TARGETED CHECKPOINT INHIBITORS AND METHODS OF USE

CROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No. 62/464,982 filed February 28, 2017 which is incorporated by reference herein in its entirety.

BACKGROUND

[0002] The selective destruction of an individual cell or a specific cell type is often desirable in a variety of clinical settings. For example, it is a primary goal of cancer therapy to specifically destroy tumor cells, while leaving healthy cells and tissues intact and undamaged. One such method is by inducing an immune response against the tumor with immunomodulators.

SUMMARY

[0003] Provided herein are inducible target-binding proteins, comprising a single polypeptide chain comprising two or more inactive immune checkpoint protein binding domains, two or more target antigen binding domains, one or more half-life extension domains, and one or more protease cleavage sites; wherein upon protease cleavage of the protease cleavage site and binding the target antigens by the target antigen binding domains, the immune check point protein binding domain becomes active and binds to an immune check point protein. In some embodiments, binding to the immune check point protein inhibits the immune checkpoint and activates a T cell. In some embodiments, the protease cleavage site is cleaved by at least one of a serine protease, a cysteine protease, an aspartate protease, a threonine protease, a glutamic acid protease, a metalloproteinase, a gelatinase, and an asparagine peptide lyase. In some embodiments, the protease cleavage site is cleaved at the site of a tumor. In some embodiments, the protease is expressed by a cell in the microenvironment of the tumor In some embodiments, the protein comprises two or more protease cleavage sites. In some embodiments, the immune checkpoint protein is Cd27, CD137, 2B4, CD155, ICOS, HVEM, CD40L, LIGHT, TEVI-1, OX40, DNAM-1, PD-L1, PD1, PD-L2, CTLA-4, CD8-, B7-H4, CD40, CEACAM1, CD48, CD70, 4 IBB, A2AR, B7H3, B7H4, BTLA, IDO, KIR, LAG3, TIM-3, or VISTA. In some embodiments, the ligand does not dimerize unless the protease cleavage site has been cleaved. In some embodiments, one or more half-life extension domains comprise a binding domain to human serum albumin. In some embodiments, one or more half-life extension domains comprise a scFv, a variable heavy domain (VH), a variable light domain (VL), a nanobody, a peptide, a ligand, a small molecule, or a Fc domain. In some embodiments, the target antigen binding domain specifically binds to a cell surface molecule. In some embodiments, the target antigen binding domain specifically binds to a tumor antigen. In some embodiments, the target antigen binding domain specifically binds to an antigen selected from at least one of EpCAM, EGFR, HER-2, HER-3, cMet, CEA, and FoIR. In some embodiments, the protein has improved pharmacokinetics as compared to an IgG to the same target antigen.

[0004] Also provided herein are polynucleotides encoding an inducible target-binding protein according to any one of the above embodiments. Further provided herein are vectors comprising polynucleotides encoding inducible target-binding proteins herein. Additionally provided herein are host cells transformed with vectors comprising polynucleotides encoding inducible target-binding proteins herein.

[0005] Also provided herein are pharmaceutical compositions comprising (i) the inducible target- binding proteins according to any one the above embodiments, the polynucleotides encoding the inducible target-binding proteins herein, the vector comprising nucleotides encoding the inducible target- binding proteins herein, or the host cell transformed with the vector comprising the polynucleotide encoding the inducible target-binding proteins herein and (ii) a pharmaceutically acceptable carrier.

[0006] Additionally provided herein are processes for the production of an inducible target-binding protein of claim 1, said process comprising culturing a host transformed or transfected with a vector comprising a nucleic acid sequence encoding an target-binding protein of any one of the above embodiments under conditions allowing the expression of the protein and recovering and purifying the produced protein from the culture.

[0007] Also provided herein are methods for the treatment or amelioration of a proliferative disease, a tumorous disease, an inflammatory disease, an immunological disorder, an autoimmune disease, an infectious disease, a viral disease, an allergic reaction, a parasitic reaction, a graft-versus-host disease or a host-versus-graft disease comprising the administration of an inducible target-binding protein of any of the above embodiments to a subject in need of such a treatment or amelioration. In some embodiments, the subject is a human. In some embodiments, the method further comprises administration of an agent in combination with the inducible target-binding protein of any one of the above embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

[0008] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

[0009] Figure 1 shows an exemplary inducible immune checkpoint protein. In this example, the anti- CTLA-4 VH and VL domains are separated by a protease cleavage site which keeps the anti -CTLA-4 VH and VL domains from folding properly and binding to CTLA-4 on a T cell. Figure 1 also shows the cleaved inducible immune checkpoint protein, where the VH and VL domains are folded such that they are able to bind to CTLA-4 and the anti-target domains are bound to the target antigen on the surface of the target cell. This example also has a half-life extension domain.

[0010] Figure 2 shows an exemplary dual-target inducible immune checkpoint protein. In this example, the anti -CTLA-4 VH and VL domains are separated by a protease cleavage site which keeps the anti- CTLA-4 VH and VL domains from folding properly and binding to CTLA-4 on a T cell. Figure 2 also shows the cleaved inducible immune checkpoint protein, where the VH and VL domains are folded such that they are able to bind CTLA-4 and each anti-target domain is bound to its target antigen on the surface of the target cell. This example also has a half-life extension domain.

[0011] Figure 3 shows an exemplary inducible immune checkpoint protein. In this example, the anti- CTLA-4 VH and VL domains are bound to protease cleavable VL and VH domains that together do not bind to CTLA-4. Once these domains are cleaved, the anti-CTLA-4 VH and VL domains are able to fold and associate with CTLA-4 on a T cell. This example also has two anti -target domains that in some cases bind to two different antigens and in some cases bind to the same antigen. This example also has a half-life extension domain.

[0012] Figure 4 shows an exemplary inducible immune checkpoint protein. In this example, the anti- OX40 VH and VL domains are separated by a protease cleavage site which keeps the anti-OX40 VH and VL domains from folding properly and binding to OX40 on a T cell. Figure 4 also shows the cleaved inducible immune checkpoint protein, where the VH and VL domains are folded such that they are able to bind to OX40 and the anti -target domains are bound to the target antigen on the surface of the target cell. This example also has a half-life extension domain.

[0013] Figure 5 shows an exemplary dual-target inducible immune checkpoint protein. In this example, the anti-OX40 VH and VL domains are separated by a protease cleavage site which keeps the anti-OX40 VH and VL domains from folding properly and binding to OX40 on a T cell. Figure 5 also shows the cleaved inducible immune checkpoint protein, where the VH and VL domains are folded such that they are able to bind OX40 and each anti-target domain is bound to its target antigen on the surface of the target cell. This example also has a half -life extension domain.

[0014] Figure 6 shows an exemplary inducible immune checkpoint protein. In this example, the anti- OX40 VH and VL domains are bound to protease cleavable VL and VH domains that together do not bind to OX40. Once these domains are cleaved, the anti-OX40 VH and VL domains are able to fold and associate with OX40 on a T cell. This example also has two anti-target domains that in some cases bind to two different antigens and in some cases bind to the same antigen. This example also has a half-life extension domain.

DETAILED DESCRIPTION

[0015] Described herein are inducible checkpoint inhibitor proteins, such as tri-specific, quad-specific antigen, and multi-specific inducible checkpoint inhibitor proteins, pharmaceutical compositions thereof, as well as nucleic acids, recombinant expression vectors, and host cells for making such inducible checkpoint inhibitor proteins. Also provided are methods of using the disclosed inducible checkpoint inhibitor proteins in the prevention and/or treatment of diseases, conditions, and disorders. The inducible checkpoint inhibitor proteins are capable of specifically binding to one or more target antigen as well as a checkpoint protein, and optionally a half-life extension domain, such as an HSA binding domain.

Binding to a checkpoint protein is only possible once activated by a protease and binding to the target antigen(s). It is to be understood that in some embodiments, protease cleavage of the protease cleavage domain occurs before target antigen binding domain binding to the target antigen. It is also to be understood that in some embodiments, protease cleavage of the protease cleavage domain occurs after target antigen binding domain binding to the target antigen. Figures 1, 2 and 3 depict three non-limiting examples of an inducible checkpoint inhibitor protein.

[0016] The inducible checkpoint inhibitor proteins descnbed herein are designed to allow specific targeting of cells expressing a target antigen by modulating an immune response. This improves specificity compared to checkpoint inhibitors which may or may not be have specific activity at a target cell, such as a tumor or cancer cell. In contrast, by activating immune checkpoint binding specifically in the microenvironment of the target cell, where the target antigen and proteases are highly expressed, the inducible checkpoint inhibitor proteins can activate an immune response towards cells expressing a target antigen in a highly specific fashion, thereby directing the immune response towards the target cell. The inducible checkpoint inhibitor proteins described herein inhibit a checkpoint protein via protease- activated binding to an immune checkpoint protein, which inhibits an immune response. Simultaneous binding of several inducible checkpoint inhibitor proteins to an immune checkpoint protein and to a target antigen expressed on the surface of particular cells causes immune activation and mediates an immune response to the particular target antigen expressing cell. Thus, inducible checkpoint inhibitor proteins are contemplated to display strong, specific and efficient target cell killing. In some embodiments, the inducible checkpoint inhibitor proteins described herein stimulate target cell killing by an immune response to eliminate pathogenic cells in protease-rich microenvironments (e.g., tumor cells, virally or bacterially infected cells, autoreactive T cells, etc). In some of such embodiments, cells are eliminated selectively, thereby reducing the potential for toxic side effects. In other embodiments, the same polypeptides could be used to enhance the elimination of endogenous cells for therapeutic effect, such as B or T lymphocytes in autoimmune disease, or hematopoietic stem cells (HSCs) for stem cell transplantation. Proteases known to be associated with diseased cells or tissues include, but are not limited to, serine proteases, cysteine proteases, aspartate proteases, threonine proteases, glutamic acid proteases, metalloproteases, asparagine peptide lyases, serum proteases, cathepsins, Cathepsin B, Cathepsin C, Cathepsin D, Cathepsin E, Cathepsm K, Cathepsin L, kallikreins, hKl, hK10, hK15, plasmin, collagenase, Type IV collagenase, stromelysin, Factor Xa, chymotrypsin-like protease, trypsin- like protease, elastase-like protease, subtilisin-like protease, actinidain, bromelain, calpain, caspases, caspase-3, Mirl-CP, papain, HIV-1 protease, HSV protease, CMV protease, chymosin, renin, pepsin, matriptase, legumain, plasmepsin, nepenthesin, metalloexopeptidases, metalloendopeptidases, matrix metalloproteases (MMP), MMP1, MMP2, MMP3, MMP8, MMP9, MMP13, MMP11, MMP14, urokinase plasminogen activator (uPA), enterokinase, prostate-specific antigen (PSA, hK3), interleukin- 1β converting enzyme, thrombin, FAP (FAP-a), dipeptidyl peptidase, and dipeptidyl peptidase IV (DPPIV/CD26).

[0017] The inducible checkpoint inhibitor proteins described herein confer further therapeutic advantages over traditional monoclonal antibodies and other checkpoint inhibitors. Toxicities are possible when, in some cases, healthy cells or tissues are targeted by an immune response elicited by a checkpoint inhibitor. One benefit to an inducible checkpoint inhibitor protein is that binding to a checkpoint inhibitor is dependent upon activation by a protease expressed by the target cell, such as a tumor cell, and binding of the antigen binding domains to one or more target antigens, for example a tumor antigen. The inducible checkpoint inhibitor proteins comprise an inactive checkpoint protein binding domain comprising VH and VL domains separated by one or more protease cleavage sites. In the protease-rich environment of the target cell, the protease cleavage sites are cleaved, allowing the VH and VL domains to fold properly and bind to an immune checkpoint protein when one or more target antigens are bound. In the absence of protease cleavage, the immune checkpoint binding domain is inactive and cannot bind to the immune checkpoint.

[0018] The inducible checkpoint inhibitor proteins described herein comprise at least one protease cleavage site comprising an amino acid sequence that is cleaved by at least one protease. In some cases, the inducible checkpoint inhibitor proteins described herein comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more protease cleavage sites that are cleaved by at least one protease. In some cases, the protease cleavage site comprises an amino acid sequence recognized by a protease is a MMP9 cleavage site comprising a polypeptide having an amino acid sequence LEATA (SEQ ID NO: 4).

[0019] The inducible checkpoint inhibitor proteins described herein confer additional therapeutic advantages over traditional monoclonal antibodies and other smaller bispecific molecules. Generally, the effectiveness of recombinant protein pharmaceuticals depends heavily on the intrinsic pharmacokinetics of the protein itself. One such additional benefit here is that the inducible checkpoint inhibitor proteins described herein have extended pharmacokinetic elimination half-time due to having a half-life extension domain, for example a binding domain specific to HSA. In this respect, the inducible checkpoint inhibitor proteins described herein have an extended serum elimination half-time of about two, three, about five, about seven, about 10, about 12, or about 14 days in some embodiments. This contrasts to other binding proteins such as BiTE or DART molecules which have relatively much shorter elimination half-times. For example, the BiTE CD19xCD3 bispecific scFv-scFv fusion molecule requires continuous intravenous infusion (i.v.) drug delivery due to its short elimination half-time. The longer intrinsic half-times of the inducible checkpoint inhibitor proteins solve this issue thereby allowing for increased therapeutic potential such as low-dose pharmaceutical formulations, decreased periodic administration and/or novel pharmaceutical compositions.

[0020] The inducible checkpoint inhibitor proteins described herein also have an optimal size for enhanced tissue penetration and distribution and enhanced reduced first pass renal clearance. Because the kidney generally filters out molecules below 50 kDa, efforts to reduce clearance in the design of protein therapeutics have focused on increasing molecular size through protein fusions, glycosylation, or the addition of polyethylene glycol polymers (i.e. , PEG). However, while increasing the size of a protein therapeutic may prevent renal clearance, the downside is that the larger size also prevents penetration of the molecule into the target tissues. The inducible checkpoint inhibitor proteins described herein avoid this by associating with albumin which will prevent renal clearance while also having a small size that allows enhanced tissue penetration, distribution, and optimal efficacy. Accordingly, the inducible checkpoint inhibitor proteins described herein, in some embodiments, have a size of about 50 kD to about 80 kD, about 50 kD to about 75 kD, about 50 kD to about 70 kD, or about 50 kD to about 65 kD. Thus, the size of the inducible checkpoint inhibitor is advantageous over IgG antibodies, which are about 150 kD, and the BiTE and DART diabody molecules, which are about 55 kD, but are not half-life extended and therefore are cleared quickly through the kidney. [0021] Another feature of the inducible checkpoint inhibitor proteins described herein is that they are of a single-polypeptide design with flexible linkage of their domains. This allows for facile production and manufacturing of the inducible checkpoint inhibitor proteins as they can be encoded by a single cDNA molecule to be easily incorporated into a vector. Further, because the inducible checkpoint inhibitor proteins described herein are a monomeric single polypeptide chain, there are no chain pairing issues or a requirement for dimerization. It is contemplated that the inducible checkpoint inhibitor proteins described herein have a reduced tendency to aggregate unlike other reported molecules, such as bispecific BiTE proteins.

[0022] In one aspect, the inducible checkpoint inhibitor proteins, in pre-activated form, comprise a single polypeptide chain comprising a first region and a second region separated by at least one protease cleavage domain (P). In an embodiment, the first region comprises an anti-immune checkpoint V _H binding domain (CV _H ) and a target antigen binding domain (Ti). In an embodiment, the second region comprises an anti -immune checkpoint V _L binding domain (CV _L ) and a target antigen binding domain (T ₂ ). In an embodiment, the inducible checkpoint inhibitor domain optionally comprises a half -life extension domain (H) in the first region. In an embodiment, the inducible checkpoint inhibitor protein optionally comprises a half-life extension domain (H) in the second region. Once activated by a protease cleaving the protease cleavage domain (P) and target antigen binding domains Ti and T ₂ binding the target antigens, the anti-immune checkpoint binding domains CV _H and CV _L are activated to bind to an immune checkpoint protein. The domains in an inducible checkpoint inhibitor protein are contemplated to be arranged in any order within each region, with a protease cleavage domain (P) in the center of the pre-activated polypeptide. Further, each region may be in any order within the pre-activated polypeptide. Thus, by way of example only, it is contemplated that exemplary domain order of the inducible checkpoint inhibitor proteins include, but are not limited to:

a) CV _H -T _J -P-T _Z -CV _L ,

b) _TL -CV _H -P-T ₂ -CV _L ,

c) CV _H -T _J -P-CV _L -T,,

d) T _J -CV _H -P-CV _L -T,,

e) H-CV _H -T P-T ₂ -CV, _L

f) CV _H -H-T P-T ₂ -CV _L ,

g) CV _H -T _J -H-P-T.-CV _L ,

h) CV _H -T _J -P-H-T.-CV _L ,

i) CV _H -T _J -P-T.-H-CV _L ,

j) CV _H -T _J -P-T.-CV _L -H,

k) H-T _! -CV _H -P-T ₂ -CV _L ,

1) T _J -H-CV _H -P-T.-CV _L ,

m) T _J -CV _H -H-P-T.-CV _L ,

n) _TI -CV _H -P-H-T ₂ -CV _L ,

o) _TI -CV _H -P-T ₂ -H-CV _L , P) _Tl -CV _H -P-T ₂ -CV _L -H,

q) H-CV _H -T _! -P-CV _L -T: 2,

r) CV _H -H-TVP-CV _L -T _Z ,

s) CV _H -T _J -H-P-CV _L -T^

t) CV _H -T _J -P-H-CV _L -T^

u) CV _H -T _I -P-CV _L -H-T ₂ ,

v) CV _H -T _J -P -CV _L -T ₂ -H,

w) H-T _! -CV _H -P-CV _L -T _J ,

x) T _J -H-CV _H -P-CV _L -T _J ,

y) T _I -CV _H -H-P-CV _L -T ₂ ,

z) T _J -CV _H -P-H-CV _L -T _I ,

aa) T _J -CV _H -P-CV _L -H-T _I , and

bb) T _J -CV _H -P-CV _L -^-H.

[0023] In one aspect, the inducible checkpoint inhibitor proteins, in pre-activated form, comprise a single polypeptide chain comprising a first region and a second region. In an embodiment, the first region comprises an anti-immune checkpoint V _H binding domain (CV _H ), an inactive anti-immune checkpoint V _L binding domain (CV _Li ) which associates with CV _H , and a target antigen binding domain (Ti), wherein CV _L , comprises at least one protease cleavage domain (P). In an embodiment, the second region comprises an anti-immune checkpoint V _L binding domain (CV _L ), an inactive anti-immune checkpoint V _H binding domain (CV _Hi ) which associates with CV _L , and a target antigen binding domain (T ₂ ), wherein CV _L , compnses at least one protease cleavage domain (Ρ). In an embodiment, the inducible checkpoint inhibitor protein optionally comprises a half-life extension domain (H) in the first region. In an embodiment, the inducible checkpoint inhibitor protein optionally comprises a half-life extension domain (H) in the second region. Once activated by a protease cleaving the protease cleavage domains (P) and target antigen binding domains Ti and T ₂ binding the target antigens, the anti-immune checkpoint binding domains CV _H and CV _L are activated to bind to an immune checkpoint. An example of this type of inducible checkpoint inhibitor protein is described in Figure 3.

[0024] In the inducible checkpoint inhibitor proteins described herein, the domains are linked by internal linkers LI, L2, L3, and L4, where LI links the first and second domain of the inducible checkpoint inhibitor proteins, L2 links the second and third domains of the inducible checkpoint inhibitor proteins, L3 links the third and fourth domains of the inducible checkpoint inhibitor proteins, and L4 links the fourth and fifth domains of the protease activated inducible checkpoint inhibitor proteins. Linkers LI, L2, L3, and L4 have an optimized length and/or amino acid composition. In some embodiments, linkers LI, L2, L3, and L4 are the same length and amino acid composition. In other embodiments, LI, L2, L3, and L4 are different. In certain embodiments, internal linkers LI, L2, L3, and/or L4 are "short", i.e., consist of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acid residues. Thus, in certain instances, the internal linkers consist of about 12 or less amino acid residues. In the case of 0 amino acid residues, the internal linker is a peptide bond. In certain embodiments, internal linkers LI, L2, L3, and/or L4 are "long", i.e., consist of 15, 20, or 25 amino acid residues. In some embodiments, these internal linkers consist of about 3 to about 15, for example 8, 9, or 10 contiguous amino acid residues. Regarding the amino acid composition of the internal linkers LI, L2, L3, and L4, peptides are selected with properties that confer flexibility to the inducible checkpoint inhibitor proteins, do not interfere with the binding domains, as well as resist cleavage from proteases. For example, glycine and serine residues generally provide protease resistance. Examples of internal linkers suitable for linking the domains in the inducible checkpoint inhibitor proteins include, but are not limited to, (GS) _n , (GGS) _n , (GGGS) _n , (GGSG)„, (GGSGG) _n , or (GGGGS)„, wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In one embodiment, internal linker LI, L2, and/or L3 is (GGGGS) ₄ or (GGGGS) ₃ .

Immune Checkpoint Binding Domain

[0025] The specificity of the response of T cells is mediated by the recognition of antigen (displayed in context of a major histocompatibility complex, MHC) by the T cell receptor complex. An added regulatory mechanism in an immune response is mediated by co-stimulatory proteins that modulate the immune response. Immune checkpoint proteins include, but are not limited, to CD27, CD40, OX40, GITR, CD137, B7, CD28, ICOS, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR, LAG3, PD-1, PD- 1L, TIM-3, and VISTA. Inhibitory immune checkpoint proteins to be inhibited in activating an immune response include, but are not limited to, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR, LAG3, PD- 1, PD-IL, TIM-3, and VISTA. In some embodiments, binding of an anti-immune checkpoint antibody to an immune checkpoint protein is regulated by a protease cleavage domain which restncts binding of the immune checkpoint antibody to the immune checkpoint protein only in the microenvironment of a diseased cell or tissue with elevated levels of proteases, for example in a tumor microenvironment.

[0026] In one aspect, the inducible checkpoint inhibitor proteins described herein comprise a domain which specifically binds to an immune checkpoint protein when activated by a protease. In one aspect, the inducible checkpoint inhibitor proteins described herein comprise two or more domains which, when activated by a protease, specifically bind to a human immune checkpoint protein. In some embodiments, the inducible checkpoint inhibitor proteins described herein comprise two or more domains which, when activated by a protease, specifically binds to A2AR. In some embodiments, the inducible checkpoint inhibitor proteins described herein comprise two or more domains which, when activated by a protease, specifically bind to B7-H3. In some embodiments, the inducible checkpoint inhibitor proteins described herein comprise two or more domains which, when activated by a protease, specifically bind to B7-H4. In some embodiments, the inducible checkpoint inhibitor proteins described herein comprise two or more domains which, when activated by a protease, specifically bind to BTLA. In some embodiments, the inducible checkpoint inhibitor proteins described herein comprise two or more domains which, when activated by a protease, specifically bind to CTLA-4. In some embodiments, the inducible checkpoint inhibitor proteins described herein comprise two or more domains which, when activated by a protease, specifically bind to IDO. In some embodiments, the inducible checkpoint inhibitor proteins described herein comprise two or more domains which, when activated by a protease, specifically bind to KIR. In some embodiments, the inducible checkpoint inhibitor proteins described herein comprise two or more domains which, when activated by a protease, specifically bind to LAG3. In some embodiments, the inducible checkpoint inhibitor proteins described herein comprise two or more domains which, when activated by a protease, specifically bind to PD-1. In some embodiments, the inducible checkpoint inhibitor proteins described herein comprise two or more domains which, when activated by a protease, specifically bind to PD-1L. In some embodiments, the inducible checkpoint inhibitor proteins described herein comprise two or more domains which, when activated by a protease, specifically bind to TIM -3. In some embodiments, the inducible checkpoint inhibitor proteins described herein comprise two or more domains which, when activated by a protease, specifically bind to VISTA.

[0027] In some embodiments, the protease cleavage site is between the anti-immune checkpoint protein VH and VL domains and prevents them from folding and binding to an immune checkpoint protein on a T cell. Once the protease cleavage site is cleaved by a protease present at the target cell, the anti -immune checkpoint protein VH and VL domains are able to fold and bind to an immune checkpoint protein on a T cell. In an alternate embodiment, the protease cleavage site is designed into a non -immune checkpoint protein binding VL and VH domain that binds to the anti-immune checkpoint protein VH and VL domains. Cleavage of the protease cleavage site by a protease present at the target cell removes the nonimmune checkpoint protein binding VL and VH domain and allows the anti -immune checkpoint protein VH and VL domain to fold and to bind an immune checkpoint protein on a T cell.

[0028] The inducible checkpoint inhibitor proteins described herein comprise a domain which specifically binds to an immune checkpoint protein when activated by a protease. In one embodiment, the domain which specifically binds to an immune checkpoint protein comprises a VH domain and a VL domain separated by at least one protease cleavage site. When the protease cleavage site is cleaved, the VH domain and the VL domain are able to fold and therefore bind to an immune checkpoint protein. In some embodiments, the protease cleavage site is in a loop region. In some embodiments, the protease cleavage site is within the VH and/or the VL domains and the protease cleavage sites are cleaved revealing the VH and/or the VL domains allowing them to fold and therefore bind to an immune checkpoint protein.

[0029] In certain embodiments, the immune checkpoint protein binding domain of the inducible checkpoint inhibitor proteins described herein exhibit not only potent immune checkpoint protein binding affinities with human immune checkpoint proteins, but show also excellent cross reactivity with the respective cynomolgus monkey immune checkpoint proteins. In some instances, the immune checkpoint protein binding domain of the inducible checkpoint inhibitor proteins is cross-reactive with an immune checkpoint protein from cynomolgus monkey. In certain instances, human ynomolgous K _D ratios for an immune checkpoint protein are between 5 and 0.2.

[0030] In some embodiments, the immune checkpoint protein binding domain of the inducible checkpoint inhibitor protein can be any domain that binds to an immune checkpoint protein including, but not limited to, domains from a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a human antibody, or a humanized antibody. In some instances, it is beneficial for the immune checkpoint protein binding domain to be derived from the same species in which the inducible checkpoint inhibitor protein will ultimately be used. For example, for use in humans, it may be beneficial for the immune checkpoint protein binding domain of the inducible checkpoint inhibitor protein to comprise immune checkpoint protein binding domain residues of a human or humanized antibody or antibody fragment.

[0031] Thus, in one aspect, the inducible checkpoint inhibitor protein comprises a humanized or human antibody or an antibody fragment, or a murine antibody or antibody fragment. In one embodiment, the humanized or human anti -immune checkpoint protein binding domain comprises one or more (e.g., all three) light chain complementary determining region 1 (LC CDR1), light chain complementary determining region 2 (LC CDR2), and light chain complementary determining region 3 (LC CDR3) of a humanized or human anti- immune checkpoint protein binding domain described herein, and/or one or more (e.g., all three) heavy chain complementary determining region 1 (HC CDR1), heavy chain complementary determining region 2 (HC CDR2), and heavy chain complementary determining region 3 (HC CDR3) of a humanized or human anti -immune checkpoint protein binding domain described herein, e.g., a humanized or human anti -immune checkpoint protein binding domain comprising one or more, e.g., all three, LC CDRs and one or more, e.g., all three, HC CDRs.

[0032] In some embodiments, the humanized or human anti -immune checkpoint protein binding domain comprises a humanized or human light chain variable region specific to an immune checkpoint protein where the light chain variable region specific to an immune checkpoint protein comprises human or non- human light chain CDRs in a human light chain framework region. In certain instances, the light chain framework region is a λ (lambda) light chain framework. In other instances, the light chain framework region is a κ (kappa) light chain framework.

[0033] In some embodiments, the humanized or human anti -immune checkpoint protein binding domain comprises a humanized or human heavy chain variable region specific to an immune checkpoint protein where the heavy chain variable region specific to an immune checkpoint protein comprises human or non-human heavy chain CDRs in a human heavy chain framework region.

[0034] In certain instances, the complementary determining regions of the heavy chain and/or the light chain are derived from known anti-immune checkpoint protein antibodies, such as, for example, pembrolizumab, nivolumab, and ipillimumab.

[0035] In one embodiment, the anti-immune checkpoint protein binding domain is a single chain variable fragment (scFv) comprising a light chain and a heavy chain of an amino acid sequence. As used herein, "single chain variable fragment" or "scFv" refers to a antibody fragment comprising a variable region of a light chain and at least one antibody fragment comprising a variable region of a heavy chain, wherein the light and heavy chain variable regions are contiguously linked via a short flexible polypeptide linker and capable of being expressed as a single polypeptide chain, and wherein the scFv retains the specificity of the intact antibody from which it is derived. In an embodiment, the anti -immune checkpoint protein binding domain comprises: a light chain variable region comprising an amino acid sequence having at least one, two or three modifications (e.g., substitutions) but not more than 30, 20, or 10 modifications (e.g., substitutions) of an amino acid sequence of a light chain variable region, or a sequence with 95-99% identity with an amino acid sequence provided herein; and/or a heavy chain variable region comprising an amino acid sequence having at least one, two, or three modifications (e.g., substitutions) but not more than 30, 20, or 10 modifications (e.g., substitutions) of an amino acid sequence of a heavy chain variable region provided herein, or a sequence with 95-99% identity to an amino acid sequence provided herein. In one embodiment, the humanized or human anti-immune checkpoint binding domain is a scFv, and a light chain variable region comprising an amino acid sequence described herein, is attached to a heavy chain variable region comprising an amino acid sequence described herein, via a scFv linker. The light chain variable region and heavy chain variable region of a scFv can be, e.g., in any of the following orientations: light chain variable region- scFv linker- heavy chain variable region or heavy chain variable region- scFv linker-light chain variable region.

[0036] In some instances, scFvs which bind to an immune checkpoint protein are prepared according to known methods. For example, scFv molecules can be produced by linking VH and VL regions together using flexible polypeptide linkers. The scFv molecules comprise a scFv linker (e.g., a Ser-Gly linker) with an optimized length and/or amino acid composition. Accordingly, in some embodiments, the length of the scFv linker is such that the VH or VL domain can associate intermolecularly with the other variable domain to form the immune checkpoint protein binding site. In certain embodiments, such scFv linkers are "short", i.e. consist of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acid residues. Thus, in certain instances, the scFv linkers consist of about 12 or less amino acid residues. In the case of 0 amino acid residues, the scFv linker is a peptide bond. In some embodiments, these scFv linkers consist of about 3 to about 15, for example 8, 9, or 10 contiguous amino acid residues. Regarding the amino acid composition of the scFv linkers, peptides are selected that confer flexibility, do not interfere with the variable domains, as well as allow inter-chain folding to bring the two variable domains together to form a functional immune checkpoint protein binding site. For example, scFv linkers comprising glycine and serine residues generally provide protease resistance. In some embodiments, linkers in a scFv comprise glycine and serine residues. The amino acid sequence of the scFv linkers can be optimized, for example, by phage -display methods to improve the immune checkpoint protein binding and production yield of the scFv. Examples of peptide scFv linkers suitable for linking a variable light chain domain and a variable heavy chain domain in a scFv include but are not limited to (GS) _n , (GGS) _n , (GGGS) _n , (GGSG) _n , (GGSGG) _n , or (GGGGS)„, wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In one embodiment, the scFv linker can be (GGGGS) ₄ or (GGGGS) ₃ . Variation in the linker length may retain or enhance activity, giving rise to superior efficacy in activity studies.

[0037] In some embodiments, immune checkpoint protein binding domain of an inducible checkpoint inhibitor protein has an affinity to an immune checkpoint protein on immune checkpoint protein expressing cells with a K _D of 1000 nM or less, 100 nM or less, 50 nM or less, 20 nM or less, 10 nM or less, 5 nM or less, 1 nM or less, or 0.5 nM or less. In some embodiments, the immune checkpoint protein binding domain of an inducible checkpoint inhibitor protein has an affinity to A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR, LAG3, PD-1, PD-1L, TIM-3, or VISTA with a K _D of 1000 nM or less, 100 nM or less, 50 nM or less, 20 nM or less, 10 nM or less, 5 nM or less, 1 nM or less, or 0.5 nM or less. In further embodiments, immune checkpoint protein binding domain of an inducible immune checkpoint protein has low affinity to an immune checkpoint protein, i.e., about 100 nM or greater.

[0038] The affinity to bind to an immune checkpoint protein can be determined, for example, by the ability of the inducible checkpoint inhibitor protein itself or its immune checkpoint protein binding domain to bind to an immune checkpoint protein coated on an assay plate; displayed on a microbial cell surface; in solution; etc. The binding activity of the inducible checkpoint inhibitor protein itself or its immune checkpoint protein binding domain of the present disclosure to an immune checkpoint protein can be assayed by immobilizing the ligand (e.g., the immune checkpoint protein) or the inducible checkpoint inhibitor protein itself or its immune checkpoint protein binding domain, to a bead, substrate, cell, etc. Agents can be added in an appropriate buffer and the binding partners incubated for a period of time at a given temperature. After washes to remove unbound material, the bound protein can be released with, for example, SDS, buffers with a high pH, and the like and analyzed, for example, by Surface Plasmon Resonance (SPR).

Protease Cleavage Domains

[0039] Protease cleavage domains are polypeptides having a sequence recognized and cleaved in a sequence-specific manner. Inducible checkpoint inhibitor proteins contemplated herein, in some cases, comprise a protease cleavage domain recognized in a sequence -specific manner by a matrix

metalloprotease (MMP), for example a MMP9. In some cases, the protease cleavage domain recognized by a MMP9 comprises a polypeptide having an ammo acid sequence PR(S/T)(L/I)(S/T) (SEQ ID NO: 3). In some cases, the protease cleavage domain recognized by a MMP9 comprises a polypeptide having an amino acid sequence LEATA (SEQ ID NO: 4). In some cases, the protease cleavage domain is recognized in a sequence-specific manner by a MMP11. In some cases, the protease cleavage domain recognized by a MMP11 comprises a polypeptide having an amino acid sequence GGAANLVRGG (SEQ ID NO: 5). In some cases, the protease cleavage domain is recognized by a protease disclosed in Table 1. In some cases, the protease cleavage domain recognized by a protease disclosed in Table 1 comprises a polypeptide having an amino acid sequence selected from a sequence disclosed in Table 1 (SEQ ID NOS: 1-42).

[0040] Proteases are proteins that cleave proteins, in some cases, in a sequence-specific manner.

Proteases include, but are not limited to, serine proteases, cysteine proteases, aspartate proteases, threonine proteases, glutamic acid proteases, metalloproteases, asparagine peptide lyases, serum proteases, cathepsins, Cathepsin B, Cathepsin C, Cathepsin D, Cathepsin E, Cathepsin K, Cathepsin L, kallikreins, hKl, hK10, hK15, plasmin, collagenase, Type IV collagenase, stromelysin, Factor Xa, chymotrypsin-like protease, trypsin-like protease, elastase-like protease, subtilisin-like protease, actinidain, bromelain, calpain, caspases, caspase-3, Mirl-CP, papain, HIV-1 protease, HSV protease, CMV protease, chymosin, renin, pepsin, matriptase, legumain, plasmepsin, nepenthesin,

metalloexopeptidases, metalloendopeptidases, matrix metalloproteases (MMP), MMP1, MMP2, MMP3, MMP8, MMP9, MMP13, MMP11, MMP14, urokinase plasminogen activator (uPA), enterokinase, prostate-specific antigen (PSA, hK3), interleukin-ΐβ converting enzyme, thrombin, FAP (FAP -a), dipeptidyl peptidase, and dipeptidyl peptidase IV (DPPIV/CD26).

Table 1: Exemplary Proteases and Protease Cleavage Domain Sequences

Caspase-3 KGSGDVEG 34

Interleukin 1β converting enzyme GWEHDG 35

Enterokinase EDDDDKA 36

FAP KQEQNPGST 37

Kallikrein 2 GKAFRR 38

Plasmin DAFK 39

Plasmin DVLK 40

Plasmin DAFK 41

TOP ALLLALL 42

[0041] Proteases are known to be secreted by some diseased cells and tissues, for example tumor or cancer cells, creating a microenvironment that is rich in proteases or a protease -rich microenvironment. In some case, the blood of a subject is rich in proteases. In some cases, cells surrounding the tumor secrete proteases into the tumor microenvironment. Cells surrounding the tumor secreting proteases include but are not limited to the tumor stromal cells, myofibroblasts, blood cells, mast cells, B cells, NK cells, regulatory T cells, macrophages, cytotoxic T lymphocytes, dendritic cells, mesenchymal stem cells, polymorphonuclear cells, and other cells. In some cases, proteases are present in the blood of a subject, for example proteases that target amino acid sequences found in microbial peptides. This feature allows for targeted therapeutics such as inducible checkpoint inhibitor proteins to have additional specificity because T cells will not be bound by the inducible checkpoint inhibitor protein except in the protease rich microenvironment of the targeted cells or tissue.

Half-Life Extension Domain

[0042] Contemplated herein are domains which extend the half-life of an inducible checkpoint inhibitor protein. Such domains are contemplated to include, but are not limited to, HSA binding domains, Fc domains, small molecules, and other half-life extension domains known in the art.

[0043] Human serum albumin (HSA) (molecular mass ~67 kDa) is the most abundant protein in plasma, present at about 50 mg/ml (600 μΜ), and has a half-life of around 20 days in humans. HSA serves to maintain plasma pH, contributes to colloidal blood pressure, functions as carrier of many metabolites and fatty acids, and serves as a major drug transport protein in plasma.

[0044] Noncovalent association with albumin extends the elimination half-time of short lived proteins. For example, a recombinant fusion of an albumin binding domain to a Fab fragment resulted in an in vivo clearance of 25- and 58-fold and a half-life extension of 26- and 37-fold when administered

intravenously to mice and rabbits respectively as compared to the administration of the Fab fragment alone. In another example, when insulin is acylated with fatty acids to promote association with albumin, a protracted effect was observed when injected subcutaneously in rabbits or pigs. Together, these studies demonstrate a linkage between albumin binding and prolonged action.

[0045] In one aspect, the inducible checkpoint inhibitor proteins described herein comprise a half-life extension domain, for example a domain which specifically binds to HSA. In some embodiments, the HSA binding domain of an inducible checkpoint inhibitor protein can be any domain that binds to HSA including, but not limited to, domains from a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a human antibody, or a humanized antibody. In some embodiments, the HSA binding domain is a single chain variable fragment (scFv), single-domain antibody such as a heavy chain variable domain (VH), a light chain variable domain (VL), and a variable domain (VHH) of camelid derived nanobody, peptide, ligand, or small molecule specific for HSA. In certain embodiments, the HSA binding domain is a single-domain antibody. In other embodiments, the HSA binding domain is a peptide. In further embodiments, the HSA binding domain is a small molecule. It is contemplated that the HSA binding domain of an inducible checkpoint inhibitor protein is fairly small and no more than 25 kD, no more than 20 kD, no more than 15 kD, or no more than 10 kD in some embodiments. In certain instances, the HSA binding is 5 kD or less if it is a peptide or small molecule.

[0046] The half-life extension domain of an inducible checkpoint inhibitor protein provides for altered pharmacodynamics and pharmacokinetics of the inducible checkpoint inhibitor protein itself. As above, the half-life extension domain extends the elimination half-time. The half-life extension domain also alters pharmacodynamic properties including alteration of tissue distribution, penetration, and diffusion of the inducible checkpoint inhibitor protein. In some embodiments, the half-life extension domain provides for improved tissue (including tumor) targeting, tissue penetration, tissue distribution, diffusion within the tissue, and enhanced efficacy as compared with a protein without a half-life extension binding domain. In one embodiment, therapeutic methods effectively and efficiently utilize a reduced amount of the inducible checkpoint inhibitor protein, resulting in reduced side effects, such as reduced non-tumor cell cytotoxicity.

[0047] Further, characteristics of the half-life extension domain, for example a HSA binding domain, include the binding affinity of the HSA binding domain for HSA. Affinity of said HSA binding domain can be selected so as to target a specific elimination half-time in a particular inducible checkpoint inhibitor protein. Thus, in some embodiments, the HSA binding domain has a high binding affinity. In other embodiments, the HSA binding domain has a medium binding affinity. In yet other embodiments, the HSA binding domain has a low or marginal binding affinity. Exemplary binding affinities include K _D concentrations at 10 nM or less (high), between 10 nM and 100 nM (medium), and greater than 100 nM (low). As above, binding affinities to HSA are determined by known methods such as Surface Plasmon Resonance (SPR).

Target Antigen Binding Domain

[0048] In addition to the described immune checkpoint inhibitor and half-life extension domains, the inducible checkpoint inhibitor proteins described herein also comprise at least two more domains that bind to one or more target antigens. It is contemplated herein that an inducible immune checkpoint inhibitor protein is cleaved, for example, in a disease-specific microenvironment or in the blood of a subject at the protease cleavage domain and that each target antigen binding domain will bind to a target antigen on a target cell, thereby activating the immune checkpoint protein binding domain to bind a T cell. At least one target antigen is involved in and/or associated with a disease, disorder or condition. In particular, a target antigen associated with a proliferative disease, a tumorous disease, an inflammatory disease, an immunological disorder, an autoimmune disease, an infectious disease, a viral disease, an allergic reaction, a parasitic reaction, a graft-versus-host disease or a host-versus-graft disease. In some embodiments, a target antigen is a tumor antigen expressed on a tumor cell Alternatively in some embodiments, a target antigen is associated with a pathogen such as a virus or bacterium. At least one target antigen may also be directed against healthy tissue.

[0049] In some embodiments, a target antigen is a cell surface molecule such as a protein, lipid or polysaccharide. In some embodiments, a target antigen is a on a tumor cell, virally infected cell, bacterially infected cell, damaged red blood cell, arterial plaque cell, or fibrotic tissue cell. It is contemplated herein that upon binding more than one target antigen, two inactive immune checkpoint protein binding domains are co -localized and form an active immune checkpoint protein binding domain on the surface of the target cell. In some embodiments, the inducible checkpoint inhibitor protein comprises more than one target antigen binding domain to activate an inactive immune checkpoint protein binding domain in the inducible checkpoint inhibitor protein. In some embodiments the inducible checkpoint inhibitor protein comprises more than one target antigen binding domain to enhance the strength of binding to the target cell. In some embodiments, more than one target antigen binding domain comprise the same target antigen binding domain. In some embodiments, more than one target antigen binding domain comprise different target antigen binding domains. For example, two different target antigen binding domains known to be dually expressed in a diseased cell or tissue, for example a tumor or cancer cell, can enhance binding or selectivity of an target antigen binding protein for a target.

[0050] Inducible checkpoint inhibitor proteins contemplated herein include at least one target antigen binding domain, wherein the target antigen binding domain binds to at least one target antigen. Target antigens, in some cases, are expressed on the surface of a diseased cell or tissue, for example a tumor or a cancer cell. Target antigens include, but are not limited to, CD19, CD20, CD33, CD30, CD64, CD123, EpCAM, EGFR, HER-2, HER-3, c-Met, LAG3, FoIR, EGFR, PSMA, VEGF, and CEA. Inducible checkpoint inhibitor proteins disclosed herein also include proteins comprising two target antigen binding domains that bind to two different target antigens known to be expressed on a diseased cell or tissue. Exemplary pairs of target antigen binding domains, include but are not limited to, EGFR/CEA, EpCAM CEA, and HER-2/HER-3. In some embodiments, a target antigen is a viral antigen. Examples of viral antigen include but are not limited to Hepatitis Viruses, Flaviviruses, Westnile Virus, Ebola Virus, Pox-Virus, Smallpox Virus, Measles Virus, Herpes Virus, Adenovirus, Papilloma Virus, Polyoma Virus, Parvovirus, Rhinovirus, Coxsackie virus, Polio Virus, Echovirus, Japanese Encephalitis virus, Dengue Virus, Tick Burne Encephalitis Virus, Yellow Fever Virus, Coronavirus, respiratory syncytial virus, parainfluenza virus, La Crosse Virus, Lassa Virus, Rabies Viruse, and Rotavirus antigens.

[0051] The design of the inducible checkpoint inhibitor proteins described herein allows the binding domain to one or more target antigens to be flexible in that the binding domain to a target antigen can be any type of binding domain, including but not limited to, domains from a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a human antibody, or a humanized antibody. In some embodiments, the binding domain to a target antigen is a single chain variable fragment (scFv), single- domain antibody such as a heavy chain variable domain (VH), a light chain variable domain (VL), and a variable domain (VHH) of camelid derived nanobody. In other embodiments, the binding domain to a target antigen is a non-Ig binding domain, i.e ., antibody mimetic, such as anticalins, affilins, affibody molecules, affimers, affitins, alphabodies, avimers, DARPins, fynomers, kunitz domain peptides, and monobodies. In further embodiments, the binding domain to one or more target antigens is a ligand or peptide that binds to or associates with one or more target antigens.

Inducible Immune Checkpoint Inhibitor Protein Pharmacokinetics

[0052] The inducible checkpoint inhibitor proteins described herein have certain advantages that would be recognized by one of skill in the art. For example inducible checkpoint inhibitor proteins described herein have improved pharmacokinetics over traditional antibody therapeutics. Improved

pharmacokinetics comprises at least one of a shallower alpha phase and higher exposure in the beta phase. Improved pharmacokinetics of inducible checkpoint inhibitor proteins herein are attributed to at least the half-life extension domain and the immune checkpoint protein binding domain. Half-life extension domains, as disclosed herein, include various polypeptides including, but not limited to, Fc domains and polypeptides binding to HSA. Immune checkpoint protein binding domains herein have unique properties which give superior pharmacokinetics. The immune checkpoint protein binding domains herein do not bind to an immune checkpoint until they are activated by at least cleavage of at least one protease cleavage domain and binding of the target antigen binding domains to target antigens. Therefore, enhanced pharmacokinetics of inducible checkpoint inhibitor proteins herein is attributed at least in part to reduced or eliminated target mediated drug disposition through immune checkpoint protein binding in the circulation of a person. Inducible checkpoint inhibitor proteins described herein, thus have a larger therapeutic window with smaller peak trough differences in exposure when compared to traditional antibody therapeutics.

Inducible Immune Checkpoint Inhibitor Protein Modifications

[0053] The inducible checkpoint inhibitor proteins described herein encompass derivatives or analogs in which (i) an amino acid is substituted with an amino acid residue that is not one encoded by the genetic code, (ii) the mature polypeptide is fused with another compound such as polyethylene glycol, or (iii) additional amino acids are fused to the protein, such as a leader or secretory sequence or a sequence for purification of the protein.

[0054] Typical modifications include, but are not limited to, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent crosslinks, formation of cystine, formation of pyroglutamate, formylation, gamma

carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination.

[0055] Modifications are made anywhere in inducible checkpoint inhibitor proteins described herein, including the peptide backbone, the amino acid side-chains, and the amino or carboxyl termini Certain common peptide modifications that are useful for modification of inducible checkpoint inhibitor proteins include glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation, blockage of either or both the amino or carboxyl group in a polypeptide by a covalent modification, and ADP-ribosylation.

Polynucleotides Encoding Inducible Immune Checkpoint Inhibitor Proteins

[0056] Also provided, in some embodiments, are polynucleotide molecules encoding an inducible checkpoint inhibitor protein described herein. In some embodiments, the polynucleotide molecules are provided as a DNA construct. In other embodiments, the polynucleotide molecules are provided as a messenger RNA transcript.

[0057] The polynucleotide molecules are constructed by known methods, such as by combining the genes encoding the three binding domains either separated by peptide linkers or, in other embodiments, directly linked by a peptide bond, into a single genetic construct operably linked to a suitable promoter, and optionally a suitable transcription terminator, and expressing it in bacteria or other appropriate expression systems such as, for example CHO cells. In the embodiments where the target binding domain is a small molecule, the polynucleotides contain genes encoding the domains that bind to an immune checkpoint protein and HSA. In the embodiments where the half-life extension domain is a small molecule, the polynucleotides contain genes encoding the domains that bind to an immune checkpoint protein and the target antigen. Depending on the vector system and host utilized, any number of suitable transcription and translation elements, including constitutive and inducible promoters, may be used. The promoter is selected such that it drives the expression of the polynucleotide in the respective host cell.

[0058] In some embodiments, the polynucleotide is inserted into a vector, preferably an expression vector, which represents a further embodiment. This recombinant vector can be constructed according to known methods. Vectors of particular interest include plasmids, phagemids, phage derivatives, virii (e.g., retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, lentiviruses, and the like), and cosmids.

[0059] A variety of expression vector/host systems may be utilized to contain and express the polynucleotide encoding the polypeptide of the described inducible immune checkpoint inhibitor protein. Examples of expression vectors for expression in E.coli are pSKK (Le Gall et al., J Immunol Methods. (2004) 285(1): 111-27) or pcDNA5 (Invitrogen) for expression in mammalian cells.

[0060] Thus, the inducible immune checkpoint inhibitor proteins as described herein, in some embodiments, are produced by introducing a vector encoding the protein as described above into a host cell and culturing said host cell under conditions whereby the protein domains are expressed, may be isolated and, optionally, further purified. Pharmaceutical Compositions

[0061] Also provided, in some embodiments, are pharmaceutical compositions comprising an inducible immune checkpoint inhibitor protein described herein, a vector comprising the polynucleotide encoding the polypeptide of the inducible immune checkpoint inhibitor proteins, or a host cell transformed by this vector and at least one pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" includes, but is not limited to, any carrier that does not interfere with the effectiveness of the biological activity of the ingredients and that is not toxic to the patient to whom it is administered.

Examples of suitable pharmaceutical carriers are well known in the art and include phosphate buffered saline solutions; water; emulsions, such as oil/water emulsions; various types of wetting agents; sterile solutions; etc. Such carriers can be formulated by conventional methods and can be administered to the subject at a suitable dose. Preferably, the compositions are sterile. These compositions may also contain excipients such as preservatives, emulsifying agents, and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents.

[0062] In some embodiments of the pharmaceutical compositions, the inducible immune checkpoint inhibitor protein described herein is encapsulated in nanoparticles. In some embodiments, the nanoparticles are fullerenes, liquid crystals, liposomes, quantum dots, superparamagnetic nanoparticles, dendrimers, or nanorods. In other embodiments of the pharmaceutical compositions, the inducible immune checkpoint inhibitor protein is attached to liposomes. In some instances, the inducible immune checkpoint inhibitor protein is conjugated to the surface of liposomes. In some instances, the inducible immune checkpoint inhibitor protein is encapsulated within the shell of a liposome. In some instances, the liposome is a catiomc liposome.

[0063] The inducible immune checkpoint inhibitor proteins described herein are contemplated for use as a medicament. Administration is effected by different ways, e.g. by intravenous, intraperitoneal, subcutaneous, intramuscular, topical, or intradermal administration. In some embodiments, the route of administration depends on the kind of therapy and the kind of compound contained in the pharmaceutical composition. The dosage regimen will be determined by the attending physician and other clinical factors. Dosages for any one patient depends on many factors, including the patient ^' s size, body surface area, age, sex, the particular compound to be administered, time and route of administration, the kind of therapy, general health, and other drugs being administered concurrently. An "effective dose" refers to amounts of the active ingredient that are sufficient to affect the course and the severity of the disease, leading to the reduction or remission of such pathology and may be determined using known methods. Methods of treatment

[0064] Also provided herein, in some embodiments, are methods and uses for stimulating the immune system of an individual in need thereof comprising administration of an inducible immune checkpoint inhibitor protein described herein In some instances, the administration of an inducible immune checkpoint inhibitor protein described herein induces and/or sustains cytotoxicity towards a cell expressing a target antigen where the cell expressing the target antigen is in a microenvironment with increased levels of protease activity. In some instances, the cell expressing a target antigen is a cancer or tumor cell, a virally infected cell, a bacterially infected cell, an autoreactive T or B cell, damaged red blood cells, arterial plaques, or fibrotic tissue. In some instances, the blood of the subject is rich in proteases.

[0065] Also provided herein are methods and uses for a treatment of a disease, disorder, or condition associated with a target antigen comprising administering to an individual in need thereof an inducible immune checkpoint inhibitor protein described herein. Diseases, disorders, or conditions associated with a target antigen include, but are not limited to, viral infection, bacterial infection, auto-immune disease, transplant rejection, atherosclerosis, or fibrosis. In other embodiments, the disease, disorder, or condition associated with a target antigen is a proliferative disease, a tumorous disease, an inflammatory disease, an immunological disorder, an autoimmune disease, an infectious disease, a viral disease, an allergic reaction, a parasitic reaction, a graft-versus-host disease, or a host-versus-graft disease. In one embodiment, the disease, disorder or condition associated with a target antigen is cancer. In one instance, the cancer is a hematological cancer. In another instance, the cancer is a solid tumor cancer.

[0066] As used herein, in some embodiments, "treatment", "treating", or "treated" refers to therapeutic treatment wherein the object is to slow (lessen) an undesired physiological condition, disorder, or disease, or to obtain beneficial or desired clinical results. For the purposes described herein, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms; diminishment of the extent of the condition, disorder, or disease; stabilization (i.e., not worsening) of the state of the condition, disorder, or disease; delay in onset or slowing of the progression of the condition, disorder, or disease; amelioration of the condition, disorder, or disease state; and remission (whether partial or total), whether detectable or undetectable, or enhancement or improvement of the condition, disorder, or disease. Treatment includes eliciting a clinically significant response without excessive levels of side effects. Treatment also includes prolonging survival as compared to expected survival if not receiving treatment. In other embodiments, "treatment", "treating", or "treated" refers to prophylactic measures, wherein the object is to delay onset of, or reduce severity of, an undesired physiological condition, disorder, or disease, such as, for example is a person who is predisposed to a disease (e.g., an individual who carries a genetic marker for a disease such as breast cancer).

[0067] In some embodiments of the methods described herein, the inducible immune checkpoint inhibitor proteins are administered in combination with an agent for treatment of the particular disease, disorder or condition. Agents include, but are not limited to, therapies involving antibodies, small molecules (e.g., chemotherapeutics), hormones (steroidal, peptide, and the like), radiotherapies (γ-rays, X-rays, and/or the directed delivery of radioisotopes, microwaves, UV radiation, and the like), gene therapies (e.g., antisense, retroviral therapy, and the like) and other immunotherapies. In some embodiments, the inducible immune checkpoint inhibitor proteins are administered in combination with anti -diarrheal agents, anti -emetic agents, analgesics, opioids, and/or non-steroidal anti-inflammatory agents. In some embodiments, the inducible immune checkpoint inhibitor proteins are administered before, during, or after surgery. Certain Definitions

[0068] As used herein, "elimination half-time" is used in its ordinary sense, as is described in Goodman and Gillman's The Pharmaceutical Basis of Therapeutics 21-25 (Alfred Goodman Gilman, Louis S. Goodman, and Alfred Gilman, eds , 6th ed. 1980). Briefly, the term is meant to encompass a quantitative measure of the time course of drug elimination. The elimination of most drugs is exponential (i.e., follows first -order kinetics), since drag concentrations usually do not approach those required for saturation of the elimination process. The rate of an exponential process may be expressed by its rate constant, k, which expresses the fractional change per unit of time, or by its half-time, ti _/2 the time required for 50% completion of the process. The units of these two constants are time ¹ and time, respectively. A first-order rate constant and the half-time of the reaction are simply related and may be interchanged accordingly. Since first-order elimination kinetics dictates that a constant fraction of drug is lost per unit time, a plot of the log of drug concentration versus time is linear at all times following the initial distribution phase (i.e. after drug absorption and distribution are complete). The half-time for drug elimination can be accurately determined from such a graph.

EXAMPLES

[0069] The following examples are given for the purpose of illustrating various embodiments of the invention and are not meant to limit the present invention in any fashion. The present examples, along with the methods described herein are presently representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. Changes therein and other uses which are encompassed within the spirit of the invention as defined by the scope of the claims will occur to those skilled in the art.

Example 1 : Construction of an Exemplary Inducible Immune Checkpoint Inhibitor Protein to CD20

Generation of a scFv CTLA-4 binding domain

[0070] The human CTLA-4 chain canonical sequence is Uniprot Accession No. P16410. Antibodies against CTLA-4 are generated via known technologies such as affinity maturation. Where murine anti- CTLA-4 antibodies are used as a starting material, humanization of murine anti-CTLA-4 antibodies is desired for the clinical setting, where the mouse-specific residues may induce a human-anti-mouse antigen (HAMA) response in subjects who receive treatment of an antigen binding protein described herein. Humanization is accomplished by grafting CDR regions from murine anti-CTLA-4 antibody onto appropriate human germline acceptor frameworks, optionally including other modifications to CDR and/or framework regions. As provided herein, antibody and antibody fragment residue numbering follows Kabat (Kabat E. A. et al, 1991; Chothia et al, 1987).

[0071] Human or humanized anti-CTLA-4 antibodies are therefore used to generate scFv sequences for CTLA-4 binding domains of an antigen-binding protein. DNA sequences coding for human or humanized VL and VH domains are obtained, and the codons for the constructs are, optionally, optimized for expression in cells from Homo sapiens. A protease cleavage site is included between the VH and VL domains. The order in which the VL and VH domains appear in the scFv is varied (i.e., VL- VH, or VH-VL orientation), and three copies of the "G4S" or "G ₄ S" subunit (G ₄ S) ₃ connect the variable domains to create the scFv domain. Anti-CTLA-4 scFv plasmid constructs can have optional Flag, His or other affinity tags, and are electroporated into HEK293 or other suitable human or mammalian cell lines and purified. Validation assays include binding analysis by FACS, kinetic analysis using Proteon, and staining of CTLA-4-expressing cells.

Generation of a scFv CD20 binding domain

[0072] CD20 is one of the cell surface proteins present on B-lymphocytes. CD20 antigen is found in normal and malignant pre-B and mature B lymphocytes, including those in over 90% of B-cell non- Hodgkin's lymphomas (NHL). The antigen is absent in hematopoietic stem cells, activated B lymphocytes (plasma cells) and normal tissue. As such, several antibodies mostly of murine origin have been described: 1F5, 2B8/C2B8, 2H7, and 1H4.

[0073] A scFv binding domain to CD20 is generated similarly to the above method for generation of a scFv binding domain to CTLA-4.

Cloning of DNA expression constructs encoding the antigen-binding protein

[0074] The anti-CTLA-4 scFv with protease cleavage site domains are used to construct an antigen binding protein in combination with an anti-CD20 scFv domain and a half-life extension domain (e.g., a HSA binding peptide or VH domain), with the domains organized as shown Figure 1. For expression of an antigen binding protein in CHO cells, coding sequences of all protein domains are cloned into a mammalian expression vector system. In brief, gene sequences encoding the CTLA-4 binding domain, half-life extension domain, and CD20 binding domain along with peptide linkers LI and L2 are separately synthesized and subcloned. The resulting constructs are then ligated together in the order of 'CD20 binding domain - LI - VH CTLA-4 binding domain - L2 - protease cleavage domain - L3 - VL CTLA-4 binding domain - L4 - anti-CD20 scFv - L5 - half-life extension domain to yield a final construct. All expression constructs are designed to contain coding sequences for an N-terminal signal peptide and a C-terminal hexahistidine (6xHis)-tag to facilitate protein secretion and purification, respectively.

Expression of antigen-binding proteins in stably transfected CHO cells

[0075] A CHO cell expression system (Flp-In®, Life Technologies), a derivative of CHO-K1 Chinese Hamster ovary cells (ATCC, CCL-61) (Kao and Puck, Proc. Natl. Acad Sci USA 1968;60(4): 1275-81), is used. Adherent cells are subcultured according to standard cell culture protocols provided by Life Technologies.

[0076] For adaption to growth in suspension, cells are detached from tissue culture flasks and placed in serum -free medium. Suspension-adapted cells are cryopreserved in medium with 10% DMSO.

[0077] Recombinant CHO cell lines stably expressing secreted antigen-binding proteins are generated by transfection of suspension-adapted cells. During selection with the antibiotic Hygromycin B viable cell densities are measured twice a week, and cells are centrifuged and resuspended in fresh selection medium at a maximal density of 0. lxlO ⁶ viable cells/mL. Cell pools stably expressing antigen-binding proteins are recovered after 2-3 weeks of selection at which point cells are transferred to standard culture medium in shake flasks. Expression of recombinant secreted proteins is confirmed by performing protein gel electrophoresis or flow cytometry. Stable cell pools are cryopreserved in DMSO containing medium.

[0078] Antigen-binding proteins are produced in 10-day fed-batch cultures of stably transfected CHO cell lines by secretion into the cell culture supernatant. Cell culture supernatants are harvested after 10 days at culture viabilities of typically >75%. Samples are collected from the production cultures every other day and cell density and viability are assessed. On day of harvest, cell culture supernatants are cleared by centrifugation and vacuum filtration before further use.

[0079] Protein expression titers and product integrity in cell culture supernatants are analyzed by SDS- PAGE.

Purification of antigen-binding proteins

[0080] Antigen-binding proteins are purified from CHO cell culture supernatants in a two-step procedure. The constructs are subjected to affinity chromatography in a first step followed by preparative size exclusion chromatography (SEC) on Superdex 200 in a second step. Samples are buffer- exchanged and concentrated by ultrafiltration to a typical concentration of >1 mg/mL. Purity and homogeneity (typically >90%) of final samples are assessed by SDS PAGE under reducing and non- reducing conditions, followed by immunoblotting using an anti-HSA or anti idiotype antibody as well as by analytical SEC, respectively. Purified proteins are stored at aliquots at -80°C until use.

Example 2: Determination of antigen affinity by flow cytometry

[0081] The antigen-binding proteins of Example 1 are tested for their binding affinities to human CTLA-4 ⁺ and CD20 ⁺ cells and cynomolgus CTLA-4 ⁺ and CD20 ⁺ cells.

[0082] CTLA-4 ⁺ and CD20 ⁺ cells are incubated with 100 μΐ of serial dilutions of the antigen -binding proteins of Example 1 and at least one protease. After washing three times with FACS buffer the cells are incubated with 0.1 mL of 10 μg/mL mouse monoclonal anti -idiotype antibody in the same buffer for 45 min on ice. After a second washing cycle, the cells are incubated with 0.1 mL of 15 μg/mL FITC- conjugated goat anti-mouse IgG antibodies under the same conditions as before. As a control, cells are incubated with the anti-His IgG followed by the FITC -conjugated goat anti-mouse IgG antibodies without the antigen-binding proteins. The cells were then washed again and resuspended in 0.2 mL of FACS buffer containing 2 μg mL propidium iodide (PI) in order to exclude dead cells. The fluorescence of lxlO ⁴ living cells is measured using a Beckman-Coulter FC500 MPL flow cytometer using the MXP software (Beckman-Coulter, Krefeld, Germany) or a Millipore Guava EasyCyte flow cytometer using the Incyte software (Merck Millipore, Schwalbach, Germany). Mean fluorescence intensities of the cell samples are calculated using CXP software (Beckman-Coulter, Krefeld, Germany) or Incyte software (Merck Millipore, Schwalbach, Germany). After subtracting the fluorescence intensity values of the cells stained with the secondary and tertiary reagents alone the values are them used for calculation of the K _D values with the equation for one-site binding (hyperbola) of the GraphPad Prism (version 6.00 for Windows, GraphPad Software, La Jolla California USA).

[0083] CTLA-4 binding affinity and crossreactivity are evaluated in titration and flow cytometric experiments on CTLA-4 ⁺ Jurkat cells and the cynomolgus CTLA-4 ⁺ HSC-F cell line (JCRB, cat.:JCRBl 164). CD20 binding and crossreactivity are assessed on the human CD20 tumor cell lines. The K _D ratio of crossreactivity is calculated using the K _D values determined on the CHO cell lines expressing either recombinant human or recombinant cynomolgus antigens.

Example 3: Cytotoxicity Assay

[0084] The antigen binding protein of Example 1 is evaluated in vitro on its mediation of T cell dependent cytotoxicity to CD20 ⁺ target cells.

[0085] Fluorescence labeled CD20 ⁺ REC-1 cells (a Mantle cell lymphoma cell line, ATCC CRL-3004) are incubated with isolated PBMC of random donors or CB 15 T-cells (standardized T-cell line) as effector cells in the presence of the antigen binding protein of Example 1 and at least one protease. After incubation for 4 h at 37°C in a humidified incubator, the release of the fluorescent dye from the target cells into the supernatant is determined in a spectrofluorimeter. Target cells incubated without the antigen binding protein of Example land target cells totally lysed by the addition of saponin at the end of the incubation serve as negative and positive controls, respectively.

[0086] Based on the measured remaining living target cells, the percentage of specific cell lysis is calculated according to the following formula: [1 -(number of living targetS( _Sam pie ₎ /number of living targetS( _Sp0 ntaneous))] x 100%. Sigmoidal dose response curves and EC ₅₀ values are calculated by non-linear regression/4-parameter logistic fit using the GraphPad Software. The lysis values obtained for a given antibody concentration are used to calculate sigmoidal dose -response curves by 4 parameter logistic fit analysis using the Prism software.

Example 4: Pharmacokinetics of Antigen-binding Proteins

[0087] The antigen binding protein of Example 1 is evaluated for half-time elimination in animal studies.

[0088] The antigen binding protein is administered to cynomolgus monkeys as a 0.5 mg/kg bolus injection into the saphenous vein. Another cynomolgus monkey group receives a comparable protein in size with binding domains to CTLA-4 and CD20, but lacking a half-life extension domain. A third and fourth group receive a protein with CTLA-4 and half-life extension domains and a protein with CD20 and half-life extension domains respectively, and both comparable in size to the antigen-binding protein. Each test group consists of 5 monkeys. Serum samples are taken at indicated time points, serially diluted, and the concentration of the proteins is determined using a binding ELISA to CTLA-4 and/or CD20.

[0089] Pharmacokinetic analysis is performed using the test article plasma concentrations. Group mean plasma data for each test article conforms to a multi -exponential profile when plotted against the time post-dosing. The data are fit by a standard two -compartment model with bolus input and first-order rate constants for distribution and elimination phases. The general equation for the best fit of the data for i.v. administration is: c(t)=Ae ^~ot +Be ^pt , where c(t) is the plasma concentration at time t, A and B are intercepts on the Y-axis, and a and β are the apparent first-order rate constants for the distribution and elimination phases, respectively. The -phase is the initial phase of the clearance and reflects distribution of the protein into all extracellular fluid of the animal, whereas the second or β-phase portion of the decay curve represents true plasma clearance. Methods for fitting such equations are well known in the art. For example, A=D/V(a-k21)/(a-p), B=D/V(p-k21)/(a-p), and a and β (for α>β) are roots of the quadratic equation: r ² +(kl2+k21+kl0)r+k21kl0=0 using estimated parameters of V=volume of distribution, klO=elimination rate, kl2=transfer rate from compartment 1 to compartment 2 and k21=transfer rate from compartment 2 to compartment 1, and D=the administered dose.

[0090] Data analysis: Graphs of concentration versus time profiles are made using KaleidaGraph (KaleidaGraph™ V. 3.09 Copyright 1986-1997. Synergy Software. Reading, Pa.). Values reported as less than reportable (LTR) are not included in the PK analysis and are not represented graphically.

Pharmacokinetic parameters are determined by compartmental analysis using WinNonlin software (WinNonlin® Professional V. 3.1 WinNonlin™ Copyright 1998-1999. Pharsight Corporation. Mountain View, Calif). Pharmacokinetic parameters are computed as described in Ritschel W A and Kearns G L, 1999, IN: Handbook Of Basic Pharmacokinetics Including Clinical Applications, 5th edition, American Pharmaceutical Assoc., Washington, D.C.

[0091] It is expected that the antigen binding protein of Example 1 has improved pharmacokinetic parameters such as an increase in elimination half-time as compared to proteins lacking a half-life extension domain.

Example 5: Xenograft Tumor Model

[0092] The antigen binding protein of Example 1 is evaluated in a xenograft model.

[0093] Female immune -deficient NOD/scid mice are sub-lethally irradiated (2 Gy) and subcutaneously inoculated with 4xl0 ⁶ Ramos RA1 cells into the right dorsal flank. When tumors reach 100 to 200 mm ³ , animals are allocated into 3 treatment groups. Groups 2 and 3 (8 animals each) are mtraperitoneally injected with 1.5xl0 ⁷ activated human T-cells. Three days later, animals from Group 3 are subsequently treated with a total of 9 intravenous doses of 50 μg antigen binding protein of Example 1 (qdx9d).

Groups 1 and 2 are only treated with vehicle. Body weight and tumor volume are determined for 30 days.

[0094] It is expected that animals treated with the antigen binding protein of Example 1 have a statistically significant delay in tumor growth in comparison to the respective vehicle-treated control group.

Example 6: Proof-of-Concept Clinical Trial Protocol for Administration of the Antigen Binding Protein of Example 1 to B-cell Lymphoma Patients

[0095] This is a Phase I/II clinical trial for studying the antigen binding protein of Example 1 as a treatment for B-cell Lymphoma.

[0096] Study Outcomes:

[0097] Primary: Maximum tolerated dose of antigen binding protein of Example 1

[0098] Secondary: To determine whether in vitro response of antigen binding protein of Example 1 is associated with clinical response

[0099] Phase I

[00100] The maximum tolerated dose (MTD) will be determined in the phase I section of the trial. 1.1 The maximum tolerated dose (MTD) will be determined in the phase I section of the trial.

1.2 Patients who fulfill eligibility criteria will be entered into the trial to antigen binding protein of Example 1.

1 3 The goal is to identify the highest dose of antigen binding protein of Example 1 that can be administered safely without severe or unmanageable side effects in participants. The dose given will depend on the number of participants who have been enrolled in the study prior and how well the dose was tolerated. Not all participants will receive the same dose.

[00101] Phase II

2.1 A subsequent phase II section will be treated at the MTD with a goal of determining if therapy with therapy of antigen binding protein of Example 1 results in at least a 20% response rate.

Primary Outcome for the Phase II— To determine if therapy of antigen binding protein of Example 1 results in at least 20% of patients achieving a clinical response (blast response, minor response, partial response, or complete response)

[00102] Eligibility:

Histologically confirmed newly diagnosed aggressive B-cell lymphoma according to the current World Health Organisation Classification, from 2001 to 2007

Any stage of disease.

Treatment with R-CHOP or R-CHOP like regimens (+/- transplant).

Age > 18 years

Karnofsky performance status > 50% or ECOG performance status 0-2

[00103] Life expectancy > 6 weeks

[00104] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.