VSIG8-based chimeric proteins

Abstract

To provide novel methods and compositions that provide effective immunotherapies.SOLUTION: The present invention relates, in part, to, chimeric proteins which include the extracellular domain of V-set and immunoglobulin domain-containing protein 8 (VSIG8) and their use in the treatment of diseases, such as immunotherapies for cancer and / or inflammatory diseases.SELECTED DRAWING: Figure 14A

Description

[Technical field]

[0001] Priority This application claims the benefit of and priority to U.S. Provisional Application No. 62 / 463,999, filed February 27, 2017, the contents of which are incorporated herein by reference in their entirety.

[0002] Description of electronically transmitted text files This application contains a sequence listing. The sequence listing was submitted electronically via EFS-Web as an ASCII text file entitled "SHK-003PC_SequenceListing_ST25". The sequence listing is 81,752 bytes in size and was created on or about February 27, 2018. The sequence listing is incorporated herein by reference in its entirety.

[0003] Technical Field The present invention relates, in part, to chimeric proteins comprising the extracellular domain of V-set and immunoglobulin domain-containing protein 8 (VSIG8) and their use in the treatment of disease, such as immunotherapy for cancer and / or inflammatory disease. [Background technology]

[0004] background Recent clinical data have demonstrated impressive patient responses to agents targeting immune co-inhibitory molecules, including, for example, the clinical trials that led to the approval of Yervoy, Keytruda, and Opdivo. These immunotherapies are collectively characterized as checkpoint inhibitors, and unfortunately, these treatments provide clinical benefit to only approximately 15-30% of cancer patients. One potential approach to improve clinical response rates for a broader population of cancer patients involves combining checkpoint inhibitor therapy with another treatment. Such combinations, when applied using multiple individual therapeutic agents, offer improved clinical benefit but are cumbersome to develop. Furthermore, many immunotherapies are associated with severe side effects that significantly narrow a patient's therapeutic window for treatment.

[0005] There remains a need for novel methods and compositions that provide effective immunotherapy, including the integration of multiple therapeutic mechanisms in a single agent. Summary of the Invention

[0006] overview Thus, the present invention provides compositions and methods for use in cancer therapy, in part, for example, by overcoming multiple inhibitory mechanisms in the tumor microenvironment and stimulating immune anti-tumor mechanisms.Similarly, the compositions and methods are used in the treatment of inflammatory diseases.

[0007] In embodiments, the chimeric proteins mask immunoinhibitory signals and / or enhance immunostimulatory signals in a single construct, hi embodiments, such immunomodulatory effects are achieved via direct receptor / ligand interactions.

[0008] For example, the present invention provides, in part, compositions and methods that allow for simultaneously inhibiting VISTA / VISIG8 signaling and stimulating OX40 / OX40L signaling in antigen presenting cells, such simultaneous VISIG8 blockade and OX40 agonism resulting in, inter alia, a general reduction in immune inhibitory cells and a shift to a more inflammatory environment, as well as increased anti-tumor effects.

[0009] In an embodiment, the present invention provides a heterologous chimeric protein comprising: (a) a first domain comprising a portion of a V set and an immunoglobulin domain-containing protein 8 (VSIG8) capable of binding to a VSIG8 ligand; (b) a second domain comprising a portion of an OX40 ligand (OX40L) capable of binding to an OX40L receptor; and (c) a linker connecting the first domain and the second domain. In an embodiment, the present invention provides a method of treating cancer with the heterologous chimeric protein. In an embodiment, the present invention provides a method of treating inflammatory disease with the heterologous chimeric protein.

[0010] In an embodiment, the present invention provides a recombinant fusion protein comprising the general structure of N-terminus-(a)-(b)-(c)-C-terminus, where (a) is a first domain comprising an extracellular domain of VSIG8 that is at least 95% identical to the amino acid sequence of SEQ ID NO:2 and can bind to VSIG8 ligand, (b) is a linker that links the first domain and the second domain and comprises a hinge-CH2-CH3 Fc domain derived from human IgG4 (e.g., 95% identical to the amino acid sequence of SEQ ID NO:25, SEQ ID NO:26, or SEQ ID NO:27), and (c) is a second domain comprising an extracellular domain of OX40 ligand (OX40L) that is at least 95% identical to the amino acid sequence of SEQ ID NO:4 and can bind to OX40L receptor. In an embodiment, the present invention provides a method of treating cancer with this heterologous chimeric protein. In an embodiment, the present invention provides a method of treating inflammatory disease with this heterologous chimeric protein.

[0011] In embodiments, the chimeric proteins of the invention are used in methods that can or involve shifting the balance of immune cells in favor of immune attack of tumors or any other undesirable cells. For example, the chimeric proteins of the invention can shift the proportion of immune cells at clinically important sites in favor of cells that can kill tumors (e.g., T cells, cytotoxic T lymphocytes, T helper cells, natural killer (NK) cells, natural killer T (NKT) cells, anti-tumor macrophages (e.g., M1 macrophages), B cells, and dendritic cells) and against cells that protect tumors (e.g., myeloid-derived suppressor cells (MDSCs), regulatory T cells (Tregs), tumor-associated neutrophils (TANs), M2 macrophages, and tumor-associated macrophages (TAMs)). In embodiments, the chimeric proteins enhance the recognition of tumor antigens by CD8+ T cells and / or enhance tumor infiltration by these T cells.

[0012] In aspects, the chimeric and / or recombinant fusion proteins of the invention are used in methods for treating cancer or inflammatory disease comprising administering to a patient in need thereof an effective amount of a pharmaceutical composition comprising the chimeric protein. In cancer treatment embodiments, for example, the chimeric and / or recombinant fusion proteins of the invention provide an immune memory response.

[0013] Embodiments include the use of the chimeric and / or recombinant fusion proteins of the invention in the manufacture of a medicament, for example, to treat cancer and / or inflammatory diseases.

[0014] Any aspect or embodiment described herein may be combined with any other aspect or embodiment disclosed herein. [Brief description of the drawings]

[0015] [Figure 1A]Without being bound by theory, the computer predicted secondary structure of the human VSIG8-Fc-OX40L chimeric protein is shown, with each of its three domains in their predicted native state. The predicted molecular weight of the chimeric protein is approximately 68.1 kDa. [Figure 1B] 1 shows the synapse formed between tumor cells and T cells by the chimeric proteins. [Diagram 2] Characterization of human VSIG8-Fc-OX40L chimeric protein by Western blot analysis under non-reducing, reducing, and reducing / deglycosylating (PNGase) conditions is shown. Specifically, each individual domain of the chimeric construct was probed with anti-VSIG8, anti-Fc, or anti-OX40L antibodies. An untreated sample of hVSIG8-Fc-OX40L chimeric protein, e.g., control, was loaded in lane 2 of all blots (without β-mercaptoethanol or PNGase). Samples in lane 3 of all blots were treated with a reducing agent, β-mercaptoethanol, and samples in lane 4 of all blots were treated with amidase peptide:N-glycosidase enzyme. The band size confirms the expected monomeric molecular weight of approximately 68.1 kDa, suggesting that the native state of the chimeric protein is a glycosylated dimer. [Diagram 3] Graphs of functional ELISA demonstrating binding of human VSIG8-Fc-OX40L to human IgG (left panel) and recombinant OX40 (right panel) are shown. [Figure 4] Graph showing in vitro cell binding of human VSIG8-Fc-OX40L to an OX40-expressing cell line (i.e., Jurkat cells engineered to overexpress human OX40). Binding was performed in vitro and analyzed by flow cytometry. [Diagram 5]1 is a graph showing the binding affinity of octet to human VSIG8-Fc-OX40L. The binding affinity of VSIG8-Fc-OX40L to OX40 was measured by biolayer interferometry (Octet) in comparison to a commercial single-sided human Fc-OX40L control or tavolixumab (anti-human OX40 antibody). The data show that VSIG8-Fc-OX40L binds to human OX40 with high affinity as measured at 767 pM (top curve: tavolixumab, middle curve: VSIG8-Fc-OX40L, bottom curve: single-sided human Fc-OX40L). [Figure 6] 1 is a graph showing an in vitro NF-κB / NIK signaling assay using human VSIG8-Fc-OX40L chimeric protein. U2OS cells from the DiscoverX NIK signaling assay were cultured with either 30 μg / mL of commercial single-sided Fc-OX40L, anti-OX40 antibody, anti-OX40L antibody, or hVSIG8-Fc-OX40L chimeric protein. Bars, from left to right, are single-sided Fc-OX40L, anti-OX40L antibody, anti-OX40 antibody, and hVSIG8-Fc-OX40L chimeric protein. [Figure 7] Graph showing Staphylococcal Enterotoxin B (SEB) superantigen activation / cytokine release assay. Human PBMCs were cultured with Staphylococcal Enterotoxin B (200 ng / mL) with or without a titration of commercial human Fc-OX40L half-side, commercial VSIG8-Fc half-side, a combination of the two half-side molecules, or human VSIG8-Fc-OX40L chimeric protein. After 3 days, supernatants were assessed using an ELISA specific for human IL2. [Figure 8] Characterization of human VSIG8-Fc-OX40L chimeric protein by Western blot analysis under non-reducing, reducing, and reducing / deglycosylating (PNGase) conditions. The band size confirms the expected monomer molecular weight of approximately 68.1 kDa, suggesting that the native state of the chimeric protein is a glycosylated dimer. [Figure 9A-C]ELISA assays demonstrating the binding affinity of different domains of mouse VSIG8-Fc-OX40L chimeric protein to their respective binding partners. Figure 9A shows the binding and detection of mVSIG8-Fc-OX40L chimeric protein to VISTA, a binding partner of VSIG8. As there is no commercially available mVSIG8-Fc standard, a standard curve was not generated. Figure 9B shows the binding and detection of the Fc portion of mVSIG8-Fc-OX40L chimeric protein to plate-bound anti-human IgG antibody. Mouse Ig (mIg) was used as standard (circle symbols). Detection was via HRP-conjugated anti-human IgG antibody. It was observed that in general in ELISA assays, detecting chimeric proteins using the central Fc region tends to underestimate the actual protein content in the sample. Thus, lower levels of VSIG8-Fc-OX40L chimeric protein (square symbols) were detected compared to the standard (circle symbols). Figure 9C shows the binding and detection of mVSIG8-Fc-OX40L chimeric protein to plate-bound recombinant mouse OX40, a binding partner of OX40L, and detection via an OX40L-specific antibody. Mouse OX40L was used as a standard (circle symbol). [Figure 10A-B] 10A-10B are graphs showing in vitro cell binding assays of mouse VSIG8-Fc-OX40L to VISTA- and OX40-expressing cell lines. Immortalized cell lines were engineered to stably express (FIG. 10A) mouse VISTA (EL4-mVISTA) or (FIG. 10B) mouse OX40 (CHOK1-mOX40). Increasing concentrations of mVSIG8-Fc-OX40L were incubated with each parental and overexpressing cell line for 2 hours. Cells were harvested, washed, and stained with antibodies for detection of chimeric protein binding by flow cytometry. All engineered cell lines bound mVSIG8-Fc-OX40L in a concentration-dependent manner with in vitro cell binding affinities of low nM. The binding affinity of mVSIG8-Fc-OX40L to CHOK1 / mOX40 was 16 nM, and the binding affinity of mVSIG8-Fc-OX40L to EL4 / mVISTA was 56 nM. [Figure 11]Graph showing Octet-based evaluation of binding affinity of mouse VSIG8-Fc-OX40L to mouse OX40-His. Either 10 μg / mL of commercially available mFc-OX40L (lower curve) or mVSIG8-Fc-OX40L chimeric protein (upper curve) bound to a penta-his biosensor coated with recombinant mouse OX40-his. [Figure 12] 1 is a graph showing an in vitro NF-κB luciferase signaling assay using mouse VSIG8-Fc-OX40L chimeric protein. Jurkat cells engineered to express NF-κB luciferase and OX40 were cultured with 18 nM of either an irrelevant protein (negative control, left bar), commercial single-sided Fc-OX40L (middle bar), or mVSIG8-Fc-OX40L (right bar). [Figure 13A-C] Immunophenotyping from tumor-bearing mice treated with murine VSIG8-Fc-OX40L. Mice were inoculated with CT26 tumors on day 0. When tumors were palpable and at least 4-6 mm in diameter, mice were treated with two doses of 150 μg mVSIG8-Fc-OX40L chimeric protein. Immune phenotyping was performed on various tissues of a subset of mice from each treatment group on day 13 after tumor implantation. The data demonstrate that mice treated with mVSIG8-Fc-OX40L chimeric protein exhibited a higher percentage of total CD4+ T cells in the spleen, tumor-draining lymph nodes (TDLN), and tumors (Figure 13A), and that this increase consisted mostly of an increase in CD4+CD25- T cells (Figure 13B). Further analysis was performed by tetramer staining to analyze the fraction of CD8+ T cells that recognize the AH1 tumor antigen naturally expressed by CT26 tumors. A higher percentage of these T cells were found to recognize the AH1 tumor antigen in mice treated with mVSIG8-Fc-OX40L compared to other groups, both in the spleen and in the tumor (Figure 13C). For each panel in Figure 13A-C, the conditions were either untreated or treated with mouse VSIG8-Fc-OX40L. [Figure 14A-C]Figure 14 shows the antitumor effect of murine VSIG8-Fc-OX40L against colorectal CT26 tumors. Balb / c mice were inoculated with CT26 tumors on day 0. After 4 days of tumor growth, when tumors reached 4-5 mm in diameter, mice were treated with either control antibody or mVSIG8-Fc-OX40L chimeric protein. Treatment was then repeated again on day 7. Figure 14A shows the individual tumor growth curves for each treatment group, Figure 14B shows the overall survival rate up to day 50 of the experiment, and Figure 14C is a table summarizing group size and treatment outcome for each group. [Figure 15] FIG. 1 is a graph showing an ELISA-based anti-drug antibody (ADA) assay of mouse VSIG8-Fc-OX40L chimeric protein. [Figure 16] Four potential configurations of an exemplary chimeric protein (PD1-Fc-OX40L) are shown. [Figure 17] Western blots of PD-1-Fc-OX40L chimeric protein run on SDS-PAGE under non-reducing conditions, reducing conditions, and reducing conditions followed by treatment with peptide-N-glycosidase F (PNGase F). [Figure 18] Chromatograph of PD-1-Fc-OX40L chimeric protein run by size exclusion chromatography (SEC). [Figure 19] Shown are SDS-PAGE and native (non-SDS) PAGE gels of PD-1-Fc-OX40L chimeric protein run under non-reducing conditions ("-") or reducing conditions ("+"). [Figure 20] A native (non-SDS) PAGE gel of PD-1-No-Fc-OX40L chimeric protein lacking the Fc domain in the linker is shown. [Figure 21] Without being bound by theory, a model of how hexamers and concatemers are formed from the chimeric proteins of the present invention is presented. [Figure 22]1 is a table showing binding linkers and Fc linkers that can be combined into exemplary modular linkers that can be combined with any of the type I and type II proteins described herein and / or the extracellular domains of the type I and type II proteins described herein to form chimeric proteins of the invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] Detailed Description The present invention is based in part on the discovery of engineered chimeric proteins that include an extracellular domain of V-set and immunoglobulin domain-containing protein 8 (VSIG8) near the amino terminus. In an embodiment, the chimeric protein further includes an extracellular domain of OX40 ligand (OX40L) near the carboxy terminus. In an embodiment, the two extracellular domains are linked by a linker. In an embodiment, the chimeric proteins of the present invention mask immune inhibitory signals on tumor cells, replacing them with immune stimulatory signals for effective treatment of cancer.

[0017] Chimeric Proteins In an embodiment, the present invention relates to a chimeric protein engineered to contain the immune inhibitory receptor V-set and extracellular domain of immunoglobulin domain-containing protein 8 (VSIG8). VSIG8 is a single-pass type I membrane protein that functions as a receptor for V-domain immunoglobulin-containing suppressor of T-cell activation (VISTA). Specifically, the human VSIG8 protein contains 414 amino acids, including a 21 amino acid signal sequence, two Ig-like V-type (immunoglobulin-like) domains containing a 242 amino acid extracellular domain (ECD), a 21 amino acid transmembrane domain, and a 130 amino acid cytoplasmic domain.

[0018] In an embodiment, the chimeric protein of the invention comprises a domain, e.g., the extracellular domain, of human VSIG8. Human VSIG8 comprises the amino acid sequence of SEQ ID NO:1 (with the amino acid sequence of the extracellular domain comprising SEQ ID NO:2).

[0019] In embodiments, a chimeric protein of the invention may comprise the extracellular domain of VSIG8 (e.g., SEQ ID NO:2) described herein, or a variant or functional fragment thereof. For example, the chimeric protein may be at least about 60%, or at least about 61%, or at least about 62%, or at least about 63%, or at least about 64%, or at least about 65%, or at least about 66%, or at least about 67%, or at least about 68%, or at least about 69%, or at least about 70%, or at least about 71%, or at least about 72%, or at least about 73%, or at least about 74%, or at least about 75%, or at least about 76%, or at least about 77%, or at least about 78%, or at least about 79%, or at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or at least about 99%, or at least about 100%, or at least about 101%, or at least about 102%, or at least about 103%, or at least about 104%, or at least about 105%, or at least about 106%, or at least about 107%, or at least about 108%, or at least about 109%, or at least about 110, or at least about 111, or at least about 112, or at least about 113, or at least about The VSIG8 or variant or functional fragment thereof may comprise the sequence of the extracellular domain of the above-mentioned VSIG8, or a variant or functional fragment thereof, having at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity thereto.

[0020] In embodiments, a chimeric protein of the invention may comprise a mutant extracellular domain of VSIG8 in which the signal peptide (e.g., as provided in SEQ ID NO: 1) is replaced with an alternative signal peptide. In embodiments, a chimeric protein of the invention may comprise a mutant extracellular domain of VSIG8 expressed from a cDNA that has been codon-optimized for expression in protein-producing cells, such as Chinese hamster ovary (CHO) or human embryonic kidney (HEK) cells.

[0021] In embodiments, the extracellular domain of VSIG8 refers to a portion of a protein that can interact with the extracellular environment. In embodiments, the extracellular domain of VSIG8 is the entire amino acid sequence of a protein that is on the outside of a cell or in the cell membrane. In embodiments, the extracellular domain of VSIG8 is a portion of the amino acid sequence of a protein that is on the outside of a cell or in the cell membrane and is required for signal transduction and / or ligand binding, as can be assayed using methods known in the art (e.g., in vitro ligand binding and / or cell activation assays).

[0022] In embodiments, the extracellular domain of VSIG8 refers to a portion of the protein that can bind to V-region immunoglobulin-containing suppressor of T cell activation (VISTA). VISTA is a negative checkpoint regulator involved in suppressing activation of resting T cells, including CD4+ or CD8+ T cells. Binding of VSIG8 to VISTA induces an inhibitory effect on T cell activation, proliferation and / or immune cytokine production. In embodiments, the chimeric protein has a K of less than about 1 μM, about 900 nM, about 800 nM, about 700 nM, about 600 nM, about 500 nM, about 400 nM, about 300 nM, about 200 nM, about 100 nM, about 90 nM, about 80 nM, about 70 nM, about 60 nM, about 55 nM, about 50 nM, about 45 nM, about 40 nM, about 35 nM, about 30 nM, about 25 nM, about 20 nM, about 15 nM, about 10 nM, or about 5 nM, or about 1 nM. DIn embodiments, the chimeric protein binds to human VISTA with a K of less than about 1 nM, about 900 pM, about 800 pM, about 700 pM, about 600 pM, about 500 pM, about 400 pM, about 300 pM, about 200 pM, about 100 pM, about 90 pM, about 80 pM, about 70 pM, about 60 pM, about 55 pM, about 50 pM, about 45 pM, about 40 pM, about 35 pM, about 30 pM, about 25 pM, about 20 pM, about 15 pM, or about 10 pM, or about 1 pM. D In embodiments, the chimeric protein binds to human VISTA with a K of about 200 pM to about 600 pM (e.g., as measured by surface plasmon resonance or biolayer interferometry). D to human VISTA (eg, as measured by surface plasmon resonance or biolayer interferometry).

[0023] In an embodiment, the chimeric protein of the present invention further comprises a domain, for example, an extracellular domain, of the immunostimulatory molecule OX40 ligand (OX40L). OX40L is a type II transmembrane glycoprotein belonging to the tumor necrosis factor (TNF) superfamily. Specifically, human OX40L protein comprises 183 amino acids, including an amino-terminal cytoplasmic domain (amino acids 1-23) and a carboxy-terminal extracellular domain (amino acids 51-183).

[0024] In an embodiment, the chimeric protein of the present invention comprises the extracellular domain of human OX40L. Human OX40L comprises the amino acid sequence of SEQ ID NO:3 (having the amino acid sequence of the extracellular domain comprising SEQ ID NO:4).

[0025] In an embodiment, the chimeric protein of the present invention may comprise the extracellular domain of OX40L (e.g., SEQ ID NO: 4) described herein, or a variant or functional fragment thereof. For example, the chimeric protein may be at least about 60%, or at least about 61%, or at least about 62%, or at least about 63%, or at least about 64%, or at least about 65%, or at least about 66%, or at least about 67%, or at least about 68%, or at least about 69%, or at least about 70%, or at least about 71%, or at least about 72%, or at least about 73%, or at least about 74%, or at least about 75%, or at least about 76%, or at least about 77%, or at least about 78%, or at least about 79%, or at least about The extracellular domain of OX40L or a variant or functional fragment thereof may comprise a sequence having 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity thereto.

[0026] In embodiments, the chimeric proteins of the invention may comprise a mutant extracellular domain of OX40L in which the signal peptide (e.g., as provided in SEQ ID NO: 3) is replaced with another signal peptide. In embodiments, the chimeric proteins of the invention may comprise a mutant extracellular domain of OX40L expressed from a cDNA that has been codon-optimized for expression in protein-producing cells, such as CHO or HEK cells.

[0027] In an embodiment, the extracellular domain of OX40L refers to the part of a protein that can interact with the extracellular environment.In an embodiment, the extracellular domain of OX40L is the entire amino acid sequence of a protein that is on the outside of a cell or in a cell membrane.In an embodiment, the extracellular domain of OX40L is the part of the amino acid sequence of a protein that is required for signal transduction and / or ligand binding that is on the outside of a cell or in a cell membrane and can be assayed using methods known in the art.

[0028] In an embodiment, the extracellular domain of OX40L refers to the part of the protein that can bind to OX40 receptor.Like other TNF superfamily members, membrane-bound OX40L exists as a homotrimer.OX40L binds to OX40, a member of the TNF receptor superfamily that is mainly expressed on CD4+ and / or CD8+ T cells and some lymphoid and non-lymphoid cells.Evidence suggests that the main function of OX40-OX40L interaction is to transmit late costimulatory signals to promote the survival and proliferation of activated T cells and prolong immune response.

[0029] In embodiments, the chimeric proteins of the invention have a K of less than about 1 μM, about 900 nM, about 800 nM, about 700 nM, about 600 nM, about 500 nM, about 400 nM, about 300 nM, about 200 nM, about 100 nM, about 90 nM, about 80 nM, about 70 nM, about 60 nM, about 55 nM, about 50 nM, about 45 nM, about 40 nM, about 35 nM, about 30 nM, about 25 nM, about 20 nM, about 15 nM, about 10 nM, or about 5 nM, or about 1 nM. D In embodiments, the chimeric protein binds to human OX40 with a K of less than about 1 nM, about 900 pM, about 800 pM, about 700 pM, about 600 pM, about 500 pM, about 400 pM, about 300 pM, about 200 pM, about 100 pM, about 90 pM, about 80 pM, about 70 pM, about 60 pM, about 55 pM, about 50 pM, about 45 pM, about 40 pM, about 35 pM, about 30 pM, about 25 pM, about 20 pM, about 15 pM, or about 10 pM, or about 1 pM. DIn embodiments, the chimeric protein binds to human OX40 with a K of about 200 pM to about 600 pM (e.g., as measured by surface plasmon resonance or biolayer interferometry). D binds to human OX40 at 100 .ANG. (as measured, for example, by surface plasmon resonance or biolayer interferometry).

[0030] In embodiments, the chimeric protein may comprise an amino acid sequence having one or more amino acid mutations relative to any of the protein sequences described herein, in embodiments, the chimeric protein comprises a sequence having about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 or more amino acid mutations relative to any one of the amino acid sequences of the chimeric proteins disclosed herein.

[0031] In embodiments, the one or more amino acid mutations may be independently selected from a substitution, an insertion, a deletion, and a truncation.

[0032] In embodiments, the amino acid mutations are amino acid substitutions, and can include conservative and / or non-conservative substitutions.

[0033] "Conservative substitutions" may be made, for example, on the basis of similarity in polarity, charge, size, solubility, hydrophobicity, hydrophilicity, and / or the amphipathic nature of the amino acid residues involved. The 20 naturally occurring amino acids can be grouped into the following six standard amino acid groups: (1) hydrophobic: Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr; Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe.

[0034] As used herein, "conservative substitution" is defined as the replacement of an amino acid with another amino acid listed in the same group of the six standard amino acid groups shown above. For example, the replacement of Asp with Glu retains one negative charge in the polypeptide so modified. In addition, glycine and proline can be substituted for each other based on their ability to disrupt α-helices.

[0035] As used herein, a "non-conservative substitution" is defined as the replacement of an amino acid with another amino acid listed in a different group of the six standard amino acid groups (1) to (6) shown above.

[0036] In embodiments, substitutions may also include non-classical amino acids (e.g., in general, designer amino acids such as selenocysteine, pyrrolysine, N-formylmethionine, β-alanine, GABA and δ-aminolevulinic acid, 4-aminobenzoic acid (PABA), D-isomers of common amino acids, 2,4-diaminobutyric acid, α-aminoisobutyric acid, 4-aminobutyric acid, Abu, 2-aminobutyric acid, γ-Abu, ε-Ahx, 6-aminohexanoic acid, Aib, 2-aminoisobutyric acid, 3-aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosme, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, β-alanine, fluoroamino acids, β-methyl amino acids, Cα-methyl amino acids, Nα-methyl amino acids, and amino acid analogs).

[0037] Mutations can also be made in the nucleotide sequence of the chimeric protein by reference to the genetic code, including taking into account codon degeneracy.

[0038] In embodiments, the chimeric proteins of the invention can be variants as described herein, for example, the chimeric proteins of the invention have an amino acid sequence similar to that of one or more of the amino acid sequences of the chimeric proteins of the invention, e.g., SEQ ID NO:5 and SEQ ID NO:6, that is at least about 60%, or at least about 61%, or at least about 62%, or at least about 63%, or at least about 64%, or at least about 65%, or at least about 66%, or at least about 67%, or at least about 68%, or at least about 69%, or at least about 70%, or at least about 71%, or at least about 72%, or at least about 73%, or at least about 74%, or at least about 75%, or at least about The nucleic acid sequence may have a sequence having 76%, or at least about 77%, or at least about 78%, or at least about 79%, or at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity.

[0039] In an embodiment, the chimeric protein comprises a linker. In an embodiment, the linker comprises at least one cysteine ​​residue capable of forming a disulfide bond. As described elsewhere herein, such at least one cysteine ​​residue capable of forming a disulfide bond is involved, without being bound by theory, in maintaining the proper multimer state of the chimeric protein and allowing efficient production.

[0040] In an embodiment, a chimeric protein of the invention comprises (a) a first domain comprising a portion of a V set and immunoglobulin domain-containing protein 8 (VSIG8), e.g., an extracellular domain of VSIG8 capable of binding to VISTA; (b) a second domain comprising a portion of OX40L, e.g., an extracellular domain of OX40L capable of binding to OX40; and, optionally, (c) a linker connecting the first and second domains.

[0041] In embodiments, a chimeric protein is a recombinant fusion protein, e.g., a single polypeptide having an extracellular domain (and optionally a linker) as described herein. For example, in embodiments, a chimeric protein is translated as a single unit in a cell. In embodiments, a chimeric protein refers to multiple polypeptides, e.g., recombinant proteins of multiple extracellular domains as described herein, that are linked in vitro (e.g., using one or more synthetic linkers as described herein) to provide a single unit. In embodiments, a chimeric protein can be chemically synthesized as one polypeptide, or each domain can be chemically synthesized separately and then combined. In embodiments, a chimeric protein is partially translated and partially chemically synthesized.

[0042] In an embodiment, the chimeric protein comprises a linker. In an embodiment, the linker can be derived from a naturally occurring multidomain protein or is an experimental linker, for example, as described in Chichili et al., (2013), Protein Sci. 22(2): 153-167, Chen et al., (2013), Adv Drug Deliv Rev. 65(10): 1357-1369, the entire contents of which are incorporated herein by reference. In an embodiment, the linker can be designed using linker designing databases and computer programs, such as those described in Chen et al., (2013), Adv Drug Deliv Rev. 65(10): 1357-1369 and Crasto et al., (2000), Protein Eng. 13(5): 309-312, the entire contents of which are incorporated herein by reference.

[0043] In embodiments, the linker is a synthetic linker, such as PEG.

[0044] In an embodiment, the linker comprises a polypeptide. In an embodiment, the polypeptide is less than about 500 amino acids in length, about 450 amino acids in length, about 400 amino acids in length, about 350 amino acids in length, about 300 amino acids in length, about 250 amino acids in length, about 200 amino acids in length, about 150 amino acids in length, or about 100 amino acids in length. For example, the linker can be less than about 100, about 95, about 90, about 85, about 80, about 75, about 70, about 65, about 60, about 55, about 50, about 45, about 40, about 35, about 30, about 25, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 12, about 11, about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, or about 2 amino acids in length. In an embodiment, the linker is flexible. In an embodiment, the linker is rigid.

[0045] In embodiments, the linker is substantially composed of glycine and serine residues (e.g., about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90%, or about 95%, or about 97%, or about 98%, or about 99%, or about 100% glycine and serine).

[0046] In an embodiment, the linker comprises a hinge region of an antibody (e.g., IgG, IgA, IgD, and IgE, including subclasses (e.g., IgG1, IgG2, IgG3, and IgG4, and IgA1 and IgA2)). The hinge region found in antibodies of the IgG, IgA, IgD, and IgE classes functions as a flexible spacer, allowing the Fab portion to move freely in space. In contrast to the constant region, the hinge domain is structurally diverse, varying in both sequence and length between immunoglobulin classes and subclasses. For example, the length and flexibility of the hinge region varies between IgG subclasses. The hinge region of IgG1 encompasses amino acids 216-231, and because it is freely flexible, the Fab fragments can rotate around their axis of symmetry and move within a sphere centered on the first of the disulfide bridges between the two heavy chains. IgG2 has a shorter hinge than IgG1, with 12 amino acid residues and four disulfide bridges. The hinge region of IgG2 lacks glycine residues, is relatively short, and contains a rigid polyproline double helix stabilized by an additional inter-heavy chain disulfide bridge. These properties limit the flexibility of the IgG2 molecule. IgG3 differs from other subclasses by its unique extended hinge region (approximately four times longer than the IgG1 hinge), which contains 62 amino acids (including 21 prolines and 11 cysteines) and forms an inflexible polyproline double helix. In IgG3, the Fab fragment is relatively far from the Fc fragment, giving the molecule more flexibility. The extended hinge in IgG3 is also responsible for its higher molecular weight compared to other subclasses. The hinge region of IgG4 is shorter than that of IgG1, and its flexibility is intermediate between that of IgG1 and IgG2. The flexibility of the hinge region has been reported to decrease in the order IgG3>IgG1>IgG4>IgG2. In embodiments, the linker is derived from human IgG4 and may contain one or more mutations to enhance dimerization (including S228P) or FcRn binding.

[0047] According to crystallographic studies, immunoglobulin hinge regions can be functionally subdivided into three regions: the upper hinge region, the core region, and the lower hinge region. See Shin et al., 1992 Immunological Reviews 130:87. The upper hinge region is H1 The upper hinge region contains amino acids from the carboxyl terminus of the heavy chain to the first residue in the hinge that restricts movement, generally the first cysteine ​​residue that forms an interchain disulfide bond between the two heavy chains. The length of the upper hinge region correlates with the flexibility of that segment of the antibody. The core hinge region contains the interchain disulfide bridges, while the lower hinge region contains the C H2 Binds to the amino terminus of the domain, C H2 The core hinge region of wild-type human IgG1 contains the sequence CPPC (SEQ ID NO: 48), which upon dimerization by disulfide bond formation results in a cyclic octapeptide that is believed to act as a pivot, thus providing flexibility. In embodiments, the linker comprises one, two, or three of the upper hinge region, core region, and lower hinge region of any antibody (e.g., IgG, IgA, IgD, and IgE, including subclasses (e.g., IgG1, IgG2, IgG3, and IgG4, and IgA1 and IgA2)). The hinge region may also contain one or more glycosylation sites, including several structurally distinct types of sites for carbohydrate attachment. For example, IgA1 contains five glycosylation sites within a 17 amino acid segment of the hinge region, conferring resistance of the hinge region polypeptide to intestinal proteases, a property believed to be advantageous for secretory immunoglobulins. In an embodiment, a linker of the invention comprises one or more glycosylation sites.

[0048] In embodiments, the linker comprises an Fc domain of an antibody (e.g., IgG, IgA, IgD, and IgE, including subclasses (e.g., IgG1, IgG2, IgG3, and IgG4, and IgA1 and IgA2). In embodiments, the linker comprises a hinge-CH2-CH3 Fc domain derived from a human IgG4 antibody. In embodiments, the linker comprises a hinge-CH2-CH3 Fc domain derived from a human IgG1 antibody. In embodiments, the Fc domain exhibits increased affinity and enhanced binding to the neonatal Fc receptor (FcRn). In embodiments, the Fc domain comprises one or more mutations that increase affinity for FcRn and enhance binding to FcRn. Without being bound by theory, it is believed that the increased affinity and enhanced binding to FcRn increases the in vivo half-life of the chimeric proteins of the invention.

[0049] In embodiments, the Fc domain linker contains one or more amino acid substitutions at amino acid residues 250, 252, 254, 256, 308, 309, 311, 416, 428, 433, or 434 (according to the Kabat numbering in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md. (1991), expressly incorporated herein by reference), or their equivalents. In embodiments, the amino acid substitution at amino acid residue 250 is with glutamine. In embodiments, the amino acid substitution at amino acid residue 252 is with tyrosine, phenylalanine, tryptophan, or threonine. In embodiments, the amino acid substitution at amino acid residue 254 is with threonine. In an embodiment, the amino acid substitution at amino acid residue 256 is with serine, arginine, glutamine, glutamic acid, aspartic acid, or threonine. In an embodiment, the amino acid substitution at amino acid residue 308 is with threonine. In an embodiment, the amino acid substitution at amino acid residue 309 is with proline. In an embodiment, the amino acid substitution at amino acid residue 311 is with serine. In an embodiment, the amino acid substitution at amino acid residue 385 is with arginine, aspartic acid, serine, threonine, histidine, lysine, alanine, or glycine. In an embodiment, the amino acid substitution at amino acid residue 386 is with threonine, proline, aspartic acid, serine, lysine, arginine, isoleucine, or methionine. In an embodiment, the amino acid substitution at amino acid residue 387 is with arginine, proline, histidine, serine, threonine, or alanine. In an embodiment, the amino acid substitution at amino acid residue 389 is with a proline, serine, or asparagine. In an embodiment, the amino acid substitution at amino acid residue 416 is with a serine.In an embodiment, the amino acid substitution at amino acid residue 428 is with leucine. In an embodiment, the amino acid substitution at amino acid residue 433 is with arginine, serine, isoleucine, proline, or glutamine. In an embodiment, the amino acid substitution at amino acid residue 434 is with histidine, phenylalanine, or tyrosine.

[0050] In an embodiment, the Fc domain linker (e.g., comprising an IgG constant region) comprises one or more mutations, such as substitutions at amino acid residues 252, 254, 256, 433, 434, or 436 (according to the Kabat numbering in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md. (1991), expressly incorporated herein by reference). In an embodiment, the IgG constant region comprises three M252Y / S254T / T256E mutations or a YTE mutation. In another embodiment, the IgG constant region comprises three H433K / N434F / Y436H mutations or a KFH mutation. In an embodiment, the IgG constant region comprises a combination of YTE and KFH mutations.

[0051] In embodiments, the modified humanized antibodies of the invention comprise an IgG constant region that contains one or more mutations at amino acid residues 250, 253, 307, 310, 380, 428, 433, 434, and 435 (according to the Kabat numbering in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md. (1991), which is expressly incorporated herein by reference). Exemplary mutations include T250Q, M428L, T307A, E380A, I253A, H310A, M428L, H433K, N434A, N434F, N434S, and H435A. In embodiments, the IgG constant region comprises an M428L / N434S mutation or an LS mutation. In an embodiment, the IgG constant region comprises N428L / N434S mutations or LS mutations. In an embodiment, the IgG constant region comprises T250Q / M428L mutations or QL mutations. In an embodiment, the IgG constant region comprises N434A mutations. In an embodiment, the IgG constant region comprises T307A / E380A / N434A mutations or AAA mutations. In an embodiment, the IgG constant region comprises I253A / H310A / H435A mutations or IHH mutations. In an embodiment, the IgG constant region comprises H433K / N434F mutations in combination. In an embodiment, the IgG constant region comprises M252Y / S254T / T256E mutations and H433K / N434F mutations.

[0052] Additional exemplary mutations in IgG constant regions are described, for example, in Robbie et al., Antimicrobial Agents and Chemotherapy (2013), 57(12):6147-6153, Dall'Acqua et al., JBC (2006), 281(33):23514-24, Dall'Acqua et al., Journal of Immunology (2002), 169:5171-80, Ko et al., Nature (2014) 514:642-645, Grevys et al., Journal of Immunology. (2015), 194(11):5497-508, and U.S. Patent No. 7,083,784, the entire contents of which are incorporated herein by reference.

[0053] In an embodiment, the Fc domain in the linker comprises SEQ ID NO:25 (see table below), or an amino acid sequence having at least 90%, or 93%, or 95%, or 97%, or 98%, or 99% identity to SEQ ID NO:25. In an embodiment, mutations are made to SEQ ID NO:25 to increase stability and / or half-life. For example, in an embodiment, the Fc domain in the linker comprises SEQ ID NO:26 (see table below), or an amino acid sequence having at least 90%, or 93%, or 95%, or 97%, or 98%, or 99% identity to SEQ ID NO:26. An exemplary Fc stabilizing mutant is S228P. Exemplary Fc half-life extending mutants are T250Q, M428L, V308T, L309P, and Q311S, and the linker may comprise one, or two, or three, or four, or five of these mutants. In embodiments, the Fc domain in the linker comprises SEQ ID NO:27 (see table below), or an amino acid sequence having at least 90%, or 93%, or 95%, or 97%, or 98%, or 99% identity to SEQ ID NO:27.

[0054] Additionally, one or more binding linkers may be used to link the Fc domain (e.g., one of SEQ ID NO:25, SEQ ID NO:26, or SEQ ID NO:27, or having at least 90%, or 93%, or 95%, or 97%, or 98%, or 99% identity thereto) and the extracellular domain in the linker. For example, any one of SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, or variants thereof may link the extracellular domain described herein and the linker described herein. Optionally, any one of SEQ ID NOs:28-74, or variants thereof, is placed between the extracellular domain described herein and the linker described herein.

[0055] The amino acid sequences of SEQ ID NOs:25-74 are provided in Table 1 below.

[0056] [Table 1] JPEG2025065443000003.jpg194158

[0057] In embodiments, the conjugated linker comprises substantially glycine and serine residues (e.g., about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90%, or about 95%, or about 97%, or about 98%, or about 99%, or about 100% glycine and serine). For example, in embodiments, the conjugated linker is (Gly4Ser) n where n is from about 1 to about 8, e.g., 1, 2, 3, 4, 5, 6, 7, or 8 (SEQ ID NOs: 49 to 56, respectively). In an embodiment, the conjugated linker sequence is GGSGGGGGGGGGGGGS (SEQ ID NO: 57). Additional exemplary conjugated linkers include the sequences LE, (Gly)8 (SEQ ID NO: 58), (Gly)6 (SEQ ID NO: 59), (EAAAK) n (n = 1 to 3) (SEQ ID NO: 60 to SEQ ID NO: 62), A(EAAAK) nA(n=2 to 5) (SEQ ID NO: 63 to SEQ ID NO: 66), A(EAAAK)4ALEA(EAAAK)4A (SEQ ID NO: 67), PAPAP (SEQ ID NO: 68), KESGSVSSEQLAQFRSLD (SEQ ID NO: 69), GSAGSAAGSGEF (SEQ ID NO: 70), and (XP) n In some embodiments, the linker is GGS.

[0058] In embodiments, the conjugated linker is one or more of GGGSE (SEQ ID NO: 71), GSESG (SEQ ID NO: 72), GSEGS (SEQ ID NO: 73), GEGGSGEGSSGEGSSSEGGGSEGGGSEGGGSEGGS (SEQ ID NO: 74), and a conjugated linker in which G, S, and E are randomly spaced at intervals of every 4 amino acids.

[0059] In an embodiment, the chimeric protein comprises a modular linker as shown in FIG.

[0060] In embodiments, the linker may be functional. For example, but not limited to, the linker may function to improve the folding and / or stability, improve expression, improve pharmacokinetics, and / or improve bioactivity of the chimeric protein of the present invention. In another example, the linker may function to target the chimeric protein to a particular cell type or location.

[0061] In embodiments, the chimeric protein exhibits enhanced stability and protein half-life. In embodiments, the chimeric protein binds to FcRn with high affinity. In embodiments, the chimeric protein has a K of about 1 nM to about 80 nM. DFor example, the chimeric protein may bind to FcRn with a K of about 1 nM, about 2 nM, about 3 nM, about 4 nM, about 5 nM, about 6 nM, about 7 nM, about 8 nM, about 9 nM, about 10 nM, about 15 nM, about 20 nM, about 25 nM, about 30 nM, about 35 nM, about 40 nM, about 45 nM, about 50 nM, about 55 nM, about 60 nM, about 65 nM, about 70 nM, about 71 nM, about 72 nM, about 73 nM, about 74 nM, about 75 nM, about 76 nM, about 77 nM, about 78 nM, about 79 nM, or about 80 nM. D In embodiments, the chimeric protein can bind to FcRn with a K of about 9 nM. D In embodiments, the chimeric protein does not substantially bind to other Fc receptors that have effector functions (i.e., other than FcRn).

[0062] In an embodiment, a chimeric protein having the formula ECD 1-binding linker 1-Fc domain-binding linker 2-ECD 2, where ECD 1 is VSIG8 and ECD 2 is OX40L, may be referred to in the present disclosure as VSIG8-Fc-OX40L. In an embodiment, the chimeric protein lacks one or both binding linkers, and such chimeric protein may also be referred to in the present disclosure as VSIG8-Fc-OX40L. These chimeric proteins may lack one or both binding linkers. Exemplary binding linkers 1s, Fc domains, and binding linkers 2s are described in Table 1 above, and modular linkers including specific binding linkers 1s, Fc domains, and binding linkers 2s useful for forming chimeric proteins are shown in FIG. 22.

[0063] In embodiments, the methods result in a memory response that may be capable of, for example, preventing relapse.

[0064] In embodiments, the chimeric protein is a fusion protein having the formula N-terminus-(a)-(b)-(c)-C-terminus, where (a) is VSIG8, (b) is a linker comprising at least a portion of an Fc domain, and (c) is OX40L, and may be referred to in the present disclosure as VSIG8-Fc-OX40L.

[0065] In an embodiment, the chimeric protein is optimized / directed against a mouse ligand / receptor. An example of such a chimeric protein is mouse VSIG8-Fc-OX40L, also referred to herein as mVSIG8-Fc-OX40L.

[0066] In embodiments, the chimeric protein is optimized / directed against a human ligand / receptor. An example of such a chimeric protein is human VSIG8-Fc-OX40L, also referred to herein as hVSIG8-Fc-OX40L.

[0067] In an embodiment, the chimeric protein of the present invention targets the VISTA / VSIG8 immune inhibitory signaling pathway. In an embodiment, the chimeric protein disrupts, blocks, reduces, and / or inhibits the transmission of immune inhibitory signals mediated by the binding of VISTA to VSIG8. In an embodiment, immune inhibitory signals refer to signals that reduce or eliminate immune responses. For example, in an oncology context, such signals may reduce or eliminate anti-tumor immunity. Under normal physiological conditions, inhibitory signals are also useful in maintaining self-tolerance (e.g., preventing autoimmunity) and to protect tissues from damage when the immune system is responding to pathogenic infections. For example, but not limited to, immune inhibitory signals can be identified by detecting increased cell proliferation, cytokine production, cell killing activity, or phagocytic activity when such inhibitory signals are blocked.

[0068] In embodiments, the chimeric proteins of the invention disrupt, block, reduce, and / or inhibit the delivery of immune inhibitory signals mediated by the binding of VISTA or other binding partners to VSIG8. In embodiments, the chimeric proteins bind to VISTA but disrupt, block, reduce, and / or inhibit inhibitory signaling to immune cells (e.g., T cells). In embodiments, the chimeric proteins act, for example, on lymphocyte cells that express VISTA and disrupt, block, reduce, and / or inhibit inhibitory signaling to immune cells (e.g., T cells).

[0069] In embodiments, the chimeric proteins of the invention can reduce, disrupt or eliminate binding of the immune inhibitory receptor / ligand pair VISTA / VSIG8 or are used in methods that include reducing, disrupting or eliminating it. In embodiments, the chimeric proteins of the invention block, reduce and / or inhibit VSIG8 and / or binding of VSIG8 to VISTA or other binding partners.

[0070] In an embodiment, the chimeric protein of the present invention targets the immunostimulatory signal mediated by the binding of OX40L to OX40. In an embodiment, the chimeric protein enhances, increases, and / or stimulates the transmission of the immunostimulatory signal mediated by the binding of OX40L to OX40. In an embodiment, the immunostimulatory signal refers to a signal that enhances immune response. For example, in the context of oncology, such a signal can enhance anti-tumor immunity. For example, but not limited to, the immunostimulatory signal can be specified by directly stimulating the proliferation, cytokine production, killing activity, or phagocytic activity of leukocytes, including a subset of T cells.

[0071] In an embodiment, the chimeric protein of the present invention enhances, increases, and / or stimulates the transmission of immune stimulatory signals mediated by the binding of OX40L to OX40. In an embodiment, the chimeric protein of the present invention comprising the extracellular domain of OX40L acts on immune cells (e.g., T cells) that express OX40, and enhances, increases, and / or stimulates stimulatory signaling to the immune cells (e.g., T cells).

[0072] In embodiments, the chimeric proteins of the invention can stimulate or enhance or are used in methods that include stimulating or enhancing binding of the immunostimulatory receptor / ligand pair OX40:OX40L. In embodiments, the chimeric proteins of the invention increase and / or stimulate binding of OX40 to one or more of OX40 and / or OX40L.

[0073] In embodiments, the chimeric protein comprises the extracellular domain of a type II protein other than OX40L. Exemplary type II proteins include 4-1BBL, CD30L, CD40L, FasL, GITRL, LIGHT, TL1A, and TRAIL. The invention further includes chimeric proteins and methods of using the following chimeric proteins: VISIG8 / 4-1BBL, VISIG8 / CD30L, VISIG8 / CD40L, VISIG8 / FasL, VISIG8 / GITRL, VISIG8 / LIGHT, VISIG8 / TL1A, and VISIG8 / TRAIL. In embodiments, the chimeric protein has one of the general structures: VISIG8-Fc-4-1BBL, VISIG8-Fc-CD30L, VISIG8-Fc-CD40L, VISIG8-Fc-FasL, VISIG8-Fc-GITRL, VISIG8-Fc-LIGHT, VISIG8-Fc-TL1A, and VISIG8-Fc-TRAIL.

[0074] The amino acid sequences of 4-1BBL, CD30L, CD40L, FasL, GITRL, LIGHT, TL1A, and TRAIL include SEQ ID NOs: 7, 9, 11, 13, 15, 17, 21, and 23, respectively.

[0075] In embodiments, the chimeric protein comprises the extracellular domain of one of 4-1BBL, CD30L, CD40L, FasL, GITRL, LIGHT, TL1A, and TRAIL, including SEQ ID NOs: 8, 10, 12, 14, 16, 18, 22, and 24, respectively. In embodiments, the chimeric protein of the invention may comprise the extracellular domain of 4-1BBL, CD30L, CD40L, FasL, GITRL, LIGHT, TL1A, or TRAIL, or a variant or functional fragment thereof, as described herein. For example, the chimeric protein may be at least about 60%, or at least about 61%, or at least about 62%, or at least about 63%, or at least about 64%, or at least about 65%, or at least about 66%, or at least about 67%, or at least about 68%, or at least about 69%, or at least about 70%, or at least about 71%, or at least about 72%, or at least about 73%, or at least about 74%, or at least about 75%, or at least about 76%, or at least about 77%, or at least about 78%, or at least about 79%, or at least about The antibody may comprise an extracellular domain sequence of 4-1BBL, CD30L, CD40L, FasL, GITRL, LIGHT, TL1A, or TRAIL, or a variant or functional fragment thereof, having about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity thereto, as provided above.

[0076] In embodiments, the chimeric proteins of the invention comprise variants of the extracellular domains described herein and have at least about 60%, or at least about 61%, or at least about 62%, or at least about 63%, or at least about 64%, or at least about 65%, or at least about 66%, or at least about 67%, or at least about 68%, or at least about 69%, or at least about 70%, or at least about 71%, or at least about 72%, or at least about 73%, or at least about 74%, or at least about 75%, similar to the known amino acid or nucleic acid sequences of the extracellular domains, e.g., one or more of SEQ ID NOs: 2, 4, 8, 10, 12, 14, 16, 18, 20, 22, and 24. or at least about 75%, or at least about 76%, or at least about 77%, or at least about 78%, or at least about 79%, or at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity.

[0077] In an embodiment, the chimeric protein of the invention comprises the extracellular domain of VSIG8 (SEQ ID NO:2).

[0078] In an embodiment, the chimeric protein of the invention comprises the extracellular domain of OX40L (SEQ ID NO: 4).

[0079] In an embodiment, the chimeric protein of the invention comprises the extracellular domain of CD40L (SEQ ID NO: 12).

[0080] In an embodiment, a chimeric protein of the invention comprises the extracellular domain of VSIG8 (SEQ ID NO: 2) and the extracellular domain of OX40L (SEQ ID NO: 4).

[0081] In an embodiment, a chimeric protein of the invention comprises the extracellular domain of VSIG8 (SEQ ID NO: 2) and the extracellular domain of CD40L (SEQ ID NO: 12).

[0082] In an embodiment, a chimeric protein of the invention comprises the hinge-CH2-CH3 domains from a human IgG4 antibody sequence (SEQ ID NO: 25, 26, or 27).

[0083] In an embodiment, the chimeric protein comprises a modular linker as shown in FIG.

[0084] In an embodiment, a chimeric protein of the invention comprises the extracellular domain of VSIG8 and the extracellular domain of OX40L using the hinge-CH2-CH3 domains from a human IgG4 antibody sequence as a linker (this VSIG8-Fc-OX40L chimera is SEQ ID NO: 5).

[0085] In an embodiment, a chimeric protein of the invention comprises the extracellular domain of VSIG8 and the extracellular domain of CD40L using the hinge-CH2-CH3 domains from a human IgG4 antibody sequence as a linker (this VSIG8-Fc-CD40L chimera is SEQ ID NO: 6).

[0086] In embodiments, the chimeric proteins of the present invention deliver immune stimulatory signals to immune cells (e.g., T cells) while masking immune inhibitory signals. In embodiments, the chimeric proteins deliver signals that have the end result of immune activation (e.g., T cell activation).

[0087] In embodiments, the chimeric proteins of the invention can promote and be used in methods that include promoting immune activation (e.g., against tumors). In embodiments, the chimeric proteins of the invention can suppress and be used in methods that include suppressing immune inhibition (e.g., allowing tumors to survive). In embodiments, the chimeric proteins of the invention provide improved immune activation and / or improved suppression of immune inhibition due to the signaling proximity provided by the chimeric nature of the construct.

[0088] In embodiments, the chimeric proteins of the invention can be used in methods that include modulating the amplification of an immune response, e.g., modulating or modulating the level of effector output. In embodiments, for example, when used to treat cancer and / or inflammatory diseases, the chimeric proteins of the invention alter the degree of immunostimulation compared to immunoinhibition to increase the amplification of T cell responses, including, but not limited to, stimulating increased levels of cytokine production, proliferation, or target killing capacity.

[0089] In an embodiment, the chimeric protein of the present invention is used in a method that can mask an inhibitory ligand and replace the immune inhibitory ligand with an immune stimulatory ligand, or that includes masking and replacing it. For example, a chimeric protein construct of the present invention comprising (i) the extracellular domain of VSIG8 and (ii) the extracellular domain of OX40L allows for the disruption of the inhibitory VISTA / VSIG8 signal and replacing it with a stimulatory OX40L / OX40 signal. Thus, the chimeric protein of the present invention is used in an embodiment that can reduce or eliminate an inhibitory immune signal and / or increase or activate an immune stimulatory signal, or that includes reducing or eliminating an inhibitory immune signal and / or increasing an immune stimulatory signal. For example, lymphocytes or other cells that carry an inhibitory signal (and thus evade an immune response) can then be replaced with a positive signal that binds on T cells that can attack tumor cells. Thus, in an embodiment, the inhibitory immune signal is masked by the construct of the present invention and the stimulatory immune signal is activated. Such beneficial properties are enhanced by the single construct approach of the chimeric protein of the present invention. For example, signal replacement can be performed nearly simultaneously, and signal replacement is tailored to be localized at clinically important sites (e.g., tumor microenvironment). In embodiments, the constructs localize positive immune stimulatory signals near sequestering or inhibitory signals to better link these functional groups. This local linking effect may explain the superior performance of the VSIG8-Fc-OX40L construct, for example, compared to the combination of a VISTA blocking antibody and an OX40 agonist antibody.

[0090] In embodiments, the chimeric proteins of the invention can enhance, restore, promote, and / or stimulate immune regulation or are used in methods that include enhancing, restoring, promoting, and / or stimulating immune regulation. In embodiments, the chimeric proteins of the invention described herein restore, promote, and / or stimulate the activity or activation of one or more immune cells against tumor cells, including, but not limited to, T cells, cytotoxic T lymphocytes, T helper cells, natural killer (NK) cells, natural killer T (NKT) cells, anti-tumor macrophages (e.g., M1 macrophages), B cells, and dendritic cells. In embodiments, the chimeric proteins of the invention enhance, restore, promote and / or stimulate T cell activity and / or activation, including by activating and / or stimulating one or more T cell intrinsic signals, including, by way of non-limiting examples, pro-survival signals; autocrine or paracrine growth signals; p38 MAPK, ERK, STAT, JAK, AKT or PI3K mediated signals; anti-apoptotic signals; and / or signals promoting and / or required for one or more of inflammatory cytokine production, or T cell migration, or T cell tumor infiltration.

[0091] In embodiments, the chimeric proteins of the invention can cause or are used in methods that include causing recruitment of one or more of T cells (including but not limited to cytotoxic T lymphocytes, T helper cells, natural killer T (NKT) cells), B cells, natural killer (NK) cells, natural killer T (NKT) cells, dendritic cells, monocytes, and macrophages (e.g., one or more of M1 and M2) into a tumor or tumor microenvironment. In embodiments, the chimeric proteins enhance recognition of tumor antigens by CD8+ T cells, particularly T cells that have infiltrated the tumor microenvironment.

[0092] In embodiments, the chimeric proteins of the invention can inhibit and / or cause a decrease in the recruitment of, or are used in methods that include inhibiting and / or causing a decrease in the recruitment of, immunosuppressive cells (e.g., myeloid-derived suppressor cells (MDSCs), regulatory T cells (Tregs), tumor-associated neutrophils (TANs), M2 macrophages, and tumor-associated macrophages (TAMs), particularly within the tumor and / or tumor microenvironment (TME). In embodiments, the therapy can alter the ratio of M1 to M2 macrophages at the tumor site and / or TME in favor of M1 macrophages.

[0093] In embodiments, the chimeric proteins of the present invention can inhibit and / or reduce T cell inactivation and / or immune resistance to tumors, and can be used in methods that include inhibiting and / or reducing the same, comprising administering to a subject an effective amount of the chimeric proteins described herein. In embodiments, the chimeric proteins of the present invention can increase serum levels of various cytokines, including, but not limited to, one or more of IFNγ, TNFα, IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, IL-17A, IL-17F, and IL-22. In embodiments, the chimeric proteins of the present invention can enhance IL-2, IL-4, IL-5, IL-10, IL-13, IL-17A, IL-22, TNFα, or IFNγ in the serum of a treated subject. Detection of such cytokine responses can provide a method for determining optimal dosing regimens for a given chimeric protein.

[0094] In an embodiment, the chimeric protein of the present invention inhibits, blocks and / or reduces cell death of anti-tumor CD8+ and / or CD4+ T cells; or stimulates, induces and / or increases cell death of tumor-promoting T cells. T cell exhaustion is a state of T cell dysfunction characterized by progressive loss of proliferation and effector functions, leading to clonal deletion. Thus, tumor-promoting T cells refer to a state of T cell dysfunction that occurs during many chronic infections, inflammatory diseases, and cancers. This dysfunction is defined by insufficient proliferation and / or effector functions, persistent expression of inhibitory receptors, and a transcriptional state that differs from that of functional effector or memory T cells. Exhaustion prevents optimal control of infections and tumors. In addition, anti-tumor CD8+ and / or CD4+ T cells refer to T cells that can mount an immune response against tumors. Exemplary tumor-promoting T cells include, but are not limited to, Tregs, CD4+ and / or CD8+ T cells expressing one or more checkpoint inhibitory receptors, Th2 cells, and Th17 cells. Checkpoint inhibitor receptors refer to receptors expressed on immune cells that prevent or inhibit uncontrolled immune responses.

[0095] In an embodiment, the chimeric proteins of the present invention can be used in methods that can increase the ratio of effector T cells to regulatory T cells. An exemplary effector T cell is ICOS + Effector T cells; cytotoxic T cells (e.g., αβ TCR, CD3 + , CD8 + , CD45RO + );CD4 + Effector T cells (e.g., αβ TCR, CD3 + , CD4 + , CCR7 + , CD62Lhi, IL-7R / CD127 + );CD8 + Effector T cells (e.g., αβ TCR, CD3 + , CD8 + , CCR7 + , CD62Lhi, IL-7R / CD127 +); effector memory T cells (e.g., CD62L low, CD44 + , TCR, CD3 + , IL-7R / CD127 + , IL-15R + , CCR7 low); central memory T cells (e.g., CCR7 + , CD62L + , CD27 + or CCR7hi, CD44 + , CD62Lhi, TCR, CD3 + , IL-7R / CD127 + , IL-15R + );CD62L + Effector T cells; early effector memory T cells (CD27 + CD62L - ) and late effector memory T cells (CD27 - CD62L - ) (TemE and TemL, respectively) + Effector memory T cells (TEM); CD127 ( + )CD25(low / -) effector T cells;CD127( - )CD25( - ) Effector T cells; CD8 + Stem cell memory effector cells (TSCM) (e.g., CD44(low)CD62L(high)CD122(high)sca( + )); TH1 effector T cells (e.g., CXCR3 + , CXCR6 + and CCR5 + ; or αβ TCR, CD3 + , CD4 + , IL-12R + , IFN-γR + , CXCR3 + ), TH2 effector T cells (e.g., CCR3 + , CCR4 + and CCR8 + ; or αβ TCR, CD3 + , CD4 + , IL-4R + , IL-33R + , CCR4 + , IL-17RB+ , CRTH2 + ); TH9 T cells (eg TCR, αβ TCR, CD3 + 、CD4 + );TH17 T cells (eg TCR, αβ TCR, CD3 + 、CD4 + IL-23R + 、CCR6 + , IL-1R + );CD4 + CD45RO + CCR7 + Effect T cells, CD4 + CD45RO + CCR7( - ) T cells that secrete IL-2, IL-4 and IFN-γ and contain T cells that secrete IL-2, IL-4 and IFN-γ. Exemplary regulatory T cells, ICOS + Controlled T cells, CD4 + CD25 + FOXP3 + Controlled T cells, CD4 + CD25 + Controlled T cells, CD4 + CD25 - Controlled T cells, CD4 + CD25 high-control T cells, TIM-3 + PD-1 + Controlled T cells, linpakal activation gene-3 (LAG-3) + Regulatory T cells, CTLA-4 / CD152 + Regulatory T cells, nippon-1 (Nrp-1) + Controlled T cells, CCR4 + CCR8 + Controller T cells, CD62L (Lセレクチン) + Regulatory T cells, CD45RB low Regulatory T cells, CD127 low Regulatory T cells, LRRC32 / GARP + Regulatory T cells, CD39 + Regulatory T cells, GITR + Regulatory T cells, LAP + Controlled T cells, 1B11 + Controlled T cells, BTLA +Regulatory T cells, type 1 regulatory T cells (Tr1 cells), T helper type 3 (Th3) cells, regulatory cells of natural killer T cell phenotype (NKTreg), CD8+ regulatory T cells, CD8 + CD28 - This includes regulatory T cells and / or regulatory T cells secreting IL-10, IL-35, TGF-β, TNF-α, Galectin-1, IFN-γ and / or MCP1.

[0096] In an embodiment, the chimeric protein of the present invention causes an increase in effector T cells (e.g., CD4+CD25- T cells). In an embodiment, the chimeric protein causes a decrease in regulatory T cells (e.g., CD4+CD25+ T cells).

[0097] In embodiments, the chimeric proteins provide a memory response that may, for example, prevent or protect animals from recurrence and / or prevent or reduce the likelihood of metastasis. Thus, animals treated with the chimeric proteins can attack tumor cells and / or prevent tumor development when subsequently exposed to relevant antigens after initial treatment with the chimeric proteins. Thus, the chimeric proteins of the present invention stimulate both active tumor destruction and immune recognition of tumor antigens, which is essential for programming a memory response that can prevent recurrence.

[0098] In embodiments, the chimeric proteins of the invention can stimulate and can be used in methods that include stimulating effector immune cells transiently for about 12 hours, about 24 hours, about 48 hours, about 72 hours, or about 96 hours, or about one week, or about two weeks or less. In embodiments, the chimeric proteins of the invention can stimulate and can be used in methods that include depleting or inhibiting regulatory or immune inhibitory cells transiently for about 12 hours, about 24 hours, about 48 hours, about 72 hours, or about 96 hours, or about one week, or about two weeks or less. In embodiments, the transient stimulation of effector T cells and / or the transient depletion or inhibition of immune inhibitory cells occurs substantially in the patient's bloodstream or in specific tissues / locations including lymphatic tissues, such as bone marrow, lymph nodes, spleen, thymus, mucosa-associated lymphoid tissue (MALT), non-lymphoid tissues, or in the tumor microenvironment.

[0099] In embodiments, the chimeric proteins of the present invention provide advantages including, but not limited to, ease of use and ease of production, since two different immunotherapeutic agents are combined into a single product allowing for a single manufacturing process instead of two independent manufacturing processes. In addition, administration of a single agent instead of two separate agents allows for easier administration and greater patient compliance.

[0100] In an embodiment, the chimeric proteins of the invention can be produced in mammalian host cells as secretable, fully functional single polypeptide chains.

[0101] In embodiments, the chimeric proteins of the present invention unexpectedly exhibit slow off-rates (Kd or K off) provides binding of the extracellular domain components to their respective binding partners. In embodiments, this provides an unexpectedly long interaction of the receptor to the ligand and vice versa. Such an effect allows for a persistent negative signal masking effect. Also, in embodiments, this delivers a longer positive signal effect, for example, to allow effector cells to be sufficiently stimulated for anti-tumor effects. For example, the chimeric proteins of the present invention, for example, via long off-rate binding, allow sufficient signaling to provide T cell proliferation and enable anti-tumor attack. As a further example, the chimeric proteins of the present invention, for example, via long off-rate binding, allow sufficient signaling to provide release of stimulatory signals, such as cytokines.

[0102] The stable synapse of cells (e.g., a cell with a negative signal and a T cell that can be stimulated to attack a tumor) promoted by the agents of the present invention provides a spatial orientation that favors tumor regression, such as positioning the T cell to attack tumor cells and / or sterically preventing tumor cells from delivering negative signals, including negative signals beyond those masked by the chimeric protein of the present invention.

[0103] In an embodiment, this is the serum T 1 / 2 Longer on-target (e.g., intratumoral) half-life (t 1 / 2 Such properties may have the combined advantage of reducing off-target toxicity associated with systemic distribution of the chimeric protein.

[0104] In embodiments, the chimeric proteins of the present invention provide a biphasic immune effect, providing a rapid response and a latent response. For example, direct T cell costimulation via OX40 or inhibition of signaling via VSIG8 may result in immediate enhancement of antitumor activity mediated by T cells (or other immune cells) and improve short-term control of tumor growth and rejection. In embodiments, the early dual interaction between immune cells and VSIG8-Fc-OX40L modifies the strength or quality of immune priming and contributes to enhanced development of long-lived memory T cell immunity. Such events may be evident in tumor models with enhanced long-term control or equilibrium of established tumors. In some cases, the immune response programmed by treatment with VSIG8-Fc-OX40L may contribute to delayed tumor rejection long-term after administration of the molecule.

[0105] Also, in embodiments, the chimeric proteins of the invention provide a synergistic therapeutic effect allowing for improved site-specific interaction of two immunotherapeutic agents.

[0106] In embodiments, the chimeric proteins of the invention offer the potential for reduced off-site and / or systemic toxicity.

[0107] In embodiments, the chimeric proteins of the invention provide reduced side effects, e.g., GI complications, relative to current immunotherapies, e.g., antibodies against checkpoint molecules described herein. Exemplary GI complications include abdominal pain, loss of appetite, autoimmune effects, constipation, cramps, dehydration, diarrhea, feeding problems, fatigue, flatulence, fluid in the abdomen or ascites, gastrointestinal (GI) dysbiosis, GI mucositis, inflammatory bowel disease, irritable bowel syndrome (IBS-D and IBS-C), nausea, pain, stool or urinary changes, ulcerative colitis, vomiting, weight gain from retained fluids, and / or weakness.

[0108] Disease; Treatment; and Patient Selection In embodiments, the present invention relates to cancer and / or tumors, e.g., the treatment or prevention of cancer and / or tumors. As explained elsewhere herein, the treatment of cancer may, in embodiments, involve modulating the immune system with the chimeric proteins of the present invention to favor immune stimulation over immune inhibition.

[0109] Cancer or tumor refers to the uncontrolled proliferation of cells and / or abnormally increased cell viability and / or inhibition of apoptosis that interferes with the normal functioning of the body's organs and systems. Benign and malignant cancers, polyps, hyperplasias, and dormant tumors or micrometastases are included. Also included are cells with abnormal proliferation that are not prevented by the immune system (e.g., virus-infected cells). Cancer can be primary or metastatic cancer. A primary cancer is an area of ​​cancer cells at the primary site that becomes clinically detectable, which can be a primary tumor. In contrast, a metastatic cancer can be the spread of disease from one organ or part to another non-adjacent organ or part. A metastatic cancer can be caused by cancer cells that acquire the ability to penetrate and invade surrounding normal tissues in a localized area, forming a new tumor, which can be a local metastasis. Cancer can also be caused by cancer cells that acquire the ability to penetrate lymphatic and / or vascular walls, after which the cancer cells can circulate via the bloodstream to other sites and tissues in the body (thereby being circulating tumor cells). Cancer may result from processes such as lymphatic or hematogenous spread. Cancer may also be caused by tumor cells that arrest at another site, re-infiltrate through the blood vessels or wall, continue to grow, and eventually form another clinically detectable tumor. Cancer may be this new tumor, which may be a metastatic (or secondary) tumor.

[0110] Cancer may be caused by metastasized tumor cells, which may be secondary or metastatic tumors. The cells of the tumor may be similar to those in the original tumor. As an example, if breast or colon cancer metastasizes to the liver, the secondary tumor, while present in the liver, is composed of abnormal breast or colon cells, not abnormal liver cells. The tumor in the liver may therefore be metastatic breast cancer or metastatic colon cancer, but not liver cancer.

[0111] Cancer can have an origin from any tissue. Cancer can originate from melanoma, colon, breast, or prostate, and thus be composed of cells that were originally skin, colon, breast, or prostate, respectively. Cancer can also be a hematological malignancy, which can be a leukemia or lymphoma. Cancer can invade tissues such as the liver, lung, bladder, or intestine.

[0112] Representative cancers and / or tumors of the present invention include basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; cancer of the brain and central nervous system; breast cancer; cancer of the iliac bone; cervical cancer; choriocarcinoma; colon and rectal cancer; connective tissue cancer; cancer of the digestive system; endometrial cancer; esophageal cancer; eye cancer; head and neck cancer; gastric cancer (including gastrointestinal cancer); glioblastoma; liver cancer; hepatoma; intraepithelial neoplasia; kidney or renal cancer; laryngeal cancer; leukemia; liver cancer; lung cancer (e.g., small cell alveolar lung cancer, non-small cell lung cancer, lung adenocarcinoma, and squamous cell carcinoma of the lung; melanoma; myeloma; neuroblastoma; oral cancer (lips, tongue, mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; cancer of the respiratory system; salivary gland cancer; sarcoma; skin cancer; squamous cell carcinoma; stomach cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; urinary system cancer; vulvar cancer; Hodgkin's and non-Hodgkin's lymphoma, and B-cell lymphomas (including low grade / follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL, intermediate grade / follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-round cell NHL; giant mass disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); hairy cell leukemia; chronic myeloblastic leukemia; and other carcinomas and sarcomas; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal blood vessel proliferation associated with nematoses, edema (such as associated with brain tumors), and Meigs' syndrome.

[0113] In embodiments, the chimeric proteins are used to treat subjects with treatment-refractory cancer. In embodiments, the chimeric proteins are used to treat subjects who are refractory to one or more immunomodulatory agents. For example, in embodiments, the chimeric proteins are used to treat subjects who do not respond to treatment or show further progression after as much as 12 weeks of treatment. For example, in embodiments, the subject is refractory to a PD-1 and / or PD-L1 and / or PD-L2 agent, including, for example, nivolumab (ONO-4538 / BMS-936558, MDX1106, OPDIVO, BRISTOL MYERS SQUIBB), pembrolizumab (KEYTRUDA, MERCK), pidilizumab (CT-011, CURE TECH), MK-3475 (MERCK), BMS 936559 (BRISTOL MYERS SQUIBB), ibrutinib (PHARMACYCLICS / ABBVIE), atezolizumab (TECENTRIQ, GENENTECH), and / or MPDL328OA (ROCHE) refractory patients. For example, in embodiments, the subject is refractory to an anti-CTLA-4 agent, e.g., an ipilimumab (YERVOY) refractory patient (e.g., a melanoma patient). Thus, in embodiments, the invention provides methods of cancer treatment that rescue patients who are unresponsive to a variety of treatments, including monotherapy with one or more immunomodulatory agents.

[0114] In embodiments, the method provides treatment with a chimeric protein to patients who are refractory to additional agents, where such "additional agents" are described elsewhere herein and include, but are not limited to, various chemotherapeutic agents described herein.

[0115] In embodiments, the chimeric proteins are used to treat, modulate, or prevent one or more inflammatory diseases or conditions. Non-limiting examples of inflammatory diseases include acne, acute inflammation, allergic rhinitis, asthma, atherosclerosis, atopic dermatitis, autoimmune diseases, autoinflammatory diseases, autosomal recessive spastic ataxia, bronchiectasis, celiac disease, chronic cholecystitis, chronic inflammation, chronic prostatitis, colitis, diverticulitis, familial eosinophilia (fe), glomerulonephritis, glycerol kinase deficiency, hidradenitis suppurativa, hypersensitivity, inflammation, inflammatory bowel disease, inflammatory pelvic disease, interstitial cystitis, laryngeal inflammatory disease, Leigh's syndrome, lichen planus, mast cell activation syndrome, mastocytosis, ocular inflammatory disease, otitis, pain, pelvic inflammatory disease, reperfusion injury, respiratory disease, restenosis, rheumatic fever, rheumatoid arthritis, rhinitis, sarcoidosis, septic shock, silicosis and other pneumonias, transplant rejection, tuberculosis, and vasculitis.

[0116] In various embodiments, the inflammatory disease is an autoimmune disease or condition such as multiple sclerosis, diabetes, lupus, celiac disease, Crohn's disease, ulcerative colitis, Guillain-Barre syndrome, scleroderma, Goodpasture's syndrome, Wegener's granulomatosis, autoimmune epilepsy, Rasmussen's encephalitis, primary biliary sclerosis, sclerosing cholangitis, autoimmune hepatitis, Addison's disease, Hashimoto's thyroiditis, fibromyalgia, Meniere's syndrome; transplant rejection (e.g., prevention of allograft rejection), pernicious anemia, rheumatoid arthritis, systemic lupus erythematosus, dermatomyositis, Sjogren's syndrome, lupus erythematosus, multiple sclerosis, myasthenia gravis, Reiter's syndrome, Graves' disease, and other autoimmune diseases.

[0117] In embodiments, the chimeric agents of the invention are used in methods of activating T cells, for example, via the extracellular domain of OX40L.

[0118] In some embodiments, the chimeric agents of the invention are used in methods to prevent cellular transduction of immunosuppressive signals via the extracellular domain of VSIG8.

[0119] Combination Therapies and Conjugations In embodiments, the present invention provides chimeric proteins and methods further comprising administering an additional agent to the subject. In embodiments, the present invention relates to co-administration and / or co-formulation. Any of the compositions described herein may be co-formulated and / or co-administered.

[0120] In embodiments, any of the chimeric proteins described herein act synergistically when co-administered with another agent and are administered at a lower dose than would typically be used if such agent were used as a monotherapy. In embodiments, any of the agents referenced herein may be used in combination with any of the chimeric proteins described herein.

[0121] In an embodiment, the chimeric protein of the present invention comprising the extracellular domain of VSIG8 as described herein is co-administered with another chimeric protein. In an embodiment, the chimeric protein of the present invention comprising the extracellular domain of VISIG8 as described herein is co-administered with another chimeric protein, for example, one that regulates an adaptive immune response. In an embodiment, the chimeric protein of the present invention comprising the extracellular domain of VSIG8 as described herein is co-administered with a chimeric protein comprising one or more of CSF1R, CD40L, PD-1, GITRL, 4-1BBL, SIRPα, TIM3, TIGIT, and LIGHT. Without being bound by theory, it is believed that a combination regimen comprising administration of a chimeric protein of the present invention that induces an adaptive immune response and one or more chimeric proteins that induce an innate immune response may provide a synergistic effect (e.g., a synergistic anti-tumor effect).

[0122] Any chimeric protein that induces an innate immune response can be utilized in the present invention. For example, the chimeric protein can be any of the chimeric proteins disclosed in U.S. Patent No. 62 / 464,002 that induce an innate immune response. In such an embodiment, the chimeric protein comprises a first extracellular domain of a type I transmembrane protein at or near the N-terminus and a second extracellular domain of a type II transmembrane protein at or near the C-terminus, where one of the first and second extracellular domains provides an immunoinhibitory signal and one of the first and second extracellular domains provides an immunostimulatory signal as disclosed in U.S. Patent No. 62 / 464,002, the entire contents of which are incorporated herein by reference. In an exemplary embodiment, the chimeric protein that induces an innate immune response is a chimeric protein that comprises an extracellular domain of CSF1R at the N-terminus and an extracellular domain of CD40L at the C-terminus. In an embodiment, the chimeric protein that induces an innate immune response is a chimeric protein that comprises an extracellular domain of SIRPα at the N-terminus and an extracellular domain of CD40L at the C-terminus.

[0123] In an embodiment, the chimeric protein of the invention comprising the extracellular domain of VSIG8 is administered to a patient who has previously been administered a chimeric protein for stimulating an innate immune response (e.g., a CSF1R-based chimeric protein). For example, the chimeric protein of the invention comprising the extracellular domain of VSIG8 can be administered after the chimeric protein for stimulating an innate immune response (e.g., a CSF1R-based chimeric protein), including 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, or 4 weeks later.

[0124] In embodiments, including but not limited to cancer applications, the present invention relates to chemotherapeutic agents as additional agents. Examples of chemotherapeutic agents are alkylating agents such as thiotepa and CYTOXAN cyclosphosphamide; alkylsulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carbocone, meturedopa and uredopa; ethylenimines and methylamelanamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylolmelamine; acetogenins (e.g., bullatacin and bullatacinone); camptothecins (including the synthetic analog topotecan); bryostatin; calistatin; CC-1065 (including its synthetic analogs adozelesin, carzelesin and bizelesin); cryptophycins (e.g., cryptophycin 1 and cryptophycin 8); dolastatins; duocarmycins (synthetic analogs, KW-2189 and CB 1-TM1); eleutherobin; pancratistatin; sarcodictin; spongiostatin; chlorambucil, chlornaphazine, colofosfamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, nobembitine, phenesterine, prednimustine, trophosfamide, nitrogen mustards such as uracil mustard; carmustine, chlorozotocin, fotemustine, romosine, nitrosoureas, such as nimustine, nimustine, and ranimnustine; antibiotics, such as the enediyne antibiotics (e.g., the calicheamicins, particularly calicheamicin gamma II and calicheamicin omega II (see, e.g., Agnew, Chem. Intl. Ed. Engl., 33:183-186 (1994)); dynemicins, including dynemicin A; bisphosphonates, such as clodronate; esperamicin;and neocarzinostatin chromophore and related chromoproteins (enediyne antibiotic chromophores), aclacinomycin, actinomycin, ausramycin, azaserine, bleomycin, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, adriamycin, doxorubicin (morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2- pyrrolino-doxorubicin and deoxydoxorubicin), mitomycins such as epirubicin, esorubicin, idarubicin, marcelomycin, and mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfilomycin, puromycin, keramycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, and zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); denopterin, methotrexate, pteropteris, and the like. purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calsterone, dromostanol propionate, epithiostanol, mepitiostane, testolactone; antiadrenal agents such as aminoglutethimide, mitotane, trilostane; Folic acid supplements such as loric acid;aceglatone;aldophosphamide glycosides;aminolevulinic acid;eniluracil;amsacrine;bestrabulil;bisantrene;edatraxate;demecolcine;diaziquone;elformitin;elliptinium acetate;epothilone;etoglucide;gallium nitrate;hydroxyurea;lentinan;lonidynin;maytansinoids such as maytansine and ansamitocin;mitoguazone;mitoxantrone;mopidamol;nitraerine;pentostatin;phenamet;pirarubicin;losoxantrone; podophyllic acid; 2-ethylhydrazide; procarbazine; PSK polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2''-trichlorotriethylamine; trichothecenes (e.g., T-2 toxin, veraculin A, roridin A, and anguidine); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, e.g., Taxol paclitaxel (Bristol-Myers Squibb Oncology, Princeton, NJ), abraxane cremophor-free, albumin-treated nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, 111.), and Taxotere docetaxel (Rhone-Poulenc Rorer, Antony, France); chlorambucil; Gemzar gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogues such as cisplatin, oxaliplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; navelbine vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (Camptosar, CPT-11) (including treatment regimens of irinotecan with 5-FU and leucovorin); topoisomerase inhibitors RFS 2000;difluoromethylornithine (DMFO);retinoids such as retinoic acid;capecitabine;combretastatin;leucovorin (LV);oxaliplatin including oxaliplatin treatment regimen (FOLFOX);lapatinib (TYKERB);Inhibitors of PKC-α, Raf, H-Raf, EGFR (e.g., erlotinib (Tarceva) and VEGF-A, which reduce cell proliferation), and pharma- ceutically acceptable salts, acids, or derivatives of any of the above, may be included. Additionally, the method of treatment may further include the use of radiation. Additionally, the method of treatment may further include the use of photodynamic therapy.

[0125] In embodiments, including but not limited to cancer applications, the additional agents of the invention are agents that block, reduce and / or inhibit PD-1 and PD-L1 or PD-L2 and / or binding of PD-1 to PD-L1 or PD-L2 (non-limiting examples include nivolumab (ONO-4538 / BMS-936558, MDX1106, OPDIVO, BRISTOL MYERS SQUIBB), pembrolizumab (KEYTRUDA, Merck), pidilizumab (CT-011, CURE TECH), MK-3475 (MERCK), BMS 936559 (BRISTOL MYERS SQUIBB), and / or PD-L1 / PD-L2 binding. SQUIBB), atezolizumab (Tecentriq, GENENTECH), MPDL328OA (ROCHE)), agents that increase and / or stimulate CD137 (4-1BB) and / or binding of CD137 (4-1BB) to one or more 4-1BB ligands (non-limiting examples include urelumab (BMS-663513 and anti-4-1BB antibodies), and agents that block, reduce and / or inhibit the activity of CTLA-4 and / or the binding of CTLA-4 to one or more of AP2M1, CD80, CD86, SHP-2, and PPP2R5A and / or the binding of OX40 to OX40L (non-limiting examples include one or more immunomodulatory agents selected from GBR830 (GLENMARK), MEDI6469 (MEDIMMUNE).

[0126] In embodiments, including but not limited to infectious disease applications, the present invention relates to an anti-infective agent as an additional agent, hi embodiments, the anti-infective agent is an antiviral agent, including but not limited to abacavir, acyclovir, adefovir, amprenavir, atazanavir, cidofovir, darunavir, delavirdine, didanosine, docosanol, efavirenz, elvitegravir, emtricitabine, enfuvirtide, etravirine, famciclovir, and foscarnet. In embodiments, the anti-infective agent is an antibacterial agent, including, but not limited to, cephalosporin antibiotics (cephalexin, cefuroxime, cefadroxil, cefazolin, cephalothin, cefaclor, cefamandole, cefoxitin, cefprozil, and ceftobiprole); fluoroquinolone antibiotics (cipro, levaquin, floxin, tequin, avelox, and norflox); tetracycline antibiotics (tetracycline, minocycline, oxytetracycline, and doxycycline); penicillin antibiotics (amoxicillin, ampicillin, penicillin V, dicloxacillin, carbenicillin, vancomycin, and methicillin); monobactam antibiotics (aztreonam); and carbapenem antibiotics (ertapenem, doripenem, imipenem / cilastatin, and meropenem). In embodiments, anti-infective agents include anti-malarials (e.g., chloroquine, quinine, mefloquine, primaquine, doxycycline, artemether / lumefantrine, atovaquone / proguanil and sulfadoxine / pyrimethamine), metronidazole, tinidazole, ivermectin, pyrantel pamoate, and albendazole.

[0127] In embodiments, including but not limited to autoimmune applications, the additional agent is an immunosuppressant. In embodiments, the immunosuppressant is an anti-inflammatory agent, such as a steroidal anti-inflammatory agent or a nonsteroidal anti-inflammatory drug (NSAID). Steroids, particularly adrenal corticosteroids and their synthetic analogs, are well known in the art. Examples of corticosteroids useful in the present invention include hydroxyltriamcinolone, alpha-methyldexamethasone, beta-methylbetamethasone, beclomethasone dipropionate, betamethasone benzoate, betamethasone dipropionate, betamethasone valerate, clobetasol valerate, desonide, desoximetasone, dexamethasone, diflorasone diacetate, diflucortolone valerate, fluadrenolone, fluclorolone acetonide, flumethasone pivalate, fluocinolone acetonide, fluocinonide, flucortine butyl ester, fluocortolone, fluprednidene (fluprednylidene) acetate, flurandrenolone, halcinonide, hydrochloride, hydroxypropyltriamcinolone ... These include, but are not limited to, cortisone acetate, hydrocortisone butyrate, methylprednisolone, triamcinolone acetonide, cortisone, cortodoxone, flucetonide, fludrocortisone, difluorosone diacetate, fluradrenolon acetonide, medrysone, amcinafide, amcinafide, betamethasone and its esters, chloroprednisone, clocortelone, clesinolone, dichlorisone, difluprednate, flucloronide, flunisolide, fluoromethalone, fluperolone, fluprednisolone, hydrocortisone, meprednisone, paramethasone, prednisolone, prednisone, beclomethasone dipropionate. NSAIDs that may be used in the present invention include, but are not limited to, salicylic acid, acetylsalicylic acid, methylsalicylic acid, glycolsalicylic acid, salicylmide, benzyl-2,5-diacetoxybenzoic acid, ibuprofen, fulindac, naproxen, ketoprofen, etofenamate, phenylbutazone, and indomethacin.In embodiments, the immunosuppressant may be a cytostatic agent such as an alkylating agent, antimetabolite (e.g., azathioprine, methotrexate), cytotoxic antibiotics, antibodies (e.g., basiliximab, daclizumab, and muromonab), anti-immunophilins (e.g., cyclosporine, tacrolimus, sirolimus), interferon, opioids, TNF binding proteins, mycophenolates, and small biologic agents (e.g., fingolimod, myriocin).

[0128] In embodiments, the chimeric proteins (and / or additional agents) described herein include derivatives that are modified, i.e., derivatives that are modified by the covalent attachment of any type of molecule to the composition, such that the covalent attachment does not prevent the activity of the composition. For example, but not limited to, derivatives include compositions that are modified by, among others, glycosylation, lipidation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, linkage to cellular ligands or other proteins, and the like. Any of a number of chemical modifications can be made by known techniques, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tulicamycin, and the like. Additionally, derivatives may contain one or more non-classical amino acids. In embodiments, the chimeric proteins (and / or additional agents) described herein further include cytotoxic agents, which in exemplary embodiments include toxins, chemotherapeutic agents, radioisotopes, and agents that cause apoptosis or cell death. Such agents may be conjugated to the compositions described herein.

[0129] The chimeric proteins (and / or additional agents) described herein can thus be post-translationally modified to add effector moieties such as chemical linkers, detectable moieties such as fluorochromes, enzymes, substrates, bioluminescent agents, radioactive agents, and chemiluminescent moieties, or functional moieties such as streptavidin, avidin, biotin, cytotoxins, cytotoxic agents, and radioactive agents.

[0130] formulation The chimeric proteins (and / or additional agents) described herein may possess sufficiently basic functional groups capable of reacting with inorganic or organic acids, or carboxyl groups capable of reacting with inorganic or organic bases, to form pharma- ceutically acceptable salts. Pharmaceutically acceptable acid addition salts are formed from pharma- ceutically acceptable acids well known in the art. Such salts include, for example, those listed in Journal of Pharmaceutical Science, 66, 2-19 (1977), which is incorporated herein by reference in its entirety, and in The Handbook of Pharmaceutical Salts; Properties, Selection, and Use, edited by PH Stahl and CG Wermuth, Verlag, Zurich (Switzerland) 2002.

[0131] In embodiments, the compositions described herein are in the form of a pharma- ceutically acceptable salt.

[0132] Any chimeric protein (and / or additional agent) described herein can also be administered to a subject as a component of a composition that includes a pharma- ceutically acceptable carrier or vehicle. Such compositions may, if necessary, include a suitable amount of a pharma- ceutical acceptable excipient to provide a suitable form for administration. The pharmaceutical excipient can be a liquid, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, and sesame oil. The pharmaceutical excipient can be, for example, saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, and urea. In addition, auxiliary agents, stabilizers, thickening agents, lubricants, and coloring agents can be used. In embodiments, the pharma- ceutical acceptable excipient is sterile when administered to a subject. Water is a useful excipient when any agent described herein is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid excipients, particularly for injectable solutions. Suitable pharmaceutical excipients also include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, etc. Any of the agents described herein, if desired, may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.

[0133] In embodiments, the compositions described herein are suspended in a saline buffer (including but not limited to TBS, PBS, and the like).

[0134] In embodiments, the chimeric proteins may be conjugated and / or fused to another agent to extend half-life or otherwise improve pharmacodynamic and pharmacokinetic properties. In embodiments, the chimeric proteins may be fused or conjugated to one or more of PEG, XTEN (e.g., as rPEG), polysialic acid (POLYXEN), albumin (e.g., human serum albumin or HAS), elastin-like protein (ELP), PAS, HAP, GLK, CTP, and transferrin, etc. In embodiments, each of the individual chimeric proteins is fused to one or more of the agents described in BioDrugs (2015) 29:215-239, the entire contents of which are incorporated herein by reference.

[0135] Administration, Dosing, and Treatment Regimens The present invention includes the described chimeric proteins (and / or additional agents) in various formulations. Any chimeric proteins (and / or additional agents) described herein may be in the form of a solution, suspension, emulsion, droplets, tablets, pills, pellets, capsules, liquid-containing capsules, powders, sustained release formulations, suppositories, emulsions, aerosols, sprays, suspensions, or any other form suitable for use. DNA or RNA constructs encoding the protein sequences may also be used. In embodiments, the composition is in the form of a capsule (see, e.g., U.S. Pat. No. 5,698,155). Other examples of suitable pharmaceutical excipients are described in Remington's Pharmaceutical Sciences 1447-1676 (ed. Alfonso R. Gennaro, 19th ed., 1995), which is incorporated herein by reference.

[0136] Optionally, the formulation containing the chimeric protein (and / or additional agent) may also contain a solubilizing agent. The agent may also be delivered by a suitable vehicle or delivery device known in the art. The combination therapy outlined herein may be co-delivered in a single delivery vehicle or delivery device. The composition for administration may optionally contain a local anesthetic, such as lignocaine, to reduce pain at the injection site.

[0137] The formulations containing the chimeric protein of the present invention (and / or additional agents) may be conveniently presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Such methods generally include the step of bringing into association the therapeutic agent with the carrier which constitutes one or more accessory ingredients. Typically, the formulations are prepared by uniformly and intimately bringing into association the therapeutic agent with liquid carriers, finely divided solid carriers, or both, and then, if necessary, shaping the product into the desired formulation (e.g., by wet or dry granulation, powder blending, or the like, followed by tableting using conventional methods known in the art).

[0138] In embodiments, any chimeric protein (and / or additional agent) described herein is formulated in accordance with routine procedures into a composition compatible with the modes of administration described herein.

[0139] Routes of administration include, for example, intradermal, intratumoral, intramuscular, intradermal, intravenous, subcutaneous, intranasal, epidural, oral, sublingual, intranasal, intracerebral, intravaginal, transdermal, rectal, by inhalation, or topically, particularly to the ear, nose, eye, or skin. In embodiments, administration is by oral or parenteral injection. In some cases, administration results in the release of any agent described herein into the bloodstream, or the agent is administered directly to the site of active disease.

[0140] Any chimeric protein (and / or additional agent) described herein can be administered orally. Such chimeric proteins (and / or additional agents) can also be administered by any other convenient route, for example, by intravenous infusion or bolus injection, by absorption through epithelial or mucosal linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and can be administered together with another biologically active agent. Administration can be systemic or local. Various delivery systems, for example, encapsulation in liposomes, microparticles, microcapsules, capsules, etc., are known and can be used for administration.

[0141] In certain embodiments, it may be desirable to administer locally to the area in need of treatment. In embodiments, for example, in the treatment of cancer, the chimeric protein (and / or additional agent) is administered to and / or targeted to the tumor microenvironment (e.g., cells, molecules, extracellular matrix, and / or blood vessels that surround and / or nourish tumor cells, including tumor vasculature; tumor infiltrating lymphocytes; fibroblast reticular cells; endothelial progenitor cells (EPCs); cancer-associated fibroblasts; pericytes; other stromal cells; components of the extracellular matrix (ECM); dendritic cells; antigen-presenting cells; T cells; regulatory T cells; macrophages; neutrophils; and other immune cells located proximal to the tumor) or lymph nodes. In embodiments, for example, in the treatment of cancer, the chimeric protein (and / or additional agent) is administered intratumorally.

[0142] In embodiments, the chimeric proteins of the present invention allow for a dual effect resulting in fewer side effects than those seen with conventional immunotherapies (e.g., treatment with one or more of OPDIVO, KEYTRUDA, YERVOY, and TECENTRIQ). For example, the chimeric proteins of the present invention reduce or prevent commonly observed immune-related adverse events, such as hypotension, colitis, hepatitis, pneumonia, rash, and rheumatic diseases, affecting various tissues and organs including the skin, gastrointestinal tract, kidneys, peripheral and central nervous systems, liver, lymph nodes, eyes, pancreas, and endocrine system. Additionally, local administration, e.g., intratumoral administration, of the present invention obviates adverse events seen with standard systemic administration, e.g., IV infusion as used in conventional immunotherapies (e.g., treatment with one or more of OPDIVO, KEYTRUDA, YERVOY, and TECENTRIQ).

[0143] Suitable dosage forms for parenteral administration (e.g., intravenous, intramuscular, intraperitoneal, subcutaneous, and intraarticular injection and infusion) include, for example, solutions, suspensions, dispersions, and emulsions. They can also be manufactured in the form of sterile solid compositions (e.g., lyophilized compositions) that can be dissolved or suspended in a sterile injection medium immediately before use. They can contain, for example, suspending or dispersing agents known in the art.

[0144] The dosage and dosing schedule of any of the chimeric proteins (and / or additional agents) described herein may depend on a variety of parameters, including, but not limited to, the disease being treated, the overall health of the subject, and the discretion of the administering physician. Any of the chimeric proteins described herein can be administered prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), simultaneously with, or after (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) administration of the additional agent to a subject in need thereof. In embodiments, any chimeric protein described herein and additional agents are administered 1 minute apart, 10 minutes apart, 30 minutes apart, less than 1 hour apart, 1 hour apart, 1 to 2 hours apart, 2 to 3 hours apart, 3 to 4 hours apart, 4 to 5 hours apart, 5 to 6 hours apart, 6 to 7 hours apart, 7 to 8 hours apart, 8 to 9 hours apart, 9 to 10 hours apart, 10 to 11 hours apart, 11 to 12 hours apart, 1 day apart, 2 days apart, 3 days apart, 4 days apart, 5 days apart, 6 days apart, 1 week apart, 2 weeks apart, 3 weeks apart, or 4 weeks apart.

[0145] In an embodiment, the present invention relates to the simultaneous administration of a chimeric protein of the present invention comprising the extracellular domain of VSIG8 and another chimeric protein that induces an innate immune response. In such an embodiment, the chimeric protein of the present invention can be administered before, simultaneously with, or after administration of the chimeric protein that induces an innate immune response. For example, the chimeric protein can be administered at intervals of 1 minute, 10 minutes, 30 minutes, less than 1 hour, 1 hour, 1 to 2 hours, 2 to 3 hours, 3 to 4 hours, 4 to 5 hours, 5 to 6 hours, 6 to 7 hours, 7 to 8 hours, 8 to 9 hours, 9 to 10 hours, 10 to 11 hours, 11 to 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, or 4 weeks. In an exemplary embodiment, the chimeric protein of the invention comprising the extracellular domain of VSIG8 and the chimeric protein that induces an innate immune response are administered at weekly intervals or every other week (i.e., administration of the chimeric protein that induces an innate immune response is followed one week later by administration of the chimeric protein of the invention comprising the extracellular domain of VSIG8, etc.).

[0146] The dosage of any chimeric protein (and / or additional agent) described herein may depend on several factors, including the severity of the condition, whether the condition is being treated or prevented, and the age, weight, and health of the subject being treated. In addition, pharmacogenomic (the effect of genotype on the pharmacokinetic, pharmacodynamic, or therapeutic efficacy profile) information for a particular subject may affect the dosage used. Also, the exact individual dosage may be adjusted somewhat depending on a variety of factors, including the particular combination of agents administered, the time of administration, the route of administration, the nature of the formulation, the rate of excretion, the particular disease being treated, the severity of the disorder, and the anatomical location of the disorder. Some variation in dosage is expected.

[0147] For administration by parenteral injection of any of the chimeric proteins (and / or additional agents) described herein, the dosage can be from about 0.1 mg to about 250 mg per day, from about 1 mg to about 20 mg per day, or from about 3 mg to about 5 mg per day. Generally, for oral or parenteral administration, the dosage of any of the agents described herein can be from about 0.1 mg to about 1500 mg per day, or from about 0.5 mg to about 10 mg per day, or from about 0.5 mg to about 5 mg per day, or from about 200 to about 1,200 mg per day (e.g., about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, about 1,000 mg, about 1,100 mg, about 1,200 mg per day).

[0148] In embodiments, administration of the chimeric proteins (and / or additional agents) described herein is by parenteral injection at a dosage of about 0.1 mg to about 1500 mg per treatment, or about 0.5 mg to about 10 mg per treatment, or about 0.5 mg to about 5 mg per treatment, or about 200 to about 1,200 mg per treatment (e.g., about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, about 1,000 mg, about 1,100 mg, about 1,200 mg per treatment).

[0149] In embodiments, a suitable dosage of the chimeric protein (and / or additional agent) ranges from about 0.01 mg / kg to about 100 mg / kg of the subject's body weight, or from about 0.01 mg / kg to about 10 mg / kg, including all values ​​and ranges therebetween, such as about 0.01 mg / kg body weight, about 0.02 mg / kg body weight, about 0.03 mg / kg body weight, about 0.04 mg / kg body weight, about 0.05 mg / kg body weight, about 0.06 mg / kg body weight, about 0.07 mg / kg body weight, about 0.08 mg / kg body weight, about 0.09 mg / kg body weight, about 0.1 mg / kg body weight, about 0.2 mg / kg body weight, about 0.3 mg / kg body weight, about 0.4 mg / kg body weight, and the like. about 0.5 mg / kg body weight, about 0.6 mg / kg body weight, about 0.7 mg / kg body weight, about 0.8 mg / kg body weight, about 0.9 mg / kg body weight, about 1 mg / kg body weight, about 1.1 mg / kg body weight, about 1.2 mg / kg body weight, about 1.3 mg / kg body weight, about 1.4 mg / kg body weight, about 1.5 mg / kg body weight, about 1.6 mg / kg body weight, about 1.7 mg / kg body weight, about 1.8 mg / kg body weight, about 1.9 mg / kg body weight, about 2 mg / kg body weight, about 3 mg / kg body weight, about 4 mg / kg body weight, about 5 mg / kg body weight, about 6 mg / kg body weight, about 7 mg / kg body weight, about 8 mg / kg body weight, about 9 mg / kg body weight, and about 10 mg / kg body weight.

[0150] In embodiments, delivery may be in vesicles, particularly liposomes (see Langer, 1990, Science 249:1527-1533; Treat et al., Liposomes in the Therapy of Infectious Disease and cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989)).

[0151] Any chimeric protein (and / or additional agent) described herein can be administered by controlled or sustained release means or by delivery devices known to those of skill in the art. Examples include, but are not limited to, those described in U.S. Patent Nos. 3,845,770, 3,916,899, 3,536,809, 3,598,123, 4,008,719, 5,674,533, 5,059,595, 5,591,767, 5,120,548, 5,073,543, 5,639,476, 5,354,556, and 5,733,556, each of which is incorporated herein by reference in its entirety. Such dosage forms may be useful to provide controlled or sustained release of one or more active ingredients using, for example, hydropropylmethylcellulose, other polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, liposomes, microspheres, or combinations thereof, to provide a desired release profile at various rates. Controlled or sustained release of an active ingredient can be stimulated by a variety of conditions, including, but not limited to, a change in pH, a change in temperature, stimulation with appropriate wavelengths of light, enzyme concentration or availability, water concentration or availability, or other physiological conditions or compounds.

[0152] In embodiments, polymeric materials can be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Florida (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, 1983, J. Macromol. Sci. Rev. Macromol. Chem. 23:61; see also Levy et al., 1985, Science 228:190; During et al., 1989, Ann. Neurol. 25:351; Howard et al., 1989, J. Neurosurg. 71:105).

[0153] In embodiments, a controlled release system can be placed near the target area to be treated, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, Medical Applications of Controlled Release, supra, Vol. 2, pp. 115-138 (1984)). Other controlled release systems discussed in the review by Langer, 1990, Science 249:1527-1533 can be used.

[0154] Administration of any of the chimeric proteins (and / or additional agents) described herein can, independently, be 1-4 times daily, or 1-4 times monthly, or 1-6 times per year, or once every 2, 3, 4, or 5 years. Administration can be daily or for a period of 1, 2, 3, 6 months, 1, 2, 3 years, or even for the life of the subject.

[0155] Dosage regimens utilizing any of the chimeric proteins (and / or additional agents) described herein can be selected according to a variety of factors, including the type, species, age, weight, sex, and condition of the subject; the severity of the condition being treated; the route of administration; the renal or hepatic function of the subject; the pharmacogenomic makeup of the individual; and the particular compound of the invention being used. Any of the chimeric proteins (and / or additional agents) described herein can be administered in a single daily dose, or the total daily dosage can be administered in divided doses two, three, or four times daily. Additionally, any of the chimeric proteins (and / or additional agents) described herein can be administered continuously, rather than intermittently, throughout the dosage regimen.

[0156] Cells and Nucleic Acids In embodiments, the present invention provides an expression vector comprising a nucleic acid encoding a chimeric protein described herein. In embodiments, the expression vector comprises DNA or RNA. In embodiments, the expression vector is a mammalian expression vector.

[0157] Both prokaryotic and eukaryotic vectors can be used to express the chimeric proteins. Prokaryotic vectors include constructs based on E. coli sequences (see, e.g., Makrides, Microbiol Rev 1996, 60:512-538). Non-limiting examples of regulatory regions that can be used for expression in E. coli include lac, trp, lpp, phoA, recA, tac, T3, T7 and λP. LNon-limiting examples of prokaryotic expression vectors may include the λgt vector series, such as λgt11 (Huynh et al., DNA Cloning Techniques, Vol. I: A Practical Approach, 1984, (D. Glover, ed.), pp. 49-78, IRL Press, Oxford), and the pET vector series (Studier et al., Methods Enzymol 1990, 185: 60-89). However, prokaryotic host vector systems cannot perform many of the post-translational processes of mammalian cells. Therefore, eukaryotic host vector systems may be particularly useful. A variety of regulatory regions can be used for the expression of chimeric proteins in mammalian host cells. For example, the SV40 early and late promoters, the cytomegalovirus (CMV) immediate early promoter, and the Rous sarcoma virus long terminal repeat (RSV-LTR) promoter can be used. Inducible promoters that may be useful in mammalian cells include, but are not limited to, the promoter associated with the metallothionein II gene, the mouse mammary tumor virus glucocorticoid-responsive long terminal repeat (MMTV-LTR), the β-interferon gene, and the hsp70 gene (see Williams et al., Cancer Res 1989, 49:2735-42; and Taylor et al., Mol Cell Biol 1990, 10:165-75). Heat shock promoters or stress promoters may also be advantageous in promoting the expression of chimeric proteins in recombinant host cells.

[0158] In embodiments, an expression vector of the invention comprises a nucleic acid encoding a chimeric protein (and / or additional agent) or its complement operably linked to an expression control region, or its complement, that is functional in a mammalian cell. The expression control region is capable of driving expression of the nucleic acid encoding the operably linked blocking agent and / or stimulating agent such that the blocking agent and / or stimulating agent is produced in a human cell transformed with the expression vector.

[0159] Expression control regions are regulatory polynucleotides (sometimes referred to herein as elements), such as promoters and enhancers, that affect the expression of operably linked nucleic acids. The expression control regions of the expression vectors of the present invention are capable of expressing operably linked encoding nucleic acids in human cells. In embodiments, the cells are tumor cells. In embodiments, the cells are non-tumor cells. In embodiments, the expression control regions confer regulatable expression to operably linked nucleic acids. A signal (sometimes referred to as a stimulus) can increase or decrease the expression of a nucleic acid operably linked to such an expression control region. Such expression control regions that increase expression in response to a signal are often referred to as inducible. Such expression control regions that decrease expression in response to a signal are often referred to as repressible. Typically, the amount of increase or decrease conferred by such elements is proportional to the amount of signal present, the greater the amount of signal, the greater the increase or decrease in expression.

[0160] In an embodiment, the present invention contemplates the use of inducible promoters that can induce transient high levels of expression in response to a cue. For example, when in the vicinity of tumor cells, cells transformed with an expression vector for a chimeric protein (and / or additional agent) that includes such expression control sequences can be induced to transiently produce high levels of the agent by exposing the transformed cells to the appropriate cue. Exemplary inducible expression control regions include those that include an inducible promoter stimulated by a cue, such as a small molecule chemical compound. Specific examples can be found, for example, in U.S. Patent Nos. 5,989,910, 5,935,934, 6,015,709, and 6,004,941, each of which is incorporated herein by reference in its entirety.

[0161] Expression control regions and locus control regions include full-length promoter sequences, such as native promoter and enhancer elements, as well as subsequences or polynucleotide variants that retain all or a portion of the full-length or unvarianded function. As used herein, the term "functional" and grammatical variations thereof, when used in reference to a nucleic acid sequence, subsequence, or fragment, means that the sequence has one or more functions of the native nucleic acid sequence (e.g., the unvariant or unmodified sequence).

[0162] "Operable linkage," as used herein, refers to a physical juxtaposition of the described components that allows them to function in their intended manner. In the example of an expression control element in operable linkage with a nucleic acid, the relationship is such that the control element regulates expression of the nucleic acid. Typically, the expression control region that regulates transcription is juxtaposed near the 5' end (i.e., "upstream") of the transcribed nucleic acid. Expression control regions can also be located at the 3' end (i.e., "downstream") of the transcribed sequence or within the transcript (e.g., in an intron). Expression control elements can be located at a distance from the transcribed sequence (e.g., 100-500, 500-1000, 2000-5000, or more nucleotides from the nucleic acid). A specific example of an expression control element is a promoter, which is usually located 5' to the transcribed sequence. Another example of an expression control element is an enhancer, which can be located 5' or 3' to the transcribed sequence or within the transcribed sequence.

[0163] Expression systems that function in human cells are well known in the art and include viral systems. In general, a promoter functional in human cells is any DNA sequence that can bind mammalian RNA polymerase and initiate downstream (3') transcription of a coding sequence into mRNA. A promoter has a transcription initiation region, usually located at the 5' end of the coding sequence, and a TATA box, typically located 25-30 base pairs upstream of the transcription initiation site. The TATA box is thought to direct RNA polymerase II to begin RNA synthesis at the correct site. A promoter also typically contains an upstream promoter element (enhancer element), typically located 100-200 base pairs upstream of the TATA box. The upstream promoter element determines the rate at which transcription is initiated and can act in either direction. Of particular use as promoters are promoters derived from mammalian viral genomes, since viral genes are often highly expressed and have a wide host range. Examples include the SV40 early promoter, mouse mammary tumor virus LTR promoter, adenovirus major late promoter, herpes simplex virus promoter, and CMV promoter.

[0164] Typically, transcription termination and polyadenylation sequences recognized by mammalian cells are regulatory regions located 3' to the translation stop codon and thus flank the coding sequence, together with the promoter element. The 3' end of the mature mRNA is formed by site-specific post-translational cleavage and polyadenylation. Examples of transcription terminator and polyadenylation signals include those derived from SV40. Introns may also be included in the expression construct.

[0165] There are various techniques available for introducing nucleic acids into living cells. Techniques suitable for the introduction of nucleic acids into mammalian cells in vitro include the use of liposomes, electroporation, microinjection, cell fusion, polymer-based systems, DEAE-dextran, viral transduction, calcium phosphate precipitation, and the like. For in vivo gene transfer, several techniques and reagents may be used, including liposomes; natural polymer-based delivery vehicles such as chitosan and gelatin. Viral vectors are also suitable for in vivo transduction. In some cases, it may be desirable to provide a targeting agent, such as an antibody or ligand specific for a tumor cell surface membrane protein. When liposomes are used, proteins that bind to cell surface membrane proteins associated with endocytosis may be used to target and / or promote uptake, for example, capsid proteins or fragments thereof that are directed to specific cell types, antibodies against proteins that undergo internalization in the circulation, proteins that target intracellular localization and enhance intracellular half-life. The technique of receptor-mediated endocytosis has been described, for example, by Wu et al., J. Biol. Chem. 262, 4429-4432 (1987); and Wagner et al., Proc. Natl. Acad. Sci. USA 87, 3410-3414 (1990).

[0166] Optionally, gene delivery agents such as integration sequences can also be used. Numerous integration sequences are known in the art (see, for example, Nunes-Duby et al., Nucleic Acids Res. 26:391-406, 1998; Sadwoski, J. Bacteriol., 165:341-357, 1986; Bestor, Cell, 122(3):322-325, 2005; Plasterk et al., TIG 15:326-332, 1999; Kootstra et al., Ann. Rev. Pharm. Toxicol., 43:413-439, 2003). These include recombinases and transposases. Examples include Cre (Sternberg and Hamilton, J. Mol. Biol., 150:467-486, 1981), lambda (Nash, Nature, 247, 543-545, 1974), Flp (Broach, et al., Cell, 29:227-234, 1982), R (Matsuzaki, et al., J. Bacteriology, 172:610-618, 1990), cpC31 (see, e.g., Groth et al., J. Mol. Biol. 335:667-678, 2004), Sleeping Beauty (sleeping beauty), transposases of the mariner family (Plasterk et al., supra), and AAV, antiviruses with components for integrating viruses such as retroviruses, and components that provide viral integration such as retroviral or lentiviral LTR sequences and AAV ITR sequences (Kootstra et al., Ann. Rev. Pharm. Toxicol., 43:413-439, 2003). In addition, direct and targeted gene integration strategies can be used to insert nucleic acid sequences encoding chimeric proteins, including CRISPR / CAS9, zinc finger, TALEN, and meganuclease gene editing technologies.

[0167] In one aspect, the present invention provides an expression vector for the expression of the chimeric protein (and / or additional agent) that is a viral vector. Many viral vectors useful for gene therapy are known (see, for example, Lundstrom, Trends Biotechnol., 21:1 17, 122, 2003). Exemplary viral vectors include those selected from antiviruses (LV), retroviruses (RV), adenoviruses (AV), adeno-associated viruses (AAV), and alphaviruses, although other viral vectors may be used. For in vivo use, viral vectors that do not integrate into the host genome are suitable for use, such as alphaviruses and adenoviruses. Exemplary types of alphaviruses include Sindbis virus, Venezuelan equine encephalitis (VEE) virus, and Semliki Forest virus (SFV). For in vitro use, viral vectors that integrate into the host genome are suitable, such as retroviruses, AAV, and antiviruses. In an embodiment, the invention provides a method of transducing human cells in vivo, comprising contacting a solid tumor in vivo with a viral vector of the invention.

[0168] In an embodiment, the invention provides a host cell comprising an expression vector comprising a chimeric protein described herein.

[0169] The expression vector can be introduced into a host cell to produce the chimeric protein of the invention. The cells can be, for example, cultured or genetically engineered in vitro. Useful mammalian host cells include, but are not limited to, cells of human, monkey, and rodent origin (see, for example, Gene Transfer and Expression: A Laboratory Manual, 1990, New York, Freeman & Co.). These include SV40 transformed monkey kidney cell lines (e.g., COS-7, ATCC CRL 1651); human embryonic kidney lines (e.g., 293, 293-EBNA, or 293 cells subcloned for growth in suspension culture, Graham et al., J Gen Virol 1977, 36:59); baby hamster kidney cells (e.g., BHK, ATCC CCL 10); Chinese hamster ovary cells DHFR (e.g., CHO, Urlaub and Chasin, Proc Natl Acad Sci USA 1980, 77:4216); DG44 CHO cells, CHO-K1 cells, mouse Sertoli cells (Mather, Biol Reprod 1980, 23:243-251); mouse fibroblasts (e.g., NIH-3T3), monkey kidney cells (e.g., CV1 ATCC CCL 70); African green monkey kidney cells (e.g., VERO-76, ATCC CRL-1587); human cervical carcinoma cells (e.g., HELA, ATCC CCL 2); canine kidney cells (e.g., MDCK, ATCC CCL 34); buffalo rat hepatocytes (e.g., BRL 3A, ATCC CRL 1442); human lung cells (e.g., W138, ATCC CCL 75); human hepatocytes (e.g., Hep G2, HB 8065); and mouse mammary carcinoma cells (e.g., MMT 060562, ATCC CCL51).Exemplary cancer cell types for expressing the chimeric proteins described herein include a mouse fibroblast cell line, NIH3T3, a mouse Lewis lung carcinoma cell line, LLC, a mouse mast cell line, P815, a mouse lymphoma cell line, EL4 and its ovalbumin transfectants, E.G7, a mouse melanoma cell line, B16F10, a mouse fibrosarcoma cell line, MC57, and human small cell lung carcinoma cell lines, SCLC#2 and SCLC#7.

[0170] Host cells can be obtained from healthy or diseased subjects, including healthy humans, cancer patients, and patients with infectious diseases, private laboratory depositories, public culture collections such as the American Type Culture Collection, or commercial suppliers.

[0171] Cells that can be used to produce the chimeric proteins of the invention in vitro, ex vivo, and / or in vivo include, but are not limited to, epithelial cells, endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytes, blood cells such as T lymphocytes, B lymphocytes, monocytes, macrophages, neutrophils, eosinophils, megakaryocytes, granulocytes, and the like, various stem or progenitor cells, particularly hematopoietic stem or progenitor cells (e.g., obtained from bone marrow), umbilical cord blood, peripheral blood, fetal liver, etc. The choice of cell type will depend on the type of tumor or infection to be treated or prevented and can be determined by one of skill in the art.

[0172] Subjects and / or Animals In embodiments, the subject and / or animal is a mammal, e.g., a human, mouse, rat, guinea pig, dog, cat, horse, cow, pig, rabbit, sheep, or a non-human primate, such as a monkey, chimpanzee, or baboon. In embodiments, the subject and / or animal is a non-mammal, e.g., a zebrafish. In embodiments, the subject and / or animal may comprise fluorescently tagged cells (e.g., with GFP). In embodiments, the subject and / or animal is a transgenic animal that comprises fluorescent cells.

[0173] In embodiments, the subject and / or animal is a human. In embodiments, the human is a pediatric human. In embodiments, the human is an adult human. In embodiments, the human is a geriatric human. In embodiments, the human may be referred to as a patient.

[0174] In certain embodiments, the human is about 0 months to about 6 months old, about 6 months to about 12 months old, about 6 months to about 18 months old, about 18 months to about 36 months old, about 1 year to about 5 years old, about 5 years to about 10 years old, about 10 years to about 15 years old, about 15 years to about 20 years old, about 20 years to about 25 years old, about 25 years to about 30 years old, about 30 years to about 35 years old, about 35 years old about 40 years old, about 40 to about 45 years old, about 45 to about 50 years old, about 50 to about 55 years old, about 55 to about 60 years old, about 60 to about 65 years old, about 65 to about 70 years old, about 70 to about 75 years old, about 75 to about 80 years old, about 80 to about 85 years old, about 85 to about 90 years old, about 90 to about 95 years old, or about 95 to about 100 years old.

[0175] In an embodiment, the subject is a non-human animal, and thus the invention relates to veterinary uses. In a particular embodiment, the non-human animal is a household pet. In another particular embodiment, the non-human animal is a livestock animal.

[0176] kit The present invention provides kits that can simplify the administration of any of the agents described herein. An exemplary kit of the present invention includes any of the compositions described herein in unit dosage form. In an embodiment, the unit dosage form is a container, such as a pre-filled syringe that can be sterilized, containing any of the agents described herein and a pharma- ceutically acceptable carrier, diluent, excipient, or vehicle. The kit may further include a label or printed instructions that describe the use of any of the agents described herein. The kit may also include a cap speculum, a local anesthetic, and a cleaning agent for the administration site. The kit may also further include one or more additional agents described herein. In an embodiment, the kit includes a container that contains an effective amount of the composition of the present invention and an effective amount of another composition, such as those described herein.

[0177] Any aspect or embodiment described herein may be combined with any other aspect or embodiment disclosed herein.

[0178] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

[0179] Working Example Example 1: Computer simulation predicted secondary structure of human VSIG8-Fc-OX40L chimeric protein A computer simulation was performed to predict the structure of the human VSIG8-Fc-OX40L chimeric protein with 603 amino acid residues, and the p-value was 5.36 × 10 -16 The molecular weight of the monomeric protein was predicted to be approximately 68.1 kDa. The structure of the chimeric protein is provided in Figure 1A. Figure 1B shows the synapse formed by the chimeric protein between tumor cells and T cells.

[0180] Secondary structure prediction of the entire sequence of the chimeric protein showed that the protein has a composition of 0% α-helix (H), 58% β-sheet (E), and 41% coil (C). Absolute global quality GDT (global distance test) and uGDT (unnormalized GDT) were also calculated for the chimeric protein, yielding an overall uGDT (GDT) of 362 (60). The three state predictions for the solvent accessibility of the protein residues were 33% exposed (E), 47% intermediate (M), and 19% buried (B).

[0181] Example 2: Characterization of human VSIG8-Fc-OX40L chimeric protein Human VSIG8-Fc-OX40L chimeric protein was constructed as described above in the detailed description and in U.S. Patent No. 62 / 464,002, the contents of which are incorporated herein by reference in their entirety. The chimeric protein was characterized by performing Western blot analysis with antibodies against each domain of the chimeric protein, i.e., anti-VSIG8, anti-FC, and anti-OX40L antibodies.

[0182] Western blots showed the presence of a predominant dimer band in the non-reducing lanes (no β-mercaptoethanol or PNGase; Figure 2, lane 2 of each blot), which was reduced to a glycosylated monomeric band in the presence of the reducing agent β-mercaptoethanol (Figure 2, lane 3 of each blot). As shown in Figure 2, lane 4 of each blot, the chimeric protein migrated as a monomer with the predicted molecular weight of approximately 68.1 kDa in the presence of both a reducing agent (β-mercaptoethanol) and the endoglycosidase peptide:N-glycosidase (PNGase).

[0183] Example 3: Characterization of the binding affinities of different domains of the human VSIG8-Fc-OX40L chimeric protein using ELISA An enzyme-linked immunosorbent assay (ELISA) assay was developed to demonstrate the binding affinity of the different domains of human VSIG8-Fc-OX40L to their respective hIgG or recombinant OX40L. Specifically, the Fc portion of the chimeric protein was captured on plate-bound human IgG and detected by HRP-conjugated anti-human IgG antibody (left panel of FIG. 3). The OX40 domain of the chimeric protein was captured on plate-bound recombinant OX40 protein and detected by anti-OX40-specific antibody (right panel of FIG. 3). It was observed that detecting the chimeric protein using the central Fc region in the ELISA assay tends to underestimate the actual protein content in the sample. Thus, a low level of hVSIG8-Fc-OX40L chimeric protein was detected compared to the standard in this assay.

[0184] Example 4: Characterization of in vitro cell binding affinity of human VSIG8-Fc-OX40L chimeric protein Cell-binding assays were performed to demonstrate the binding affinity of different domains of the human VSIG8-Fc-OX40L chimeric protein to their respective binding partners on the surface of mammalian cell membranes.

[0185] For cell binding assays, an immortalized cell line was engineered to stably express human OX40 (Jurkat / hOX40). Increasing concentrations of VSIG8-Fc-OX40L chimeric protein were incubated with the overexpressing (Jurkat / hOX40) cell line for 2 hours. Cells were harvested, washed, and stained with antibodies for detection of chimeric protein binding by flow cytometry.

[0186] As shown in Figure 4, the VSIG8-Fc-OX40L chimeric protein bound to OX40 present on the cell surface in a concentration-dependent manner and with low nM affinity. Specifically, as shown in Figure 4, the cell binding assay showed that VSIG8-Fc-OX40L bound to OX40 with an affinity of approximately 30 nM (EC 50 We demonstrated that it binds to OX40 by (calculation).

[0187] Example 5: Characterization of the binding affinity of human VSIG8-Fc-OX40L chimeric protein by surface plasmon resonance (SPR) The binding affinity of the OX40L domain of the human VSIG8-Fc-OX40L chimeric protein was measured by surface plasmon resonance (SPR) using a BioRad ProteOn XPR 360 system. Specifically, the affinity of the chimeric protein to human OX40 was determined and compared to recombinant control protein and tavoricumab (anti-human OX40 antibody). The results are shown in Figure 5 and in the table below. [Table 2]

[0188] The VSIG8-Fc-OX40L chimeric protein was found to bind to hOX40 with high affinity. In particular, the off-rate of the hVSIG8-Fc-OX40L chimeric protein was found to be much slower than that of the control Fc-OX40L protein. For example, the off-rate of the OX40L chimeric protein was 10-fold slower than that of the Fc-OX40L protein.

[0189] Example 6. Induction of OX40 signaling in vitro Activation of human OX40 leads to induction of a signaling cascade involving activation of both NF-κB and NIK. Figure 6 shows exemplary data from an in vitro NF-κB / NIK signaling assay using human VSIG8-Fc-OX40L chimeric protein. U2OS cells from the DiscoverX NIK signaling assay were cultured with titrations of either commercial single-sided hFc-OX40L, anti-OX40L antibody (oxelumab), anti-OX40 antibody (tavoricumab), or human VSIG8-Fc-OX40L chimeric protein. Relative luciferase units (RLU) indicate the relative intensity of NF-κB / NIK signaling activated after treatment with the indicated regimen. VSIG8-Fc-OX40L is shown to have signaling activated more potently through NF-κB and NIK and to a greater extent than any of the other indicated regimens.

[0190] Example 7: Functional activity of human VSIG8-Fc-OX40L chimeric protein in a superantigen cytokine release assay Another functional assay performed to characterize the functional activity of human VSIG8-Fc-OX40L chimeric protein is the superantigen cytokine release assay. In this assay, human peripheral blood leukocytes were activated with 200 ng / ml Staphylococcal enterotoxin B (SEB) in the presence of various concentrations of test agents, i.e., single-sided human Fc-OX40L, commercial single-sided VSIG8-Fc, a combination of two single-sided molecules, or human VSIG8-Fc-OX40L chimeric protein. After 3 days, the supernatants were evaluated using an ELISA specific for human IL2.

[0191] As shown in Figure 7, the amount of IL-2 secreted into the culture supernatant was monitored as a functional readout of the ability of the test agent to block inhibitory signaling events or costimulate immune activation signals. At concentrations above 0.5 nM, the VSIG8-Fc-OX40L chimeric protein induced IL-2 secretion at higher levels than any other test agent or drug combination and at any concentration (up to 50 nM). Media and IgG controls were used. Taken together, these results suggest that the hVSIG8-Fc-OX40L chimeric protein functionally activates primary human white blood cells in vitro.

[0192] Example 8: Characterization of the binding affinities of different domains of the VSIG8-Fc-OX40L chimeric protein using ELISA An ELISA (enzyme-linked immunosorbent assay) assay was developed to demonstrate the binding affinity of the different domains of mouse VSIG8-Fc-OX40L to their respective binding partners (i.e., VISTA, human IgG (hIgG), or OX40). Specifically, the VSIG8 domain of the mVSIG8-Fc-OX40L chimeric protein was captured onto plate-bound recombinant mouse VISTA protein and detected with an OX40L-specific antibody. The Fc portion of the chimeric protein was captured onto plate-bound hIgG and detected with an HRP-conjugated anti-hIgG antibody. The OX40L domain of the chimeric protein was captured onto plate-bound recombinant mouse OX40 protein and detected with an OX40L-specific antibody.

[0193] As shown in Figure 8, the different domains of the mVSIG8-Fc-OX40L chimeric protein effectively interacted with their respective binding partners with high affinity. Nevertheless, it was observed that detecting the chimeric protein using the central Fc region in an ELISA assay tends to underestimate the actual protein content in the sample. Thus, low levels of VSIG8-Fc-OX40L chimeric protein were detected in this assay compared to the standard. The size of the band confirmed the expected monomer molecular weight of approximately 68.1 kDa, suggesting that the native state of the chimeric protein is a glycosylated dimer.

[0194] Example 9: Characterization of mouse VSIG8-Fc-OX40L chimeric protein The mouse VSIG8-Fc-OX40L chimeric protein was constructed as described above in the detailed description and in US Pat. No. 62 / 464,002, the contents of which are incorporated herein by reference in their entirety.

[0195] 9A-9C show ELISA assays demonstrating the binding affinity of different domains of mouse VSIG8-Fc-OX40L chimeric protein to their respective binding partners. Specifically, the VSIG8 domain of mVSIG8-Fc-OX40L chimeric protein was captured to plate-bound recombinant mouse VISTA protein (binding partner of VSIG8) and detected by HRP-conjugated anti-mouse IgG antibody (FIG. 9A). A standard curve was not generated because a commercially available mVSIG8-Fc standard was not available. The Fc portion of the chimeric protein was captured to plate-bound mouse IgG and detected by HRP-conjugated anti-mouse IgG antibody (FIG. 9B). The OX40L domain of the chimeric protein was captured to plate-bound recombinant mouse OX40 protein and detected by OX40L-specific antibody (FIG. 9C). FIG. 9A-9C show that the different domains of mVSIG8-Fc-OX40L chimeric protein effectively interacted with their respective binding partners with high affinity. Nevertheless, it was observed that detecting chimeric proteins using the central Fc region in an ELISA assay tends to underestimate the actual protein content in the sample, and therefore lower levels of mVSIG8-Fc-OX40L chimeric protein were detected in this assay compared to the standard.

[0196] Example 10: Characterization of in vitro cell binding affinity of VSIG8-Fc-OX40L chimeric protein Cell-binding assays were performed to demonstrate the binding affinity of different domains of the mouse VSIG8-Fc-OX40L chimeric protein to their respective binding partners on the surface of mammalian cell membranes.

[0197] For cell binding assays, immortalized cell lines were engineered to stably express mouse VISTA (EL4-mVISTA) and mouse receptor OX40 (CHOK1-mOX40). Increasing concentrations of mVSIG8-Fc-OX40L were incubated with each parental cell line (control) and overexpressing cell line for 2 hours. Cells were harvested, washed, and stained with antibodies for detection of chimeric protein binding by flow cytometry.

[0198] As shown in Figures 10A and 10B, the mVSIG8-Fc-OX40L chimeric protein bound to mVISTA and mOX40 present on the cell surface in a concentration-dependent manner and with low nM affinity. Specifically, as shown in Figure 10A, the EL4 parental cell line (lower curve) did not respond to increasing concentrations of the mVSIG8-Fc-OX40L chimeric protein because it did not overexpress VISTA. In comparison, the EL4-mVISTA cell line (upper curve), which was engineered to overexpress mVISTA, bound to mVSIG8-FC-OX40L in a concentration-dependent manner. Similarly, as shown in Figure 10B, the CHOK1 parental cell line (lower curve) did not respond to increasing concentrations of the mVSIG8-Fc-OX40L chimeric protein because it did not overexpress mOX40. In contrast, the CHOK1-mOX40 cell line engineered to overexpress mOX40 (upper curve) bound mVSIG8-Fc-OX40L in a concentration-dependent manner. Cell binding assays also showed that mVSIG8-Fc-OX40L bound to mOX40 with an affinity of 16 nM and to mVISTA with an affinity of 56 nM (EC 50 (Calculated).

[0199] Example 11: Characterization of the binding affinity of mouse VSIG8-Fc-OX40L chimeric protein by surface plasmon resonance (SPR) The binding affinity of the OX40L domain of the mouse VSIG8-Fc-OX40L chimeric protein was measured by surface plasmon resonance (SPR) using a BioRad ProteOn XPR 360 system. Specifically, the affinity of the chimeric protein for mouse OX40 was determined and compared to a commercially available recombinant control protein (mFc-OX40L). The results are shown in FIG. 11. The mVSIG8-Fc-OX40L chimeric protein was found to bind to mOX40 with high affinity.

[0200] Example 12: Induction of OX40 signaling in vitro Activation of mouse OX40 leads to induction of a signaling cascade involving activation of both NF-κB and NIK. Figure 12 shows exemplary data from an in vitro NF-κB / NIK signaling assay using mouse VSIG8-Fc-OX40L chimeric protein. U2OS cells from the DiscoverX NIK signaling assay were cultured with titrations of either an irrelevant protein, commercial single-sided hFc-OX40L, or mVSIG8-Fc-OX40L chimeric protein. Relative luciferase units (RLU) indicate the relative strength of NF-κB / NIK signaling activated after treatment with the indicated regimen. VSIG8-Fc-OX40L is shown to have signaling potently activated through NF-κB and NIK and to a greater extent than any of the other indicated regimens.

[0201] Example 13: Functional assay of VSIG8-Fc-OX40L chimeric protein In vivo functional assays were performed to demonstrate the functional activity of murine VSIG8-Fc-OX40L chimeric protein. Mice were inoculated with CT26 tumors on day 0. When tumors were palpable and at least 4-6 mm in diameter, mice were treated with two doses of 150 μg mVSIG8-Fc-OX40L chimeric protein. Immunophenotyping was performed on various tissues collected from mice on day 13 after tumor implantation.

[0202] Figures 13A-C show the results from in vivo functional assays. Immune profiling was performed on tumor-bearing mice treated with mVSIG8-Fc-OX40L chimeric protein. As shown in Figure 13A, mice treated with mVSIG8-Fc-OX40L chimeric protein showed a higher percentage of total CD4+ T cells in the spleen, peripheral lymph nodes, and tumors when compared to untreated control mice. In the spleen and tumors, this increase in the CD4+ T cell population was mostly due to an increase in CD4+CD25- effector T cells, suggesting that activation of non-regulatory T cells is involved (Figure 13B). Treated mice also showed a lower percentage of CD4+CD25+ regulatory T cells, suggesting that regulatory T cells may be suppressed by the chimeric protein (Figure 13B).

[0203] The ability of chimeric protein to stimulate the recognition of tumor antigen by CD8+ T cells was also analyzed. Specifically, FIG. 13C shows tetramer staining analysis to determine the fraction of CD8+ T cells that recognized the AH1 tumor antigen naturally expressed by CT26 tumor. In the spleen, a higher percentage of CD8+ T cells was found to recognize the AH1 tumor antigen in mice treated with mVSIG8-Fc-OX40L chimeric protein, when compared with untreated control mice. In particular, a much higher percentage of AH1 tetramer-positive CD8+ T cells was observed in tumor infiltrating lymphocytes (TILs) of mice treated with chimeric protein, when compared with untreated control mice.

[0204] Example 14: Characterization of the in vivo antitumor activity of VSIG8-Fc-OX40L chimeric protein The in vivo antitumor activity of the VSIG8-Fc-OX40L chimeric protein was analyzed using a CT26 mouse colorectal tumor model.

[0205] In one set of experiments, Balb / c mice were inoculated with CT26 tumor cells on day 0 and / or received a second inoculation of CT26 tumor cells on day 30. After 4 days of tumor growth, when tumors reached a diameter of 4-5 mm, mice were treated with either a control antibody or 150 μg of murine VSIG8-Fc-OX40L chimeric protein. Treatment was repeated on day 7. Analysis of tumor size evolution was performed over a 45-day period following tumor inoculation.

[0206] As shown in FIG. 14A, untreated mice developed large tumors, while most of the mice treated with mVSIG8-Fc-OX40L chimeric protein did not develop tumors of detectable size. FIG. 14B shows the overall survival rate of mice over 50 days after tumor inoculation. All of the untreated mice died within 21 days after tumor inoculation, while mice treated with mVSIG8-Fc-OX40L chimeric protein showed 100% survival rate at 30 days after tumor inoculation. At 50 days after tumor inoculation, more than 75% of the mVSIG8-Fc-OX40L-treated mice remained alive. FIG. 14C summarizes the treatment results of each group. As shown in FIG. 14C, treatment with chimeric protein also resulted in significantly higher tumor rejection than treatment with control antibody. Notably, some mice showed long-term tumor stabilization, consistent with a balancing effect between tumor growth and antitumor immunity. This period extended well beyond the last treatment with chimeric protein, suggesting that memory immunity may have been activated. Interestingly, some mice in which immune balance was observed showed delayed complete tumor rejection. More specifically, treatment with the chimeric protein led to the rejection of the primary tumor and the complete rejection of tumor cells administered during rechallenge, suggesting that memory T cells may be involved.

[0207] The above data suggest that treatment with VSIG8-Fc-OX40L chimeric protein results in an immune memory effect in vivo, such that treated animals can subsequently attack tumor cells and / or prevent tumor development upon rechallenge after the first treatment with the chimeric protein.

[0208] Example 15: ELISA-based anti-drug antibody assay of mouse VSIG8-Fc-OX40L chimeric protein An ELISA-based anti-drug antibody (ADA) assay was performed with mouse VSIG8-Fc-OX40L chimeric protein. High-binding ELISA plates were coated with either animal serum (blue line; starting undiluted "neat"), then diluted 1:2, 1:4, and 1:8) or recombinant mouse Fc (black line; starting at 5 μg / mL, then diluted 1:2, 1:4, 1:8, 1:16, and 1:32) and probed with 10 μg / mL of mVSIG8-Fc-OX40L chimeric protein. Serum was collected from mice that rejected both primary and secondary CT26 tumors, and then loaded with a third dose of 150 μg of mVSIG8-Fc-OX40L chimeric protein by intraperitoneal (IP) injection on day 91. Serum was collected one week after day 98. The chimeric proteins were detected using goat anti-mouse OX40L antibody followed by a goat anti-HRP tertiary antibody. 450 ) and the nonlinear fits of each curve are shown in FIG.

[0209] Example 16: Characterization of the contribution of the Fc domain in the linker to the functionality of the chimeric protein In this example, the contribution of the Fc domain in the linker to the functionality of the chimeric protein of the invention was assayed, where PD1-Fc-OX40L was used as a model for Fc-containing chimeric proteins, and therefore the data presented below are relevant to the chimeric protein of the invention.

[0210] In the native state, PD1 exists as a monomer, while OX40L tends to dimerize due to electrostatic interactions between the OX40L domains. The Fc domains bind to each other via disulfide bonds. In summary, several intermolecular interactions may contribute to the quaternary structure of PD1-Fc-OX40L. There are at least four potential structures of PD1-Fc-OX40L, and the chimeric protein exists as a monomer, dimer, trimer, or hexamer. See Figure 16.

[0211] The presence of monomeric and dimeric structures of the chimeric protein was tested by exposing the chimeric protein to reducing and non-reducing conditions and then running the protein on SDS-PAGE. Under non-reducing conditions (reduced: "-"), the chimeric protein migrated at approximately 200 kDa on SDS-PAGE. Here, Western blots were probed with antibodies against PD1, Fc, or OX40L, as shown in the left, middle, and right blots of FIG. 17, respectively. Since the predicted monomeric molecular weight of the chimeric protein is 57.6 kDa, the 200 kDa species was expected to be at least a dimer. However, under reducing conditions (reduced: "+"), which reduces disulfide bonds (e.g., between Fc domains), the chimeric protein migrated at approximately 100 kDa on SDS-PAGE. Since the 100 kDa species was larger than expected, the extra mass was expected to be due to glycosylation. Finally, the chimeric protein was treated with peptide-N-glycosidase F (PNGaseF "+") and run on SDS-PAGE under reducing conditions. Under these conditions, the chimeric protein migrated at approximately 57.6 kDa. These data suggest that the chimeric protein is glycosylated and exists naturally at least as a dimer, with dimerization likely resulting from disulfide bonds between the Fc domains.

[0212] SDS-PAGE gel methods do not accurately predict molecular weights for proteins with high charge and / or large molecular weight. Therefore, the chimeric proteins were next characterized using size-exclusion chromatography (SEC). Unlike SDS-PAGE, where the negative charge SDS reduces charge-based interactions between peptides, SEC does not use detergents or reducing agents. When the PD1-Fc-OX40L chimeric protein was subjected to SEC, there was no peak around 200 kDa. This suggests that in nature, the chimeric protein does not exist as a dimer. Instead, a peak with a size of over 670 kDa was detected. See Figure 18. This data and previous data suggest that the PD1-Fc-OX40L chimeric protein exists as a hexamer in its native state.

[0213] As shown above, when run on SDS-PAGE under non-reducing or reducing conditions, SDS in the sample and / or running buffer converts the hexameric PD1-Fc-OX40L chimeric protein to predominantly dimers or monomers, respectively, with or without reducing agents. See Figure 19 (left gel). When run on native PAGE lacking SDS, in the absence of reducing agents, the chimeric protein exists as a hexamer. However, when run on native PAGE in the presence of reducing agents (which reduce disulfide bonds), the chimeric protein migrated heavier than expected. As shown in Figure 19 (right gel, lane #2), the chimeric protein was virtually unable to migrate from the loading well. This data suggests that the chimeric protein oligomerized into higher order proteins. Therefore, disulfide bonds appear to be important in controlling higher order oligomerization in chimeric proteins.

[0214] To further confirm this, chimeric proteins lacking the Fc domain, e.g., "PD1-No-Fc-OX40L", were constructed. Such chimeric proteins do not have the disulfide bonds that occur between the Fc domains in the aforementioned chimeric proteins. As shown in FIG. 20, when the chimeric proteins lacking the Fc domain were run on native PAGE, virtually none of the proteins migrated from the loading well (lanes #1-#4 show increasing loading concentrations of PD1-No Fc-OX40L), again suggesting that the "Fc-free" chimeric proteins formed concatemer-like complexes containing multiple proteins. Thus, removal of the Fc domain in the chimeric proteins results in the formation of protein aggregates. These data indicate that disulfide bonds, e.g., between the Fc domains on different chimeric proteins, stabilize the chimeric proteins and ensure that they exist as hexamers, respectively, and not as higher-order proteins / concatamers. In other words, the Fc domain surprisingly brings order to the chimeric protein complex. Lanes #1 to #4 contain 2.5 μg of PD1-No-Fc-OX40L, 5 μg of PD1-No-Fc-OX40L, 7.5 μg of PD1-No-Fc-OX40L, and 10 μg of PD1-No-Fc-OX40L, respectively.

[0215] Shown in Figure 21 is a model summarizing the above data and illustrating how hexamers and concatemers are formed from the chimeric proteins of the present invention. The exemplary chimeric protein (PD1-Fc-OX40L) spontaneously forms hexamers (due to electrostatic interactions between the OX40L domains and dimerization through the Fc domain). However, under reducing conditions for the PD1-Fc-OX40L protein, due to the absence of the regulating effect of disulfide bonds between the Fc domains and the absence of an Fc domain in PD1-No-Fc-OX40L, these latter chimeric proteins form concatemers.

[0216] In addition, chimeric proteins were constructed in which the Fc domain (described herein) was replaced with ficolin (which lacks the cysteine ​​residues necessary for disulfide bonds between the chimeric proteins). Similar to the chimeric proteins without Fc and those containing Fc run in the presence of reducing agents on native PAGE (both formed aggregates that did not migrate in the gel), the chimeric proteins containing ficolin also appeared to form higher order lattices and did not migrate in the gel. These data strengthen the conclusion that disulfide bonds are important for proper folding and function of the chimeric proteins of the invention.

[0217] Finally, chimeric proteins were prepared with a coiled Fc domain (CCDFc). In functional evaluation, little of the purified protein was delivered.

[0218] Therefore, inclusion of an Fc domain in the linker of the chimeric proteins, which can form disulfide bonds between the chimeric proteins, helps to avoid the formation of insoluble and possibly non-functional protein concatemers and / or aggregates.

[0219] Example 17: Generation of additional VSIG8-containing chimeric proteins containing the extracellular domains of other type II proteins In this example, additional chimeric proteins of the present invention are described. Such additional chimeric proteins are made in the same manner as the VSIG8-Fc-OX40L chimeric protein is made, for example, as described above in the detailed description and in U.S. Patent No. 62 / 464,002, the contents of which are incorporated herein by reference in their entirety.

[0220] These additional chimeric proteins have the general formula: ECD 1-binding linker 1-Fc domain-binding linker 2-ECD 2, where ECD 1 is the extracellular domain of VSIG8 and ECD 2 is the extracellular domain of a type II protein other than OX40L. Exemplary type II proteins include 4-1BBL, CD30L, CD40L, FasL, GITRL, LIGHT, TL1A and TRAIL. These chimeric proteins may lack one or both of the binding linkers. Exemplary binding linkers 1, Fc domains and binding linkers 2 are listed in Table 1 above. Modular linkers useful for forming chimeric proteins and including specific binding linkers 1, Fc domains and binding linkers 2 are shown in FIG. 22.

[0221] Alternatively, additional chimeric proteins are fusion proteins having the general formula: N-terminus -(a)-(b)-(c)-C-terminus, where (a) is VSIG8, (b) is a linker comprising at least a portion of an Fc domain, and (c) is an extracellular domain of a type II protein other than OX40L. Exemplary type II proteins include 4-1BBL, CD30L, CD40L, FasL, GITRL, LIGHT, TL1A, and TRAIL. Exemplary linkers are listed in Table 1 above. A modular linker useful for forming chimeric proteins and comprising a specific binding linker 1, an Fc domain, and a binding linker 2 is shown in FIG. 22.

[0222] The amino acid sequences of 4-1BBL, CD30L, CD40L, FasL, GITRL, LIGHT, TL1A and TRAIL comprise SEQ ID NOs: 7, 9, 11, 13, 15, 17, 21, and 23, respectively. The amino acid sequences of the extracellular domains of 4-1BBL, CD30L, CD40L, FasL, GITRL, LIGHT, TL1A and TRAIL comprise SEQ ID NOs: 8, 10, 12, 14, 16, 18, 22, and 24, respectively. The amino acid sequence of VSIG8 comprises SEQ ID NO: 1, and the extracellular domain of VSIG8 comprises SEQ ID NO: 2. The chimeric proteins can comprise variants of the above sequences, for example, at least about 95% identity to the above sequences.

[0223] Accordingly, the present invention further includes the following additional chimeric proteins and methods using the additional chimeric proteins: VSIG8-Fc-4-1BBL, VSIG8-Fc-CD30L, VSIG8-Fc-CD40L, VSIG8-Fc-FasL, VSIG8-Fc-GITRL, VSIG8-Fc-LIGHT, VSIG8-Fc-TL1A, and VSIG8-Fc-TRAIL (e.g., in the treatment of cancer and / or the treatment of inflammatory diseases).

[0224] The additional chimeric proteins are characterized as described above for CSF1R-Fc-CD40L in Examples 1-15, but with reagents (e.g., binding partners, recombinant target cells, and cancer cell / tumor types) that are specific for the additional chimeric proteins rather than those required to characterize VSIG8-FC-OX40L. Thus, using VSIG8-Fc-4-1BBL as an example, characterization of VSIG8-Fc-4-1BBL similar to Example 2 can be performed using anti-VSIG8, anti-Fc, and anti-4-1BBL antibodies rather than the anti-VSIG8, anti-Fc, and anti-OX40L antibodies required for VSIG8-Fc-OX40L.

[0225] Similar to the VSIG8-Fc-OX40L chimeric protein, the additional chimeric proteins are effective in treating cancer and / or treating inflammatory diseases by blocking VSIG8 (inhibiting immune inhibitory signaling) and enhancing, increasing, and / or stimulating the delivery of immune stimulatory signals through activation of one of the receptors / ligands of 4-1BBL, CD30L, CD40L, FasL, GITRL, LIGHT, TL1A, and TRAIL. Furthermore, the additional chimeric proteins are effective in treating cancer and / or inflammatory diseases without the toxicity resulting from treatments that include multiple antibodies, e.g., VISTA blocking antibodies and agonistic antibodies against one of the receptors / ligands of 4-1BBL, CD30L, CD40L, FasL, GITRL, LIGHT, TL1A, and TRAIL.

[0226] (Additional Note) The present disclosure includes the following aspects. <1> (a) a first domain that comprises a portion of the V set and an immunoglobulin domain-containing protein 8 (VSIG8) capable of binding to a VSIG8 ligand; (b) a second domain comprising a portion of an OX40 ligand (OX40L) capable of binding to an OX40L receptor; and (c) a linker connecting the first domain and the second domain A heterologous chimeric protein comprising: <2> The first domain comprises substantially all of the extracellular domain of VSIG8 and the second domain comprises substantially all of the extracellular domain of OX40L. <1> A heterologous chimeric protein as described in claim 1. <3> The heterologous chimeric protein can inhibit immunosuppressive signals. <1> or <2> A heterologous chimeric protein as described in claim 1. <4> The heterologous chimeric protein is (a) capable of reducing, disrupting, or eliminating an immune inhibitory signal when a portion of VSIG8 binds to its ligand; and / or (b) a portion of OX40L can increase, stimulate, or activate an immune stimulatory signal when bound to its receptor; <1> or <2> A heterologous chimeric protein as described in claim 1. <5> The VSIG8 ligand is V-domain immunoglobulin-containing suppressor of T-cell activation (VISTA); <1> ~ <4> A heterologous chimeric protein according to any one of claims 1 to 4. <6> The OX40L receptor is OX40; <1> ~ <5> A heterologous chimeric protein according to any one of claims 1 to 4. <7> The heterologous chimeric protein can simultaneously bind to a VSIG8 ligand and an OX40L receptor, the VSIG8 ligand being VISTA and the OX40L receptor being OX40; <1> ~ <6> A heterologous chimeric protein according to any one of claims 1 to 4. <8> The chimeric protein is capable of simultaneously binding to recombinant human OX40 and human VISTA in vitro; <1> ~ <7> A heterologous chimeric protein according to any one of claims 1 to 4. <9> The linker is a polypeptide selected from a flexible amino acid sequence, an IgG hinge region, or an antibody sequence. <1> ~ <8> A heterologous chimeric protein according to any one of claims 1 to 4. <10> the linker comprises a hinge-CH2-CH3 Fc domain from IgG4; <9> A heterologous chimeric protein as described in claim 1. <11> the hinge-CH2-CH3 Fc domain is derived from human IgG4; <10> A heterologous chimeric protein as described in claim 1. <12> the chimeric protein is expressed by a mammalian host cell as a secretable, functional single polypeptide chain; <1> ~ <11> A heterologous chimeric protein according to any one of claims 1 to 4. <13> The portion of VSIG8 is at least 95% identical to the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2. <1> ~ <12> A heterologous chimeric protein according to any one of claims 1 to 4. <14> The portion of OX40L is at least 95% identical to the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4; <1> ~ <13> A heterologous chimeric protein according to any one of claims 1 to 4. <15> The linker comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:25, SEQ ID NO:26, or SEQ ID NO:27; <1> ~ <14> A heterologous chimeric protein according to any one of claims 1 to 4. <16> The linker comprises one or more bond linkers, such as bond linkers independently selected from SEQ ID NOs: 28-74; <1> ~ <15> A heterologous chimeric protein according to any one of claims 1 to 4. <17> the linker comprises two or more linked linkers, each independently selected from SEQ ID NOs: 28-74, one linked linker being at the N-terminus of the hinge-CH2-CH3 Fc domain and another linked linker being at the C-terminus of the hinge-CH2-CH3 Fc domain; <16> A heterologous chimeric protein as described in claim 1. <18> The chimeric protein is a recombinant fusion protein. <1> ~ <17> A heterologous chimeric protein according to any one of claims 1 to 4. <19> the chimeric protein enhances tumor antigen recognition by CD8+ T cells; <1> ~ <18> A heterologous chimeric protein according to any one of claims 1 to 4. <20> The chimeric protein optionally depletes VISTA in serum. <1> ~ <19> A heterologous chimeric protein according to any one of claims 1 to 4. <21> The chimeric protein exhibits enhanced anti-tumor effects compared to OX40 agonist antibody and / or VSIG8 antagonist antibody. <1> ~ <20> A heterologous chimeric protein according to any one of claims 1 to 4. <22> The heterologous chimeric protein can increase a subpopulation of CD4+ and / or CD8+ T cells or prevent a decrease in a subpopulation of CD4+ and / or CD8+ T cells. <1> ~ <21> A heterologous chimeric protein according to any one of claims 1 to 4. <23> The heterologous chimeric protein can enhance tumor killing activity by T cells. <1> ~ <22> A heterologous chimeric protein according to any one of claims 1 to 4. <24> The heterologous chimeric protein can provide a sustained immunomodulatory effect. <1> ~ <23> A heterologous chimeric protein according to any one of claims 1 to 4. <25> The heterologous chimeric protein is capable of causing activation of antigen-presenting cells. <1> ~ <24> A heterologous chimeric protein according to any one of claims 1 to 4. <26> The heterologous chimeric protein can enhance the ability of an antigen-presenting cell to present an antigen. <1> ~ <25> A heterologous chimeric protein according to any one of claims 1 to 4. <27> the chimeric protein shifts the ratio of immune cells in favor of cells that can kill tumors as opposed to cells that protect tumors; <1> ~ <26> A heterologous chimeric protein according to any one of claims 1 to 4. <28> the chimeric protein increases the ratio of effector T cells to regulatory T cells; <1> ~ <27> A heterologous chimeric protein according to any one of claims 1 to 4. <29> The chimeric protein provides a sustained masking effect of immune inhibitory signals. <1> ~ <28> A heterologous chimeric protein according to any one of claims 1 to 4. <30> The chimeric protein is a serum t 1 / 2 Longer on-target (e.g., intratumoral) half-life (t 1 / 2 ) to provide <1> ~ <29> A heterologous chimeric protein according to any one of claims 1 to 4. <31> The chimeric protein reduces toxicity compared to treatment with antibodies against VSIG8 and OX40. <1> ~ <30> A heterologous chimeric protein according to any one of claims 1 to 4. <32> The chimeric protein is capable of forming stable synapses between cells. <1> ~ <31> A heterologous chimeric protein according to any one of claims 1 to 4. <33> Stable synapses between the cells provide a spatial orientation that is favorable for tumor regression. <32> A heterologous chimeric protein as described in claim 1. <34> the spatial orientation positions the T cells to attack tumor cells and / or sterically prevents tumor cells from delivery of negative signals, including negative signals other than those masked by the chimeric protein of the invention; <32> or <33> A heterologous chimeric protein as described in claim 1. <35> Binding of either or both of the extracellular domains to their respective binding partners results in a slow off-rate (K off ) occurs in <1> ~ <34> A heterologous chimeric protein according to any one of claims 1 to 4. <36> The longer the interaction, the longer the positive signaling effect. <35> A heterologous chimeric protein as described in claim 1. <37> The longer positive signaling effect allows effector cells to be sufficiently stimulated for anti-tumor effects. <36> A heterologous chimeric protein as described in claim 1. <38> The long interaction provides T cell proliferation and enables anti-tumor attack. <36> or <37> A heterologous chimeric protein as described in claim 1. <39> The long interaction allows for sufficient signal transduction to provide for the release of a stimulatory signal. <36> ~ <38> A heterologous chimeric protein according to any one of claims 1 to 4. <40> The stimulatory signal is a cytokine. <39> A heterologous chimeric protein as described in claim 1. <41> <1> An expression vector comprising a nucleic acid encoding a heterologous chimeric protein described in any one of claims 1 to 40. <42> <41> A host cell comprising the expression vector described in . <43> <1> A pharmaceutical composition comprising a therapeutically effective amount of a heterologous chimeric protein described in any one of claims 1 to 40. <44> A method for treating cancer or an inflammatory disease, comprising administering to a subject in need thereof <43> 23. A method comprising administering an effective amount of the pharmaceutical composition described in claim 22. <45> A method of modulating an immune response in a patient, comprising administering to a subject in need thereof <43> 23. A method comprising administering an effective amount of the pharmaceutical composition described in claim 22. <46> T cells of the patient are activated. <44> or <45> The method according to <47> the patient has a tumor and one or more tumor cells are prevented from transmitting an immunosuppressive signal; <44> or <45> The method according to <48> 1. A method of treating cancer or an inflammatory disease comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition comprising: (a) a first domain comprising a portion of the colony-stimulating factor 1 receptor (VSIG8) capable of binding to a VSIG8 ligand; (b) a second domain comprising a portion of CD40 ligand (OX40L) capable of binding to the OX40L receptor; and (c) a linker connecting the first domain and the second domain The method comprises administering to a subject a heterologous chimeric protein comprising: <49> wherein the subject's T cells are activated upon binding of the second domain of the heterologous chimeric protein; and (a) one or more tumor cells are prevented from transmitting an immunosuppressive signal upon binding of the first domain of the heterologous chimeric protein; (b) a quantifiable cytokine response in the peripheral blood of the subject is achieved; and / or (c) tumor growth is reduced in a subject in need thereof compared to a subject treated with an OX40 blocking antibody and / or a VISTA blocking antibody. <48> The method according to <50> The method stimulates OX40 signaling and activates antigen presenting cells. <44> The method according to any one of claims 1 to 49. <51> the method reduces the amount or activity of regulatory T cells (Tregs) compared to an untreated subject or a subject treated with an agent that targets one of OX40 / OX40L and VISTA / VSIG8. <44> ~ <50> 2. The method according to claim 1 . <52> the method increases priming of effector T cells in the draining lymph nodes of the subject compared to an untreated subject or a subject treated with an agent that targets one of OX40 / OX40L and VISTA / VSIG8. <44> ~ <51> 2. The method according to claim 1 . <53> the method results in an overall reduction in immunosuppressive cells and a shift to a more inflammatory tumor environment compared to an untreated subject or a subject treated with an agent that targets one of OX40 / OX40L and VISTA / VSIG8. <44> ~ <52> 2. The method according to claim 1 . <54> For use as medicine <1> ~ <40> A heterologous chimeric protein according to any one of claims 1 to 4. <55> For use in the treatment of cancer <1> ~ <40> A heterologous chimeric protein according to any one of claims 1 to 4. <56> For use in the treatment of inflammatory diseases <1> ~ <40> A heterologous chimeric protein according to any one of claims 1 to 4. <57> In pharmaceutical manufacturing <1> ~ <40> Use of a heterologous chimeric protein according to any one of claims 1 to 5. <58> 1. A recombinant fusion protein comprising the general structure N-terminus-(a)-(b)-(c)-C-terminus, wherein (a) is a first domain that comprises the extracellular domain of VSIG8 that is at least 95% identical to the amino acid sequence of SEQ ID NO:2 and is capable of binding to a VSIG8 ligand; (b) is a linker that links the first domain and the second domain and includes a hinge-CH2-CH3 Fc domain derived from human IgG4 and, optionally, a binding linker sequence of any of SEQ ID NOs: 28 to 74; and (c) is a second domain that is at least 95% identical to the amino acid sequence of SEQ ID NO: 4 and includes the extracellular domain of CD40 ligand (OX40L) that can bind to the OX40L receptor; Recombinant fusion proteins. <59> The linker comprises a sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 25, SEQ ID NO: 26, or SEQ ID NO: 27; <58> A recombinant fusion protein as described in claim 1. <60> For use as a medicine, <58> or <59> A recombinant fusion protein as described in claim 1. <61> For use in the treatment of cancer <58> ~ <60> 2. A recombinant fusion protein according to any one of claims 1 to 11. <62> For use in the treatment of inflammatory diseases, <58> ~ <60> 2. A recombinant fusion protein according to any one of claims 1 to 11. <63> In the manufacture of pharmaceuticals, <58> ~ <60> 2. Use of a recombinant fusion protein according to any one of claims 1 to 11.

Claims

[Claim 1] (a) a first domain comprising a portion of VSIG8 (V-set and immunoglobulin domain-containing protein 8) capable of binding a VSIG8 ligand; (b) a second domain comprising a portion of an OX40 ligand (OX40L) capable of binding an OX40L receptor; and (c) a linker connecting the first domain and the second domain A heterologous chimeric protein comprising:

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