CHIMERIC PROTEINS IN AUTOIMMUNITY

TECHNICAL FIELD

The present invention relates to, inter alia, compositions and methods, including chimeric proteins that find use in the treatment of disease, such as in immunotherapies for treating autoimmunity.

DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY

This application contains a sequence listing. It has been submitted electronically via EFS-Web as an ASCII text file entitled “SHK-018PC_SequenceListing_ST25”. The sequence listing is 243,589 bytes in size, and was prepared on or about Aug. 30, 2020. The sequence listing is hereby incorporated by reference in its entirety.

BACKGROUND

The most important function of an immune system is to protect a subject against infection by foreign agents. Thus, a healthy immune system must discriminate between the healthy tissue (“self”) and foreign agents (“non-self”). Non-limiting examples of foreign agents can be microorganisms, pollen, and transplanted tissues from another individual. Autoimmune disease occurs when an immune system mounts an attack against healthy tissue since the system does not recognize the healthy tissue as “self”. Unfortunately, once an autoimmune disease has been diagnosed, it becomes or has become a chronic problem. A standard treatment for autoimmune diseases is a generalized suppression of the immune system. Unfortunately, such non-specific therapies may inhibit the immune system's ability to recognize and attack actual foreign agents which places the subject at risk for infections and cancerous malignancies. Accordingly, there is an unmet need for autoimmune therapies that effectively treat autoimmune disease yet minimized risk for infections and malignancy outgrowth.

SUMMARY

In various aspects, the present invention provides for compositions and methods that are useful for immunotherapies for treating an autoimmune disease. For instance, the present invention, in part, relates to specific chimeric proteins comprising two domains that each or both domains decrease self-directed immune system activity when bound to its ligand/receptor. Importantly, when each or both domains decreases immune system activity by activating an immune inhibitory signal or inhibiting an immune activating signal. Accordingly, the present chimeric proteins, compositions, and methods overcome various deficiencies in bi-specific agents directed to treat autoimmunity.

An aspect of the present invention is a chimeric protein of a general structure of: N terminus-(a)-(b)-(c)-C terminus in which (a) is a first domain comprising a portion of the extracellular domain of a transmembrane protein, a secreted protein, or a membrane-anchored extracellular protein, (c) is a second domain comprising a portion of the extracellular domain of a transmembrane protein, a secreted protein, or a membrane-anchored extracellular protein, and (b) is a linker adjoining the first domain and the second domain. In this aspect, either or both of the first domain and the second domain decreases self-directed immune system activity when bound to its ligand/receptor.

Another aspect of the present invention is a chimeric protein comprising: (a) a first domain comprising a portion of VSIG4 that is capable of binding a VSIG4 ligand/receptor, (b) a second domain comprising a portion of IL2 that is capable of binding an IL2 receptor, and (c) a linker linking the first domain and the second domain and comprising a hinge-CH2-CH3 Fc domain.

Yet another aspect of the present invention is a chimeric protein comprising: (a) a first domain comprising a portion of CTLA4 that is capable of binding a CTLA4 ligand/receptor, (b) a second domain comprising a portion of IL2 that is capable of binding an IL2 receptor, and (c) a linker linking the first domain and the second domain and comprising a hinge-CH2-CH3 Fc domain. In embodiments, the IL2 receptor is a high-affinity IL2 receptor that is expressed by regulatory T cells, e.g., the portion of IL2 comprises one or more mutations relative to a corresponding portion of wild-type IL2 which provides preferential binding to the high-affinity IL2 receptor that is expressed by regulatory T cells.

In an aspect, the present invention provides a chimeric protein comprising: (a) a first domain comprising a portion of CTLA4 that is capable of binding a CTLA4 ligand/receptor, (b) a second domain comprising a portion of PD-L1 that is capable of binding PD-1, and (c) a linker linking the first domain and the second domain and comprising a hinge-CH2-CH3 Fc domain.

In another aspect, the present invention provides a chimeric protein comprising: (a) a first domain comprising a portion of B7H3 that is capable of binding a B7H3 ligand/receptor, (b) a second domain comprising a portion of PD-L1 that is capable of binding PD-1, and (c) a linker linking the first domain and the second domain and comprising a hinge-CH2-CH3 Fc domain.

In yet another aspect, the present invention provides a chimeric protein comprising: (a) a first domain comprising a portion of B7H4 that is capable of binding a B7H4 ligand/receptor, (b) a second domain comprising a portion of PD-L1 that is capable of binding PD-1, and (c) a linker linking the first domain and the second domain and comprising a hinge-CH2-CH3 Fc domain.

An aspect of the present invention is a chimeric protein comprising: (a) a first domain comprising a portion of ICOSL that is capable of binding an ICOSL ligand/receptor, (b) a second domain comprising a portion of PD-L1 that is capable of binding PD-1, and (c) a linker linking the first domain and the second domain and comprising a hinge-CH2-CH3 Fc domain.

Another aspect of the present invention is chimeric protein comprising: (a) a first domain comprising a portion of ILDR2 that is capable of binding an ILDR2 ligand/receptor, (b) a second domain comprising a portion of PD-L1 that is capable of binding PD-1, and (c) a linker linking the first domain and the second domain and comprising a hinge-CH2-CH3 Fc domain.

Yet another aspect of the present invention is chimeric protein comprising: (a) a first domain comprising a portion of BTNL2 that is capable of binding a BTNL2 ligand/receptor, (b) a second domain comprising a portion of PD-L1 that is capable of binding PD-1, and (c) a linker linking the first domain and the second domain and comprising a hinge-CH2-CH3 Fc domain.

In an aspect, the present invention provides a chimeric protein comprising: (a) a first domain comprising a portion of PD-L1 that is capable of binding PD-1, (b) a second domain comprising a portion of BTNL2 that is capable of binding a BTNL2 ligand/receptor, and (c) a linker linking the first domain and the second domain and comprising a hinge-CH2-CH3 Fc domain.

In another aspect, the present invention provides a chimeric protein comprising: (a) a first domain comprising a portion of CSF3 that is capable of binding a CSF3 ligand/receptor, (b) a second domain comprising a portion of TL1A that is capable of binding a TL1A ligand/receptor, and (c) a linker linking the first domain and the second domain and comprising a hinge-CH2-CH3 Fc domain.

In yet another aspect, the present invention provides a chimeric protein comprising: (a) a first domain comprising a portion of CTLA4 that is capable of binding a CTLA4 ligand/receptor, (b) a second domain comprising a portion of TL1A that is capable of binding a TL1A ligand/receptor, and (c) a linker linking the first domain and the second domain and comprising a hinge-CH2-CH3 Fc domain.

Another aspect of the present invention is a chimeric protein comprising: (a) a first domain comprising a portion of CTLA4 that is capable of binding a CTLA4 ligand/receptor, (b) a second domain comprising a portion of SEMA3E that is capable of binding a SEMA3E ligand/receptor, and (c) a linker linking the first domain and the second domain and comprising a hinge-CH2-CH3 Fc domain.

Another aspect of the present invention is a chimeric protein comprising: (a) a first domain comprising a portion of MadCAM that is capable of binding a MadCAM ligand/receptor, (b) a second domain comprising a portion of CCL25 that is capable of binding a CCL25 ligand/receptor, and (c) a linker linking the first domain and the second domain and comprising a hinge-CH2-CH3 Fc domain.

Another aspect of the present invention is a chimeric protein comprising: (a) a first domain comprising a portion of TNFR2 that is capable of binding a TNFR2 ligand/receptor, (b) a second domain comprising a portion of TGFβ that is capable of binding a TGFβ ligand/receptor, and (c) a linker linking the first domain and the second domain and comprising a hinge-CH2-CH3 Fc domain.

Another aspect of the present invention is a chimeric protein comprising: (a) a first domain comprising an extracellular domain of IL-6R that is capable of binding a IL-6R ligand/receptor, (b) a second domain comprising a portion of IL-35 that is capable of binding a IL-35 ligand/receptor, and (c) a linker linking the first domain and the second domain and comprising a hinge-CH2-CH3 Fc domain. In embodiments, the first domain comprises an extracellular domain of IL-6ST and/or IL-6R. In embodiments, the second domain comprises a portion of EBI3 and/or IL-12A. In embodiments, the chimeric protein is heterodimeric.

Another aspect of the present invention is a chimeric protein comprising: (a) a first domain comprising an extracellular domain of integrin α4β7 that is capable of binding an integrin α4β7 ligand/receptor, (b) a second domain comprising a portion of IL-35 that is capable of binding a IL-35 ligand/receptor, and (c) a linker linking the first domain and the second domain and comprising a hinge-CH2-CH3 Fc domain. In embodiments, the first domain comprises an extracellular domain of integrin α4 and/or integrin β7. In embodiments, the second domain comprises a portion of EBI3 and/or IL-12A. In embodiments, the chimeric protein is heterodimeric.

The chimeric protein of any of the above aspects or embodiments may be a recombinant fusion protein.

The chimeric protein of any of the above aspects or embodiments may be used as a medicament in the treatment of an autoimmune disease, e.g., selected from ankylosing spondylitis, diabetes mellitus, Grave's disease, Hashimoto's thyroiditis, hypersensitivity reactions (e.g., allergies, hay fever, asthma, and acute edema cause type I hypersensitivity reactions), inflammatory bowel diseases (e.g., colitis ulcerosa and Crohn's disease), multiple sclerosis, psoriasis, psoriasis, rheumatoid arthritis, sarcoidosis, Sjögren's syndrome, systemic lupus erythematosus, and vasculitis.

The present invention includes the use of the chimeric protein of any of the above aspects or embodiments in the manufacture of a medicament.

An aspect of the present invention is an expression vector comprising a nucleic acid encoding the chimeric protein of any of the above aspects or embodiments.

Another aspect of the present invention is a host cell comprising the expression vector of the preceding aspect.

Yet another aspect of the present invention is a pharmaceutical composition comprising a therapeutically effective amount of the chimeric protein of any of the herein disclosed aspects or embodiments.

An aspect of the present invention is a method of treating an autoimmune disease comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition comprising a therapeutically effective amount of the chimeric protein of any of the herein disclosed aspects or embodiments.

Any aspect or embodiment disclosed herein can be combined with any other aspect or embodiment as disclosed herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A to FIG. 1C show schematic illustrations of proteins that may be used in chimeric proteins of the present disclosure. FIG. 1A shows a Type I transmembrane protein (left protein) and Type II transmembrane protein (right proteins); these proteins differ in that Type I proteins have their amino terminus (“N-”), which comprises its ligand/receptor binding site, directed extracellularly whereas Type II proteins have their carboxy terminus (“C-”), which comprises its ligand/receptor binding site, directed extracellularly. FIG. 1B shows two membrane-anchored extracellular proteins; the illustrated proteins have a ligand/receptor binding site at its amino terminus (“N-”) and is membrane anchored via its carboxy terminus (left protein) or have a ligand/receptor binding site at its carboxy terminus (“C-”) and is membrane anchored via its amino terminus (right protein); however, membrane-anchored extracellular proteins may be membrane anchored via other locations along the protein's amino acid sequence. FIG. 1C shows two secreted proteins (which lack a transmembrane domain or a membrane anchorage); the left protein has its ligand/receptor binding site at it amino terminus (“N-”) and the right protein has its ligand/receptor binding site at its carboxy terminus (“C-”).

FIG. 2A to FIG. 2D show schematic illustrations of chimeric proteins of the present disclosure. FIG. 2A shows a chimeric protein comprising a first domain with a ligand/receptor binding site at its amino terminus and a second domain with a ligand/receptor binding site at its carboxy terminus. Non-limiting examples of this configuration of chimeric protein include a chimeric protein comprising a portion of a Type I transmembrane protein as its first domain and a portion of a Type II transmembrane protein as its second domain and a chimeric protein comprising a portion of a Type I transmembrane protein as its first domain and a portion of a secreted protein as its second domain. FIG. 2B shows a chimeric protein comprising a first domain with a ligand/receptor binding site at its amino terminus and a second domain with a ligand/receptor binding site at its amino terminus. Non-limiting examples of this configuration of chimeric protein include a chimeric protein comprising a portion of a Type I transmembrane protein as its first domain and a portion of a Type I transmembrane protein as its second domain and a chimeric protein comprising a portion of a Type I transmembrane protein as its first domain and a portion of a secreted protein as its second domain. FIG. 2C shows a chimeric protein comprising a first domain with a ligand/receptor binding site at its carboxy terminus and a second domain with a ligand/receptor binding site at its carboxy terminus. Non-limiting examples of this configuration of chimeric protein include a chimeric protein comprising a portion of a membrane anchored protein as its first domain and a portion of secreted protein as its second domain and a chimeric protein comprising a portion of secreted protein as its first domain and a portion of a Type II transmembrane protein as its second domain. FIG. 2D shows a chimeric protein comprising a first domain with a ligand/receptor binding site at its carboxy terminus and a second domain with a ligand/receptor binding site at its amino terminus. Non-limiting examples of this configuration of chimeric protein include a chimeric protein comprising a portion of secreted protein as its first domain and a portion of a membrane anchored protein as its second domain and a chimeric protein comprising a portion of Type II transmembrane protein as its first domain and a portion of a Type I transmembrane protein as its second domain.

FIG. 3 is a schematic of a CSF3- and TL1A-based chimeric protein of the present disclosure and shows characterization of a murine CSF3-Fc-TL1A chimeric protein by western blot demonstrating the chimeric proteins native state and tendency to form a multimer. Untreated samples (i.e., without reducing agent or deglycosylation agent) of the CSF3-Fc-TL1A chimeric protein, e.g., control, were loaded into lane 2 in all the blots. Samples in lane 3 were treated with the reducing agent, 3-mercaptoethanol. Samples in lane 4 were treated with a deglycosylation agent and the reducing agent. Each individual domain of the chimeric protein was probed using an anti-CSF3, anti-Fc, or anti-TL1A antibody, respectively.

FIG. 4A to FIG. 4D show ELISA assays demonstrating the binding affinity of the CSF3 domain of mCSF3-Fc-TL1A (FIG. 4A and FIG. 4B), the Fc domain of mCSF3-Fc-TL1A (FIG. 4C), and of the TL1A domain of mCSF3-Fc-TL1A (FIG. 4D) for their respective binding partners.

FIG. 5 is a graph demonstrating the in vivo ability of the mCSF3-Fc-TL1A chimeric protein to increase the frequency of regulatory T cells (Treg) relative to blood stem cells.

FIG. 6 is a graph demonstrating the in vivo ability of the mCSF3-Fc-TL1A chimeric protein to increase the frequency of regulatory T cells (Treg) relative to other CD4+ T cells. Here, the treatments administered, from left to right, are: control (PBS), anti-DR3 antibody (100 μg), G-CSF (10 μg manufactured by LSbio), G-CSF (10 μg manufactured by Biolegend), G-CSF (50 μg manufactured by Biolegend), a combination of the anti-DR3 antibody (100 μg) and G-CSF (10 μg manufactured by Biolegend), or the mCSF3-Fc-TL1A chimeric protein (at 100 μg or 300 μg).

FIG. 7A is a schematic of a VISG4- and IL2-based chimeric protein of the present disclosure. FIG. 7B shows characterization of a murine VSIG4-Fc-IL2 chimeric protein by western blot. Untreated samples (i.e., without reducing agent or deglycosylation agent “NR”) of the mVSIG4-Fc-IL2 chimeric protein, samples treated with the reducing agent, β-mercaptoethanol (“R”), and samples treated with a deglycosylation agent and the reducing agent (“DG”) are shown. The blot was probed using an anti-Fc antibody. FIG. 7C shows an ELISA of the mVSIG4-Fc-IL2 chimeric protein captured by its Fc domain.

FIG. 8A is a schematic of a PD-L1- and BTNL2-based chimeric protein of the present disclosure. FIG. 8B shows characterization of a murine PD-L1-Fc-BTNL2 chimeric protein by western blot. FIG. 8C shows an ELISA of the mPD-L1-Fc-BTNL2 chimeric protein captured by its Fc domain.

FIG. 9A is a schematic of a CTLA4- and SEMA3E-based chimeric protein of the present disclosure. FIG. 9B shows characterization of a human CTLA4-Fc-SEMA3E chimeric protein by western blot probed using an anti-Fc antibody. FIG. 9C shows an ELISA of the hCTLA4-Fc-SEMA3E chimeric protein captured by its Fc domain.

FIG. 10A is a schematic of an ILDR2- and PD-L1-based chimeric protein of the present disclosure. FIG. 10B shows characterization of a human ILDR2-Fc-PD-L1 chimeric protein by western blot probed using an anti-Fc antibody. FIG. 10C shows an ELISA of the h ILDR2-Fc-PD-L1 chimeric protein captured by its Fc domain.

FIG. 11A and FIG. 11B show chromatographs for the human IL-6R-Fc-IL-35 chimeric proteins run on size exclusion chromatography (SEC).

FIG. 12 shows the induction of apoptosis as measured by a luciferase assay in DS-1 cells cultured for 24 hours in the presence of increasing molar ratios of the indicated molecules to IL-6. Caspase 3/7 activity was plotted.

FIG. 13A to FIG. 13G show the change in the relative levels of mRNA, as measured by qRT-PCR, of EBI3 (FIG. 13A), IL-12A (FIG. 13B), FOXP3 (FIG. 13C), TOP2A (FIG. 13D), TGF-β (FIG. 13E), IL-10 (FIG. 13F), and IL-6 (FIG. 13G) in purified, naïve splenic CD4 T cells that were cultured in the presence of anti-CD3/anti-CD28 beads and vehicle alone control (Unstim), IL-35, the human IL-6R-Fc-IL-35 chimeric protein, an unrelated control chimeric protein, IL-2, or TGF-β/Retinoic Acid (RA) for 9 days.

FIG. 14A and FIG. 14B show the effect of the CD4 T cells generated as described for FIG. 13A to FIG. 13G on the proliferation of syngeneic CD8 cells when mixed at CD4: CD8 ratio of 2:1 (FIG. 14A) or 0.5:1 (FIG. 14B).

FIG. 15A shows a schematic of an IL-6R- and IL-35-based chimeric protein of the present disclosure. FIG. 15B shows characterization of a murine IL-6R-Fc-IL-35 chimeric protein by western blot demonstrating the chimeric proteins native state and tendency to form a multimer. Untreated samples (i.e., without reducing agent or deglycosylation agent) of the IL-6R-Fc-IL-35 chimeric protein were loaded into lane NR in each blot. Samples in lane R were treated with β-mercaptoethanol, a reducing agent. Samples in lane DG were treated with a deglycosylation agent and the reducing agent. Each individual domain of the chimeric protein was probed using an anti-IL-6ST, anti-IL-6R, anti-Fc, anti-EBI3, or anti-IL-12A antibodies, respectively.

FIG. 16A and FIG. 16B show the results of sandwich ELISA performed on the MSD platform to determine the relative abundance of the heterodimeric IL-6R-Fc-IL-35 chimeric protein in the purified preparations. For FIG. 16A, an anti-IL-6ST antibody was coated on plates. Increasing amounts of the IL-6R-Fc-IL-35 chimeric protein or the TNFR2-Fc-TGFβ chimeric protein were added to the plate for capture by the plate-bound anti-IL-6ST antibody. The binding was detected using an anti-IL-12A antibody. For FIG. 16B, an anti-IL-6R antibody was coated on plates. Increasing amounts of the IL-6R-Fc-IL-35 chimeric protein or the TNFR2-Fc-TGFβ chimeric protein were added to the plate for capture by the plate-bound anti-IL-6R antibody. The binding was detected using an anti-IL-27B antibody.

FIG. 17A shows a schematic of a MadCAM- and CCL25-based chimeric protein of the present disclosure. FIG. 17B shows characterization of a murine MadCAM-Fc-CCL25 chimeric protein by western blot demonstrating the chimeric proteins native state and tendency to form a multimer. Untreated samples (i.e., without reducing agent or deglycosylation agent) of the MadCAM-Fc-CCL25 chimeric protein were loaded into lane NR in each blot. Samples in lane R were treated with 3-mercaptoethanol, a reducing agent. Samples in lane DG were treated with a deglycosylation agent and the reducing agent. Each individual domain of the chimeric protein was probed using an anti-MadCAM, anti-Fc, or anti-CCL25 antibodies, respectively.

FIG. 18A and FIG. 18B show the results of sandwich ELISA performed on the MSD platform to determine the relative abundance of the heterodimeric MadCAM-Fc-CCL25 chimeric protein in the purified preparations. For FIG. 18A, an anti-CCL25 antibody was coated on plates. Increasing amounts of the MadCAM-Fc-CCL25 chimeric protein or the TNFR2-Fc-TGFβ chimeric protein were added to the plate for capture by the plate-bound anti-CCL25 antibody. The binding was detected using an anti-MadCAM antibody. For FIG. 18B, an anti-MadCAM antibody was coated on plates. Increasing amounts of the MadCAM-Fc-CCL25 chimeric protein or the TNFR2-Fc-TGFβ chimeric protein were added to the plate for capture by the plate-bound anti-MadCAM antibody. The binding was detected using an anti-CCL25 antibody.

FIG. 19A shows a schematic of an α4β7- and IL-35-based chimeric protein of the present disclosure. FIG. 19B shows characterization of a murine α4β7-Fc-IL-35 chimeric protein by western blot demonstrating the chimeric proteins native state and tendency to form a multimer. Untreated samples (i.e., without reducing agent or deglycosylation agent) of the α4β7-Fc-IL-35 chimeric protein were loaded into lane NR in each blot. Samples in lane R were treated with 3-mercaptoethanol, a reducing agent. Samples in lane DG were treated with a deglycosylation agent and the reducing agent. Each individual domain of the chimeric protein was probed using an anti-α4, anti-β7, anti-Fc, anti-EBI3 or anti-IL-12A antibodies, respectively.

FIG. 20A and FIG. 20B show the results of sandwich ELISA performed on the MSD platform to determine the relative abundance of the hetrodimeric α4β7-Fc-IL-35 chimeric protein in the purified preparations. For FIG. 20A, an anti-α4 antibody was coated on plates. Increasing amounts of the α4β7-Fc-IL-35 chimeric protein or the TNFR2-Fc-TGFβ chimeric protein were added to the plate for capture by the plate-bound anti-α4 antibody. The binding was detected using an anti-IL27B antibody. For FIG. 20B, an anti-IL-12A antibody was coated on plates. Increasing amounts of the α4β7-Fc-IL-35 chimeric protein or the TNFR2-Fc-TGFβ chimeric protein were added to the plate for capture by the plate-bound anti-IL-12A antibody. The binding was detected using an anti-β7 antibody.

FIG. 21A shows a schematic of a TNFR2- and TGFβ-based chimeric protein of the present disclosure. FIG. 21B shows characterization of a murine TNFR2-Fc-TGFβ chimeric protein by western blot demonstrating the chimeric proteins native state and tendency to form a multimer. Untreated samples (i.e., without reducing agent or deglycosylation agent) of the TNFR2-Fc-TGFβ chimeric protein were loaded into lane NR in each blot. Samples in lane R were treated with β-mercaptoethanol, a reducing agent. Samples in lane DG were treated with a deglycosylation agent and the reducing agent. Each individual domain of the chimeric protein was probed using an anti-TNFR2, anti-Fc, or anti-TGFβ antibodies, respectively.

FIG. 22A and FIG. 22B show the results of sandwich ELISA performed on the MSD platform to determine the relative abundance of the hetrodimeric TNFR2-Fc-TGFβ chimeric protein in the purified preparations. For FIG. 22A, an anti-TGFβ antibody was coated on plates. Increasing amounts of the TNFR2-Fc-TGFβ chimeric protein or the MadCAM-Fc-CCL chimeric protein were added to the plate for capture by the plate-bound anti-TGFβ antibody. The binding was detected using an anti-TNFR2 antibody. For FIG. 22B, an anti-TNFR2 antibody was coated on plates. Increasing amounts of the TNFR2-Fc-TGFβ chimeric protein or the MadCAM-Fc-CCL chimeric protein were added to the plate for capture by the plate-bound anti-TNFR2 antibody. The binding was detected using an anti-TGFβ antibody.

FIG. 23 shows a t-distributed stochastic neighbor embedding (t-SNE) plot illustrating dextran sodium sulfate (DSS) induced colitis in mice. Mice were untreated or received 3% DSS in their drinking water ad libitum for 8 days starting day 0. On Day 9, DSS containing drinking water was replaced with unmodified drinking water. On day 11, all animals were sacrificed, and mesenteric lymph nodes (MLN) were harvested. Cells from MLN were subjected to flow cytometry using a 15-parameter FACS panel to phenotypically characterize the cellular composition of the MLN. The data were analyzed on FlowJo, and was subjected to dimensionality reduction with t-SNE and phenotypic populations mapped with X-Shift.

FIG. 24A to FIG. 24C illustrate the phenotypic differences in cells from MLN of mice induced to have colitis using DSS and treated with the chimeric proteins of the present disclosure. Mice were left untreated or untreated as discussed for FIG. 23. On days 0, 1, and 2, experimental treatment group animals were administered 100 μg of the indicated therapeutic molecule, once daily, intraperitoneally. Control animals were administered 100 μg of murine IgG. Cells from MLN were subjected to flow cytometry using the 15-parameter FACS panel to phenotypically characterize the cellular composition of the MLN. The data were analyzed on FlowJo, and was subjected to dimensionality reduction with t-SNE and phenotypic populations mapped with X-Shift. FIG. 24A shows the t-SNE density plot overlays. FIG. 24B illustrates the differences between the density plot overlays. For example, the treatment with the MadCAM-Fc-CCL25, IL-6R-Fc-IL-35, and TNFR2-Fc-TGFβ chimeric proteins decreased the cells within the area marked with lighter outlined shapes and increased the cells within the area marked with a black outlined shape. FIG. 24C is a table showing the differences in relative abundance of the indicated cell types.

FIG. 25 illustrates that the TNFR2-Fc-TGFβ chimeric protein protects cells from TNF-α mediated apoptosis in L929 cells, which are known to be highly sensitive to TNF-α induced apoptosis. Fixed numbers of L929 cells were incubated in microtiter plates with 10 ng/ml of TNF-α for 24 hours. Increasing molar ratios of the TNFR2-Fc-TGFβ chimeric protein or an irrelevant chimeric protein (OH) that is not known to protect cells from apoptosis were titrated into the plates. After 24 hours, the cells were assessed for cell death using the Caspase 3/7 CytoGlo system on the Promega GloMax Luminometer.

FIG. 26 shows a plot of body weights of mice that induced to have colitis using 2,4,6-trinitrobenzenesulfonic acid (TNBS). 2.5% in ethanol was administered on day 0. The negative control animals were administered colonic instillation of ethanol alone. 100 μg of the TNFR2-Fc-TGFβ or CLTA4-Fc-TL1A chimeric proteins, or vehicle only were administered on days 0, 1, and 2, once daily, intraperitoneally.

FIG. 27A to FIG. 27C illustrate the phenotypic differences in the cells from MLN of control mice, mice induced to have colitis by colonic instillation of TNBS as discussed for FIG. 26, and treated with the TNFR2-Fc-TGFβ chimeric protein as discussed for FIG. 26. FIG. 27A shows the t-SNE density plot of mice induced to have colitis by colonic instillation of TNBS treated with the TNFR2-Fc-TGFβ chimeric protein. FIG. 27B illustrates the differences between the density plot overlays. FIG. 27C is a table showing the differences in relative abundance of the indicated cell types.

FIG. 28A to FIG. 28F illustrate the relative change in the relative levels of mRNA, as measured by qRT-PCR, of TLR5 (FIG. 28A), IL-17A (FIG. 28B), IL-4 (FIG. 28C), IL-1B (FIG. 28D), CCL-2 (FIG. 28E), and IL-6 (FIG. 28F) in the cells from MLN of control mice, mice induced to have colitis by colonic instillation of TNBS as discussed for FIG. 26, and mice induced to have colitis by colonic instillation of TNBS treated with the TNFR2-Fc-TGFβ chimeric protein as discussed for FIG. 26.

FIG. 29A to FIG. 29C illustrate the phenotypic differences in the CD4 T cells were isolated from the spleens of FoxP3 RFP knock-in mice (FIR mice), that were cultured for 5 days with activating anti-CD3/anti-CD28 beads in the presence of a IL4, TGFβ, MadCAM-fc-CCL25, TNFR2-fc-TGFβ, α4β7-fc-IL35, or IL6R-fc-IL35. FIG. 29A shows the t-SNE density plot of showing eight distinct phenotypic populations of CD4 cells. FIG. 29B illustrates the differences between the density plot overlays when the CD4 cell were treated with the indicated protein. FIG. 29C is a table showing the differences in relative abundance of the indicated cell types.

DETAILED DESCRIPTION

The present invention is based, in part, on the discovery that chimeric proteins can be engineered from a first domain comprising an extracellular domain of a first transmembrane protein, a first secreted protein, or a first membrane-anchored extracellular protein and a second domain comprising an extracellular domain of a second transmembrane protein, a second secreted protein, or a second membrane-anchored extracellular protein. In these chimeric proteins, either or both of the first domain and the second domain decreases self-directed immune system activity when bound to its ligand/receptor. Accordingly, the present invention find use in the treatment of an autoimmune disease, which occurs when a subject's own antigens become targets for an immune response.

The present chimeric proteins provide advantages including, without limitation, ease of use and ease of production. This is because two distinct immunotherapy agents are combined into a single product which may allow 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. Further, in contrast to, for example, monoclonal antibodies, which are large multimeric proteins containing numerous disulfide bonds and post-translational modifications such as glycosylation, the present chimeric proteins are easier and more cost effective to manufacture.

Importantly, since a chimeric protein of the present invention comprises two ligand/receptor binding domains, it is capable of, via two cellular pathways, decreasing immune system activity by activating an immune inhibitory signal and/or by inhibiting an immune activating signal. This dual-action is more likely to provide any anti-autoimmune effect in a subject. Moreover, since the chimeric proteins and methods using the chimeric proteins operate by multiple distinct pathways, they can be efficacious, at least, in patients who do not respond, respond poorly, or become resistant to treatments that target one of the pathways. Thus, a patient who is a poor responder to treatments acting via one of the two pathways, can receive a therapeutic benefit by targeting multiple pathways.

Chimeric Proteins

In embodiments, the portion of the first domain is capable of binding the native ligand/receptor for the transmembrane protein, the secreted protein, or the membrane-anchored extracellular protein.

In embodiments, the portion of the second domain is capable of binding the native ligand/receptor for the transmembrane protein, the secreted protein, or the membrane-anchored extracellular protein.

In embodiments, the first domain comprises substantially the entire extracellular domain of the transmembrane protein, substantially the entire secreted protein, or substantially the entire membrane-anchored extracellular protein.

In embodiments, the second domain comprises substantially the entire extracellular domain of the transmembrane protein, substantially the entire secreted protein, or substantially the entire membrane-anchored extracellular protein.

In embodiments, the binding of the portion of the first domain to its ligand/receptor decreases immune system activity by activating an immune inhibitory signal or inhibiting an immune activating signal.

In embodiments, the binding of the portion of the second domain to its ligand/receptor decreases immune system activity by activating an immune inhibitory signal or by inhibiting an immune activating signal.

In embodiments, the portion of the first domain comprises a transmembrane protein, a secreted protein, or a membrane-anchored extracellular protein selected from B7H3, B7H4, BTNL2, CTLA4, CSF3, ICOSL, ILDR2, PD-L1, TNFR2, and VSIG4.

In embodiments, the portion of the second domain comprises a transmembrane protein, a secreted protein, or a membrane-anchored extracellular protein selected from BTNL2, IL2, PD-L1, SEMA3E, IL-35, CCL25, TGFβ, and TL1A.

In embodiments, the first domain comprises a portion of VSIG4 and the second domain comprises a portion of IL2.

In embodiments, the first domain comprises a portion of CTLA4 and the second domain comprises a portion of IL2, e.g., the portion of IL2 comprises one or more mutations relative to a corresponding portion of wild-type IL2 wherein the one or more mutations provide preferential binding to a high-affinity IL2 receptor that is expressed by regulatory T cells.

In embodiments, the first domain comprises a portion of CTLA4 and the second domain comprises a portion of PD-L1.

In embodiments, the first domain comprises a portion of B7H3 and the second domain comprises a portion of PD-L1.

In embodiments, the first domain comprises a portion of B7H4 and the second domain comprises a portion of PD-L1.

In embodiments, the first domain comprises a portion of ICOSL and the second domain comprises a portion of PD-L1.

In embodiments, the first domain comprises a portion of ILDR2 and the second domain comprises a portion of PD-L1.

In embodiments, the first domain comprises a portion BTNL2 of and the second domain comprises a portion of PD-L1 or the first domain comprises a portion of PD-L1 and the second domain comprises a portion of BTNL2.

In embodiments, the first domain comprises a portion of CSF3 and the second domain comprises a portion of TL1A.

In embodiments, the first domain comprises a portion of CTLA4 and the second domain comprises a portion of TL1A.

In embodiments, the first domain comprises a portion of CTLA4 and the second domain comprises a portion of SEMA3E.

In embodiments, the first domain comprises a portion of TNFR2 and the second domain comprises an extracellular domain of a transmembrane protein selected from 4-1BBL, APRIL, BAFF, BTNL2, CD28, CD30L, CD40L, CD70, C-type lectin domain (CLEC) family members, FasL, GITRL, LIGHT, LTa, LTa1b2, NKG2A, NKG2C, NKG2D, OX40L, RANKL, TL1A, TNFa, and TRAIL; in embodiments, the second domain comprises an extracellular domain of GITRL or TL1A. In embodiments, the CLEC family member is selected from AlCL/CLEC-2B, ASGR1/ASGPR1, ASGR2, C1q R1/CD93, CD161, CD161/NK1.1, CD23/Fc epsilon RII, CD302/CLEC13A, CD72, CD94, Chondrolectin, CLEC-1, CLEC10A/CD301, CLEC12B, CLEC14A, CLEC16A, CLEC17A, CLEC18A, CLEC18B, CLEC18C, CLEC-2/CLEC1B, CLEC-2A, CLEC3A, CLEC3B/Tetranectin, CLEC4B2/mDCAR1, CLEC4D/CLECSF8, CLEC4E, CLEC4F/CLECSF13, CLEC9a, CLECL1/DCAL-1, CL-K1/COLEC11, CL-L1/COLEC10, CL-P1/COLEC12, DCAR/CLEC4B, DCIR/CLEC4A, DCIR4/CLEC4A1, DC-SIGN/CD209, DC-SIGN+FDC-SIGNR, DC-SIGNR/CD299, DC-SIGNR/CD299, DEC-205/CD205, Dectin-1/CLEC7A, Dectin-2/CLEC6A, DLEC/CLEC4C/BDCA-2, Ficolin-1, Ficolin-2, Ficolin-3, Klre-1, KLRG2, Langerin/CD207, Layilin, LOX-1/OLR1, LSECtin/CLEC4G, MBL, MBL-1, MBL-2, MDL-1/CLEC5A, MGL1/2 (CD301a/b), MGL1/CD301a, MGL2/CD301b, MGL2/CD301b, MICL/CLEC12A, MMR/CD206, Mrc2, NKG2A/CD159a, NKG2A/NKG2B Isoform 2, NKG2C/CD159c, NKG2D/CD314, NKG2E, NKG2H, NKp80/KLRF1, OCIL/CLEC2d, OCILRP2/CLEC2i, PLA2R1, QBRICK/FREM1, Reg1, Reg1A, Reg1B, Reg2, Reg3A, Reg3B, Reg3D, Reg3G, Reg4, SCGF/CLEC11a, SFTPA1, SIGNR1/CD209b, SIGNR3/CD209d, SIGNR4/CD209e, SIGNR7/CD209g, and SP-D.

In embodiments, the first domain comprises a portion of CTLA4 and the second domain comprises an extracellular domain of a transmembrane protein selected from 4-1BBL, APRIL, BAFF, BTNL2, CD28, CD30L, CD40L, CD70, C-type lectin domain (CLEC) family members, FasL, GITRL, LIGHT, LTa, LTa1b2, NKG2A, NKG2C, NKG2D, OX40L, RANKL, TL1A, TNFa, and TRAIL; in embodiments, the second domain comprises an extracellular domain of GITRL or TL1A. In embodiments, the CLEC family member is selected from AlCL/CLEC-2B, ASGR1/ASGPR1, ASGR2, C1q R1/CD93, CD161, CD161/NK1.1, CD23/Fc epsilon RII, CD302/CLEC13A, CD72, CD94, Chondrolectin, CLEC-1, CLEC10A/CD301, CLEC12B, CLEC14A, CLEC16A, CLEC17A, CLEC18A, CLEC18B, CLEC18C, CLEC-2/CLEC1B, CLEC-2A, CLEC3A, CLEC3B/Tetranectin, CLEC4B2/mDCAR1, CLEC4D/CLECSF8, CLEC4E, CLEC4F/CLECSF13, CLEC9a, CLECL1/DCAL-1, CL-K1/COLEC11, CL-L1/COLEC10, CL-P1/COLEC12, DCAR/CLEC4B, DCIR/CLEC4A, DCIR4/CLEC4A1, DC-SIGN/CD209, DC-SIGN+FDC-SIGNR, DC-SIGNR/CD299, DC-SIGNR/CD299, DEC-205/CD205, Dectin-1/CLEC7A, Dectin-2/CLEC6A, DLEC/CLEC4C/BDCA-2, Ficolin-1, Ficolin-2, Ficolin-3, Klre-1, KLRG2, Langerin/CD207, Layilin, LOX-1/OLR1, LSECtin/CLEC4G, MBL, MBL-1, MBL-2, MDL-1/CLEC5A, MGL1/2 (CD301a/b), MGL1/CD301a, MGL2/CD301b, MGL2/CD301b, MICL/CLEC12A, MMR/CD206, Mrc2, NKG2A/CD159a, NKG2A/NKG2B Isoform 2, NKG2C/CD159c, NKG2D/CD314, NKG2E, NKG2H, NKp80/KLRF1, OCIL/CLEC2d, OCILRP2/CLEC2i, PLA2R1, QBRICK/FREM1, Reg1, Reg1A, Reg1B, Reg2, Reg3A, Reg3B, Reg3D, Reg3G, Reg4, SCGF/CLEC11a, SFTPA1, SIGNR1/CD209b, SIGNR3/CD209d, SIGNR4/CD209e, SIGNR7/CD209g, and SP-D.

In embodiments, the binding of either or both of the first domain and the second domains to its ligand/receptor occurs with slow off rates (Koff), which provides a long interaction of a receptor and its ligand. In embodiments, the long interaction provides a prolonged decrease in immune system activity which comprises sustained activation of an immune inhibitory signal and/or a sustained inhibition of an immune activating signal. In embodiments, the sustained activation of the immune inhibitory signal and/or the sustained inhibition of the immune activating signal reduces the activity or proliferation of an immune cell, e.g., a B cell or a T cell. In embodiments, the sustained activation of the immune inhibitory signal and/or the sustained inhibition of the immune activating signal decreases synthesis and/or decreases release of a pro-inflammatory cytokine. In embodiments, the sustained activation of the immune inhibitory signal and/or the sustained inhibition of the immune activating signal increases synthesis and/or increases release of an anti-inflammatory cytokine. In embodiments, the sustained activation of the immune inhibitory signal and/or the sustained inhibition of the immune activating signal decreases antibody production and/or decreases secretion of antibodies by a B cell, e.g., an antibody that recognizes a self-antigen. In embodiments, the sustained activation of the immune inhibitory signal and/or the sustained inhibition of the immune activating signal decreases the activity of and/or decreases the number of T cytotoxic cells, e.g., which recognize a self-antigen and kill cells presenting or expressing the self-antigen. In embodiments, the sustained activation of the immune inhibitory signal and/or the sustained inhibition of the immune activating signal increases the activity and/or increases the number of T regulatory cells.

In embodiments, the linker is a polypeptide selected from a flexible amino acid sequence, an IgG hinge region, and an antibody sequence.

In embodiments, the linker comprises at least one cysteine residue capable of forming a disulfide bond and/or comprises a hinge-CH2-CH3 Fc domain, e.g., a hinge-CH2-CH3 Fc domain is derived from IgG (e.g., IgG1, IgG2, IgG3, and IgG4), IgA (e.g., IgA1 and IgA2), IgD, or IgE. In embodiments, the IgG is IgG4, e.g., a human IgG4. In embodiments, the linker comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

In embodiments, the hinge-CH2-CH3 Fc domain comprises at least one cysteine residue capable of forming a disulfide bond. In embodiments, the hinge-CH2-CH3 Fc domain is derived from IgG (e.g., IgG1, IgG2, IgG3, and IgG4), IgA (e.g., IgA1 and IgA2), IgD, or IgE. In embodiments, the IgG is IgG4, e.g., a human IgG4. In embodiments, the linker comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

In a chimeric protein of the present invention, the chimeric protein is a recombinant fusion protein, e.g., a single polypeptide having the extracellular domains disclosed herein. For example, in embodiments, the chimeric protein is translated as a single unit in a prokaryotic cell, a eukaryotic cell, or a cell-free expression system.

In embodiments, the present chimeric protein is producible in a mammalian host cell as a secretable and fully functional single polypeptide chain.

In embodiments, chimeric protein refers to a recombinant protein of multiple polypeptides, e.g., multiple extracellular domains disclosed herein, that are combined (via covalent or no-covalent bonding) to yield a single unit, e.g., in vitro (e.g., with one or more synthetic linkers disclosed herein).

In embodiments, the chimeric protein is chemically synthesized as one polypeptide or each domain may be chemically synthesized separately and then combined. In embodiments, a portion of the chimeric protein is translated and a portion is chemically synthesized.

Transmembrane proteins typically consist of an extracellular domain, one or a series of transmembrane domains, and an intracellular domain. Without wishing to be bound by theory, the extracellular domain of a transmembrane protein is responsible for interacting with a soluble receptor or ligand or membrane-bound receptor or ligand (i.e., a membrane of an adjacent cell). Without wishing to be bound by theory, the trans-membrane domain(s) is responsible for localizing the transmembrane protein to the plasma membrane. Without wishing to be bound by theory, the intracellular domain of a transmembrane protein is responsible for coordinating interactions with cellular signaling molecules to coordinate intracellular responses with the extracellular environment (or visa-versa). Illustrations of transmembrane proteins are shown in FIG. 1A.

In contrast to transmembrane proteins, membrane-anchored extracellular proteins lack a transmembrane domain that spans, at least part, of a cell's lipid bilayer. Instead, these proteins are associated with the extracellular face of a cell's membrane. The association may be a result of hydrophobic interactions between the bilayer and exposed nonpolar residues at the surface of a protein, by specific non-covalent binding interactions with regulatory lipids, or through their attachment to covalently bound lipid anchors (including the lipids glycosylphosphatidylinositol (GPI) and cholesterol). Alternately, membrane-anchored extracellular proteins may indirectly be associated with the cell's lipid bilayer via another protein that is directly associated with the membrane, including transmembrane proteins. Illustrations of membrane-anchored extracellular proteins are shown in FIG. 1B.

A secreted protein can be defined as a protein which is actively transported out of the cell. Medically important secreted proteins include cytokines, coagulation factors, enzymes, growth factors, hormones, and other signaling molecules.

Often secreted proteins have an amino terminal comprising a signal sequence consisting of 6 to 12 amino acids with hydrophobic side chains. The signal sequence, at least, permits packaging of secreted proteins into vesicles which, when fused with the cell's membrane, the secreted protein leaves the cell. Illustrations of secreted proteins are shown in FIG. 1C.

FIG. 2A to FIG. 2D show schematic illustrations of chimeric proteins of the present invention. FIG. 2A shows a chimeric protein comprising a first domain with a ligand/receptor binding site at its amino terminus and a second domain with a ligand/receptor binding site at its carboxy terminus. Non-limiting examples of chimeric proteins of the present invention which may have this configuration include CSF3-Fc-TL1A, CTLA4-Fc-IL2, CTLA4-Fc-SEMA3E, CTLA4-Fc-TL1A, PD-L1-Fc-BTNL2, and VSIG4-Fc-IL2. FIG. 2B shows a chimeric protein comprising a first domain with a ligand/receptor binding site at its amino terminus and a second domain with a ligand/receptor binding site at its amino terminus. Non-limiting examples of chimeric proteins of the present invention which may have this configuration include B7H3-Fc-PD-L1, B7H4-Fc-PD-L1, CTLA4-Fc-IL2, CTLA4-Fc-PD-L1, CTLA4-Fc-SEMA3E, ICOSL-Fc-PD-L1, ILDR2-Fc-PD-L1, and VSIG4-Fc-IL2. FIG. 2C shows a chimeric protein comprising a first domain with a ligand/receptor binding site at its carboxy terminus and a second domain with a ligand/receptor binding site at its carboxy terminus. Non-limiting examples of chimeric proteins of the present invention which may have this configuration include CSF3-Fc-TL1A. FIG. 2D shows a chimeric protein comprising a first domain with a ligand/receptor binding site at its carboxy terminus and a second domain with a ligand/receptor binding site at its amino terminus. Non-limiting examples of chimeric proteins of the present invention which may have this configuration include BTNL2-Fc-PD-L1.

Chimeric proteins of the present invention have a first domain which is sterically capable of binding its ligand/receptor and/or a second domain which is sterically capable of binding its ligand/receptor. This means that there is sufficient overall flexibility in the chimeric protein and/or physical distance between a first domain (or portion thereof) and the rest of the chimeric protein such that the ligand/receptor binding domain of the first domain is not sterically hindered from binding its ligand/receptor and/or there is sufficient physical distance between a second domain (or portion thereof) and the rest of the chimeric protein such that the ligand/receptor binding domain of the second domain is not sterically hindered from binding its ligand/receptor. This flexibility and/or physical distance (which is herein referred to as “slack”) may be normally present in the first and/or second domain(s), normally present in the linker, and/or normally present in the chimeric protein (as a whole). Alternately, or additionally, the chimeric protein may be modified by including one or more additional amino acid sequences (e.g., the joining linkers described below) or synthetic linkers (e.g., a polyethylene glycol (PEG) linker) which provide additional slack needed to avoid steric hindrance. Further description of linkers useful in the present invention, and especially the linkers of SEQ ID NO: 1 to SEQ ID NO: 3, are included in the next section of this disclosure entitled “Linkers”.

In embodiments, the chimeric protein is capable of contemporaneously binding the CSF3 receptor and the TL1A receptor. In embodiments, the CSF3 receptor is granulocyte colony-stimulating factor receptor (G-CSF-R) also known as CD114 (Cluster of Differentiation 114) and the TL1A receptor is TNFRSF25/DR3 or TNFRSF21/DR6/DcR3. CSF3 is a cytokine that controls the production, differentiation, and function of two related white cell populations of the blood, the granulocytes and the monocytes-macrophages and is capable of suppressing many autoimmune diseases like Crohn's disease, Type-1 diabetes, Myasthenia gravis and experimental autoimmune thyroiditis. TL1A binding to its receptor promotes, at least, expansion of activated and regulatory T cells, which can decrease an autoimmune response. Accordingly, a chimeric protein comprising a portion of CSF3 which includes its receptor-binding domain and the extracellular domain of TL1A is capable of contemporaneously stimulating granulocytosis and mobilizing stem cells from the bone marrow (via CSF3) and inhibiting an immune activating signal (via TL1A). In embodiments, this chimeric protein is referred to herein as CSF3-Fc-TL1A.

In embodiments, the chimeric proteins of the present invention comprise variants of a portion of CSF3 which includes its receptor-binding domain. As examples, the variant may 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%, 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 with the known amino acid sequence of the portion of CSF3, e.g., human CSF3, which comprises its receptor-binding domain.

In embodiments, the portion of CSF3 comprising its receptor-binding domain has the following amino acid sequence:

(SEQ ID NO: 57)

ATPLGPASSLPQSFLLKCLEQVRKIQGDGAALQEKLVSECATYKLCHPEE

LVLLGHSLGIPWAPLSSCPSQALQLAGCLSQLHSGLFLYQGLLQALEGIS

PELGPTLDTLQLDVADFATTIWQQMEELGMAPALQPTQGAMPAFASAFQR

RAGGVLVASHLQSFLEVSYRVLRHLAQP.

In embodiments, a chimeric protein comprises a variant of the portion of CSF3 comprising its receptor-binding domain. As examples, the variant may 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%, 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 with SEQ ID NO: 57.

In embodiments, the first domain of a chimeric protein comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 57.

One of ordinary skill may select variants of the known amino acid sequence of CSF3 by consulting the literature, e.g., Aritomi et al., “Atomic structure of the GCSF-receptor complex showing a new cytokine-receptor recognition scheme.” Nature 401:713-717 (1999); Tamada et al., “Homodimeric cross-over structure of the human granulocyte colony-stimulating factor (GCSF) receptor signaling complex.” Proc. Natl. Acad. Sci. U.S.A. 103:3135-3140 (2006); Zink et al., “Secondary structure of human granulocyte colony-stimulating factor derived from NMR spectroscopy.” FEBS Lett. 314 (3), 435-439 (1992); and Battacharya et al., “GM-CSF: An Immune Modulatory Cytokine that can Suppress Autoimmunity.” Cytokine. 2015 October; 75(2): 261-271, each of which is incorporated by reference in its entirety.

In embodiments, the chimeric proteins of the present invention comprise variants of the extracellular domain, which includes the receptor-binding domain, of TL1A. As examples, the variant may 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%, 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 with the known amino acid sequence of the extracellular domain of TL1A, e.g., human TL1A.

In embodiments, the extracellular domain of TL1A has the following amino acid sequence:

(SEQ ID NO: 58)

SQLRAQGEACVQFQALKGQEFAPSHQQVYAPLRADGDKPRAHLTVVRQTP

TQHFKNQFPALHWEHELGLAFTKNRMNYTNKFLLIPESGDYFIYSQVTFR

GMTSECSEIRQAGRPNKPDSITVVITKVIDSYPEPTQLLMGTKSVCEVGS

NWFQPIYLGAMFSLQEGDKLMVNVSDISLVDYTKEDKTFFGAFLL.

In embodiments, a chimeric protein comprises a variant of the extracellular domain of TL1A. As examples, the variant may 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%, 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 with SEQ ID NO: 58.

In embodiments, the second domain of a chimeric protein comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 58.

One of ordinary skill may select variants of the known amino acid sequence of TL1A by consulting the literature, e.g., Yue et al., “TL1, a novel tumor necrosis factor-like cytokine, induces apoptosis in endothelial cells. Involvement of activation of stress protein kinases (stress-activated protein kinase and p38 mitogen-activated protein kinase) and caspase-3-like protease.” J. Biol. Chem. 274 (3), 1479-1486 (1999); Richard et al., “Reduced monocyte and macrophage TNFSF15/TL1A expression is associated with susceptibility to inflammatory bowel disease.” PLoS Genet. 14 (9), e1007458 (2018); Migone et al., “TL1A is a TNF-like ligand for DR3 and TR6/DcR3 and functions as a T cell costimulator.” Immunity 16:479-492(2002); Jin et al., “X-ray crystal structure of TNF ligand family member TL1A at 2.1A.” Biochem. Biophys. Res. Commun. 364:1-6(2007); Zhan et al., “Decoy strategies: the structure of TL1A:DcR3 complex.” Structure 19:162-171(2011); Khan et al., “TL1A-Ig induces transplantation tolerance”, J Immunol May 1, 2013, 190 (1 Supplement) 113.2; Khan et al., “Cloning, Expression, and Functional Characterization of TL1A-Ig” J Immunol Feb. 15, 2013, 190 (4) 1540-1550; and Schreiber et al. “Therapeutic Treg expansion in mice by TNFRSF25 prevents allergic lung inflammation” Clin Invest. 2010; 120(10):3629-3640, each of which is incorporated by reference in its entirety.

In embodiments, a chimeric protein of the present invention comprises: (1) a first domain comprising the amino acid sequence of SEQ ID NO: 57, (b) a second domain comprises the amino acid sequence of SEQ ID NO: 58, and (c) a linker comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

In embodiments, a CSF3-Fc-TL1A chimeric protein of the present invention has the following amino acid sequence:

(SEQ ID NO: 59)

ATPLGPASSLPQSFLLKCLEQVRKIQGDGAALQEKLVSECATYKLCHPEE

LVLLGHSLGIPWAPLSSCPSQALQLAGCLSQLHSGLFLYQGLLQALEGIS

PELGPTLDTLQLDVADFATTIWQQMEELGMAPALQPTQGAMPAFASAFQR

RAGGVLVASHLQSFLEVSYRVLRHLAQPSKYGPPCPPCPAPEFLGGPSVF

LFPPKPKDQLMISRTPEVICVVVDVSQEDPEVQFNWYVDGVEVHNAKTKP

REEQFNSTYRWSVLTVLHQDWLSGKEYKCKVSSKGLPSSIEKTISNATGQ

PREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK

TTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVLHEALHNHYTQKSLS

LSLGKIEGRMDSQLRAQGEACVQFQALKGQEFAPSHQQVYAPLRADGDKP

RAHLTWRQTPTQHFKNQFPALHWEHELGLAFTKNRMNYTNKFLLIPESGD

YFIYSQVTFRGMTSECSEIRQAGRPNKPDSITWITKVIDSYPEPTQLLMG

TKSVCEVGSNWFQPIYLGAMFSLQEGDKLMVNVSDISLVDYTKEDKTFFG

AFLL.

In embodiments, a chimeric protein comprises a variant of a CSF3-Fc-TL1A chimeric protein. As examples, the variant may 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%, 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 with SEQ ID NO: 59.

In embodiments, the chimeric protein is capable of contemporaneously binding the CTLA4 ligand and the TL1A receptor. In embodiments, the CTLA4 ligand is CD80 or CD86 and the TL1A receptor is TNFRSF25/DR3 or TNFRSF21/DR6/DcR3. CTLA4 acts as an “off” switch when bound to its ligand on the surface of antigen-presenting cells (APCs). CTLA4 is a protein receptor that functions as an immune checkpoint and downregulates immune responses. When TL1A binds to its receptor promotes, at least, expansion of activated and regulatory T cells. Accordingly, a chimeric protein comprising the extracellular domains of CTLA4 and TL1A is capable of contemporaneously competitively inhibiting an immune activating signal (via CTLA4) and activating an immune receptor TNFRSF25 (via TL1A), which stimulates regulatory T cell proliferation. In embodiments, this chimeric protein is referred to herein as CTLA4-Fc-TL1A.

In embodiments, the chimeric proteins of the present invention comprise variants of the extracellular domain, which includes the ligand/receptor binding domain, of CTLA4. As examples, the variant may 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%, 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 with the known amino acid sequence of the extracellular domain of CTLA4, e.g., human CTLA4.

In embodiments, the extracellular domain of CTLA4 has the following amino acid sequence:

(SEQ ID NO: 60)

KAMHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVC

AATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMY

PPPYYLGIGNGTQIYVIDPEPCPDSD.

In embodiments, a chimeric protein comprises a variant of the extracellular domain of CTLA4. As examples, the variant may 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%, 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 with SEQ ID NO: 60.

In embodiments, the first domain of a chimeric protein comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 60.

One of ordinary skill may select variants of the known amino acid sequence of CTLA4 by consulting the literature, e.g., Linsley et al., “CTLA-4 is a second receptor for the B cell activation antigen B7.” J. Exp. Med. 174 (3), 561-569 (1991); Dariavach et al., “Human Ig superfamily CTLA-4 gene: chromosomal localization and identity of protein sequence between murine and human CTLA-4 cytoplasmic domains”. Eur. J. Immunol. 18 (12), 1901-1905 (1988); Teft et al., “A molecular perspective of CTLA-4 function.” Annu. Rev. Immunol. 24:65-97 (2006); Ramagopal et al., “Structural basis for cancer immunotherapy by the first-in-class checkpoint inhibitor ipilimumab.” Proc. Natl. Acad. Sci. U.S.A. 114:E4223-E4232 (2017); Schwartz et al., “Structural basis for co-stimulation by the human CTLA-4/B7-2 complex.” Nature 410:604-608 (2001); Stamper et al., “Crystal structure of the B7-1/CTLA-4 complex that inhibits human immune responses.” Nature 410:608-611 (2001); and Yu et al., “Rigid-body ligand recognition drives cytotoxic T-lymphocyte antigen 4 (CTLA-4) receptor triggering.” J. Biol. Chem. 286:6685-6696 (2011), each of which is incorporated by reference in its entirety.

In embodiments, the extracellular domain of TL1A has the following amino acid sequence:

In embodiments, the second domain of a chimeric protein comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 58.

In embodiments, a chimeric protein of the present invention comprises: (1) a first domain comprising the amino acid sequence of SEQ ID NO: 60, (b) a second domain comprises the amino acid sequence of SEQ ID NO: 58, and (c) a linker comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

In embodiments, a CTLA4-Fc-TL1A chimeric protein of the present invention has the following amino acid sequence:

(SEQ ID NO: 61)

KAMHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVC

AATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMY

PPPYYLGIGNGTQIYVIDPEPCPDSDSKYGPPCPPCPAPEFLGGPSVFLF

PPKPKDQLMISRTPEVTCWVDVSQEDPEVQFNWYVDGVEVHNAKTKPREE

QFNSTYRVVSVLTVLHQDWLSGKEYKCKVSSKGLPSSIEKTISNATGQPR

EPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT

PPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVLHEALHNHYTQKSLSLS

LGKIEGRMDSQLRAQGEACVQFQALKGQEFAPSHQQVYAPLRADGDKPRA

HLTVVRQTPTQHFKNQFPALHWEHELGLAFTKNRMNYTNKFLLIPESGDY

FIYSQVTFRGMTSECSEIRQAGRPNKPDSITVVITKVTDSYPEPTQLLMG

TKSVCEVGSNWFQPIYLGAMFSLQEGDKLMVNVSDISLVDYTKEDKTFFG

AFLL.

In embodiments, a chimeric protein comprises a variant of a CTLA4-Fc-TL1A chimeric protein. As examples, the variant may 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%, 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 with SEQ ID NO: 61.

In embodiments, the chimeric protein is capable of contemporaneously binding the CTLA4 ligand and the PD-L1 receptor. In embodiments, the CTLA4 ligand is CD80 or CD86 and the PD-L1 receptor is PD-1. CTLA4 acts as an “off” switch when bound to its ligand on the surface of antigen-presenting cells (APCs). CTLA4 is a protein receptor that functions as an immune checkpoint and downregulates immune responses. PD-L1 plays a critical role in induction and maintenance of immune tolerance to self, in part, by acting as a ligand for the inhibitory receptor PD-1; it modulates the activation threshold of T-cells and limits T-cell effector response, including cytotoxic T lymphocytes (CTLs) effector function Accordingly, a chimeric protein comprising the extracellular domains of CTLA4 and PD-L1 is capable of contemporaneously competitively inhibiting an immune activating signal (via CTLA4) and activating an immune inhibitory signal (via PD-L1). In embodiments, this chimeric protein is referred to herein as CTLA4-Fc-PD-L1.

In embodiments, the extracellular domain of CTLA4 has the following amino acid sequence:

(SEQ ID NO: 60)

KAMHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVC

AATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMY

PPPYYLGIGNGTQIYVIDPEPCPDSD.

In embodiments, the first domain of a chimeric protein comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 60.

In embodiments, the chimeric proteins of the present invention comprise variants of the extracellular domain, which includes the receptor-binding domain, of PD-L1. As examples, the variant may 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%, 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 with the known amino acid sequence of PD-L1, e.g., human PD-L1.

In embodiments, the extracellular domain of PD-L1 has the following amino acid sequence:

(SEQ ID NO: 62)

FTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFV

HGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISY

GGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWT

SSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEE

NHTAELVIPELPLAHPPNER.

In embodiments, a chimeric protein comprises a variant of the extracellular domain of PD-L1. As examples, the variant may 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%, 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 with SEQ ID NO: 62.

In embodiments, the second domain of a chimeric protein comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 62.

One of ordinary skill may select variants of the known amino acid sequence of PD-L1 by consulting the literature, e.g., Freeman et al., “Engagement of the PD-1 immunoinhibitory receptor by a novel B7-family member leads to negative regulation of lymphocyte activation.” J. Exp. Med. 192:1027-1034 (2000); Burr, et al, “CMTM6 maintains the expression of PD-L1 and regulates anti-tumour immunity.” Nature 549 (7670), 101-105 (2017); Lin et al., “The PD-1/PD-L1 complex resembles the antigen-binding Fv domains of antibodies and T cell receptors.” Proc. Natl. Acad. Sci. U.S.A. 105 (8), 3011-3016 (2008); and Zak et al., “Structure of the Complex of Human Programmed Death 1, PD-1, and Its Ligand PD-L1.” Structure 23 (12), 2341-2348 (2015), each of which is incorporated by reference in its entirety.

In embodiments, a chimeric protein of the present invention comprises: (1) a first domain comprising the amino acid sequence of SEQ ID NO: 60, (b) a second domain comprises the amino acid sequence of SEQ ID NO: 62, and (c) a linker comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

In embodiments, a CTLA4-Fc-PD-L1 chimeric protein of the present invention has the following amino acid sequence:

(SEQ ID NO: 63)

KAMHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVC

AATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMY

PPPYYLGIGNGTQIYVIDPEPCPDSDSKYGPPCPPCPAPEFLGGPSVFLF

PPKPKDQLMISRTPEVICVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPRE

EQFNSTYRVVSVLTVLHQDWLSGKEYKCKVSSKGLPSSIEKTISNATGQP

REPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT

TPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVLHEALHNHYTQKSLSL

SLGKIEGRMDFTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWE

MEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQD

AGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAE

GYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFY

CTFRRLDPEENHTAELVIPELPLAHPPNER.

In embodiments, a chimeric protein comprises a variant of a CTLA4-Fc-PD-L1 chimeric protein. As examples, the variant may 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%, 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 with SEQ ID NO: 63.

In embodiments, the chimeric protein is capable of contemporaneously binding the B7H3 receptor and the PD-L1 receptor. In embodiments, the PD-L1 receptor is PD-1; however, the B7H3 receptor has not been characterized. B7H3 (CD276) is an important immune checkpoint member of the B7 and CD28 families, many of whom interact with known checkpoint markers including CTLA4, PD-1, and CD28. B7-H3 plays an important role in the inhibition of T-cell function. PD-L1 plays a critical role in induction and maintenance of immune tolerance to self, in part, by acting as a ligand for the inhibitory receptor PD-1; it modulates the activation threshold of T-cells and limits T-cell effector response, including cytotoxic T lymphocytes (CTLs) effector function Accordingly, a chimeric protein comprising the extracellular domains of B7H3 and PD-L1 is capable of contemporaneously activating an immune inhibitory signal (via B7H3 receptors) and activating an immune inhibitory signal (via PD-L1). In embodiments, this chimeric protein is referred to herein as B7H3-Fc-PD-L1.

In embodiments, the chimeric proteins of the present invention comprise variants of the extracellular domain, which includes the receptor-binding domain, of B7H3. As examples, the variant may 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%, 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 with the known amino acid sequence of the extracellular domain of B7H3, e.g., human B7H3.

In embodiments, the extracellular domain of B7H3 has the following amino acid sequence:

(SEQ ID NO: 64)

LEVQVPEDPVVALVGTDATLCCSFSPEPGFSLAQLNLIWQLTDTKQLVHS

FAEGQDQGSAYANRTALFPDLLAQGNASLRLQRVRVADEGSFTCFVSIRD

FGSAAVSLQVAAPYSKPSMTLEPNKDLRPGDTVTITCSSYQGYPEAEVFW

QDGQGVPLIGNVITSQMANEQGLFDVHSILRWLGANGTYSCLVRNPVLQQ

DAHSSVTITPQRSPTGAVEVQVPEDPVVALVGTDATLRCSFSPEPGFSLA

QLNLIWQLTDTKQLVHSFTEGRDQGSAYANRTALFPDLLAQGNASLRLQR

VRVADEGSFTCFVSIRDFGSAAVSLQVAAPYSKPSMTLEPNKDLRPGDTV

TITCSSYRGYPEAEVFWQDGQGVPLIGNVITSQMANEQGLFDVHSVLRWL

GANGTYSCLVRNPVLQQDAHGSVTITGQPMTFPPEA.

In embodiments, a chimeric protein comprises a variant of the extracellular domain of B7H3. As examples, the variant may 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%, 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 with SEQ ID NO: 64.

In embodiments, the first domain of a chimeric protein comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 64.

One of ordinary skill may select variants of the known amino acid sequence of B7H3 by consulting the literature, e.g., Chapoval et al., “B7-H3: a costimulatory molecule for T cell activation and IFN-gamma production.” Nat. Immunol. 2:269-274 (2001); Steinberger et al., “Molecular characterization of human 4Ig-B7-H3, a member of the B7 family with four Ig-like domains.” J. Immunol. 172:2352-2359 (2004); Wang et al., “B7-H3 promotes acute and chronic allograft rejection.” Eur. J. Immunol. 35:428-438 (2005); Castellanos et al., “B7-H3 role in the immune landscape of cancer” Am J Clin Exp Immunol. 2017; 6(4): 66-75; and Vigdorovich et al., “Structure and T cell inhibition properties of B7 family member, B7-H3.” Structure. 2013 May 7; 21(5):707-17, each of which is incorporated by reference in its entirety.

In embodiments, the extracellular domain of PD-L1 has the following amino acid sequence:

(SEQ ID NO: 62)

FTITAPKDLYVVEYGSNVTMECRFPVERELDLLALVVYWEKEDEQVIQFV

AGEEDLKPQHSNFRGRASLPKDQLLKGNAALQITDVKLQDAGVYCCIISY

GGADYKRITLKVNAPYRKINQRISVDPATSEHELICQAEGYPEAEVIWTN

SDHQPVSGKRSVTTSRTEGMLLNVTSSLRVNATANDVFYCTFWRSQPGQN

HTAELIIPELPATHPPQNRTH.

In embodiments, the second domain of a chimeric protein comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 62.

In embodiments, a chimeric protein of the present invention comprises: (1) a first domain comprising the amino acid sequence of SEQ ID NO: 64, (b) a second domain comprises the amino acid sequence of SEQ ID NO: 62, and (c) a linker comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

In embodiments, a B7H3-Fc-PD-L1 chimeric protein of the present invention has the following amino acid sequence:

(SEQ ID NO: 65)

LEVQVPEDPVVALVGTDATLCCSFSPEPGFSLAQLNLIWQLTDTKQLVHS

FAEGQDQGSAYANRTALFPDLLAQGNASLRLQRVRVADEGSFTCFVSIRD

FGSAAVSLQVAAPYSKPSMTLEPNKDLRPGDTVTITCSSYQGYPEAEVFW

QDGQGVPLTGNVTTSQMANEQGLFDVHSILRVVLGANGTYSCLVRNPVLQ

QDAHSSVTITPQRSPTGAVEVQVPEDPWALVGTDATLRCSFSPEPGFSLA

QLNLIWQLTDTKQLVHSFTEGRDQGSAYANRTALFPDLLAQGNASLRLQR

VRVADEGSFTCFVSIRDFGSAAVSLQVAAPYSKPSMTLEPNKDLRPGDTV

TITCSSYRGYPEAEVFWQDGQGVPLTGNVTTSQMANEQGLFDVHSVLRVV

LGANGTYSCLVRNPVLQQDAHGSVTITGQPMTFPPEASKYGPPCPPCPAP

EFLGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGV

EVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLSGKEYKCKVSSKGLPSSI

EKTISNATGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWE

SNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVLHEAL

HNHYTQKSLSLSLGKIEGRMDFTVTVPKDLYVVEYGSNMTIECKFPVEKQ

LDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNA

ALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPV

TSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTL

RINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNER.

In embodiments, a chimeric protein comprises a variant of a B7H3-Fc-PD-L1 chimeric protein. As examples, the variant may 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%, 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 with SEQ ID NO: 65.

In embodiments, the chimeric protein is capable of contemporaneously binding the B7H4 receptor and the PD-L1 receptor. In embodiments, the B7H4 ligand is CD28 and MIM 186760 and the PD-L1 receptor is PD-1. B7H4 is an immune checkpoint molecule that negatively regulates T-cell-mediated immune response by inhibiting T-cell activation, proliferation, cytokine production and development of cytotoxicity. PD-L1 plays a critical role in induction and maintenance of immune tolerance to self, in part, by acting as a ligand for the inhibitory receptor PD-1; it modulates the activation threshold of T-cells and limits T-cell effector response, including cytotoxic T lymphocytes (CTLs) effector function Accordingly, a chimeric protein comprising the extracellular domains of B7H4 and PD-L1 is capable of contemporaneously activating an immune inhibitory signal (via B7H4 receptors) and activating an immune inhibitory signal (via PD-L1). In embodiments, this chimeric protein is referred to herein as B7H4-Fc-PD-L1.

In embodiments, the chimeric proteins of the present invention comprise variants of the extracellular domain, which includes the ligand-binding domain, of B7H4. As examples, the variant may 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%, 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 with the known amino acid sequence of the extracellular domain of B7H4, e.g., human B7H4.

In embodiments, the extracellular domain of B7H4 has the following amino acid sequence:

(SEQ ID NO: 66)

LIIGFGISGRHSITVTTVASAGNIGEDGILSCTFEPDIKLSDIVIQWLKE

GVLGLVHEFKEGKDELSEQDEMFRGRTAVFADQVIVGNASLRLKNVQLTD

AGTYKCYIITSKGKGNANLEYKTGAFSMPEVNVDYNASSETLRCEAPRWF

PQPTVVWASQVDQGANFSEVSNTSFELNSENVTMKVVSVLYNVTINNTYS

CMIENDIAKATGDIKVTESEIKRRSH.

In embodiments, a chimeric protein comprises a variant of the extracellular domain of B7H4. As examples, the variant may 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%, 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 with SEQ ID NO: 66.

In embodiments, the first domain of a chimeric protein comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 66.

One of ordinary skill may select variants of the known amino acid sequence of B7H4 by consulting the literature, e.g., Salceda et al., “The immunomodulatory protein B7-H4 is overexpressed in breast and ovarian cancers and promotes epithelial cell transformation.” Exp. Cell Res. 306:128-141 (2005); Kryczek et al., “B7-H4 expression identifies a novel suppressive macrophage population in human ovarian carcinoma.” J. Exp. Med. 203:871-881 (2006); and Vigdorovich et al., “Structure and cancer immunotherapy of the B7 family member B7x”, Cell Rep (2014) 9 p. 1089-98; each of which is incorporated by reference in its entirety.

In embodiments, the extracellular domain of PD-L1 has the following amino acid sequence:

In embodiments, the second domain of a chimeric protein comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 62.

In embodiments, a chimeric protein of the present invention comprises: (1) a first domain comprising the amino acid sequence of SEQ ID NO: 66, (b) a second domain comprises the amino acid sequence of SEQ ID NO: 62, and (c) a linker comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

In embodiments, a B7H4-Fc-PD-L1 chimeric protein of the present invention has the following amino acid sequence:

(SEQ ID NO: 67)

LIIGFGISGRHSITVTTVASAGNIGEDGILSCTFEPDIKLSDIVIQWLKE

GVLGLVHEFKEGKDELSEQDEMFRGRTAVFADQVIVGNASLRLKNVQLTD

AGTYKCYIITSKGKGNANLEYKTGAFSMPEVNVDYNASSETLRCEAPRWF

PQPTVVWASQVDQGANFSEVSNTSFELNSENVTMKVVSVLYNVTINNTYS

CMIENDIAKATGDIKVTESEIKRRSHSKYGPPCPPCPAPEFLGGPSVFLF

PPKPKDQLMISRTPEVICVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPRE

EQFNSTYRVVSVLTVLHQDWLSGKEYKCKVSSKGLPSSIEKTISNATGQP

REPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT

TPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVLHEALHNHYTQKSLSL

SLGKIEGRMDFTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWE

MEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQD

AGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAE

GYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFY

CTFRRLDPEENHTAELVIPELPLAHPPNER.

In embodiments, a chimeric protein comprises a variant of a B7H4-Fc-PD-L1 chimeric protein. As examples, the variant may 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%, 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 with SEQ ID NO: 67.

In embodiments, the chimeric protein is capable of contemporaneously binding the ILDR2 receptor and the PD-L1 receptor. In embodiments, the PD-L1 receptor is PD-1; however, the ILDR2 ligand/receptor is presently unknown. ILDR2 is B7-like protein with robust T cell inhibitory activity. PD-L1 plays a critical role in induction and maintenance of immune tolerance to self, in part, by acting as a ligand for the inhibitory receptor PD-1; it modulates the activation threshold of T-cells and limits T-cell effector response, including cytotoxic T lymphocytes (CTLs) effector function Accordingly, a chimeric protein comprising the extracellular domains of ILDR2 and PD-L1 is capable of contemporaneously activating an immune inhibitory signal (via ILDR2) and activating an immune inhibitory signal (via PD-L1). In embodiments, this chimeric protein is referred to herein as ILDR2-Fc-PD-L1.

In embodiments, the chimeric proteins of the present invention comprise variants of the extracellular domain, which includes the ligand/receptor-binding domain, of ILDR2. As examples, the variant may 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%, 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 with the known amino acid sequence of the extracellular domain of ILDR2, e.g., human ILDR2.

In embodiments, the extracellular domain of ILDR2 has the following amino acid sequence:

(SEQ ID NO: 70)

LQVTVPDKKKVAMLFQPTVLRCHFSTSSHQPAVVQWKFKSYCQDRMGESL

GMSSTRAQSLSKRNLEWDPYLDCLDSRRTVRVVASKQGSTVTLGDFYRGR

EITIVHDADLQIGKLMWGDSGLYYCIITTPDDLEGKNEDSVELLVLGRTG

LLADLLPSFAVEIMPE.

In embodiments, a chimeric protein comprises a variant of the extracellular domain of ILDR2. As examples, the variant may 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%, 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 with SEQ ID NO: 70.

In embodiments, the first domain of a chimeric protein comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 70.

One of ordinary skill may select variants of the known amino acid sequence of ILDR2 by consulting the literature, e.g., Hecht et al., “ILDR2 Is a Novel B7-like Protein That Negatively Regulates T Cell Responses”. J Immunol. 2018 Mar. 15; 200(6):2025-2037; Podojil et al., “ILDR2-Fc Is a Novel Regulator of Immune Homeostasis and Inducer of Antigen-Specific Immune Tolerance.” J Immunol. 2018 Mar. 15; 200(6):2013-2024; and Watanabe et al., “ILDR2: An Endoplasmic Reticulum Resident Molecule Mediating Hepatic Lipid Homeostasis.” PLoS One. 2013; 8(6): e67234, each of which is incorporated by reference in its entirety.

In embodiments, the extracellular domain of PD-L1 has the following amino acid sequence:

In embodiments, the second domain of a chimeric protein comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 62.

In embodiments, a chimeric protein of the present invention comprises: (1) a first domain comprising the amino acid sequence of SEQ ID NO: 70, (b) a second domain comprises the amino acid sequence of SEQ ID NO: 62, and (c) a linker comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

In embodiments, an ILDR2-Fc-PD-L1 chimeric protein of the present invention has the following amino acid sequence:

(SEQ ID NO: 71)

LQVTVPDKKKVAMLFQPTVLRCHFSTSSHQPAVVQWKFKSYCQDRMGESL

GMSSTRAQSLSKRNLEWDPYLDCLDSRRTVRVVASKQGSTVTLGDFYRGR

EITIVHDADLQIGKLMWGDSGLYYCIITTPDDLEGKNEDSVELLVLGRTG

LLADLLPSFAVEIMPEVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTIT

LTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVS

ELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPP

KEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSY

FVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGIIEGRMDF

TITAPKDLYVVEYGSNVTMECRFPVERELDLLALVVYWEKEDEQVIQFVA

GEEDLKPQHSNFRGRASLPKDQLLKGNAALQITDVKLQDAGVYCCIISYG

GADYKRITLKVNAPYRKINQRISVDPATSEHELICQAEGYPEAEVIWTNS

DHQPVSGKRSVTTSRTEGMLLNVTSSLRVNATANDVFYCTFWRSQPGQNH

TAELIIPELPATHPPQNRTH.

In embodiments, a chimeric protein comprises a variant of an ILDR2-Fc-PD-L1 chimeric protein. As examples, the variant may 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%, 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 with SEQ ID NO: 71.

In embodiments, the chimeric protein is capable of contemporaneously binding the BTNL2 receptor and the PD-L1 receptor. In embodiments, the PD-L1 receptor is PD-1; however, the BTNL2 ligand/receptor is presently unknown. BTNL2 may be involved in immune surveillance, serving as a negative T-cell regulator by decreasing T-cell proliferation and cytokine release. PD-L1 plays a critical role in induction and maintenance of immune tolerance to self, in part, by acting as a ligand for the inhibitory receptor PD-1; it modulates the activation threshold of T-cells and limits T-cell effector response, including cytotoxic T lymphocytes (CTLs) effector function Accordingly, a chimeric protein comprising the extracellular domains of BTNL2 and PD-L1 is capable of contemporaneously activating an immune inhibitory signal (via BTNL2) and activating an immune inhibitory signal (via PD-L1). In embodiments, this chimeric protein is referred to herein as BTNL2-Fc-PD-L1.

In embodiments, the chimeric proteins of the present invention comprise variants of the extracellular domain, which includes the ligand/receptor-binding domain, of BTNL2. As examples, the variant may 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%, 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 with the known amino acid sequence of the extracellular domain of BTNL2, e.g., human BTNL2.

In embodiments, the extracellular domain of BTNL2 has the following amino acid sequence:

(SEQ ID NO: 72)

KQSEDFRVIGPAHPILAGVGEDALLTCQLLPKRTTMHVEVRWYRSEPSTP

VFVHRDGVEVTEMQMEEYRGWVEWIENGIAKGNVALKIHNIQPSDNGQYW

CHFQDGNYCGETSLLLKVAGLGSAPSIHMEGPGESGVQLVCTARGWFPEP

QVYWEDIRGEKLLAVSEHRIQDKDGLFYAEATLVVRNASAESVSCLVHNP

VLTEEKGSVISLPEKLQTELASLKVNGPSQPILVRVGEDIQLTCYLSPKA

NAQSMEVRWDRSHRYPAVHVYMDGDHVAGEQMAEYRGRTVLVSDAIDEGR

LTLQILSARPSDDGQYRCLFEKDDVYQEASLDLKVVSLGSSPLITVEGQE

DGEMQPMCSSDGWFPQPHVPWRDMEGKTIPSSSQALTQGSHGLFHVQTLL

RVTNISAVDVTCSISIPFLGEEKIATFSLSGW.

In embodiments, a chimeric protein comprises a variant of the extracellular domain of BTNL2. As examples, the variant may 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%, 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 with SEQ ID NO: 72.

In embodiments, the first domain of a chimeric protein comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 72.

One of ordinary skill may select variants of the known amino acid sequence of BTNL2 by consulting the literature, e.g., Valentonyte et al., “Sarcoidosis is associated with a truncating splice site mutation in BTNL2.” Nat. Genet. 37:357-364 (2005); Nguyen et al., “BTNL2, a butyrophilin-like molecule that functions to inhibit T cell activation.” J Immunol. 2006 Jun. 15; 176(12):7354-60; Arnett et al., “BTNL2, a butyrophilin/B7-like molecule, is a negative costimulatory molecule modulated in intestinal inflammation.” J. Immunol. 178 (3), 1523-1533 (2007); Rhodes et al., “Regulation of Immunity by Butyrophilins.” Annu Rev Immunol. 2016 May 20; 34:151-72; and Cui et al., “In vivo administration of recombinant BTNL2-Fc fusion protein ameliorates graft-versus-host disease in mice.” Cell Immunol. 2019 January; 335:22-29, each of which is incorporated by reference in its entirety.

In embodiments, the extracellular domain of PD-L1 has the following amino acid sequence:

In embodiments, the second domain of a chimeric protein comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 62.

In embodiments, a chimeric protein of the present invention comprises: (1) a first domain comprising the amino acid sequence of SEQ ID NO: 72, (b) a second domain comprises the amino acid sequence of SEQ ID NO: 62, and (c) a linker comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

In embodiments, a BTNL2-Fc-PD-L1 chimeric protein of the present invention has the following amino acid sequence:

(SEQ ID NO: 73)

KQSEDFRVIGPAHPILAGVGEDALLTCQLLPKRTTMHVEVRWYRSEPSTP

VFVHRDGVEVTEMQMEEYRGWVEWIENGIAKGNVALKIHNIQPSDNGQYW

CHFQDGNYCGETSLLLKVAGLGSAPSIHMEGPGESGVQLVCTARGWFPEP

QVYWEDIRGEKLLAVSEHRIQDKDGLFYAEATLVVRNASAESVSCLVHNP

VLTEEKGSVISLPEKLQTELASLKVNGPSQPILVRVGEDIQLTCYLSPKA

NAQSMEVRWDRSHRYPAVHVYMDGDHVAGEQMAEYRGRTVLVSDAIDEGR

LTLQILSARPSDDGQYRCLFEKDDVYQEASLDLKVVSLGSSPLITVEGQE

DGEMQPMCSSDGWFPQPHVPWRDMEGKTIPSSSQALTQGSHGLFHVQTLL

RVTNISAVDVTCSISIPFLGEEKIATFSLSGWSKYGPPCPPCPAPEFLGG

PSVFLFPPKPKDQLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNA

KTKPREEQFNSTYRVVSVLTVLHQDWLSGKEYKCKVSSKGLPSSIEKTIS

NATGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQP

ENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVLHEALHNHYT

QKSLSLSLGKIEGRMDFTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAA

LIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQIT

DVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHE

LTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTT

TNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNER.

In embodiments, a chimeric protein comprises a variant of a BTNL2-Fc-PD-L1 chimeric protein. As examples, the variant may 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%, 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 with SEQ ID NO: 73.

In embodiments, the chimeric protein is capable of contemporaneously binding the BTNL2 receptor and the PD-L1 receptor. In embodiments, the PD-L1 receptor is PD-1; however, the BTNL2 ligand/receptor is presently unknown. BTNL2 may be involved in immune surveillance, serving as a negative T-cell regulator by decreasing T-cell proliferation and cytokine release. PD-L1 plays a critical role in induction and maintenance of immune tolerance to self, in part, by acting as a ligand for the inhibitory receptor PD-1; it modulates the activation threshold of T-cells and limits T-cell effector response, including cytotoxic T lymphocytes (CTLs) effector function Accordingly, a chimeric protein comprising the extracellular domains of BTNL2 and PD-L1 is capable of contemporaneously activating an immune inhibitory signal or competitively inhibiting an immune activating signal (via BTNL2) and activating an immune inhibitory signal (via PD-L1). In embodiments, this chimeric protein is referred to herein as PD-L1-Fc-BTNL2.

In embodiments, the extracellular domain of PD-L1 has the following amino acid sequence:

In embodiments, the second domain of a chimeric protein comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 62.

In embodiments, the extracellular domain of BTNL2 has the following amino acid sequence:

In embodiments, the first domain of a chimeric protein comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 72.

In embodiments, a PD-L1-Fc-BTNL2 chimeric protein of the present invention has the following amino acid sequence:

(SEQ ID NO: 74)

FTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFV

HGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISY

GGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWT

SSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEE

NHTAELVIPELPLAHPPNERSKYGPPCPPCPAPEFLGGPSVFLFPPKPKD

QLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNST

YRVVSVLTVLHQDWLSGKEYKCKVSSKGLPSSIEKTISNATGQPREPQVY

TLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD

SDGSFFLYSRLTVDKSRWQEGNVFSCSVLHEALHNHYTQKSLSLSLGKIE

GRMDKQSEDFRVIGPAHPILAGVGEDALLTCQLLPKRTTMHVEVRWYRSE

PSTPVFVHRDGVEVTEMQMEEYRGWVEWIENGIAKGNVALKIHNIQPSDN

GQYWCHFQDGNYCGETSLLLKVAGLGSAPSIHMEGPGESGVQLVCTARGW

FPEPQVYWEDIRGEKLLAVSEHRIQDKDGLFYAEATLWRNASAESVSCLV

HNPVLTEEKGSVISLPEKLQTELASLKVNGPSQPILVRVGEDIQLTCYLS

PKANAQSMEVRWDRSHRYPAVHVYMDGDHVAGEQMAEYRGRTVLVSDAID

EGRLTLQILSARPSDDGQYRCLFEKDDVYQEASLDLKVVSLGSSPLITVE

GQEDGEMQPMCSSDGWFPQPHVPWRDMEGKTIPSSSQALTQGSHGLFHVQ

TLLRVTNISAVDVTCSISIPFLGEEKIATFSLSGW.

In embodiments, a chimeric protein comprises a variant of a PD-L1-Fc-BTNL2 chimeric protein. As examples, the variant may 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%, 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 with SEQ ID NO: 74.

In embodiments, the chimeric protein is capable of contemporaneously binding the CTLA4 ligand and the IL2 receptor. In embodiments, the CTLA4 ligand is CD80 or CD86 and IL2 binds to the IL2 receptor, which has three forms, generated by different combinations of three different proteins, often referred to as “chains”: IL2Rα, IL2Rβ, and IL2Rγ. CTLA4 acts as an “off” switch when bound to its ligand on the surface of antigen-presenting cells (APCs). CTLA4 is a protein receptor that functions as an immune checkpoint and downregulates immune responses. Certain “High Affinity” IL2 expands and activates Tregs which help prevent autoimmunity and control inflammation. Accordingly, a chimeric protein comprising the portion of IL2 capable binding its receptor and the extracellular domain of CTLA4 is capable of contemporaneously competitively inhibiting an immune activating signal (via CTLA4) and stimulating regulatory T cells (via IL2). In embodiments, this chimeric protein is referred to herein as CTLA4-Fc-IL2.

In embodiments, the extracellular domain of CTLA4 has the following amino acid sequence:

(SEQ ID NO: 60)

KAMHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVC

AATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMY

PPPYYLGIGNGTQIYVIDPEPCPDSD.

In embodiments, the first domain of a chimeric protein comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 60.

In embodiments, the chimeric proteins of the present invention comprises variants of IL2 comprising its receptor-binding domain. As examples, the variant may 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%, 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 with the known amino acid sequence of the portion IL2, e.g., human IL2, comprising its receptor-binding domain.

In embodiments, the portion of IL2 comprising its receptor-binding domain, relevant to the present invention, has one the following amino acid sequences:

(SEQ ID NO: 75)

APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKA

TELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSE

TTFMCEYACETATIVEFLNRWITFSQSIISTLT;

(SEQ ID NO: 76)

APTSSSTKKTQLQLEHLLLHLQMILNGINNYKNPKLTRMLTFKFYMPKKA

TELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSE

TTFMCEYADETATIVEFLNRWITFSLSIISTLT;

(SEQ ID NO: 77)

APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKA

TELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSE

TTFMCEYADETATIVEFLNRWITFCQSIISTLT;

and

(SEQ ID NO: 78)

APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKA

TELKHLQCLEEELKPLEEVLNLAQSKNFHFDPRDVVSNINVFVLELKGSE

TTFMCEYADETATIVEFLNRWITFCQSIISTLT

The IL2 variants of SEQ ID NO: 75 and SEQ ID NO: 76 are “high affinity” IL2s, which is preferentially expressed by regulatory T cells.

In embodiments, a chimeric protein comprises a variant of a portion of IL2 comprising its receptor-binding domain. As examples, the variant may 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%, 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 with one of SEQ ID NO: 75 to SEQ ID NO: 78.

In embodiments, the second domain of a chimeric protein comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of one of SEQ ID NO: 75 to SEQ ID NO: 78.

One of ordinary skill may select variants of the known amino acid sequence of IL2 by consulting the literature, e.g., Taniguchi et al., “Structure and expression of a cloned cDNA for human interleukin-2.” Nature 302 (5906), 305-310 (1983); Brandhuber et al., “Three-dimensional structure of interleukin-2” Science 238 (4834), 1707-1709 (1987); Bazan “Unraveling the structure of IL-2” Science 257 (5068), 410-413 (1992); Mot et al., “Secondary structure of human interleukin 2 from 3D heteronuclear NMR experiments.” Biochemistry 31 (33), 7741-7744 (1992); Wang et al., “Structure of the quaternary complex of interleukin-2 with its alpha, beta, and gammac receptors.” Science 310 (5751), 1159-1163 (2005); and Stauber et al., “Crystal structure of the IL-2 signaling complex: paradigm for a heterotrimeric cytokine receptor.” Proc. Natl. Acad. Sci. U.S.A. 103 (8), 2788-2793 (2006), each of which is incorporated by reference in its entirety.

In embodiments, a chimeric protein of the present invention comprises: (1) a first domain comprising the amino acid sequence of SEQ ID NO: 60, (b) a second domain comprises the amino acid sequence of one of SEQ ID NO: 75 to SEQ ID NO: 78, and (c) a linker comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

In embodiments, a CTLA4-Fc-IL2 chimeric protein of the present invention has the one of following amino acid sequences:

(SEQ ID NO: 79)

KAMHVAQPAVVLASSRGIASFVCEYASPGKATEVR

VTVLRQADSQVTEVCAATYMMGNELTFLDDSICTG

TSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYY

LGIGNGTQIYVIDPEPCPDSDSKYGPPCPPCPAPE

FLGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSQ

EDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVV

SVLTVLHQDWLSGKEYKCKVSSKGLPSSIEKTISN

ATGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFY

PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS

RLTVDKSRWQEGNVFSCSVLHEALHNHYTQKSLSL

SLGKIEGRMDAPTSSSTKKTQLQLEHLLLDLQMIL

NGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLE

EELKPLEEVLNLAQSKNFHLRPRDLISRINVIVLE

LKGSETTFMCEYACETATIVEFLNRWITFSQSIIS

TLT;

(SEQ ID NO: 80)

KAMHVAQPAVVLASSRGIASFVCEYASPGKATEVR

VTVLRQADSQVTEVCAATYMMGNELTFLDDSICTG

TSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYY

LGIGNGTQIYVIDPEPCPDSDSKYGPPCPPCPAPE

FLGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSQ

EDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVV

SVLTVLHQDWLSGKEYKCKVSSKGLPSSIEKTISN

ATGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFY

PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS

RLTVDKSRWQEGNVFSCSVLHEALHNHYTQKSLSL

SLGKIEGRMDAPTSSSTKKTQLQLEHLLLHLQMIL

NGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLE

EELKPLEEVLNLAQSKNFHLRPRDLISRINVIVLE

LKGSETTFMCEYADETATIVEFLNRWITFSLSIIS

TLT;

(SEQ ID NO: 81)

KAMHVAQPAVVLASSRGIASFVCEYASPGKATEVR

VTVLRQADSQVTEVCAATYMMGNELTFLDDSICTG

TSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYY

LGIGNGTQIYVIDPEPCPDSDSKYGPPCPPCPAPE

FLGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSQ

EDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVV

SVLTVLHQDWLSGKEYKCKVSSKGLPSSIEKTISN

ATGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFY

PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS

RLTVDKSRWQEGNVFSCSVLHEALHNHYTQKSLSL

SLGKIEGRMDAPTSSSTKKTQLQLEHLLLDLQMIL

NGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLE

EELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLE

LKGSETTFMCEYADETATIVEFLNRWITFCQSIIS

TLT;

or

(SEQ ID NO: 82)

KAMHVAQPAVVLASSRGIASFVCEYASPGKATEVR

VTVLRQADSQVTEVCAATYMMGNELTFLDDSICTG

TSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYY

LGIGNGTQIYVIDPEPCPDSDSKYGPPCPPCPAPE

FLGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSQ

EDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVV

SVLTVLHQDWLSGKEYKCKVSSKGLPSSIEKTISN

ATGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFY

PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS

RLTVDKSRWQEGNVFSCSVLHEALHNHYTQKSLSL

SLGKIEGRMDAPTSSSTKKTQLQLEHLLLDLQMIL

NGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLE

EELKPLEEVLNLAQSKNFHFDPRDVVSNINVFVLE

LKGSETTFMCEYADETATIVEFLNRWITFCQSIIS

TLT

In embodiments, a chimeric protein comprises a variant of a CTLA4-Fc-IL2 chimeric protein. As examples, the variant may 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%, 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 with one of SEQ ID NO: 79 to SEQ ID NO: 82.

In embodiments, the chimeric protein is capable of contemporaneously binding the CTLA4 ligand and the SEMA3E receptor. In embodiments, the CTLA4 ligand is CD80 or CD86 and the SEMA3E receptor is PlexinD1. CTLA4 acts as an “off” switch when bound to its ligand on the surface of antigen-presenting cells (APCs). CTLA4 is a protein receptor that functions as an immune checkpoint and downregulates immune responses. SEMA3E may act as a secreted chemorepellent in neutrophil migration and recent in vitro and in vivo experimental evidence demonstrates a key regulator role of SEMA3E on airway inflammation, hyperresponsiveness and remodeling in allergic asthma. Accordingly, a chimeric protein comprising the extracellular domains of CTLA4 and a portion of SEMA3E which includes its receptor-binding domain is capable of contemporaneously competitively inhibiting an immune activating signal (via CTLA4) and activating an immune inhibitory signal (via SEMA3E). In embodiments, this chimeric protein is referred to herein as CTLA4-Fc-SEMA3E.

In embodiments, the extracellular domain of CTLA4 has the following amino acid sequence:

(SEQ ID NO: 60)

KAMHVAQPAVVLASSRGIASFVCEYASPGKATEVR

VTVLRQADSQVTEVCAATYMMGNELTFLDDSICTG

TSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYY

LGIGNGTQIYVIDPEPCPDSD.

In embodiments, the first domain of a chimeric protein comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 60.

In embodiments, the chimeric proteins of the present invention comprise variants of the portion of SEMA3E which includes the receptor-binding domain. As examples, the variant may 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%, 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 with the known amino acid sequence of the portion of SEMA3E, e.g., human SEMA3E, which comprises its receptor-binding domain.

In embodiments, the portion of SEMA3E which comprises the receptor-binding domain has the following amino acid sequence:

(SEQ ID NO: 83)

ADTTHPRLRLSHKELLNLNRTSIFHSPFGFLDLHTM

LLDEYQERLFVGGRDLVYSLSLERISDGYKEIHWP

STALKMEECIMKGKDAGECANYVRVLHHYNRTHLL

TCGTGAFDPVCAFIRVGYHLEDPLFHLESPRSERG

RGRCPFDPSSSFISTLIGSELFAGLYSDYWSRDAA

IFRSMGRLAHIRTEHDDERLLKEPKFVGSYMIPDN

EDRDDNKVYFFFTEKALEAENNAHAIYTRVGRLCV

NDVGGQRILVNKWSTFLKARLVCSVPGMNGIDTYF

DELEDVFLLPTRDHKNPVIFGLFNTTSNIFRGHAI

CVYHMSSIRAAFNGPYAHKEGPEYHWSVYEGKVPY

PRPGSCASKVNGGRYGTTKDYPDDAIRFARSHPLM

YQAIKPAHKKPILVKTDGKYNLKQIAVDRVEAEDG

QYDVLFIGTDNGIVLKVITIYNQEMESMEEVILEE

LQIFKDPVPIISMEISSKRQQLYIGSASAVAQVRF

HHCDMYGSACADCCLARDPYCAWDGISCSRYYPTG

THAKRRFRRQDVRHGNAAQQCFGQQFVGDALDKTE

EHLAYGIENNSTLLECTPRSLQAKVIWFVQKGRET

RKEEVKTDDRVVKMDLGLLFLRLHKSDAGTYFCQT

VEHSFVHTVRKITLEWEEEKVEDMFNKDDEEDRHH

RMPCPAQSSISQGAKPWYKEFLQLIGYSNFQRVEE

YCEKVWCTDRKRKKLKMSPSKWKYANPQEKKLRSK

PEHYRLPRHTLDS.

In embodiments, a chimeric protein comprises a variant of the portion of SEMA3E which includes the receptor-binding domain. As examples, the variant may 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%, 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 with SEQ ID NO: 83.

In embodiments, the second domain of a chimeric protein comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 83.

One of ordinary skill may select variants of the known amino acid sequence of SEMA3E by consulting the literature, e.g., Lalani et al., “SEMA3E mutation in a patient with CHARGE syndrome.” J. Med. Genet. 41 (7), e94 (2004); Ota et al., “Complete sequencing and characterization of 21,243 full-length human cDNAs.” Nat. Genet. 36:40-45(2004); Movassagh et al., “The regulatory role of semaphorin 3E in allergic asthma” Int. J. Biochem. Cell Biol. 106, 68-73 (2019); Movassagh et al., “Chemorepellent Semaphorin 3E Negatively Regulates Neutrophil Migration In Vitro and In Vivo.” J. Immunol. 198 (3), 1023-1033 (2017); Siebold and Jones “Structural insights into semaphorins and their receptors” Semin. Cell Dev. Biol., 24 (2013), pp. 139-145, each of which is incorporated by reference in its entirety.

In embodiments, a chimeric protein of the present invention comprises: (1) a first domain comprising the amino acid sequence of SEQ ID NO: 60, (b) a second domain comprises the amino acid sequence of SEQ ID NO: 83, and (c) a linker comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

In embodiments, a CTLA4-Fc-SEMA3E chimeric protein of the present invention has the following amino acid sequence:

(SEQ ID NO: 84)

KAMHVAQPAVVLASSRGIASFVCEYASPGKATEVR

VTVLRQADSQVTEVCAATYMMGNELTFLDDSICTG

TSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYY

LGIGNGTQIYVIDPEPCPDSDSKYGPPCPPCPAPE

FLGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSQ

EDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVV

SVLTVLHQDWLSGKEYKCKVSSKGLPSSIEKTISN

ATGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFY

PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS

RLTVDKSRWQEGNVFSCSVLHEALHNHYTQKSLSL

SLGKIEGRMDADTTHPRLRLSHKELLNLNRTSIFH

SPFGFLDLHTMLLDEYQERLFVGGRDLVYSLSLER

ISDGYKEIHWPSTALKMEECIMKGKDAGECANYVR

VLHHYNRTHLLTCGTGAFDPVCAFIRVGYHLEDPL

FHLESPRSERGRGRCPFDPSSSFISTLIGSELFAG

LYSDYWSRDAAIFRSMGRLAHIRTEHDDERLLKEP

KFVGSYMIPDNEDRDDNKVYFFFTEKALEAENNAH

AIYTRVGRLCVNDVGGQRILVNKWSTFLKARLVCS

VPGMNGIDTYFDELEDVFLLPTRDHKNPVIFGLFN

TTSNIFRGHAICVYHMSSIRAAFNGPYAHKEGPEY

HWSVYEGKVPYPRPGSCASKVNGGRYGTTKDYPDD

AIRFARSHPLMYQAIKPAHKKPILVKTDGKYNLKQ

IAVDRVEAEDGQYDVLFIGTDNGIVLKVITIYNQE

MESMEEVILEELQIFKDPVPIISMEISSKRQQLYI

GSASAVAQVRFHHCDMYGSACADCCLARDPYCAWD

GISCSRYYPTGTHAKRRFRRQDVRHGNAAQQCFGQ

QFVGDALDKTEEHLAYGIENNSTLLECTPRSLQAK

VIWFVQKGRETRKEEVKTDDRVVKMDLGLLFLRLH

KSDAGTYFCQTVEHSFVHTVRKITLEVVEEEKVED

MFNKDDEEDRHHRMPCPAQSSISQGAKPWYKEFLQ

LIGYSNFQRVEEYCEKVWCTDRKRKKLKMSPSKWK

YANPQEKKLRSKPEHYRLPRHTLDS.

In embodiments, a chimeric protein comprises a variant of a CTLA4-Fc-SEMA3E chimeric protein. As examples, the variant may 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%, 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 with SEQ ID NO: 84.

In embodiments, the chimeric protein is capable of contemporaneously binding the VSIG4 ligand and the IL2 receptor. In embodiments, the VSIG4 ligand is C3b or an unidentified T-cell ligand or receptor and IL2 binds to the IL2 receptor, which has three forms, generated by different combinations of three different proteins, often referred to as “chains”: a (alpha) (also called IL2Rα, CD25, or Tac antigen), β (beta) (also called IL2Rβ, or CD122), and γ (gamma) (also called IL2Rγ, γc, common gamma chain, or CD132). VSIG4 is a phagocytic receptor, strong negative regulator of T-cell proliferation and IL2 production; it is a potent inhibitor of the alternative complement pathway convertases. Low-dose IL2 has been shown to expand and activate Tregs which helps prevent autoimmunity and control inflammation. Accordingly, a chimeric protein comprising the extracellular domains of VSIG4 and IL2 is capable of contemporaneously activating an immune inhibitory signal (via VSIG4) and activating regulatory T cells (via IL2). In embodiments, this chimeric protein is referred to herein as VSIG4-Fc-IL2.

In embodiments, the chimeric proteins of the present invention comprise variants of the extracellular domain, which includes the receptor-binding domain, of VSIG4. As examples, the variant may 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%, 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 with the known amino acid sequence of the extracellular domain of VSIG4, e.g., human VSIG4.

In embodiments, the extracellular domain of VSIG4 has the following amino acid sequence:

(SEQ ID NO: 85)

RPILEVPESVTGPWKGDVNLPCTYDPLQGYTQVLVK

WLVQRGSDPVTIFLRDSSGDHIQQAKYQGRLHVSHK

VPGDVSLQLSTLEMDDRSHYTCEVTWQTPDGNQVVR

DKITELRVQKLSVSKPTVTTGSGYGFTVPQGMRISL

QCQARGSPPISYIWYKQQTNNQEPIKVATLSTLLFK

PAVIADSGSYFCTAKGQVGSEQHSDIVKFVVKDSSK

LLKTKTEAPTTMTYPLKATSTVKQSWDWTTDMDGYL

GETSAGPGKSLP.

In embodiments, a chimeric protein comprises a variant of the extracellular domain of VSIG4. As examples, the variant may 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%, 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 with SEQ ID NO: 85.

In embodiments, the first domain of a chimeric protein comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 85.

One of ordinary skill may select variants of the known amino acid sequence of VSIG4 by consulting the literature, e.g., Vogt et al., “VSIG4, a B7 family-related protein, is a negative regulator of T cell activation.” J. Clin. Invest. 116:2817-2826 (2006); Wiesmann et al., “Structure of C3b in complex with CRIg gives insights into regulation of complement activation.” Nature 444:217-220 (2006); and Zhang and Henzel “Signal peptide prediction based on analysis of experimentally verified cleavage sites.” Protein Sci. 13 (10), 2819-2824 (2004), each of which is incorporated by reference in its entirety.

In embodiments, the chimeric proteins of the present invention comprise variants of the receptor-binding domain, of IL2. As examples, the variant may 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%, 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 with the known amino acid sequence of the portion of IL2, e.g., human IL2, which comprises its receptor-binding domain.

In embodiments, the portion of IL2 comprising its receptor-binding domain, relevant to the present invention, has one the following amino acid sequences:

(SEQ ID NO: 75)

APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLT

RMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQ

SKNFHLRPRDLISRINVIVLELKGSETTFMCEYACET

ATIVEFLNRWITFSQSIISTLT;

(SEQ ID NO: 76)

APTSSSTKKTQLQLEHLLLHLQMILNGINNYKNPKLT

RMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQ

SKNFHLRPRDLISRINVIVLELKGSETTFMCEYADET

ATIVEFLNRWITFSLSIISTLT;

(SEQ ID NO: 77)

APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLT

RMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQ

SKNFHLRPRDLISNINVIVLELKGSETTFMCEYADET

ATIVEFLNRWITFCQSIISTLT;

and

(SEQ ID NO: 78)

APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLT

RMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQ

SKNFHFDPRDWSNINVFVLELKGSETTFMCEYADETA

TIVEFLNRWITFCQSIISTLT

The IL2 variants of SEQ ID NO: 75 and SEQ ID NO: 76 are “high affinity” IL2s, which is preferentially expressed by regulatory T cells.

In embodiments, a chimeric protein comprises a variant of the receptor-binding domain of IL2. As examples, the variant may 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%, 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 with one of SEQ ID NO: 75 to SEQ ID NO: 78.

In embodiments, the second domain of a chimeric protein comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of one of SEQ ID NO: 75 to SEQ ID NO: 78.

In embodiments, a chimeric protein of the present invention comprises: (1) a first domain comprising the amino acid sequence of SEQ ID NO: 85, (b) a second domain comprises the amino acid sequence of one of SEQ ID NO: 75 to SEQ ID NO: 78, and (c) a linker comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

In embodiments, a VSIG4-Fc-IL2 chimeric protein of the present invention has the one of following amino acid sequences:

(SEQ ID NO: 86)

RPILEVPESVTGPWKGDVNLPCTYDPLQGYTQVLV

KWLVQRGSDPVTIFLRDSSGDHIQQAKYQGRLHVS

HKVPGDVSLQLSTLEMDDRSHYTCEVTWQTPDGNQ

VVRDKITELRVQKLSVSKPTVTTGSGYGFTVPQGM

RISLQCQARGSPPISYIWYKQQTNNQEPIKVATLS

TLLFKPAVIADSGSYFCTAKGQVGSEQHSDIVKFV

VKDSSKLLKTKTEAPTTMTYPLKATSTVKQSWDWT

TDMDGYLGETSAGPGKSLPSKYGPPCPPCPAPEFL

GGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSQED

PEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSV

LTVLHQDWLSGKEYKCKVSSKGLPSSIEKTISNAT

GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPS

DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRL

TVDKSRWQEGNVFSCSVLHEALHNHYTQKSLSLSL

GKIEGRMDAPTSSSTKKTQLQLEHLLLDLQMILNG

INNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEE

LKPLEEVLNLAQSKNFHLRPRDUSRINVIVLELKG

SETTFMCEYACETATIVEFLNRWITFSQSIISTLT;

(SEQ ID NO: 87)

RPILEVPESVTGPWKGDVNLPCTYDPLQGYTQVLV

KWLVQRGSDPVTIFLRDSSGDHIQQAKYQGRLHVS

HKVPGDVSLQLSTLEMDDRSHYTCEVTWQTPDGNQ

VVRDKITELRVQKLSVSKPTVTTGSGYGFTVPQGM

RISLQCQARGSPPISYIWYKQQTNNQEPIKVATLS

TLLFKPAVIADSGSYFCTAKGQVGSEQHSDIVKFV

VKDSSKLLKTKTEAPTTMTYPLKATSTVKQSWDWT

TDMDGYLGETSAGPGKSLPSKYGPPCPPCPAPEFL

GGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSQED

PEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSV

LTVLHQDWLSGKEYKCKVSSKGLPSSIEKTISNAT

GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPS

DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRL

TVDKSRWQEGNVFSCSVLHEALHNHYTQKSLSLSL

GKIEGRMDAPTSSSTKKTQLQLEHLLLHLQMILNG

INNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEE

LKPLEEVLNLAQSKNFHLRPRDUSRINVIVLELKG

SETTFMCEYADETATIVEFLNRWITFSLSIISTLT;

(SEQ ID NO: 88)

RPILEVPESVTGPWKGDVNLPCTYDPLQGYTQVLV

KWLVQRGSDPVTIFLRDSSGDHIQQAKYQGRLHVS

HKVPGDVSLQLSTLEMDDRSHYTCEVTWQTPDGNQ

VVRDKITELRVQKLSVSKPTVTTGSGYGFTVPQGM

RISLQCQARGSPPISYIWYKQQTNNQEPIKVATLS

TLLFKPAVIADSGSYFCTAKGQVGSEQHSDIVKFV

VKDSSKLLKTKTEAPTTMTYPLKATSTVKQSWDWT

TDMDGYLGETSAGPGKSLPSKYGPPCPPCPAPEFL

GGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSQED

PEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSV

LTVLHQDWLSGKEYKCKVSSKGLPSSIEKTISNAT

GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPS

DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRL

fVDKSRWQEGNVFSCSVLHEALHNHYTQKSLSLSLG

KIEGRMDAPTSSSTKKTQLQLEHLLLDLQMILNGI

NNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEEL

KPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKG

SETTFMCEYADETATIVEFLNRWITFCQSIISTLT;

or

(SEQ ID NO: 89)

RPILEVPESVTGPWKGDVNLPCTYDPLQGYTQVLV

KWLVQRGSDPVTIFLRDSSGDHIQQAKYQGRLHVS

HKVPGDVSLQLSTLEMDDRSHYTCEVTWQTPDGNQ

VVRDKITELRVQKLSVSKPTVTTGSGYGFTVPQGM

RISLQCQARGSPPISYIWYKQQTNNQEPIKVATLS

TLLFKPAVIADSGSYFCTAKGQVGSEQHSDIVKFV

VKDSSKLLKTKTEAPTTMTYPLKATSTVKQSWD

WTTDMD

GYLGETSAGPGKSLPSKYGPPCPPCPAPEFLGGPS

VFLFPPKPKDQLMISRTPEVTCVVVDVSQEDPEVQ

FNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVL

HQDWLSGKEYKCKVSSKGLPSSIEKTISNATGQPR

EPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAV

EWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDK

SRWQEGNVFSCSVLHEALHNHYTQKSLSLSLGKIE

GRMDAPTSSSTKKTQLQLEHLLLDLQMILNGINNY

KNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPL

EEVLNLAQSKNFHFDPRDWSNINVFVLELKGSETT

FMCEYADETATIVEFLNRWITFCQSIISTLT

In embodiments, a chimeric protein comprises a variant of a VSIG4-Fc-IL2 chimeric protein. As examples, the variant may 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%, 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 with one of SEQ ID NO: 86 to SEQ ID NO: 89.

In embodiments, the chimeric protein is capable of contemporaneously binding the CTLA4 ligand and a ligand/receptor of a Type II transmembrane protein selected from BTNL2, C-type lectin domain (CLEC) family members, GITRL, TL1A, IL-10, TGF-beta, In embodiments, the CLEC family member is selected from AlCL/CLEC-2B, ASGR1/ASGPR1, ASGR2, C1q R1/CD93, CD161, CD161/NK1.1, CD23/Fc epsilon RII, CD302/CLEC13A, CD72, CD94, Chondrolectin, CLEC-1, CLEC10A/CD301, CLEC12B, CLEC14A, CLEC16A, CLEC17A, CLEC18A, CLEC18B, CLEC18C, CLEC-2/CLEC1B, CLEC-2A, CLEC3A, CLEC3B/Tetranectin, CLEC4B2/mDCAR1, CLEC4D/CLECSF8, CLEC4E, CLEC4F/CLECSF13, CLEC9a, CLECL1/DCAL-1, CL-K1/COLEC11, CL-L1/COLEC10, CL-P1/COLEC12, DCAR/CLEC4B, DCIR/CLEC4A, DCIR4/CLEC4A1, DC-SIGN/CD209, DC-SIGN+FDC-SIGNR, DC-SIGNR/CD299, DC-SIGNR/CD299, DEC-205/CD205, Dectin-1/CLEC7A, Dectin-2/CLEC6A, DLEC/CLEC4C/BDCA-2, Ficolin-1, Ficolin-2, Ficolin-3, Klre-1, KLRG2, Langerin/CD207, Layilin, LOX-1/OLR1, LSECtin/CLEC4G, MBL, MBL-1, MBL-2, MDL-1/CLEC5A, MGL1/2 (CD301a/b), MGL1/CD301a, MGL2/CD301b, MGL2/CD301b, MICL/CLEC12A, MMR/CD206, Mrc2, NKG2A/CD159a, NKG2A/NKG2B Isoform 2, NKG2C/CD159c, NKG2D/CD314, NKG2E, NKG2H, NKp80/KLRF1, OCIL/CLEC2d, OCILRP2/CLEC2i, PLA2R1, QBRICK/FREM1, Reg1, Reg1A, Reg1B, Reg2, Reg3A, Reg3B, Reg3D, Reg3G, Reg4, SCGF/CLEC11a, SFTPA1, SIGNR1/CD209b, SIGNR3/CD209d, SIGNR4/CD209e, SIGNR7/CD209g, and SP-D. In embodiments, this chimeric protein is referred to herein as CTLA4-Fc-Type II.

In embodiments, the chimeric proteins of the present invention comprise variants of the extracellular domain, which includes the ligand-binding domain, of CTLA4. As examples, the variant may 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%, 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 with the known amino acid sequence of the extracellular domain of CTLA4, e.g., human CTLA4.

In embodiments, the extracellular domain of CTLA4 has the following amino acid sequence:

(SEQ ID NO: 60)

KAMHVAQPAVVLASSRGIASFVCEYASPGKATEVR

VTVLRQADSQVTEVCAATYMMGNELTFLDDSICTG

TSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYY

LGIGNGTQIYVIDPEPCPDSD.

In embodiments, the first domain of a chimeric protein comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 60.

In embodiments, the chimeric proteins of the present invention comprise variants of the extracellular domain of a herein-described Type II transmembrane protein, i.e., selected from 4-1BBL, APRIL, BAFF, BTNL2, CD28, CD30L, CD40L, CD70, C-type lectin domain (CLEC) family members, FasL, GITRL, LIGHT, LTa, LTa1b2, NKG2A, NKG2C, NKG2D, OX40L, RANKL, TL1A, TNFa, and TRAIL. In embodiments, the CLEC family member is selected from AlCL/CLEC-2B, ASGR1/ASGPR1, ASGR2, C1q R1/CD93, CD161, CD161/NK1.1, CD23/Fc epsilon RII, CD302/CLEC13A, CD72, CD94, Chondrolectin, CLEC-1, CLEC10A/CD301, CLEC12B, CLEC14A, CLEC16A, CLEC17A, CLEC18A, CLEC18B, CLEC18C, CLEC-2/CLEC1B, CLEC-2A, CLEC3A, CLEC3B/Tetranectin, CLEC4B2/mDCAR1, CLEC4D/CLECSF8, CLEC4E, CLEC4F/CLECSF13, CLEC9a, CLECL1/DCAL-1, CL-K1/COLEC11, CL-L1/COLEC10, CL-P1/COLEC12, DCAR/CLEC4B, DCIR/CLEC4A, DCIR4/CLEC4A1, DC-SIGN/CD209, DC-SIGN+FDC-SIGNR, DC-SIGNR/CD299, DC-SIGNR/CD299, DEC-205/CD205, Dectin-1/CLEC7A, Dectin-2/CLEC6A, DLEC/CLEC4C/BDCA-2, Ficolin-1, Ficolin-2, Ficolin-3, Klre-1, KLRG2, Langerin/CD207, Layilin, LOX-1/OLR1, LSECtin/CLEC4G, MBL, MBL-1, MBL-2, MDL-1/CLEC5A, MGL1/2 (CD301a/b), MGL1/CD301a, MGL2/CD301b, MGL2/CD301b, MICL/CLEC12A, MMR/CD206, Mrc2, NKG2A/CD159a, NKG2A/NKG2B Isoform 2, NKG2C/CD159c, NKG2D/CD314, NKG2E, NKG2H, NKp80/KLRF1, OCIL/CLEC2d, OCILRP2/CLEC2i, PLA2R1, QBRICK/FREM1, Reg1, Reg1A, Reg1B, Reg2, Reg3A, Reg3B, Reg3D, Reg3G, Reg4, SCGF/CLEC11a, SFTPA1, SIGNR1/CD209b, SIGNR3/CD209d, SIGNR4/CD209e, SIGNR7/CD209g, and SP-D. The amino acid sequence of the herein-described Type II transmembrane protein are publically available, see, e.g., at the World Wide Web (www) uniprot.org and at the World Wide Web (www) ncbi.nlm.nih.gov/protein and in one or more of WO2018/157162; WO2018/157165; WO2018/157164; WO2018/157163; and WO2017/059168, the contents relevant to this embodiment are incorporated herein by reference in its entirety. Moreover, many of the herein-described Type II transmembrane proteins have been structurally characterized, e.g., by predictive algorithms and/or x-ray crystallography; again see (www) uniprot.org; the contents relevant to this embodiment are incorporated herein by reference in its entirety.

Based on the published amino acid sequences and structural characterizations, a skilled artisan could readily determine sequence variants of the herein-described Type II transmembrane protein which retain (or enhance) the native ligand/receptor binding affinity or the Type II transmembrane protein. Examples of such variants may 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%, 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 with the known amino acid sequence of the extracellular domain of a herein-described Type II transmembrane protein, e.g., the human Type II transmembrane protein.

In embodiments, a chimeric protein of the present invention comprises: (1) a first domain comprising the amino acid sequence of SEQ ID NO: 60 or a variant thereof, as described above, (b) a second domain comprises the amino acid sequence of a portion of the extracellular domain of a herein-described Type II transmembrane protein, or a variant thereof, as described above, and (c) a linker comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

In embodiments, the chimeric protein is capable of contemporaneously binding the TNFR2 ligand and a ligand/receptor of a Type II transmembrane protein selected from BTNL2C-type lectin domain (CLEC) family members, GITRL TL1A, IL-10, TGF-beta. In embodiments, the CLEC family member is selected from AlCL/CLEC-2B, ASGR1/ASGPR1, ASGR2, C1q R1/CD93, CD161, CD161/NK1.1, CD23/Fc epsilon RII, CD302/CLEC13A, CD72, CD94, Chondrolectin, CLEC-1, CLEC10A/CD301, CLEC12B, CLEC14A, CLEC16A, CLEC17A, CLEC18A, CLEC18B, CLEC18C, CLEC-2/CLEC1B, CLEC-2A, CLEC3A, CLEC3B/Tetranectin, CLEC4B2/mDCAR1, CLEC4D/CLECSF8, CLEC4E, CLEC4F/CLECSF13, CLEC9a, CLECL1/DCAL-1, CL-K1/COLEC11, CL-L1/COLEC10, CL-P1/COLEC12, DCAR/CLEC4B, DCIR/CLEC4A, DCIR4/CLEC4A1, DC-SIGN/CD209, DC-SIGN+FDC-SIGNR, DC-SIGNR/CD299, DC-SIGNR/CD299, DEC-205/CD205, Dectin-1/CLEC7A, Dectin-2/CLEC6A, DLEC/CLEC4C/BDCA-2, Ficolin-1, Ficolin-2, Ficolin-3, Klre-1, KLRG2, Langerin/CD207, Layilin, LOX-1/OLR1, LSECtin/CLEC4G, MBL, MBL-1, MBL-2, MDL-1/CLEC5A, MGL1/2 (CD301a/b), MGL1/CD301a, MGL2/CD301b, MGL2/CD301b, MICL/CLEC12A, MMR/CD206, Mrc2, NKG2A/CD159a, NKG2A/NKG2B Isoform 2, NKG2C/CD159c, NKG2D/CD314, NKG2E, NKG2H, NKp80/KLRF1, OCIL/CLEC2d, OCILRP2/CLEC2i, PLA2R1, QBRICK/FREM1, Reg1, Reg1A, Reg1B, Reg2, Reg3A, Reg3B, Reg3D, Reg3G, Reg4, SCGF/CLEC11a, SFTPA1, SIGNR1/CD209b, SIGNR3/CD209d, SIGNR4/CD209e, SIGNR7/CD209g, and SP-D. In embodiments, this chimeric protein is referred to herein as TNFR2-Fc-Type II.

In embodiments, TNFR2 is the receptor that binds tumor necrosis factor-alpha (TNFα), which is a cytokine produced by lymphocytes and macrophages, that mediates the immune response by attracting additional white blood cells to sites of inflammation and through additional molecular mechanisms that initiate and amplify inflammation. TNFR2's binding to TNFα, helps decrease excess inflammation cause by, as examples, autoimmune diseases such as ankylosing spondylitis, juvenile rheumatoid arthritis, psoriasis, psoriatic arthritis, rheumatoid arthritis, and, potentially, in a variety of other disorders mediated by excess TNFα.

In embodiments, the chimeric proteins of the present invention comprise variants of the extracellular domain, which includes the ligand-binding domain, of TNFR2. As examples, the variant may 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%, 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 with the known amino acid sequence with the known amino acid sequence of the extracellular domain of TNFR2, e.g., human TNFR2.

In embodiments, the extracellular domain of TNFR2 has the following amino acid sequence:

(SEQ ID NO: 90)

LPAQVAFTPYAPEPGSTCRLREYYDQTAQMCCSKC

SPGQHAKVFCTKTSDTVCDSCEDSTYTQLWNWVPE

CLSCGSRCSSDQVETQACTREQNRICTCRPGWYCA

LSKQEGCRLCAPLRKCRPGFGVARPGTETSDVVCK

PCAPGTFSNTTSSTDICRPHQICNVVAIPGNASMD

AVCTSTSPTRSMAPGAVHLPQPVSTRSQHTQPTPE

PSTAPSTSFLLPMGPSPPAEGSTGD.

In embodiments, a chimeric protein comprises a variant of the extracellular domain of TNFR2. As examples, the variant may 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%, 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 with SEQ ID NO: 90.

In embodiments, the first domain of a chimeric protein comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 90.

One of ordinary skill may select variants of the known amino acid sequence of TNFR2 by consulting the literature, e.g., Kohno et al., “A second tumor necrosis factor receptor gene product can shed a naturally occurring tumor necrosis factor inhibitor.” Proc. Natl. Acad. Sci. U.S.A. 87 (21), 8331-8335 (1990); Smith et al., “A receptor for tumor necrosis factor defines an unusual family of cellular and viral proteins.” Science 248 (4958), 1019-1023 (1990); Loetscher et al., “Purification and partial amino acid sequence analysis of two distinct tumor necrosis factor receptors from HL60 cells.” J. Biol. Chem. 265 (33), 20131-20138 (1990); Dembic, et al., “Two human TNF receptors have similar extracellular, but distinct intracellular, domain sequences.” Cytokine 2 (4), 231-237 (1990); Pennica et al., “Biochemical properties of the 75-kDa tumor necrosis factor receptor. Characterization of ligand binding, internalization, and receptor phosphorylation.” J. Biol. Chem. 267 (29), 21172-21178 (1992); and Park et al., “Structural basis for self-association and receptor recognition of human TRAF2.” Nature 398 (6727), 533-538 (1999), each of which is incorporated by reference in its entirety.

In embodiments, a chimeric protein of the present invention comprises: (1) a first domain comprising the amino acid sequence of SEQ ID NO: 90 or a variant thereof, as described above, (b) a second domain comprises the amino acid sequence of a portion of the extracellular domain of a herein-described Type II transmembrane protein, or a variant thereof, as described above, and (c) a linker comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

In embodiments, the chimeric protein is capable of contemporaneously binding the MadCAM receptor/ligand and the CCL25 receptor/ligand. In embodiments, the MadCAM receptor is leukocyte beta7 integrin LPAM-1 (alpha4/beta7), L-selectin, and VLA-4 (alpha4/beta1) on myeloid cells to direct leukocytes into mucosal and inflamed tissues. MadCAM is a member of the immunoglobulin superfamily and is similar to ICAM-1 and VCAM-1. CCL25, also known as TECK (Thymus-Expressed Chemokine), is a small cytokine of the CC chemokine family. It is chemotactic for thymocytes, macrophages, and dendritic cells. CCL25 elicits its effects by binding to the chemokine receptor CCR9. Accordingly, a chimeric protein comprises an extracellular domain of MadCAM, which includes its receptor-binding domain and a portion of CCL25, which is capable of contemporaneously. In embodiments, this chimeric protein is referred to herein as MadCAM-Fc-CCL25.

In embodiments, the chimeric proteins of the present invention comprise variants of a portion of an extracellular domain of MadCAM which includes its receptor-binding domain. As examples, the variant may 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%, 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 with the known amino acid sequence of the portion of an extracellular domain of MadCAM, e.g., human MadCAM, which comprises its receptor-binding domain.

In embodiments, the portion of an extracellular domain of MadCAM comprising its receptor-binding domain has the following amino acid sequence:

(SEQ ID NO: 91)

QSLQVKPLQVEPPEPVVAVALGASRQLTCRLACADR

GASVQWRGLDTSLGAVQSDTGRSVLIVRNASLSAA

GTRVCVGSCGGRTFQHTVQLLVYAFPDQLTVSPAA

LVPGDPEVACTAHKVTPVDPNALSFSLLVGGQELE

GAQALGPEVQEEEEEPQGDEDVLFRVTERWRLPPL

GTPVPPALYCQATMRLPGLELSHRQAIPVLHSPTS

PEPPDTTSPESPDTTSPESPDTTSQEPPDTTSPEP

PDKTSPEPAPQQGSTHTPRSPGSTRTRRPEISQAG

PTQGEVIPTGSSKPAGDQ.

In embodiments, a chimeric protein comprises a variant of the portion of an extracellular domain of MadCAM comprising its receptor-binding domain. As examples, the variant may 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%, 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 with SEQ ID NO: 91.

In embodiments, the first domain of a chimeric protein comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 91.

One of ordinary skill may select variants of the known amino acid sequence of an extracellular domain of MadCAM by consulting the literature, e.g., Tan et al., “The structure of immunoglobulin superfamily domains 1 and 2 of MAdCAM-1 reveals novel features important for integrin recognition.” Structure 6: 793-801 (1998); Dando et al., “A Reassessment of the Madcam-1 Structure and its Role in Integrin Recognition.” Acta Crystallogr D Biol Crystallogr 58: 233 (2002), each of which is incorporated by reference in its entirety.

In embodiments, the chimeric proteins of the present invention comprise variants of a portion of CCL25, which includes the receptor-binding domain, of CCL25. As examples, the variant may 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%, 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 with the known amino acid sequence of the extracellular domain of CCL25, e.g., human CCL25.

In embodiments, the extracellular domain of CCL25 has the following amino acid sequence:

(SEQ ID NO: 92)

MNLWLLACLVAGFLGAWAPAVHTQGVFEDCCLAYH

YPIGWAVLRRAWTYRIQEVSGSCNLPAAIFYLPKR

HRKVCGNPKSREVQRAMKLLDARNKVFAKLHHNTQ

TFQAGPHAVKKLSSGNSKLSSSKFSNPISSSKRNV

SLLISANSGL.

In embodiments, a chimeric protein comprises a variant of CCL25. As examples, the variant may 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%, 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 with SEQ ID NO: 92.

In embodiments, the second domain of a chimeric protein comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 92.

One of ordinary skill may select variants of the known amino acid sequence of CCL25 by consulting the protein structure homology-model of CCL25 which is available at SWISS-MODEL repository. Bienert et al., “The SWISS-MODEL Repository—new features and functionality.” Nucleic Acids Research, 45(D1): D313-D319 (2017), which is incorporated by reference in its entirety.

In embodiments, a chimeric protein of the present invention comprises: (1) a first domain comprising the amino acid sequence of SEQ ID NO: 91, (b) a second domain comprises the amino acid sequence of SEQ ID NO: 92, and (c) a linker comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

In embodiments, a MadCAM-Fc-CCL25 chimeric protein of the present invention has the following amino acid sequence (the extracellular domain of MadCAM is indicated by underline, and the portion of CCL25 is shown in a boldface font):

(SEQ ID NO: 93)

MEWSWVFLFFLSVITGVHSQSLQVKPLQVEPPEPVV

AVALGASRQLTCRLACADRGASVQVRGLDTSLG

AVQSDTGRSVLIVRNASLSAAGTRVCVGSCGGRTF

QHTVQLLVYAFPDQLTVSPAALVPGDPEVACTAHK

VTPVDPNALSFSLLVGGQELEGAQALGPEVQEEEE

EPQGDEDVLFRVTERWRLPPLGTPVPPALYCQATM

RLPGLELSHRQAIPVLHSPTSPEPPDTTSPESPDT

TSPESPDTTSQEPPDTTSPEPPDKTSPEPAPQQGST

HTPRSPGSTRTRRPEISQAGPTQGEVIPTGSSKPAG

DQSKYGPPCPPCPAPEFLGGPSVFLFPPKPKDQLMI

SRTPEVICVVVDVSQEDPEVQFNWYVDGVEVHNAK

TKPREEQFNSTYRVVSVLTVLHQDWLSGKEYKCK

VSSKGLPSSIEKTISKAKGQPREPQVYTLPPSQEE

MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT

TPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVLH

EALHNHYTQKSLSLSLGKIEGRMDQGVFEDCCLAY

HYPIGWAVLRRAWTYRIQEVSGSCNLPAAIFYLPK

RHRKVCGNPKSREVQRAMKLLDARNKVFAKLHHN

TQTFQAGPHAVKKLSSGNSKLSSSKFSNPISSSKR

NVSLLISANSGL.

In embodiments, a chimeric protein comprises a variant of a MadCAM-Fc-CCL25 chimeric protein. As examples, the variant may 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%, 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 with SEQ ID NO: 93.

In embodiments, the chimeric protein is capable of contemporaneously binding the IL-6R receptor/ligand and the IL-35 receptor/ligand. In embodiments, the IL-6R ligand is IL-6. IL-6 plays a pathological role in chronic inflammation and autoimmunity. In some embodiments, the IL-6R part of the chimeric protein comprises IL-6RA (which is also known as IL-6R subunit alpha) and IL-6ST (which is also known as IL-6R subunit beta). IL-6R binds IL-6 with low affinity, but does not transduce a signal. Binding of IL-6R and IL-6ST to IL-6 leads to signal activation. Accordingly, in some embodiments, the IL-6R portion of the chimeric protein comprises an extracellular domain of IL-6RA and/or an extracellular domain of IL-6ST. IL-35 is a Treg-restricted inhibitory cytokine and is capable of exhibiting its suppressive activities in a range of autoimmune diseases. Human IL-35 comprises two subunits: EBI3, which is also known as Interleukin-27 subunit beta, and IL-12A, which is also known as Interleukin-12 subunit alpha. Accordingly, in some embodiments, the IL-35 portion of the chimeric protein comprises a portion of EBI3 and/or a portion of IL-12A. Accordingly, the chimeric protein comprises an extracellular domain of IL-6R which includes its receptor-binding domain and a portion of IL-35, which is capable of contemporaneously inhibiting IL-6 and stimulating IL-35. In embodiments, this chimeric protein is referred to herein as IL-6R-Fc-IL-35.

In embodiments, the chimeric proteins of the present invention comprise variants of an extracellular domain of IL-6RA which includes its receptor-binding domain. As examples, the variant may 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%, 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 with the known amino acid sequence of the extracellular domain of IL-6RA, e.g., human IL-6RA, which comprises its receptor-binding domain.

In embodiments, the extracellular domain of IL-6RA comprising its receptor-binding domain has the following amino acid sequence (the gene is IL6R, the protein is also known as IL6RA or CD126):

(SEQ ID NO: 94)

APRRCPAQEVARGVLTSLPGDSVILTCPGVEPEDN

ATVHWVLRKPAAGSHPSRWAGMGRRLLLRSVQLHD

SGNYSCYRAGRPAGTVHLLVDVPPEEPQLSCFRKS

PLSNVVCEWGPRSTPSLITKAVLLVRKFQNSPAEDF

QEPCQYSQESQKFSCQLAVPEGDSSFYIVSMCVAS

SVGSKFSKTQTFQGCGILQPDPPANITVTAVARNPR

WLSVTWQDPHSWNSSFYRLRFELRYRAERSKTFTT

WMVKDLQHHCVIHDAWSGLRHVVQLRAQEEFGQG

EWSEWSPEAMGTPWTESRSPPAENEVSTPMQALTT

NKDDDNILFRDSANATSLPVQDSSSVPLP.

In embodiments, a chimeric protein comprises a variant of the extracellular domain of IL-6RA comprising its receptor-binding domain. As examples, the variant may 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%, 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 with SEQ ID NO: 94.

In embodiments, the first domain of a chimeric protein comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 94.

One of ordinary skill may select variants of the known amino acid sequence of IL-6RA by consulting the literature, e.g., Varghese et al., Structure of the extracellular domains of the human interleukin-6 receptor alpha-chain.” Proc Natl Acad Sci USA 99: 15959-15964 (2002); Boulanger et al., “Hexameric Structure and Assembly of the Interleukin-6/IL-6 alpha-Receptor/gp130 Complex.” Science 300: 2101-2104 (2003); Hecht et al., “The solution structure of the membrane-proximal cytokine receptor domain of the human interleukin-6 receptor.” Biol Chem 387: 1255-1259 (2006), each of which is incorporated by reference in its entirety.

In embodiments, the IL-6RA part of the chimeric protein further comprises IL-6ST, which is also known as IL-6RA subunit beta.

In embodiments, the chimeric proteins of the present invention comprise variants of an extracellular domain of IL-6ST which includes its receptor-binding domain. As examples, the variant may 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%, 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 with the known amino acid sequence of the extracellular domain of IL-6ST, e.g., human IL-6ST, which comprises its receptor-binding domain.

In embodiments, the extracellular domain of IL-6ST comprising its receptor-binding domain has the following amino acid sequence (the gene is IL6ST, the protein is also known as IL6RB, gp130 or CD130):

(SEQ ID NO: 95)

ELLDPCGYISPESPVVQLHSNFTAVCVLKEKCMDY

FHVNANYIVWKTNHFTIPKEQYTIINRTASSVTFT

DIASLNIQLTCNILTFGQLEQNVYGITIISGLPPE

KPKNLSCIVNEGKKMRCEWDGGRETHLETNFTLKS

EWATHKFADCKAKRDTPTSCTVDYSTVYFVNIEVW

VEAENALGKVTSDHINFDPVYKVKPNPPHNLSVIN

SEELSSILKLTWTNPSIKSVIILKYNIQYRTKDAS

TWSQIPPEDTASTRSSFTVQDLKPFTEYVFRIRCM

KEDGKGYWSDWSEEASGITYEDRPSKAPSFWYKID

PSHTQGYRTVQLVWKTLPPFEANGKILDYEVTLTR

WKSHLQNYTVNATKLTVNLTNDRYLATLTVRNLVG

KSDAAVLTIPACDFQATHPVMDLKAFPKDNMLWVE

WTTPRESVKKYILEWCVLSDKAPCITDWQQEDGTV

HRTYLRGNLAESKCYLITVTPVYADGPGSPESIKA

YLKQAPPSKGPTVRTKKVGKNEAVLEWDQLPVDVQ

NGFIRNYTIFYRTIIGNETAVNVDSSHTEYTLSSL

TSDTLYMVRMAAYTDEGGKDGPEFTFTTPKFAQGE

IE.

In embodiments, a chimeric protein comprises a variant of the extracellular domain of IL-6ST comprising its receptor-binding domain. As examples, the variant may 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%, 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 with SEQ ID NO: 95.

In embodiments, the first domain of a chimeric protein comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 95.

One of ordinary skill may select variants of the known amino acid sequence of IL-6ST by consulting the literature, e.g., Kernebeck et al., “The signal transducer gp130: solution structure of the carboxy-terminal domain of the cytokine receptor homology region.” Protein Sci 8: 5-12 (1999); Boulanger et al., “Hexameric Structure and Assembly of the Interleukin-6/IL-6 alpha-Receptor/gp130 Complex.” Science 300: 2101-2104 (2003); Hecht et al., “The solution structure of the membrane-proximal cytokine receptor domain of the human interleukin-6 receptor.” Biol Chem 387: 1255-1259 (2006); Bravo et al., “Crystal structure of a cytokine-binding region of gp130.” EMBO J 17: 1665-1674 (1998); Chow et al., “Structure of an extracellular gp130 cytokine receptor signaling complex.” Science 291: 2150-2150 (2001); Xu et al., “Crystal structure of the entire ectodomain of gp130: insights into the molecular assembly of the tall cytokine receptor complexes.” J Biol Chem 285: 21214-21218 (2010), each of which is incorporated by reference in its entirety.

In embodiments, the chimeric proteins of the present invention comprise variants of the extracellular domain, which includes the receptor-binding domain, of IL-35. As examples, the variant may 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%, 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 with the known amino acid sequence of the extracellular domain of IL-35, e.g., human IL-35.

Human IL-35 comprises two subunits: EBI3, which is also known as Interleukin-27 subunit beta, and IL-12A, which is also known as Interleukin-12 subunit alpha. Accordingly, in some embodiments, the IL-35 portion of the chimeric protein comprises a portion of EBI3 and/or a portion of IL-12A.

In embodiments, the portion of EBI3 has the following amino acid sequence (also known as IL27B):

(SEQ ID NO: 96)

MTPQLLLALVLWASCPPCSGRKGPPAALTLPRVQC

RASRYPIAVDCSWTLPPAPNSTSPVSFIATYRLGM

AARGHSWPCLQQTPTSTSCTITDVQLFSMAPYVLNV

TAVHPWGSSSSFVPFITEHIIKPDPPEGVRLSPLAE

RQLQVQWEPPGSWPFPEIFSLKYWIRYKRQGAARFH

RVGPIEATSFILRAVRPRARYYVQVAAQDLTDYGEL

SDWSLPATATMSLGK.

In embodiments, a chimeric protein comprises a variant of the portion of EBI3. As examples, the variant may 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%, 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 with SEQ ID NO: 96.

In embodiments, the second domain of a chimeric protein comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 96.

One of ordinary skill may select variants of the known amino acid sequence of EBI3 by consulting the protein structure homology-model of CCL25 which is available at SWISS-MODEL repository. Bienert et al., “The SWISS-MODEL Repository—new features and functionality.” Nucleic Acids Research, 45(D1): D313-D319 (2017), which is incorporated by reference in its entirety.

In embodiments, the portion of IL-12A has the following amino acid sequence:

(SEQ ID NO: 97)

MCPARSLLLVATLVLLDHLSLARNLPVATPDPGMFPCLHHSQNLLRAVSN

MLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSR

ETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPK

RQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLH

AFRIRAVTIDRVMSYLNAS.

In embodiments, a chimeric protein comprises a variant of the portion of IL-12A. As examples, the variant may 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%, 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 with SEQ ID NO: 97.

In embodiments, the second domain of a chimeric protein comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 97.

One of ordinary skill may select variants of the known amino acid sequence of IL12A by consulting the literature, e.g., Yoon et al., “Charged residues dominate a unique interlocking topography in the heterodimeric cytokine interleukin-12.” EMBO J 19: 3530-3541 (2000); Luo et al., “Structural basis for the dual recognition of IL-12 and IL-23 by ustekinumab.” J Mol Biol 402: 797-812 (2010), each of which is incorporated by reference in its entirety.

In embodiments, a chimeric protein of the present invention comprises: (1) a first domain comprising the amino acid sequence of SEQ ID NO: 94, (b) a second domain comprises the amino acid sequence of SEQ ID NO: 96, and (c) a linker comprises an amino acid sequence that is at least 96% identical to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

In embodiments, a chimeric protein of the present invention comprises: (1) a first domain comprising the amino acid sequence of SEQ ID NO: 95, (b) a second domain comprises the amino acid sequence of SEQ ID NO: 97, and (c) a linker comprises an amino acid sequence that is at least 96% identical to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

In embodiments, a chimeric protein of the present invention comprises: (1) a first domain comprising the amino acid sequence of SEQ ID NO: 95, (b) a second domain comprises the amino acid sequence of SEQ ID NO: 96, and (c) a linker comprises an amino acid sequence that is at least 96% identical to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

In embodiments, a chimeric protein of the present invention comprises: (1) a first domain comprising the amino acid sequence of SEQ ID NO: 94, (b) a second domain comprises the amino acid sequence of SEQ ID NO: 97, and (c) a linker comprises an amino acid sequence that is at least 96% identical to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

In embodiments, the IL-6R-Fc-IL-35 comprises a heterodimer of an Alpha Chain and a Beta Chain, wherein the Alpha Chain comprises: (1) a first domain comprising the amino acid sequence of SEQ ID NO: 94, (b) a second domain comprises the amino acid sequence of SEQ ID NO: 96, and (c) a linker comprises an amino acid sequence that is at least 96% identical to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3; and the Beta Chain comprises (1) a first domain comprising the amino acid sequence of SEQ ID NO: 95, (b) a second domain comprises the amino acid sequence of SEQ ID NO: 97, and (c) a linker comprises an amino acid sequence that is at least 96% identical to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

In embodiments, the IL-6R-Fc-IL-35 comprises a heterodimer of an Alpha Chain and a Beta Chain, wherein the Alpha Chain comprises: 1) a first domain comprising the amino acid sequence of SEQ ID NO: 95, (b) a second domain comprises the amino acid sequence of SEQ ID NO: 96, and (c) a linker comprises an amino acid sequence that is at least 96% identical to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3; and the Beta Chain comprises (1) a first domain comprising the amino acid sequence of SEQ ID NO: 94, (b) a second domain comprises the amino acid sequence of SEQ ID NO: 97, and (c) a linker comprises an amino acid sequence that is at least 96% identical to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

In embodiments, an IL-6R-Fc-IL-35 chimeric protein of the present invention has two chains: an Alpha Chain and a Beta Chain. In embodiments, an Alpha Chain (gp130-Fc-IL-12A or IL-6ST-Fc-IL-12A) of the IL-6R-Fc-IL-35 chimeric protein of the present invention has the following sequence (the extracellular domain of IL-6ST (gp130) is indicated by underline, and the portion of IL-12A is shown in a boldface font):

(SEQ ID NO: 98)

MEWSWVFLFFLSVITGVHSELLDPCGYISPESPWQLHSNFTAVCVLKEKC

MDYFHVNANYIVWKINHFTIPKEQYTIINRTASSVTFTDIASLNIQLTCN

ILTFGQLEQNVYGITIISGLPPEKPKNLSCIVNEGKKMRCEWDGGRETHL

ETNFTLKSEWATHKFADCKAKRDTPTSCTVDYSTVYFVNIEVWVEAENAL

GKVTSDHINFDPVYKVKPNPPHNLSVINSEELSSILKLTWTNPSIKSVII

LKYNIQYRTKDASTWSQIPPEDTASTRSSFTVQDLKPFTEYVFRIRCMKE

DGKGYWSDWSEEASGITYEDRPSKAPSFWYKIDPSHTQGYRTVQLVWKTL

PPFEANGKILDYEVTLTRWKSHLQNYTVNATKLTVNLTNDRYLATLTVRN

LVGKSDAAVLTIPACDFQATHPVMDLKAFPKDNMLWVEWTTPRESVKKYI

LEWCVLSDKAPCITDWQQEDGTVHRTYLRGNLAESKCYLITVTPVYADGP

GSPESIKAYLKQAPPSKGPTVRTKKVGKNEAVLEWDQLPVDVQNGFIRNY

TIFYRTIIGNETAVNVDSSHTEYTLSSLTSDTLYMVRMAAYTDEGGKDGP

EFTFTTPKFAQGEIEGSGSRKGGKRGSKYGPPCPPCPAPEFLGGPSVFLF

PPKPKDQLMISRTPEVICWVDVSQEDPEVQFNWYVDGVEVHNAKTKPREE

QFNSTYRWSVLTVLHQDWLSGKEYKCKVSSKGLPSSIEKTISNATGQPRE

PQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP

PVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVLHEALHNHYTQKSLSLSL

GKDEGGEDGSGSRNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLE

FYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSC

LASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNML

AVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTID

RVMSYLNAS.

In embodiments, a Beta Chain (IL-6RA-Fc-IL27B or IL-6RA-Fc-EBI3) of the IL-6R-Fc-IL-35 chimeric protein of the present invention has the following sequence (the extracellular domain of IL-6RA is indicated by underline, and the portion of IL27B (EBI3) is shown in a boldface font):

(SEQ ID NO: 99)

MEWSWVFLFFLSVTTGVHSLAPRRCPAQEVARGVLTSLPGDSVTLTCPGV

EPEDNATVHWVLRKPAAGSHPSRWAGMGRRLLLRSVQLHDSGNYSCYRAG

RPAGTVHLLVDVPPEEPQLSCFRKSPLSNWCEWGPRSTPSLTTKAVLLVR

KFQNSPAEDFQEPCQYSQESQKFSCQLAVPEGDSSFYIVSMCVASSVGSK

FSKTQTFQGCGILQPDPPANITVTAVARNPRWLSVTWQDPHSWNSSFYRL

RFELRYRAERSKTFTTWMVKDLQHHCVIHDAWSGLRHWQLRAQEEFGQGE

WSEWSPEAMGTPWTESRSPPAENEVSTPMQALTINKDDDNILFRDSANAT

SLPVQDSSSVPLPGSGSDEGGEDGSKYGPPCPPCPAPEFLGGPSVFLFPP

KPKDQLMISRTPEVICVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQ

FNSTYRWSVLTVLHQDWLSGKEYKCKVSSKGLPSSIEKTISNATGQPREP

QVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP

VLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVLHEALHNHYTQKSLSLSLG

KRKGGKRGSGSRKGPPAALTLPRVQCRASRYPIAVDCSWTLPPAPNSTSP

VSFIATYRLGMAARGHSWPCLQQTPTSTSCTITDVQLFSMAPYVLNVTAV

HPWGSSSSFVPFITEHIIKPDPPEGVRLSPLAERQLQVQWEPPGSWPFPE

IFSLKYWIRYKRQGAARFHRVGPIEATSFILRAVRPRARYYVQVAAQDLT

DYGELSDWSLPATATMSLGK.

In embodiments, a chimeric protein comprises a variant of a IL-6R-Fc-IL-35 chimeric protein. As examples, the variant may 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%, 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 with SEQ ID NO: 98 and/or SEQ ID NO: 99.

In embodiments, the chimeric protein is capable of contemporaneously binding the TNFR2 receptor/ligand and the TGFβ receptor/ligand. In embodiments, the TNFR2 ligand is TNFα. Transforming growth factor beta (TGFβ) is an inducer of the regulatory T cells, and thus, has a crucial role in maintaining immune homeostasis. Accordingly, in embodiments, the chimeric protein comprising an extracellular domain of TNFR2 which includes its receptor-binding domain and the portion of TGFβ. In embodiments, this chimeric protein is referred to herein as TNFR2-Fc-TGFβ.

In embodiments, the chimeric proteins of the present invention comprise variants of the extracellular domain, which includes the receptor-binding domain, of TNFR2. As examples, the variant may 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%, 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 with the known amino acid sequence of the extracellular domain of TNFR2, e.g., human TNFR2.

In embodiments, the extracellular domain of TNFR2 has the following amino acid sequence:

(SEQ ID NO: 100)

LPAQVAFTPYAPEPGSTCRLREYYDQTAQMCCSKCSPGQHAKVFCTKTSD

TVCDSCEDSTYTQLWNWVPECLSCGSRCSSDQVETQACTREQNRICTCRP

GWYCALSKQEGCRLCAPLRKCRPGFGVARPGTETSDVVCKPCAPGIFSNI

TSSTDICRPHQICNVVAIPGNASMDAVCTSTSPTRSMAPGAVHLPQPVST

RSQHTQPTPEPSTAPSTSFLLPMGPSPPAEGSTGD.

In embodiments, the second domain of a chimeric protein comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 100.

One of ordinary skill may select variants of the known amino acid sequence of TNFR2 by consulting the literature, e.g., Park et al., “Structural basis for self-association and receptor recognition of human TRAF2.” Nature 398: 533-538 (1999); Mukai et al. “Solution of the Structure of the TNF-TNFR2 Complex” Sci Signal 3: ra83-ra83 (2010), each of which is incorporated by reference in its entirety.

In embodiments, the chimeric proteins of the present invention comprise variants of a portion of TGFβ which includes its receptor-binding domain. As examples, the variant may 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%, 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 with the known amino acid sequence of the portion of TGFβ, e.g., human TGFβ, which comprises its receptor-binding domain.

In embodiments, the portion of TGFβ comprising its receptor-binding domain has the following amino acid sequence:

(SEQ ID NO: 101)

MPPSGLRLLLLLLPLLWLLVLTPGRPAAGLSTCKTIDMELVKRKRIEAIR

GQILSKLRLASPPSQGEVPPGPLPEAVLALYNSTRDRVAGESAEPEPEPE

ADYYAKEVTRVLMVETHNEIYDKFKQSTHSIYMFFNTSELREAVPEPVLL

SRAELRLLRLKLKVEQHVELYQKYSNNSWRYLSNRLLAPSDSPEWLSFDV

TGWRQWLSRGGEIEGFRLSAHCSCDSRDNTLQVDINGFTTGRRGDLATIH

GMNRPFLLLMATPLERAQHLQSSRHRRALDTNYCFSSTEKNCCVRQLYID

FRKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGAS

AAPCCVPQALEPLPIVYYVGRKPKVEQLSNMIVRSCKCS.

In embodiments, a chimeric protein comprises a variant of the portion of TGFβ comprising its receptor-binding domain. As examples, the variant may 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%, 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 with SEQ ID NO: 101.

In embodiments, the first domain of a chimeric protein comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 101.

One of ordinary skill may select variants of the known amino acid sequence of TGFβ by consulting the literature, e.g., Hinck et al., “Transforming growth factor beta 1: three-dimensional structure in solution and comparison with the X-ray structure of transforming growth factor beta 2.” Biochemistry 35: 8517-8534 (1996); Hinck et al., “Transforming growth factor beta 1: three-dimensional structure in solution and comparison with the X-ray structure of transforming growth factor beta 2.” Biochemistry 35: 8517-8534 (1996); Radaev et al., “Ternary complex of transforming growth factor-beta1 reveals isoform-specific ligand recognition and receptor recruitment in the superfamily.” J Biol Chem 285: 14806-14814 (2010); Zhao et al., “Prodomain-growth factor swapping in the structure of pro-TGF-beta 1.” J Biol Chem 293: 1579-1589 (2018), each of which is incorporated by reference in its entirety.

In embodiments, a chimeric protein of the present invention comprises: (1) a first domain comprising the amino acid sequence of SEQ ID NO: 100, (b) a second domain comprises the amino acid sequence of SEQ ID NO: 101, and (c) a linker comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

In embodiments, a TNFR2-Fc-TGFβ chimeric protein of the present invention has the following amino acid sequence (the extracellular domain of TNFR2 is indicated by underline, and the portion of TGFβ is shown in a boldface font):

(SEQ ID NO: 102)

MEFGLSWVFLVAIIKGVQCLPAQVAFTPYAPEPGSTCRLREYYDQTAQMC

CSKCSPGQHAKVFCTKTSDTVCDSCEDSTYTQLWNWVPECLSCGSRCSSD

QVETQACTREQNRICTCRPGWYCALSKQEGCRLCAPLRKCRPGFGVARPG

TETSDVVCKPCAPGIFSNITSSTDICRPHQICNVVAIPGNASMDAVCTST

SPTRSMAPGAVHLPQPVSTRSQHTQPTPEPSTAPSTSFLLPMGPSPPAEG

STGDSKYGPPCPPCPAPEFLGGPSVFLFPPKPKDQLMISRTPEVICVVVD

VSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRWSVLTVLHQDWLSG

KEYKCKVSSKGLPSSIEKTISNATGQPREPQVYTLPPSQEEMTKNQVSLT

CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSR

WQEGNVFSCSVLHEALHNHYTQKSLSLSLGKIEGRMDALDTNYCFSSTEK

NCCVRQLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLA

LYNQHNPGASAAPCCVPQALEPLPIVYYVGRKPKVEQLSNMIVRSCKCS.

In embodiments, a chimeric protein comprises a variant of a TNFR2-Fc-TGFβ chimeric protein. As examples, the variant may 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%, 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 with SEQ ID NO: 102.

In embodiments, the chimeric protein is capable of contemporaneously binding the integrin α4β7 receptor/ligand and the IL-35 receptor/ligand. In embodiments, the α4β7 receptor is mucosal vascular addressing cell adhesion molecule-1 (MadCAM-1) or vascular cell adhesion molecule-1 (VCAM-1). Integrin α4β7 comprises two subunits α4 and β7. Accordingly, in some embodiments, the IL-35 portion of the chimeric protein comprises an extracellular domain of α4 and/or an extracellular domain of β7. IL-35 is a Treg-restricted inhibitory cytokine and is capable of exhibiting its suppressive activities in a range of autoimmune diseases. Human IL-35 comprises two subunits: EBI3, which is also known as Interleukin-27 subunit beta, and IL-12A, which is also known as Interleukin-12 subunit alpha. Accordingly, in some embodiments, the IL-35 portion of the chimeric protein comprises a portion of EBI3 and/or a portion of IL-12A. Accordingly, the chimeric protein comprising an extracellular domain of α4β7 which includes its receptor-binding domain and a portion of IL-35. In embodiments, this chimeric protein is referred to herein as α4β7-Fc-IL-35.

In embodiments, the chimeric proteins of the present invention comprise variants of a portion of integrin α4, which includes its receptor-binding domain. As examples, the variant may 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%, 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 with the known amino acid sequence of the portion of integrin α4, e.g., human integrin α4, which comprises its receptor-binding domain.

In embodiments, the portion of integrin α4 comprising its receptor-binding domain has the following amino acid sequence:

(SEQ ID NO: 103)

NVDTESALLYQGPHNTLFGYSVVLHSHGANRWLLVGAPTANWLANASVIN

PGAIYRCRIGKNPGQICEQLQLGSPNGEPCGKTCLEERDNQWLGVTLSRQ

PGENGSIVTCGHRWKNIFYIKNENKLPTGGCYGVPPDLRTELSKRIAPCY

QDYVKKFGENFASCQAGISSFYTKDLIVMGAPGSSYWTGSLFVYNITTNK

YKAFLDKQNQVKFGSYLGYSVGAGHFRSQHTTEVVGGAPQHEQIGKAYIF

SIDEKELNILHEMKGKKLGSYFGASVCAVDLNADGFSDLLVGAPMQSTIR

EEGRVFVYINSGSGAVMNAMETNLVGSDKYAARFGESIVNLGDIDNDGFE

DVAIGAPQEDDLQGAIYIYNGRADGISSTFSQRIEGLQISKSLSMFGQSI

SGQIDADNNGYVDVAVGAFRSDSAVLLRTRPVVIVDASLSHPESVNRTKF

DCVENGWPSVCIDLTLCFSYKGKEVPGYIVLFYNMSLDVNRKAESPPRFY

FSSNGTSDVITGSIQVSSREANCRTHQAFMRKDVRDILTPIQIEAAYHLG

PHVISKRSTEEFPPLQPILQQKKEKDIMKKTINFARFCAHENCSADLQVS

AKIGFLKPHENKTYLAVGSMKTLMLNVSLFNAGDDAYETTLHVKLPVGLY

FIKILELEEKQINCEVTDNSGWQLDCSIGYIYVDHLSRIDISFLLDVSSL

SRAEEDLSITVHATCENEEEMDNLKHSRVTVAIPLKYEVKLTVHGFVNPT

SFVYGSNDENEPETCMVEKMNLTFHVINTGNSMAPNVSVEIMVPNSFSPQ

TDKLFNILDVQTTTGECHFENYQRVCALEQQKSAMQTLKGIVRFLSKTDK

RLLYCIKADPHCLNFLCNFGKMESGKEASVHIQLEGRPSILEMDETSALK

FEIRATGFPEPNPRVIELNKDENVAHVLLEGLHHQRPKRYFT.

In embodiments, a chimeric protein comprises a variant of the portion of integrin α4 comprising its receptor-binding domain. As examples, the variant may 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%, 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 with SEQ ID NO: 103.

In embodiments, the first domain of a chimeric protein comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 103.

One of ordinary skill may select variants of the known amino acid sequence of integrin α4 by consulting the literature, e.g., Yu et al., “Structural specializations of α4b7, an Integrin that Mediates Rolling Adhesion” J Cell Biol 196: 131-146 (2012), which is incorporated by reference in its entirety.

In embodiments, the chimeric proteins of the present invention comprise variants of a portion of integrin β7, which includes its receptor-binding domain. As examples, the variant may 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%, 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 with the known amino acid sequence of the portion of integrin β7, e.g., human integrin β7, which comprises its receptor-binding domain.

In embodiments, the portion of integrin β7 comprising its receptor-binding domain has the following amino acid sequence:

(SEQ ID NO: 104)

ELDAKIPSTGDATEWRNPHLSMLGSCQPAPSCQKCILSHPSCAWCKQLNF

TASGEAEARRCARREELLARGCPLEELEEPRGQQEVLQDQPLSQGARGEG

ATQLAPQRVRVTLRPGEPQQLQVRFLRAEGYPVDLYYLMDLSYSMKDDLE

RVRQLGHALLVRLQEVTHSVRIGFGSFVDKTVLPFVSTVPSKLRHPCPTR

LERCQSPFSFHHVLSLTGDAQAFEREVGRQSVSGNLDSPEGGFDAILQAA

LCQEQIGWRNVSRLLVFTSDDTFHTAGDGKLGGIFMPSDGHCHLDSNGLY

SRSTEFDYPSVGQVAQALSAANIQPIFAVTSAALPVYQELSKLIPKSAVG

ELSEDSSNWQLIMDAYNSLSSTVTLEHSSLPPGVHISYESQCEGPEKREG

KAEDRGQCNHVRINQTVTFWVSLQATHCLPEPHLLRLRALGFSEELIVEL

HTLCDCNCSDTQPQAPHCSDGQGHLQCGVCSCAPGRLGRLCECSVAELSS

PDLESGCRAPNGTGPLCSGKGHCQCGRCSCSGQSSGHLCECDDASCERHE

GILCGGFGRCQCGVCHCHANRTGRACECSGDMDSCISPEGGLCSGHGRCK

CNRCQCLDGYYGALCDQCPGCKTPCERHRDCAECGAFRTGPLATNCSTAC

AHTNVTLALAPILDDGWCKERTLDNQLFFFLVEDDARGTVVLRVRPQEKG

ADH.

In embodiments, a chimeric protein comprises a variant of the portion of integrin β7 comprising its receptor-binding domain. As examples, the variant may 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%, 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 with SEQ ID NO: 104.

In embodiments, the first domain of a chimeric protein comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 104.

One of ordinary skill may select variants of the known amino acid sequence of integrin β7 by consulting the literature, e.g., Yu et al., “Structural specializations of α4b7, an Integrin that Mediates Rolling Adhesion” J Cell Biol 196: 131-146 (2012), which is incorporated by reference in its entirety.

In embodiments, the portion of EBI3 has the following amino acid sequence (also known as IL27B):

(SEQ ID NO: 96)

MTPQLLLALVLWASCPPCSGRKGPPAALTLPRVQCRASRYPIAVDCSWTL

PPAPNSTSPVSFIATYRLGMAARGHSWPCLQQTPTSTSCTITDVQLFSMA

PYVLNVTAVHPWGSSSSFVPFITEHIIKPDPPEGVRLSPLAERQLQVQWE

PPGSWPFPEIFSLKYWIRYKRQGAARFHRVGPIEATSFILRAVRPRARYY

VQVAAQDLTDYGELSDWSLPATATMSLGK.

In embodiments, the second domain of a chimeric protein comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 96.

In embodiments, the portion of IL-12A has the following amino acid sequence:

In embodiments, the second domain of a chimeric protein comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 97.

In embodiments, a chimeric protein of the present invention comprises: (1) a first domain comprising the amino acid sequence of SEQ ID NO: 103, (b) a second domain comprises the amino acid sequence of SEQ ID NO: 96, and (c) a linker comprises an amino acid sequence that is at least 96% identical to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

In embodiments, a chimeric protein of the present invention comprises: (1) a first domain comprising the amino acid sequence of SEQ ID NO: 104, (b) a second domain comprises the amino acid sequence of SEQ ID NO: 97, and (c) a linker comprises an amino acid sequence that is at least 96% identical to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

In embodiments, a chimeric protein of the present invention comprises: (1) a first domain comprising the amino acid sequence of SEQ ID NO: 104, (b) a second domain comprises the amino acid sequence of SEQ ID NO: 96, and (c) a linker comprises an amino acid sequence that is at least 96% identical to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

In embodiments, a chimeric protein of the present invention comprises: (1) a first domain comprising the amino acid sequence of SEQ ID NO: 103, (b) a second domain comprises the amino acid sequence of SEQ ID NO: 97, and (c) a linker comprises an amino acid sequence that is at least 96% identical to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

In embodiments, the α4β7-Fc-IL-35 comprises a heterodimer of an Alpha Chain and a Beta Chain, wherein the Alpha Chain comprises: (1) a first domain comprising the amino acid sequence of SEQ ID NO: 103, (b) a second domain comprises the amino acid sequence of SEQ ID NO: 96, and (c) a linker comprises an amino acid sequence that is at least 96% identical to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3; and the Beta Chain comprises (1) a first domain comprising the amino acid sequence of SEQ ID NO: 104, (b) a second domain comprises the amino acid sequence of SEQ ID NO: 97, and (c) a linker comprises an amino acid sequence that is at least 96% identical to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

In embodiments, the α4β7-Fc-IL-35 comprises a heterodimer of an Alpha Chain and a Beta Chain, wherein the Alpha Chain comprises: 1) a first domain comprising the amino acid sequence of SEQ ID NO: 104, (b) a second domain comprises the amino acid sequence of SEQ ID NO: 96, and (c) a linker comprises an amino acid sequence that is at least 96% identical to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3; and the Beta Chain comprises (1) a first domain comprising the amino acid sequence of SEQ ID NO: 103, (b) a second domain comprises the amino acid sequence of SEQ ID NO: 97, and (c) a linker comprises an amino acid sequence that is at least 96% identical to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

In embodiments, an α4β7-Fc-IL-35 chimeric protein of the present invention has two chains: an Alpha Chain and a Beta Chain. In embodiments, an Alpha Chain (ITG4A-Fc-IL-12A or integrin α4-Fc-IL-12A) of the α4β7-Fc-IL-35 chimeric protein of the present invention has the following sequence (the extracellular domain of ITG4A (integrin α4) is indicated by underline, and the portion of IL-12A is shown in a boldface font):

(SEQ ID NO: 105)

MEFGLSWVFLVAIIKGVQCYNVDTESALLYQGPHNTLFGYSWLHSHGANR

WLLVGAPTANWLANASVINPGAIYRCRIGKNPGQTCEQLQLGSPNGEPCG

KTCLEERDNQWLGVTLSRQPGENGSIVTCGHRWKNIFYIKNENKLPTGGC

YGVPPDLRTELSKRIAPCYQDYVKKFGENFASCQAGISSFYTKDLIVMGA

PGSSYWTGSLFVYNITTNKYKAFLDKQNQVKFGSYLGYSVGAGHFRSQHT

TEWGGAPQHEQIGKAYIFSIDEKELNILHEMKGKKLGSYFGASVCAVDLN

ADGFSDLLVGAPMQSTIREEGRVFVYINSGSGAVMNAMETNLVGSDKYAA

RFGESIVNLGDIDNDGFEDVAIGAPQEDDLQGAIYIYNGRADGISSTFSQ

RIEGLQISKSLSMFGQSISGQIDADNNGYVDVAVGAFRSDSAVLLRTRGS

GSRKGGKRGSKYGPPCPPCPAPEFLGGPSVFLFPPKPKDQLMISRTPEVI

CWVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQ

DWLSGKEYKCKVSSKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKN

QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLT

VDKSRWQEGNVFSCSVLHEALHNHYTQKSLSLSLGKDEGGEDGSGSRNLP

VATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITK

DKTSTVEACLPLELTKNESCLNSRETSFIINGSCLASRKTSFMMALCLSS

IYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSET

VPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS.

In embodiments, a Beta Chain (ITGB7-Fc-IL27B or integrin β7-Fc-EBI3) of the α4β7-Fc-IL-35 chimeric protein of the present invention has the following sequence (the extracellular domain of ITG4A (integrin α4) is indicated by underline, and the portion of IL27B (EBI3) is shown in a boldface font):

(SEQ ID NO: 106)

MEFGLSWVFLVAIIKGVQCMVALPMVLVLLLVLSRGESELDAKIPSTGDA

TEWRNPHLSMLGSCQPAPSCQKCILSHPSCAWCKQLNFTASGEAEARRCA

RREELLARGCPLEELEEPRGQQEVLQDQPLSQGARGEGATQLAPQRVRVT

LRPGEPQQLQVRFLRAEGYPVDLYYLMDLSYSMKDALERVRQLGHALLVR

LQEVTHSVRIGFGSFVDKTVLPFVSTVPSKLRHPCPTRLERCQSPFSFHH

VLSLTGDAQAFEREVGRQSVSGNLDSPEGGFDAILQAALCQEQIGWRNVS

RLLVFTSDDTFHTAGDGKLGGIFMPSDGHCHLDSNGLYSRSTEFDYPSVG

QVAQALSAANIQPIFAVISAALPVYQELSKLIPKSAVGELSEDSSNWQLI

MDAYNSLSSTVTLEHSSLPPGVHISYESQCEGPEKREGKAEDRGQCNHVR

INQTVTFWVSLQATHCLPEPHLLRLRALGFSEELIVELHTLCGSGSDEGG

EDGSKYGPPCPPCPAPEFLGGPSVFLFPPKPKDQLMISRTPEVICWVDVS

QEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRWSVLTVLHQDWLSGKE

YKCKVSSKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCL

VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQ

EGNVFSCSVLHEALHNHYTQKSLSLSLGKRKGGKRGSGSRKGPPAALTLP

RVQCRASRYPIAVDCSWTLPPAPNSTSPVSFIATYRLGMAARGHSWPCLQ

QTPTSTSCTITDVQLFSMAPYVLNVTAVHPWGSSSSFVPFITEHIIKPDP

PEGVRLSPLAERQLQVQWEPPGSWPFPEIFSLKYWIRYKRQGAARFHRVG

PIEATSFILRAVRPRARYYVQVAAQDLTDYGELSDWSLPATATMSLGK.

In embodiments, a chimeric protein comprises a variant of an α4β7-Fc-IL-35 chimeric protein. As examples, the variant may 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%, 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 with SEQ ID NO: 105 and/or SEQ ID NO: 106.

In any herein-disclosed aspect and embodiment, the chimeric protein may comprise an amino acid sequence having one or more amino acid mutations relative to any of the protein sequences disclosed herein. In embodiments, the one or more amino acid mutations may be independently selected from substitutions, insertions, deletions, and truncations.

In embodiments, the amino acid mutations are amino acid substitutions, and may include conservative and/or non-conservative substitutions. “Conservative substitutions” may be made, for instance, 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. As used herein, “conservative substitutions” are defined as exchanges of an amino acid by another amino acid listed within the same group of the six standard amino acid groups shown above. For example, the exchange of Asp by Glu retains one negative charge in the so modified polypeptide. In addition, glycine and proline may be substituted for one another based on their ability to disrupt α-helices. As used herein, “non-conservative substitutions” are defined as exchanges of an amino acid by another amino acid listed in a different group of the six standard amino acid groups (1) to (6) shown above.

In embodiments, the substitutions may also include non-classical amino acids (e.g., selenocysteine, pyrrolysine, N-formylmethionine β-alanine, GABA and δ-Aminolevulinic acid, 4-aminobenzoic acid (PABA), D-isomers of the common amino acids, 2,4-diaminobutyric acid, α-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, γ-Abu, ε-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosme, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, β-alanine, fluoro-amino acids, designer amino acids such as β methyl amino acids, C α-methyl amino acids, N α-methyl amino acids, and amino acid analogs in general).

Mutations may also be made to the nucleotide sequences of the chimeric proteins by reference to the genetic code, including taking into account codon degeneracy.

In embodiments, a chimeric protein is capable of binding murine ligand(s)/receptor(s).

In embodiments, a chimeric protein is capable of binding human ligand(s)/receptor(s).

In embodiments, each extracellular domain (or variant thereof) of the chimeric protein binds to its cognate receptor or ligand with a K_Dof about 1 nM to about 5 nM, for example, about 1 nM, about 1.5 nM, about 2 nM, about 2.5 nM, about 3 nM, about 3.5 nM, about 4 nM, about 4.5 nM, or about 5 nM. In embodiments, the chimeric protein binds to a cognate receptor or ligand with a K_Dof about 5 nM to about 15 nM, for example, about 5 nM, about 5.5 nM, about 6 nM, about 6.5 nM, about 7 nM, about 7.5 nM, about 8 nM, about 8.5 nM, about 9 nM, about 9.5 nM, about 10 nM, about 10.5 nM, about 11 nM, about 11.5 nM, about 12 nM, about 12.5 nM, about 13 nM, about 13.5 nM, about 14 nM, about 14.5 nM, or about 15 nM.

In embodiments, each extracellular domain (or variant thereof) of the chimeric protein binds to its cognate receptor or ligand with a K_Dof 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 150 nM, about 130 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 (as measured, for example, by surface plasmon resonance or biolayer interferometry). In embodiments, the chimeric protein binds to human CSF1 with a K_Dof less than about 1 nM, about 900 μM, about 800 μM, about 700 μM, about 600 μM, about 500 μM, about 400 μM, about 300 μM, about 200 μM, about 100 μM, about 90 μM, about 80 μM, about 70 μM, about 60 μM about 55 μM about 50 μM about 45 μM, about 40 μM, about 35 μM, about 30 μM, about 25 μM, about 20 μM, about 15 μM, or about 10 μM, or about 1 μM (as measured, for example, by surface plasmon resonance or biolayer interferometry).

As used herein, a variant of an extracellular domain is capable of binding the receptor/ligand of a native extracellular domain. For example, a variant may include one or more mutations in an extracellular domain which do not affect its binding affinity to its receptor/ligand; alternately, the one or more mutations in an extracellular domain may improve binding affinity for the receptor/ligand; or the one or more mutations in an extracellular domain may reduce binding affinity for the receptor/ligand, yet not eliminate binding altogether. In embodiments, the one or more mutations are located outside the binding pocket where the extracellular domain interacts with its receptor/ligand. In embodiments, the one or more mutations are located inside the binding pocket where the extracellular domain interacts with its receptor/ligand, as long as the mutations do not eliminate binding altogether. Based on the skilled artisan's knowledge and the knowledge in the art regarding receptor-ligand binding, s/he would know which mutations would permit binding and which would eliminate binding.

In embodiments, the chimeric protein exhibits enhanced stability, high-avidity binding characteristics, prolonged off-rate for target binding and protein half-life relative to single-domain fusion protein or antibody controls.

A chimeric protein of the present invention may comprise more than two extracellular domains. For example, the chimeric protein may comprise three, four, five, six, seven, eight, nine, ten, or more extracellular domains. A second extracellular domain may be separated from a third extracellular domain via a linker, as disclosed herein. Alternately, a second extracellular domain may be directly linked (e.g., via a peptide bond) to a third extracellular domain. In embodiments, a chimeric protein includes extracellular domains that are directly linked and extracellular domains that are indirectly linked via a linker, as disclosed herein.

Linkers

In embodiments, the chimeric protein comprises a linker.

In embodiments, the linker comprising at least one cysteine residue capable of forming a disulfide bond. The at least one cysteine residue is capable of forming a disulfide bond between a pair (or more) of chimeric proteins. Without wishing to be bound by theory, such disulfide bond forming is responsible for maintaining a useful multimeric state of chimeric proteins. This allows for efficient production of the chimeric proteins; it allows for desired activity in vitro and in vivo.

In a chimeric protein of the present invention, the linker is a polypeptide selected from a flexible amino acid sequence, an IgG hinge region, or an antibody sequence.

In embodiments, the linker is derived from naturally-occurring multi-domain proteins or is an empirical linker as described, for example, 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 hereby incorporated by reference. In embodiments, the linker may 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 hereby incorporated by reference.

In embodiments, the linker comprises a polypeptide. In embodiments, the polypeptide is less than about 500 amino acids long, about 450 amino acids long, about 400 amino acids long, about 350 amino acids long, about 300 amino acids long, about 250 amino acids long, about 200 amino acids long, about 150 amino acids long, or about 100 amino acids long. For example, the linker may 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 long. In embodiments, the linker is not a single amino acid linker, e.g., without limitation, the linker is greater than one amino acid long. In embodiments, the linker has a length of greater than 1-6 amino acids, e.g., without limitation, the linker is greater than seven amino acids long. In embodiments, the linker comprises more than a single glycine residue.

In embodiments, the linker is flexible.

In embodiments, the linker is rigid.

In embodiments, the linker is substantially comprised 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% glycines and serines).

In embodiments, the linker comprises a hinge region of an antibody (e.g., of IgG, IgA, IgD, and IgE, inclusive of subclasses (e.g., IgG1, IgG2, IgG3, and IgG4, and IgA1, and IgA2)). The hinge region, found in IgG, IgA, IgD, and IgE class antibodies, acts as a flexible spacer, allowing the Fab portion to move freely in space. In contrast to the constant regions, the hinge domains are structurally diverse, varying in both sequence and length among immunoglobulin classes and subclasses. For example, the length and flexibility of the hinge region varies among the IgG subclasses. The hinge region of IgG1 encompasses amino acids 216-231 and, because it is freely flexible, the Fab fragments can rotate about their axes of symmetry and move within a sphere centered at the first of two inter-heavy chain disulfide bridges. IgG2 has a shorter hinge than IgG1, with 12 amino acid residues and four disulfide bridges. The hinge region of IgG2 lacks a glycine residue, is relatively short, and contains a rigid poly-proline double helix, stabilized by extra inter-heavy chain disulfide bridges. These properties restrict the flexibility of the IgG2 molecule. IgG3 differs from the other subclasses by its unique extended hinge region (about four times as long as the IgG1 hinge), containing 62 amino acids (including 21 prolines and 11 cysteines), forming an inflexible poly-proline double helix. In IgG3, the Fab fragments are relatively far away from the Fc fragment, giving the molecule a greater flexibility. The elongated hinge in IgG3 is also responsible for its higher molecular weight compared to the 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 regions reportedly decreases in the order IgG3>IgG1>IgG4>IgG2. In embodiments, the linker may be derived from human IgG4 and contain one or more mutations to enhance dimerization (including S228P) or FcRn binding.

According to crystallographic studies, the immunoglobulin hinge region can be further subdivided functionally 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 includes amino acids from the carboxyl end of CH1 to the first residue in the hinge that restricts motion, 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 segmental flexibility of the antibody. The core hinge region contains the inter-heavy chain disulfide bridges, and the lower hinge region joins the amino terminal end of the C_H2domain and includes residues in C_H2. Id. The core hinge region of wild-type human IgG1 contains the sequence CPPC (SEQ ID NO: 24) which, when dimerized by disulfide bond formation, results in a cyclic octapeptide believed to act as a pivot, thus conferring flexibility. In embodiments, the present linker comprises, one, or two, or three of the upper hinge region, the core region, and the lower hinge region of any antibody (e.g., of IgG, IgA, IgD, and IgE, inclusive of subclasses (e.g., IgG1, IgG2, IgG3, and IgG4, and IgA1 and IgA2)). The hinge region may also contain one or more glycosylation sites, which include a number of 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, considered an advantageous property for a secretory immunoglobulin. In embodiments, the linker of the present invention comprises one or more glycosylation sites.

In embodiments, the linker comprises an Fc domain of an antibody (e.g., of IgG, IgA, IgD, and IgE, inclusive of subclasses (e.g., IgG1, IgG2, IgG3, and IgG4, and IgA1 and IgA2)).

In a chimeric protein of the present invention, the linker comprises a hinge-CH2-CH3 Fc domain derived from IgG4. In embodiments, the linker comprises a hinge-CH2-CH3 Fc domain derived from a human IgG4. In embodiments, the linker comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of any one of SEQ ID NO: 1 to SEQ ID NO: 3, e.g., at least 95% identical to the amino acid sequence of SEQ ID NO: 2. In embodiments, the linker comprises one or more joining linkers, such joining linkers independently selected from SEQ ID NO: 4 to SEQ ID NO: 50 (or a variant thereof). In embodiments, the linker comprises two or more joining linkers each joining linker independently selected from SEQ ID NO: 4 to SEQ ID NO: 50 (or a variant thereof); wherein one joining linker is N terminal to the hinge-CH2-CH3 Fc domain and another joining linker is C terminal to the hinge-CH2-CH3 Fc domain.

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 for and enhanced binding to the neonatal Fc receptor (FcRn). In embodiments, the Fc domain includes one or more mutations that increases the affinity and enhances binding to FcRn. Without wishing to be bound by theory, it is believed that increased affinity and enhanced binding to FcRn increases the in vivo half-life of the present chimeric proteins.

In embodiments, the Fc domain in a linker contains one or more amino acid substitutions at amino acid residue 250, 252, 254, 256, 308, 309, 311, 416, 428, 433 or 434 (in accordance with Kabat numbering, as in as 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 equivalents thereof. In embodiments, the amino acid substitution at amino acid residue 250 is a substitution with glutamine. In embodiments, the amino acid substitution at amino acid residue 252 is a substitution with tyrosine, phenylalanine, tryptophan or threonine. In embodiments, the amino acid substitution at amino acid residue 254 is a substitution with threonine. In embodiments, the amino acid substitution at amino acid residue 256 is a substitution with serine, arginine, glutamine, glutamic acid, aspartic acid, or threonine. In embodiments, the amino acid substitution at amino acid residue 308 is a substitution with threonine. In embodiments, the amino acid substitution at amino acid residue 309 is a substitution with proline. In embodiments, the amino acid substitution at amino acid residue 311 is a substitution with serine. In embodiments, the amino acid substitution at amino acid residue 385 is a substitution with arginine, aspartic acid, serine, threonine, histidine, lysine, alanine or glycine. In embodiments, the amino acid substitution at amino acid residue 386 is a substitution with threonine, proline, aspartic acid, serine, lysine, arginine, isoleucine, or methionine. In embodiments, the amino acid substitution at amino acid residue 387 is a substitution with arginine, proline, histidine, serine, threonine, or alanine. In embodiments, the amino acid substitution at amino acid residue 389 is a substitution with proline, serine or asparagine. In embodiments, the amino acid substitution at amino acid residue 416 is a substitution with serine. In embodiments, the amino acid substitution at amino acid residue 428 is a substitution with leucine. In embodiments, the amino acid substitution at amino acid residue 433 is a substitution with arginine, serine, isoleucine, proline, or glutamine. In embodiments, the amino acid substitution at amino acid residue 434 is a substitution with histidine, phenylalanine, or tyrosine.

In embodiments, the Fc domain linker (e.g., comprising an IgG constant region) comprises one or more mutations such as substitutions at amino acid residue 252, 254, 256, 433, 434, or 436 (in accordance with Kabat numbering, as in as 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 embodiments, the IgG constant region includes a triple M252Y/S254T/T256E mutation or YTE mutation. In embodiments, the IgG constant region includes a triple H433K/N434F/Y436H mutation or KFH mutation. In embodiments, the IgG constant region includes an YTE and KFH mutation in combination.

In embodiments, the linker comprises an IgG constant region that contains one or more mutations at amino acid residues 250, 253, 307, 310, 380, 428, 433, 434, and 435 (in accordance with Kabat numbering, as in as 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). Illustrative mutations include T250Q, M428L, T307A, E380A, 1253A, H310A, M428L, H433K, N434A, N434F, N4345, and H435A. In embodiments, the IgG constant region comprises a M428L/N434S mutation or LS mutation. In embodiments, the IgG constant region comprises a T250Q/M428L mutation or QL mutation. In embodiments, the IgG constant region comprises an N434A mutation. In embodiments, the IgG constant region comprises a T307A/E380A/N434A mutation or AAA mutation. In embodiments, the IgG constant region comprises an 1253A/H310A/H435A mutation or IHH mutation. In embodiments, the IgG constant region comprises a H433K/N434F mutation. In embodiments, the IgG constant region comprises a M252Y/S254T/T256E and a H433K/N434F mutation in combination.

Additional exemplary mutations in the IgG constant region 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. Pat. No. 7,083,784, the entire contents of which are hereby incorporated by reference.

An illustrative Fc stabilizing mutant is S228P. Illustrative Fc half-life extending mutants are T250Q, M428L, V308T, L309P, and Q311S and the present linkers may comprise 1, or 2, or 3, or 4, or 5 of these mutants.

In embodiments, the chimeric protein binds to FcRn with high affinity. In embodiments, the chimeric protein may bind to FcRn with a K_Dof about 1 nM to about 80 nM. For example, the chimeric protein may bind to FcRn with a K_Dof 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. In embodiments, the chimeric protein may bind to FcRn with a K_Dof about 9 nM. In embodiments, the chimeric protein does not substantially bind to other Fc receptors (i.e. other than FcRn) with effector function.

In embodiments, the Fc domain in a linker has the amino acid sequence of SEQ ID NO: 1 (see Table 1, below), or at least 90%, or 93%, or 95%, or 97%, or 98%, or 99% identity thereto. In embodiments, mutations are made to SEQ ID NO: 1 to increase stability and/or half-life. For instance, in embodiments, the Fc domain in a linker comprises the amino acid sequence of SEQ ID NO: 2 (see Table 1, below), or at least 90%, or 93%, or 95%, or 97%, or 98%, or 99% identity thereto. For instance, in embodiments, the Fc domain in a linker comprises the amino acid sequence of SEQ ID NO: 3 (see Table 1, below), or at least 90%, or 93%, or 95%, or 97%, or 98%, or 99% identity thereto.

Further, one or more joining linkers may be employed to connect an Fc domain in a linker (e.g., one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or at least 90%, or 93%, or 95%, or 97%, or 98%, or 99% identity thereto) and the extracellular domains. For example, any one of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or variants thereof may connect an extracellular domain as disclosed herein and an Fc domain in a linker as disclosed herein. Optionally, any one of SEQ ID NO: 4 to SEQ ID NO: 50, or variants thereof are located between an extracellular domain as disclosed herein and an Fc domain as disclosed herein.

In embodiments, the present chimeric proteins may comprise variants of the joining linkers disclosed in Table 1, below. For instance, a linker may 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%, 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 with the amino acid sequence of any one of SEQ ID NO: 4 to SEQ ID NO: 50.

In embodiments, the first and second joining linkers may be different or they may be the same.

Without wishing to be bound by theory, including a linker comprising at least a part of an Fc domain in a chimeric protein, helps avoid formation of insoluble and, likely, non-functional protein concatenated oligomers and/or aggregates. This is in part due to the presence of cysteines in the Fc domain which are capable of forming disulfide bonds between chimeric proteins.

In embodiments, a chimeric protein may comprise one or more joining linkers, as disclosed herein, and lack a Fc domain linker, as disclosed herein.

In embodiments, the first and/or second joining linkers are independently selected from the amino acid sequences of SEQ ID NO: 4 to SEQ ID NO: 50 and are provided in Table 1 below:

TABLE 1

Illustrative linkers (Fc domain linkers and joining linkers)

SEQ

ID

NO.
Sequence

1
APEFLGGPSVFLFPPKPKDILMISRTPEVICWVDVSQEDPEVQFNWYVDGVEVHNAKTKPRE

EQFNSTYRWSVLTVLHQDWLSGKEYKCKVSSKGLPSSIEKTISNATGQPREPQVYTLPPSQE

EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSSWQ

EGNVFSCSVMHEALHNHYTQKSLSLSLGK

2
APEFLGGPSVFLFPPKPKDQLMISRTPEVICWVDVSQEDPEVQFNWYVDGVEVHNAKTKPREE

QFNSTYRWSVLTTPHSDWLSGKEYKCKVSSKGLPSSIEKTISNATGQPREPQVYTLPPSQEEM

TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSSWQEGN

VFSCSVLHEALHNHYTQKSLSLSLGK

3
APEFLGGPSVFLFPPKPKDQLMISRTPEVICWVDVSQEDPEVQFNWYVDGVEVHNAKTKPREE

QFNSTYRWSVLTVLHQDWLSGKEYKCKVSSKGLPSSIEKTISNATGQPREPQVYTLPPSQEEM

TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGN

VFSCSVLHEALHNHYTQKSLSLSLGK

4
SKYGPPCPSCP

5
SKYGPPCPPCP

6
SKYGPP

7
IEGRMD

8
GGGVPRDCG

9
IEGRMDGGGGAGGGG

10
GGGSGGGS

11
GGGSGGGGSGGG

12
EGKSSGSGSESKST

13
GGSG

14
GGSGGGSGGGSG

15
EAAAKEAAAKEAAAK

16
EAAAREAAAREAAAREAAAR

17
GGGGSGGGGSGGGGSAS

18
GGGGAGGGG

19
GS or GGS or LE

20
GSGSGS

21
GSGSGSGSGS

22
GGGGSAS

23
APAPAPAPAPAPAPAPAPAP

24
CPPC

25
GGGGS

26
GGGGSGGGGS

27
GGGGSGGGGSGGGGS

28
GGGGSGGGGSGGGGSGGGGS

29
GGGGSGGGGSGGGGSGGGGSGGGGS

30
GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS

31
GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS

32
GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS

33
GGSGGSGGGGSGGGGS

34
GGGGGGGG

35
GGGGGG

36
EAAAK

37
EAAAKEAAAK

38
EAAAKEAAAKEAAAK

39
AEAAAKEAAAKA

40
AEAAAKEAAAKEAAAKA

41
AEAAAKEAAAKEAAAKEAAAKA

42
AEAAAKEAAAKEAAAKEAAAKEAAAKA

43
AEAAAKEAAAKEAAAKEAAAKALEAEAAAKEAAAKEAAAKEAAAKA

44
PAPAP

45
KESGSVSSEQLAQFRSLD

46
GSAGSAAGSGEF

47
GGGSE

48
GSESG

49
GSEGS

50
GEGGSGEGSSGEGSSSEGGGSEGGGSEGGGSEGGS

In embodiments, the joining linker substantially comprises 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% glycines and serines). For example, in embodiments, the joining 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 NO: 25 to SEQ ID NO: 32, respectively). In embodiments, the joining linker sequence is GGSGGSGGGGSGGGGS (SEQ ID NO: 33). Additional illustrative joining linkers include, but are not limited to, linkers having the sequence LE, (EAAAK)_n(n=1-3) (SEQ ID NO: 36 to SEQ ID NO: 38), A(EAAAK)_nA (n=2-5) (SEQ ID NO: 39 to SEQ ID NO: 42), A(EAAAK)₄ALEA(EAAAK)₄A (SEQ ID NO: 43), PAPAP (SEQ ID NO: 44), KESGSVSSEQLAQFRSLD (SEQ ID NO: 45), GSAGSAAGSGEF (SEQ ID NO: 46), and (XP)_n, with X designating any amino acid, e.g., Ala, Lys, or Glu. In embodiments, the joining linker is GGS. In embodiments, a joining linker has the sequence (Gly)_nwhere n is any number from 1 to 100, for example: (Gly)₈(SEQ ID NO: 34) and (Gly)₆(SEQ ID NO: 35).

In embodiments, the joining linker is one or more of GGGSE (SEQ ID NO: 47), GSESG (SEQ ID NO: 48), GSEGS (SEQ ID NO: 49), GEGGSGEGSSGEGSSSEGGGSEGGGSEGGGSEGGS (SEQ ID NO: 50), and a joining linker of randomly placed G, S, and E every 4 amino acid intervals.

In embodiments, where a chimeric protein comprises a first domain, one joining linker preceding an Fc domain, a second joining linker following the Fc domain, and a second domain, the chimeric protein may comprise the following structure:

- First Domain-Joining Linker 1-Fc Domain-Joining Linker 2-Second Domain

The combination of a first joining linker, an Fc Domain linker, and a second joining linker is referend to herein as a “modular linker”. In embodiments, a chimeric protein comprises a modular linker as shown in Table 2:

TABLE 2

Illustrative modular linkers

Modular Linker =

Joining

Joining
Joining Linker 1 +

Linker 1
Fc
Linker 2
Fc + Joining Linker 2

SKYGPPCPSCP
APEFLGGPSVFLFPPKPKDTLMIS
IEGRMD
SKYGPPCPSCPAPEFLGGPSVFL

(SEQ ID NO: 4)
RTPEVICWVDVSQEDPEVQFN
(SEQ ID NO: 7)
FPPKPKDILMISRTPEVICVVVDV

WYVDGVEVHNAKTKPREEQFNS

SQEDPEVQFNWYVDGVEVHNAK

TYRWSVLTVLHQDWLSGKEYKC

TKPREEQFNSTYRWSVLTVLHQ

KVSSKGLPSSIEKTISNATGQPRE

DWLSGKEYKCKVSSKGLPSSIEK

PQVYTLPPSQEEMTKNQVSLTCL

TISNATGQPREPQVYTLPPSQEE

VKGFYPSDIAVEWESNGQPENNY

MTKNQVSLTCLVKGFYPSDIAVE

KTTPPVLDSDGSFFLYSRLTVDKS

WESNGQPENNYKTTPPVLDSDG

SWQEGNVFSCSVMHEALHNHYT

SFFLYSRLTVDKSSWQEGNVFSC

QKSLSLSLGK (SEQ ID NO: 1)

SVMHEALHNHYTQKSLSLSLGKIE

GRMD (SEQ ID NO: 51)

SKYGPPCPSCP
APEFLGGPSVFLFPPKPKDQLMIS
IEGRMD
SKYGPPCPSCPAPEFLGGPSVFL

(SEQ ID NO: 4)
RTPEVICWVDVSQEDPEVQFN
(SEQ ID NO: 7)
FPPKPKDQLMISRTPEVICVVVD

WYVDGVEVHNAKTKPREEQFNS

VSQEDPEVQFNWYVDGVEVHNA

TYRWSVLTTPHSDWLSGKEYKC

KTKPREEQFNSTYRWSVLTTPH

KVSSKGLPSSIEKTISNATGQPRE

SDWLSGKEYKCKVSSKGLPSSIE

PQVYTLPPSQEEMTKNQVSLTCL

KTISNATGQPREPQVYTLPPSQE

VKGFYPSDIAVEWESNGQPENNY

EMTKNQVSLTCLVKGFYPSDIAV

KTTPPVLDSDGSFFLYSRLTVDKS

EWESNGQPENNYKTTPPVLDSD

SWQEGNVFSCSVLHEALHNHYT

GSFFLYSRLTVDKSSWQEGNVFS

QKSLSLSLGK (SEQ ID NO: 2)

CSVLHEALHNHYTQKSLSLSLGKI

EGRMD (SEQ ID NO: 52)

SKYGPPCPSCP
APEFLGGPSVFLFPPKPKDQLMIS
IEGRMD
SKYGPPCPSCPAPEFLGGPSVFL

(SEQ ID NO: 4)
RTPEVICWVDVSQEDPEVQFN
(SEQ ID NO: 7)
FPPKPKDQLMISRTPEVICVVVD

WYVDGVEVHNAKTKPREEQFNS

VSQEDPEVQFNWYVDGVEVHNA

TYRWSVLTVLHQDWLSGKEYKC

KTKPREEQFNSTYRWSVLTVLH

KVSSKGLPSSIEKTISNATGQPRE

QDWLSGKEYKCKVSSKGLPSSIE

PQVYTLPPSQEEMTKNQVSLTCL

KTISNATGQPREPQVYTLPPSQE

VKGFYPSDIAVEWESNGQPENNY

EMTKNQVSLTCLVKGFYPSDIAV

KTTPPVLDSDGSFFLYSRLTVDKS

EWESNGQPENNYKTTPPVLDSD

RWQEGNVFSCSVLHEALHNHYT

GSFFLYSRLTVDKSRWQEGNVFS

QKSLSLSLGK (SEQ ID NO: 3)

CSVLHEALHNHYTQKSLSLSLGKI

EGRMD (SEQ ID NO: 53)

SKYGPPCPPCP
APEFLGGPSVFLFPPKPKDTLMIS
IEGRMD
SKYGPPCPPCPAPEFLGGPSVFL

(SEQ ID NO: 5)
RTPEVICWVDVSQEDPEVQFN
(SEQ ID NO: 7)
FPPKPKDILMISRTPEVICVVVDV

WYVDGVEVHNAKTKPREEQFNS

SQEDPEVQFNWYVDGVEVHNAK

TYRWSVLTVLHQDWLSGKEYKC

TKPREEQFNSTYRWSVLTVLHQ

KVSSKGLPSSIEKTISNATGQPRE

DWLSGKEYKCKVSSKGLPSSIEK

PQVYTLPPSQEEMTKNQVSLTCL

TISNATGQPREPQVYTLPPSQEE

VKGFYPSDIAVEWESNGQPENNY

MTKNQVSLTCLVKGFYPSDIAVE

KTTPPVLDSDGSFFLYSRLTVDKS

WESNGQPENNYKTTPPVLDSDG

SWQEGNVFSCSVMHEALHNHYT

SFFLYSRLTVDKSSWQEGNVFSC

QKSLSLSLGK (SEQ ID NO: 1)

SVMHEALHNHYTQKSLSLSLGKIE

GRMD (SEQ ID NO: 54)

SKYGPPCPPCP
APEFLGGPSVFLFPPKPKDQLMIS
IEGRMD
SKYGPPCPPCPAPEFLGGPSVFL

(SEQ ID NO: 7)
RTPEVICWVDVSQEDPEVQFN
(SEQ ID NO: 5)
FPPKPKDQLMISRTPEVICVVVD

WYVDGVEVHNAKTKPREEQFNS

VSQEDPEVQFNWYVDGVEVHNA

TYRWSVLTTPHSDWLSGKEYKC

KTKPREEQFNSTYRWSVLTTPH

KVSSKGLPSSIEKTISNATGQPRE

SDWLSGKEYKCKVSSKGLPSSIE

PQVYTLPPSQEEMTKNQVSLTCL

KTISNATGQPREPQVYTLPPSQE

VKGFYPSDIAVEWESNGQPENNY

EMTKNQVSLTCLVKGFYPSDIAV

KTTPPVLDSDGSFFLYSRLTVDKS

EWESNGQPENNYKTTPPVLDSD

SWQEGNVFSCSVLHEALHNHYT

GSFFLYSRLTVDKSSWQEGNVFS

QKSLSLSLGK (SEQ ID NO: 2)

CSVLHEALHNHYTQKSLSLSLGKI

EGRMD (SEQ ID NO: 55)

SKYGPPCPPCP
APEFLGGPSVFLFPPKPKDQLMIS
IEGRMD
SKYGPPCPPCPAPEFLGGPSVFL

(SEQ ID NO: 5)
RTPEVICWVDVSQEDPEVQFN
(SEQ ID NO: 7)
FPPKPKDQLMISRTPEVICVVVD

WYVDGVEVHNAKTKPREEQFNS

VSQEDPEVQFNWYVDGVEVHNA

TYRWSVLTVLHQDWLSGKEYKC

KTKPREEQFNSTYRWSVLTVLH

KVSSKGLPSSIEKTISNATGQPRE

QDWLSGKEYKCKVSSKGLPSSIE

PQVYTLPPSQEEMTKNQVSLTCL

KTISNATGQPREPQVYTLPPSQE

VKGFYPSDIAVEWESNGQPENNY

EMTKNQVSLTCLVKGFYPSDIAV

KTTPPVLDSDGSFFLYSRLTVDKS

EWESNGQPENNYKTTPPVLDSD

RWQEGNVFSCSVLHEALHNHYT

GSFFLYSRLTVDKSRWQEGNVFS

QKSLSLSLGK (SEQ ID NO: 3)

CSVLHEALHNHYTQKSLSLSLGKI

EGRMD (SEQ ID NO: 56)

In embodiments, the present chimeric proteins may comprise variants of the modular linkers disclosed in Table 2, above. For instance, a linker may 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%, 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 with the amino acid sequence of any one of SEQ ID NO: 51 to SEQ ID NO: 56.

In embodiments, the linker may be flexible, including without limitation highly flexible. In embodiments, the linker may be rigid, including without limitation a rigid alpha helix. Characteristics of illustrative joining linkers is shown below in Table 3:

TABLE 3

Characteristics of illustrative joining linkers

Joining Linker Sequence
Characteristics

SKYGPPCPPCP (SEQ ID NO: 5)
IgG4 Hinge Region

IEGRMD (SEQ ID NO: 7)
Linker

GGGVPRDCG (SEQ ID NO: 8)
Flexible

GGGSGGGS (SEQ ID NO: 10)
Flexible

GGGSGGGGSGGG (SEQ ID NO: 11)
Flexible

EGKSSGSGSESKST (SEQ ID NO: 12)
Flexible +

soluble

GGSG (SEQ ID NO: 13)
Flexible

GGSGGGSGGGSG (SEQ ID NO: 14)
Flexible

EAAAKEAAAKEAAAK (SEQ ID NO: 15)
Rigid Alpha Helix

EAAAREAAAREAAAREAAAR
Rigid Alpha Helix

(SEQ ID NO: 16)

GGGGSGGGGSGGGGSAS
Flexible

(SEQ ID NO: 17)

GGGGAGGGG (SEQ ID NO: 18)
Flexible

GS (SEQ ID NO: 19)
Highly flexible

GSGSGS (SEQ ID NO: 20)
Highly flexible

GSGSGSGSGS (SEQ ID NO: 21)
Highly flexible

GGGGSAS (SEQ ID NO: 22)
Flexible

APAPAPAPAPAPAPAPAPAP
Rigid

(SEQ ID NO: 23)

In embodiments, the linker may be functional. For example, without limitation, the linker may function to improve the folding and/or stability, improve the expression, improve the pharmacokinetics, and/or improve the bioactivity of the present chimeric protein. In another example, the linker may function to target the chimeric protein to a particular cell type or location.

In embodiments, a chimeric protein comprises only one joining linkers.

In embodiments, a chimeric protein lacks joining linkers.

In embodiments, the linker is a synthetic linker such as polyethylene glycol (PEG).

In embodiments, a chimeric protein has a first domain which is sterically capable of binding its ligand/receptor and/or the second domain which is sterically capable of binding its ligand/receptor. Thus, there is enough overall flexibility in the chimeric protein and/or physical distance between an extracellular domain (or portion thereof) and the rest of the chimeric protein such that the ligand/receptor binding domain of the extracellular domain is not sterically hindered from binding its ligand/receptor. This flexibility and/or physical distance (which is referred to as “slack”) may be normally present in the extracellular domain(s), normally present in the linker, and/or normally present in the chimeric protein (as a whole). Alternately, or additionally, an amino acid sequence (for example) may be added to one or more extracellular domains and/or to the linker to provide the slack needed to avoid steric hindrance. Any amino acid sequence that provides slack may be added. In embodiments, the added amino acid sequence comprises the sequence (Gly)_nwhere n is any number from 1 to 100. Additional examples of addable amino acid sequence include the joining linkers described in Table 1 and Table 3. In embodiments, a polyethylene glycol (PEG) linker may be added between an extracellular domain and a linker to provide the slack needed to avoid steric hindrance. Such PEG linkers are well known in the art.

Pharmaceutical Composition

Aspects of the present invention include a pharmaceutical composition comprising a therapeutically effective amount of the chimeric protein of any of the herein disclosed aspects or embodiments.

In embodiments, the chimeric protein in the pharmaceutical composition has a general structure of: N terminus-(a)-(b)-(c)-C terminus in which (a) is a first domain comprising a portion of the extracellular domain of a transmembrane protein, a secreted protein, or a membrane-anchored extracellular protein, (c) is a second domain comprising a portion of the extracellular domain of a transmembrane protein, a secreted protein, or a membrane-anchored extracellular protein, and (b) is a linker adjoining the first domain and the second domain. In this aspect, either or both of the first domain and the second domain decreases self-directed immune system activity when bound to its ligand/receptor.