CHIMERIC PROTEINS COMPRISING EXTRACELLULAR DOMAINS AND USES THEREOF

PRIORITY

This application claims the benefit of, and priority to, U.S. Provisional Application No. 62/724,593, filed August 29, 2018, and .U.S. Provisional Application No. 62/724,595, filed August 29, 2018; the contents of each of which is herein incorporated by reference in its entirety.

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 immunotherapies for cancer and inflammation.

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-019PCSequenceListing_ST25”. The sequence listing is 113,111 bytes in size, and was created on August 28, 2019. The sequence listing is hereby incorporated by reference in its entirety.

BACKGROUND

The immune system is central to the body's response to foreign entities that can cause disease and to the body's response to cancer cells. However, many cancers have developed mechanisms to avoid the immune system by, for instance, delivering or propagating immune inhibitory signals. Thus, there remains a need to develop therapeutics that, at least, reverse immune inhibitory signals and/or directly stimulate and/or activate a patient's anti-cancer immune response.

SUMMARY

Accordingly, in various aspects, the present invention provides for chimeric proteins, compositions, and methods that are useful in cancer immunotherapy. For instance, the present invention, in part, relates to specific chimeric proteins that provide immune activating or co-stimulatory signals and/or specific chimeric proteins that reverse or suppress immune inhibitory signals. Importantly, the present invention provides for improved chimeric proteins that can maintain a stable and reproducible multimeric state. Accordingly, the present compositions and methods overcome various deficiencies in producing bi-specific agents.

The present invention relates to chimeric proteins comprising extracellular domains of transmembrane proteins, e.g., single-pass transmembrane proteins. Some transmembrane proteins have their extracellular domain located at its amino terminus and others transmembrane proteins have their extracellular domain located at its carboxy terminus. Some transmembrane proteins act as receptors and other transmembrane proteins act as ligands. The extracellular domain of a transmembrane protein relevant to the present invention contains functional domains that are responsible for interacting with other binding partners (either ligands or receptors) in the extracellular environment (see, e.g., FIG. 1A).

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 an extracellular domain of a first transmembrane protein, (b) is a linker domain adjoining the first and second domains, and (c) is a second domain comprising an extracellular domain of a second transmembrane protein. In this aspect, each transmembrane protein in its native state comprises an extracellular domain having a proximal region and a distal region, wherein the proximal region is directly or indirectly attached to its transmembrane domain, and the linker domain adjoins the first domain and the second domain via the proximal region of the extracellular domain for the first transmembrane protein or via the proximal region of the extracellular domain for the second transmembrane protein but not via the proximal regions for both transmembrane proteins. See, by way of non-limiting examples, FIG. 1B and FIG. 1C.

In embodiments, the first transmembrane protein and the second transmembrane protein are independently selected from the group consisting of: 2B4, 4-1 BB, ACVRI b, ACVR2A, ACVR2B, AXL, BCMA, BTLA, BTNL2, CD2, CD27, CD30, CD31 , CD31 , CD40, CD48(SLAMF2), CD58(LFA3), CD137, CD160, CD200, CD226(PTAUDNAMI), CD244, CD247, CSF 1 R(CD 115), CTLA-4, DcR3, Fas, FGFR3, Fn14, GITR, HVEM, ICOS, ICOSL, KIR2DL1 , KIR2DL2, KIR2DL3, KIR3DL1 , KIRDL2, LAG3, LAP, LAYN, LTbR, NKG2A, NKp46(NCR1 ), NKp80(KLRF1 ), NTB-A, 0X40, PD- 1 , PD-L1 , PD-L2, PVR, RANK, SIGLEC7, SIGLEC9, SIRPo(CD172a), SLAMF6, TACI, TGFBR2, TIGIT, TI M-3, TMIGD2, TNFR1 , TNFR2, TNFRSF25, TNFRSF4, TRAIL-R, VISTA, and VSIG8.

In embodiments, the first transmembrane protein is PD-1 and the second transmembrane protein is TGFBR2.

In embodiments, the first transmembrane protein is PD-1 and the second transmembrane protein is LAG3.

In embodiments, the first transmembrane protein and the second transmembrane protein are independently selected from the group consisting of: 4-1 BBL, APRIL, BAFF, BTNL2, CD28, CD30L, CD40L, CD70, C-type lectin domain (CLEC) family members, FasL, GITRL, LIGHT, LTa, LTa1 b2, NKG2A, NKG2C, NKG2D, OX40L, RANKL, TL1A, TNFa, and TRAIL.

Another aspect of the present invention is a chimeric protein comprising: (a) a first domain comprising a portion of PD- 1 that is capable of binding PD-L1 or PD-L2, (b) a second domain comprising a portion of TGFBR2 that is capable of binding TGF , 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 sequence that is at least 95% identical to SEQ ID NO: 62 and capable of binding PD-L1 or PD-L2, (b) a second domain comprising a sequence that is at least 95% identical to SEQ ID NO: 66 and capable of binding TGF , 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 the amino acid sequence of SEQ ID NO: 62; (b) a second domain comprising the amino acid sequence of SEQ ID NO: 66; and (c) a linker linking the first domain and the amino acid sequence of SEQ ID NO: 2.

In any of the herein-disclosed aspects or embodiments, the chimeric protein may be a recombinant fusion protein.

An 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.

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 shows a schematic illustration of single-pass transmembrane (TM) proteins useful in the present invention. FIG. 1B and FIG. 1C show chimeric proteins of the present invention engineered from the extracellular domain (ECD) of two transmembrane proteins that are adjoined via a linker domain. The extracellular domains may include the entire amino acid sequence of candidate transmembrane protein which is typically localized outside the cell membrane, or any portion thereof which retains binding to the intended receptor or ligand. Moreover, the chimeric protein comprises sufficient overall flexibility and/or physical distance between domains such that a first extracellular domain is sterically capable of binding its receptor/ligand and/or a second extracellular domain is sterically capable of binding its receptor/ligand. FIG. 1D shows a chimeric protein having the proximal regions for both of its ECDs attached to the same linker.

FIG. 2 shows immune inhibitory and immune stimulatory signaling that is relevant to the present invention (from Mahoney, Nature Reviews Drug Discovery 2015: 14;561-585).

FIG. 3A and FIG. 3B show Western blots of, respectively, the murine PD-1 -Fc-TGFBR2 and the human PD-1-Fc- TGFBR2 that were probed with anti-PD-1 , anti-Fc, and anti-TGFBR2 antibodies, under non-reducing and reducing conditions, and ± the deglycosylase PNGase F. FIG. 3C provides ELISA data showing the murine PD-1 -Fc-TGFBR2 chimeric protein's ability to bind recombinant mPD-L1 and recombinant mTGFBeta and be bound by an anti-mouse- IgG antibody. FIG. 3D provides ELISA data showing the human PD-1 -Fc-TGFBR2 chimeric protein's ability to bind recombinant hPD-L1 and recombinant hTGFBeta and be bound by an anti-human-lgG antibody. FIG. 3E shows competitive ELISA data demonstrating that the hPD-1 -Fc-TGFBR2 chimeric protein can outcompete recombinant human TGFBR2 (rhTGFBR2) protein for binding to recombinant TGFBeta.

FIG. 4A and FIG. 4B demonstrate the hPD-1 -Fc-TGFBR2 chimeric protein's ability to block apoptosis in the human lymphoma cell line RAMOS when exposed to TGFBeta. FIG. 5A shows Western blots of the human PD-1 -Fc-LAG3 that was probed with anti-PD-1 , anti-Fc, and anti-LAG3 antibodies, under non-reducing and reducing conditions, and ± the deglycosylase PNGase F.

FIG. 5B provides ELISA data showing the human PD-1-Fc-LAG3 chimeric protein's ability to bind recombinant hPD- L1 and recombinant hLAG3 and be bound by an anti-human-lgG antibody.

FIG. 6 shows Western blots of the murine LAG3-1 -Fc-TIM-3 that was probed with anti-LAG3, anti-Fc, and anti-TIM-3 antibodies, under non-reducing and reducing conditions, and ± the deglycosylase PNGase F.

FIG. 7A and FIG. 7B show evidence that an illustrative chimeric protein (LAG3-Fc-TI M-3) is effective in killing cancer cells in vivo.

FIG. 8A and FIG. 8B show evidence that an illustrative chimeric protein (AXL-Fc-TIGIT) is effective in killing cancer cells in vivo and in promoting survival in treated subjects.

DETAILED DESCRIPTION

The present invention is based, in part, on the discovery that chimeric proteins can be engineered from the extracellular, or effector, regions of two immune-modulating transmembrane proteins. Binding of a chimeric protein's first domain to the ligand/receptor of the first transmembrane protein can modulate (e.g., activate or inhibit) an immunosuppressive signal or modulate an immune stimulatory signal and binding of a chimeric protein's second domain to the ligand/receptor of the second transmembrane protein can modulate an immunosuppressive signal or modulate an immune stimulatory signal. Thus, the chimeric proteins of the present invention find use, at least, in the treatment of a disease, such as cancer and inflammation by reversing or suppressing immune inhibitory signals and/or by providing immune activating or co-stimulatory signals.

Features of the present invention are illustrated in FIG. 1A to FIG. 1C.

FIG. 1 A shows two generic, single-pass transmembrane (TM) proteins, identified as a "first TM protein” and a "second TM protein”. Each of the illustrated TM proteins, in its native state, comprises three domains: a cytoplasmic domain which contacts the cell's cytosol, a transmembrane domain which spans the cell's cell membrane, and an extracellular domain (ECD) which resides outside the cell, e.g., into the extracellular compartment.

Each ECD has a distal region and a proximal region. These two regions are not defined by any amino acid sequence, motif, or domain function. Instead, they are used herein to delineate a region of the ECD's amino acid sequence that is naturally distant, along the protein's primary structure, from the protein's TM domain (/.a, the distal region of the ECD) and a region of the ECD's amino acid sequence that is naturally near the protein's TM domain (/.a, the proximal region of the ECD). The distal region of the first TM protein's ECD and the proximal region of its ECD are shown in FIG. 1A. Although not identified, the second TM protein has domains and regions as annotated in the first TM protein. In FIG. 1 A, the solid black or checkerboard semi-circle of each ECD represents a portion of each ECD that is capable of binding its ligand/receptor ("ligand/receptor binding portion”). In FIG. 1A, the black line between the ligand/receptor binding portion and the TM domain represents a portion of the proximal region of ECD that is distinct from the ligand/receptor binding portion. This distinct portion is not directly responsible for ligand/receptor binding. Thus, an ECD may retain the ability to bind its ligand/receptor when a distinct portion is omitted; this allows design of a chimeric protein which lacks the entire native ECD yet has the ability to bind a ligand/receptor.

Although not shown, other TM proteins may have a distinct portion that is located distal to the ECD's ligand/receptor binding portion (relative to TM domain). In such TM proteins, with respect to FIG. 1A, there would be a black line extending beyond the solid black or checkerboard semi-circle. Other TM proteins have a first distinct portion that is located distal to the ECD's ligand/receptor binding portion and a second distinct portion that is located proximal to the ECD's ligand/receptor binding portion; in such TM proteins, the ligand/receptor binding portion is centrally located within the ECD.

In certain TM proteins, the entire ECD may be considered a ligand/receptor binding portion; thus, the ECD lacks a distinct portion. For such TM proteins, a chimeric protein of the present invention would include the entire native ECD such that the chimeric protein is able to bind its ligand/receptor.

As shown in FIG. 1A, the TM proteins in their native states, each comprise an ECD having a proximal region and a distal region, with the proximal region being directly or indirectly attached to its TM domain. In the chimeric proteins of the present invention, the linker domain adjoins the first domain and the second domain via the proximal region of the ECD for the first TM protein or via the proximal region of the ECD for the second TM protein. Notably, the linker domain does not adjoin the first and second domains via the proximal regions for both TM proteins. Features of this aspect are illustrated in FIG. 1B and FIG. 1C.

Each of the illustrative chimeric proteins of FIG. 1B and FIG. 1C comprise a first domain comprising an ECD of a first TM protein, a linker domain adjoining the first and second domains, and a second domain comprising an ECD of a second TM protein. In FIG. 1B, the first domain comprises an ECD for a first TM protein, which is shown in solid black and is lacking an ECD distinct portion; the proximal region of the first TM protein's ECD is attached to the linker domain (diagonal hatched line). In FIG. 1C the second domain comprises an ECD for a second TM protein, which is shown in checkerboard and includes an ECD distinct portion (black line); the proximal region of the second TM protein's ECD is attached to the linker domain (diagonal hatched line). In the embodiments respectively shown in FIG. 1B and in FIG. 1C, only the proximal region of the ECD for the first TM protein is attached to the linker domain and only the proximal region of the ECD for the second TM protein is attached to the linker domain. In other words, the proximal regions for both ECDs are not attached to the same linker domain. Chimeric proteins having the proximal regions for both ECDs attached to the same linker would appear as shown in FIG. 1D. In the embodiment shown in FIG. 1 B, the amino to carboxy order of a chimeric protein includes the distal region of the ECD for the first TM protein and then the proximal region of the ECD for the first TM protein - which together form a first domain, the linker domain, the distal region of the ECD for the second TM protein and then the proximal region of the ECD for the second TM protein (including a distinct portion) -- which together form a second domain. Here, the carboxy terminus of the proximal region of the ECD for the first TM protein is attached to the linker. In the alternate embodiment shown in FIG. 1C, the amino to carboxy order of a chimeric protein includes the proximal region of the ECD for the first TM protein and then the distal region of the ECD for the first TM protein -- which together form a first domain, the linker domain, the proximal region of the ECD for the second TM protein (including a distinct portion) and then the distal region of the ECD for the second TM protein -- which together form a second domain. Here, the amino terminus of the proximal region of the ECD for the second TM protein is attached to the linker.

In the two illustrative TM proteins shown in FIG. 1 A, the ligand/receptor binding portions are located at the extracellular free end of the proteins. In FIG. 1B, with respect to the chimeric protein, only the first domain's ligand/receptor binding domain is at a free end and the second domain's ligand/receptor binding domain is adjacent to a linker and not at a free end. In the chimeric protein of FIG. 1C, only the second domain's ligand/receptor binding domain is at a free end and the first domain's ligand/receptor binding domain is adjacent to a linker and not at a free end. Accordingly, in the chimeric proteins of the present invention, at least one of the ligand/receptor binding domains is not at a free end; this is in contrast to the two illustrative TM proteins shown in FIG. 1A and in FIG. 1D, in which each ligand/receptor binding domain is at a free end.

Chimeric Proteins

Chimeric proteins of the present invention comprise the extracellular, or effector, regions of two immune-modulating transmembrane proteins. In embodiments, the chimeric proteins reverse or suppress immune inhibitory signals and/or provide immune activating or co-stimulatory signals, at least in the treatment of cancer.

A potentially fruitful method for treating cancer involves combinations of agents with synergistic and/or complementary mechanisms of action. A currently used regimen combines OPDIVO (nivolumab; an anti-PD-1 antibody) and YERVOY (ipilimumab; an anti-CTLA-4 antibody). Although this regimen has proven to be effective in treating certain cancers, it suffers from high toxicity to the patient and a huge cost, e.g., about US$ 257,000 for a course of treatment. Indeed, some of the greatest issues/challenges expected from similar combination therapies is huge costs (which may not be covered and/or reimbursed by third party health payers), the requirement for large clinical trials due to the need to assess safety and dosing for each agent individually and for the combined agents; and possibly higher toxicity profiles from the combinations.

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.

Additionally, since a chimeric protein has two immune-modulating domains, it can operate via two distinct immune pathways and, preferably, act on two separate cell types; thus, this dual-action is more likely to provide any/enhanced anti-tumor effect in a patient. Moreover, since the methods 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.

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 an extracellular domain of a first transmembrane protein, (b) is a linker domain adjoining the first and second domains, and (c) is a second domain comprising an extracellular domain of a second transmembrane protein. In this aspect, each transmembrane protein in its native state comprises an extracellular domain having a proximal region and a distal region, wherein the proximal region is directly or indirectly attached to its transmembrane domain, and the linker domain adjoins the first domain and the second domain via the proximal region of the extracellular domain for the first transmembrane protein or via the proximal region of the extracellular domain for the second transmembrane protein but not via the proximal regions for both transmembrane proteins.

An extracellular domain refers to a portion of a transmembrane protein which is capable of interacting with the extracellular environment; it typically is sufficient for binding to a ligand or receptor and is effective in transmitting a signal to a cell. An extracellular domain may be the entire amino acid sequence of a transmembrane protein which is normally present at the exterior of a cell or of the cell membrane. An extracellular domain may be that portion of an amino acid sequence of a transmembrane protein which is external of a cell or of the cell membrane and is needed for signal transduction and/or ligand binding as may be assayed using methods know in the art (e.g., in vitro ligand binding and/or cellular activation assays).

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 (/. e., a membrane of an adjacent cell). Without wishing to be bound by theory, the transmembrane 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).

In embodiments, the first transmembrane protein and the second transmembrane protein are different proteins.

In embodiments, the first domain comprises substantially the entire extracellular domain of the first transmembrane protein and/or the second domain comprises substantially the entire extracellular domain of the second transmembrane protein. In embodiments, the first domain comprises substantially the entire extracellular domain of the first transmembrane protein and the second domain comprises substantially the entire extracellular domain of the second transmembrane protein.

In embodiments, the first domain is capable of binding a ligand/receptor of the first transmembrane protein and/or the second domain is capable of binding a ligand/receptor of the second transmembrane protein. In embodiments, the first domain is capable of binding a ligand/receptor of the first transmembrane protein and the second domain is capable of binding a ligand/receptor of the second transmembrane protein.

In embodiments, the first domain is capable of binding the ligand/receptor of the first transmembrane protein similarly to the native domain's ability to bind the ligand/receptor and/or the second domain is capable of binding the ligand/receptor of the second transmembrane protein similarly to the native domain's ability to bind the ligand/receptor.

In embodiments, the binding of the first domain to the ligand/receptor of the first transmembrane protein activates an immunosuppressive signal and/or binding the second domain to the ligand/receptor of the second transmembrane protein activates an immunosuppressive signal. In embodiments, the binding of the first domain to the ligand/receptor of the first transmembrane protein activates an immunosuppressive signal and/or binding the second domain to the ligand/receptor of the second transmembrane protein inhibits an immunosuppressive signal. In embodiments, the binding of the first domain to the ligand/receptor of the first transmembrane protein inhibits an immunosuppressive signal and/or binding the second domain to the ligand/receptor of the second transmembrane protein activates an immunosuppressive signal. In embodiments, the binding of the first domain to the ligand/receptor of the first transmembrane protein inhibits an immunosuppressive signal and/or binding the second domain to the ligand/receptor of the second transmembrane protein inhibits an immunosuppressive signal.

In embodiments, the binding of the first domain to the ligand/receptor of the first transmembrane protein activates an immune stimulatory signal and/or binding the second domain to the ligand/receptor of the second transmembrane protein activates an immune stimulatory signal. In embodiments, the binding of the first domain to the ligand/receptor of the first transmembrane protein activates an immune stimulatory signal and/or binding the second domain to the ligand/receptor of the second transmembrane protein inhibits an immune stimulatory signal. In embodiments, the binding of the first domain to the ligand/receptor of the first transmembrane protein inhibits an immune stimulatory signal and/or binding the second domain to the ligand/receptor of the second transmembrane protein activates an immune stimulatory signal. In embodiments, the binding of the first domain to the ligand/receptor of the first transmembrane protein inhibits an immune stimulatory signal and/or binding the second domain to the ligand/receptor of the second transmembrane protein inhibits an immune stimulatory signal.

In embodiments, the binding of the first domain to the ligand/receptor of the first transmembrane protein activates an immunosuppressive signal and binding the second domain to the ligand/receptor of the second transmembrane protein activates an immune stimulatory signal. In embodiments, the binding of the first domain to the ligand/receptor of the first transmembrane protein activates an immunosuppressive signal and binding the second domain to the ligand/receptor of the second transmembrane protein inhibits an immune stimulatory signal. In embodiments, the binding of the first domain to the ligand/receptor of the first transmembrane protein inhibits an immunosuppressive signal and binding the second domain to the ligand/receptor of the second transmembrane protein activates an immune stimulatory signal. In embodiments, the binding of the first domain to the ligand/receptor of the first transmembrane protein inhibits an immunosuppressive signal and binding the second domain to the ligand/receptor of the second transmembrane protein inhibits an immune stimulatory signal.

In embodiments, the binding of the first domain to the ligand/receptor of the first transmembrane protein activates an immune stimulatory signal and binding the second domain to the ligand/receptor of the second transmembrane protein activates an immunosuppressive signal. In embodiments, the binding of the first domain to the ligand/receptor of the first transmembrane protein activates an immune stimulatory signal and binding the second domain to the ligand/receptor of the second transmembrane protein inhibits an immunosuppressive signal. In embodiments, the binding of the first domain to the ligand/receptor of the first transmembrane protein inhibits an immune stimulatory signal and binding the second domain to the ligand/receptor of the second transmembrane protein activates an immunosuppressive signal. In embodiments, the binding of the first domain to the ligand/receptor of the first transmembrane protein inhibits an immune stimulatory signal and binding the second domain to the ligand/receptor of the second transmembrane protein inhibits an immunosuppressive signal.

In embodiments, the chimeric protein is capable of forming a stable synapse between cells. In embodiments, the stable synapse between cells provides spatial orientation that favors tumor reduction and/or reduces inflammation. In embodiments, the spatial orientation positions T cells to attack tumor cells and/or sterically prevents a tumor cell from delivering negative signals, including negative signals beyond those masked by the chimeric protein of the invention.

In embodiments, the chimeric protein is capable of forming a stable synapse between cells. In embodiments, the stable synapse between cells provides spatial orientation that favors tumor reduction.

In embodiments, the binding of the first domain to the ligand/receptor of the first transmembrane protein and/or binding the second domain to the ligand/receptor of the second transmembrane protein occurs with slow off rates (K _0ff ), which provides a long interaction of a receptor and its ligand. In embodiments, the long interaction delivers a longer positive signal effect and/or provides a sustained negative signal masking effect, e.g., which provides for immune cell proliferation and allows for anti-tumor attack and/or allows sufficient signal transmission to provide release of stimulatory signals, e.g., a cytokine.

In embodiments, the chimeric protein is capable of increasing or preventing a decrease in a sub-population of CD4+ and/or CD8+ T cells.

In embodiments, the chimeric protein is capable of directly and/or indirectly enhancing tumor-killing activity by T cells.

In embodiments, the chimeric protein is capable of directly and/or indirectly causing activation of antigen presenting cells.

In embodiments, the chimeric protein is capable directly and/or indirectly enhancing the ability of antigen presenting cells to present antigen.

In embodiments, the chimeric protein is capable of providing a sustained immunomodulatory effect.

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

In embodiments, the linker domain comprises at least one cysteine residue capable of forming a disulfide bond and/or comprises a hinge-CH2-CH3 Fc domain. In embodiments, the hinge-CH2-CH3 Fc domain is derived from IgG (e.g., lgG1 , lgG2, lgG3, and lgG4), IgA (e.g., lgA1 and lgA2), IgD, or IgE. In embodiments, the lgG4 is a human lgG4. In embodiments, the linker domain 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 first transmembrane protein and the second transmembrane protein are independently selected from the group consisting of: PD-1 , TGFBR2, LAG3, 2B4, 4-1 BB, ACVRI b, ACVR2A, ACVR2B, AXL, BCMA, BTLA, BTNL2, CD2, CD27, CD30, CD31 , CD31 , CD40, CD48(SLAMF2), CD58(LFA3), CD137, CD160, CD200, CD226(PTAUDNAMI), CD244, CD247, CSF1 R(CD 115), CTLA-4, DcR3, Fas, FGFR3, Fn14, GITR, HVEM, ICOS, ICOSL, KIR2DL1 , KIR2DL2, KIR2DL3, KIR3DL1 , KIRDL2, LAP, LAYN, LTbR, NKG2A, NKp46(NCR1 ), NKp80(KLRF1 ), NTB-A, 0X40, PD-L1 , PD-L2, PVR, RANK, SIGLEC7, SIGLEC9, SIRPo(CD172a), SLAMF6, TACI, TIGIT, TIM-3, TMIGD2, TNFR1 , TNFR2, TNFRSF25, TNFRSF4, TRAIL-R, VISTA, and VSIG8.

In embodiments, the first transmembrane protein or the second transmembrane protein is PD-1. In embodiments, the first transmembrane protein is PD-1 and the second transmembrane protein is selected from TGFBR2, LAG3, AXL, CTLA-4, ICOSL, LAP, SIGLEC7, SIGLEC9, SIRPo(CD 172a), TIGIT, and TI M-3. In embodiments, the first transmembrane protein is selected from AXL, CTLA-4, ICOSL, LAG3, LAP, SIGLEC7, SIGLEC9, SIRPa(CD172a), TGFBR2, TIGIT, and TI M-3 and the second transmembrane protein is PD-1.

In embodiments, the first transmembrane protein is PD-1 and the second transmembrane protein is TGFBR2. In embodiments, the first transmembrane protein is PD-1 and the second transmembrane protein is LAG3.

In embodiments, the first transmembrane protein or the second transmembrane protein is TIM-3. In embodiments, the first transmembrane protein is TIM-3 and the second transmembrane protein is selected from AXL, CTLA-4, ICOSL, LAG3, LAP, PD-1 , SIGLEC7, SIGLEC9, SIRPo(CD172a), TGFBR2, and TIGIT. In embodiments, the first transmembrane protein is selected from AXL, CTLA-4, ICOSL, LAG3, LAP, PD-1, SIGLEC7, SIGLEC9, SIRPa(CD172a), TGFBR2, and TIGIT and the second transmembrane protein is TIM-3.

In embodiments, the first transmembrane protein or the second transmembrane protein is AXL. In embodiments, the first transmembrane protein is AXL and the second transmembrane protein is selected from TIGIT, CTLA-4, ICOSL, LAG3, LAP, PD-1 , SIGLEC7, SIGLEC9, SIRPo(CD172a), TGFBR2, and TIM-3. In embodiments, the first transmembrane protein is selected from CTLA-4, ICOSL, LAG3, LAP, PD-1 , SIGLEC7, SIGLEC9, SIRPa(CD172a), TGFBR2, TIGIT, and TIM-3 and the second transmembrane protein is AXL.

In embodiments, the first transmembrane protein is AXL and the second transmembrane protein is TIGIT.

In embodiments, the first transmembrane protein or the second transmembrane protein is TGFBR2. In embodiments, the first transmembrane protein is TGFBR2 and the second transmembrane protein is selected from AXL, CTLA-4, ICOSL, LAG3, LAP, PD-1, SIGLEC7, SIGLEC9, SIRPo(CD172a), TIGIT, and TIM-3. In embodiments, the first transmembrane protein is selected from AXL, CTLA-4, ICOSL, LAG3, LAP, PD-1 , SIGLEC7, SIGLEC9, SIRPa(CD172a), TIGIT, and TIM-3 and the second transmembrane protein is TGFBR2.

In embodiments, the first transmembrane protein or the second transmembrane protein is LAP. In embodiments, the first transmembrane protein is LAP and the second transmembrane protein is selected from AXL, CTLA-4, ICOSL, LAG3, PD-1 , SIGLEC7, SIGLEC9, SIRPo(CD172a), TGFBR2, TIGIT, and TIM-3. In embodiments, the first transmembrane protein is selected from AXL, CTLA-4, ICOSL, LAG3, PD-1 , SIGLEC7, SIGLEC9, SIRPa(CD172a), TGFBR2, TIGIT, and TIM-3 and the second transmembrane protein is LAP.

In embodiments, the first transmembrane protein or the second transmembrane protein is LAG3. In embodiments, the first transmembrane protein is LAG3 and the second transmembrane protein is selected from TIM-3, AXL, CTLA-4, ICOSL, LAP, PD-1 , SIGLEC7, SIGLEC9, SIRPo(CD172a), TGFBR2, and TIGIT. In embodiments, the first transmembrane protein is selected from PD-1 , AXL, CTLA-4, ICOSL, LAP, SIGLEC7, SIGLEC9, SIRPa(CD172a), TGFBR2, TIGIT, and TIM-3 and the second transmembrane protein is LAG3.

In embodiments, the first transmembrane protein is LAG3 and the second transmembrane protein is TIM-3.

In embodiments, the first transmembrane protein or the second transmembrane protein is SIRPa(CD172a). In embodiments, the first transmembrane protein is SIRPa(CD172a) and the second transmembrane protein is selected from TGFBR2, AXL, CTLA-4, ICOSL, LAG3, LAP, PD-1 , SIGLEC7, SIGLEC9, SIRPo(CD172a), and TIGIT. In embodiments, the first transmembrane protein is selected from AXL, CTLA-4, ICOSL, LAG3, LAP, PD-1 , SIGLEC7, SIGLEC9, SIRPa(CD172a), TGFBR2, and TIGIT and the second transmembrane protein is TIM-3.

In embodiments, the first transmembrane protein is SIRPa(CD172a) and the second transmembrane protein is TGFBR2.

In embodiments, the first transmembrane protein is PD-L1 and the second transmembrane protein is independently selected from the group consisting of: BTNL2, 4-1 BB(CD137), AITR(CD357), BAFFR(CD268), BCMA, CD27, CD270, CD271 , CD30, CD40(CD134), DR6, EDAR, EDAR2(XEDAR), Fas(CD95), FN14(TWEAK-R), GITR, HVEM, LT R, NGFR(CD271), OCIF(TR1), 0X40, RANK(TRANCE-R), RELT, TACI(CD267), TNFR1, TNFR2, TNFRSF13A(CD269), TNFRSF4, TNFRSF25(DR3), TRAIL-R1 (DR4), TRAIL-R2(DR5), TRAIL-R3(DCR1), TRAIL-R4(DCR2), and TROY.

In embodiments, the first transmembrane protein is PD-L1 and the second transmembrane protein is BTNL2.

In embodiments, the first transmembrane protein is PD-L1 and the second transmembrane protein is CTLA-4.

In embodiments, the first transmembrane protein is independently selected from the group consisting of: 4- 1 BB(CD137), AITR(CD357), BAFFR(CD268), BCMA, CD27, CD270, CD271 , CD30, CD40(CD134), DR6, EDAR, EDAR2(XEDAR), Fas(CD95), FN14(TWEAK-R), GITR, HVEM, LT R, NGFR(CD271), OCIF(TR1), 0X40, RANK(TRANCE-R), RELT, TACI(CD267), TNFR1 , TNFR2, TNFRSF13A(CD269), TNFRSF4, TNFRSF25(DR3), TRAIL-R1 (DR4), TRAIL-R2(DR5), TRAIL-R3(DCR1), TRAIL-R4(DCR2), and TROY and the second transmembrane protein is PD-L1.

In embodiments, the first transmembrane protein is CTLA-4 and the second transmembrane protein is PD-L1.

In embodiments, the first transmembrane protein is CTLA-4 and the second transmembrane protein is independently selected from the group consisting of: BTNL2, 4-1 BB(CD137), AITR(CD357), BAFFR(CD268), BCMA, CD27, CD270, CD271 , CD30, CD40(CD134), DR6, EDAR, EDAR2(XEDAR), Fas(CD95), FN14(TWEAK-R), GITR, HVEM, LT R, NGFR(CD271), OCIF(TR1), 0X40, RANK(TRANCE-R), RELT, TACI(CD267), TNFR1, TNFR2, TNFRSF13A(CD269), TNFRSF4, TNFRSF25(DR3), TRAIL-R1 (DR4), TRAIL-R2(DR5), TRAIL-R3(DCR1), TRAIL-R4(DCR2), and TROY.

In embodiments, the first transmembrane protein is independently selected from the group consisting of: 4- 1 BB(CD137), AITR(CD357), BAFFR(CD268), BCMA, CD27, CD270, CD271 , CD30, CD40(CD134), DR6, EDAR, EDAR2(XEDAR), Fas(CD95), FN14(TWEAK-R), GITR, HVEM, LT R, NGFR(CD271), OCIF(TR1), 0X40, RANK(TRANCE-R), RELT, TACI(CD267), TNFR1 , TNFR2, TNFRSF13A(CD269), TNFRSF4, TNFRSF25(DR3), TRAIL-R1 (DR4), TRAIL-R2(DR5), TRAIL-R3(DCR1), TRAIL-R4(DCR2), and TROY and the second transmembrane protein is CTLA-4.

In embodiments, a chimeric protein comprising, at least, one or more of 4-1 BB, CD40, CTLA4, GITR, ICOS, 0X40, PD-L1 , or PD-L2 may be useful in in reducing inflammation and autoimmunity. In embodiments, the first transmembrane protein and the second transmembrane protein are independently selected from the group consisting of: 4-1 BBL, 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 chimeric proteins of the present invention comprise variants of the extracellular domains of the transmembrane proteins disclosed herein. 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 any of the disclosed extracellular domains of the transmembrane proteins disclosed herein, e.g., human extracellular domains.

In embodiments, a chimeric protein of the present invention comprises the extracellular domain of PD-1 (or a variant thereof) and the extracellular domain of TGFBR2 (or a variant thereof).

In embodiments, a chimeric protein of the present invention comprises the extracellular domain of TGFBR2 (or a variant thereof) and the extracellular domain of PD-1 (or a variant thereof).

In embodiments, a chimeric protein of the present invention comprises the extracellular domain of PD-1 (or a variant thereof) and the extracellular domain of LAP (or a variant thereof).

In embodiments, a chimeric protein of the present invention comprises the extracellular domain of LAP (or a variant thereof) and the extracellular domain of PD-1 (or a variant thereof).

In embodiments, a chimeric protein of the present invention comprises the extracellular domain of PD-1 (or a variant thereof) and the extracellular domain of LAG3 (or a variant thereof).

In embodiments, a chimeric protein of the present invention comprises the extracellular domain of LAG3 (or a variant thereof) and the extracellular domain of PD-1 (or a variant thereof).

In embodiments, a chimeric protein of the present invention comprises the extracellular domain of PD-1 (or a variant thereof) and the extracellular domain of TIM-3 (or a variant thereof).

In embodiments, a chimeric protein of the present invention comprises the extracellular domain of TIM-3 (or a variant thereof) and the extracellular domain of PD-1 (or a variant thereof). In embodiments, a chimeric protein of the present invention comprises the extracellular domain of LAG3 (or a variant thereof) and the extracellular domain of TIM-3 (or a variant thereof).

In embodiments, a chimeric protein of the present invention comprises the extracellular domain of TIM-3 (or a variant thereof) and the extracellular domain of LAG3 (or a variant thereof).

In embodiments, a chimeric protein of the present invention comprises the extracellular domain of AXL (or a variant thereof) and the extracellular domain of TIGIT (or a variant thereof).

In embodiments, a chimeric protein of the present invention comprises the extracellular domain of TIGIT (or a variant thereof) and the extracellular domain of AXL (or a variant thereof).

In embodiments, a chimeric protein of the present invention comprises the extracellular domain of SIRPa(CD172a) (or a variant thereof) and the extracellular domain of TGFBR2 (or a variant thereof).

In embodiments, a chimeric protein of the present invention comprises the extracellular domain of PD-L1 (or a variant thereof) and the extracellular domain of BTNL2 (or a variant thereof).

In any herein-disclosed aspect and embodiment, the chimeric protein may comprise one or more variant amino acid sequences each of which has at least one amino acid mutation relative to an amino acid sequence disclosed herein. In embodiments, the at least one amino acid mutation may be independently selected from a substitution, insertion, deletion, and truncation.

In embodiments, amino acid mutations are amino acid substitutions, and may include conservative and/or non conservative substitutions. "Conservative substitutions” may be made, for instance, based on 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, lie; (2) neutral hydrophilic: Cys, Ser, Thr; Asn, Gin; (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 a-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, substitutions may also include non-classical amino acids (e.g., selenocysteine, pyrrolysine, N- formylmethionine b-alanine, GABA and d-Aminolevulinic acid, 4-aminobenzoic acid (PABA), D-isomers of the common amino acids, 2,4-diaminobutyric acid, a-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, y-Abu, e-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, b-alanine, fluoro-amino acids, designer amino acids such as b methyl amino acids, C a-methyl amino acids, N a-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 (in an un-mutated form or as a variant) is capable of binding murine ligand(s)/receptor(s).

In embodiments, a chimeric protein (in an un-mutated form or as a variant) 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 KD of 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 KD of 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 1 1 nM, about 1 1.5 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 KD of less than about 1 mM, 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 KD of less than about 1 nM, about 900 pM, about 800 pM, about 700 pM, about 600 pM, about 500 pM, about 400 pM, about 300 pM, about 200 pM, about 100 pM, about 90 pM, about 80 pM, about 70 pM, about 60 pM about 55 pM about 50 pM about 45 pM, about 40 pM, about 35 pM, about 30 pM, about 25 pM, about 20 pM, about 15 pM, or about 10 pM, or about 1 pM (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 extracellular domain of human AXL has the following amino acid sequence:

APRGTQAEESPFVGNPGNITGARGLTGTLRCQLQVQGEPPEVHWLRDGQILELADSTQTQ VPLG

EDEQDDWIVVSQLRITSLQLSDTGQYQCLVFLGHQTFVSQPGYVGLEGLPYFLEEPE DRTVAANT

PFNLSCQAQGPPEPVDLLWLQDAVPLATAPGHGPQRSLHVPGLNKTSSFSCEAHNAK GVTTSRT

ATITVLPQQPRNLHLVSRQPTELEVAWTPGLSGIYPLTHCTLQAVLSDDGMGIQAGE PDPPEEPLT

SQASVPPHQLRLGSLHPHTPYHIRVACTSSQGPSSWTHWLPVETPEGVPLGPPENIS ATRNGSQA

FVHWQEPRAPLQGTLLGYRLAYQGQDTPEVLMDIGLRQEVTLELQGDGSVSNLTVCV AAYTAAG

DGPWSLPVPLEAWRPGQAQPVHQLVKEPSTPAFSWPWW (SEQ ID NO: 57).

In embodiments, a chimeric protein used in methods of the present invention comprises a variant of the extracellular domain of AXL. 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. One of ordinary skill may select variants of the known amino acid sequence of AXL by consulting the literature, e.g., Janssen et al "A novel putative tyrosine kinase receptor with oncogenic potential” Oncogene 6 (1 1 ), 21 13-2120 (1991 ); Craven et al "Receptor tyrosine kinases expressed in metastatic colon cancer” Int. J. Cancer 60 (6), 791 -797 (1995); Sasaki et al "Structural basis for Gas6-Axl signaling” EMBO J. 25 (1), 80-87 (2006), each of which is incorporated by reference in its entirety.

In embodiments, the extracellular domain of human CTLA-4 has the following amino acid sequence:

KAMHVAQPAWLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMMGNELT FLDD SICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEPCPDS D (SEQ ID NO: 58).

In embodiments, a chimeric protein used in methods of the present invention comprises a variant of the extracellular domain of CTLA-4. 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. One of ordinary skill may select variants of the known amino acid sequence of CTLA-4 by consulting the literature, e.g., Harper etal" CTLA-4 and CD28 activated lymphocyte molecules are closely related in both mouse and human as to sequence, message expression, gene structure, and chromosomal location” J. Immunol. 147 (3), 1037-1044 (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); and Linsley et al "CTLA-4 is a second receptor for the B cell activation antigen B7” J. Exp. Med. 174 (3), 561 -569 (1991 ), each of which is incorporated by reference in its entirety.

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

DTGEKEVRAMVGSDVELSCACPEGSRFDLNDVYVYWGTSESKTWTYHIPGNSSLENVDSR YRN

RALMSPAGMLRGDFSLRLFNVTPCDECKFHCLVLSaSLGFCEVLSVEVTLHVAANFS VPWSAPH

SPSGDELTFTCTSI NGYPRPNVYWI NKTDNSLLDGALGNDTVFLNMRGLYDWSVLRIARTPSVNI

GCCI ENVLLCCNLTVGSCTGNDIGERDKITENPVSTGEKNAAT (SEC ID NO: 59).

In embodiments, a chimeric protein used in methods of the present invention comprises a variant of the extracellular domain of ICOSL. 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 SEC ID NO: 59. One of ordinary skill may select variants of the known amino acid sequence of ICOSL by consulting the literature, e.g., Wang et al "Costimulation of T cells by B7-H2, a B7-like molecule that binds ICOS” Blood 96 (8), 2808-2813 (2000); Yoshinaga et al "Characterization of a new human B7-related protein: B7RP-1 is the ligand to the co-stimulatory protein ICOS” Int. Immunol. 12 (10), 1439-1447 (2000); and Ling ef a/“Cutting edge: identification of GL50, a novel B7-like protein that functionally binds to ICOS receptor” J. Immunol. 164 (4), 1653-1657 (2000), each of which is incorporated by reference in its entirety.

In embodiments, the extracellular domain of human LAG-3 has the following amino acid sequence:

VPWWAQEGAPAQLPCSPTIPLQDLSLLRRAGVTWQHQPDSGPPAAAPGHPLAPGPHPAAP SSW

GPRPRRYTVLSVGPGGLRSGRLPLQPRVQLDERGRQRGDFSLWLRPARRADAGEYRA AVHLRD

RALSCRLRLRLGQASMTASPPGSLRASDWVILNCSFSRPDRPASVHWFRNRGQGRVP VRESPH

HHLAESFLFLPQVSPMDSGPWGCILTYRDGFNVSIMYNLTVLGLEPPTPLTVYAGAG SRVGLPCRL

PAGVGTRSFLTAKWTPPGGGPDLLVTGDNGDFTLRLEDVSQAQAGTYTCHI HLQEQQLNATVTLA

IITVTPKSFGSPGSLGKLLCEVTPVSGQERFVWSSLDTPSQRSFSGPWLEAQEAQLL SQPWQCQL

YQGERLLGAAVYFTELSSPGAQRSGRAPGALPAGHL (SEQ ID NO: 60).

In embodiments, a chimeric protein used in methods of the present invention comprises a variant of the extracellular domain of LAG-3. 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. One of ordinary skill may select variants of the known amino acid sequence of LAG-3 by consulting the literature, e.g., Triebel et al, "LAG-3, a novel lymphocyte activation gene closely related to CD4” J. Exp. Med. 171 (5), 1393-1405 (1990), the contents of which is incorporated by reference in its entirety.

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

LSTCKTIDMELVKRKRI EAIRGQILSKLRLASPPSQGEVPPGPLPEAVLALYNSTRDRVAGESAEPE PEPEADYYAKEVTRVLMVETHNEIYDKFKQSTHSIYMFFNTSELREAVPEPVLLSRAELR LLRLKLK VEQHVELYQKYSNNSWRYLSNRLLAPSDSPEWLSFDVTGWRQWLSRGGEIEGFRLSAHCS CDS RDNTLQVDINGFTTGRRGDLATIHGMNRPFLLLMATPLERAQHLQSSRHRR (SEQ ID NO: 61 ).

In embodiments, a chimeric protein used in methods of the present invention comprises a variant of the extracellular domain of LAP. 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. One of ordinary skill may select variants of the known amino acid sequence of LAP by consulting the literature, e.g., Derynck et al "Intron-exon structure of the human transforming growth factor- beta precursor gene” Nucleic Acids Res. 15 (7), 3188-3189 (1987); Derynck et al "Human transforming growth factor- beta complementary DNA sequence and expression in normal and transformed cells” Nature 316 (6030), 701 -705 (1985); and Bourdrel et al "Recombinant human transforming growth factor-beta 1 : expression by Chinese hamster ovary cells, isolation, and characterization” Protein Expr. Purif. 4 (2), 130-140 (1993), each of which is incorporated by reference in its entirety.

In embodiments, the extracellular domain of human PD-1 has the following amino acid sequence:

LDSPDRPWNPPTFSPALLWTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPED RSQ

PGQDCRFRVTQLPNGRDFHMSWRARRNDSGTYLCGAISLAPKAQI KESLRAELRVTERRAEVPT

AHPSPSPRPAGQFQ (SEQ ID NO: 62).

In embodiments, a chimeric protein used in methods of the present invention comprises a variant of the extracellular domain of PD-1. 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. One of ordinary skill may select variants of the known amino acid sequence of PD-1 by consulting the literature, e.g. Zhang et al "Structural and Functional Analysis of the Costimulatory Receptor Programmed Death-1” Immunity. 2004 Mar; 20(3):337-47; Lin et a/ "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. 2008 Feb 26; 105(8):301 1 -6; Zak et al "Structure of the Complex of Human Programmed Death 1 , PD-1 , and Its Ligand PD-L1”, Structure. 2015 Dec 1 ;23(12):2341-2348; and Cheng et al "Structure and Interactions of the Human Programmed Cell Death 1 Receptor”, J Biol Chem. 2013 Apr 26;288(17): 1 1771 -85, each of which is incorporated by reference in its entirety.

In embodiments, the ligand for PD-1 is PD-L1 or PD-L2. In embodiments, the extracellular domain of human SIGLEC7 has the following amino acid sequence:

QKSNRKDYSLTMQSSVTVQEGMCVHVRCSFSYPVDSQTDSDPVHGYWFRAGNDISWK APVATN

NPAWAVQEETRDRFHLLGDPQTKNCTLSIRDARMSDAGRYFFRMEKGNI KWNYKYDQLSVNVTA

LTHRPNILIPGTLESGCFQNLTCSVPWACEQGTPPMISWMGTSVSPLHPSTTRSSVL TLIPQPQHH

GTSLTCQVTLPGAGVTTNRTIQLNVSYPPQNLTVTVFQGEGTASTALGNSSSLSVLE GQSLRLVCA

VDSNPPARLSWTWRSLTLYPSQPSNPLVLELQVHLGDEGEFTCRAQNSLGSQHVSLN LSLQQEY

TGKMRPVSGVLL (SEQ ID NO: 63)

In embodiments, a chimeric protein used in methods of the present invention comprises a variant of the extracellular domain of SIGLEC7. 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. One of ordinary skill may select variants of the known amino acid sequence of SIGLEC7 by consulting the literature, e.g., Falco et al "Identification and molecular cloning of p75/AIRM1 , a novel member of the sialoadhesin family that functions as an inhibitory receptor in human natural killer cells” J. Exp. Med. 190 (6), 793-802 (1999); Alphey et a/“High resolution crystal structures of Siglec-7. Insights into ligand specificity in the Siglec family” J. Biol. Chem. 278 (5), 3372-3377 (2003); and Dimasi et al "Structure of the saccharide-binding domain of the human natural killer cell inhibitory receptor p75/AIRM1” Acta Crystallogr. D Biol. Crystallogr. 60 (Pt 2), 401 -403 (2004), each of which is incorporated by reference in its entirety.

In embodiments, the extracellular domain of human SIGLEC9 has the following amino acid sequence:

QTSKLLTMQSSVTVQEGLCVHVPCSFSYPSHGWIYPGPWHGYWFREGANTDQDAPVATNN PA

RAVWEETRDRFHLLGDPHTKNCTLSIRDARRSDAGRYFFRMEKGSIKWNYKHHRLSV NVTALTHR

PNILIPGTLESGCPQNLTCSVPWACEQGTPPMISWIGTSVSPLDPSTTRSSVLTLIP QPQDHGTSLT

CQVTFPGASVTTNKTVHLNVSYPPQNLTMTVFQGDGTVSTVLGNGSSLSLPEGQSLR LVCAVDAV

DSNPPARLSLSWRGLTLCPSQPSNPGVLELPWVHLRDAAEFTCRAQNPLGSQQVYLN VSLQSKA

TSGVTQG (SEQ ID NO: 64):

In embodiments, a chimeric protein used in methods of the present invention comprises a variant of the extracellular domain of SIGLEC9. 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. One of ordinary skill may select variants of the known amino acid sequence of SIGLEC9 by consulting the literature, e.g., Angata and Varki "Cloning, characterization, and phylogenetic analysis of siglec-9, a new member of the CD33-related group of siglecs. Evidence for co-evolution with sialic acid synthesis pathways” J. Biol. Chem. 275 (29), 22127-22135 (2000); and Foussias et al "Identification and molecular characterization of a novel member of the siglec family (SIGLEC9)” Genomics 67 (2), 171 -178 (2000), each of which is incorporated by reference in its entirety.

In embodiments, the extracellular domain of human SIRPa(CD172a) has the following amino acid sequence:

EEELQVIQPDKSVLVAAGETATLRCTATSLIPVGPIQWFRGAGPGRELIYNQKEGHFPRV TTVSDLT

KRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSAPWSG PAARATP

QHTVSFTCESHGFSPRDITLKWFKNGNELSDFQTNVDPVGESVSYSIHSTAKWLTRE DVHSQVIC

EVAHVTLQGDPLRGTANLSETIRVPPTLEVTQQPVRAENQVNVTCQVRKFYPQRLQL TWLENGNV

SRTETASTVTENKDGTYNWMSWLLVNVSAHRDDVKLTCQVEHDGQPAVSKSHDLKVS AHPKEQ

GSNTAAENTGSNERNIY (SEQ ID NO: 65):

In embodiments, a chimeric protein used in methods of the present invention comprises a variant of the extracellular domain of SIRPa(CD 172a). 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. One of ordinary skill may select variants of the known amino acid sequence of SIRPa(CD172a) by consulting the literature, e.g., LEE, et al., "Novel Structural Determinants of SIRPa that Mediate Binding of CD47," The Journal of Immunology, 179, 7741 -7750, 2007 and HATHERLEY, et al., "The Structure of the Macrophage Signal Regulatory Protein a (SIRPa) Inhibitory Receptor Reveals a Binding Face Reminiscent of That Used by T Cell Receptors," The Journal Of Biological Chemistry, Vol. 282, No. 19, pp. 14567- 14575, 2007, each of which is incorporated by reference in its entirety.

In embodiments, the extracellular domain of human TGFBR2 has the following amino acid sequence:

TIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQE VCVAV WRKNDENITLETVCHDPKLPYHDFILEDAASPKCI MKEKKKPGETFFMCSCSSDECNDNII FSEEYN TSNPDLLLVIFQ (SEQ ID NO: 66):

In embodiments, a chimeric protein used in methods of the present invention comprises a variant of the extracellular domain of TGFBR2. 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. One of ordinary skill may select variants of the known amino acid sequence of TGFBR2 by consulting the literature, e.g., Lin et al "Expression cloning of the TGF-beta type II receptor, a functional transmembrane serine/threonine kinase” Cell 68 (4), 775-785 (1992); Hart et al "Crystal structure of the human TbetaR2 ectodomain--TGF-beta3 complex” Nat. Struct. Biol. 9 (3), 203-208 (2002); Boesen et a/ "The 1.1 A crystal structure of human TGF-beta type I I receptor ligand binding domain” Structure 10 (7), 913-919 (2002); and Radaev et al "Ternary complex of transforming growth factor-betal reveals isoform-specific ligand recognition and receptor recruitment in the superfamily” J. Biol. Chem. 285 (19), 14806-14814 (2010), each of which is incorporated by reference in its entirety.

In embodiments, the ligand for TGFBR2 is TGF .

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

MMTGTIETTGNISAEKGGSI ILQCHLSSTTAQVTQVNWEQQDQLLAICNADLGWHISPSFKDRVAP GPGLGLTLQSLTVNDTGEYFCIYHTYPDGTYTGRIFLEVLESSVAEHGARFQIP (SEQ ID NO: 67):

In embodiments, a chimeric protein used in methods of the present invention comprises a variant of the extracellular domain of TIGIT. 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. One of ordinary skill may select variants of the known amino acid sequence of TIGIT by consulting the literature, e.g., Yu, et a/ "The surface protein TIGIT suppresses T cell activation by promoting the generation of mature immunoregulatory dendritic cells” Nat. Immunol. 10 (1 ), 48-57 (2009) and Stengel et al "Structure of TIGIT immunoreceptor bound to poliovirus receptor reveals a cell-cell adhesion and signaling mechanism that requires cis-trans receptor clustering” Proc. Natl. Acad. Sci. U.S.A. 109 (14), 5399-5404 (2012), each of which is incorporated by reference in its entirety.

In embodiments, the extracellular domain of human TI M-3 has the following amino acid sequence:

SEVEYRAEVGQNAYLPCFYTPAAPGNLVPVCWGKGACPVFECGNWLRTDERDVNYWTSRY WL NGDFRKGDVSLTIENVTLADSGIYCCRIQIPGIMNDEKFNLKLVI KPAKVTPAPTRQRDFTAAFPRML TTRGHGPAETQTLGSLPDI NLTQISTLANELRDSRLANDLRDSGATIRIG (SEQ ID NO: 68):

In embodiments, a chimeric protein used in methods of the present invention comprises a variant of the extracellular domain of TIM-3. 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: 68. One of ordinary skill may select variants of the known amino acid sequence of TIM-3 by consulting the literature, e.g. Cao, et al., "T Cell Immunoglobulin Mucin-3 Crystal Structure Reveals a Galectin-9-lndependent Ligand-Binding Surface,” Immunity 26, pp. 31 1 -321 , 2007 and Freeman, et al., "TIM genes: a family of cell surface phosphatidylserine receptors that regulate innate and adaptive immunity,” Immunol Rev., 235(1 ), pp. 172-189, 2010, each of which is incorporated by reference in its entirety.

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

FTITAPKDLYWEYGSNVTMECRFPVERELDLLALWYWEKEDEQVIQFVAGEEDLKPQ HSNFRGRASLPK

DQLLKGNAALQITDVKLQDAGVYCCIISYGGADYKRITLKVNAPYRKI NQRISVDPATSEHELICQAEGYPEAE VIWTNSDHQPVSGKRSVTTSRTEGMLLNVTSSLRVNATANDVFYCTFWRSQPGQNHTAEL I IPELPATHPP QNRTH (SEQ ID NO: 75).

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: 75.

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: 75.

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

KQSEDFRVIGPAHPILAGVGEDALLTCQLLPKRTTMHVEVRWYRSEPSTPVFVHRDG VEVTEMQMEEYRGW

VEWI ENGIAKGNVALKI HNIQPSDNGQYWCHFQDGNYCGETSLLLKVAGLGSAPSI HMEGPGESGVQLVCTA

RGWFPEPQVYWEDIRGEKLLAVSEHRIQDKDGLFYAEATLWRNASAESVSCLVHNPV LTEEKGSVISLPEKL

QTELASLKVNGPSQPILVRVGEDIQLTCYLSPKANAQSMEVRWDRSHRYPAVHVYMD GDHVAGEQMAEYR

GRTVLVSDAIDEGRLTLQILSARPSDDGQYRCLFEKDDVYQEASLDLKWSLGSSPLI TVEGQEDGEMQPMC

SSDGWFPQPHVPWRDMEGKTIPSSSQALTQGSHGLFHVQTLLRVTNISAVDVTCSIS IPFLGEEKIATFSLSG

W (SEQ ID NO: 76).

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: 76.

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: 76.

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 Jan;335:22-29, each of which is incorporated by reference in its entirety.

Another aspect of the present invention is a chimeric protein comprising: (a) a first domain comprising a portion of PD- 1 that is capable of binding PD-L1 or PD-L2, (b) a second domain comprising a portion of TGFBR2 that is capable of binding TGF , and (c) a linker linking the first domain and the second domain and comprising a hinge-CH2-CH3 Fc domain. 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., lgG1 , lgG2, lgG3, and lgG4), IgA (e.g., lgA1 and lgA2), IgD, or IgE. In embodiments, the lgG4 is a human lgG4. In embodiments, the linker domain 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. The term "at least 95% identical” includes 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 or reference amino acid sequence.

Yet another aspect of the present invention is a chimeric protein comprising: (a) a first domain comprising a sequence that is at least 95% identical to SEQ ID NO: 62 and capable of binding PD-L1 or PD-L2, (b) a second domain comprising a sequence that is at least 95% identical to SEQ ID NO: 66 and capable of binding TGF , 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 comprising a sequence that is at least 97% identical to SEQ ID NO: 62. In embodiments, the second domain comprising a sequence that is at least 97% identical to SEQ ID NO: 66. In embodiments, the first domain comprises SEQ ID NO: 62 and/or the second domain comprises SEQ ID NO: 66. In embodiments, the linker comprises an amino acid sequence that is at least 95% identical, e.g., at least 97% identical, to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. The term "at least 95% identical” includes 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 or reference amino acid sequence.

In an aspect, the present invention provides a chimeric protein comprising: (a) a first domain comprising the amino acid sequence of SEQ ID NO: 62; (b) a second domain comprising the amino acid sequence of SEQ ID NO: 66; and (c) a linker linking the first domain and the amino acid sequence of SEQ ID NO: 2.

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.

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.

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 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 herein 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, 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. In embodiments, the chimeric protein of the present invention comprises an extracellular domain of one or more of the immune-modulating agents described in Mahoney, Nature Reviews Drug Discovery 2015: 14; 561-585, the entire contents of which are hereby incorporated by reference.

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 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., lgG1, lgG2, lgG3, and lgG4, and lgA1, and lgA2)). 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 lgG1 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. lgG2 has a shorter hinge than lgG1, with 12 amino acid residues and four disulfide bridges. The hinge region of lgG2 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 lgG2 molecule. lgG3 differs from the other subclasses by its unique extended hinge region (about four times as long as the lgG1 hinge), containing 62 amino acids (including 21 prolines and 11 cysteines), forming an inflexible poly-proline double helix. In lgG3, the Fab fragments are relatively far away from the Fc fragment, giving the molecule a greater flexibility. The elongated hinge in lgG3 is also responsible for its higher molecular weight compared to the other subclasses. The hinge region of lgG4 is shorter than that of lgG1 and its flexibility is intermediate between that of lgG1 and lgG2. The flexibility of the hinge regions reportedly decreases in the order lgG3>lgG1>lgG4>lgG2. In embodiments, the linker may be derived from human lgG4 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 a!., 1992 Immunological Reviews 130:87. The upper hinge region includes amino acids from the carboxyl end of Cm 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 CH2 domain and includes residues in CH2. Id. The core hinge region of wild-type human lgG1 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., lgG1, lgG2, lgG3, and lgG4, and lgA1 and lgA2)). 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, lgA1 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., lgG1, lgG2, lgG3, and lgG4, and lgA1 and lgA2)).

In a chimeric protein of the present invention, the linker comprises a hinge-CH2-CH3 Fc domain derived from lgG4. In embodiments, the linker comprises a hinge-CH2-CH3 Fc domain derived from a human lgG4. 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 lgG1 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, I253A, H310A, M428L, H433K, N434A, N434F, N434S, 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 I253A/H310A/H435A mutation or I HH 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(1 1):5497-508, and U.S. Patent 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 Q31 1 S 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 KD of about 1 nM to about 80 nM. For example, the chimeric protein may bind to FcRn with a KD of about 1 nM, about 2 nM, about 3 nM, about 4 nM, about 5 nM, about 6 nM, about 7 nM, about 8 nM, about 9 nM, about 10 nM, about 15 nM, about 20 nM, about 25 nM, about 30 nM, about 35 nM, about 40 nM, about 45 nM, about 50 nM, about 55 nM, about 60 nM, about 65 nM, about 70 nM, about 71 nM, about 72 nM, about 73 nM, about 74 nM, about 75 nM, about 76 nM, about 77 nM, about 78 nM, about 79 nM, or about 80 nM. In embodiments, the chimeric protein may bind to FcRn with a KD of about 9 nM. In embodiments, the chimeric protein does not substantially bind to other Fc receptors (/. 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 an 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)

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) _n A (n = 2-5) (SEQ ID NO: 39 to SEQ ID NO: 42), A(EAAAK) ₄ ALEA(EAAAK) ₄ A (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)„ where 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 an extracellular domain (ECD) of a first transmembrane protein, one joining linker preceding an Fc domain, a second joining linker following the Fc domain, and an ECD of a second transmembrane protein, the chimeric protein may comprise the following structure: ECD of the first transmembrane protein - Joining Linker 1 - Fc Domain - Joining Linker 2 - ECD of the second transmembrane protein

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

n 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

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) _n where 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.

Below are illustrative chimeric proteins that comprise the following structure:

ECD of the first transmembrane protein - Joining Linker 1 - Fc Domain - Joining Linker 2 - ECD of the second transmembrane protein

A chimeric protein of the present invention may comprise the extracellular domain of PD-1 (or a variant thereof), a linker, and the extracellular domain of TGFBR2 (or a variant thereof). In embodiments, the linker comprises a hinge- CH2-CH3 Fc domain, e.g., from an lgG1 or from lgG4, including human lgG1 or lgG4. Thus, in embodiments, a chimeric protein of the present invention comprises the extracellular domain of PD-1 , linker comprising a hinge-CH2-CH3 Fc domain, and the extracellular domain of TGFBR2 (or a variant thereof). Such a chimeric protein may be referred to herein as "PD-1-FC-TGFBR2”.

In embodiments, a PD-1 -Fc-TGFBR2 chimeric protein of the present invention has the following amino acid sequence:

LDSPDRPWNPPTFSPALLWTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAF PEDRSQPGQDCR

FRVTQLPNGRDFHMSWRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVP TAHPSPSPRPAGQ

FQSKYGPPCPPCPAPEFLGGPSVFLFPPKPKDQLMISRTPEVTCVWDVSQEDPEVQF NWYVDGVEVHNA

KTKPREEQFNSTYRWSVLTVLHQDWLSGKEYKCKVSSKGLPSSI EKTISNATGQPREPQVYTLPPSQEEM

TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKS RWQEGNVFSCSVL

HEALHNHYTQKSLSLSLGKIEGRMDTIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFC DVRFSTCDNQKSCM

SNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCI MKEKKKPGETFFMCSCSSD

ECNDNI IFSEEYNTSNPDLLLVI FQ (SEQ ID NO: 70).

In embodiments, a chimeric protein comprises a variant of a PD-1 -Fc-TGFBR2 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: 70 and/or a human equivalent of the PD-1 -Fc-TGFBR2 chimeric protein.

A chimeric protein of the present invention may comprise the extracellular domain of PD-1 (or a variant thereof), a linker, and the extracellular domain of LAP (or a variant thereof). In embodiments, the linker comprises a hinge-CH2- CH3 Fc domain, e.g., from an lgG1 or from lgG4, including human lgG1 or lgG4. Thus, in embodiments, a chimeric protein of the present invention comprises the extracellular domain of PD-1 (or a variant thereof), a linker comprising a hinge-CH2-CH3 Fc domain, and the extracellular domain of LAP (or a variant thereof). Such a chimeric protein may be referred to herein as "PD-1 -Fc-LAP”.

In embodiments, a PD-1-Fc-LAP chimeric protein of the present invention has the following amino acid sequence:

MWVRQVPWSFTWAVLQLSWQSGWLLEVPNGPWRSLTFYPAWLTVSEGANATFTCSLS NWSEDLMLNWN

RLSPSNQTEKQAAFCNGLSQPVQDARFQIIQLPNRHDFHMNILDTRRNDSGIYLCGA ISLHPKAKIEESPGAE

LWTERILETSTRYPSPSPKPEGRFQVPRDCGCKPCICTVPEVSSVFI FPPKPKDVLTITLTPKVTCWVDISK

DDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNS AAFPAPI EKTISKT

KGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQP IMDTDGSYFVYSKL

NVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGII EGRMDLSTCKTIDMELVKRKRIEAIRGQILSKLRL

ASPPSQGEVPPGPLPEAVLALYNSTRDRVAGESADPEPEPEADYYAKEVTRVLMVDR NNAIYEKTKDISHSI

YMFFNTSDIREAVPEPPLLSRAELRLQRLKSSVEQHVELYQKYSNNSWRYLGNRLLT PTDTPEWLSFDVTG

WRQWLNQGDGIQGFRFSAHCSCDSKDNKLHVEINGISPKRRGDLGTIHDMNRPFLLL MATPLERAQHLHS

SRHRR (SEQ ID NO: 69).

In embodiments, a chimeric protein comprises a variant of a PD-1 -Fc-LAP 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 and/or a human equivalent of the PD-1 -Fc-LAP chimeric protein.

A chimeric protein of the present invention may comprise the extracellular domain of LAP (or a variant thereof), a linker, and the extracellular domain of PD-1 (or a variant thereof). In embodiments, the linker comprises a hinge-CH2-CH3 Fc domain, e.g., from an lgG1 or from lgG4, including human lgG1 or lgG4. Thus, in embodiments, a chimeric protein of the present invention comprises the extracellular domain of LAP, linker comprising a hinge-CH2-CH3 Fc domain, and the extracellular domain of PD-1 (or a variant thereof). Such a chimeric protein may be referred to herein as "LAP-Fc- PD-1”.

A chimeric protein of the present invention may comprise the extracellular domain of TGFBR2 (or a variant thereof), a linker, and the extracellular domain of PD-1 (or a variant thereof). In embodiments, the linker comprises a hinge-CH2- CH3 Fc domain, e.g., from an lgG1 or from lgG4, including human lgG1 or lgG4. Thus, in embodiments, a chimeric protein of the present invention comprises the extracellular domain of TGFBR2, linker comprising a hinge-CH2-CH3 Fc domain, and the extracellular domain of PD-1 (or a variant thereof). Such a chimeric protein may be referred to herein as "TGFBR2-FC-PD-1”.

A chimeric protein of the present invention may comprise the extracellular domain of PD-1 (or a variant thereof), a linker, and the extracellular domain of LAG3 (or a variant thereof). In embodiments, the linker comprises a hinge-CH2- CH3 Fc domain, e.g., from an lgG1 or from lgG4, including human lgG1 or lgG4. Thus, in embodiments, a chimeric protein of the present invention comprises the extracellular domain of PD-1 , linker comprising a hinge-CH2-CH3 Fc domain, and the extracellular domain of LAG3 (or a variant thereof). Such a chimeric protein may be referred to herein as“PD-1-FC-LAG3”.

In embodiments, a PD-1-Fc-LAG3 chimeric protein of the present invention has the following amino acid sequence:

LDSPDRPWNPPTFSPALLWTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAF PEDRSQPGQDCR

FRVTQLPNGRDFHMSWRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVP TAHPSPSPRPAGQ

FQSKYGPPCPPCPAPEFLGGPSVFLFPPKPKDQLMISRTPEVTCVWDVSQEDPEVQF NWYVDGVEVHNA

KTKPREEQFNSTYRWSVLTVLHQDWLSGKEYKCKVSSKGLPSSIEKTISNATGQPRE PQVYTLPPSQEEM

TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKS RWQEGNVFSCSVL

HEALHNHYTQKSLSLSLGKIEGRMDVPWWAQEGAPAQLPCSPTIPLQDLSLLRRAGV TWQHQPDSGPPA

AAPGHPLAPGPHPAAPSSWGPRPRRYTVLSVGPGGLRSGRLPLQPRVQLDERGRQRG DFSLWLRPARRA

DAGEYRAAVHLRDRALSCRLRLRLGQASMTASPPGSLRASDWVILNCSFSRPDRPAS VHWFRNRGQGRV

PVRESPHHHLAESFLFLPQVSPMDSGPWGCILTYRDGFNVSIMYNLTVLGLEPPTPL TVYAGAGSRVGLPC

RLPAGVGTRSFLTAKWTPPGGGPDLLVTGDNGDFTLRLEDVSQAQAGTYTCHIHLQE QQLNATVTLAIITVT

PKSFGSPGSLGKLLCEVTPVSGQERFVWSSLDTPSQRSFSGPWLEAQEAQLLSQPWQ CQLYQGERLLGA

AVYFTELSSPGAQRSGRAPGALPAGHL (SEQ ID NO: 71).

In embodiments, a chimeric protein comprises a variant of a PD-1-Fc-LAG3 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 and/or a human equivalent of the PD-1 -Fc-LAG3 chimeric protein.

A chimeric protein of the present invention may comprise the extracellular domain of PD-1 (or a variant thereof), a linker, and the extracellular domain of TI M-3 (or a variant thereof). In embodiments, the linker comprises a hinge-CH2- CH3 Fc domain, e.g., from an lgG1 or from lgG4, including human lgG1 or lgG4. Thus, in embodiments, a chimeric protein of the present invention comprises the extracellular domain of PD-1 , linker comprising a hinge-CH2-CH3 Fc domain, and the extracellular domain of TIM-3 (or a variant thereof). Such a chimeric protein may be referred to herein as“PD-l -Fc-TI M-3”.

In embodiments, a PD-1-Fc-TI M-3 chimeric protein of the present invention has the following amino acid sequence:

LDSPDRPWNPPTFSPALLWTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAF PEDRSQPGQDCR

FRVTQLPNGRDFHMSWRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVP TAHPSPSPRPAGQ

FQSKYGPPCPPCPAPEFLGGPSVFLFPPKPKDQLMISRTPEVTCVWDVSQEDPEVQF NWYVDGVEVHNA

KTKPREEQFNSTYRWSVLTVLHQDWLSGKEYKCKVSSKGLPSSI EKTISNATGQPREPQVYTLPPSQEEM

TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKS RWQEGNVFSCSVL

HEALHNHYTQKSLSLSLGKIEGRMDSEVEYRAEVGQNAYLPCFYTPAAPGNLVPVCW GKGACPVFECGNV

VLRTDERDVNYWTSRYWLNGDFRKGDVSLTIENVTLADSGIYCCRIQIPGIMNDEKF NLKLVIKPAKVTPAPT

RQRDFTAAFPRMLTTRGHGPAETQTLGSLPDI NLTQISTLANELRDSRLANDLRDSGATIRIG (SEQ ID NO:

72).

In embodiments, a chimeric protein comprises a variant of a PD-1 -Fc-TI M-3 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 I D NO: 72 and/or a human equivalent of the PD-1 -Fc-TI M-3 chimeric protein. A chimeric protein of the present invention may comprise the extracellular domain of LAG3 (or a variant thereof), a linker, and the extracellular domain of PD-1 (or a variant thereof). In embodiments, the linker comprises a hinge-CH2- CH3 Fc domain, e.g., from an lgG1 or from lgG4, including human lgG1 or lgG4. Thus, in embodiments, a chimeric protein of the present invention comprises the extracellular domain of LAG3, linker comprising a hinge-CH2-CH3 Fc domain, and the extracellular domain of PD-1 (or a variant thereof). Such a chimeric protein may be referred to herein as“LAG3-FC-PD-1”.

A chimeric protein of the present invention may comprise the extracellular domain of TI M-3 (or a variant thereof), a linker, and the extracellular domain of PD-1 (or a variant thereof). In embodiments, the linker comprises a hinge-CH2- CH3 Fc domain, e.g., from an lgG1 or from lgG4, including human lgG1 or lgG4. Thus, in embodiments, a chimeric protein of the present invention comprises the extracellular domain of TIM-3, linker comprising a hinge-CH2-CH3 Fc domain, and the extracellular domain of PD-1 (or a variant thereof). Such a chimeric protein may be referred to herein as“TIM-3-Fc-PD-1”.

A chimeric protein of the present invention may comprise the extracellular domain of LAG3 (or a variant thereof), a linker, and the extracellular domain of TI M-3 (or a variant thereof). In embodiments, the linker comprises a hinge-CH2- CH3 Fc domain, e.g., from an lgG1 or from lgG4, including human lgG1 or lgG4. Thus, in embodiments, a chimeric protein of the present invention comprises the extracellular domain of LAG3, linker comprising a hinge-CH2-CH3 Fc domain, and the extracellular domain of TIM-3 (or a variant thereof). Such a chimeric protein may be referred to herein as“LAG3-FC-TI M-3”.

In embodiments, a LAG3-Fc-TIM-3 chimeric protein of the present invention has the following amino acid sequence:

VPWWAQEGAPAQLPCSPTIPLQDLSLLRRAGVTWQHQPDSGPPAAAPGHPLAPGPHP AAPSSWGPRPR

RYTVLSVGPGGLRSGRLPLQPRVQLDERGRQRGDFSLWLRPARRADAGEYRAAVHLR DRALSCRLRLRL

GQASMTASPPGSLRASDWVILNCSFSRPDRPASVHWFRNRGQGRVPVRESPHHHLAE SFLFLPQVSPMD

SGPWGCILTYRDGFNVSIMYNLTVLGLEPPTPLTVYAGAGSRVGLPCRLPAGVGTRS FLTAKWTPPGGGPD

LLVTGDNGDFTLRLEDVSQAQAGTYTCHIHLQEQQLNATVTLAIITVTPKSFGSPGS LGKLLCEVTPVSGQER

FVWSSLDTPSQRSFSGPWLEAQEAQLLSQPWQCQLYQGERLLGAAVYFTELSSPGAQ RSGRAPGALPAG

HLSKYGPPCPPCPAPEFLGGPSVFLFPPKPKDQLMISRTPEVTCVWDVSQEDPEVQF NWYVDGVEVHNA

KTKPREEQFNSTYRWSVLTVLHQDWLSGKEYKCKVSSKGLPSSI EKTISNATGQPREPQVYTLPPSQEEM

TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKS RWQEGNVFSCSVL

HEALHNHYTQKSLSLSLGKIEGRMDSEVEYRAEVGQNAYLPCFYTPAAPGNLVPVCW GKGACPVFECGNV

VLRTDERDVNYWTSRYWLNGDFRKGDVSLTIENVTLADSGIYCCRIQIPGIMNDEKF NLKLVIKPAKVTPAPT

RQRDFTAAFPRMLTTRGHGPAETQTLGSLPDI NLTQISTLANELRDSRLANDLRDSGATIRIG (SEQ ID NO:

73). In embodiments, a chimeric protein comprises a variant of a LAG3-Fc-TI M-3 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: 72 and/or a human equivalent of the LAG3-Fc-TIM-3 chimeric protein.

A chimeric protein of the present invention may comprise the extracellular domain of TI M-3 (or a variant thereof), a linker, and the extracellular domain of LAG3 (or a variant thereof). In embodiments, the linker comprises a hinge-CH2- CH3 Fc domain, e.g., from an lgG1 or from lgG4, including human lgG1 or lgG4. Thus, in embodiments, a chimeric protein of the present invention comprises the extracellular domain of TIM-3, linker comprising a hinge-CH2-CH3 Fc domain, and the extracellular domain of LAG3 (or a variant thereof). Such a chimeric protein may be referred to herein as“TIM-3-FC-LAG3”.

A chimeric protein of the present invention may comprise the extracellular domain of AXL (or a variant thereof), a linker, and the extracellular domain of TIGIT (or a variant thereof). In embodiments, the linker comprises a hinge-CH2-CH3 Fc domain, e.g., from an lgG1 or from lgG4, including human lgG1 or lgG4. Thus, in embodiments, a chimeric protein of the present invention comprises the extracellular domain of AXL, linker comprising a hinge-CH2-CH3 Fc domain, and the extracellular domain of TIGIT (or a variant thereof). Such a chimeric protein may be referred to herein as "AXL- Fc-TIGIT”.

In embodiments, an AXL-Fc-TIGIT chimeric protein of the present invention has the following amino acid sequence:

APRGTQAEESPFVGNPGNITGARGLTGTLRCQLQVQGEPPEVHWLRDGQILELADST QTQVPLGEDEQDD

WIWSQLRITSLQLSDTGQYQCLVFLGHQTFVSQPGYVGLEGLPYFLEEPEDRTVAAN TPFNLSCQAQGPP

EPVDLLWLQDAVPLATAPGHGPQRSLHVPGLNKTSSFSCEAHNAKGVTTSRTATITV LPQQPRNLHLVSRQ

PTELEVAWTPGLSGIYPLTHCTLQAVLSNDGMGIQAGEPDPPEEPLTSQASVPPHQL RLGSLHPHTPYHIRV

ACTSSQGPSSWTHWLPVETPEGVPLGPPENISATRNGSQAFVHWQEPRAPLQGTLLG YRLAYQGQDTPE

VLMDIGLRQEVTLELQGDGSVSNLTVCVAAYTAAGDGPWSLPVPLEAWRPGQAQPVH QLVKEPSTPAFSW

PWWSKYGPPCPPCPAPEFLGGPSVFLFPPKPKDQLMISRTPEVTCVWDVSQEDPEVQ FNWYVDGVEVH

NAKTKPREEQFNSTYRWSVLTVLHQDWLSGKEYKCKVSSKGLPSSIEKTISNATGQP REPQVYTLPPSQE EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSR WQEGNVFSCS VLHEALHNHYTQKSLSLSLGKIEGRMDMMTGTIETTGNISAEKGGSIILQCHLSSTTAQV TQVNWEQQDQLL AICNADLGWHISPSFKDRVAPGPGLGLTLQSLTVNDTGEYFCIYHTYPDGTYTGRIFLEV LESSVAEHGARF QIP (SEQ ID NO: 74).

In embodiments, a chimeric protein comprises a variant of an AXL-Fc-TIGIT 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 and/or a human equivalent of the AXL-Fc-TIGIT chimeric protein.

A chimeric protein of the present invention may comprise the extracellular domain of TIGIT (or a variant thereof), a linker, and the extracellular domain of AXL (or a variant thereof). In embodiments, the linker comprises a hinge-CH2- CH3 Fc domain, e.g., from an lgG1 or from lgG4, including human lgG1 or lgG4. Thus, in embodiments, a chimeric protein of the present invention comprises the extracellular domain of TIGIT, linker comprising a hinge-CH2-CH3 Fc domain, and the extracellular domain of AXL (or a variant thereof). Such a chimeric protein may be referred to herein as“TIGIT-Fc-AXL”.

A chimeric protein of the present invention may comprise the extracellular domain of SIRPa(CD172a) (or a variant thereof), a linker, and the extracellular domain of TGFBR2 (or a variant thereof). In embodiments, the linker comprises a hinge-CH2-CH3 Fc domain, e.g., from an lgG1 or from lgG4, including human lgG1 or lgG4. Thus, in embodiments, a chimeric protein of the present invention comprises the extracellular domain of SIRPa(CD172a), linker comprising a hinge-CH2-CH3 Fc domain, and the extracellular domain of TGFBR2 (or a variant thereof). Such a chimeric protein may be referred to herein as "SIRPa(CD172a)-Fc-TGFBR2”.

In embodiments, a SIRPa(CD172a)-Fc-TGFBR2 chimeric protein of the present invention has the following amino acid sequence:

MEWSWVFLFFLSVTTGVHSEEELQVIQPDKSVLVAAGETATLRCTATSLIPVGPIQW FRGAGPGRELIYNQK

EGHFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGT ELSVRAKPSAPWSG

PAARATPQHTVSFTCESHGFSPRDITLKWFKNGNELSDFQTNVDPVGESVSYSIHST AKWLTREDVHSQVI

CEVAHVTLQGDPLRGTANLSETIRVPPTLEVTQQPVRAENQVNVTCQVRKFYPQRLQ LTWLENGNVSRTET ASTVTENKDGTYNWMSWLLVNVSAHRDDVKLTCQVEHDGQPAVSKSHDLKVSAHPKEQGS NTAAENTGS

NERNIYSKYGPPCPPCPAPEFLGGPSVFLFPPKPKDQLMISRTPEVTCVWDVSQEDP EVQFNWYVDGVEV

HNAKTKPREEQFNSTYRWSVLTVLHQDWLSGKEYKCKVSSKGLPSSI EKTISNATGQPREPQVYTLPPSQ

EEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTV DKSRWQEGNVFSC

SVLHEALHNHYTQKSLSLSLGKIEGRMDTIPPHVQKSVNNDMIVTDNNGAVKFPQLC KFCDVRFSTCDNQK

SCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCI MKEKKKPGETFFMCS

CSSDECNDNII FSEEYNTSNPDLLLVI FQ (SEQ ID NO: 77).

In embodiments, a chimeric protein comprises a variant of a SIRPa(CD172a)-Fc-TGFBR2 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: 77 and/or a human equivalent of the SIRPa(CD172a)-Fc-TGFBR2 chimeric protein.

A chimeric protein of the present invention may comprise the extracellular domain of PD L1 (or a variant thereof), a linker, and the extracellular domain of BTNL2 (or a variant thereof). In embodiments, the linker comprises a hinge-CH2- CH3 Fc domain, e.g., from an lgG1 or from lgG4, including human lgG1 or lgG4. Thus, in embodiments, a chimeric protein of the present invention comprises the extracellular domain of PD L1 , linker comprising a hinge-CH2-CH3 Fc domain, and the extracellular domain of BTNL2 (or a variant thereof). Such a chimeric protein may be referred to herein as“PD L1 -Fc-BTNL2”.

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

FTVTVPKDLYWEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKV QHSSYRQRARLLKD

QLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILWDPVT SEHELTCQAEGYPK

AEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRI NTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPN

ERSKYGPPCPPCPAPEFLGGPSVFLFPPKPKDQLMISRTPEVTCVWDVSQEDPEVQF NWYVDGVEVHNA

KTKPREEQFNSTYRWSVLTVLHQDWLSGKEYKCKVSSKGLPSSI EKTISNATGQPREPQVYTLPPSQEEM

TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKS RWQEGNVFSCSVL

HEALHNHYTQKSLSLSLGKIEGRMDKQSEDFRVIGPAHPILAGVGEDALLTCQLLPK RTTMHVEVRWYRSE PSTPVFVHRDGVEVTEMQMEEYRGWVEWIENGIAKGNVALKIHNIQPSDNGQYWCHFQDG NYCGETSLLL

KVAGLGSAPSIHMEGPGESGVQLVCTARGWFPEPQVYWEDIRGEKLLAVSEHRIQDK DGLFYAEATLWRN

ASAESVSCLVHNPVLTEEKGSVISLPEKLQTELASLKVNGPSQPILVRVGEDIQLTC YLSPKANAQSMEVRW

DRSHRYPAVHVYMDGDHVAGEQMAEYRGRTVLVSDAIDEGRLTLQILSARPSDDGQY RCLFEKDDVYQEA

SLDLKWSLGSSPLITVEGQEDGEMQPMCSSDGWFPQPHVPWRDMEGKTIPSSSQALT QGSHGLFHVQTL

LRVTNISAVDVTCSISIPFLGEEKIATFSLSGW (SEQ ID NO: 78).

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: 78 and/or a human equivalent of the PD L1-Fc-BTNL2 chimeric protein.

Diseases, Methods of Treatment, and Mechanisms of Action

A chimeric protein disclosed herein may be used in the treatment of cancer and/or in the treatment of an inflammatory disease.

Aspects of the present invention provide methods of treating cancer. The methods comprise a step of administering to a subject in need thereof an effective amount of a pharmaceutical composition which comprises a chimeric protein as disclosed herein.

In embodiments, the present invention pertains to cancers and/or tumors; for example, the treatment or prevention of cancers and/or tumors. As disclosed elsewhere herein, the treatment of cancer involves, in embodiments, modulating the immune system with the present chimeric proteins to favor of blocking or preventing immune inhibition (e.g., by a cancer cell) over immune stimulation.

In some aspects, the present chimeric agents are used in methods of preventing the cellular transmission of an immunosuppressive signal.

In aspects providing methods of treating cancer, treating an inflammatory disease, or modulating a patient's immune response, the methods comprise a step of administering to a subject in need thereof an effective amount of a pharmaceutical composition comprising a chimeric protein as disclosed herein. In embodiments, inhibition of the patient's T cells is reduced. In embodiments, the patient has a tumor and one or more tumor cells are prevented from transmitting an immunosuppressive signal. In embodiments, the method reduces the amount or activity of regulatory T cells (Tregs) as compared to untreated subjects or subjects treated with antibodies directed to the first transmembrane protein, the second transmembrane protein, and/or their respective ligands or receptors. In embodiments, the method increases priming of effector T cells in draining lymph nodes of the subject as compared to untreated subjects or subjects treated with antibodies directed to the first transmembrane protein, the second transmembrane protein, and/or their respective ligands or receptors. In embodiments, the method causes an overall decrease in immunosuppressive cells and a shift toward a more inflammatory tumor environment as compared to untreated subjects or subjects treated with antibodies directed to the first transmembrane protein, the second transmembrane protein, and/or their respective ligands or receptors.

In embodiments, the extracellular domain may be used to produce a soluble protein to competitively inhibit signaling by that receptor's ligand. In embodiments, the extracellular domains of the transmembrane protein provide immune inhibitory signals. In embodiments, an immune inhibitory signal refers to a signal that diminishes or eliminates an immune response. For example, in the context of oncology, such signals may diminish or eliminate antitumor immunity. Under normal physiological conditions, inhibitory signals are useful in the maintenance of self-tolerance (e.g., prevention of autoimmunity) and also to protect tissues from damage when the immune system is responding to pathogenic infection. For instance, without limitation, immune inhibitory signal may be identified by detecting an increase in cellular proliferation, cytokine production, cell killing activity or phagocytic activity when such an inhibitory signal is blocked.

In embodiments, the chimeric protein of the invention provides a localized trap or sequester of immune inhibitory signals.

In embodiments, the present chimeric proteins are capable of, and can be used in methods comprising, suppressing immune inhibition (e.g., that allows tumors to survive).

In embodiments, the present chimeric proteins are capable of, or can be used in methods comprising, modulating the amplitude of an immune response, e.g., modulating the level of effector output. In embodiments, e.g., when used for the treatment of a cancer and/or an inflammatory disease, the present chimeric proteins alter the extent of immune stimulation as compared to immune inhibition to increase the amplitude of a T cell response, including, without limitation, stimulating increased levels of cytokine production, proliferation or target killing potential.

In embodiments, the present chimeric proteins are capable of, or find use in methods involving, masking an inhibitory ligand on the surface of a tumor cell. Accordingly, the present chimeric proteins, in embodiments are capable of, or find use in methods involving, reducing or eliminating an inhibitory immune signal. It is often desirable to enhance immune stimulatory signal transmission to boost an immune response, for instance to enhance a patient's anti-tumor immune response.

In embodiments, the chimeric protein of the present invention comprises extracellular domains of transmembrane proteins which have immune stimulatory properties, which enhance, increase, and/or stimulate the transmission of an immune stimulatory signal, by way of non-limiting example, the binding of the transmembrane protein with its ligand/receptor.

In embodiments, the chimeric protein comprises an immune stimulatory signal which is an extracellular domain of a ligand of an immune stimulatory signal; this may act on a T cell that bears a cognate receptor of the immune stimulatory signal.

In embodiments, the extracellular domain may be used to provide artificial signaling.

In embodiments, the extracellular domain of the transmembrane protein is an immune stimulatory signal.

In embodiments, the present invention pertains to cancers and/or tumors; for example, the treatment or prevention of cancers and/or tumors. As disclosed elsewhere herein, the treatment of cancer involves, in embodiments, modulating the immune system with the present chimeric proteins to favor of increasing or activating immune stimulatory signals. In embodiments, the method reduces the amount or activity of regulatory T cells (Tregs) as compared to untreated subjects or subjects treated with antibodies directed to the first transmembrane protein, the second transmembrane protein, and/or their respective ligands or receptors. In embodiments, the method increases priming of effector T cells in draining lymph nodes of the subject as compared to untreated subjects or subjects treated with antibodies directed to the first transmembrane protein, the second transmembrane protein, and/or their respective ligands or receptors. In embodiments, the method causes an overall decrease in immunosuppressive cells and a shift toward a more inflammatory tumor environment as compared to untreated subjects or subjects treated with antibodies directed to the first transmembrane I protein, the second transmembrane protein, and/or their respective ligands or receptors.

In embodiments, the present chimeric proteins are capable of, or can be used in methods comprising, modulating the amplitude of an immune response, e.g., modulating the level of effector output. In embodiments, e.g., when used for the treatment of cancer, the present chimeric proteins alter the extent of immune stimulation as compared to immune inhibition to increase the amplitude of a T cell response, including, without limitation, stimulating increased levels of cytokine production, proliferation or target killing potential. In embodiments, the patient's T cells are activated and/or stimulated by the chimeric protein, with the activated T cells being capable of dividing and/or secreting cytokines.

Cancers or tumors refer to an uncontrolled growth of cells and/or abnormal increased cell survival and/or inhibition of apoptosis which interferes with the normal functioning of the bodily organs and systems. Included are benign and malignant cancers, polyps, hyperplasia, as well as dormant tumors or micrometastases. Also, included are cells having abnormal proliferation that is not impeded by the immune system (e.g., virus-infected cells). The cancer may be a primary cancer or a metastatic cancer. The primary cancer may be an area of cancer cells at an originating site that becomes clinically detectable, and may be a primary tumor. In contrast, the metastatic cancer may be the spread of a disease from one organ or part to another non-adjacent organ or part. The metastatic cancer may be caused by a cancer cell that acquires the ability to penetrate and infiltrate surrounding normal tissues in a local area, forming a new tumor, which may be a local metastasis. The cancer may also be caused by a cancer cell that acquires the ability to penetrate the walls of lymphatic and/or blood vessels, after which the cancer cell is able to circulate through the bloodstream (thereby being a circulating tumor cell) to other sites and tissues in the body. The cancer may be due to a process such as lymphatic or hematogeneous spread. The cancer may also be caused by a tumor cell that comes to rest at another site, re-penetrates through the vessel or walls, continues to multiply, and eventually forms another clinically detectable tumor. The cancer may be this new tumor, which may be a metastatic (or secondary) tumor.

The cancer may be caused by tumor cells that have metastasized, which may be a secondary or metastatic tumor. The cells of the tumor may be like those in the original tumor. As an example, if a breast cancer or colon cancer metastasizes to the liver, the secondary tumor, while present in the liver, is made up of abnormal breast or colon cells, not of abnormal liver cells. The tumor in the liver may thus be a metastatic breast cancer or a metastatic colon cancer, not liver cancer.

The cancer may have an origin from any tissue. The cancer may originate from melanoma, colon, breast, or prostate; thus, the cancer may comprise cells that were originally skin, colon, breast, or prostate tissue, respectively. The cancer may also be a hematological malignancy, which may be leukemia or lymphoma. The cancer may invade a tissue such as liver, lung, bladder, or intestinal.

Representative cancers and/or tumors of the present invention include, but are not limited to, a basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and central nervous system cancer; breast cancer; cancer of the peritoneum; cervical cancer; choriocarcinoma; colon and rectum cancer; connective tissue cancer; cancer of the digestive system; endometrial cancer; esophageal cancer; eye cancer; cancer of the head and neck; gastric cancer (including gastrointestinal cancer); glioblastoma; hepatic carcinoma; hepatoma; intra-epithelial neoplasm; kidney or renal cancer; larynx cancer; leukemia; liver cancer; lung cancer (e.g., small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung); melanoma; myeloma; neuroblastoma; oral cavity cancer (lip, tongue, mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; cancer of the respiratory system; salivary gland carcinoma; sarcoma; skin cancer; squamous cell cancer; stomach cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; cancer of the urinary system; vulval cancer; lymphoma including Hodgkin's and non-Hodgkin's lymphoma, as well as B-cell lymphoma (including low grade/follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate grade/follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's Macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblastic leukemia; as well as other carcinomas and sarcomas; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with phakomatoses, edema (such as that associated with brain tumors), and Meigs' syndrome.

In embodiments, the chimeric protein is used to treat a subject that has a treatment-refractory cancer. In embodiments, the chimeric protein is used to treat a subject that is refractory to one or more immune-modulating agents. For example, in embodiments, the chimeric protein is used to treat a subject that presents no response to treatment, or even progress, after 12 weeks or so of treatment. For instance, in embodiments, the subject is refractory to a PD-1 and/or PD-L1 and/or PD-L2 agent, including, for example, nivolumab (ONO-4538/BMS-936558, MDX1106, OPDIVO, BRISTOL MYERS SQUIBB), pembrolizumab (KEYTRUDA, MERCK), MK-3475 (MERCK), BMS 936559 (BRISTOL MYERS SQUIBB), Ibrutinib (PHARMACYCLICS/ABBVIE), atezolizumab (TECENTRIQ, GENENTECH), and/or MPDL3280A (ROCHE)-refractory patients. For instance, in embodiments, the subject is refractory to an anti-CTLA-4 agent, e.g., ipilimumab (YERVOY)-refractory patients (e.g., melanoma patients). Accordingly, in embodiments the present invention provides methods of cancer treatment that rescue patients that are non-responsive to various therapies, including monotherapy of one or more immune-modulating agents.

In embodiments, the present invention provides chimeric proteins which target a cell or tissue within the tumor microenviroment. In embodiments, the cell or tissue within the tumor microenvironment expresses one or more targets or binding partners of the chimeric protein. The tumor microenvironment refers to the cellular milieu, including cells, secreted proteins, physiological small molecules, and blood vessels in which the tumor exists. In embodiments, the cells or tissue within the tumor microenvironment are one or more of: tumor vasculature; tumor-infiltrating lymphocytes; fibroblast reticular cells; endothelial progenitor cells (EPC); cancer-associated fibroblasts; pericytes; other stromal cells; components of the extracellular matrix (ECM); dendritic cells; antigen presenting cells; T-cells; regulatory T cells; macrophages; neutrophils; and other immune cells located proximal to a tumor. In embodiments, the present chimeric protein targets a cancer cell. In embodiments, the cancer cell expresses one or more of targets or binding partners of the chimeric protein.

In embodiments, the present methods provide treatment with the chimeric protein in a patient who is refractory to an additional agent, such "additional agents” being disclosed elsewhere herein, inclusive, without limitation, of the various chemotherapeutic agents disclosed herein.

In embodiments, the present chimeric proteins are capable of, or find use in methods involving, enhancing, restoring, promoting and/or stimulating immune modulation. In embodiments, the present chimeric proteins disclosed herein, restore, promote and/or stimulate the activity or activation of one or more immune cells against tumor cells including, but not limited to: T cells, cytotoxic T lymphocytes, T helper cells, natural killer (NK) cells, natural killer T (NKT) cells, anti-tumor macrophages (e.g., M1 macrophages), B cells, and dendritic cells. In embodiments, the present chimeric proteins enhance, restore, promote and/or stimulate the activity and/or activation of T cells, including, by way of a non limiting example, activating and/or stimulating one or more T- cell intrinsic signals, including a pro-survival signal; an autocrine or paracrine growth signal; a p38 MAPK-, ERK-, STAT-, JAK-, AKT- or PI3K-mediated signal; an anti- apoptotic signal; and/or a signal promoting and/or necessary for one or more of: proinflammatory cytokine production or T cell migration or T cell tumor infiltration.

In embodiments, the present chimeric proteins are capable of, or find use in methods involving, causing an increase of one or more of T cells (including without limitation cytotoxic T lymphocytes, T helper cells, natural killer T (NKT) cells), B cells, natural killer (NK) cells, natural killer T (NKT) cells, dendritic cells, monocytes, and macrophages (e.g., one or more of M1 and M2) into a tumor or the tumor microenvironment. In embodiments, the chimeric protein enhances recognition of tumor antigens by CD8+ T cells, particularly those T cells that have infiltrated into the tumor microenvironment. In embodiments, the present chimeric protein induces CD19 expression and/or increases the number of CD19 positive cells (e.g., CD19 positive B cells). In embodiments, the present chimeric protein induces IL- 15Ra expression and/or increases the number of IL-15Ra positive cells (e.g., IL-15Ra positive dendritic cells).

In embodiments, the present chimeric proteins are capable of, or find use in methods involving, inhibiting and/or causing a decrease in immunosuppressive cells (e.g., myeloid-derived suppressor cells (MDSCs), regulatory T cells (Tregs), tumor associated neutrophils (TANs), M2 macrophages, and tumor associated macrophages (TAMs)), and particularly within the tumor and/or tumor microenvironment (TME). In embodiments, the present therapies may alter the ratio of M1 versus M2 macrophages in the tumor site and/or TME to favor M1 macrophages.

In embodiments, the present chimeric proteins are capable of, and can be used in methods comprising, inhibiting and/or reducing T cell inactivation and/or immune tolerance to a tumor, comprising administering an effective amount of a chimeric protein disclosed herein to a subject.

In embodiments, the present chimeric proteins are able to increase the serum levels of various cytokines or chemokines including, but not limited to, one or more of IFNy, TNFa, IL-2, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-13, IL-15, IL-17A, IL- 17F, and IL-22, CCL2, CCL3, CCL4, CXCL8, CXCL9, CXCL10, CXCL11 and CXCL12. In embodiments, the present chimeric proteins are capable of enhancing IL-2, IL-4, IL-5, IL-10, IL-13, IL-17A, IL-22, TNFa or IFNy in the serum of a treated subject. In embodiments, administration of the present chimeric protein is capable of enhancing TNFa secretion. In a specific embodiment, administration of the present chimeric protein is capable of enhancing superantigen mediated TNFa secretion by leukocytes. Detection of such a cytokine response may provide a method to determine the optimal dosing regimen for the indicated chimeric protein.

In a chimeric protein of the present invention, the chimeric protein is capable of increasing or preventing a decrease in a sub-population of CD4+ and/or CD8+ T cells. In a chimeric protein of the present invention, the chimeric protein is capable of enhancing tumor killing activity by T cells, e.g., indirectly enhancing tumor killing activity by T cells.

In embodiments, the present chimeric proteins inhibit, block and/or reduce cell death of an anti-tumor CD8+ and/or CD4+ T cell; or stimulate, induce, and/or increase cell death of a pro-tumor T cell. T cell exhaustion is a state of T cell dysfunction characterized by progressive loss of proliferative and effector functions, culminating in clonal deletion. Accordingly, a pro-tumor T cell refers to a state of T cell dysfunction that arises during many chronic infections, inflammatory diseases, and cancer. This dysfunction is defined by poor proliferative and/or effector functions, sustained expression of inhibitory receptors and a transcriptional state distinct from that of functional effector or memory T cells. Exhaustion prevents optimal control of infection and tumors. Illustrative pro-tumor T cells include, but are not limited to, Tregs, CD4+ and/or CD8+ T cells expressing one or more checkpoint inhibitory receptors, Th2 cells and Th17 cells. Checkpoint inhibitory receptors refer to receptors expressed on immune cells that prevent or inhibit uncontrolled immune responses. In contrast, an anti-tumor CD8+ and/or CD4+ T cell refers to T cells that can mount an immune response to a tumor.

In embodiments, the present chimeric proteins are capable of, and can be used in methods comprising, increasing a ratio of effector T cells to regulatory T cells. Illustrative effector T cells include ICOS- effector T cells; cytotoxic T cells {e.g., ab TCR, CD3 ⁺ , CD8 ⁺ , CD45RO); CD4 ⁺ effector T cells {e.g., ab TCR, CD3 ⁺ , CD4 ⁺ , CCR7 ⁺ , CD62Lhi, IL 7R/CD 127"-); CD8- effector T cells {e.g., ab TCR, CD3-, CD8-, CCR7 ⁺ , CD62Lhi, IL7R/CD127 ⁺ ); effector memory T cells {e.g., CD62Llow, CD44-, TCR, CD3-, IL7R/CD127 ⁺ , IL-15R-, CCR7low); central memory T cells {e.g., CCR7 ⁺ , CD62L-, CD27-; or CCR7hi, CD44 ⁺ , CD62Lhi, TCR, CD3 ⁺ , IL-7R/CD127 ^÷ , IL-15R ⁺ ); CD62L ⁺ effector T cells; CD8 ⁺ effector memory T cells (TEM) including early effector memory T cells (CD27- CD62L-) and late effector memory T cells (CD27- CD62L-) (TemE and TemL, respectively); CD127( ⁺ )CD25(low/-) effector T cells; CD127( )CD25( ) effector T cells; CD8 ⁺ stem cell memory effector cells (TSCM) {e.g., CD44(low)CD62L(high)CD 122(high)sca( ⁺ )); TH1 effector T-cells {e.g., CXCR3 ⁺ , CXCR6 ⁺ and CCR5 ⁺ ; or ab TCR, CD3 ⁺ , CD4 ⁺ , IL-12R ⁺ , I FNyR-, CXCR3 ⁺ ), TH2 effector T cells {e.g., CCR3", CCR4 ⁺ and CCR8 ⁺ ; or ab TCR, CD3 ⁺ , CD4 ⁺ , IL-4R ⁺ , IL-33R ⁺ , CCR4 ⁺ , IL-17RB ⁺ , CRTH2 ⁺ ); TH9 effector T cells {e.g., ab TCR, CD3 ⁺ , CD4 ⁺ ); TH17 effector T cells {e.g., ab TCR, CD3 ⁺ , CD4 ⁺ , IL-23R ^÷ , CCR6 ⁺ , IL-1 R ^÷ ); CD4 ⁺ CD45RO ⁺ CCR7 ⁺ effector T cells, CD4 ⁺ CD45RO ⁺ CCR7( ) effector T cells; and effector T cells secreting IL-2, IL-4 and/or I FN-g. Illustrative regulatory T cells include ICOS ⁺ regulatory T cells, CD4 ⁺ CD25 ⁺ FOXP3 ⁺ regulatory T cells, CD4-CD25-· regulatory T cells, CD4-CD25- regulatory T cells, CD4 ⁺ CD25high regulatory T cells, TI M-3 ⁺ PD-1 ⁺ regulatory T cells, lymphocyte activation gene-3 (LAG-3) ⁺ regulatory T cells, CTLA-4/CD152- regulatory T cells, neuropilin-1 (Nrp-1 ) ⁺ regulatory T cells, CCR4 ⁺ CCR8 ⁺ regulatory T cells, CD62L (L-selectin)- regulatory T cells, CD45RBIow regulatory T cells, CD127low regulatory T cells, LRRC32/GARP- regulatory T cells, CD39- regulatory T cells, GITR- regulatory T cells, LAP- regulatory T cells, 1 B1 1 - regulatory T cells, BTLA- regulatory T cells, type 1 regulatory T cells (Tr1 cells), T helper type 3 (Th3) cells, regulatory cell of natural killer T cell phenotype (NKTregs), CD8- regulatory T cells, CD8-CD28- regulatory T cells and/or regulatory T-cells secreting IL-10, IL-35, TGF-b, TNF-a, Galectin-1, IFN-g and/or MCP1.

In embodiments, the chimeric protein of the invention causes an increase in effector T cells (e.g., CD4+CD25- T cells).

In embodiments, the chimeric protein causes a decrease in regulatory T cells (e.g., CD4+CD25+ T cells).

In embodiments, the chimeric protein generates a memory response which may be capable of preventing relapse or protecting the animal from a recurrence and/or preventing, or reducing the likelihood of, metastasis. Thus, an animal treated with the chimeric protein is later able to attack tumor cells and/or prevent development of tumors when rechallenged after an initial treatment with the chimeric protein. Accordingly, a chimeric protein of the present invention stimulates both active tumor destruction and also immune recognition of tumor antigens, which are essential in programming a memory response capable of preventing relapse.

In embodiments, the chimeric protein is capable of causing activation of antigen presenting cells, e.g., indirectly capable of causing activation of antigen presenting cells.

In embodiments, the chimeric protein is capable enhancing the ability of antigen presenting cells to present antigen, e.g., indirectly capable enhancing the ability of antigen presenting cells to present antigen.

In embodiments, the present chimeric proteins are capable of, and can be used in methods comprising, transiently depleting or inhibiting regulatory or immune suppressive and/or transiently stimulating effector T cells for longer than about 12 hours, about 24 hours, about 48 hours, about 72 hours or about 96 hours or about 1 week or about 2 weeks. In embodiments, the transient depletion or inhibition of immune inhibitory cells and/or the transient stimulation of effector T cells occurs substantially in a patient's bloodstream or in a particular tissue/location including lymphoid tissues such as for example, the bone marrow, lymph-node, spleen, thymus, mucosa-associated lymphoid tissue (MALT), non-lymphoid tissues, or in the tumor microenvironment.

In a chimeric protein of the present invention, the present chimeric protein unexpectedly provides binding of the extracellular domain components to their respective binding partners with slow off rates (Kd or K _0ff ). In embodiments, this provides an unexpectedly long interaction of the receptor to ligand and vice versa. Such an effect allows for a sustained negative signal masking effect and/or a longer positive signal effect, e.g., increase in or activation of immune stimulatory signals. For example, the present chimeric protein, e.g., via the long off rate binding allows sufficient signal transmission to provide immune cell proliferation, allow for anti-tumor attack, allows sufficient signal transmission to provide release of stimulatory signals, e.g., cytokines.

In a chimeric protein of the present invention, the chimeric protein is capable of forming a stable synapse between cells. The stable synapse of cells promoted by the chimeric proteins (e.g., between cells bearing negative signals) provides spatial orientation to favor tumor reduction - such as positioning the T cells to attack tumor cells and/or sterically preventing the tumor cell from delivering negative signals, including negative signals beyond those masked by the chimeric protein of the invention. In embodiments, this provides longer on-target (e.g., intra-tumoral) half-life (h _/2 ) as compared to serum ti _/2 of the chimeric proteins. Such properties could have the combined advantage of reducing off-target toxicities associated with systemic distribution of the chimeric proteins.

In embodiments, the chimeric protein is capable of providing a sustained immunomodulatory effect.

The present chimeric proteins provide synergistic therapeutic effects (e.g., anti-tumor effects) as it allows for improved site-specific interplay of two immunotherapy agents. In embodiments, the present chimeric proteins provide the potential for reducing off-site and/or systemic toxicity.

In embodiments, the present chimeric protein exhibit enhanced safety profiles. In embodiment, the present chimeric protein exhibit reduced toxicity profiles. For example, administration of the present chimeric proteins may result in reduced side effects such as one or more of diarrhea, inflammation (e.g., of the gut), or weight loss, which occur following administration of antibodies directed to the ligand(s)/receptor(s) targeted by the extracellular domains of the present chimeric proteins. In embodiments, the present chimeric protein provides improved safety, as compared to antibodies directed to the ligand(s)/receptor(s) targeted by the extracellular domains of the present chimeric proteins, yet, without sacrificing efficacy.

In embodiments, the present chimeric proteins provide reduced side effects, e.g., Gl complications, relative to current immunotherapies, e.g., antibodies directed to I igand (s)/receptor(s) targeted by the extracellular domains of the present chimeric proteins. Illustrative Gl complications include abdominal pain, appetite loss, autoimmune effects, constipation, cramping, dehydration, diarrhea, eating problems, fatigue, flatulence, fluid in the abdomen or ascites, gastrointestinal (Gl) dysbiosis, Gl mucositis, inflammatory bowel disease, irritable bowel syndrome (IBS-D and IBS-C), nausea, pain, stool or urine changes, ulcerative colitis, vomiting, weight gain from retaining fluid, and/or weakness.

Other aspects of the present invention provide methods of treating an inflammatory disease. The methods comprise a step of administering to a subject in need thereof an effective amount of a pharmaceutical composition which comprises a chimeric protein as disclosed herein. A patient in need of such treatment may have or be at risk for having an abnormally elevated immune system activity and/or an autoimmune disorder.

In embodiments, the chimeric protein for treating an inflammatory disease, disclosed herein, is useful for treating autoimmune diseases, alloimmune responses, or any other disease, disorder or condition that involves a T cell response in a patient in need thereof. Generally, these are conditions in which the immune system of an individual (e.g., activated T cells) attacks the individual's own tissues and cells, or implanted tissues, cells, or molecules (as in a graft or transplant). Non-limiting examples of diseases and disorders that can be treated according to the methods disclosed herein, include, e.g., autoimmune disease or disorder (e.g., IBD and rheumatoid arthritis), transplant rejection, graft-versus-host disease (GVHD), inflammation, asthma, allergies, and chronic infection. In embodiments, the chimeric protein for treating an inflammatory disease, disclosed herein, is useful for improving tolerance to transplanted cells and tissues, as well as graft versus host disease,

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

In embodiments, a patient in need of treatment of an inflammatory disease, has received or will receive an allogenic cell, tissue, and/or organ transplant. Thus, the patient in need may receive the chimeric protein, in part, to help prevent graft-versus-host disease (GVHD), transplant rejection, and T cell proliferative disorder.

In embodiments, the inflammatory disease is an autoimmune disease or condition. Autoimmune diseases or disorders occur when a patient's own antigens become targets for an immune response. In embodiments, the treatment of an autoimmune disease or disorder may involve modulating the immune system with the present chimeric proteins to favor immune inhibition over immune stimulation. Examples of an autoimmune disease or disorder includes Addison's disease, autoimmune epilepsy, Autoimmune hepatitis, celiac disease, Crohn's disease, dermatomyositis, diabetes mellitus, Fibromyalgia, Goodpasture's syndrome, Grave's disease., Guillain-Barre syndrome, Hashimoto's thyroiditis, hypersensitivity reactions (e.g., allergies, hay fever, asthma, and acute edema cause Type I hypersensitivity reactions), lupus, lupus erythematosus, Menier's syndrome, multiple sclerosis, myasthenia gravis, pernicious anemia, Primary biliary sclerosis, Rasmussen's encephalitis, Reiter's syndrome, rheumatoid arthritis, scleroderms, Sclerosing cholangitis, Sjogren's syndrome, systemic lupus erythematosus, ulcerative colitis, Wegener's granulomatosis, and vasculitis.

In embodiments, the chimeric protein for treating an inflammatory disease, disclosed herein, is also useful for reducing inflammatory reactions caused by exposure to immune checkpoint inhibitors and/or adoptive cell therapy.

In some aspects, the present chimeric agents are used to eliminate intracellular pathogens. In some aspects, the present chimeric agents are used to treat one or more infections. In embodiments, the present chimeric proteins are used in methods of treating viral infections (including, for example, HIV and HCV), parasitic infections (including, for example, malaria), and bacterial infections. In embodiments, the infections induce immunosuppression. For example, HIV infections often result in immunosuppression in the infected subjects. Accordingly, as disclosed elsewhere herein, the treatment of such infections may involve, in embodiments, modulating the immune system with the present chimeric proteins to favor immune stimulation over blocking or preventing immune inhibition (e.g., by a cancer cell).

Alternatively, the present invention provides methods for treating infections that induce immunoactivation. For example, intestinal helminth infections have been associated with chronic immune activation. In these embodiments, the treatment of such infections may involve modulating the immune system with the present chimeric proteins to favor immune inhibition over immune stimulation.

In embodiments, the present invention provides methods of treating viral infections including, without limitation, acute or chronic viral infections, for example, of the respiratory tract, of papilloma virus infections, of herpes simplex virus (HSV) infection, of human immunodeficiency virus (HIV) infection, and of viral infection of internal organs such as infection with hepatitis viruses. In embodiments, the viral infection is caused by a virus of family Flaviviridae. In embodiments, the virus of family Flaviviridae is selected from Yellow Fever Virus, West Nile virus, Dengue virus, Japanese Encephalitis Virus, St. Louis Encephalitis Virus, and Hepatitis C Virus. In embodiments, the viral infection is caused by a virus of family Picornaviridae, e.g., poliovirus, rhinovirus, coxsackievirus. In embodiments, the viral infection is caused by a member of Orthomyxoviridae, e.g., an influenza virus. In embodiments, the viral infection is caused by a member of Retroviridae, e.g., a lentivirus. In embodiments, the viral infection is caused by a member of Paramyxoviridae, e.g., respiratory syncytial virus, a human parainfluenza virus, rubulavirus (e.g., mumps virus), measles virus, and human metapneumovirus. In embodiments, the viral infection is caused by a member of Bunyaviridae, e.g., Hantavirus. In embodiments, the viral infection is caused by a member of Reoviridae, e.g., a rotavirus.

In embodiments, the present invention provides methods of treating parasitic infections such as protozoan or helminths infections. In embodiments, the parasitic infection is by a protozoan parasite. In embodiments, the oritiziab parasite is selected from intestinal protozoa, tissue protozoa, or blood protozoa. Illustrative protozoan parasites include, but are not limited to, Entamoeba hystolytica, Giardia lamblia, Cryptosporidium muris, Trypanosomatida gambiense, Trypanosomatida rhodesiense, Trypanosomatida crusi, Leishmania mexicana, Leishmania braziliensis, Leishmania tropica, Leishmania donovani, Toxoplasma gondii, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, Plasmodium falciparum, Trichomonas vaginalis, and Histomonas meleagridis. In embodiments, the parasitic infection is by a helminthic parasite such as nematodes (e.g., Adenophorea). In embodiments, the parasite is selected from Secementea (e.g., Trichuris trichiura, Ascaris lumbricoides, Enterobius vermicularis, Ancylostoma duodenale, Necator americanus, Strongyloides stercoralis, Wuchereria bancrofti, Dracunculus medinensis). In embodiments, the parasite is selected from trematodes (e.g., blood flukes, liver flukes, intestinal flukes, and lung flukes). In embodiments, the parasite is selected from Schistosoma mansoni, Schistosoma haematobium, Schistosoma japonicum, Fasciola hepatica, Fasciola gigantica, Fieterophyes, Paragonimus westermani. In embodiments, the parasite is selected from cestodes (e.g., Taenia solium, Taenia saginata, Flymenolepis nana, Echinococcus granulosus).

In embodiments, the present invention provides methods of treating bacterial infections. In embodiments, the bacterial infection is by gram-positive bacteria, gram-negative bacteria, aerobic and/or anaerobic bacteria. In embodiments, the bacteria is selected from, but not limited to, Staphylococcus, Lactobacillus, Streptococcus, Sarcina, Escherichia, Enterobacter, Klebsiella, Pseudomonas, Acinetobacter, Mycobacterium, Proteus, Campylobacter, Citrobacter, Nisseria, Baccillus, Bacteroides, Peptococcus, Clostridium, Salmonella, Shigella, Serratia, Flaemophilus, Brucella and other organisms. In embodiments, the bacteria is selected from, but not limited to, Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas acidovorans, Pseudomonas alcaligenes, Pseudomonas putida, Stenotrophomonas maltophilia, Burkholderia cepacia, Aeromonas hydrophilia, Escherichia coli, Citrobacter freundii, Salmonella typhimurium, Salmonella typhi, Salmonella paratyphi, Salmonella enteritidis, Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Enterobacter cloacae, Enterobacter aerogenes, Klebsiella pneumoniae, Klebsiella oxytoca, Serratia marcescens, Francisella tularensis, Morganella morganii, Proteus mirabilis, Proteus vulgaris, Providencia alcalifaciens, Providencia rettgeri, Providencia stuartii, Acinetobacter baumannii, Acinetobacter calcoaceticus, Acinetobacter haemolyticus, Yersinia enterocolitica, Yersinia pestis, Yersinia pseudotuberculosis, Yersinia intermedia, Bordetella pertussis, Bordetella parapertussis, Bordetella bronchiseptica, Flaemophilus influenzae, Flaemophilus parainfluenzae, Flaemophilus haemolyticus, Flaemophilus parahaemolyticus, Flaemophilus ducreyi, Pasteurella multocida, Pasteurella haemolytica, Branhamella catarrhalis, Flelicobacter pylori, Campylobacter fetus, Campylobacter jejuni, Campylobacter coli, Borrelia burgdorferi, Vibrio cholerae, Vibrio parahaemolyticus, Legionella pneumophila, Listeria monocytogenes, Neisseria gonorrhoeae, Neisseria meningitidis, Kingella, Moraxella, Gardnerella vaginalis, Bacteroides fragilis, Bacteroides distasonis, Bacteroides 3452A homology group, Bacteroides vulgatus, Bacteroides ovalus, Bacteroides thetaiotaomicron, Bacteroides uniformis, Bacteroides eggerthii, Bacteroides splanchnicus, Clostridium difficile, Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium leprae, Corynebacterium diphtheriae, Corynebacterium ulcerans, Streptococcus pneumoniae, Streptococcus agalactiae, Streptococcus pyogenes, Enterococcus faecalis, Enterococcus faecium, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Staphylococcus intermedius, Staphylococcus hyicus subsp. hyicus, Staphylococcus haemolyticus, Staphylococcus hominis, or Staphylococcus saccharolyticus.

Combination Therapies and Conjugation

In embodiments, the invention provides for chimeric proteins and methods that further comprise administering an additional agent to a subject. In embodiments, the invention pertains to co-administration and/or co-formulation. Any of the compositions disclosed herein may be co-formulated and/or co-administered. In embodiments, any chimeric protein disclosed herein acts synergistically when co-administered with another agent and is administered at doses that are lower than the doses commonly employed when such agents are used as monotherapy. In embodiments, any agent referenced herein may be used in combination with any of the chimeric proteins disclosed herein.

In aspects and embodiments of the present invention, the patient in need of a cancer treatment comprising a chimeric protein, as disclosed herein, has been treated with, is contemporaneously treated with, or is subsequently treated with another anti-cancer therapy, as disclosed herein.

The other anti-cancer therapy may comprise radiotherapy.

The other anti-cancer therapy may comprise an antibody or an antibody fragment, i.e., an antibody-based immunotherapy. In embodiments, the antibody or antibody fragment is selected from one or more of a monoclonal antibody, polyclonal antibody, antibody fragment, Fab, Fab', Fab'-SH, F(ab')2, Fv, single chain Fv, diabody, linear antibody, bispecific antibody, multispecific antibody, chimeric antibody, humanized antibody, human antibody, and fusion protein comprising the antigen-binding portion of an antibody. In embodiments, the antibody or antibody fragment is selected from Adcetris (Brentuximab Vedotin), Ado-Trastuzumab Emtansine, Alemtuzumab, Arzerra (Ofatumumab), Atezolizumab (TECENTRIQ, GENENTECFI), Avastin (Bevacizumab), Avelumab, Bavencio (Avelumab), Besponsa (Inotuzumab Ozogamicin), Bevacizumab, Bexxar (Tositumomab), Blinatumomab, Blincyto (Blinatumomab), BMS 936559 (BRISTOL MYERS SQUIBB), Brentuximab Vedotin, Campath (Alemtuzumab), Cetuximab, Cinqair (Reslizumab), Cyramza (Ramucirumab), Daratumumab, Darzalex (Daratumumab), Denosumab, Dinutuximab, Durvalumab, Elotuzumab, Empliciti (Elotuzumab), Erbitux (Cetuximab), Folfiri-Bevacizumab, Folfiri- Cetuximab, Gazyva (Obinutuzumab), GBR 830 (GLENMARK), Gemtuzumab Ozogamicin, Herceptin (Trastuzumab), Ibritumomab Tiuxetan, Imfinzi (Durvalumab), Inotuzumab Ozogamicin, Ipilimumab, Kadcyla (Ado-trastuzumab Emtansine), Keytruda (Pembrolizumab), Lartruvo (Olaratumab), MEDI6469 (MEDIMMUNE)., MK-3475 (MERCK), MPDL3280A (ROCHE)), Mylotarg (Gemtuzumab Ozogamicin), Necitumumab, Nivolumab (ONO-4538/B MS-936558, MDX1106, OPDIVO, BRISTOL MYERS SQUIBB), Obinutuzumab, Ofatumumab, Olaratumab, Opdivo (Nivolumab), Opdivo (Nivolumab), or Zevalin (Ibritumomab Tiuxetan). In embodiments, Panitumumab, Pembrolizumab (KEYTRUDA, Merck), Perjeta (Pertuzumab), Pertuzumab, Portrazza (Necitumumab), Prolia (Denosumab), Ramucirumab, Rituxan (Rituximab), Rituximab and Hyaluronidase Human, Siltuximab, Sylvant (Siltuximab), Tecentriq (Atezolizumab), the antibody is Keytruda (Pembrolizumab), Trastuzumab, Unituxin (Dinutuximab), Urelumab (BMS- 663513 and anti-4-1 BB antibody), Vectibix (Panitumumab), Xgeva (Denosumab), Yervoy (Ipilimumab), and Yervoy (Ipilimumab).

In embodiments, the other anti-cancer therapy is one or more immune-modulating agents selected from an agent that blocks, reduces and/or inhibits PD-1 and PD-L1 or PD-L2 and/or the binding of PD-1 with PD-L1 or PD-L2 (by way of non-limiting example, one or more of nivolumab (ONO-4538/B MS-936558, MDX1106, OPDIVO, BRISTOL MYERS SQUI BB), pembrolizumab (KEYTRUDA, Merck), MK-3475 (MERCK), BMS 936559 (BRISTOL MYERS SQUI BB), atezolizumab (TECENTRIQ, GENENTECH), MPDL3280A (ROCHE)), an agent that increases and/or stimulates CD137 (4-1 BB) and/or the binding of CD137 (4-1 BB) with one or more of 4-1 BB ligand (by way of non-limiting example, urelumab (BMS-663513 and anti-4-1 BB antibody), and an agent that blocks, reduces and/or inhibits the activity of CTLA-4 and/or the binding of CTLA-4 with one or more of AP2M1 , CD80, CD86, SHP-2, and PPP2R5A and/or the binding of 0X40 with OX40L (by way of non-limiting example GBR 830 (GLENMARK), MEDI6469 (MEDIMMUNE).

The other anti-cancer therapy may include a synthetic polypeptide comprising at least one domain capable of binding an immune checkpoint molecule. In embodiments, the immune checkpoint molecule is selected from PD-1 , PD-L1 , PD-L2, ICOS, ICOSL, and CTLA-4.

The other anti-cancer therapy may be surgery to excise the cancer, i.e., tumor.

The other anti-cancer therapy may include a cell-based immuno-oncology therapy, e.g., chimeric antigen receptor T cell (CAR-T), including wherein the CAR-T secretes the chimeric protein either continuously or in response to specific tumor antigen recognition.

The other anti-cancer therapy may include administration of one more chemotherapeutic agents.

In aspects and embodiments of the present invention, the one or more chemotherapeutic agent selected from 5-FU (Fluorouracil), Abemaciclib, Abiraterone Acetate, Abitrexate (Methotrexate), Abraxane (Paclitaxel Albumin-stabilized Nanoparticle Formulation), ABVD, ABVE, ABVE-PC, AC, Acalabrutinib, AC-T, ADE, Adriamycin (Doxorubicin), Afatinib Dimaleate, Afinitor (Everolimus), Afinitor Difsperz (Everolimus), Akynzeo (Netupitant and Palonosetron), Aldara (Imiquimod), Aldesleukin, Alecensa (Alectinib), Alectinib, Alimta (PEMETREXED), Aliqopa (Copanlisib Hydrochloride), Alkeran (Melphalan), Aloxi (Palonosetron Hydrochloride), Alunbrig (Brigatinib), Ambochlorin (Chlorambucil), Amboclorin (Chlorambucil), Amifostine, Aminolevulinic Acid, Anastrozole, Aprepitant, Aredia (Pamidronate), Arimidex (Anastrozole), Aromasin (Exemestane), Arranon (Nelarabine), Arsenic Trioxide, Asparaginase Erwinia chrysanthemi, Axicabtagene Ciloleucel, Axitinib, Azacitidine, BEACOPP, Becenum (Carmustine), Beleodaq (Belinostat), Belinostat, Bendamustine Hydrochloride, BEP, Bexarotene, Bicalutamide, BiCNU (Carmustine), Blenoxane (Bleomycin), Bortezomib, Bosulif (Bosutinib), Bosutinib, Brigatinib, BuMel, Busulfan, Busulfex (Busulfan)C, Cabazitaxel, Cabometyx (Cabozantinib), Cabozantinib-S-Malate, CAF, Calquence (Acalabrutinib), Camptosar (Irinotecan Hydrochloride), Capecitabine, CAPOX, Caprelsa (Vandetanib), Carac (Fluorouracil— T opical), Carboplatin, CARBOPLATI N-TAXOL, Carfilzomib, Carmubris (Carmustine), Carmustine, Casodex (Bicalutamide), CeeNU (Lomustine), CEM, Ceritinib, Cerubidine (Daunorubicin), Cervarix (Recombinant HPV Bivalent Vaccine), CEV, Chlorambucil, CHLORAMBUCIL- PREDNISONE, CHOP, Cisplatin, Cladribine, Clafen (Cyclophosphamide), Clofarabine, Clofarex (Clofarabine), Clolar (Clofarabine), CMF, Cobimetinib, Cometriq (Cabozantinib), Copanlisib Hydrochloride, COPDAC, COPP, COPP-ABV, Cosmegen (Dactinomycin), Cotellic (Cobimetinib), Crizotinib, CVP, Cyclophosphamide, Cyfos (Ifosfamide), Cytarabine, Cytarabine Liposome, Cytosar-U (Cytarabine), Cytoxan (Cyclophosphamide), Cytoxan (Cytoxan), Dabrafenib, Dacarbazine, Dacogen (Decitabine), Dactinomycin, Dasatinib, Daunorubicin Hydrochloride, Daunorubicin Hydrochloride and Cytarabine Liposome, DaunoXome (Daunorubicin Lipid Complex), Decadron (Dexamethasone), Decitabine, Defibrotide Sodium, Defitelio (Defibrotide Sodium), Degarelix, Denileukin Diftitox, DepoCyt (Cytarabine Liposome), Dexamethasone, Dexamethasone Intensol (Dexamethasone), Dexpak Taperpak (Dexamethasone), Dexrazoxane Hydrochloride, Docefrez (Docetaxel), Docetaxel, Doxil (Doxorubicin Hydrochloride Liposome), Doxorubicin Hydrochloride, Doxorubicin Hydrochloride Liposome, Dox-SL (Doxorubicin Hydrochloride Liposome), Droxia (Hydroxyurea), DTIC (Decarbazine), DTIC-Dome (Dacarbazine), Efudex (Fluorouracil— Topical), Eligard (Leuprolide), Elitek (Rasburicase), Ellence (Ellence (epirubicin)), Eloxatin (Oxaliplatin), Elspar (Asparaginase), Eltrombopag Olamine, Emcyt (Estramustine), Emend (Aprepitant), Enasidenib Mesylate, Enzalutamide, Epirubicin Hydrochloride, EPOCH, Eribulin Mesylate, Erivedge (Vismodegib), Erlotinib Hydrochloride, Erwinaze (Asparaginase Erwinia chrysanthemi), Ethyol (Amifostine), Etopophos (Etoposide Phosphate), Etoposide, Etoposide Phosphate, Eulexin (Flutamide), Evacet (Doxorubicin Hydrochloride Liposome), Everolimus, Evista (Raloxifene Hydrochloride), Evomela (Melphalan Hydrochloride), Exemestane, Fareston (Toremifene), Farydak (Panobinostat), Faslodex (Fulvestrant), FEC, Femara (Letrozole), Filgrastim, Firmagon (Degarelix), FloPred (Prednisolone), Fludara (Fludarabine), Fludarabine Phosphate, Fluoroplex (Fluorouracil), Fluorouracil, Flutamide, Folex (Methotrexate), Folex PFS (Methotrexate), FOLFIRI, FOLFIRINOX, FOLFOX, Folotyn (Pralatrexate), FUDR (FUDR (floxuridine)), FU-LV, Fulvestrant, Gardasil (Recombinant HPV Quadrivalent Vaccine), Gardasil 9 (Recombinant HPV Nonavalent Vaccine), Gefitinib, Gemcitabine Hydrochloride, GEMCITABINE-CISPLATIN, GEMCITABINE-OXALIPLATIN, Gemzar (Gemcitabine), Gilotrif (Afatinib Dimaleate), Gilotrif (Afatinib), Gleevec (Imatinib Mesylate), Gliadel (Carmustine), Glucarpidase, Goserelin Acetate, Halaven (Eribulin Mesylate), Hemangeol (Propranolol Hydrochloride), Hexalen (Altretamine), HPV Bivalent Vaccine, Recombinant, HPV Nonavalent Vaccine, Recombinant, HPV Quadrivalent Vaccine, Recombinant, Hycamtin (Topotecan Hydrochloride), Hycamtin (Topotecan), Hydrea (Hydroxyurea), Hydroxyurea, Hyper-CVAD, Ibrance (Palbociclib), Ibrutinib, ICE, lclusig (Ponatinib), Idamycin PFS (Idarubicin), Idarubicin Hydrochloride, Idelalisib, Idhifa (Enasidenib), Ifex (Ifosfamide), Ifosfamide, Ifosfamidum (Ifosfamide), Imatinib Mesylate, Imbruvica (Ibrutinib), Imiquimod, Imlygic (Talimogene Laherparepvec), Inlyta (Axitinib), Iressa (Gefitinib), Irinotecan Hydrochloride, Irinotecan Hydrochloride Liposome, Istodax (Romidepsin), Ixabepilone, Ixazomib Citrate, Ixempra (Ixabepilone), Jakafi (Ruxolitinib Phosphate), Jakafi (Ruxolitinib), JEB, Jevtana (Cabazitaxel), Keoxifene (Raloxifene Hydrochloride), Kepivance (Palifermin), Kisqali (Ribociclib), Kyprolis (Carfilzomib), Lanreotide Acetate, Lanvima (Lenvatinib), Lapatinib Ditosylate, Lenalidomide, Lenvatinib Mesylate, Lenvima (Lenvatinib Mesylate), Letrozole, Leucovorin Calcium, Leukeran (Chlorambucil), Leukine (Sargramostim), Leuprolide Acetate, Leustatin (Cladribine), Levulan (Aminolevulinic Acid), Linfolizin (Chlorambucil), LipoDox (Doxorubicin Hydrochloride Liposome), Lomustine, Lonsurf (Trifluridine and Tipiracil), Lupron (Leuprolide), Lynparza (Olaparib), Lysodren (Mitotane), Marqibo (Vincristine Sulfate Liposome), Marqibo Kit (Vincristine Lipid Complex), Matulane (Procarbazine), Mechlorethamine Hydrochloride, Megace (Megestrol), Megestrol Acetate, Mekinist (Trametinib), Melphalan, Melphalan Hydrochloride, Mercaptopurine, Mesnex (Mesna), Metastron (Strontium-89 Chloride), Methazolastone (Temozolomide), Methotrexate, Methotrexate LPF (Methotrexate), Methylnaltrexone Bromide, Mexate (Methotrexate), Mexate-AQ (Methotrexate), Midostaurin, Mitomycin C, Mitoxantrone Hydrochloride, Mitozytrex (Mitomycin C), MOPP, Mostarina (Prednimustine), Mozobil (Plerixafor), Mustargen (Mechlorethamine), Mutamycin (Mitomycin), Myleran (Busulfan), Mylosar (Azacitidine), Nanoparticle Paclitaxel (Paclitaxel Albumin-stabilized Nanoparticle Formulation), Navelbine (Vinorelbine), Nelarabine, Neosar (Cyclophosphamide), Neratinib Maleate, Nerlynx (Neratinib), Netupitant and Palonosetron Hydrochloride, Neulasta (filgrastim), Neulasta (pegfilgrastim), Neupogen (filgrastim), Nexavar (Sorafenib), Nilandron (Nilutamide), Nilotinib, Nilutamide, Ninlaro (Ixazomib), Nipent (Pentostatin), Niraparib Tosylate Monohydrate, Nolvadex (Tamoxifen), Novantrone (Mitoxantrone), Nplate (Romiplostim), Odomzo (Sonidegib), OEPA, OFF, Olaparib, Omacetaxine Mepesuccinate, Oncaspar (Pegaspargase), Oncovin (Vincristine), Ondansetron Hydrochloride, Onivyde (Irinotecan Hydrochloride Liposome), Ontak (Denileukin Diftitox), Onxol (Paclitaxel), OPPA, Orapred (Prednisolone), Osimertinib, Oxaliplatin, Paclitaxel, Paclitaxel Albumin-stabilized Nanoparticle Formulation, PAD, Palbociclib, Palifermin, Palonosetron Hydrochloride, Palonosetron Hydrochloride and Netupitant, Pamidronate Disodium, Panobinostat, Panretin (Alitretinoin), Paraplat (Carboplatin), Pazopanib Hydrochloride, PCV, PEB, Pediapred (Prednisolone), Pegaspargase, Pegfilgrastim, Pemetrexed Disodium, Platinol (Cisplatin), PlatinolAQ (Cisplatin), Plerixafor, Pomalyst (Pomalidomide), Ponatinib Hydrochloride, Pralatrexate, Prednisone, Procarbazine Hydrochloride, Proleukin (Aldesleukin), Promacta (Eltrombopag Olamine), Propranolol Hydrochloride, Purinethol (Mercaptopurine), Purixan (Mercaptopurine), Radium 223 Dichloride, Raloxifene Hydrochloride, Rasburicase, R- CHOP, R-CVP, Reclast (Zoledronic acid), Recombinant Human Papillomavirus (HPV) Bivalent Vaccine, Recombinant Human Papillomavirus (HPV) Nonavalent Vaccine, Recombinant Human Papillomavirus (HPV) Quadrivalent Vaccine, Regorafenib, Relistor (Methylnaltrexone Bromide), R-EPOCH, Revlimid (Lenalidomide), Rheumatrex (Methotrexate), Ribociclib, R-ICE, Rolapitant Hydrochloride, Romidepsin, Romiplostim, Rubex (Doxorubicin), Rubidomycin (Daunorubicin Hydrochloride), Rubraca (Rucaparib), Rucaparib Camsylate, Ruxolitinib Phosphate, Rydapt (Midostaurin), Sandostatin (Octreotide), Sandostatin LAR Depot (Octreotide), Sclerosol Intrapleural Aerosol (Talc), Soltamox (Tamoxifen), Somatuline Depot (Lanreotide Acetate), Sonidegib, Sorafenib Tosylate, Sprycel (Dasatinib), STANFORD V, Sterapred (Prednisone), Sterapred DS (Prednisone), Sterile Talc Powder (Talc), Steritalc (Talc), Sterecyst (Prednimustine), Stivarga (Regorafenib), Sunitinib Malate, Supprelin LA (Histrelin), Sutent (Sunitinib Malate), Sutent (Sunitinib), Synribo (Omacetaxine Mepesuccinate), Tabloid (Thioguanine), TAC, Tafinlar (Dabrafenib), Tagrisso (Osimertinib), Talc, Talimogene Laherparepvec, Tamoxifen Citrate, Tarabine PFS (Cytarabine), Tarceva (Erlotinib), Targretin (Bexarotene), Tasigna (Decarbazine), Tasigna (Nilotinib), Taxol (Paclitaxel), Taxotere (Docetaxel), Temodar (Temozolomide), Temozolomide, Temsirolimus, Tepadina (Thiotepa), Thalidomide, Thalomid (Thalidomide), TheraCys BCG (BCG), Thioguanine, Thioplex (Thiotepa), Thiotepa, TICE BCG (BCG), Tisagenlecleucel, Tolak (Fluorouracil- Topical), Toposar (Etoposide), Topotecan Hydrochloride, Toremifene, Torisel (Temsirolimus), Totect (Dexrazoxane Hydrochloride), TPF, Trabectedin, Trametinib, Treanda (Bendamustine hydrochloride), Trelstar (Triptorelin), Trexall (Methotrexate), Trifluridine and Tipiracil Hydrochloride, Trisenox (Arsenic trioxide), Tykerb (lapatinib), Uridine Triacetate, VAC, Valrubicin, Valstar (Valrubicin Intravesical), Valstar (Valrubicin), VAMP, Vandetanib, Vantas (Histrelin), Varubi (Rolapitant), VelP, Velban (Vinblastine), Velcade (Bortezomib), Velsar (Vinblastine Sulfate), Vemurafenib, Venclexta (Venetoclax), Vepesid (Etoposide), Verzenio (Abemaciclib), Vesanoid (Tretinoin), Viadur (Leuprolide Acetate), Vidaza (Azacitidine), Vinblastine Sulfate, Vincasar PFS (Vincristine), Vincrex (Vincristine), Vincristine Sulfate, Vincristine Sulfate Liposome, Vinorelbine Tartrate, VIP, Vismodegib, Vistogard (Uridine Triacetate), Voraxaze (Glucarpidase), Vorinostat, Votrient (Pazopanib), Vumon (Teniposide), Vyxeos (Daunorubicin Hydrochloride and Cytarabine Liposome), W, Wellcovorin (Leucovorin Calcium), Wellcovorin IV (Leucovorin), Xalkori (Crizotinib), XELIRI, Xeloda (Capecitabine), XELOX, Xofigo (Radium 223 Dichloride), Xtandi (Enzalutamide), Yescarta (Axicabtagene Ciloleucel), Yondelis (Trabectedin), Zaltrap (Ziv-Aflibercept), Zanosar (Streptozocin), Zarxio (Filgrastim), Zejula (Niraparib), Zelboraf (Vemurafenib), Zinecard (Dexrazoxane Hydrochloride), Ziv-Aflibercept, Zofran (Ondansetron Hydrochloride), Zoladex (Goserelin), Zoledronic Acid, Zolinza (Vorinostat), Zometa (Zoledronic acid), Zortress (Everolimus), Zydelig (Idelalisib), Zykadia (Ceritinib), Zytiga (Abiraterone Acetate), and Zytiga (Abiraterone).

In embodiments, any chimeric protein disclosed herein may be used in combination with any of the anti-cancer therapy disclosed herein.

In embodiments, any chimeric protein disclosed herein acts synergistically when co-administered with another anti cancer therapy (e.g., radiotherapy, an immunotherapy, and/or a chemotherapeutic agent); resulting in, for example, the other anti-cancer therapy is administered at doses that are lower than the doses commonly employed when the other anti-cancer therapy is are used as monotherapy. In embodiments, the chimeric protein, as disclosed herein, reduces the number of administrations of the co-administered anti-cancer therapy.

In aspects and embodiments of the present invention, a patient in need of a cancer treatment comprising a chimeric protein, as disclosed herein, is or is predicted to be poorly responsive or is non-responsive to an immunotherapy, e.g., an anti-cancer immunotherapy, as disclosed herein. Moreover, in embodiments, a patient in need of an anti-cancer agent, as disclosed herein, is or may is predicted to be poorly responsive or non-responsive to an immune checkpoint immunotherapy. The immune checkpoint molecule may be selected from PD-1, PD-L1, PD-L2, ICOS, ICOSL, and CTLA-4.

In embodiments, inclusive of, without limitation, infectious disease applications, the present invention pertains to anti- infectives as additional agents. In embodiments, the anti-infective is an anti-viral agent including, but not limited to, Abacavir, Acyclovir, Adefovir, Amprenavir, Atazanavir, Cidofovir, Darunavir, Delavirdine, Didanosine, Docosanol, Efavirenz, Elvitegravir, Emtricitabine, Enfuvirtide, Etravirine, Famciclovir, and Foscarnet. In embodiments, the anti- infective is an anti-bacterial agent including, but not limited to, cephalosporin antibiotics (cephalexin, cefuroxime, cefadroxil, cefazolin, cephalothin, cefaclor, cefamandole, cefoxitin, cefprozil, and ceftobiprole); fluoroquinolone antibiotics (cipro, Levaquin, floxin, tequin, avelox, and norflox); tetracycline antibiotics (tetracycline, minocycline, oxytetracycline, and doxycycline); penicillin antibiotics (amoxicillin, ampicillin, penicillin V, dicloxacillin, carbenicillin, vancomycin, and methicillin); monobactam antibiotics (aztreonam); and carbapenem antibiotics (ertapenem, doripenem, imipenem/cilastatin, and meropenem). In embodiments, the anti-infectives include anti-malarial agents {e.g., chloroquine, quinine, mefloquine, primaquine, doxycycline, artemether/lumefantrine, atovaquone/proguanil and sulfadoxine/pyrimethamine), metronidazole, tinidazole, ivermectin, pyrantel pamoate, and albendazole.

In aspects and embodiments of the present invention, the patient in need of treatment for an inflammatory disease or disorder, has been treated with, is contemporaneously treated with, or is subsequently treated with another agent for treating an inflammatory disease or disorder. Examples of such other agents include a steroidal anti-inflammatory agent, a non-steroidal anti-inflammatory agent (NSAID), and/or an immunosuppressive drug.

Examples of a NSAID include salicylic acid, acetyl salicylic acid, methyl salicylate, glycol salicylate, salicylmides, benzyl-2, 5-diacetoxybenzoic acid, ibuprofen, fulindac, naproxen, ketoprofen, etofenamate, phenylbutazone, and indomethacin.

Examples of a steroidal anti-inflammatory agents includes corticosteroids selected from hydroxyltriamcinolone, alpha- methyl dexamethasone, beta-methyl betamethasone, beclomethasone dipropionate, betamethasone benzoate, betamethasone dipropionate, betamethasone valerate, clobetasol valerate, desonide, desoxymethasone, dexamethasone, diflorasone diacetate, diflucortolone valerate, fluadrenolone, fluclorolone acetonide, flumethasone pivalate, fluosinolone acetonide, fluocinonide, flucortine butylester, fluocortolone, fluprednidene (fluprednylidene) acetate, flurandrenolone, halcinonide, hydrocortisone acetate, hydrocortisone butyrate, methylprednisolone, triamcinolone acetonide, cortisone, cortodoxone, flucetonide, fludrocortisone, difluorosone diacetate, fluradrenolone acetonide, medrysone, amcinafel, amcinafide, betamethasone and the balance of its esters, chloroprednisone, clocortelone, clescinolone, dichlorisone, difluprednate, flucloronide, flunisolide, fluoromethalone, fluperolone, fluprednisolone, hydrocortisone, meprednisone, paramethasone, prednisolone, prednisone, beclomethasone dipropionate.

A steroidal anti-inflammatory agent may likewise have activity as an immunosuppressive drug.

Other examples of immunosuppressive drug include cytostatics such as alkylating agents, antimetabolites (e.g., azathioprine, methotrexate), cytotoxic antibiotics, antibodies (e.g., basiliximab, daclizumab, and muromonab), anti- immunophilins (e.g., cyclosporine, tacrolimus, sirolimus), inteferons, opioids, TNF binding proteins, mycophenolates, and small biological agents (e.g., fingolimod, myriocin).

In embodiments, a patient in need of an agent for treating an autoimmune disease or disorder, has been treated with, is contemporaneously treated with, or is subsequently treated with a steroidal anti-inflammatory agent, a non-steroidal anti-inflammatory agent, and/or an immunosuppressive drug, as disclosed elsewhere herein. In embodiments, the chimeric proteins disclosed herein, include derivatives that are modified, i.e., by the covalent attachment of any type of molecule to the composition such that covalent attachment does not prevent the activity of the composition. For example, but not by way of limitation, derivatives include composition that have been modified by, inter alia, glycosylation, lipidation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any of numerous chemical modifications can be carried out by known techniques, including, but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Additionally, the derivative can contain one or more non-classical amino acids. In still other embodiments, the chimeric proteins (and/or additional agents) disclosed herein further comprise a cytotoxic agent, comprising, in illustrative embodiments, a toxin, a chemotherapeutic agent, a radioisotope, and an agent that causes apoptosis or cell death. Such agents may be conjugated to a composition disclosed herein.

The chimeric proteins disclosed herein may thus be modified post-translationally to add effector moieties such as chemical linkers, detectable moieties such as for example fluorescent dyes, enzymes, substrates, bioluminescent materials, radioactive materials, and chemiluminescent moieties, or functional moieties such as for example streptavidin, avidin, biotin, a cytotoxin, a cytotoxic agent, and radioactive materials.

Pharmaceutical composition

Aspects of the present invention include a pharmaceutical composition comprising a therapeutically effective amount of a chimeric protein as disclosed herein.

The chimeric proteins (and/or additional agents) disclosed herein can possess a sufficiently basic functional group, which can react with an inorganic or organic acid, or a carboxyl group, which can react with an inorganic or organic base, to form a pharmaceutically acceptable salt. A pharmaceutically acceptable acid addition salt is formed from a pharmaceutically acceptable acid, as is well known in the art. Such salts include the pharmaceutically acceptable salts listed in, for example, Journal of Pharmaceutical Science, 66, 2-19 (1977) and The Handbook of Pharmaceutical Salts; Properties, Selection, and Use. P. H. Stahl and C. G. Wermuth (eds.), Verlag, Zurich (Switzerland) 2002, which are hereby incorporated by reference in their entirety.

In embodiments, the compositions disclosed herein are in the form of a pharmaceutically acceptable salt.

Further, any chimeric protein (and/or additional agents) disclosed herein can be administered to a subject as a component of a composition, e.g., pharmaceutical composition, that comprises a pharmaceutically acceptable carrier or vehicle. Such pharmaceutical compositions can optionally comprise a suitable amount of a pharmaceutically acceptable excipient so as to provide the form for proper administration. Pharmaceutical excipients can be liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. The pharmaceutical excipients can be, for example, saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea and the like. In addition, auxiliary, stabilizing, thickening, lubricating, and coloring agents can be used. In embodiments, the pharmaceutically acceptable excipients are sterile when administered to a subject. Water is a useful excipient when any agent disclosed herein is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid excipients, specifically for injectable solutions. Suitable pharmaceutical excipients also include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. Any agent disclosed herein, if desired, can also comprise minor amounts of wetting or emulsifying agents, or pH buffering agents.

In embodiments, the compositions, e.g., pharmaceutical compositions, disclosed herein are resuspended in a saline buffer (including, without limitation TBS, PBS, and the like).

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

The present invention includes the disclosed chimeric protein (and/or additional agents) in various formulations of pharmaceutical composition. Any chimeric protein (and/or additional agents) disclosed herein can take the form of solutions, suspensions, emulsion, drops, tablets, pills, pellets, capsules, capsules containing liquids, powders, sustained-release formulations, suppositories, emulsions, aerosols, sprays, suspensions, or any other form suitable for use. DNA or RNA constructs encoding the protein sequences may also be used. In embodiments, the composition is in the form of a capsule (see, e.g., U.S. Patent No. 5,698,155). Other examples of suitable pharmaceutical excipients are described in Remington’s Pharmaceutical Sciences 1447-1676 (Alfonso R. Gennaro eds., 19th ed. 1995), incorporated herein by reference.

Where necessary, the pharmaceutical compositions comprising the chimeric protein (and/or additional agents) can also include a solubilizing agent. Also, the agents can be delivered with a suitable vehicle or delivery device as known in the art. Combination therapies outlined herein can be co-delivered in a single delivery vehicle or delivery device. Compositions for administration can optionally include a local anesthetic such as, for example, lignocaine to lessen pain at the site of the injection.

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

In embodiments, any chimeric protein (and/or additional agents) disclosed herein is formulated in accordance with routine procedures as a pharmaceutical composition adapted for a mode of administration disclosed herein.

Administration, Dosing, and Treatment Regimens

Routes of administration include, for example: intradermal, intratumoral, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, oral, sublingual, intranasal, intracerebral, intravaginal, transdermal, rectally, by inhalation, or topically, particularly to the ears, nose, eyes, or skin.

As examples, administration results in the release of chimeric protein (and/or additional agents) disclosed herein into the bloodstream (via enteral or parenteral administration), or alternatively, the chimeric protein (and/or additional agents) is administered directly to the site of active disease.

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

In specific embodiments, it may be desirable to administer locally to the area in need of treatment. In embodiments, for instance in the treatment of cancer, the chimeric protein (and/or additional agents) are administered in the tumor microenvironment (e.g., cells, molecules, extracellular matrix and/or blood vessels that surround and/or feed a tumor cell, inclusive of, for example, tumor vasculature; tumor-infiltrating lymphocytes; fibroblast reticular cells; endothelial progenitor cells (EPC); cancer-associated fibroblasts; pericytes; other stromal cells; components of the extracellular matrix (ECM); dendritic cells; antigen presenting cells; T-cells; regulatory T cells; macrophages; neutrophils; and other immune cells located proximal to a tumor) or lymph node and/or targeted to the tumor microenvironment or lymph node. In embodiments, for instance in the treatment of cancer, the chimeric protein (and/or additional agents) are administered intratumorally.

In embodiments, the present chimeric protein allows for a dual effect that provides less side effects than are seen in conventional immunotherapy (e.g., treatments with one or more of OPDIVO, KEYTRUDA, YERVOY, and TECENTRIQ). For example, the present chimeric proteins reduce or prevent commonly observed immune-related adverse events that affect various tissues and organs including the skin, the gastrointestinal tract, the kidneys, peripheral and central nervous system, liver, lymph nodes, eyes, pancreas, and the endocrine system; such as hypophysitis, colitis, hepatitis, pneumonitis, rash, and rheumatic disease. Further, the present local administration, e.g., intratumorally, obviate adverse event seen with standard systemic administration, e.g., IV infusions, as are used with conventional immunotherapy (e.g., treatments with one or more of OPDIVO, KEYTRUDA, YERVOY, and TECENTRIQ).

Dosage forms suitable for parenteral administration (e.g., intravenous, intramuscular, intraperitoneal, subcutaneous and intra-articular injection and infusion) include, for example, solutions, suspensions, dispersions, emulsions, and the like. They may also be manufactured in the form of sterile solid compositions (e.g., lyophilized composition), which can be dissolved or suspended in sterile injectable medium immediately before use. They may contain, for example, suspending or dispersing agents known in the art.

The dosage of any chimeric protein (and/or additional agents) disclosed herein as well as the dosing schedule can depend on various parameters, including, but not limited to, the disease being treated, the subject's general health, and the administering physician's discretion. Any chimeric protein disclosed herein, can be administered prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concurrently with, or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of an additional agent, to a subject in need thereof.

In embodiments, a chimeric protein and an additional agent(s) are administered 1 minute apart, 10 minutes apart, 30 minutes apart, less than 1 hour apart, 1 hour apart, 1 hour to 2 hours apart, 2 hours to 3 hours apart, 3 hours to 4 hours apart, 4 hours to 5 hours apart, 5 hours to 6 hours apart, 6 hours to 7 hours apart, 7 hours to 8 hours apart, 8 hours to

9 hours apart, 9 hours to 10 hours apart, 10 hours to 1 1 hours apart, 1 1 hours to 12 hours apart, 1 day apart, 2 days apart, 3 days apart, 4 days apart, 5 days apart, 6 days apart, 1 week apart, 2 weeks apart, 3 weeks apart, or 4 weeks apart.

In embodiments, the present invention relates to the co-administration of a chimeric protein which induces an innate immune response and another chimeric protein which induces an adaptive immune response. In such embodiments, the chimeric protein which induces an innate immune response may be administered before, concurrently with, or subsequent to administration of the chimeric protein which induces an adaptive immune response. For example, the chimeric proteins may be administered 1 minute apart, 10 minutes apart, 30 minutes apart, less than 1 hour apart, 1 hour apart, 1 hour to 2 hours apart, 2 hours to 3 hours apart, 3 hours to 4 hours apart, 4 hours to 5 hours apart, 5 hours to 6 hours apart, 6 hours to 7 hours apart, 7 hours to 8 hours apart, 8 hours to 9 hours apart, 9 hours to 10 hours apart,

10 hours to 1 1 hours apart, 1 1 hours to 12 hours apart, 1 day apart, 2 days apart, 3 days apart, 4 days apart, 5 days apart, 6 days apart, 1 week apart, 2 weeks apart, 3 weeks apart, or 4 weeks apart. In an illustrative embodiment, the chimeric protein which induces an innate immune response and the chimeric protein which induces an adaptive response are administered 1 week apart, or administered on alternate weeks (/. e., administration of the chimeric protein inducing an innate immune response is followed 1 week later with administration of the chimeric protein which induces an adaptive immune response and so forth).

The dosage of any chimeric protein (and/or additional agents) disclosed herein can depend on several factors including the severity of the condition, whether the condition is to be treated or prevented, and the age, weight, and health of the subject to be treated. Additionally, pharmacogenomic (the effect of genotype on the pharmacokinetic, pharmacodynamic or efficacy profile of a therapeutic) information about a particular subject may affect dosage used. Furthermore, the exact individual dosages can be adjusted somewhat depending on a variety of factors, including the specific combination of the agents being administered, the time of administration, the route of administration, the nature of the formulation, the rate of excretion, the particular disease being treated, the severity of the disorder, and the anatomical location of the disorder. Some variations in the dosage can be expected.

For administration of any chimeric protein (and/or additional agents) disclosed herein by parenteral injection, the dosage may be about 0.1 mg to about 250 mg per day, about 1 mg to about 20 mg per day, or about 3 mg to about 5 mg per day. Generally, when orally or parenterally administered, the dosage of any agent disclosed herein may be about 0.1 mg to about 1500 mg per day, or about 0.5 mg to about 10 mg per day, or about 0.5 mg to about 5 mg per day, or about 200 to about 1 ,200 mg per day (e.g., about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, about 1 ,000 mg, about 1 , 100 mg, about 1 ,200 mg per day).

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

In embodiments, a suitable dosage of the chimeric protein (and/or additional agents) is in a range of about 0.01 mg/kg to about 100 mg/kg of body weight ,or about 0.01 mg/kg to about 10 mg/kg of body weight of the subject, for example, about 0.01 mg/kg, about 0.02 mg/kg, about 0.03 mg/kg, about 0.04 mg/kg, about 0.05 mg/kg, about 0.06 mg/kg, about 0.07 mg/kg, about 0.08 mg/kg, about 0.09 mg/kg, about 0.1 mg/kg, about 0.2 mg/kg, about 0.3 mg/kg, about 0.4 mg/kg, about 0.5 mg/kg, about 0.6 mg/kg, about 0.7 mg/kg, about 0.8 mg/kg, about 0.9 mg/kg, about 1 mg/kg, about 1.1 mg/kg, about 1.2 mg/kg, about 1.3 mg/kg, about 1.4 mg/kg, about 1.5 mg/kg, about 1.6 mg/kg, about 1.7 mg/kg, about 1.8 mg/kg, 1.9 mg/kg, about 2 mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about 6 mg/kg, about 7 mg/kg, about 8 mg/kg, about 9 mg/kg, about 10 mg/kg body weight, inclusive of all values and ranges therebetween. In another embodiment, delivery can be in a vesicle, in particular a liposome (see Langer, 1990, Science 249: 1527- 1533; Treat et al., in Liposomes in Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989).

A chimeric protein (and/or additional agents) disclosed herein can be administered by controlled-release or sustained- release means or by delivery devices that are well known to those of ordinary skill in the art. Examples include, but are not limited to, those described in U.S. Patent Nos. 3,845,770; 3,916,899; 3,536,809; 3,598, 123; 4,008,719; 5,674,533; 5,059,595; 5,591 ,767; 5, 120,548; 5,073,543; 5,639,476; 5,354,556; and 5,733,556, each of which is incorporated herein by reference in its entirety. Such dosage forms can be useful for providing controlled- or sustained-release of one or more active ingredients using, for example, hydropropylmethyl cellulose, other polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, liposomes, microspheres, or a combination thereof to provide the desired release profile in varying proportions. Controlled- or sustained-release of an active ingredient can be stimulated by various conditions, including but not limited to, changes in pH, changes in temperature, stimulation by an appropriate wavelength of light, concentration or availability of enzymes, concentration or availability of water, or other physiological conditions or compounds.

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

In another embodiment, a controlled-release system can be placed in proximity of the target area to be treated, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 1 15-138 (1984)). Other controlled-release systems discussed in the review by Langer, 1990, Science 249: 1527-1533) may be used.

Administration of any chimeric protein (and/or additional agents) disclosed herein can, independently, be one to four times daily or one to four times per month or one to six times per year or once every two, three, four or five years. Administration can be for the duration of one day or one month, two months, three months, six months, one year, two years, three years, and may even be for the life of the subject.

The dosage regimen utilizing any chimeric protein (and/or additional agents) disclosed herein can be selected in accordance with a variety of factors including type, species, age, weight, sex and medical condition of the subject; the severity of the condition to be treated; the route of administration; the renal or hepatic function of the subject; the pharmacogenomic makeup of the individual; and the specific compound of the invention employed. Any chimeric protein (and/or additional agents) disclosed herein can be administered in a single daily dose, or the total daily dosage can be administered in divided doses of two, three or four times daily. Furthermore, any chimeric protein (and/or additional agents) disclosed herein can be administered continuously rather than intermittently throughout the dosage regimen.

Cells and Nucleic Acids

Another aspect of the present invention is an expression vector comprising a nucleic acid encoding the chimeric protein of any of the herein-disclosed aspects or embodiments.

In any of the herein-disclosed aspects or embodiments, the chimeric protein may be 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.

Constructs could be produced by cloning these three fragments (the extracellular domain of a first transmembrane protein, followed by a linker sequence, followed by the extracellular domain of a second transmembrane protein) into a vector (plasmid, viral or other). Accordingly, in embodiments, the present chimeric proteins are engineered as such.

Aspects of the present invention provide an expression vector comprising a nucleic acid which encodes a chimeric protein as disclosed herein. The expression vector comprises a nucleic acid encoding the chimeric protein disclosed herein. In embodiments, the expression vector comprises DNA or RNA. In embodiments, the expression vector is a mammalian expression vector.

Both prokaryotic and eukaryotic vectors can be used for expression of the chimeric protein. Prokaryotic vectors include constructs based on £. coli sequences (see, e.g., Makrides, Microbiol Rev 1996, 60:512-538). Non-limiting examples of regulatory regions that can be used for expression in £. coli include lac, trp, Ipp, phoA, recA, tac, T3, T7 and APL. Non-limiting examples of prokaryotic expression vectors may include the Agt vector series such as Agt11 (Huynh et al., in "DNA Cloning Techniques, Vol. I: A Practical Approach,” 1984, (D. Glover, ed.), pp. 49-78, IRL Press, Oxford), and the pET vector series (Studier et al., Methods Enzymol 1990, 185:60-89). Prokaryotic host-vector systems cannot perform much of the post-translational processing of mammalian cells, however. Thus, eukaryotic host- vector systems may be particularly useful. A variety of regulatory regions can be used for expression of the chimeric proteins in mammalian host cells. For example, the SV40 early and late promoters, the cytomegalovirus (CMV) immediate early promoter, and the Rous sarcoma virus long terminal repeat (RSV-LTR) promoter can be used. Inducible promoters that may be useful in mammalian cells include, without limitation, promoters associated with the metallothionein II gene, mouse mammary tumor virus glucocorticoid responsive long terminal repeats (MMTV-LTR), the b-interferon gene, and the hsp70 gene (see, Williams et al., Cancer Res 1989, 49:2735-42; and Taylor et al., Mol Cell Biol 1990, 10:165-75). Heat shock promoters or stress promoters also may be advantageous for driving expression of the chimeric proteins in recombinant host cells.

In embodiments, expression vectors of the invention comprise a nucleic acid encoding the chimeric proteins, or a complement thereof, operably linked to an expression control region, or complement thereof, that is functional in a mammalian cell. The expression control region is capable of driving expression of the operably linked blocking and/or stimulating agent encoding nucleic acid such that the blocking and/or stimulating agent is produced in a human cell transformed with the expression vector.

Expression control regions are regulatory polynucleotides (sometimes referred to herein as elements), such as promoters and enhancers, that influence expression of an operably linked nucleic acid. An expression control region of an expression vector of the invention is capable of expressing operably linked encoding nucleic acid in a human cell. In embodiments, the cell is a tumor cell. In another embodiment, the cell is a non-tumor cell. In embodiments, the expression control region confers regulatable expression to an operably linked nucleic acid. A signal (sometimes referred to as a stimulus) can increase or decrease expression of a nucleic acid operably linked to such an expression control region. Such expression control regions that increase expression in response to a signal are often referred to as inducible. Such expression control regions that decrease expression in response to a signal are often referred to as repressible. Typically, the amount of increase or decrease conferred by such elements is proportional to the amount of signal present; the greater the amount of signal, the greater the increase or decrease in expression.

In embodiments, the present invention contemplates the use of inducible promoters capable of effecting high level of expression transiently in response to a cue. For example, when in the proximity of a tumor cell, a cell transformed with an expression vector for the chimeric protein (and/or additional agents) comprising such an expression control sequence is induced to transiently produce a high level of the agent by exposing the transformed cell to an appropriate cue. Illustrative inducible expression control regions include those comprising an inducible promoter that is stimulated with a cue such as a small molecule chemical compound. In other examples, the chimeric protein is expressed by a chimeric antigen receptor containing cell or an in vitro expanded tumor infiltrating lymphocyte, under the control of a promoter which is sensitive to antigen recognition by the cell, and leads to local secretion of the chimeric protein in response to tumor antigen recognition. Particular examples can be found, for example, in U.S. Patent Nos. 5,989,910, 5,935,934, 6,015,709, and 6,004,941 , each of which is incorporated herein by reference in its entirety.

Expression control regions and locus control regions include full-length promoter sequences, such as native promoter and enhancer elements, as well as subsequences or polynucleotide variants which retain all or part of full-length or non-variant function. As used herein, the term "functional" and grammatical variants thereof, when used in reference to a nucleic acid sequence, subsequence or fragment, means that the sequence has one or more functions of native nucleic acid sequence (e.g., non-variant or unmodified sequence). As used herein, "operable linkage” refers to a physical juxtaposition of the components so described as to permit them to function in their intended manner. In the example of an expression control element in operable linkage with a nucleic acid, the relationship is such that the control element modulates expression of the nucleic acid. Typically, an expression control region that modulates transcription is juxtaposed near the 5' end of the transcribed nucleic acid (i.e., "upstream”). Expression control regions can also be located at the 3' end of the transcribed sequence (i.e., "downstream”) or within the transcript (e.g., in an intron). Expression control elements can be located at a distance away from the transcribed sequence (e.g., 100 to 500, 500 to 1000, 2000 to 5000, or more nucleotides from the nucleic acid). A specific example of an expression control element is a promoter, which is usually located 5' of the transcribed sequence. Another example of an expression control element is an enhancer, which can be located 5' or 3' of the transcribed sequence, or within the transcribed sequence.

Expression systems that function in human cells are well known in the art; these include viral systems. Generally, a promoter functional in a human cell is any DNA sequence capable of binding mammalian RNA polymerase and initiating the downstream (3') transcription of a coding sequence into mRNA. A promoter will have a transcription-initiating region, which is usually placed proximal to the 5' end of the coding sequence, and typically a TATA box located 25-30 base pairs upstream of the transcription initiation site. The TATA box is thought to direct RNA polymerase II to begin RNA synthesis at the correct site. A promoter will also typically contain an upstream promoter element (enhancer element), typically located within 100 to 200 base pairs upstream of the TATA box. An upstream promoter element determines the rate at which transcription is initiated; they can act in either orientation. Of particular use as promoters are the promoters from mammalian viral genes, since the viral genes are often highly expressed and have a broad host range. Examples include the SV40 early promoter, mouse mammary tumor virus LTR promoter, adenovirus major late promoter, herpes simplex virus promoter, and the CMV promoter.

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

There is a variety of techniques available for introducing nucleic acids into viable cells. Techniques suitable for the transfer of nucleic acid into mammalian cells in vitro include the use of liposomes, electroporation, microinjection, cell fusion, polymer-based systems, DEAE-dextran, viral transduction, the calcium phosphate precipitation method, etc. For in vivo gene transfer, a number of techniques and reagents may also be used, including liposomes; natural polymer- based delivery vehicles, such as chitosan and gelatin; viral vectors are also suitable for in vivo transduction. In some situations, it is desirable to provide a targeting agent, such as an antibody or ligand specific for a tumor cell surface membrane protein. Where liposomes are employed, proteins which bind to a cell surface membrane protein associated with endocytosis may be used for targeting and/or to facilitate uptake, e.g., capsid proteins or fragments thereof tropic for a particular cell type, antibodies for proteins which undergo internalization in cycling, proteins that target intracellular localization and enhance intracellular half-life. The technique of receptor-mediated endocytosis is described, for example, by Wu et al., J. Biol. Chem. 262, 4429-4432 (1987); and Wagner et al., Proc. Natl. Acad. Sci. USA 87, 3410- 3414 (1990).

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

In embodiments, the expression vectors for the expression of the chimeric proteins (and/or additional agents) are viral vectors. Many viral vectors useful for gene therapy are known (see, e.g., Lundstrom, Trends Biotechnol., 21 : 1 17, 122, 2003. Illustrative viral vectors include those selected from Antiviruses (LV), retroviruses (RV), adenoviruses (AV), adeno-associated viruses (AAV), and a-viruses, though other viral vectors may also be used. For in vivo uses, viral vectors that do not integrate into the host genome are suitable for use, such as a viruses and adenoviruses. Illustrative types of a-viruses include Sindbis virus, Venezuelan equine encephalitis (VEE) virus, and Semliki Forest virus (SFV). For in vitro uses, viral vectors that integrate into the host genome are suitable, such as retroviruses, AAV, and Antiviruses. In embodiments, the invention provides methods of transducing a human cell in vivo, comprising contacting a solid tumor in vivo with a viral vector of the invention.

Aspects of the present invention include a host cell comprising the expression vector which encodes the chimeric protein disclosed herein.

Expression vectors can be introduced into host cells for producing the present chimeric proteins. Cells may be cultured in vitro or genetically engineered, for example. Useful mammalian host cells include, without limitation, cells derived from humans, monkeys, and rodents (see, for example, Kriegler in "Gene Transfer and Expression: A Laboratory Manual,” 1990, New York, Freeman & Co.). These include monkey kidney cell lines transformed by SV40 (e.g., COS- 7, ATCC CRL 1651 ); human embryonic kidney lines (e.g., 293, 293-EBNA, or 293 cells subcloned for growth in suspension culture, Graham et al., J Gen Virol 1977, 36:59); baby hamster kidney cells (e.g., BHK, ATCC CCL 10); Chinese hamster ovary-cells-DHFR {e.g., CHO, Urlaub and Chasin, Proc Natl Acad Sci USA 1980, 77:4216); DG44 CHO cells, CHO-K1 cells, mouse sertoli cells (Mather, Biol Reprod 1980, 23:243-251 ); mouse fibroblast cells {e.g., NIH-3T3), monkey kidney cells {e.g., CV1 ATCC CCL 70); African green monkey kidney cells, {e.g., VERO-76, ATCC CRL-1587); human cervical carcinoma cells {e.g., HELA, ATCC CCL 2); canine kidney cells {e.g., MDCK, ATCC CCL 34); buffalo rat liver cells {e.g., BRL 3A, ATCC CRL 1442); human lung cells {e.g., W138, ATCC CCL 75); human liver cells {e.g., Hep G2, HB 8065); and mouse mammary tumor cells {e.g., MMT 060562, ATCC CCL51 ). Illustrative cancer cell types for expressing the chimeric proteins disclosed herein include mouse fibroblast cell line, NI H3T3, mouse Lewis lung carcinoma cell line, LLC, mouse mastocytoma cell line, P815, mouse lymphoma cell line, EL4 and its ovalbumin transfectant, E.G7, mouse melanoma cell line, B16F10, mouse fibrosarcoma cell line, MC57, and human small cell lung carcinoma cell lines, SCLC#2 and SCLC#7.

Host cells can be obtained from normal or affected subjects, including healthy humans, cancer patients, and patients with an infectious disease, private laboratory deposits, public culture collections such as the American Type Culture Collection (ATCC), or from commercial suppliers.

Cells that can be used for production of the present chimeric proteins in vitro, ex vivo, and/or in vivo include, without limitation, epithelial cells, endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytes; blood cells such as T lymphocytes, chimeric antigen receptor expressing T cells, tumor infiltrating lymphocytes, B lymphocytes, monocytes, macrophages, neutrophils, eosinophils, megakaryocytes, granulocytes; various stem or progenitor cells, in particular hematopoietic stem or progenitor cells {e.g., as obtained from bone marrow), umbilical cord blood, peripheral blood, and fetal liver. The choice of cell type depends on the type of tumor or infectious disease being treated or prevented, and can be determined by one of skill in the art.

Production and purification of Fc-containing macromolecules (such as monoclonal antibodies) has become a standardized process, with minor modifications between products. For example, many Fc containing macromolecules are produced by human embryonic kidney (HEK) cells (or variants thereof) or Chinese Hamster Ovary (CHO) cells (or variants thereof) or in some cases by bacterial or synthetic methods. Following production, the Fc containing macromolecules that are secreted by HEK or CHO cells are purified through binding to Protein A columns and subsequently‘polished’ using various methods. Generally speaking, purified Fc containing macromolecules are stored in liquid form for some period of time, frozen for extended periods of time or in some cases lyophilized. In embodiments, production of the chimeric proteins contemplated herein may have unique characteristics as compared to traditional Fc containing macromolecules. In certain examples, the chimeric proteins may be purified using specific chromatography resins, or using chromatography methods that do not depend upon Protein A capture. In embodiments, the chimeric proteins may be purified in an oligomeric state, or in multiple oligomeric states, and enriched for a specific oligomeric state using specific methods. Without being bound by theory, these methods could include treatment with specific buffers including specified salt concentrations, pH and additive compositions. In other examples, such methods could include treatments that favor one oligomeric state over another. The chimeric proteins obtained herein may be additionally‘polished’ using methods that are specified in the art. In embodiments, the chimeric proteins are highly stable and able to tolerate a wide range of pH exposure (between pH 3-12), are able to tolerate a large number of freeze/thaw stresses (greater than 3 freeze/thaw cycles) and are able to tolerate extended incubation at high temperatures (longer than 2 weeks at 40 degrees C). In embodiments, the chimeric proteins are shown to remain intact, without evidence of degradation, deamidation, etc. under such stress conditions.

Subjects and/or Animals

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

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

In certain embodiments, the human has an age in a range of from about 0 months to about 6 months old, from about 6 to about 12 months old, from about 6 to about 18 months old, from about 18 to about 36 months old, from about 1 to about 5 years old, from about 5 to about 10 years old, from about 10 to about 15 years old, from about 15 to about 20 years old, from about 20 to about 25 years old, from about 25 to about 30 years old, from about 30 to about 35 years old, from about 35 to about 40 years old, from about 40 to about 45 years old, from about 45 to about 50 years old, from about 50 to about 55 years old, from about 55 to about 60 years old, from about 60 to about 65 years old, from about 65 to about 70 years old, from about 70 to about 75 years old, from about 75 to about 80 years old, from about 80 to about 85 years old, from about 85 to about 90 years old, from about 90 to about 95 years old or from about 95 to about 100 years old.

In embodiments, the subject is a non-human animal, and therefore the invention pertains to veterinary use. In a specific embodiment, the non-human animal is a household pet. In another specific embodiment, the non-human animal is a livestock animal.

Kits and Medicaments

Aspects of the present invention provide kits that can simplify the administration of any agent disclosed herein.

An illustrative kit of the invention comprises any chimeric protein and/or pharmaceutical composition disclosed herein in unit dosage form. In embodiments, the unit dosage form is a container, such as a pre-filled syringe, which can be sterile, containing any agent disclosed herein and a pharmaceutically acceptable carrier, diluent, excipient, or vehicle. The kit can further comprise a label or printed instructions instructing the use of any agent disclosed herein. The kit may also include a lid speculum, topical anesthetic, and a cleaning agent for the administration location. The kit can also further comprise one or more additional agent disclosed herein. In embodiments, the kit comprises a container containing an effective amount of a composition of the invention and an effective amount of another composition, such those disclosed herein.

Aspects of the present invention include use of a chimeric protein as disclosed herein in the manufacture of a medicament, e.g., a medicament for treatment of cancer and/or treatment of an inflammatory disease.

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

The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.

EXAMPLES

Example 1: Production and Characterization of the Murine and Human PD-1-Fc-TGFBR2 Chimeric Protein.

Constructs were created that combined the coding sequences for the extracellular domains (ECDs) of PD-1 and TGFBR2 and an antibody Fc domain. Constructs were created for both human and mouse proteins; these constructs were used to express the human PD-1 -Fc-TGFBR2 chimeric protein and the murine PD-1 -Fc-TGFBR2 chimeric protein.

Mammalian cells were transfected with the mPD-1 -Fc-TGFBR2 expressing construct or the hPD-1 -Fc-TGFBR2 expressing construct, and the secreted proteins were purified from conditioned media by affinity chromatography. The purified proteins were analyzed for the presence of each individual domain by Western blotting using anti-PD-1 , anti- Fc and anti-TGFBR2 antibodies (FIG. 3A for mPD-1 -Fc-TGFBR2 and FIG. 3B for hPD-1-Fc-TGFBR2). These blots revealed a glycosylated protein that formed a dimer under non-reducing conditions by SDS-PAGE. The reduced and deglycosylated form of the protein migrated at the predicted monomeric molecular weight of 50 kDa for the murine chimeric protein (FIG. 3A) and at the predicted monomeric molecular weight of 65 kDa for the human chimeric protein (FIG. 3B).

To determine whether the mPD-1-Fc-TGFBR2 chimeric protein was capable of binding both PD-L1 and TGFBeta, functional ELISA assays were used. These assays quantitatively demonstrated binding of mPD-1 -Fc-TGFBR2's PD- 1 -domain to recombinant mPD-L1 (FIG. 3C, top left), its TGFBR2-domain to recombinant mTGFBeta (FIG. 3C, bottom left), and its Fc domain to be bound by an anti-mouse-lgG antibody (FIG. 3C, top right). Similar functional ELISA assays were performed to determine whether or not the hPD-1 -Fc-TGFBR2 chimeric protein was capable of binding both PD-L1 and TGFBeta. These assays quantitatively demonstrated binding of hPD-1 -Fc- TGFBR2's PD-1 -domain to recombinant hPD-L1 (FIG. 3D, top left), its TGFBR2-domain to recombinant hTGFBeta (FIG. 3D, bottom left), and its Fc domain to be bound by an anti-human-lgG antibody (FIG. 3D, top right).

A functional ELISA evaluated whether hPD-1 -Fc-TGFBR2 could outcompete a recombinant human TGFBR2 (rhTGFBR2) control for binding to recombinant TGFBeta. FIG. 3E (left) shows quantified binding of TGFbl to ELISA- plate bound recombinant human TGFBR2. Soluble human hPD-1-Fc-TGFBR2 efficiently outcompeted the binding to recombinant TGFBeta by ELISA-plate bound rhTGFBR2's (FIG. 3E, right).

The hPD-1 -Fc-TGFBR2 chimeric protein's ability to block apoptosis was assayed. As shown in FIG. 4A, apoptosis can be induced in the human lymphoma cell line RAMOS when exposed to TGFb. The amount of apoptosis, as indicated by caspase 3/7-luciferase activity, was assayed at four time points (6, 24, 48, and 72 hours) after RAMOS cell exposure to TGFb. As shown, when RAMOS cells were exposed to 1 ng/mL TGFb, detection at 72 hours provided maximal luminescence. This treatment regimen was used to determine the hPD-1-Fc-TGFBR2 chimeric protein's ability to prevent or reduce apoptosis in RAMOS cells (FIG. 4B).

In the experiment disclosed in FIG. 4B, RAMOS cells were either untreated, treated with 1 ng/mL TGFb, or the same concentration of TGFb in the presence of the TGFbR blocker ALK5, a dose titration of the human PD-1 -Fc-TGFBR2 chimeric protein (identified as "ARC”), an anti-TGFb blocking antibody, human IgG control, or recombinant human PD- 1 protein. After 72 hours, cleaved caspase 3/7 luciferase-activity (Promega) was assessed using a luminometer. TGFb was consistently spiked in at 1 ng/mL and the test reagents were used at 1x, 10x, and 100x of this concentration (i.e., 1 ng/mL, 10 ng/mL, and 100 ng/mL). As shown in FIG. 4B, hPD-1-Fc-TGFBR2 at 100x (100 ng/mL) was as effective in reducing TGFb-induced apoptosis as the TGFbR blocker ALK5 (at 10 mM) and as the anti-TGFb blocking antibody (at 100x, i.e., 100 ng/mL).

The in vivo ability of the PD-1-Fc-TGFBR2 chimeric protein target and reduce tumor volume is determined. Here, mice are inoculated with tumor cells, e.g., without limitation, one of CT26 (murine colon carcinoma), MC38 (murine colon adenocarcinoma), B16.F10 (murine melanoma) A20 (lymphoma), and WEHI3 (leukemia) cells, into one flank. Once the tumors have reached sufficient volume (e.g., volume of about 100 mm ³ ), mice are administered a various concentrations of the mPD-1-Fc-TGFBR2 chimeric protein or suitable controls, e.g., vehicle, Fc fusion proteins comprising mPD-1 or Fc fusion proteins comprising mTGFBR2 or antibodies that bind PD-1 or a PD-1 ligand, and antibodies that bind TGFBR2 or TGFBeta. Beginning about eight days after inoculation, tumor volumes are measured and changes between starting tumor volumes are determined; tumor volumes are measured periodically thereafter until the end of the experiment (e.g., 20, 25, 30, 35 or more days or until euthanizing because of tumor size). At each time point, the number of surviving treated mice is determined and Kaplan-Meier plots of the percent survival each days after tumor inoculation resulting from treatments are created. In various experiments, mice that reject the tumor may be re-challenged with a secondary tumor on the opposing flank, and primary/secondary tumors continue to be measured as well as lethality of the mice.

The therapeutic activity of the treatments may further be assayed. In particular, changes in pharmacodynamic biomarkers showing tumor rejection are determined by cytokine elevations in serum (in vivo) or changes in pharmacodynamic biomarkers in vitro in immune-related cells incubated with the super-antigen Staphylococcal enterotoxin B (SEB assay) or when cultured in AIM V media. Exemplary pharmacodynamic biomarkers include IFNy, IL-2, IL-4, IL-5, IL-6, and IL-17A. Also, changes in the number of peripheral lymphocytes (and ratios of types of lymphocytes) is measured and quantified over time.

Additionally, separate groups of inoculated mice are euthanized six or twenty-four hours after administration of the mPD-1 -Fc-TGFBR2 chimeric protein or suitable control and their spleens excised, dissociated and assessed by flow cytometry for populations of activated CD4+ or CD8+ dendritic cells (as examples of splenic immune cell types) to determine the extent of T cell activation and proliferation resulting from the mPD-1 -Fc-TGFBR2 chimeric protein treatments.

Example 2: Production and Characterization of the Human PD-1-Fc-LAG3 Chimeric Protein.

Constructs were created that combined the coding sequences for the extracellular domains (ECDs) of human PD-1 and LAG3 and an antibody Fc domain. These constructs were used to express the human PD-1 -Fc-LAG3 chimeric protein.

Mammalian cells were transfected with the hPD-1 -Fc-LAG3 expressing construct, and the secreted protein was purified from conditioned media by affinity chromatography. The purified proteins were analyzed for the presence of each individual domain by Western blotting using anti-PD-1 , anti-Fc, and anti-LAG3 antibodies (FIG. 5A). These blots revealed a glycosylated protein that formed a dimer under non-reducing conditions by SDS-PAGE. The reduced and deglycosylated form of the protein migrated at the predicted monomeric molecular weight of 100 kDa for the murine chimeric protein (FIG. 5A).

To determine whether hPD-1 -Fc-LAG3 chimeric protein was capable of binding both PD-L1 and LAG3, functional ELISA assays were used. These assays quantitatively demonstrated binding of the hPD-1-Fc-LAG3 chimeric protein's PD-1 -domain to recombinant hPD-L1 (FIG. 5B, top left), its LAG3-domain to recombinant HLA-DR (FIG. 5B, bottom left), and its Fc domain to be bound by an anti-human-lgG antibody (FIG. 5B, top right).

Example 3: Production and Characterization of the Murine LAG3-Fc-TIM-3 Chimeric Protein.

Constructs were created that combined the coding sequences for the extracellular domains (ECDs) of murine LAG3 and TIM-3 and an antibody Fc domain. These constructs were used to express the murine LAG3-1 -Fc-TI M-3 chimeric protein. Mammalian cells were transfected with the mLAG3-1-Fc-TIM-3 expressing construct, and the secreted protein was purified from conditioned media by affinity chromatography. The purified proteins were analyzed for the presence of each individual domain by Western blotting using anti-LAG3, anti-Fc, and anti-TIM-3 antibodies (FIG. 6). These blots revealed a glycosylated protein that formed a dimer under non-reducing conditions by SDS-PAGE. The reduced and deglycosylated form of the protein migrated at the predicted monomeric molecular weight of approximately 100 kDa.

The in vivo activity of an illustrative chimeric protein of the present invention (LAG3-Fc-TIM-3) in killing cancer cells was determined. Here, mice were inoculated subcutaneously on the rear flank with either MC38 (murine colon adenocarcinoma; FIG. 7A) cells or B16.F10 (murine melanoma; FIG. 7B) cells. Five days later, mice were given three IP injections of 300 mg of the LAG3-Fc-TIM3 chimeric protein. Nineteen days after tumor inoculation, tumor volumes were measured. As shown in FIG. 7A and FIG. 7B, fourteen days after treatment with the chimeric protein, tumor bearing mice had their tumor volumes greatly reduced.

These data demonstrate that an illustrative chimeric protein of the present invention is capable of killing cancer cells in vivo.

Example 4: Another illustrative chimeric protein (AXL-Fc-TIGIT) promotes survival of cancer-bearing animals in vivo

In this example, in vivo activity of another illustrative chimeric protein of the present invention (AXL-Fc-TIGIT) in promoting survival of tumor-bearing mice was determined.

Here, mice were inoculated with CT26 mouse colorectal tumors and tumor-bearing mice were treated with the AXL- Fc-TIGIT chimeric protein or vehicle.

As shown in FIG. 8A and FIG. 8B, mice treated with the chimeric protein had reduced tumor volume (FIG. 8A) and extended survival (FIG. 8B) relative to vehicle.

Example 5: Functional in vivo properties of chimeric proteins of the present invention

The in vivo ability of the chimeric proteins of the present invention (e.g., PD1-Fc-LAP, PD1-Fc-TGFBR2, PD1-Fc-LAG3, PD1-Fc-TIM3, LAG3-Fc-TIM3, AXL-Fc-TIGIT, SIRPa-Fc-TGFBR2, and PDL1-Fc-BTNL2) to target and reduce tumor volume is determined. Here, mice are inoculated with tumor cells, e.g., without limitation, one of CT26 (murine colon carcinoma), MC38 (murine colon adenocarcinoma), B16.F10 (murine melanoma), A20 (lymphoma), and WEHI3 (leukemia) cells, into one flank. Once the tumors have reached sufficient volume (e.g., volume of about 100 mm ³ ), mice are administered various concentrations of the murine chimeric protein or suitable controls, e.g., vehicle, Fc fusion proteins comprising the extracellular domain of the first transmembrane protein (as included in the chimeric protein), Fc fusion proteins comprising the extracellular domain of the second transmembrane protein (as included in the chimeric protein) or antibodies that bind the extracellular domain of the first transmembrane protein or the ligand/receptor of extracellular domain of the first transmembrane protein, or antibodies that bind the extracellular domain of the second transmembrane protein or the ligand/receptor of extracellular domain of the second transmembrane protein. Beginning about eight days after inoculation, tumor volumes are measured and changes between starting tumor volumes are determined; tumor volumes are measured periodically thereafter until the end of the experiment (e.g., 20, 25, 30, 35 or more days or until euthanizing because of tumor size). At each time point, the number of surviving treated mice is determined and Kaplan-Meier plots of the percent survival each days after tumor inoculation resulting from treatments are created. In various experiments, mice that reject the tumor may be re challenged with a secondary tumor on the opposing flank, and primary/secondary tumors continue to be measured as well as lethality of the mice.

The therapeutic activity of the treatments may further be assayed. In particular, changes in pharmacodynamic biomarkers showing tumor rejection are determined by cytokine elevations in serum (in vivo) or changes in pharmacodynamic biomarkers in vitro in immune-related cells incubated with the super-antigen Staphylococcal enterotoxin B (SEB assay) or when cultured in AIM V media. Exemplary pharmacodynamic biomarkers include IFNy, IL-2, IL-4, IL-5, IL-6, and IL-17A. Also, changes in the number of peripheral lymphocytes (and ratios of types of lymphocytes) is measured and quantified over time.

Additionally, separate groups of inoculated mice are euthanized six or twenty-four hours after administration of the murine chimeric protein or suitable control and their spleens excised, dissociated and assessed by flow cytometry for populations of activated CD4+ or CD8+ dendritic cells (as examples of splenic immune cell types) to determine the extent of T cell activation and proliferation resulting from the chimeric protein treatments.

INCORPORATION BY REFERENCE

All patents and publications referenced herein are hereby incorporated by reference in their entireties.

Specifically, additional teachings related to the present invention are found, in one or more of WO2018/157162; WO2018/157165; WO2018/157164; WO2018/157163; and WO2017/059168, the contents of each of which is incorporated herein by reference in its entirety.

The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention.

As used herein, all headings are simply for organization and are not intended to limit the disclosure in any manner. The content of any individual section may be equally applicable to all sections.

EQUIVALENTS

While the invention has been disclosed in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth and as follows in the scope of the appended claims.

Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific embodiments disclosed specifically herein. Such equivalents are intended to be encompassed in the scope of the following claims.