asp and its inverse to maintain a negative charge; Ser for Thr to maintain a free -OH; and Gln for Asn to maintain a free -NH2. Similarly, bioactive analogues of proteins involving the deletion or addition of one or more adjacent or non-adjacent amino acids that do not eliminate the functional activity of the protein are also considered.

[0037] Generally, the outer portions of the CDR, such as SEQ ID NOs. 1, 2, and 3, are adaptable through mutation while maintaining anti-B7H3 functionality, i.e., specifically binding to B7H3. Therefore, anti-B7H3 proteins may contain one, two, or all three of the CDRs from SEQ ID NOs. 1, 2, and 3, i.e., the amino acids from SEQ ID NOs. 4 (CDR1), 5 (CDR2), and 6 (CDR3).

[0038] Anti-B7H3 proteins as described herein may include proteins having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence similarity to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

[0039] Anti-B7H3 proteins as described herein may include proteins having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

[0040] Variants of the sequences disclosed herein include proteins, or full-length proteins, that involve substitutions, deletions, or insertions into the protein backbone, resulting in at least approximately 70% homology to the original protein across the corresponding portion. Greater deviations from homology are acceptable if similar amino acids, i.e., conserved amino acid substitutions, are negligible as changes within the sequence. Examples of conserved substitutions include amino acids having the same or similar properties. Exemplary conservative amino acid substitutions include changes from alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartic acid to glutamic acid; cysteine ​​to serine; glutamine to asparagine; glutamic acid to aspartic acid; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine, glutamine, or glutamic acid; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine, or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; valine to isoleucine, and leucine.

[0041] In some embodiments, the anti-B7H3 protein may include further sequences, such as amino acids added to the C-terminus or N-terminus of the anti-B7H3 protein. Such modifications may facilitate purification by capture in a column, use of antibodies, or recovery when recombinantly expressed in microorganisms. Such tags include, for example, a histidine-rich tag (e.g., SEQ ID NO: 8) that enables purification of the protein in a nickel column and / or a leader sequence (e.g., SEQ ID NO: 7) that can transport the recombinantly expressed protein to the membrane of the cell in which it is recombinantly expressed. Such gene modification techniques and suitable further sequences are well known in molecular biology techniques. In some embodiments, the C-terminal and / or N-terminal modifications may be cleaved from the anti-B7H3 protein, for example, before being incorporated into a pharmaceutical composition. In other embodiments, retention of the C-terminal or N-terminal modifications may be desired for a given application, i.e., to facilitate immobilization to a substrate.

[0042] In another embodiment, the disclosure describes a multispecific compound comprising a target domain containing an anti-B7H3 protein. The anti-B7H3 protein comprises at least one CDR from SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, for example, the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6. The multispecific compound further comprises an immune cell binding domain operably linked to the target domain.

[0043] The terms "multispecific compound" and "multispecific protein" refer to a "fusion molecule" or "fusion protein" that is a biologically active polypeptide containing two or more binding domains, covalently linked (e.g., fused) by recombinant, chemical, or other preferred methods, in the presence or absence of further effector molecules. For example, one binding domain may be linked to another binding domain via a peptide linker sequence. Alternatively, a peptide linker may be used to assist in the construction of the fusion molecule.

[0044] As used herein, the term “operably linked” refers to direct or indirect covalent bonding between domains of a multispecific compound. Thus, two operably linked domains may be directly and covalently coupled to one another. Conversely, two operably linked domains may be linked by mutual covalent bonding to an intervening portion (e.g., a flanking sequence or linker). Two domains can be considered operably linked if, for example, they are separated by a third domain in the presence or absence of one or more intervening flanking sequences.

[0045] The domains of multispecific compounds may be operable binding states constructed using one or more linkers. The term "linker," as used herein, refers to any bond, small molecule, peptide sequence, or other medium that physically connects to a domain. Linkers may be sensitive to, or substantially resistant to, acid-induced cleavage, photo-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, and disulfide bond cleavage under conditions necessary for the compound or antibody to maintain its activity. Linkers are classified into those chemical motifs well known in the art, including disulfide groups, hydrazines or peptides (cleavable), or thioester groups (incleavable). Linkers also include charged linkers, as known in the art, and their hydrophilic forms.

[0046] Suitable linkers for ligating the domains of multispecific anti-B7H3 compounds may include natural linkers, empirical linkers, or combinations of natural and empirical linkers. Natural linkers are naturally occurring between protein domains and originate from multi-domain proteins. For example, by utilizing the properties of natural linkers, such as length, hydrophobicity, amino acid residues, and / or secondary structure, desirable properties can be conferred to multi-domain compounds containing natural linkers that ligate functional domains.

[0047] The study of linkers in natural multi-domain proteins has led to the generation of numerous empirical linkers with diverse sequences and conformations for the construction of recombinant fusion proteins. These empirical linkers can be classified into three types: flexible linkers, rigid linkers, and cleavable linkers. Flexible linkers can generate some degree of movement or interaction in linked domains. They typically contain small, non-polar (e.g., Gly) or polar (e.g., Ser or Thr) amino acids that provide flexibility and allow for the mobility of linked functional domains. Rigid linkers can maintain their independent functions by cleverly preserving fixed distances between domains, resulting in efficient separation of protein domains and / or significantly reducing interference between functional domains. Cleavable linkers may allow for the control of functional domain release in vivo. By utilizing intrinsic in vivo processes, cleavable linkers can be cleaved under specific conditions, such as the presence of reducing agents or proteases. This type of linker can reduce steric hindrance after linker cleavage, improve biological activity, and / or enable independent action / metabolism of each domain of the recombinant fusion protein.

[0048] An example linker is shown in the amino acid sequences of SEQ ID NOs. 12-18.

[0049] In one exemplary application, an anti-B7H3 polypeptide can be incorporated into a multispecific NK engager compound that includes at least an anti-B7H3 protein and an NK engager domain.

[0050] Natural killer (NK) cells are cytotoxic lymphocytes of the innate immune system with the ability to perform immune surveillance. Like T cells, NK cells deliver membrane-permeable and apoptosis-inducing stored granzymes and perforin granules. Unlike T cells, NK cells do not require antigen priming and recognize targets by associating with activating receptors in the absence of MHC recognition.

[0051] NK cells express CD16, an activating receptor involved in antibody-dependent cell-mediated cytotoxicity (ADCC) that binds to the Fc portion of IgG antibodies. NK cells are regulated by IL-15, which can induce increased antigen-dependent cytotoxicity, lymphokine-activated killer activity, and / or mediate responses to interferon (IFN), tumor necrosis factor (TNF), and / or granulocyte-macrophage colony-stimulating factor (GM-CSF). IL-15 can also drive NK cell proliferation and survival, thereby enhancing NK cell expansion and persistence. All of these IL-15 activating functions contribute to improved cancer defense.

[0052] Therapeutically, adoptive transplantation of NK cells, when combined with, for example, lymphocyte depletion chemotherapy or IL-2 to stimulate NK cell survival and in vivo expansion, can induce remission in subjects with refractory acute myeloid leukemia (AML). This therapy may be limited by the lack of antigen specificity and the IL-2-mediated induction of regulatory T (Treg) cells that suppress NK cell proliferation and function. NK cell-based immunotherapy can be enhanced by creating reagents that drive the antigen specificity, expansion, and / or persistence of NK cells, while bypassing the negative effect of Treg inhibition.

[0053] Therefore, in one aspect, this disclosure is B7H3 + This paper describes the design, construction, and use of a triplicate molecule comprising two domains capable of driving NK cell-mediated killing of tumor cells, and an intramolecular NK activation domain capable of generating an NK cell self-sustaining signal. The triplicate molecule drives NK cell proliferation and / or, for example, B7H3 + Cancer cells or B7H3 + This may enhance NK cell-driven cytotoxicity against cancer cell-derived cell lines.

[0054] In one aspect, this disclosure describes a triple-specific killer-engager molecule that generally includes a target domain that selectively targets B7H3, an NK cell-engager domain (e.g., CD16, CD16+CD2, CD16+DNAM, NKp46, CD16+NKp46, NKG2D, NK2C), and an NK activation domain (e.g., IL-15, IL-12, IL-18, IL-21, or other NK cell-enhancing cytokines, chemokines, and / or activating molecules), where each domain is operably linked to the other domains. As used herein, the terms “selectively targeting” and “selectively binding” refer, for example, to the ability to distinguish between two or more options having some degree of differential affinity for a particular target. In particular, in relation to triple-specific compounds, the term “operably linked” includes domains linked by mutual covalent bonds to intervening parts (e.g., flanking sequences, multiple flanking sequences, and / or other functional domains). Therefore, the two domains may be considered operably linked if, for example, they are separated by a third functional domain in the presence or absence of one or more intervening flanking sequences.

[0055] The target domain is, for example, a target in tumor cells, cancer stroma, or immobilized B7H3 + B7H3 in cells, etc. + The target domain may include any portion that selectively binds to the target. Therefore, the target domain may include, for example, an anti-B7H3 antibody. In some embodiments, the anti-B7H3 antibody may include an anti-B7H3 polypeptide as detailed herein. In one exemplary embodiment, the anti-B7H3 polypeptide may include one or more complementarity-determining regions (CDRs) of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3. In some embodiments, the anti-B7H3 protein may include two or all three CDRs of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3. In some embodiments, the anti-B7H3 protein may include SEQ ID NO: 1, SEQ ID NO: 2, or a variant thereof (e.g., SEQ ID NO: 3). Preferred alternative variants are described herein.

[0056] The NK-binding domain may include any portion that binds to and / or activates NK cells, and / or any portion that blocks the inhibition of NK cells. In some embodiments, the NK-binding domain may include an antibody that selectively binds to components on the surface of NK cells. In other embodiments, the NK-binding domain may include a ligand or small molecule that selectively binds to components on the surface of NK cells. Therefore, for brevity, with respect to an antibody that selectively binds to components on the surface of NK cells, it includes any antibody fragment exhibiting the described binding properties. Similarly, with respect to a ligand that selectively binds to components on the surface of NK cells, it includes any fragment of the ligand exhibiting the described binding properties.

[0057] In some embodiments, the NK-binding domain may selectively bind to receptors located at least partially on the surface of NK cells. In certain embodiments, the NK-binding domain may bind to NK cells, thereby bringing them into spatial proximity to targets to which the target domain selectively binds. However, in certain embodiments, the NK-binding domain may selectively bind to receptors that activate NK cells and therefore also have an activating function. As described above, activation of the CD16 receptor can induce antibody-dependent cell-mediated cytotoxicity. Therefore, in certain embodiments, the NK-binding domain may include at least a portion of an anti-CD16 receptor antibody effective in selectively binding to the CD16 receptor. In other embodiments, the NK-engager cell domain may interfere with mechanisms that inhibit NK cells. In such embodiments, the NK-engager domain may include, for example, domains that block anti-PD1 / PDL1, anti-NKG2A, anti-TIGIT, anti-killer immunoglobulin receptor (KIR), and / or any other inhibitors.

[0058] The NK-binding domain can be designed to have the desired degree of NK selectivity and, consequently, the desired immunobinding properties. For example, CD16 has been identified as the Fc receptor for FcγRIIIa (CD16a) and FcγRIIIb (CD16b). These receptors bind to the Fc portion of IgG antibodies, which then activate antibody-dependent cell-mediated cytotoxicity in NK cells. Anti-CD16 antibodies selectively bind to NK cells but can also bind to neutrophils. Anti-CD16a antibodies selectively bind to NK cells but do not bind to neutrophils. A triple-specific killer-engager compound containing an NK-binding domain that includes an anti-CD16a antibody can bind to NK cells but not to neutrophils. Therefore, in situations where it is desirable for the antibody to bind to NK cells but not to neutrophils, the NK-binding domain of the triple-specific killer-engager compound can be designed to include an anti-CD16a antibody.

[0059] In some embodiments, the NK cell-binding domain may involve the use of a humanized CD16 engager derived from an animal nanobody. While the scFv has a heavy and light variable chain component linked by a linker, the nanobody consists of a single monomeric variable chain, i.e., a variable heavy or variable light chain, which has the ability to specifically bind to a target. The single-domain antibody (sdAb) may be derived from an antibody of any suitable animal, such as a camelid (e.g., llama or camel) or a cartilaginous fish. Compared to larger antibody fragments, single-domain antibodies may offer superior physical stability, deep groove binding ability, and increased production yield.

[0060] In one exemplary embodiment, an sdAb-based NK engager molecule may contain a humanized CD16 nanobody derived from a llama nanobody (GeneBank sequence EF561291; Behar et al., 2008. Protein Eng Des Sel. 21(1):1-10) designated EF91. To confirm the functionality of the molecule, the CDR was cloned into a humanized camelid scaffold (Vincke et al., 2009. J Biol Chem. 284(5):3273-3284) and the CD16 engager (SEQ ID NO: 19) was humanized. The use of a humanized camelid sdAb in the NK-binding domain of a triple-specific killer engager compound may increase drug yield, stability, and / or enhance the efficacy of NK cell-mediated antibody-dependent cytotoxicity (ADCC).

[0061] The NK-binding domain may include any antibody or other ligand that selectively binds to CD16, while the NK-binding domain is described herein in relation to various embodiments in which the NK-binding domain includes anti-CD16 sdAb or anti-CD16 scFv. Furthermore, the NK-binding domain may include antibodies or ligands that selectively bind to any NK cell receptor, such as cytotoxic receptor 2B4, low-affinity Fc receptor CD16, killer immunoglobulin-like receptor (KIR), CD2, NKG2A, TIGIT, NKG2C, LIR-1, and / or DNAM-1.

[0062] In one embodiment, the immune cells are T cells or natural killer (NK) cells. In another embodiment, the immune cells are NK cells; and the immune cell binding domain includes a ligand or antibody that specifically binds to CD16. In some embodiments, the antibody that specifically binds to CD16 includes scFv, F(ab)2, Fab, or a single-domain antibody.

[0063] As explained in more detail above, “antibody” generally refers to an immunoglobulin or a fragment thereof, and therefore includes monoclonal antibodies and fragments thereof (e.g., scFv, Fab, F(ab')2, Fv, sdAb, or other modified forms of antibodies, including humanized forms of antibodies or fragments thereof). Thus, for brevity, with respect to antibodies that selectively bind to B7H3, this includes any antibody or antibody fragment exhibiting the described binding properties. Similarly, with respect to antibodies that selectively bind to CD16 (or any other NK cell receptor), this includes any antibody or antibody fragment exhibiting the described binding properties. In some embodiments, the immune cell binding domain includes a ligand or antibody that specifically binds to CD16, such as an antibody fragment having the amino acid sequence shown in SEQ ID NO: 19.

[0064] In some embodiments, the NK-binding domain and the target domain may be linked using any one of the linkers shown in SEQ ID NOs: 12-18. In certain embodiments, the bispecific anti-B7H3 compound may contain the amino acid sequence of SEQ ID NO: 20 or SEQ ID NO: 21. In some embodiments, the bispecific anti-B7H3 compound may contain the amino acid sequence of SEQ ID NO: 20 or SEQ ID NO: 21, having a deletion of the leader sequence, a deletion of the VDE linker and HID tag, or any deletion of the leader sequence, the VDE linker and the HIS tag.

[0065] In another embodiment, the multispecific anti-B7H3 compound further comprises an immune cell activation domain. In some embodiments, the immune cells may be NK cells, and the immune cell activation domain comprises an NK-activating cytokine or a functional portion thereof.

[0066] The NK activation domain may contain an "immune cell activation domain," such as an amino acid sequence that activates NK cells, promotes NK cell maintenance, or otherwise enhances NK cell activity. For example, NK cells are responsive to various cytokines, including IL-15, that are involved in NK cell homeostasis, proliferation, survival, activation, and / or development. IL-15 and IL-2 share several signaling components, including IL-2 / IL-15Rβ (CD122) and the common gamma chain (CD132). Unlike IL-2, IL-15 bypasses Treg inhibition in the immune response while enabling NK cell activation without stimulating Tregs. In addition to promoting NK cell homeostasis and proliferation, IL-15 can rescue functional defects in NK cells that may occur under post-transplant conditions. IL-15 also has CD8 + It may stimulate T cell function and further enhance their immunotherapeutic capabilities. Furthermore, based on preclinical studies, the toxic properties of IL-15 may be preferable to those of low-dose IL-2.

[0067] Therefore, the NK activation domain may be, or derived from, one or more cytokines capable of activating and / or maintaining NK cells. As used herein, the term “derived from” refers to an amino acid fragment of a cytokine (e.g., IL-15) sufficient to provide NK cells whose activity is activated and / or maintained. In embodiments comprising two or more NK activation domains, the NK activation domains may be provided in series or in any other combination. In addition, each cytokine-based NK activation domain may comprise the entire amino acid sequence of a cytokine, or it may be an amino acid fragment, independently of the properties of other NK activation domains contained in the triple-specific killer-engager compound. Exemplary cytokines that may be the base of an NK activation domain include, for example, IL-15, IL-18, IL-12, and IL-21. Therefore, while triple-specific killer-engager compounds are detailed herein in relation to exemplary model embodiments where the NK activation domain is derived from IL-15, they may also be designed using any suitable cytokine or an NK activation domain derived therefrom.

[0068] To simplify this explanation, with respect to the NK activation domain, by identifying the cytokine on which it is based, it includes either the entire amino acid sequence of the cytokine, any suitable amino acid fragment of the cytokine, and / or a modified version of the cytokine containing one or more amino acid substitutions. Therefore, with respect to the "IL-15" NK activation domain, it includes an NK activation domain containing the entire amino acid sequence of IL-15, an NK activation domain containing a fragment of IL-15 (e.g., SEQ ID NO: 11), a functional variant thereof, or an NK activation domain containing amino acid substitutions compared to the wild-type IL-15 amino acid sequence. For example, the NK activation domain may contain a fragment of IL-15 containing an N-to-D or N-to-A amino acid substitution at position 72 of SEQ ID NO: 11. Position 72 of SEQ ID NO: 11 simply refers to the position of the amino acid substitution, unrelated to any particular fragment of IL-15 that may be used as the NK activation domain. Therefore, the NK activation domain may contain a fragment of IL-15 other than the fragment shown in SEQ ID NO: 11, which may have an N-to-D or N-to-A amino acid substitution at the position of the alternative IL-15 fragment corresponding to position 72 of SEQ ID NO: 11.

[0069] In some embodiments, the triple-specific anti-B7H3 compound includes a domain linked by one or more linkers shown in SEQ ID NOs: 12-18, or a combination of linkers shown in SEQ ID NOs: 12-18. In a specific exemplary embodiment, the NK-binding domain is linked to the NK-activating domain using the linker of SEQ ID NO: 14, while the NK-activating domain is linked to the target domain using the linker of SEQ ID NO: 15 (e.g., SEQ ID NOs: 22-25).

[0070] In another aspect, the disclosure describes isolated nucleic acid sequences encoding any embodiment of an anti-B7H3 compound, which is one of the compounds described herein. In some embodiments, the isolated nucleic acid is one of the nucleic acid sequences of SEQ ID NOs: 26-33. Assuming an amino acid sequence of any anti-B7H3 polypeptide, or a multispecific anti-B7H3 compound containing an anti-B7H3 polypeptide, those skilled in the art can determine the entire range of polynucleotides encoding that amino acid sequence using conventional methods.

[0071] As used herein, the terms “nucleic acid” or “oligonucleotide” refer 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, antisense DNA strands, shRNA, ribozymes, nucleic acid conjugates, and oligonucleotides. Nucleic acids may be single-stranded, double-stranded, linear, or covalently cyclic closed molecules. Nucleic acids can be isolated. The term “isolated nucleic acid” means that the nucleic acid has been (i) amplified in vitro, for example, via polymerase chain reaction (PCR), (ii) recombinantly produced by cloning, (iii) purified, for example, by cleavage and separation by gel electrophoresis, (iv) synthesized, for example, by chemical synthesis, or (vi) extracted from a sample. Nucleic acids may be introduced into cells, i.e., transfected. When cells are transfected with RNA, the RNA may be modified by modification, capping, or stabilization of polyadenylation.

[0072] As used herein, "amplified DNA" or "PCR product" refers to an amplified fragment of DNA of a defined size. Various techniques are available and well known in the art for detecting PCR products. Methods for detecting PCR products include, but are not limited to, gel electrophoresis using agarose or polyacrylamide gel with ethidium bromide staining (DNA intercalant), labeled probes (radioactive or non-radioactive labeling, Southern blotting), labeled deoxyribonucleotides (for direct incorporation of radioactive or non-radioactive labeling), or silver staining for direct visualization of amplified PCR products; restriction endonuclease digestion dependent on agarose or polyacrylamide gel or high-performance liquid chromatography (HPLC); dot blotting using hybridization of amplified DNA against specific labeled probes (radioactive or non-radioactive labeling); high-pressure liquid chromatography with ultraviolet detection; electrochemiluminescence coupled with potential-initiated chemical reactions / photon detection; and direct sequencing, oligoligation assay (OLA), PCR, qPCR, DNA sequencing, fluorescence, gel electrophoresis, magnetic beads, allele-specific primer extension (ASPE), and / or direct hybridization using radioactive or fluorescently labeled deoxyribonucleotides to determine the precise order of nucleotides containing the DNA fragment of interest.

[0073] Generally, nucleic acids can be extracted, isolated, amplified, or analyzed by various techniques, such as those described in Green and Sambrook, Molecular Cloning: A Laboratory Manual (Fourth Edition), Cold Spring Harbor Laboratory Press, Woodbury, NY, 2,028 pages (2012); or in U.S. Patent No. 7,957,913; U.S. Patent No. 7,776,616; U.S. Patent No. 5,234,809; U.S. Patent Publication No. 2010 / 0285578; and U.S. Patent Publication No. 2002 / 0190663. Examples of nucleic acid analysis include, but are not limited to, sequencing and DNA-protein interactions. Sequencing may be performed by any method known in the art. DNA sequencing techniques include classical dideoxy sequencing reactions (Sanger method) using labeled terminators or primers and gel separation in a slab or capillary, as well as next-generation sequencing methods such as synthetic sequencing using reversibly terminated labeled nucleotides, pyrosequencing, 454 sequencing, Illumina / Solexa sequencing, allele-specific hybridization with a library of labeled oligonucleotide probes, synthetic sequencing using allele-specific hybridization with a library of labeled clones and subsequent ligation, real-time monitoring of the incorporation of labeled nucleotides during the polymerization step, Polony sequencing, and SOLiD sequencing. The separated molecules may be sequenced by sequential or single extension reactions using polymerase or ligase, and by single or sequential differential hybridization with a library of probes.

[0074] In another embodiment, this disclosure describes a protein encoded by any of the nucleic acid sequences described herein. In some embodiments, the protein may have an amino acid sequence comprising the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, or any protein having 90% or more amino acid identity with them.

[0075] In another aspect, this disclosure describes a host cell containing any of the isolated nucleic acid sequences and / or proteins described herein.

[0076] By introducing the nucleic acid constructs of the present invention into host cells to be modified, the expression of chimeric proteins within the cells can be enabled, thereby creating genetically modified cells. Various methods are known in the art and are suitable for introducing nucleic acids into cells, including viral and nonviral mediated techniques. Typical nonviral mediated techniques include, but are not limited to, electroporation, calcium phosphate mediated introduction, nucleofection, sonoporation, heat shock, magnetofection, liposome mediated introduction, microinjection, microprojectile mediated introduction (nanoparticles), cationic polymer mediated introduction (DEAE-dextran, polyethyleneimine, polyethylene glycol (PEG), etc.), or cell fusion. Other transfection methods include specialized transfection reagents such as Lipofectamine (Thermo Fisher Scientific, Inc., Waltham, MA), HILYMAX (Dojindo Molecular Technologies, Inc., Rockville, MD), FUGENE (Promega Corp., Madison, WI), JETPEI (Polyplus Transfection, Illkirch, France), EFFECTENE (Qiagen, Hilden, Germany), and DreamFect (OZ Biosciences, Inc. USA, San Diego, CA).

[0077] By introducing the nucleic acid constructs described herein into host cells to be modified, intracellular expression of the protein encoded by the nucleic acid can be enabled. Various host cells are known in the art and are suitable for protein expression. Typical cells used for transfection and protein expression include, but are not limited to, bacterial cells, eukaryotic cells, yeast cells, insect cells, or plant cells, such as Escherichia coli (E. coli), Bacillus, Streptomyces, Pichia pastoris, Salmonella typhimurium, Drosophila S2, Spodoptera SJ9, CHO, COS (e.g., COS-7), 3T3-F442A, HeLa, HUVEC, HUAEC, NIH3T3, Jurkat, 293, 293H, or 293F.

[0078] In some embodiments, the host cell is a T cell, an NK cell, or a macrophage.

[0079] In further embodiments, this disclosure describes a pharmaceutical composition comprising any one of the multispecific anti-B7H3 compounds described herein and a pharmaceutically acceptable carrier.

[0080] Multispecific anti-B7H3 compounds, such as the triple-specific killer-engager compounds described herein, may be formulated with a pharmaceutically acceptable carrier. As used herein, “carrier” includes any solvent, dispersion medium, medium, coating agent, diluent, antimicrobial agent, and / or antifungal agent, isotonic agent, absorption retarder, buffer, carrier solution, suspension, colloid, etc. The use of such media and / or agents in pharmaceutically active substances is well known in the art. Any conventional media or agent should be considered for use in a therapeutic composition unless it is compatible with the active ingredient. Additional active ingredients may be incorporated into the composition. As used herein, “pharmaceutically acceptable” means a material that is not biologically or otherwise unsuitable; that is, the material may be administered to an individual on the condition that it does not cause any undesirable biological effects or interact in a harmful manner with any other components of the pharmaceutical composition in which it is contained, together with the triple-specific killer-engager compound.

[0081] Therefore, multispecific compounds such as triple-specific killer-engager compounds may be formulated into pharmaceutical compositions. Pharmaceutical compositions may be formulated in various forms adapted to preferred routes of administration. Thus, compositions may be administered via known routes, including, for example, oral, parenteral (e.g., intradermal, transdermal, subcutaneous, intramuscular, intravenous, intraperitoneal, etc.), or topical (e.g., intranasal, intrapulmonary, intramammary, intravaginal, intrauterine, intradermal, transrectal, etc.). Pharmaceutical compositions may be administered to mucosal surfaces (e.g., by spray or aerosol) for example, by administration to the nasal or respiratory mucosa. Compositions may also be administered via sustained-release or delayed-release.

[0082] Therefore, multispecific compounds such as triple-specific killer-engager compounds may be provided in any preferred form, including, but not limited to, solutions, suspensions, emulsions, sprays, aerosols, or mixtures. The composition may be delivered in a formulation having any pharmaceutically acceptable excipient, carrier, or medium. For example, the formulation may be delivered in conventional topical dosage forms such as creams, ointments, aerosol formulations, non-aerosol sprays, gels, and lotions. The formulation may further contain one or more additives, such as adjuvants, skin penetration enhancers, colorants, fragrances, flavorings, humectants, and thickeners.

[0083] Formulations may be presented in unit dosage forms for convenience and may be prepared by methods well known in the pharmaceutical art. Methods for preparing compositions having pharmaceutically acceptable carriers include the step of associating a multispecific or tripspecific killer-engager compound with a carrier constituting one or more accessory components. Generally, formulations may be prepared by associating an active molecule uniformly and / or tightly with a liquid carrier, a pulverized solid carrier, or both, and then, if necessary, shaping the product into a desired formulation.

[0084] The dosage of multispecific or triplicate killer-engager compounds is not limited but can vary depending on various factors, including the specific triplicate killer-engager compound used, the subject's weight, physical condition, and / or age, and / or route of administration. Therefore, the absolute weight of the triplicate killer-engager compound in a given unit dosage form can vary considerably and depends on factors such as the subject's species, age, weight, physical condition, and / or method of administration. Consequently, it is generally impractical to state the amount that constitutes an effective dose of a multispecific or triplicate killer-engager compound for all conceivable applications. However, those skilled in the art can easily determine an appropriate dose by taking such factors into full consideration.

[0085] In some embodiments, the method can include administering a multispecific compound or a trispecific killer engager compound sufficient to provide, for example, a dose of from about 100 ng / kg to about 50 mg / kg. However, in some embodiments, the method may be carried out by administering the multispecific compound or the trispecific killer engager compound at a dose outside of this range. In some of these embodiments, the method includes administering a multispecific compound or a trispecific killer engager compound sufficient to provide a dose of from about 10 μg / kg to about 5 mg / kg, such as from about 100 μg / kg to about 1 mg / kg.

[0086] Alternatively, the dose may be calculated using the actual body weight obtained immediately prior to the start of the treatment course. In the case of a dose calculated in this manner, the body surface area (m 2 ) is calculated using the Du Bois formula: m 2 = (body weight kg 0.425 × height cm 0.725 ) × 0.007184 before the start of the treatment course.

[0087] In some embodiments, the method can include administering a multispecific compound or a trispecific killer engager compound sufficient to provide a dose of from about 0.01 mg / m 2 to about 10 mg / m 2 .

[0088] In another aspect, the disclosure describes a method that includes administering to a subject a multispecific compound that includes a target domain that, in an effective amount, includes one of the anti-B7H3 proteins described herein for inducing NK-mediated killing of cells; and an NK-binding domain operably linked to the anti-B7H3 protein.

[0089] In another aspect, the present disclosure describes a method for stimulating NK cell expansion in vivo, comprising administering to a subject an effective amount of a multispecific compound comprising a target domain containing one of the anti-B7H3 proteins described herein; and an NK-binding domain operably linked to the anti-B7H3 protein.

[0090] In another aspect, the disclosure describes a method for killing target cells in a subject. Generally, the method involves administering an effective amount of an anti-B7H3 multispecific compound to the subject in order to induce NK-mediated killing of target cells. "Treat" or a variation thereof means to reduce, limit the progression of, remit, or restore to some extent the symptoms or signs associated with a disease. As used herein, "remit" means any reduction in the range, severity, frequency, and / or likelihood of symptoms or clinical signs that characterize a particular disease; "symptoms" means any subjective evidence of a disease or disease in a subject; and "signs" or "clinical signs" means objective physical findings associated with a particular disease that can be observed by a person other than the subject.

[0091] "Treatment" may be therapeutic or prophylactic. "Therapeutic" and its variations refer to treatment that relieves one or more pre-existing symptoms or clinical signs associated with the condition. "Prophylactic" and its variations refer to treatment that limits, to some extent, the onset and / or appearance of symptoms or clinical signs of the condition. Generally, "therapeutic" treatment is initiated after the condition has appeared in the subject, while "prophylactic" treatment is initiated before the condition appears in the subject. Thus, in certain embodiments, the method may include prophylactic treatment for subjects at risk of developing the condition. "At risk" refers to subjects that may or may not actually have the described risk. Thus, for example, a subject "at risk" of developing a particular condition is a subject that has one or more signs compared to an individual lacking one or more signs that has or is at increased risk of developing the particular condition, regardless of whether the subject has the condition or exhibits any symptoms or clinical signs that it would manifest. Exemplary signs of the condition may include, for example, genetic predisposition, ancestry, age, sex, geographical location, lifestyle, or medical history. Treatment may also be continued after symptoms have subsided, for example, to prevent or delay relapse.

[0092] In some cases, treatment may involve administering an anti-B7H3 multispecific compound to a subject so that the anti-B7H3 multispecific compound can stimulate endogenous NK cells in vivo. By using the anti-B7H3 multispecific compound as part of an in vivo treatment, NK cells may become antigen-specific and may undergo simultaneous co-stimulation, enhanced survival, and expansion. In other cases, the anti-B7H3 multispecific compound can be used in vitro as an adjuvant for NK cell adoptive transplantation therapy. The terms “administer of ~” and / or “administer” should be understood to mean providing a therapeutically effective dose of the pharmaceutical composition to a subject in need of treatment. The route of administration may be enteral, topical, or parenteral. Such methods of administration include, but are not limited to, intradermal, subcutaneous, intravenous, intraperitoneal, intraarterial, subarachnoid, intra-articular, intraorbital, intracardiac, intradermal, transdermal, transtracheal, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, intrasternal, oral, sublingual, rectal, vaginal, and nasal / ocular administration, as well as infusion, inhalation, and spraying. The terms "parenteral administration" and "administered parenterally" as used herein mean modes of administration other than enteral and topical administration.

[0093] Therefore, anti-B7H3 multispecific compounds may be administered before, during, or after the subject first exhibits symptoms or clinical signs of the condition. Treatment initiated before the subject first exhibits symptoms or clinical signs associated with the condition may result in a reduced likelihood of the subject experiencing clinical evidence of the condition, a reduced severity of symptoms and / or clinical signs of the condition, and / or complete recovery of the condition, compared to a subject not administered the anti-B7H3 multispecific compound. Treatment initiated after the subject first exhibits symptoms or clinical signs associated with the condition may result in a reduced severity of symptoms and / or clinical signs of the condition, and / or complete recovery of the condition, compared to a subject not administered the composition.

[0094] The anti-B7H3 multispecific compound may be any embodiment of the above anti-B7H3 multispecific compound having a target domain that selectively binds to a suitable target cell population. In some cases, the target cells may include tumor cells, and the method may include treating cancer associated with tumor cells. Therefore, in some embodiments, the method may include relieving at least one symptom or clinical sign of the tumor.

[0095] In embodiments in which the target cells include tumor cells, the method may further include surgically removing the tumor and / or reducing the size of the tumor through chemotherapy (e.g., chemotherapy) and / or radiotherapy. Exemplary tumors that can be treated include tumors associated with prostate cancer, lung cancer, colon cancer, rectal cancer, bladder cancer, melanoma, kidney cancer, renal cancer, oral cancer, pharyngeal cancer, pancreatic cancer, uterine cancer, thyroid cancer, skin cancer, head and neck cancer, cervical cancer, ovarian cancer and / or hematopoietic cancer.

[0096] Therefore, in some embodiments, the treatment of the subject includes subjects who have cancer or are at risk of having cancer. Generally, the method involves administering to a subject an effective amount of a multispecific compound comprising a target domain containing one of the anti-B7H3 proteins described herein and an NK-binding domain operably linked to the anti-B7H3 protein. As used herein, the term “cancer” refers to a group of diseases characterized by abnormal, uncontrolled cell proliferation that begins at one site (primary site) and has the ability to invade and spread to other sites (secondary sites, metastases) and to differentiate from benign tumors into cancers (malignant tumors). Substantially any organ can be affected, meaning that more than 100 types of cancer can affect humans. Cancer can be caused by many factors, including genetic predisposition, viral infection, exposure to ionizing radiation, exposure to environmental pollutants, tobacco and / or alcohol use, obesity, poor diet, lack of physical activity, or any combination thereof. As used herein, “neoplasm” or “tumor” (and its grammatical variations) means a new abnormal growth of tissue, which may be benign or cancerous. In related embodiments, neoplasm means a neoplastic disease or disorder, including, but not limited to, various cancers. For example, such cancers may include prostate cancer, pancreatic cancer, biliary tract cancer, colon cancer, rectal cancer, liver cancer, kidney cancer, lung cancer, testicular cancer, breast cancer, ovarian cancer, brain cancer, and head and neck cancers, melanoma, sarcoma, multiple myeloma, leukemia, lymphoma, etc.

[0097] Exemplary cancers categorized by the National Cancer Institute include: acute lymphoblastic leukemia (adult); acute lymphoblastic leukemia (childhood); acute myeloid leukemia (adult); adrenocortical carcinoma (adrenal carcinoma); adrenocortical carcinoma (childhood); AIDS-related lymphoma; AIDS-related malignancies; anal cancer; cerebellar astrocytoma (childhood); cerebral astrocytoma (childhood); extrahepatic cholangiocarcinoma (bile duct cancer); bladder cancer (childhood); bone cancer, osteosarcoma / malignant fibrous histiocytoma; brainstem glioma (childhood); brain tumor (adult); brain tumor, brainstem glioma (childhood); cerebellar astrocytoma (childhood); brain tumor, cerebral astrocytoma / malignant glioma (childhood); brain Tumors, ependymoma, pediatric; brain tumors, medulloblastoma, pediatric; brain tumors, supratentorial primitive neuroectodermal tumor, pediatric; brain tumors, visual pathway and hypothalamic glioma, pediatric; brain tumors, pediatric (other); breast cancer; breast cancer and pregnancy; breast cancer, pediatric; breast cancer, male; bronchial adenoma / carcinoid, pediatric; carcinoid tumor, pediatric; carcinoid tumor, gastrointestinal; cancer, adrenal cortex; cancer, islet cell; unknown primary cancer; central nervous system lymphoma, primary; cerebellar astrocytoma, pediatric; cerebral astrocytoma / malignant glioma, pediatric; cervical cancer; childhood cancer; chronic lymphocytic leukemia; chronic myeloid leukemia; chronic bone marrow Proliferative disorders; clear cell sarcoma of the tendon sheath; colon cancer; colorectal cancer, pediatric; cutaneous T-cell lymphoma; endometrial cancer; ependymoma, pediatric; epithelial cancer, ovarian; esophageal cancer; esophageal cancer, pediatric; Ewing family tumors; extracranial germ cell tumors, pediatric; extragonadal germ cell tumors; extrahepatic bile duct cancer; eye cancer, intraocular melanoma; eye cancer, retinoblastoma; gallbladder cancer; stomach cancer; stomach cancer, pediatric; gastrointestinal carcinoid tumors; germ cell tumors, extracranial, pediatric; germ cell tumors, extragonadal; germ cell tumors, ovarian; gestational trophoblastic neoplasm; glioma; pediatric brainstem; glioma; pediatric visual pathway and hypothalamus; hair - Cellular leukemia; head and neck cancer; hepatocellular (liver) cancer, adult (primary); hepatocellular (liver) cancer, pediatric (primary); Hodgkin's lymphoma, adult; Hodgkin's lymphoma, pediatric; Hodgkin's lymphoma during pregnancy; hypopharyngeal cancer; hypothalamic and visual pathway glioma, pediatric; intraocular melanoma; islet cell carcinoma (endocrine pancreas); Kaposi's sarcoma; renal cancer; laryngeal cancer; laryngeal cancer, pediatric; leukemia, acute lymphoblastic, adult; leukemia, acute lymphoblastic, pediatric; leukemia, acute myeloid, adult; leukemia, acute myeloid, pediatric; leukemia, chronic lymphocytic; leukemia, chronic myeloid; leukemia, hairy cell;Lip and oral cancer; liver cancer, adult (primary); liver cancer, pediatric (primary); lung cancer, non-small cell; lung cancer, small cell; lymphoblastic leukemia, adult acute; lymphoblastic leukemia, pediatric acute; lymphocytic leukemia, chronic; lymphoma, AIDS-related; lymphoma, central nervous system (primary); lymphoma, cutaneous T cell; lymphoma, Hodgkin, adult; lymphoma, Hodgkin, pediatric; lymphoma, Hodgkin, pregnant; lymphoma, non-Hodgkin, adult; lymphoma, non-Hodgkin, pediatric; lymphoma, non-Hodgkin, pregnant; lymphoma, primary central nervous system; macroglobulinemia, Waldenström; male Female breast cancer; malignant mesothelioma, adult; malignant mesothelioma, child; malignant thymoma; medulloblastoma, child; melanoma; intraocular melanoma; Merkel cell carcinoma; malignant mesothelioma; occult primary metastatic squamous cell carcinoma of the neck; multiple endocrine neoplasia syndrome, child; multiple myeloma / plasmacytic neoplasm; mycosis fungoides; myeloplastic syndrome; chronic myeloid leukemia; acute myeloid leukemia, child; multiple myeloma; chronic myeloproliferative disorders; nasal cavity / paranasal sinus cancer; nasopharyngeal cancer; nasopharyngeal cancer, child; neuroblastoma; non-Hodgkin lymphoma, adult; non-Hodgkin lymphoma, child; non-Hodgkin lymphoma, pregnant; non-small cell lung Cancer; oral cancer, pediatric; oral and lip cancer; oropharyngeal cancer; osteosarcoma / malignant fibrous histiocytoma of bone; ovarian cancer, pediatric; ovarian epithelial carcinoma; ovarian germ cell tumor; low-grade ovarian tumor; pancreatic cancer; pancreatic cancer, pediatric; pancreatic cancer, islet cell; paranasal sinus / nasal cavity cancer; parathyroid cancer; penile cancer; pheochromocytoma; pineal and supratentorial primitive neuroectodermal tumors, pediatric; pituitary tumors; plasma cell neoplasms / multiple myeloma; pleuroblastoma; breast cancer during pregnancy; Hodgkin lymphoma during pregnancy; non-Hodgkin lymphoma during pregnancy; primary central nervous system lymphoma; primary liver cancer, adult; primary liver cancer, pediatric; prostate cancer; rectal cancer Renal cell carcinoma (kidney cancer); Renal cell carcinoma, pediatric; Transitional cell carcinoma of the renal pelvis and ureter; Retinoblastoma; Rhabdomyosarcoma, pediatric; Salivary gland cancer; Salivary gland cancer, pediatric; Sarcoma, Ewing family of tumors; Sarcoma, Kaposi's sarcoma; Sarcoma (osteosarcoma) / Malignant fibrous histiocytoma of bone; Sarcoma, rhabdomyosarcoma, pediatric; Sarcoma, soft tissue, adult; Sarcoma, soft tissue, pediatric; Sézary syndrome; Skin cancer; Skin cancer, pediatric; Skin cancer (melanoma); Skin cancer, Merkel cells; Small cell lung cancer; Small intestine cancer; Soft tissue sarcoma, adult; Soft tissue sarcoma, pediatric; Latent primary squamous cell carcinoma of the neck, metastatic; Gastric cancer;This includes gastric cancer (stomach cancer), pediatric; supratentorial primitive neuroectodermal tumor, pediatric; T-cell lymphoma, cutaneous; testicular cancer; thymoma, pediatric; malignant thymoma; thyroid cancer; thyroid cancer, pediatric; transitional cell carcinoma of the renal pelvis and ureter; gestational trophoblastic neoplasm, pediatric; cancer of unknown primary site, pediatric; rare cancers in children; transitional cell carcinoma of the ureter and renal pelvis; urethral cancer; uterine sarcoma; vaginal cancer; glioma of the visual pathway and hypothalamus, pediatric; vulvar cancer; Waldenström macroglobulinemia; and Wilms' tumor.

[0098] In some embodiments, cancer includes, or may be related to, prostate cancer, lung cancer, colon cancer, rectal cancer, bladder cancer, melanoma, kidney cancer, renal cancer, oral cancer, pharyngeal cancer, pancreatic cancer, uterine cancer, thyroid cancer, skin cancer, head and neck cancer, cervical cancer, ovarian cancer, and / or hematopoietic cancer.

[0099] In one embodiment, the multispecific compound is administered before, simultaneously with, or after chemotherapy, surgical resection of a tumor, or radiotherapy.

[0100] In some embodiments, the multispecific or triplespecific killer-engager compound may be administered, for example, in single to multiple doses per week, but in some embodiments, the method may be carried out by administering the triplespecific killer-engager compound at a frequency outside this range. In certain embodiments, the multispecific or triplespecific killer-engager compound may be administered from about once per month to about five times per week.

[0101] In some embodiments, the method further includes administering one or more additional therapeutic agents. The one or more additional therapeutic agents may be administered before, after, and / or concurrently with the administration of the multispecific compound or triplicate killer-engager compound. The multispecific compound or triplicate killer-engager compound and the additional therapeutic agents may be co-administered. As used herein, “co-administered” means that two or more components of a combination are administered such that the therapeutic or prophylactic effect of the combination outweighs the therapeutic or prophylactic effect of any of the components administered individually. The two components may be co-administered simultaneously or sequentially. Components co-administered simultaneously may be provided in one or more pharmaceutical compositions. Sequential co-administration of two or more components includes cases where the components are administered such that each component may be present at the treatment site simultaneously. Alternatively, sequential co-administration of two components may include cases where at least one component is excluded from the treatment site, but at least one cellular effect (e.g., cytokine production, activation of a specific cell population) resulting from the administration of that component persists at the treatment site until one or more additional components are administered to the treatment site. Thus, the co-administered combination may include components that, under certain conditions, are never present in the chemical mixture containing each other. In other embodiments, a multispecific or triplicate killer-engager compound and an additional therapeutic agent may be administered as part of a mixture or cocktail. In some embodiments, the administration of a multispecific or triplicate killer-engager compound may, when compared to the administration of one or more other therapeutic agents alone, allow for the efficacy of other modes of therapy at lower doses, thereby reducing the potential, severity, and / or extent of toxicity observed when higher doses of one or more other therapeutic agents are administered.

[0102] The term "chemotherapeutic agent" as used herein refers to any therapeutic agent used to treat cancer. Examples of chemotherapeutic agents include, but are not limited to, actinomycin, azacitidine, azathioprine, bleomycin, bortezomib, carboplatin, capecitabine, cisplatin, chlorambucil, cyclophosphamide, cytarabine, daunorubicin, docetaxel, doxifluridine, doxorubicin, epirubicin, epotilon, etoposide, fluorouracil, gemcitabine, hydroxyurea, idarubicin, and imma. Tinib, Irinotecan, Mechloretamine, Mercaptopurine, Methotrexate, Mitoxantrone, Oxaliplatin, Paclitaxel, Pemetrexed, Teniposide, Thioguanine, Topotecan, Barrubicin, Vinblastine, Vincristine, Vindesine, Vinorelbine, Panitumamab, Erbitux (trademark) (Cetuximab), Matuzumab, IMC-IIF8, TheraCIM hR3, denosumab, Avastin (trademark) (bevacizumab), Humira (trademark) (adalimumab), Herceptin (trademark) (trastuzumab), Remicade (trademark) (infliximab), rituximab, Synagis (trademark) (palivizumab), Mylotarg (trademark) (gemtuzumab oxogamicin), Raptiva (trademark) (efalizumab), Tysabri (trademark) (natalizumab), Zenapax (trademark) (dacliximab), NeutroSpec (trademark) (technetium (99mTc) phanoresomab), tocilizumab, ProstaScint (trademark) (indium -Ill-labeled capromab pendetide), Bexxar (trademark) (tositumomab), Zevalin (trademark) (yttrium-90 conjugated ibritumomab tiuxetan (IDEC-Y2B8)), Xolair (trademark) (omalizumab), MabThera (trademark) (rituximab), ReoPro (trademark) (abcisimab), MabCampath (trademark) (aremutuzumab), Simulect (trademark) (basiliximab), LeukoScan (trademark) (thresomab), CEA-Scan (trademark) (alcitumomab), Verluma (trademark) (nofetumomab), Panorex (trademark) (edrecolomab),Alemtuzumab, CDP870, Natalizumab Gilotrif (trademark) (afatinib), Lynparza (trademark) (olaparib), Perjeta (trademark) (pertuzumab), Otdivo (trademark) (nivolumab), Bosulif (trademark) (bosutinib), Cabometyx (trademark) (cabozantinib), Ogivri (trademark) (trastuzumab-dkst), Sutent (trademark) (sunitinib malate), Adcetris (trademark) (brentuximab vedotin), Alecensa (trademark) Examples include (alecinib), Calquence(trademark)(acalabrutinib), Yescarta(trademark)(siloleucel), Verzenio(trademark)(abemaciclib), Keytruda(trademark)(pembrolizumab), Aliqopa(trademark)(copanlisib), Nerlynx(trademark)(neratinib), Imfinzi(trademark)(durvalumab), Darzalex(trademark)(daratumumab), Tecentriq(trademark)(atezolizumab), and Tarceva(trademark)(erlotinib). Examples of immunotherapy agents, though not limited to them, include interleukins (Il-2, Il-7, Il-12), cytokines (interferon, G-CSF, imiquimod), chemokines (CCL3, CCl26, CXCL7), and immunomodulatory imide drugs (thalidomide and its analogues).

[0103] In some aspects, chemotherapy involves altretamine, amsacrin, L-asparaginase, colaspase, bleomycin, busulfan, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, cyclophosphamide, cytophosphan, cytarabine, dacarbazine, dactinomycin, daunorubicin, docetaxel, doxorubicin, epirubicin, etoposide, fluorouracil, fludarabine, fotemustine, ganciclovir, gemciclovir The following are selected from the group consisting of Tabin, hydroxyurea, idarubicin, ifosfamaide, irinotecan, lomustine, melphalan, mercaptopurine, methotrexate, mitoxantrone, mitomycin C, nimustine, oxaliplatin, paclitaxel, pemetrexed, procarbazine, larcitrexed, temozolomide, teniposide, thioguanine, thiotepa, topotecan, vinblastine, vincristine, vindesine, and vinorelbine.

[0104] In some embodiments, the method may involve administering a sufficient amount of a multispecific or trispecific killer-engager compound as described herein, and administering at least one additional therapeutic agent, which exhibits a therapeutic synergistic effect. In some aspects of the method of the present invention, measurements of the response to treatment observed after administration of both a multispecific or trispecific killer-engager compound as described herein and an additional therapeutic agent are improved compared to the same measurements of the response to treatment observed after administration of either a multispecific or trispecific killer-engager compound or an additional therapeutic agent alone.

[0105] The term "subject" as used herein refers to any individual or subject on which the Method is carried out. In many embodiments, the subject is a human, but the subject may be any non-human animal. Preferred non-human animals include, but are not limited to, rodents (including mice, rats, hamsters, or guinea pigs), cats, dogs, rabbits, livestock (including cows, horses, goats, sheep, pigs, chickens, etc.), or vertebrates (including monkeys, chimpanzees, orangutans, or gorillas).

[0106] In some embodiments of this model, the anti-B7H3 multispecific compound may include an immune cell activation domain containing IL-15 or a functional portion thereof, operably linked to an NK-binding domain. In some embodiments, the multispecific anti-B7H3 compound may have an amino acid sequence such as that described in any one of SEQ ID NOs. 20-25.

[0107] In another aspect, this disclosure describes a chimeric antigen receptor compound comprising one of the anti-B7H3 proteins described herein. A chimeric antigen receptor (also known as a CAR, chimeric immune receptor, chimeric T cell receptor, or artificial T cell receptor) is a receptor protein that has been modified to confer a new ability to target specific proteins to T cells. The receptor is chimeric because it combines both antigen-binding and T cell-activating functions into a single receptor.

[0108] CAR-T cell therapy uses T cells that have been modified to possess a CAR for cancer treatment. The premise of CAR-T immunotherapy is to modify T cells to recognize cancer cells, thereby more effectively targeting and destroying them. T cells are collected from a donor (autologous or allogeneic), genetically modified, and then the resulting CAR-T cells are injected into the target to attack the target's tumor. CAR-T cells can originate from T cells in the target's own blood (autologous) or from T cells of a donor (allogeneic). Once isolated from the individual, these T cells are genetically modified to express a specific CAR, which is programmed to target antigens present on the surface of the tumor. For safety reasons, CAR-T cells are modified to be specific to antigens expressed on tumors that are not expressed on healthy cells. After the CAR-T cells are injected into the target, they act as a "living drug" against cancer cells. When CAR-T cells come into contact with their targeted antigens on a cell, they bind to the antigen, become activated, proliferate, and become cytotoxic. CAR-T cells destroy cells through several mechanisms, including broad-spectrum stimulated cell proliferation, which increases their toxicity to other living cells (cytotoxicity), and by inducing increased secretion of factors that can affect other cells (e.g., cytokines, interleukins, and / or growth factors).

[0109] In another aspect, this disclosure describes targeted therapeutic compounds comprising a target domain and a therapeutic domain linked to the target domain. The target domain includes any embodiment of the anti-B7H3 protein described herein. In some embodiments, the targeted therapeutic compound can provide immunotherapy and therefore may be a targeted immunotherapy compound. In some embodiments, the therapeutic domain may include a drug, a therapeutic radioisotope, a toxin, a cytokine, or a chemokine.

[0110] As used herein, the term “drug” refers to any chemical substance that, when administered to a living organism, produces a biological effect. A pharmaceutical is a chemical substance used to treat, cure, prevent or diagnose a disease, or to promote health. Drugs can be obtained through extraction from medicinal plants or by organic synthesis. Pharmaceuticals may be used for chronic disorders, for a limited duration or on a regular basis.

[0111] A "radioisotope" or "radionic nuclide" is an atom with excess nuclear energy that makes it unstable. This excess energy can be used in one of three ways: radiation from the nucleus as gamma rays; transfer of its electrons to one of its atoms to emit it as a conversion electron; or use to create and emit new particles (alpha or beta particles) from the nucleus. During these processes, the radionuclide is said to undergo radioactive decay. These emissions are considered ionizing radiation because they are strong enough to liberate electrons from other atoms. Radioactive decay can produce a stable nuclide or a new, unstable radionuclide that may sometimes undergo further decay.

[0112] As used herein, the term “toxin” refers to a substance harmful to cells. Toxins can be small molecules, peptides, or proteins that can cause disease or cell death upon contact with or absorption by body tissue. The toxicity of toxins can vary greatly. Toxins are primarily secondary metabolites, which are organic compounds that often assist organisms in matters of defense rather than being directly involved in the growth, development, or reproduction of organisms. In some applications, toxins may be used therapeutically by targeting the toxic effect on one or more undesirable cells (e.g., tumor cells).

[0113] Cytokines are a broad category of small molecules (approximately 5–20 kDa) involved in cellular signaling. Cytokines are peptides that can traverse the cellular lipid bilayer and cannot enter the cytoplasm, yet they are involved in autocrine, paracrine, and endocrine signaling as immunomodulators. Cytokines include chemokines, interferons, interleukins, lymphokines, and tumor necrosis factor, but generally do not include hormones or growth factors (although some overlap in terminology is observed). Cytokines are produced by a wide range of cells, including immune cells such as macrophages, B lymphocytes, T lymphocytes, mast cells, endothelial cells, fibroblasts, and various stromal cells. Cytokines regulate the equilibrium between humoral and cell-based immune responses and modulate the maturation, growth, and responsiveness of specific cell populations.

[0114] In further embodiments, this disclosure describes targeted imaging compounds comprising a target domain and an imaging domain linked to the target domain. The target domain includes any one embodiment of the anti-B7H3 protein described herein. The imaging domain may include any portion capable of generating a detectable signal. Exemplary imaging portions include, but are not limited to, colorimetric, fluorescent, radioactive, magnetic, or enzymatic labeling.

[0115] In yet another aspect, the disclosure describes a capture assay device comprising one arbitrary embodiment of the anti-B7H3 protein described herein, immobilized on a substrate. For example, the anti-B7H3 protein described herein can be incorporated into cell and / or ligand capture techniques, such as ELISA-based assays. The substrate for immobilizing the anti-B7H3 protein may include, for example, cell culture plates or dishes, glass slides, or any other support that can be used to perform assays requiring the immobilized anti-B7H3 protein.

[0116] In the foregoing description and the following claims, the term “and / or” means one or all of the listed elements, or any combination of two or more of the listed elements; “comprises,” “comprising,” and their variations should be interpreted as unrestricted, i.e., additional elements or steps are optional and may or may not be present; unless otherwise specified, “a,” “an,” “the,” and “at least one” are interchangeable and mean one or more; and an enumeration of numerical ranges by endpoints includes all numbers contained within that range (e.g., 1-5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).

[0117] In the foregoing description, specific embodiments may be described separately for clarity. Unless otherwise explicitly stated, a specific embodiment may include a combination of compatible features described herein, relating to one or more embodiments.

[0118] In the case of any method disclosed herein that includes separate steps, the steps may be performed in any possible order. Furthermore, any combination of two or more steps may be performed simultaneously, as appropriate.

[0119] Examples of examining tripspecific compounds containing B7H3-binding proteins, as considered for the applications discussed, are presented below. These examples are provided to further illustrate embodiments of the present invention, but are not intended to limit the scope of the invention. While they are typical where they may be used, other procedures, methods, or techniques known to those skilled in the art may be used instead. [Examples]

[0120] Example 1 Construction of the cam1615B7H3 triple-specific killer engagement compound The CDR region from anti-CD16 in a camelid (llama) (Behar et al., Protein Eng Des Sel. 2008;21(1):1-10.doi:10.1093 / protein / gzm064.PubMed PMID:18073223) was spliced ​​into a universal humanized heavy chain scaffold (Vincke et al., J Biol Chem. 2009;284(5):3273-84.doi:10.1074 / jbc.M806889200.PubMed PMID:19010777). Using this new humanized camelid sequence, a triple-specific killer engager for sdAb B7H3 was constructed. Using Hi-Fi DNA cloning technology, hybrid coding regions encoding cam16, (SGGGG)4 linker (SEQ ID NO: 27), wild-type rhIL-15, whitlow linker, and the described anti-B7H3 sdAb (CD276-new-2, SEQ ID NO: 2 in Figure 1B) were synthesized. The Biomedical Genomics Center, University of Minnesota, St. Paul, MN, validated the gene sequence and in-frame accuracy of the constructs. The gene products were amplified, and the vectors were transfected into Expi-293 cells. After 4–7 days, the supernatant was isolated and enriched using an HIS-column in the Akta Pure platform. Protein purity was determined by SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis) stained with Simply Blue Safe (Invitrogen, Carlsbad, CA).

[0121] cancer cell lines MA-148 (Geller et al., 2013. Cytotherapy 15(10):1297-1306) is a high-grade human epithelial serous ovarian cancer cell line. In in vivo experiments, the cell line was transfected with a luciferase reporter construct using a transfection reagent (lipofectamine reagent, Invitrogen, Carlsbad, CA), and selective pressure was applied with 10 μg / mL blastosidine. Ovarian cancer cells OVCAR-8 (RRID: CVCL_1629) were obtained from the DTP, DCTD Tumor Repository, supported by the Biological Testing Branch, Developmental Therapeutic Program, NCI, NIH. Other cell lines, including C4-2 (prostate; RRID: CVCL_4782), DU145 (prostate; RRID: CVCL_0105), LNCaP (prostate; RRID: CVCL_0395), PC-3 (prostate; RRID: CVCL_0035), A549 (lung; RRID: CVCL_0023), and NCI-H460 (lung; RRID: CVCL_0459), were obtained from the American Type Culture Collection. All lines were maintained in RPMI1640 RPMI supplemented with 10-20% fetal bovine serum (FBS) and 2 mmol / L L-glutamine. The lines were incubated at 37°C in a humid atmosphere containing 5% CO2. When adherent cells were confluent (over 90%), they were passaged using trypsin-EDTA for dissociation. For cell counting, a standard hemocytometer was used. For measurement by trypan blue exclusion, only cells with a viability of >95% were used in the experiment.

[0122] Cell products Peripheral blood mononuclear cells (PBMCs) were obtained from healthy volunteers or subjects after obtaining informed consent and approval from an Institutional Review Board (IRB), in accordance with the guidelines of the Committee on the Use of Human Subjects in Research and the Declaration of Helsinki. The cells were pelletized, red blood cells were lysed, and the cells were cryopreserved in 10% DMSO / 90% FBS and stored under liquid nitrogen.

[0123] Evaluation of cytotoxicity and NK cell activation Antibody-dependent cell-mediated cytotoxicity (ADCC) was measured by flow cytometry assays, evaluating degranulation and intracellular IFN-γ production mediated by CD107a (lysosomal membrane protein LAMP-1). After thawing, PBMCs or ascites cells derived from normal donors and controls were incubated overnight (37°C, 5% CO2) in RPMI1640 medium (RPMI-10) supplemented with 10% fetal bovine serum. The following morning, they were washed twice with RPMI-10 and then suspended in tumor target cells or culture medium. The cells were then incubated with a trispecific killer enhancer compound or control at 37°C for 10 minutes. Next, anti-human CD107a monoclonal antibody conjugated with fluorescein isothiocyanate (FITC) (BD Biosciences, San Jose, CA) was added and incubated for 1 hour. After incubation, GOLGISTOP (1:1,500, BD Biosciences, San Jose, CA) and GOLGIPLUG (1:1,000, BD Biosciences, San Jose, CA) were added over 3 hours (37°C, 5% CO2). After washing with phosphate-buffered saline, the cells were stained with PE / Cy7 conjugate anti-CD56 mAb, APC / Cy7 conjugate anti-CD16 mAb, and PE-CF594 conjugate anti-CD3 mAb (BioLegend, Inc., San Diego, CA). The cells were incubated at 4°C for 15 minutes, washed, and fixed with 2% paraformaldehyde. Next, cells were permeabilized with intracellular palm buffer (BioLegend, Inc., San Diego, CA), and IFN-gamma (IFN-γ) production was evaluated via detection using aBV650 conjugate anti-human IFN-γ antibody (BioLegend, Inc., San Diego, CA). The samples were washed and evaluated using an LSRII flow cytometer (BD Biosciences, San Jose, CA).

[0124] Real-time tumor killing assay Tumor killing was evaluated in real time using the INCUCYTE platform (Essen Biosciences, Inc., Ann Arbor, MI). 40,000 CD3 cells concentrated with magnetic beads were used. - CD56 + NK effector cells were seeded together with OVCAR8 spheroids stably expressing GFP into 96-well ULA plate tissue culture-treated microplates with a clear-bottom polystyrene base (Corning, Flintshire, UK), allowing 20,000 cells to establish spheroids within 3 days prior to co-culture. The described treatment was then added at a concentration of 30 nM, and the plates were placed in an INCUCYTE S3 platform (Essen Biosciences, Inc., Ann Arbor, MI) housed inside a cell incubator at 37°C / 5% CO2. Images from three technical replicates were acquired hourly over 120 hours using a 4x objective lens and then analyzed using INCUCYTE basic software (Essen Biosciences, Inc., Ann Arbor, MI). Graph readouts represent the integrated GFP fluorescence intensity of the spheroids, normalized to tumor alone at the start (0 hours).

[0125] statistical analysis All statistical tests were performed using PRISM software (GraphPad Software, Inc., La Jolla, CA). In all in vitro studies, significance was calculated using one-way ANOVA with repeated measures compared to the cam1615B7H3 group. The bars represent the mean ± standard error of the mean. Statistical significance is calculated as follows: * P<0.05, ** P<0.01, *** P<0.001, and **** This is expressed as P < 0.0001.

[0126] Example 2 A second-generation triple-specific killer-engager compound capable of both antibody-dependent cytotoxicity (ADCC) and NK cell expansion was constructed by modifying a previously reported triple-specific killer-engager compound platform (Vallera et al., Clin Cancer Res. 2016;22(14):3440-50.doi:10.1158 / 1078-0432.CCR-15-2710.PubMed PMID:26847056;PMCID:PMC4947440;US Patent Application Publication No. 2018 / 0282386A1). In the exemplary construct, a crosslinking agent between two modified adjacent regions of wild-type human IL-15 is inserted between two antibody fragments: an N-terminal VHH humanized camelid anti-CD16 fragment and a C-terminal anti-B7H3 VHH, or into a single-domain antibody, to create an anti-B7H3 trispecific killer engager, a schematic diagram of which is shown in Figure 1.

[0127] To evaluate the ability of exemplary anti-B7H3 protein-sdAb to target the activity of triple-specific killer engagers, anti-B7H3 triple-specific NK engager molecules were used to induce NK cell activity against B7H3-containing targets: prostate cancer cell lines, lung cancer cell lines, and ovarian cancer cell lines. Generally, triple-specific NK engager molecules recruit and activate NK cells against their target cell populations. NK activation is, for example, associated with the degranulation of natural killer (NK) cells (CD107a + It can be measured using a flow cytometry assay that detects NK cells and measures inflammatory cytokine production (e.g., IFN-γ).

[0128] Exemplary anti-B7H3 protein sequences were incorporated into a triple-specific killer engager scaffold and compared to triple-specific killer engagers incorporating the anti-B7H3 scFv sequence. NK cell degranulation (percent CD107a + (Measured as NK cells) and production of interferon-gamma (IFN-γ) +The percentage of NK cells (measured as a percentage) was evaluated in various cancer cell lines in the presence of various concentrations of trispecific killer promoters by flow cytometry as described in Example 1. Specifically, after incubating the trispecific killer promoter compound for 5 hours, NK cell degranulation and inflammatory cytokine production (IFN-γ) were measured by flow cytometry. The results presented represent the mean of N=3 independent experiments.

[0129] Exemplary anti-B7H3 proteins incorporated into a triple-specific killer-engager molecule, when co-cultured with peripheral blood mononuclear cells (PBMCs) at a concentration of 30 nM, enhanced NK cell degranulation and IFN-γ production in prostate cancer cells compared to PBMCs incubated alone (see Figures 2A and 2D), as shown in PC3 and DU145 cells (see Figures 2B, 2C, 2E, and 2F), and LnCAP and C4-2 cells (see Figures 3A, 3B, 3C, and 3D).

[0130] Figures 2 and 3 provide data demonstrating that a triplicate NK engager molecule containing an exemplary anti-B7H3 sdAb protein induces NK cell activity in a target cell population expressing B7H3. The anti-B7H3 triplicate NK engager induced NK cell degranulation and inflammatory cytokine production in multiple prostate cancer cell lines. In both NK activation measurements, the sdAb-containing triplicate NK engager (black bars) induced a greater response than the anti-B7H3 triplicate NK engager molecule containing anti-B7H3 scFv (gray bars).

[0131] Exemplary anti-B7H3 proteins incorporated into the triple-specific killer-engager molecule also enhanced NK cell degranulation and IFN-γ production in lung cancer cells when co-cultured with PBMCs at a concentration of 30 nM, as shown in A549 and NCI-H460 cells (see Figures 4C, 4F, 5A, and 5D), and ovarian cancer cells, as shown in OVCAR8 and MA148 cells (see Figures 5B, 5C, 5E, and 5F), compared to PBMCs incubated alone (see Figures 4A and 4D) or PBMCs co-cultured with C4-2 cells (see Figures 4B and 4E).

[0132] Figures 4 and 5 provide data demonstrating that a triplicate NK engager molecule containing exemplary anti-B7H3 sdAb protein induces NK cell activity against target cell populations expressing additional B7H3: prostate cancer cell line C4-2, lung cancer cell line A549 and NCI-H460, and ovarian cancer cell line OVCAR8 and MA148. Again, the triplicate NK engager containing sdAb (black bars) induced a greater response than the anti-B7H3 triplicate NK engager molecule containing anti-B7H3 scFv (gray bars).

[0133] Exemplary anti-B7H3 proteins incorporated into a triple-specific killer-engager molecule were demonstrated to enhance NK cell degranulation in the prostate, independently of the dose (see Figures 6A, 6B, and 6C), at three tested doses: 0.3, 3, and 30 nM (see Figures 6D, 6E, and 6F), compared to the absence of effect when PBMCs were incubated alone.

[0134] Similar effects were observed when IFN-γ production was evaluated; exemplary anti-B7H3 proteins incorporated into a triple-specific killer-engager molecule enhanced IFN-γ production in the prostate gland at three tested doses: 0.3; 3 and 30 nM (see Figures 7D, 7E, and 7F), independently of the dose (see Figures 7A, 7B, and 7C), compared to the absence of effect when PBMCs were incubated alone.

[0135] Figures 6 and 7 provide data demonstrating that triple-specific NK engager activity is target-dependent. Background NK activity is low in the absence of B7H3-expressing target cells. NK cell degranulation and IFN-γ production are induced when B7H3-expressing target cells are co-cultured with PBMC NK cells and triple-specific engager molecules containing anti-B7H3. Again, triple-specific NK engagers containing sdAb (black bars) induced a greater response than anti-B7H3 triple-specific NK engager molecules containing anti-B7H3 scFv (gray bars).

[0136] Next, the ability of a triplicate killer engager incorporating an exemplary anti-B7H3 protein to enhance cytolytic activity against ovarian cancer spheroids was evaluated in vitro, as described in Example 1. Specifically, in a spheroid assay, the exemplary anti-B7H3 protein was incorporated into the triplicate killer engager skeleton, and its activity was compared to that of NK cells alone. 20,000 GFP-expressing OVCAR8 cells were seeded in the wells of a 96-well ULA plate and allowed to form for 3 days. Then, 40,000 enriched NK cells were added either alone or with 30 nM of the triplicate killer engager. Images showing GFP intensity measurements were acquired hourly for 120 hours. It should be noted that cell death induced a transient increase in green fluorescence. Three technical replicates were performed for each of the three biological replicates. As shown in Figure 8 and further quantified in Figure 9, the triple-specific killer engager incorporating exemplary anti-B7H3 proteins enhanced cytolytic activity against ovarian cancer spheroids compared to the activity of NK cells alone.

[0137] Figures 8 and 9 provide data demonstrating the ability of a triply specific NK engager containing anti-B7H3-sdAb to kill ovarian cancer cells using an advanced imaging-based cytolysis assay. In this assay, 20,000 OVCAR8 ovarian cancer cells were stably transduced with a GFP cassette (to provide green fluorescence), then seeded in ultra-low adhesion (ULA) 96-well plates and allowed to form spheroids for 3 days. After 3 days, cells were added either without (tumor alone), with 40,000 NK cells (tumor + NK) accompanied by small amounts of cytokines to maintain survival, or with 40,000 NK cells and a 30 nM triply specific NK engager containing anti-B7H3-sdAb. Spheroid killing, measured as a decrease in the green fluorescence intensity of the spheroids, was observed over a period of 5 days (120 hours). When NK cells are treated with a trispecific NK engager molecule containing anti-B7H3 sdAb, they kill spheroids far more effectively than in the absence of trispecificity, suggesting that this trispecificity can induce three-dimensional tumorigenetic killing.

[0138] The complete disclosures of all patents, patent applications, and publications cited herein, as well as electronically available materials (including, for example, the submission of nucleotide sequences to GenBank and RefSeq, the submission of amino acid sequences to SwissProt, PIR, PRF, and PDB, and translations from annotated code areas in GenBank and RefSeq), are incorporated by reference in their entirety. In the event of any inconsistency between the disclosures of this application and the disclosures of any documents incorporated by reference herein, the disclosures of this application shall prevail. The above-mentioned detailed descriptions and examples are given solely for the purpose of clarifying understanding. No unnecessary limitations should be inferred from them. The present invention is not limited to the exact details shown and described, and variations that are obvious to those skilled in the art are included within the scope of the invention as defined by the claims.

[0139] Unless otherwise defined, all scientific and technical terms used herein have the same meanings as those commonly understood by those skilled in the art to which this invention belongs.

[0140] Unless otherwise indicated, all numbers used herein and in the claims, such as quantities and molecular weights of components, should be understood in all cases as being modified by the term "approximately." Therefore, unless otherwise indicated, the numerical parameters described herein and in the claims are approximations that may vary depending on the desired properties sought to be obtained by the present invention. Each numerical parameter should be interpreted, at least in light of the number of significant figures reported and by applying common rounding techniques, and not in any attempt to limit the principle of equivalents to the scope of the claims.

[0141] Although the numerical ranges and parameters representing the broad scope of the present invention are approximations, the numerical values ​​shown in the specific examples are reported as accurately as possible. However, all numerical values ​​inherently include the range that inevitably arises from the standard deviation found in each of those test measurements.

[0142] All headings should be used for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading (unless otherwise specified).

[0143] Although the present invention has been described with reference to the above embodiments, it will be understood that modifications and variations are included in the spirit and scope of the invention. Accordingly, the present invention is limited to the following claims.

[0144] Sequence listing free text Sequence ID 1 [ka] Sequence ID 2 [ka] Sequence ID 3 [ka] Sequence ID 4 SYWMY Sequence ID 5 INRDGSATWY ADSVKGRFT Sequence ID 6 DPDNYSSDEM VPY Sequence ID 7 MKWVTFISLL FLFSSAYS Sequence ID 8 VDEHHHHHHH HHH Sequence ID 9 [ka] Sequence ID 10 [ka] Sequence ID 11 [ka] Sequence ID 12 PSGQAGAAAS ESLFVSNHAY Sequence ID 13 EASGGPE Sequence ID 14 SGGGGSGGGG SGGGGSGGGG Sequence ID 15 GSTSGSGKPG SGEGSTKG Sequence ID 16 EPKSSDKTHT SPPSPEL Sequence ID 17 RATPSHNSHQ VPSAGGPTAN SGTSG Sequence ID 18 SSGGGGSGGG GGGSSRSSL Sequence ID 19 [ka] CDR1: Amino Acids 31-35 CDR2: Amino Acids 51-69 CDR3: Amino Acids 97-111 Sequence ID 20 [ka] Amino Acids 1-18: Leader Amino acids 19-140: Humanized camCD16 Amino acids 141-162: Linker Amino acids 163-284: Anti-B7H3 clone 1 Amino acids 285-297: VDE linker, 10XHis tag Sequence ID 21 [ka] Amino Acids 1-18: Leader Amino acids 19-140: Humanized camCD16 Amino acids 141-162: Linker Amino acids 163-284: Anti-B7H3 clone 2 Amino acids 285-297: VDE linker, 10XHis tag Sequence ID 22 [ka] Amino acids 1-122: Humanized camCD16 Amino acids 123-142: Linker Amino acids 143-258: IL-15 fragments Amino acids 259-276: Linker Amino acids 277-498: camB7H3 clone 1 Sequence ID 23 [ka] Amino acids 1-122: Humanized camCD16 Amino acids 123-142: Linker Amino acids 143-258: IL-15 fragments Amino acids 259-276: Linker Amino acids 277-398: camB7H3 clone 2 Sequence ID 24 [ka] Amino Acids 1-18: Leader Amino acids 19-140: Humanized camCD16 Amino acids 141-162: Linker Amino acids 163-276: IL-15 fragments Amino acids 277-294: Linker Amino acids 295-416: camB7H3 clone 1 Amino acids 417-429: VDE linker and 10Xhis tag Sequence ID 25 [ka] Amino Acids 1-18: Leader Amino acids 19-140: Humanized camCD16 Amino acids 141-162: Linker Amino acids 163-276: IL-15 fragments Amino acids 277-294: Linker Amino acids 295-416: camB7H3 clone 2 Amino acids 417-429: VDE linker and 10Xhis tag Sequence ID 26 [ka] Sequence ID 27 [ka] Sequence ID 28 [ka] Sequence ID 29 [ka] Sequence ID 30 [ka] Nucleotides 1-9: Kozak sequence Nucleotides 10-900: Encode sequence number 20 Sequence ID 31 [ka] Nucleotides 1-9: Kozak sequence Nucleotides 10-900: Encode sequence number 21 Sequence ID 32 [ka] Nucleotides 1-9: Kozak sequence Nucleotides 10-1296: Encode sequence number 24 Sequence ID 33 [ka] Nucleotides 1-9: Kozak sequence Nucleotides 10-1296: Encode sequence number 25

Claims

1. An anti-B7H3 polypeptide comprising at least one of SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6, or a functional variant thereof.

2. An anti-B7H3 polypeptide comprising SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, the CDR region of SEQ ID NO: 1, the CDR region of SEQ ID NO: 2, the CDR region of SEQ ID NO: 3, or a functional variant thereof.

3. Sequence ID 4, Sequence ID 5, Sequence ID 6, CDR area of ​​Sequence ID 1 CDR area of ​​sequence number 2, The CDR area of ​​sequence number 3, or Its functional variant A target domain containing an anti-B7H3 polypeptide; and An immune cell binding domain operably linked to the aforementioned target domain. A multispecific compound containing [specific compound name].

4. The multispecific compound according to claim 3, wherein the immune cells are T cells or natural killer (NK) cells.

5. The aforementioned immune cells are NK cells; and The immune cell binding domain includes a ligand or antibody that specifically binds to CD16. The multispecific compound according to claim 4.

6. The antibody that specifically binds to CD16 is scFv, F(ab) ２ The multispecific compound according to claim 5, comprising a Fab or a single-domain antibody (sdAb).

7. The immune cell binding domain includes SEQ ID NO: 19; and The target domain includes SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, A multispecific compound according to any one of claims 3 to 6.

8. The multispecific compound according to claim 7, wherein the target domain and the immune cell engager domain are linked by Sequence ID No.

14.

9. The multispecific compound according to claim 7, comprising amino acids 19-294 of SEQ ID NO: 20 or amino acids 19-284 of SEQ ID NO:

21.

10. A multispecific compound according to any one of claims 3 to 9, further comprising an immune cell activation domain.

11. The immune cells include NK cells; and The immune cell activation domain includes a cytokine or a functional portion thereof. The multispecific compound according to claim 10.

12. The multispecific compound according to claim 11, wherein the cytokine is IL-15 or a functional variant thereof.

13. Sequence ID 19; Sequence ID 11 is operably linked to Sequence ID 19; and A target domain operably linked to Sequence ID No. 19 and Sequence ID No. 11, the target domain comprising Sequence ID No. 1, Sequence ID No. 2, or Sequence ID No.

3. A multispecific compound according to any one of claims 10 to 12, comprising:

14. Sequence ID 19 and Sequence ID 11 are linked by Sequence ID 14; and Sequence ID 11 is linked to the target domain, and 1 or 2 is linked by Sequence ID 15. The multispecific compound according to claim 13.

15. The multispecific compound according to claim 14, as described in Sequence ID No. 22 or Sequence ID No.

23.

16. The multispecific compound according to any one of claims 12 to 15, wherein the functional variant of IL-15 comprises an N72D or N72A amino acid substitution compared to SEQ ID NO:

11.

17. An isolated nucleic acid sequence encoding a multispecific compound according to any one of claims 3 to 16.

18. The isolated nucleic acid sequence according to claim 17, which is one of sequence numbers 26 to 33, or a sequence having 90% identity with one of sequence numbers 26 to 33.

19. A protein encoded by any one of the nucleic acid sequences described in claim 17.

20. The protein according to claim 19, comprising the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, or any amino acid sequence having 90% or more identity thereto.

21. A host cell comprising the isolated nucleic acid according to claim 17 or claim 18.

22. The host cell according to claim 21, which is a T cell, an NK cell, or a macrophage.

23. The multispecific compound according to any one of claims 3 to 16; and Pharmacologically acceptable carriers A pharmaceutical composition containing the following:

24. To induce natural killer (NK)-mediated cellular killing, the compound must be multispecific in an effective amount. A target domain comprising the anti-B7H3 polypeptide described in claim 1 or claim 2; and NK binding domain operably linked to the target domain A method comprising administering a multispecific compound containing the above to a target.

25. The method according to claim 24, wherein the multispecific compound further comprises an immune cell activation domain containing IL-15 or a functional portion thereof, operably linked to the NK-binding domain.

26. The method according to claim 24, wherein the multispecific compound is as described in any one of Sequence IDs 20 to 25.

27. A method for stimulating the expansion of natural killer (NK) cells in vivo, A target domain comprising the anti-B7H3 polypeptide described in claim 1 or claim 2; and NK-binding domain operably linked to the anti-B7H3 polypeptide A method comprising administering a multispecific compound containing the compound to a subject in an effective amount.

28. The method according to claim 27, wherein the multispecific compound further comprises an immune cell activation domain containing IL-15 or a functional portion thereof, operably linked to the NK-binding domain.

29. The method according to claim 27, wherein the multispecific compound is as described in any one of Sequence ID No. 20 to 25.

30. A method of treating a person who has cancer or is at risk of having cancer, A target domain comprising the anti-B7H3 polypeptide described in claim 1 or claim 2; and NK-binding domain operably linked to the anti-B7H3 polypeptide A method comprising administering a multispecific compound containing the above to the subject in an effective amount.

31. The method according to claim 30, wherein the multispecific compound further comprises an immune cell activation domain containing IL-15 or a functional portion thereof, operably linked to the NK-binding domain.

32. The method according to claim 30, wherein the multispecific compound is as described in any one of Sequence ID No. 20 to 25.

33. The method according to claim 30, wherein cancer cells express B7H3.

34. The method according to claim 30, wherein the cancer includes prostate cancer, lung cancer, colon cancer, rectal cancer, bladder cancer, melanoma, kidney cancer, oral cancer, pharyngeal cancer, pancreatic cancer, uterine cancer, thyroid cancer, skin cancer, head and neck cancer, cervical cancer, ovarian cancer, or hematopoietic cancer.

35. The method according to claim 30, wherein the multispecific compound is administered before, simultaneously with, or after chemotherapy, surgical resection of a tumor, or radiotherapy.

36. The aforementioned chemotherapy regimens include altretamine, amsacrin, L-asparaginase, colaspase, bleomycin, busulfan, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, cyclophosphamide, cytophosphan, cytarabine, dacarbazine, dactinomycin, daunorubicin, docetaxel, doxorubicin, epirubicin, etoposide, fluorouracil, fludarabine, fotemustine, ganciclovir, and gem. The method according to claim 35, comprising cytabine, hydroxyurea, idarubicin, ifosphamide, irinotecan, lomustine, melphalan, mercaptopurine, methotrexate, mitoxantrone, mitomycin C, nimustine, oxaliplatin, paclitaxel, pemetrexed, procarbazine, larcitrexed, temozolomide, teniposide, thioguanine, thiotepa, topotecan, vinblastine, vincristine, vindesine, or vinorelbine.

37. A chimeric antigen receptor compound comprising the anti-B7H3 polypeptide described in claim 1 or claim 2.

38. A target domain comprising the anti-B7H3 polypeptide described in claim 1 or claim 2; and Immunotherapy domain linked to the aforementioned target domain Targeted immunotherapy compounds including these compounds.

39. A target domain comprising the anti-B7H3 polypeptide described in claim 1 or claim 2; and Therapeutic domain linked to the aforementioned target domain Targeted therapeutic compounds including these compounds.

40. The targeted therapeutic compound according to claim 37, wherein the therapeutic domain comprises a drug, a therapeutic radioisotope, a toxin, a cytokine, or a chemokine.

41. A target domain comprising the anti-B7H3 polypeptide described in claim 1 or claim 2; and Imaging domain linked to the target domain Targeted imaging compounds containing these compounds.

42. The targeted imaging compound according to claim 41, wherein the imaging domain includes a colorimetric label, a fluorescent label, a radioactive label, a magnetic label, or an enzyme label.

43. A capture assay device comprising an anti-B7H3 polypeptide according to claim 1 or claim 2, immobilized on a substrate.

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