Multifunctional PD1 fusion proteins

Abstract

The disclosure provides a fusion protein comprising one or more of programmed cell death protein 1 (PD1) sequence, cluster of differentiation 80 (CD80) sequence, an IgG Fc region and compositions thereof. The disclosure also relates to methods for using the fusion proteins, in the treatment of checkpoint pathway-related diseases, disorders, and conditions including cell proliferative diseases such as cancer, as well as diseases, disorders, and conditions associated with immune deficiency, immune insufficiency, and / or subjects who are immune compromised.

Description

Atty Ref.67176WO01 MULTIFUNCTIONAL PD1 FUSION PROTEINS CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This patent application claims the benefit of U.S. Provisional Patent Application Nos. 63 / 544,982, filed October 20, 2023 and 63 / 705,582, filed October 10, 2024, which are incorporated by reference in their entirety herein. INCORPORATION BY REFERENCE OF SEQUENCE LISTING

[0002] This application contains a Sequence listing that has been submitted in a computer readable format and is hereby incorporated by reference in its entirety. The XML file, created on October 9, 2024, is named 67176US02_SequenceListing.xml, and is 57,344 bytes in size. BACKGROUND

[0003] Programmed cell death protein 1 (PD1 or CD279), is a membrane protein found on the surface of T cells, B cells and macrophages that functions as an immune checkpoint that down- regulates the immune system and prevents auto-immune diseases. In humans, PD1 has 288 amino acids and is encoded by the PDCD1 gene. The protein includes an extracellular Ig like V- type domain followed by a single-pass transmembrane domain and an intracellular domain. When bound to its two known ligands, PDL-1 and PDL-2, PD1 suppresses T-cell activation and subsequently negatively regulates immune response. Tumor cells are known to express PDL-1 and evade immune surveillance through the same immune checkpoint mechanism. As such, PD- 1 / PDL-1 binding inhibitors, such as anti-PD-1 antibodies and anti-PDL-1 antibodies, have been pursued as potential cancer therapies, and in some cases have been shown to restore immune response against tumor cells. A similar inhibitory effect on T cell function against tumors has also been reported for PDL-2.

[0004] The transformative success of antibodies targeting the PD1 (programmed death 1) / PD-L1 (B7-H1 B7 homolog 1) pathway (anti-PD therapy) has revolutionized cancer treatment. However, only a fraction of patients with solid tumors and some hematopoietic malignancies respond to anti-PD therapy. The tumor microenvironment (TME) is a major location forAtty. Ref.67176WO01 resistance to occur. T cells in the TME require TCR / MHC (signal 1) engagement and co- stimulatory receptor engagement to achieve full activation. Conditional co-stimulation can enhance T cell activation and proliferation, potentiating T cell-directed therapeutics particularly in cold tumors. It is also important for cancer therapy to enhance anti-tumor activity by specifically destroying tumor cells while minimizing peripheral toxicity. It is crucial that only T cells in the presence of the target tumor cells receive a costimulatory signal. However, agonism of costimulatory receptors with monospecific full-length antibodies is likely nondiscriminatory with regards to TILs (Tumor-infiltrating lymphocytes) and peripheral T cells and autoantigen- reactive T cells that contribute to autoimmune toxicities. For instance, Urelumab, a monospecific, nondiscriminatory, pan-4-lBB agonist antibody, exhibited significant liver toxicity in early phase clinical trials1. Thus, there remains a need for novel immune response enhancing compositions for the treatment of cancers. SUMMARY

[0005] In one aspect, the disclosure relates to a fusion protein comprising a first binding domain that binds to PDL1 and / or PDL2, a first linker region, an IgG Fc region, a second linker region, and a second binding domain that binds to CD28 and / or CTLA4. In embodiments of this aspect, the first binding domain comprises an extracellular domain of PD1 or a variant thereof. In embodiments of this aspect, the first linker region and / or the second linker region comprises SEQ ID NO.: 2, SEQ ID NO.: 4, or SEQ ID NO.: 13. In embodiments of this aspect, the IgG Fc region comprises the human IgG Fc region and / or a functional Fc variant thereof. In embodiments of this aspect, the second binding domain comprises an extracellular domain of CD80 or a variant thereof.

[0006] In another aspect, the disclosure relates to a fusion protein comprising a PD1 sequence, a linker region, and an IgG Fc region.

[0007] In another aspect, the disclosure relates to a fusion protein comprising a CD80 sequence, a linker region, and an IgG Fc region.

[0008] In another aspect, the disclosure relates to a fusion protein comprising a PD1 sequence, a first linker region, an IgG Fc region, a second linker region, and CD80 sequence.

[0009] In some embodiments of any of the above aspects, the PD1 sequence is attached to the Fc region at the N-terminal region.Atty. Ref.67176WO01

[0010] In some embodiments of any of the above aspects, the PD1 sequence is attached to the Fc region at the C-terminal region.

[0011] In some embodiments of any of the above aspects, the CD80 sequence is attached to the Fc region at the N-terminal region.

[0012] In some embodiments of any of the above aspects, the CD80 sequence is attached to the Fc region at the C-terminal region.

[0013] In some embodiments of any of the above aspects, the fusion protein comprises two PD1 sequences. In some embodiments of any of the above aspects, the fusion protein comprises two CD80 sequences. In some further embodiments, the PD1 sequences are attached to the Fc region at the N-terminal region, and the CD80 sequences are attached to the Fc region at the C-terminal region. In yet further embodiments, the CD80 sequences are attached to the Fc region at the N- terminal region, and the PD1 sequences are attached to the Fc region at the C-terminal region.

[0014] In embodiments of the above aspect, the fusion protein can comprise SEQ ID NO.: 1, SEQ ID NO.: 3, SEQ ID NO.: 5, SEQ ID NO.: 6, SEQ ID NO.: 7, SEQ ID NO.: 8, SEQ ID NO.: 9, SEQ ID NO.: 10, SEQ ID NO.: 11, SEQ ID NO.: 12, SEQ ID NO.: 14, or SEQ ID NO.: 15. In some further embodiments, the PD1 sequence comprises SEQ ID NO.: 1 or SEQ ID NO.: 12. In yet further embodiments, the CD80 sequence comprises SEQ ID NO.: 5.

[0015] In another aspect, the disclosure provides a pharmaceutical composition comprising the fusion protein according to any one of the above aspects and embodiments, in combination with one or more pharmaceutically acceptable excipients, diluents, or carriers.

[0016] In another aspect, the disclosure provides a method of treating cancer comprising administering to a human patient in need thereof an effective amount of the fusion protein or the pharmaceutical composition of any one or more of the above aspects and embodiments.

[0017] In another aspect, the disclosure provides a method of enhancing or inducing an immune response comprising administering to a human patient in need thereof an effective amount of the fusion protein or the pharmaceutical composition of any one or more of the above aspects and embodiments.

[0018] In certain embodiments of the above aspects, the fusion protein can comprise a PD1 sequence comprising a variant sequence of the structure: [PD-1 N-terminal sequence]-Xaa1Xaa2Xaa3Xaa4Xaa5-[PD-1 C-terminal sequence], whereinAtty. Ref.67176WO01 Xaa1Xaa2Xaa3Xaa4Xaa5comprise five variable amino acid residues that comprise at least one amino acid residue that is non-native to a PD-1 loop-1 sequence; and the PD-1 N-terminal sequence and the PD-1 C-terminal sequence comprise at least 80% homology to a wild type PD-1 sequence.

[0019] In some further embodiments, the PD-1 protein variant sequence comprises a structure wherein, Xaa1 comprises M, V, I, L, or E; Xaa2 comprises S, L, H, Y, L, G, D, T, or H; Xaa3 comprises P, E, S, T, D, K, Q, R, V, or A; Xaa4comprises S, T, G, N, D, F, R, H, R, or E; Xaa5comprises N, or G; Xaa6 comprises an amino acid residue that is not cysteine; and Xaa7 comprises a non-polar amino acid residue.

[0020] In yet further embodiments, the fusion protein can comprise a structure wherein, the N- terminal sequence comprises an amino acid sequence that is N-terminal to the loop 1 region of a native PD-1 sequence; and the C-terminal sequence comprises an amino acid sequence that is C- terminal to the loop 1 region of a native PD-1 sequence; or one or both of the N-terminal sequence and / or the C-terminal sequence comprises an amino acid sequence that is at least 80% homologous to a native N-terminal sequence and / or a C-terminal PD-1 sequence.

[0021] Various aspects of the present disclosure will now be described, by way of illustration and example only, and with reference to the attached illustrative Figures. Other aspects and embodiments will become apparent to those of skill in the art in light of the description and claims that follow. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] FIG. 1 illustrates the design formats of the present invention.

[0023] FIGS. 2A-2H illustrates the binding analysis of fusion proteins. FIG. 2A illustrates the binding of V1 and V2 to human CTLA4 protein. FIG. 2B illustrates the binding of V1 and V2 to human CD28 protein. FIG. 2C illustrates binding of V1 and V2 to human PD-L1 protein. FIG. 2D illustrates binding of V1 and V2 to human PD-L2 protein. FIG. 2E illustrates binding of CD80-Fc and Fc-CD80 to human CTLA4 protein. FIG. 2F illustrates binding of CD80-Fc and Fc-CD80 to human CD28 protein. FIG. 2G illustrates binding of PD1-Fc and Fc-PD1 to human PD-L1 protein. FIG. 2H illustrates binding of PD1-Fc and Fc-PD1 to human PD-L2 protein.Atty. Ref.67176WO01

[0024] FIGS. 3A-3D illustrates fusion proteins enhance cytokine release in SEB and MLR assay. FIG. 3A illustrates IFN-γ detection in the supernatant of superantigen stimulatory assay. FIG. 3B illustrates IL-2 detection in the supernatant of superantigen stimulatory assay. FIG. 3C illustrates IL-2 detection in the supernatant of mixed lymphocyte reaction assay. FIG. 3D illustrates IFN-γ detection in the supernatant of mixed lymphocyte reaction assay.

[0025] FIG. 4 illustrates a schematic structure of V5 and Fc-mPD1.

[0026] FIGS. 5A-5D illustrates in vitro protein based binding. FIG. 5A illustrates binding of V5 to human PD-L1 protein. FIG. 5B illustrates binding of V5 to human PD-L2 protein. FIG. 5C illustrates binding of V5 to human CTLA4 protein. FIG. 5D illustrates binding of V5 to human CD28 protein.

[0027] FIGS. 6A-6D illustrates in vitro cell based binding. FIG. 6A illustrates the binding of V5 to HEK293 T cells stably expressing human PD-L1. MFI: median fluorescent intensity. FIG. 6B illustrates the binding of V5 to HEK293 T cells stably expressing human PD-L2. FIG. 6C illustrates the binding of V5 to HEK293 T cells stably expressing human CD28. FIG. 6D illustrates the binding of V5 to HEK293 T cells stably expressing human CTLA4.

[0028] FIGS. 7A-7B illustrates two targets bridging. FIG.7A illustrates V5 bridging human PD- L1 and human CTLA4. FIG.7B illustrates V5 bridging human PD-L1 and human CD28.

[0029] FIGS. 8A-8B illustrates in vitro receptor blocking analysis. FIG. 8A illustrates V5 blocking the interaction between PD1 and PD-L1. FIG. 8B illustrates V5 blocking the interaction between PD1 and PD-L2.

[0030] FIGS. 9A-9I illustrates the activation of T cell and antitumor activity in vitro. FIG. 9A illustrates T cell proliferation assay without signal I (OKT3). FIG. 9B illustrates T cell proliferation assay with signal I (OKT3). FIG. 9C illustrates V5 promoting IFN-γsecretion in the Superantigen stimulatory assay. FIG. 9D illustrates V5 promoting IL-2 secretion in the Superantigen stimulatory assay. FIG. 9E illustrates V5 promoting IL-2 secretion in the Mixed lymphocyte reaction assay. FIG. 9F illustrates V5 promoting IFN-γsecretion in the Mixed lymphocyte reaction assay. FIG. 9G illustrates CMV recall assay. FIG. 9H illustrates tumor specific T cell killing of MDA-MB-231-LUC tumor cell. FIG. 9I illustrates tumor specific T cell killing of NCI-H292-LUC tumor cell.

[0031] FIGS. 10A-10F illustrates multifunctional fusion protein suppresses tumors in vivo. FIG. 10A illustrates V5 generated a greater antitumor effect than the combination treatment in MC38Atty. Ref.67176WO01 tumor xenograft model. FIG. 10B illustrates weight monitoring during treatment in MC38 tumor xenograft model. FIG. 10C illustrates V5 generated antitumor effect in Hepa1-6 tumor xenograft model. FIG. 10D illustrates V5 generated antitumor effect in Pan02 tumor xenograft model. FIG. 10E illustrates V5 generated antitumor effect in PD-L1 knock out MC38 tumor xenograft model. V5 induced immunological memory in preventing tumor recurrence. FIG. 10F illustrates after tumor inoculation, MC38-bearing C57BL / 6 J mice were treated with V5 twice a week for four times. After treatment, cured mice were re-challenged with MC38 tumor cells. DETAILED DESCRIPTION

[0032] PD1 is an inhibitory immune receptor, which plays critical roles in T cell co-inhibition and exhaustion upon binding to its ligands PD-L1 and PD-L2. To date, PD-L1 and PD1 inhibitors are the main anti-PD therapy approaches, through anti-PD-L1 or anti-PD monoclonal antibodies to block the interaction between PD1 and PD-L1. In addition to blocking antibodies, soluble PD1 can act as a trap to block PD1 immunosuppressive signal as well. The soluble extracellular region of PD-1 binds to PD-L1 and PD-L2, preventing the ligands from binding to PD1 on the cell membrane. However, to achieve enhanced efficacy in immunotherapy, a PD-1 decoy with higher binding affinity to PD-L1 is desirable.

[0033] CD80 is a costimulatory molecule known for its role in T cell activation and in regulation of the activity of normal and malignant B cells. It was the first ligand identified for CD28 and later for CTLA4. CD80 is expressed transiently on activated B cells, macrophages, and DCs. CD80 is a major natural agonist ligand of CD28. CD80 is often downregulated on most of the cancer cells, and losing CD80 will allow cancer cells escaping the attack from the immune system and subsequent imparted anergy and apoptosis in TILs.

[0034] PD1 is a co-inhibitory receptor that suppresses T cell activation and is an important cancer immunotherapy target. Upon activation by its ligand PD-L1, PD1 is thought to suppress signaling through the T cell receptor (TCR). But in the 2017, Enfu Hui et. al demonstrated that the co-receptor CD28 is strongly preferred over the TCR as a target for dephosphorylation by PD1–recruited Shp2 phosphatase. CD28, but not the TCR, is preferentially dephosphorylated in response to PD1 activation by PD-L1 in an intact cell system. These results reveal that PD1 suppresses T cell function primarily by inactivating CD28 signaling, suggesting thatAtty. Ref.67176WO01 costimulatory pathways play key roles in regulating effector T cell function and responses to anti–PD-L1 / PD1 therapy.

[0035] Building on these observations, the disclosure provides various aspects and embodiments relating to a multifunctional fusion protein platform that comprises fusions of the extracellular domains of PD1 and CD80 with Fc region of IgG through flexible linkers. In accordance with the various aspects and embodiments illustrated herein, the fusion protein can bind to PD-L1, PD-L2, CD28 and CTLA4, can block the interaction between receptors and ligands, and can provide immunological activation signals. The illustrative examples and embodiments demonstrate, e.g., using in vitro assays, that the multifunctional fusion protein enhances anti- tumor activity by providing a costimulatory signal to T cells against tumor cells. The illustrative examples and embodiments demonstrate, e.g., using mouse tumor burden models, that the multifunctional fusion protein displays high tumor suppression efficacy on not only PD-L1 positive tumor cell lines, but also PD-L1 knock out tumor cell lines. These are merely exemplary embodiments of the multifunctional fusion proteins encompassed by the disclosure.

[0036] Before continuing to describe the subject matter of disclosure in further detail, it is to be understood that this disclosure is not limited to specific proteins, nucleic acids, compositions, or process steps, as such can vary while still falling within the scope of the description provided herein. It must be noted that, as used in this specification and the appended claims, the singular form “a”, “an” and “the” include plural referents unless the context clearly dictates otherwise.

[0037] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention is related. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei- Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary Of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press, provide one of skill with a general dictionary of many of the terms used in this invention.

[0038] Amino acids can be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, can be referred to by their commonly accepted single-letter codes.Atty. Ref.67176WO01 A. Fusion proteins

[0039] As used herein, the term “fusion protein” refers to a polypeptide construct generated through the joining of distinct polypeptide sequences and / or expression of two or more genes encoding distinct polypeptides. Typically, the translation of the fusion gene results in a single polypeptide with functional properties derived from each of the original polypeptides. The polypeptides can be fused directly or joined via a linker or hinge region, which may also comprise a sequence of amino acids.

[0040] Described herein are novel fusion proteins. The fusion proteins can comprise combinations of two or more sequence regions comprising PD1, CD80, and an IgG Fc region. In some embodiments, the fusion protein comprises at least an Fc region fused to at least one copy of one or both of PD1 and / or CD80. As used herein, the term “programmed cell death protein 1” or “PD1” refers to any PD1 polypeptide sequence(s) or derivative(s) thereof having amino acid sequence identity, or substantial sequence identity, to a sequence found within a wild-type PD1. For example, PD1 can refer to an amino acid sequence of a recombinant or non- recombinant polypeptide having an amino acid sequence of: i) a native allelic variant of a PD1 polypeptide, ii) a biologically active fragment of a PD1 polypeptide (e.g., the ECD), or iii) a biologically active variant (i.e., mutant) or analog of a PD1 polypeptide. The PD1 that comprises at least a portion of the fusion protein can be obtained or derived from any mammalian species, or obtained from a recombinant expression system (e.g., yeast, bacteria, etc.). The PD1 sequence may be glycosylated, partially glycosylated, or non-glycosylated. In some embodiments, PD-1 refers to a soluble fragment or ECD fragment comprising a portion of the amino acid sequence disclosed as NCBI Reference Sequence: NP_005009.2 (SEQ ID NO.: 16). In some embodiments, PD1 refers to a sequence comprising SEQ ID NO.: 1 or SEQ ID NO.: 12.

[0041] In some embodiments, PD1 refers to a mutation or variant of a native PD1 amino acid sequence, such as the exemplary mutant PD1 sequences illustrated herein. Non-limiting examples of such mutant or variant PD1 sequences can comprise a structure: [PD1 N-terminal sequence]-Xaa1Xaa2Xaa3Xaa4Xaa5-[PD1 C-terminal sequence], wherein Xaa1Xaa2Xaa3 Xaa4Xaa5 comprise five variable amino acid residues that comprise at least one amino acid residue that is non-native to a PD1 loop-1 sequence; andAtty. Ref.67176WO01 the PD1 N-terminal sequence and the PD1 C-terminal sequence comprise at least 80% homology to a wild type PD-1 sequence.

[0042] In some further embodiments, the mutant or variant PD1 protein comprises a sequence wherein Xaa1comprises M, V, I, L, or E; Xaa2comprises S, L, H, Y, L, G, D, T, or H; Xaa3comprises P, E, S, T, D, K, Q, R, V, or A; Xaa4 comprises S, T, G, N, D, F, R, H, R, or E; Xaa5 comprises N, or G; Xaa6 comprises an amino acid residue that is not cysteine; and Xaa7 comprises a non-polar amino acid residue.

[0043] In yet further embodiments, the mutant or variant PD1 protein sequence comprises an N- terminal sequence having an amino acid sequence that is N-terminal to the loop 1 region of a native PD-1 sequence; and a C-terminal sequence having an amino acid sequence that is C- terminal to the loop 1 region of a native PD-1 sequence; or one or both of the N-terminal sequence and / or the C-terminal sequence comprises an amino acid sequence that is at least 80% homologous to a native N-terminal sequence and / or a C-terminal PD-1 sequence.

[0044] In some embodiments, Xaa6comprises a nucleophilic amino acid residue that is not cysteine. In some embodiments, Xaa6 comprises alanine, glycine, threonine or serine. In some embodiments, Xaa6 comprises alanine, glycine, or serine. In some embodiments Xaa6 comprises serine.

[0045] In some other embodiments, Xaa7comprises glycine, alanine, valine, leucine, isoleucine or methionine. In some further embodiments, Xaa7 comprises valine, leucine, or isoleucine. In yet further embodiments, Xaa7comprises leucine.

[0046] In some further embodiments, the disclosure provides a mutant or variant PD1 protein comprising the amino acid sequence: NPPTFSPALL VVTEGDNATF TCSFSNTSES FVLNWYR Xaa1Xaa2Xaa3Xaa4Xaa5QTDKLAAFPE DRSQPGQD Xaa6RFRVTQLPNG RDFHMSVVRA RRNDSGTYLC GAISLAPK Xaa7 QIKESLRAEL RVTERRAE (SEQ ID NO: 43); wherein, Xaa1 comprises M, V, I, L, or E; Xaa2 comprises S, L, H, Y, L, G, D, T, or H; Xaa3 comprises P, E, S, T, D, K, Q, R, V, or A; Xaa4comprises S, T, G, N, D, F, R, H, R, or E; Xaa5comprises N, or G; Xaa6 comprises an amino acid that is not cysteine; and Xaa7 comprises a non- polar amino acid residue.Atty. Ref.67176WO01

[0047] In yet further embodiments, the disclosure provides a protein comprising an amino acid sequence as described in Table 2: Table 2. Amino acid sequences of PD-1 mutants PD1 MutantSequence (SEQ ID NO.) 1 NPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRVLXGGQTDKLAAFPEDRSQPG QDSRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKLQIKESLRAELRVTERR AE (SEQ ID NO.: 17) 2 NPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRIHADGQTDKLAAFPEDRSQPG QDSRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKLQIKESLRAELRVTERR AE (SEQ ID NO.: 18) 3 NPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRIDVRGQTDKLAAFPEDRSQPG QDSRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKLQIKESLRAELRVTERR AE (SEQ ID NO.: 19) 4 NPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRVDKDGQTDKLAAFPEDRSQPG QDSRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKLQIKESLRAELRVTERR AE (SEQ ID NO.: 20) 5 NPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRVSETGQTDKLAAFPEDRSQPG QDSRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKLQIKESLRAELRVTERR AE (SEQ ID NO.: 21) 6 NPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRITEEGQTDKLAAFPEDRSQPGQ DSRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKLQIKESLRAELRVTERRA E (SEQ ID NO.: 22) 7 NPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRVTDGGQTDKLAAFPEDRSQPG QDSRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKLQIKESLRAELRVTERR AE (SEQ ID NO.: 23) 8 NPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRVDERGQTDKLAAFPEDRSQPG QDSRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKLQIKESLRAELRVTERR AE (SEQ ID NO.: 24) 9 NPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRVDRHGQTDKLAAFPEDRSQPG QDSRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKLQIKESLRAELRVTERR AE (SEQ ID NO.: 25) 10 NPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRVHRGGQTDKLAAFPEDRSQPG QDSRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKLQIKESLRAELRVTERR AE (SEQ ID NO.: 26) 11 NPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRIDQTGQTDKLAAFPEDRSQPG QDSRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKLQIKESLRAELRVTERR AE (SEQ ID NO.: 27) 12 NPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRISDSGQTDKLAAFPEDRSQPGQ DSRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKLQIKESLRAELRVTERRA E (SEQ ID NO.: 28) 13 NPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRVTPGGQTDKLAAFPEDRSQPG QDSRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKLQIKESLRAELRVTERR AE (SEQ ID NO.: 29) 14 NPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRVTSGGQTDKLAAFPEDRSQPG QDSRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKLQIKESLRAELRVTERR AE (SEQ ID NO.: 30) 15 NPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRLTERGQTDKLAAFPEDRSQPG QDSRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKLQIKESLRAELRVTERR AE (SEQ ID NO.: 31)Atty. Ref.67176WO01 16 NPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRLDKFGQTDKLAAFPEDRSQPG QDSRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKLQIKESLRAELRVTERR AE (SEQ ID NO.: 32) 17 NPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRIGEDGQTDKLAAFPEDRSQPG QDSRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKLQIKESLRAELRVTERR AE (SEQ ID NO.: 33) 18 NPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRVLDGGQTDKLAAFPEDRSQPG QDSRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKLQIKESLRAELRVTERR AE (SEQ ID NO.: 34) 19 NPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRIYDNGQTDKLAAFPEDRSQPG QDSRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKLQIKESLRAELRVTERR AE (SEQ ID NO.: 35) 20 NPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRVHTSGQTDKLAAFPEDRSQPG QDSRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKLQIKESLRAELRVTERR AE (SEQ ID NO.: 36) 21 NPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRVSSTGQTDKLAAFPEDRSQPG QDSRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKLQIKESLRAELRVTERR AE (SEQ ID NO.: 37) 22 NPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRVLPGGQTDKLAAFPEDRSQPG QDSRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKLQIKESLRAELRVTERR AE (SEQ ID NO.: 38) 23 NPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRESSSGQTDKLAAFPEDRSQPGQ DSRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKLQIKESLRAELRVTERRA E (SEQ ID NO.: 39) 24 NPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRLSETGQTDKLAAFPEDRSQPG QDSRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKLQIKESLRAELRVTERR AE (SEQ ID NO.: 40) C93S NPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPG QDSRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERR AE (SEQ ID NO.: 41) C93S / A132L NPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPG QDSRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKLQIKESLRAELRVTERR AE (SEQ ID NO.: 42)

[0048] As used herein, "PD-L1" (or programmed cell death 1 ligand 1, B7-H1, CD274, PDCD1LG1, PDCD1L1, PDCD1LG1, PDL1, hPD-L1, and the like), refers to any PD-1 ligand 1 amino acid sequence. As used herein, "PD-L2" (or programmed cell death 1 ligand 2, B7-DC, CD273, PDCD1LG2, Btdc, PDCD1L2, PDL2, bA574F11.2 and the like), refers to any PD-1 ligand 2 amino acid sequence.

[0049] As used herein, the term “cluster of differentiation 80” (or CD80 or B7-1) refers to a CD80 polypeptide or derivative thereof having identity, or substantial amino acid sequence identity to a sequence found within a wild-type CD80 amino acid sequence. In some embodiments, CD80 refers to a soluble fragment or ECD fragment of a CD80 sequence that comprises SEQ ID NO.: 5. In embodiments, the CD80 sequence can exert immune system costimulatory activity (e.g., T cell activation, regulation of B cells). For example, CD80 can refer to an amino acid sequence of aAtty. Ref.67176WO01 recombinant or non-recombinant polypeptide having an amino acid sequence of: i) a native allelic variant of a CD80 polypeptide, ii) a biologically active fragment of a CD80 polypeptide (e.g., the soluble ECD), or iii) a biologically active variant (i.e., mutant) or analog of a CD80 polypeptide. The CD80 that comprises at least a portion of the fusion protein can be isolated from any mammalian species or obtained from a recombinant expression system (e.g., yeast, bacteria, etc.). The CD80 sequence may be glycosylated, partially glycosylated, or non-glycosylated.

[0050] The fusion proteins falling within the various aspects and embodiments of the disclosure are referred to herein in several different ways. In some instances, the fusion protein can be referred to as, "XX-Fc", where "XX" refers to a particular protein (i.e., PD1 or CD80), and typically a soluble and / or an extracellular domain of protein "XX" that is fused to an N-terminal portion of Fc. In some other instances, the fusion protein can be referred to as, "Fc-XX", where XX again refers to a particular protein (i.e., PD1 or CD80), typically a soluble and / or an extracellular domain of protein "XX" that is fused to a C-terminal portion of Fc. In some instances, "XX-Fc-YY" refers to a fusion having two distinct protein domains, "XX" and "YY" (e.g., soluble and / or extracellular domains of PD1 and CD80) and which indicates that the XX domain is fused to an N-terminal portion of Fc, and the YY domain fused to a C-terminal portion of Fc (or XX-Fc). In some instances, an "m" that precedes a protein name (e.g., mPD-L1, mPD- L2, mCTLA4, and mCD28) refers to a murine form of the protein (e.g., murine PD-L1, PD-L2, CTLA4, and CD28). In some instances, the term "mut" that precedes a protein name (e.g., mutPD1) refers to a mutated sequence of the protein (e.g., mutated extracellular domain of PD- 1), and which can have modified functional activity (e.g., reduced, the same, or enhanced activity or binding affinity) relative to the native sequence. Further, the abbreviation "V#" (e.g., V1, V2, etc.) provides an indicator of a version or the reference sequence (e.g., V1 is version 1, V2 is version 2, etc.).

[0051] In one aspect, the fusion protein comprises a PD1 sequence, a linker or hinge region, an Fc region, a linker or hinge region, and a CD80 sequence. In other aspects, a fusion protein can comprise a PD1 sequence, a linker or hinge region, and an Fc region. In still other aspects, a fusion protein can comprise a CD80 sequence, a linker or hinge region, and an Fc region. In some embodiments of any of these aspects, the fusion protein can comprises less than the full- length sequence of one or both the PD1 and / or the CD80 sequences (e.g., the PD1 and / or CD80 sequence can comprise a soluble fragment, a functional fragment, and / or an ECD of one or bothAtty. Ref.67176WO01 PD1 or CD80). In embodiments, the Fc region of the fusion protein can be fused to two copies of a PD1 and a CD80 sequence (e.g., ECD of one or both PD1 or CD80). In embodiments, the fusion protein can further comprise a signal peptide at the N-terminus of the fusion protein, which may be processed (e.g., cleaved) when the fusion protein is expressed or produced in certain host cells (e.g., mammalian host cells such as 293F or CHO-K1). In some embodiments, the PD1 polypeptide sequence of the fusion protein comprises a human PD1 soluble extracellular domain sequence or a functional derivative thereof. In some embodiments, the CD80 polypeptide sequence of the fusion protein comprises a human CD80 soluble extracellular domain sequence or a functional derivative thereof.

[0052] In some embodiments, and as described above, the fusion protein comprises the PD1 or the C80 sequence disposed toward either the N-terminus or C-terminus of the Fc region. In some embodiments, the fusion protein comprises the PD1 or the C80 sequence disposed toward the N-terminus of the Fc region, with the other of the sequences (PD1 or CD80) disposed toward the C-terminus of the Fc, with linker or hinge regions disposed between the protein regions (see, e.g., FIG. 1).

[0053] Fusion proteins constructs within the scope of the disclosure typically comprise the separate protein sequences joined by the fusion of the C-terminal region (e.g., C-terminus) of one polypeptide or protein (PD1 or CD80) to the N-terminal region (e.g., N-terminus) of a hinge or linker peptide sequence and the fusion of the N-terminal region (e.g., N-terminus) of a second polypeptide or protein (PD1 or CD80) to the C-terminal region (e.g., C-terminus) of the 1stprotein-hinge or linker complex. The hinge or linker peptide comprises an amino acid sequence that provides a covalent linkage between the protein domains (PD1 or CD80), and can be immediately C-terminal to the last amino acid of the PD1 or CD80. The hinge or linker region can be any peptide linker that is generally known and used in the art to generate fusion proteins. Some non-limiting examples of linker regions include, the linker sequences illustrated by SEQ ID NO.: 2, SEQ ID NO.: 4, and SEQ ID NO.: 13). In some embodiments the hinge or linker is derived from the hinge region of an immunoglobulin such as, for example, an IgA, IgD, IgE, IgG, or IgM amino acid sequence. As used herein, “hinge region” refers to a polypeptide comprising an amino acid sequence that shares sequence identity or similarity with a portion of an immunoglobulin hinge region sequence. Accordingly, a “hinge region” encompasses fragments of the immunoglobulin hinge region that allow the linked polypeptides to achieve aAtty. Ref.67176WO01 biologically active conformation. Hinge regions in some example embodiments of the present disclosure share at least 70-80% sequence identity to a wild-type immunoglobulin hinge region amino acid sequence, preferably, greater than about 90% sequence identify. In some particular aspects, the hinge region comprises a hinge region from, or derived from, human IgD or IgG. Human IgD, which lacks cysteine and is the longest immunoglobulin hinge, provides increased fusion protein flexibility. Such flexibility can increase engagement or binding of the fusion protein with its target or targets (e.g., PD1 ligands, CD80 receptors, etc.).

[0054] As used herein, “signal peptide” typically refers to a 5-30 amino acid peptide present at the N-terminus of the fusion protein. In some aspects, the fusion protein comprises an N-terminal signal peptide derived from an immunoglobulin amino acid sequence.

[0055] In some aspects, the fusion proteins disclosed herein can be characterized by one or more of the following structural and / or functional properties: a. at least one amino acid sequence comprising: (i) a PD1 sequence of SEQ ID NO.: 1 or SEQ ID NO.: 12; and / or (ii) a CD80 sequence of SEQ ID NO.: 5; b. an amino acid sequence comprising: (i) a PD1 sequence of SEQ ID NO.: 1 or SEQ ID NO.: 12; and / or (ii) a CD80 sequence of SEQ ID NO.: 5, and (iii) an IgG Fc region; c. an amino acid sequence comprising: SEQ ID NO.: 6, SEQ ID NO.: 7, SEQ ID NO.: 8, SEQ ID NO.: 9, SEQ ID NO.: 10, SEQ ID NO.: 11, SEQ ID NO.: 14 or SEQ ID NO.: 15; d. binding activity for CTLA4; e. binding activity for CD28; f. binding activity for PD-L1; g. binding activity for PD-L2; h. ability to block binding of PD-L1 and / or PD-L2 to native PD1; i. activate Tcell and antitumor activity; j. suppress tumor growth; and / or k. induce or enhance immune response.

[0056] In some aspects, the fusion protein comprises SEQ ID NO.: 6, SEQ ID NO.: 9, or SEQ ID NO: 14.

[0057] As one of ordinary skill will appreciate, the sequences disclosed herein can be modified to some degree without compromising the ability of the fusion protein to exert activity orAtty. Ref.67176WO01 function that is associated with the underlying sequence (e.g., ligand binding specificity / affinity, target receptor binding, immunological activity, etc.). In some aspects, sequence variants retain the ability to (i) bind to one or any combination of PD-L1, PD-L2, CD28 and CTLA4; (ii) block the interaction between receptors and ligands; (iii) provide immunological activation signals (e.g., costimulatory signals to T cells); (iv) exhibit or enhance anti-tumor activity; and / or (v) slow, inhibit or stop tumor growth.

[0058] As used herein, sequence “variants” refer to a fusion protein amino acid sequence comprising at least one amino acid insertion, deletion, and / or substitution, wherein the resulting fusion protein maintains one or more of its functional characteristics as described herein. An amino acid insertion variant can be characterized by the insertion of one or more amino acids between two existing amino acids. An amino acid deletion variant can be characterized by the deletion of one or more amino acids from the fusion protein sequence. An amino acid substitution can be characterized by at least one amino acid in the sequence being replaced by another amino acid. In embodiments relating to substitutions, the amino acid substitution(s) may be a conservative substitution, (i.e. an amino acid from one family of amino acids (acidic, basic, non-polar, and uncharged, based on side chain characteristics, including size) is substituted with an amino acid from the same family).

[0059] With regard to amino acid sequences, variants (e.g., substitution, deletion, addition) can be considered as "similar" to the original polypeptide or to have a certain "percent identity" or "percent homology" to the original polypeptide, and include those polypeptides wherein one or more amino acid residues of a polypeptide are removed and replaced with alternative residues, deleted from the reference polypeptide sequence, and / or inserted into the reference polypeptide sequence. In embodiments, the substitutions in amino acid sequences are conservative in nature, however, the disclosure embraces substitutions that are also non-conservative, as long as one or more functional characteristics exhibited by the reference polypeptide are maintained or improved.

[0060] For amino acid sequences, sequence identity and / or similarity can be determined by using standard techniques known in the art such as, for example, the local sequence identity algorithm of Smith and Waterman, 1981, Adv. Appl. Math. 2:482, the sequence identity alignment algorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48:443, the search for similarity method of Pearson and Lipman, 1988, Proc. Nat. Acad. Sci. U.S.A. 85:2444, computerizedAtty. Ref.67176WO01 implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.), the Best Fit sequence program described by Devereux et al., 1984, Nucl. Acid Res. 12:387-395, using the default settings, or by inspection. Various alignment parameters can be set according to known methods (e.g., "Current Methods in Sequence Comparison and Analysis," Macromolecule Sequencing and Synthesis, Selected Methods and Applications, pp 127-149 (1988), Alan R. Liss, Inc.). Additional useful algorithms include PILEUP, which can align multiple sequences from a group of related sequences using progressive, pairwise alignments; BLAST, including gapped BLAST, WU-BLAST-2 (see, e.g., Altschul et al., 1990, J. Mol. Biol. 215:403-410; Altschul et al., 1993, Nucl. Acids Res. 25:3389-3402; Altschul et al., 1997, Nucleic Acids Res. 25:3389- 3402; Altschul et al., 1996, Methods in Enzymology 266:460-480; and Karin et al., 1993, Proc. Natl. Acad. Sci. U.S.A. 90:5873-5787).

[0061] Generally, the amino acid homology, similarity, or identity between sequences are at least 80%, including at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and from 99% to almost 100% identity. Similarly, the "percent (%) nucleic acid sequence identity" with respect to the nucleic acid sequences described herein refers to the percentage of nucleotide residues in a candidate sequence that are identical with the nucleotides disclosed herein, in the gene or coding sequence of the related polypeptide. Specific methods can include the default parameters of algorithms such as, for example, BLASTN (WU-BLAST-2). B. Labels, Conjugates, and Moieties

[0062] The fusion proteins disclosed herein can be conjugated to a therapeutic agent, solid support, affinity agent, or a detectable agent. Fusion proteins of the disclosure can be conjugated to labels wherein the fusion protein and / or its associated targets(s) can be detected. Labels include, without limitation, a chromophore, a fluorophore, a fluorescent protein, a phosphorescent dye, a tandem dye, a particle, a hapten, an enzyme and a radioisotope.

[0063] In certain aspects, the fusion proteins are conjugated to a fluorophore. The choice of the fluorophore attached to the fusion protein will determine the absorption and fluorescence emission properties of the conjugated fusion protein. Physical properties of a fluorophore label can include, but are not limited to, spectral characteristics (absorption, emission and stokes shift), fluorescence intensity, lifetime, polarization and photo-bleaching rate, or combination thereof.Atty. Ref.67176WO01 All of these physical properties can be used to distinguish one fluorophore from another, and thereby allow for multiplexed analysis. Other desirable properties of the fluorescent label can include cell permeability and low toxicity, for example if labeling of the fusion protein is to be performed in a cell or an organism (e.g., a living animal).

[0064] In certain aspects, the conjugated label can comprise an enzyme. Enzymes are desirable labels in some embodiments because amplification of the detectable signal can be obtained and result in increased assay sensitivity. The enzyme itself does not produce a detectable response but functions to break down a substrate when it is contacted by an appropriate substrate such that the converted substrate produces a fluorescent, colorimetric or luminescent signal. Enzymes can amplify the detectable signal because one enzyme on a labeling reagent can result in multiple substrates being converted to a detectable signal. The enzyme substrate is selected to yield the preferred measurable product, e.g. colorimetric, fluorescent or chemiluminescence. Such substrates are extensively used in the art and are well known by one skilled in the art and include for example, oxidoreductases such as horseradish peroxidase and a substrate such as 3,3’- diaminobenzidine (DAB); phosphatase enzymes such as an acid phosphatase, alkaline and a substrate such as 5-bromo-6-chloro-3-indolyl phosphate (BCIP); glycosidases, such as beta- galactosidase, beta-glucuronidase or beta-glucosidase and a substrate such as 5-bromo-4-chloro- 3-indolyl beta-D-galactopyranoside (X-gal); additional enzymes include hydrolases such as cholinesterases and peptidases, oxidases such as glucose oxidase and cytochrome oxidases, and reductases for which suitable substrates are known.

[0065] Enzymes and their appropriate substrates that produce chemiluminescence are suitable for some assays. These include, but are not limited to, natural and recombinant forms of luciferases and aequorins. Chemiluminescence-producing substrates for phosphatases, glycosidases and oxidases such as those containing stable dioxetanes, luminol, isoluminol and acridinium esters are additionally useful.

[0066] In another aspect, haptens, such as biotin, are also utilized as labels. Biotin is useful because it can function in an enzyme system to further amplify the detectable signal, and it can function as a tag to be used in affinity chromatography for isolation purposes. For detection purposes, an enzyme conjugate that has affinity for biotin is used, such as avidin-HRP. Subsequently a peroxidase substrate is added to produce a detectable signal.Atty. Ref.67176WO01

[0067] Haptens also include hormones, naturally occurring and synthetic drugs, pollutants, allergens, affector molecules, growth factors, chemokines, cytokines, lymphokines, amino acids, peptides, chemical intermediates, nucleotides and the like.

[0068] Enzymes and their appropriate substrates that produce chemiluminescence are suitable for some assays. These include, but are not limited to, natural and recombinant forms of luciferases and aequorins. Chemiluminescence-producing substrates for phosphatases, glycosidases and oxidases such as those containing stable dioxetanes, luminol, isoluminol and acridinium esters are additionally useful.

[0069] In another aspect, haptens, such as biotin, are also utilized as labels. Biotin is useful because it can function in an enzyme system to further amplify the detectable signal, and it can function as a tag to be used in affinity chromatography for isolation purposes. For detection purposes, an enzyme conjugate that has affinity for biotin is used, such as avidin-HRP. Subsequently a peroxidase substrate is added to produce a detectable signal.

[0070] Haptens also include hormones, naturally occurring and synthetic drugs, pollutants, allergens, affector molecules, growth factors, chemokines, cytokines, lymphokines, amino acids, peptides, chemical intermediates, nucleotides and the like.

[0071] In certain aspects, fluorescent proteins can be conjugated to the fusion protein as a label. Examples of fluorescent proteins include green fluorescent protein (GFP) and the phycobiliproteins and derivatives thereof. The fluorescent proteins, especially phycobiliprotein, are particularly useful for creating tandem dye labeled labeling reagents. These tandem dyes comprise a fluorescent protein and a fluorophore for the purposes of obtaining a larger stokes shift wherein the emission spectra is farther shifted from the wavelength of the fluorescent protein’s absorption spectra.

[0072] In certain aspects, the label is a radioactive isotope. Examples of suitable radioactive materials include, but are not limited to, iodine (121I,123I,125I,131I), carbon (14C), sulfur (35S), tritium (3H), indium (111In,112In,113mIn,115mIn,), technetium (99Tc,99mTc), thallium (201Ti), gallium (68Ga,67Ga), palladium (103Pd), molybdenum (99Mo), xenon (135Xe), fluorine (18F),153SM,177Lu,159Gd,149Pm,140La,175Yb,166Ho,90Y,47Sc,186Re,188Re,142Pr,105Rh, and97Ru.

[0073] In some aspects, drugs (e.g., other active agents) can be conjugated to the fusion protein. For example, a fusion protein can be conjugated to a therapeutic moiety or agent, such as an immunotherapy drug or a chemotherapeutic agent. In some embodiments, the fusion protein canAtty. Ref.67176WO01 bind to a target cell, and deliver the therapeutic to the cell. Any therapeutic agent known in the art can be conjugated to the fusion protein. In certain embodiments, drugs and other molecules can be conjugated to a fusion protein via site-specific conjugation. Similarly, the fusion protein disclosed herein may be used in combination with one or more other therapeutic interventions that may be used in the treatment of conditions including various cancers and / or immune response deficiency in a subject. C. Polynucleotides Encoding Fusion Proteins & Recombinant Cells Producing Fusion Proteins

[0074] The disclosure provides methods for producing fusion proteins. In certain aspects, the disclosure provides for recombinant methods of generating a fusion protein. In some embodiments, recombinant nucleic acids encoding for the fusion protein can be operably linked to one or more regulatory nucleotide sequences in an expression construct. In some embodiments, the nucleic acid sequences encoding the signal peptide, first or second polypeptide sequences, or the linker / hinge region can be cloned in the same expression vector in a defined orientation or can be cloned in different vectors. If expression is carried out using one vector, the coding genes can have their own genetic elements (e.g., promoter, RBS, leader, stop, polyA, etc.) or they can be cloned with one single set of genetic elements, but connected with a cistron element. Regulatory nucleotide sequences can be appropriate for a host cell used for expression. Numerous types of appropriate expression vectors and suitable regulatory sequences are known in the art for a variety of host cells. Typically, one or more regulatory nucleotide sequences can include, but are not limited to, promoter sequences, leader or signal sequences, ribosomal binding sites, transcriptional start and termination sequences, translational start and termination sequences, and enhancer or activator sequences. Any known constitutive or inducible promoter are contemplated for use with the aspects and embodiments included in the disclosure. The promoters can be either naturally occurring promoters, or hybrid promoters that combine elements of more than one promoter. An expression construct can be present in a cell on an episome, such as a plasmid, or the expression construct can be inserted in a chromosome.

[0075] In certain aspects, an expression vector contains a selectable marker gene to allow the selection of transformed host cells. Selectable marker genes are known and can vary with the host cell used. In certain aspects, the disclosure relates to an expression vector comprising aAtty. Ref.67176WO01 nucleotide sequence encoding a polypeptide that is operably linked to at least one regulatory sequence. Regulatory sequences are generally known and can be selected to direct expression of the encoded polypeptide. Accordingly, the term regulatory sequence includes promoters, enhancers, and other expression control elements. Exemplary, non-limiting regulatory sequences are described in Goeddel; Gene Expression Technology: Methods in Enzymology, Academic Press, San Diego, CA (1990). It should be understood that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed and / or the type of protein (e.g., fusion protein) to be expressed. Moreover, the vector's copy number, the ability to control that copy number and the expression of any other protein encoded by the vector, such as antibiotic markers, can be considered.

[0076] In some embodiments relating to methods of producing a fusion protein, a host cell can be transfected with one or more expression vector(s) encoding a fusion protein, and can be cultured under appropriate conditions to allow expression of the polypeptide(s) to occur. The fusion protein can be secreted and isolated from cells and / or cell culture media containing the fusion protein. Alternatively, the fusion protein can be retained in the cytoplasm or in a membrane fraction and the cells harvested and lysed, and the fusion protein subsequently purified and isolated. A cell culture includes host cells, media and other byproducts. Any suitable media for cell culture can be used in methods of production. Fusion proteins can be isolated from cell culture medium, host cells, or both using common techniques for purifying proteins, including, for example, ion-exchange chromatography, gel filtration chromatography, ultrafiltration, electrophoresis, and immunoaffinity purification. In certain aspects, the fusion protein can be produced with a domain (e.g., a His-tag) that facilitates its purification.

[0077] A recombinant nucleic acid can be produced by ligating the cloned gene, or a portion thereof, into a vector suitable for expression in either prokaryotic cells, eukaryotic cells (yeast, avian, insect or mammalian), or both. Expression vehicles for production of a recombinant polypeptide include plasmids and other vectors. For instance, suitable vectors include plasmids of the types: pBR322-derived plasmids, pEMBL-derived plasmids, pEX-derived plasmids, pBTac-derived plasmids and pUC-derived plasmids for expression in prokaryotic cells, such as E. coli. In certain aspects, mammalian expression vectors contain both prokaryotic sequences to facilitate the propagation of the vector in bacteria, and one or more eukaryotic transcription units that are expressed in eukaryotic cells. The pcDNAI / amp, pcDNAI / neo, pRc / CMV, pSV2gpt,Atty. Ref.67176WO01 pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo and pHyg derived vectors are examples of mammalian expression vectors suitable for transfection of eukaryotic cells. Some of these vectors are modified with sequences from bacterial plasmids, such as pBR322, to facilitate replication and drug resistance selection in both prokaryotic and eukaryotic cells. Alternatively, derivatives of viruses such as the bovine papilloma virus (BPV-1), or Epstein-Barr virus (pHEBo, pREP-derived and p205) can be used for transient expression of proteins in eukaryotic cells. The various methods employed in the preparation of the plasmids and transformation of host organisms are well known in the art. For other suitable expression systems for both prokaryotic and eukaryotic cells, as well as general recombinant procedures, see Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press, 1989) Chapters 16 and 17. In some instances, it can be desirable to express the recombinant polypeptide(s) by the use of a baculovirus expression system. Examples of such baculovirus expression systems include pVL-derived vectors (such as pVL1392, pVL1393 and pVL941), pAcUW-derived vectors (such as pAcUW1), and pBlueBac-derived vectors (such as the ß-gal containing pBlueBac III).

[0078] Techniques for making fusion genes are well known. Essentially, the joining of various nucleic acid fragments coding for different polypeptide sequences is performed in accordance with conventional techniques, employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation. In another aspect, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive nucleic acid fragments which can subsequently be annealed to generate a chimeric gene sequence (see, for example, Current Protocols in Molecular Biology, eds. Ausubel et al., John Wiley & Sons: 1992).

[0079] In some aspects, an expression vector expressing any of the nucleic acids described above can be used to express a fusion protein in a host cell. For example, a fusion protein can be expressed in bacterial cells such as E. coli, insect cells (e.g., using a baculovirus expression system), yeast, or mammalian cells. Other suitable host cells are known to those skilled in the art.Atty. Ref.67176WO01

[0080] Once the expression vector is transferred to a host cell by conventional techniques, the transfected cells are then cultured by conventional techniques to produce a fusion protein. Thus, the disclosure includes host cells containing a polynucleotide encoding a fusion protein, operably linked to a heterologous promoter. In certain aspects, if the fusion protein is encoded from different vectors, the vectors can be co-expressed in the host cell for expression of the entire fusion protein. In certain aspects, the fusion protein is expressed from a single promoter. In certain aspects, the fusion protein is expressed from multiple promoters. In certain aspects, the fusion protein is encoded on a single vector. In certain aspects, the fusion protein is encoded on multiple vectors.

[0081] Mammalian cell lines available as hosts for expression of fusion proteins are well known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC), including but not limited to Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), human epithelial kidney 293 cells, and a number of other cell lines. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the fusion protein or portion thereof expressed. To this end, eukaryotic host cells that possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product can be used. Such mammalian host cells include but are not limited to CHO, HEK293, VERY, BHK, Hela, COS, MDCK, 293, 3T3, W138, BT483, Hs578T, HTB2, BT2O and T47D, NS0 (a murine myeloma cell line that does not endogenously produce any functional immunoglobulin chains), SP20, CRL7O3O and HsS78Bst cells.

[0082] In certain aspects, fusion proteins of the disclosure are stably expressed in a cell line. Stable expression can be used for long-term, high-yield production of recombinant proteins. For example, cell lines that stably express the fusion protein can be generated. Host cells can be transformed with an appropriately engineered vector comprising expression control elements (e.g., promoter, enhancer, transcription terminators, polyadenylation sites, etc.), and a selectable marker gene. Following the introduction of the foreign DNA, cells can be allowed to grow for 1- 2 days in an enriched media, and then are switched to a selective media. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells that stably integratedAtty. Ref.67176WO01 the plasmid into their chromosomes to grow and form foci, which in turn can be cloned and expanded into cell lines. Methods for producing stable cell lines with a high yield are well known in the art and reagents are generally available commercially.

[0083] In certain aspects, fusion proteins of the disclosure are transiently expressed in a cell line. Transient transfection is a process in which the nucleic acid introduced into a cell does not integrate into the genome or chromosomal DNA of that cell, but is maintained as an extrachromosomal element, e.g. as an episome, in the cell. Transcription processes of the nucleic acid of the episome are not affected and a protein encoded by the nucleic acid of the episome is produced.

[0084] The cell line, either stable or transiently transfected, is maintained in cell culture medium and conditions well known in the art that result in the expression and production of fusion protein. In certain aspects, the mammalian cell culture media is based on commercially available media formulations, including, for example, DMEM or Ham's F12. In other aspects, the cell culture media is modified to support increases in both cell growth and biologic protein expression. As used herein, the terms “cell culture medium,” “culture medium,” and “medium formulation” refer to a nutritive solution for the maintenance, growth, propagation, or expansion of cells in an artificial in vitro environment outside of a multicellular organism or tissue. Cell culture medium can be optimized for a specific cell culture use, including, for example, cell culture growth medium that is formulated to promote cellular growth or cell culture production medium which is formulated to promote recombinant protein production. The terms “nutrient”, “ingredient”, and “component” are used interchangeably herein to refer to the constituents that make up a cell culture medium.

[0085] Once a fusion protein has been produced, it can be purified by any method known in the art for purification of a protein complex, for example, by chromatography (e.g., ion exchange, affinity, and size column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins.

[0086] When using recombinant techniques, the fusion protein can be produced intracellularly, in the periplasmic space, or directly secreted into the medium. If the protein is produced intracellularly, as a first step, the particulate debris, either host cells or lysed fragments, is removed, for example, by centrifugation or ultrafiltration. Where the protein is secreted into the medium, supernatants from such expression systems are generally first concentrated using aAtty. Ref.67176WO01 commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit. A protease inhibitor such as PMSF can be included in any of the foregoing steps to inhibit proteolysis and antibiotics can be included to prevent the growth of adventitious contaminants.

[0087] The composition prepared from the cells can be purified using, for example, hydroxylapatite chromatography, hydrophobic interaction chromatography, ion exchange chromatography, gel electrophoresis, dialysis, and / or affinity chromatography either alone or in combination with other purification steps. The matrix to which the affinity ligand is attached is most often agarose, but other matrices are available. Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose. Other techniques for protein purification such as fractionation on an ion-exchange column, ethanol precipitation, Reverse Phase HPLC, chromatography on silica, chromatography on heparin, SEPHAROSE chromatography on an anion or cation exchange resin (such as a polyaspartic acid column), chromatofocusing, SDS- PAGE, and ammonium sulfate precipitation are also available.

[0088] Regardless of how a fusion protein is purified, to confirm functional binding activity, binding assays can be performed (before and / or after purification). For example, ELISA assays, including dual ELISA assays can be used. In some aspects, a first binding target is coated on a well, and binding to this target immobilizes the fusion protein. A tagged second binding target is added to the well, and detected. Only fusion proteins that are both immobilized via binding to the first binding target and bound to the second binding target will be detected.

[0089] In some aspects, the disclosure provides for recombinant cell lines expressing the fusion protein that can be deposited and maintained with an international depository institution that is authorized under the provisions of the Budapest Treaty (i.e., an International Depositary Authority, IDA). D. Pharmaceutical Formulations

[0090] In certain aspects, the disclosure provides pharmaceutical compositions. Such pharmaceutical compositions can also be compositions comprising a fusion protein as disclosed herein and a pharmaceutically acceptable excipient. In certain aspects, the pharmaceutical compositions of the disclosure are used as a medicament (i.e., in methods of treating orAtty. Ref.67176WO01 preventing a disease or condition (e.g., lymphopenia), in a subject in need of treatment or preventative treatment). In some embodiments, pharmaceutical compositions can be compositions comprising a nucleic acid molecule that encodes a fusion protein as disclosed herein.

[0091] In certain aspects, a fusion protein (or a polynucleotide encoding the fusion protein) can be formulated with a pharmaceutically acceptable carrier, excipient or stabilizer, as pharmaceutical compositions. In certain aspects, such pharmaceutical compositions are suitable for administration to a human, or a non-human mammal or animal, via any one or more route of administration using methods known in the art. The route and / or mode of administration will vary depending upon the desired results. The term “pharmaceutically acceptable carrier” means one or more non-toxic materials that do not interfere with the effectiveness of the biological activity of the active ingredients. Such preparations can routinely contain salts, buffering agents, preservatives, compatible carriers, and optionally other therapeutic agents. Such pharmaceutically acceptable preparations can also contain compatible solid or liquid fillers, diluents or encapsulating substances, which are suitable for administration into a human. Other contemplated carriers, excipients, and / or additives, which can be utilized in the formulations described herein include, for example, flavoring agents, antimicrobial agents, sweeteners, antioxidants, antistatic agents, lipids, protein excipients such as serum albumin, gelatin, casein, salt-forming counterions such as sodium and the like. These and additional known pharmaceutical carriers, excipients and / or additives suitable for use in the formulations described herein are known in the art, e.g., as listed in “Remington: The Science & Practice of Pharmacy”, 21sted., Lippincott Williams & Wilkins, (2005), and in the “Physician’s Desk Reference”, 60thed., Medical Economics, Montvale, N.J. (2005). Pharmaceutically acceptable carriers can be selected that are suitable for the mode of administration, solubility and / or stability desired or required.

[0092] The formulations described herein comprise active agents (i.e., one or more fusion proteins as disclosed herein) in a concentration resulting in a w / v appropriate for a desired dose. In certain aspects, the active agent is present in a formulation at a concentration of about 1 mg / ml to about 200 mg / ml, about 1 mg / ml to about 100 mg / ml, about 1 mg / ml to about 50 mg / ml, or about 1 mg / ml to about 25 mg / ml. In certain aspects, the concentration of the active agent in aAtty. Ref.67176WO01 formulation can vary from about 0.1% to about 75% by total weight. In certain aspects, the concentration of the active agent is in the range of 0.003 to 1.0 molar.

[0093] When used for in vivo administration, the formulations should be sterile. Formulations can be sterilized by various sterilization methods, including sterile filtration, radiation, etc. In one aspect, the formulation is filter-sterilized with a presterilized 0.22-micron filter. Sterile compositions for injection can be formulated according to conventional pharmaceutical practice as described in “Remington: The Science & Practice of Pharmacy”, 21sted., Lippincott Williams & Wilkins, (2005).

[0094] Therapeutic compositions are encompassed by the pharmaceutical formulations described herein, and can formulated for particular routes of administration, such as oral, nasal, pulmonary, topical (including buccal and sublingual), rectal, vaginal and / or parenteral administration. The phrases “parenteral administration” and “administered parenterally” as used herein refer to modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.

[0095] The formulations can be in unit dosage form and prepared by any known method. Actual dosage levels of the active ingredients in the pharmaceutical compositions can be varied to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient (e.g., “a therapeutically effective amount”). The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions employed, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and / or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts. Suitable dosages can range from about 0.0001 to about 100 mg / kg of body weight or greater, for example about 0.1, 1, 10, or 50 mg / kg of body weight, with about 1 to about 10 mg / kg of body weight being suitable.Atty. Ref.67176WO01

[0096] In some embodiments of the disclosure, the formulations can be suitable for research use. The concentration of active agent in such formulations, as well as the presence or absence of excipients and / or pyrogens, can be modified or selected based on the particular application and intended use.

[0097] The fusion proteins described herein can be used in methods that prevent, treat, or mitigate cell proliferative diseases such as cancer, as well as diseases, disorders, and conditions associated with immune deficiency, immune insufficiency, and / or subjects who are immune compromised or immunocompromised. As used herein, “immunocompromised” refers to a subject having a weakened immune system or a reduced ability to fight infections or other diseases, due to a genetic disorder or disease, an infection, an environmental disorder or disease, or other environmental factors. Terms related to immunocompromised include, “immunodeficiency” and “immunodeficient” which refer to a physiological state in which a subject’s immune system cannot produce an adequate immune response to fight infection and / or disease. In some aspects, immunodeficiency increases the subject’s susceptibility to microbial infection (e.g. bacterial, viral, fungal, or protozoan infection).

[0098] In some aspects, the fusion proteins of the disclosure can be used to induce or enhance an immune response by, for example, increasing white blood counts, increasing lymphocyte counts, and / or increasing T cell subpopulations in a subject in need thereof. In other aspects, the fusion proteins can be used to protect an immunocompromised subject or a subject at risk of becoming immunocompromised from developing an immunodeficiency or associated disease or disorder. Such methods can be carried out by administering to the patient a therapeutically effective dose of the fusion protein of the disclosure, or pharmaceutical compositions comprising such fusion proteins.

[0099] In another aspect, the disclosure relates to methods of treating cancer comprising administering to a patient in need thereof an effective amount of a fusion protein in accordance with the above aspects and embodiments. In some embodiments, the method comprises the administration in further combination (e.g., simultaneous, separate, or sequential combination) with an effective amount of one or more cancer therapies (e.g., chemotherapeutic agents, surgical resection, immune-oncology agents, or ionizing radiation, and the like).

[0100] In one aspect, the disclosure provides a method of treating cancer in a patient comprising administering a fusion protein of the disclosure to the patient. The methods can treat a variety ofAtty. Ref.67176WO01 cancers and, in particular, those cancers that are characterized by production of one or both of PDL-1 and / or PDL-2. Some non-limiting examples of such cancers include cancers that are responsive to an immunotherapy (e.g., cancers that involve a checkpoint pathway) such as, for example, lung cancer, liver cancer, brain cancer, bladder cancer, breast cancer, cervical cancer, skin cancer, melanoma, sarcoma, head and neck cancer, esophageal cancer, stomach cancer, renal cancer, leukemia, lymphoma, myeloma, glioblastoma, neuroblastoma, ovarian cancer, prostate cancer, pancreatic cancer, uterine (endometrial) cancer, or colorectal cancer. In some embodiments, the method comprises administering the fusion protein of the disclosure to the patient prior to the administration of one or more other therapies such as, for example, chemotherapeutic agent(s) and / or ionizing radiation.

[0101] In another aspect, the disclosure provides a method for treating an immunocompromised patient or stimulating an immune response in a patient in need thereof, comprising administering a fusion protein in accordance with the disclosure to the patient. In embodiments, the method comprises an immunotherapy.

[0102] As used herein, “treat”, “treating”, or “treatment” refer to administering a fusion protein or composition as described herein to a subject in order to eliminate, reduce, or attenuate the clinical symptoms of lymphopenia or immunodeficiency; arrest, inhibit, reverse, or slow the progression of lymphopenia or immunodeficiency in a subject; prevent, reverse, or slow the loss of lymphocyte counts in the blood of a subject; stimulate the recovery of lymphocyte counts in the blood of a subject; stimulate the proliferation and / or differentiation of lymphocytes in a subject; and / or stimulate the proliferation and / or differentiation of white blood cells in a subject.

[0103] In some aspects, the disclosure provides methods for preventing lymphopenia or immunodeficiency in a subject. In some further aspects of the methods relating to preventing disease, the subject can be at risk for developing lymphopenia or immunodeficiency. By “being at risk” or having an “increased risk”, a subject is identified as having a higher than normal chance of developing lymphopenia or immunodeficiency as compared to the general population. In some embodiments, a subject who has had, or who currently has cancer, HIV, hepatitis B, hepatitis C, sepsis, aplastic anemia, lymphoma, an inherited immune disorder, tuberculosis, renal failure, autoimmune disorders such as lupus, rheumatoid arthritis, and myasthenia gravis, heart failure, or is otherwise considered susceptible to being or becoming immunocompromised has an increased risk of developing lymphopenia or immunodeficiency. At risk subjects can alsoAtty. Ref.67176WO01 include subjects receiving chemotherapy, radiation treatment, steroids, aderenocorticotropic hormone, and / or immunosuppressive medications as well as those having undergone stem cell, tissue, and / or organ transplantation.

[0104] The term “immunotherapy” relates to a treatment involving a specific immune response, specifically, stimulation or recovery of immune cell populations. In the context of the present disclosure, terms such as “protect”, “prevent”, “prophylactic”, “preventative”, or “protective” relate to the prevention of the occurrence of or the exacerbation of lymphopenia or immunodeficiency in a subject. Persons having lymphopenia or immunodeficiency, immunocompromised persons, or persons at risk of developing lymphopenia, immunodeficiency, or becoming immunocompromised, as described above, would be considered candidates for immunotherapy.

[0105] A prophylactic administration of an immunotherapy, for example, a prophylactic administration of a fusion protein or a composition comprising a fusion protein as disclosed herein, can in certain embodiments protect the recipient from developing lymphopenia or immunodeficiency. In some embodiments, prophylactic administration can reduce the severity of lymphopenia or immunodeficiency.

[0106] A therapeutic administration of an immunotherapy, for example, a therapeutic administration of a fusion protein or a composition comprising a fusion protein as disclosed herein, can inhibit or attenuate the progression of lymphopenia, arrest or recover loss of white blood cell and / or lymphocyte counts, and / or inhibit, reduce, arrest, or alleviate clinical symptoms of lymphopenia.

[0107] The terms “subject”, “individual”, or “patient” are interchangeable, and relate to vertebrates, preferably mammals. For example, mammals in the context of the disclosure are humans, non-human primates, domesticated animals such as dogs, cats, sheep, cattle, goats, pigs, horses etc., laboratory animals such as mice, rats, rabbits, guinea pigs, etc., as well as animals in captivity such as animals in zoos. The term "animal" as used herein also includes humans. The term "subject" can also include a patient, i.e., an animal, an in particular embodiments, a human having lymphopenia, immunodeficiency, or an immunocompromised person.

[0108] The fusion proteins and compositions described herein can be administered via any conventional route, including by injection or infusion. In some embodiments, the administrationAtty. Ref.67176WO01 can be carried out, for example, orally, intravenously, intraperitonealy, intramuscularly, subcutaneously, or transdermally.

[0109] The fusion proteins and the compositions comprising them are administered in effective amounts. An "effective amount" includes an amount that achieves a desired reaction or a desired effect and can be in the form of a single dose or as multiple doses. In the case of treatment of lymphopenia or immunodeficiency, the desired reaction relates to inhibition or attenuation of lymphopenia; recovery, interruption, or prevention of further loss of immune cell populations (e.g. white blood cells and lymphocytes); and / or alleviation or attenuation of clinical symptoms. A desired reaction can also be delay of onset or prevention of onset of lymphopenia or immunodeficiency.

[0110] An effective amount of a composition of the disclosure will depend on the severity of lymphopenia or immunodeficiency, underlying cause (i.e. disease or condition) of lymphopenia or immunodeficiency, the individual parameters of the patient, including age, physiological condition, size and weight, the duration of treatment, the type of an accompanying therapy (if present), the specific route of administration and similar factors. Accordingly, the doses of the compositions of the disclosure administered can depend on various combinations of such parameters. In embodiments in which an initial amount administered to a patient is insufficient, further administration with higher amounts, more frequent doses, or a different / more localized route of administration can be used. F. Kits

[0111] Another aspect of the present disclosure is a kit. In one aspect, a kit comprises any of the compositions or pharmaceutical compositions of a nucleic acid, polypeptide, expression vector, or host cell described above, and instructions or a label directing appropriate use or administration. Optionally, a kit can also include one or more containers and / or a syringe or other device to facilitate delivery or use. The disclosure contemplates that all or any subset of the components for conducting research assays and / or for administering therapeutically effective amounts of a fusion protein can be enclosed in the kit. Similarly, the kit can include instructions for making a polypeptide by, for example culturing a host cell that expresses a nucleic acid that encodes a fusion protein of the disclosure under suitable conditions. By way of additional example, a kit for therapeutic administration of a fusion protein of the disclosure can comprise aAtty. Ref.67176WO01 solution containing a pharmaceutical formulation of the fusion protein, or a lyophilized preparation of the fusion protein, and instructions for administering the composition to a patient in need thereof and / or for reconstituting the lyophilized product.

[0112] The present disclosure also encompasses a finished packaged and labeled pharmaceutical product. This article of manufacture includes the appropriate unit dosage form in an appropriate vessel or container such as a glass vial or other container that is hermetically sealed. In the case of dosage forms suitable for parenteral administration the active ingredient, e.g., an above- described fusion protein, is sterile and suitable for administration as a particulate free solution. In certain aspects, the formulation is suitable for intravenous administration, such as for intravenous infusion to a human or animal.

[0113] In a specific aspect, the formulations of the disclosure are formulated in single dose vials as a sterile liquid. Exemplary containers include, but are not limited to, vials, bottles, pre-filled syringes, IV bags, and blister packs (comprising one or more pills). Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human diagnosis and / or administration.

[0114] As with any pharmaceutical product, the packaging material and container are designed to protect the stability of the product during storage and shipment. Further, the products of the disclosure include instructions for use or other informational material that advise the physician, technician, or patient on how to appropriately prevent or treat the disease or disorder in question. In other words, the article of manufacture includes instruction means indicating or suggesting a dosing regimen including, but not limited to, actual doses, monitoring procedures, etc., and other monitoring information.

[0115] A kit for research purposes can comprise a solution containing a fusion protein or a lyophilized preparation of a fusion protein of the disclosure, wherein the fusion protein binds specifically to one or more targets. The fusion protein can be labeled according to methods known in the art and described herein, including but not limited to labels such as small molecule fluorescent tags, proteins such as biotin, GFP or other fluorescent proteins, or epitope sequences such as his or myc. Similarly, primary antibodies used for detecting the fusion protein can be included in the kit. Primary antibodies can be directed to sequences on the fusion protein or to labels, tags, or epitopes with which the fusion protein is labeled. Primary antibodies can, in turn,Atty. Ref.67176WO01 be labeled for detection, or, if further amplification of the signal is desired, the primary antibodies can be detected by secondary antibodies, which can also be included in the kit. In some aspects, kits for research use can resemble kits intended therapeutic uses but further include a label specifying that the kit and its use is restricted to research purposes only. G. Modification and / or Engineering

[0116] The disclosed fusion proteins can also be used to produce therapeutic immunoconjugates, wherein the fusion protein is conjugated with one or more therapeutic agents. For example, fusion proteins as described herein can be used in the production of protein-drug conjugates. Drugs that can be used include, without limitation, large and small molecule active agents such as, for example, chemotherapeutics, immune system modulators, growth factors, (e.g., colony- stimulating factors (CSFs)), antiviral agents, antibiotic agents, antifungal agents, antiparasitic agents, and gamma globulin, and the like. EXAMPLES Example 1: Design of the fusion proteins

[0117] Several illustrative examples of fusion proteins falling within the scope of the disclosure are provided below. Table 1 details the sequences for the fusion protein domain components, human PD1extracellular domain (PD1-ECD), CD80-ECD, linkers and Fc region. As illustrated in FIG. 1, the ECDs of PD1 and CD80 were connected to the N- and C-terminal domains of wildtype human IgG1 Fc region through different linkers (e.g., V1 and V2). As comparative control molecules fusions of both individual ECDs of PD1 and CD80 were fused individually to the Fc domain. All fusion proteins were produced by transient transfection of expression plasmids into Expi293F cells. Table 1: Example amino acid sequences for fusion proteins. Sequences PD1 NPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQT DKLAAFPEDRSQPGQDSRFRVTQLPNGRDFHMSVVRARRNDS GTYLCGAISLAPKAQIKESLRAELRVTERRAE (SEQ ID NO.: 1) Linker 1 EPKSSDKTHTCPPCP (SEQ ID NO.: 2) Fc EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVT CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRAtty. Ref.67176WO01 VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP REPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK (SEQ ID NO.: 3) Linker 2 ESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKK KEKEKEEQEERETKTP (SEQ ID NO.: 4) CD80 VIHVTKEVKEVATLSCGHNVSVEELAQTRIYWQKEKKMVLTM MSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLK YEKDAFKREHLAEVTLSVKADFPTPSISDFEIPTSNIRRIICSTSG GFPEPHLSWLENGEELNAINTTVSQDPETELYAVSSKLDFNMT TNHSFMCLIKYGHLRVNQTFNWNTTKQEH (SEQ ID NO.: 5) V1 NPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQT DKLAAFPEDRSQPGQDSRFRVTQLPNGRDFHMSVVRARRNDS GTYLCGAISLAPKAQIKESLRAELRVTERRAEEPKSSDKTHTCP PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR DELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGKESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGR GGEEKKKEKEKEEQEERETKTPVIHVTKEVKEVATLSCGHNVS VEELAQTRIYWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITN NLSIVILALRPSDEGTYECVVLKYEKDAFKREHLAEVTLSVKA DFPTPSISDFEIPTSNIRRIICSTSGGFPEPHLSWLENGEELNAINT TVSQDPETELYAVSSKLDFNMTTNHSFMCLIKYGHLRVNQTFN WNTTKQEH (SEQ ID NO.: 6) PD1-Fc NPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQT DKLAAFPEDRSQPGQDSRFRVTQLPNGRDFHMSVVRARRNDS GTYLCGAISLAPKAQIKESLRAELRVTERRAEEPKSSDKTHTCP PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR DELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK (SEQ ID NO.: 7) Fc-CD80 EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVT CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP REPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGKESPKAQASSVPTAQPQAEGSLAKA TTAPATTRNTGRGGEEKKKEKEKEEQEERETKTPVIHVTKEVK EVATLSCGHNVSVEELAQTRIYWQKEKKMVLTMMSGDMNIW PEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLKYEKDAFKR EHLAEVTLSVKADFPTPSISDFEIPTSNIRRIICSTSGGFPEPHLSWAtty. Ref.67176WO01 LENGEELNAINTTVSQDPETELYAVSSKLDFNMTTNHSFMCLI KYGHLRVNQTFNWNTTKQEH (SEQ ID NO.: 8) V2 VIHVTKEVKEVATLSCGHNVSVEELAQTRIYWQKEKKMVLTM MSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLK YEKDAFKREHLAEVTLSVKADFPTPSISDFEIPTSNIRRIICSTSG GFPEPHLSWLENGEELNAINTTVSQDPETELYAVSSKLDFNMT TNHSFMCLIKYGHLRVNQTFNWNTTKQEHEPKSSDKTHTCPP CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR DELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGKESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGR GGEEKKKEKEKEEQEERETKTPNPPTFSPALLVVTEGDNATFT CSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDSRFR VTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLR AELRVTERRAE (SEQ ID NO.: 9) CD80-Fc VIHVTKEVKEVATLSCGHNVSVEELAQTRIYWQKEKKMVLTM MSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLK YEKDAFKREHLAEVTLSVKADFPTPSISDFEIPTSNIRRIICSTSG GFPEPHLSWLENGEELNAINTTVSQDPETELYAVSSKLDFNMT TNHSFMCLIKYGHLRVNQTFNWNTTKQEHEPKSSDKTHTCPP CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR DELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK (SEQ ID NO.: 10) Fc-PD1 EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVT CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP REPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGKESPKAQASSVPTAQPQAEGSLAKA TTAPATTRNTGRGGEEKKKEKEKEEQEERETKTPNPPTFSPALL VVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPED RSQPGQDSRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISL APKAQIKESLRAELRVTERRAE (SEQ ID NO.: 11) Example 2: Binding Analysis of Fusion Proteins

[0118] It is established that the extracellular domain of PD1 binds to both ligands, PD-L1 and PD-L2, and that the extracellular domain of CD80 binds to both CTLA4 and CD28. The binding characteristics of the fusion proteins (e.g., V1 and V2; (SEQ ID NOs.: 6 and 9, respectively)) were determined using protein based ELISA.Atty. Ref.67176WO01

[0119] Briefly, ELISA plates are pre-coated with PD-L1 (Cat# 10084-H08H, Sino biological), PD-L2 (Cat# 10292-H08H, Sino biological), CD28 (Cat# 11524-HCCH, Sino biological), and CTLA-4 (Cat# 11159-H08H, Sino Biological) antigens individually at a concentration of 2 μg / mL and incubated overnight at 4°C. Prior to use, the plates were blocked with PBST (Solarbio) containing 3% milk. Subsequently, different concentrations of samples (V1 or V2) are added to the coated ELISA plates and incubated at room temperature for 1 hour. The binding of V1 and V2 to the target antigen is detected using an HRP-conjugated Goat anti-human Fc antibody (Cat# A18829, Thermo Fisher Scientific). Absorbance is measured at 450 nm using an ELISA microplate reader (TECAN Infinite M200 Pro) and the EC50 value is calculated in GraphPad Prism Version 8. FIGS. 2A-2D and Table 2 illustrate and summarize the binding characteristics of fusions V1 and V2 to each of tested binding partners (PD-L1, PD-L2, CTLA4, and CD28). As the data demonstrates, the V2 fusion format appears to have a stronger binding profile to each CTLA4 and CD28 as compared to the V1 fusion (FIGs. 2A, 2B), while V1 appears to have a stronger binding profile to antigens PD-L1 and PD-L2 as compared to the V2 fusion (FIGs. 2C, 2D). Table 2. EC50 of V1 and V2 binding to each target. Target V1 EC50 (nM) V2 EC50 (nM) CTLA4 6.59 0.341 CD28 ND 105 PD-L1 11.3 26.1 PD-L2 2.57 9.84

[0120] Additional structural studies of the fusion format using individual CD80 and PD1 domains identified that including the CD80 extracellular domain fused at the C-terminus of Fc has a measurable effect on the binding affinity characteristics to both CD28 and CTLA4. The influence of the fusion position of the PD1 extracellular domain on the binding affinity is within an acceptable range (see, FIGs. 2E- 2H, Table 3). Table 3: EC50 of Single PD1 or CD80 Fc Fusion Proteins to Each Target Target EC50 (nM)CD80-Fc Fc-CD80 PD1-Fc Fc-PD1CTLA-4 0.3131 10.29 N / A N / A CD28 387 ND N / A N / A PD-L1 N / A N / A 12.75 16.04Atty. Ref.67176WO01 PD-L2 N / A N / A 0.8596 5.134 Example 3:Enhance Cytokine Release in SEB and MLR Assay

[0121] Superantigen stimulatory assay (e.g., SEB, Staphylococcal entertoxin B) and Mixed Lymphocyte Reaction assay (MLR) were used to evaluate the T cell activation potency of various fusion proteins.

[0122] Briefly, in the SEB assay, fresh PBMCs are added to a 96-well plate (1.5 × 105per well) with RPMI-1640 medium containing 10 % FBS and a series of concentrations (2500 ng / ml, 83.3 ng / ml, 2.77 ng / ml and 0.093 ng / ml) of staphylococcal enterotoxin B (SEB, Cat# BT202, Toxin Technology). Samples containing the fusion protein constructs are added at 400 nM final concentration and cell cultures are incubated at 37℃ with 5 % CO2. IL-2 and IFN-γ secretion levels in the cell supernatant are evaluated by ELISA after incubation for 72h and 120h respectively. FIGS. 3A-3B illustrate that V2 generally stimulated a higher level of secretion for IFN-γ and IL-2 relative to V1, indicating its potential as a stronger T cell activator.

[0123] In brief summary of the MLR assay, it utilizes CD4+ T cells that are isolated from peripheral blood mononuclear cells (PBMC) using a Human CD4+ T Cell Enrichment Kit (Cat# 17952, Stemcell). Monocytes from another donor are induced to dendritic cells (DC) by IL-4 and GM-CSF cytokines for 6 days, followed by 1 μg / mL lipopolysaccharide (LPS, Cat# L2630, Sigma) stimulation for 24 h. In a 96-well plate, 1x 104DC cells and 1 x 105CD4+ T cells are added per well and incubated with V1, V2, or other proteins (including controls) at final concentrations of 3.5, 35, and 350 nM. The cell supernatant is collected to detect IL-2 levels (after 72 h) and IFN-γ levels (after 120 h). The measurement of cytokines was performed according to the instructions provided in the ELISA kit (R&D Systems). As shown in FIGs. 3C- 3D, V1 and V2 did not exhibit a significant difference in stimulating cytokines secretion in the MLR assay. Example 4: Optimization of the Fusion Protein for Target Binding

[0124] In an effort to improve the activity of the multifunctional protein V2, a PD1 extracellular domain mutant sequence ("mut PD1") was used in place of wild type PD1, and the sequence of "linker2" was changed to a "linker3" sequence which includes a flexible peptide sequence rich in glycine and serine (see, FIG. 4 and Table 4). In our hands, the mutant PD1 construct has beenAtty. Ref.67176WO01 shown to have a 10-fold higher affinity relative to wild type PD1 for both PD-L1 and PD-L2 (data not shown). (cite high affinity PD1 patent number here). Table 4: Amino Acid Sequences of Each Element of V5 Sequences mut PD1 NPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRLTERGQT DKLAAFPEDRSQPGQDSRFRVTQLPNGRDFHMSVVRARRNDS GTYLCGAISLAPKLQIKESLRAELRVTERRAE (SEQ ID NO.: 12) Linker3 GGGGSGGGGSGGGGS (SEQ ID NO.: 13) V5 VIHVTKEVKEVATLSCGHNVSVEELAQTRIYWQKEKKMVLTM MSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLK YEKDAFKREHLAEVTLSVKADFPTPSISDFEIPTSNIRRIICSTSG GFPEPHLSWLENGEELNAINTTVSQDPETELYAVSSKLDFNMT TNHSFMCLIKYGHLRVNQTFNWNTTKQEHEPKSSDKTHTCPP CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR DELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGKGGGGSGGGGSGGGGSNPPTFSPALLVVTEGDNATFT CSFSNTSESFVLNWYRLTERGQTDKLAAFPEDRSQPGQDSRFR VTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKLQIKESLR AELRVTERRAE (SEQ ID NO.: 14) Fc- EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVT mutPD1 CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP REPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSNPPTFSPAL LVVTEGDNATFTCSFSNTSESFVLNWYRLTERGQTDKLAAFPE DRSQPGQDSRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAI SLAPKLQIKESLRAELRVTERRAE (SEQ ID NO.: 15) Example 5: In vitro protein binding.

[0125] Protein based ELISA and antigen overexpression cell lines are used to evaluate the binding ability of fusion protein V5, described in Example 4. Briefly, ELISA plates are pre- coated with PD-L1 (Cat# 10084-H08H, Sino biological), PD-L2 (Cat# 10292-H08H, Sino biological), CD28 (Cat# 11524-HCCH, Sino biological), and CTLA-4 (Cat# 11159-H08H, Sino Biological) antigens individually at a concentration of 2 μg / mL and incubated overnight at 4℃. The next day, plates are blocked with PBST (Solarbio) containing 3% milk. Subsequently,Atty. Ref.67176WO01 different concentrations of V5 are added to the coated ELISA plates and incubated at room temperature for 1 hour. The binding of V5 to the target antigen is detected using an HRP- conjugated Goat anti-human Fc antibody (Cat# A18829, Thermo Fisher Scientific). Absorbance is measured at 450 nm using an ELISA microplate reader (TECAN Infinite M200 Pro) and the EC50 value is calculated in GraphPad Prism Version 8. The EC50 values is illustrated in FIGS. 5A-5D. The EC50 values were calculated and summarized in Table 5. As the data shows, V5 can bind to hPD-L1, hPD-L2, hCTLA4, and hCD28 with EC50of 0.2921 nM, 0.7028 nM, 60.11 nM, and 1.836 nM respectively. Table 5. EC50 of V5 to each target. AntigenEC50（nM）hPD-L1 0.2921 hPD-L2 0.7028 hCTLA-4 1.836 hCD28 60.11 Example 6: In vitro cell binding

[0126] HEK293T cells are engineered to overexpress PD-L1, PD-L2, CD28 or CTLA4 as HEK293T PD-L1, HEK293T PD-L2, HEK293T CD28 and HEK293T CTLA4 cells for detecting the binding ability of V5 on the cell surface. Briefly, the engineered cells (1.5×105per well) are seeded into a 96-well U-bottom plate and incubated with different concentrations of V5 or an isotype antibody as negative control (from 0.002 μg / mL to 1000 μg / mL) at 4℃ for 1 h. After washing three times with wash buffer (2% BSA in PBS), FITC-labeled anti-human IgG antibody (Cat# 31529, Thermo Fisher Scientific) is added and incubated at 4 ℃ in the dark for 0.5 h. The cell-bound V5 is detected by flow cytometry. As the data illustrates (e.g., FIGS. 6A-6D and Table 6), V5 can specifically bind cell surface targets PD-L1, PD-L2, CD28 and CTLA4, with EC50 of 0.5356 ug / ml, 1.506 ug / mL, 20.1 ug / mL, and 0.6848 ug / mL respectively. Table 6. EC50 of V5 binding to T cells stably expressing human PD-L1, PD-L2, CD28, and CTLA4.Atty. Ref.67176WO01 Target Cell R squared EC50 (µg / mL) 293T-hPD-L1 0.9987 0.5356 293T-hPD-L2 0.9981 1.506 293T-hCD28 0.9955 20.1 293T-hCTLA-4 0.9882 0.6848 Example 7: Binding Kinetic Study

[0127] The affinity kinetics of V5 to human and mouse PD-L1(Cat# 50010-M08H, Sino biological), PD-L2(Cat# 50804-M08H, Sino biological), CD28(Cat# 50103-M08H, Sino biological) and CTLA-4(Cat# 50503-M08H, Sino biological) antigens are analyzed by surface plasmon resonance (SPR) analysis (Biacore 8K, GE Healthcare Life Sciences). The running buffer HBS-EP (Cat# BR100669, Cytiva) and immobilization buffer (10 mmol / L sodium acetate, pH 4.5-5.5, Cytiva) and regeneration buffer (10 mmol / L Glycine-HCl, pH1.5, Cytiva) are used throughout the entire experiment process. Human and mouse PD-L1, PD-L2 and CTLA4 antigens are immobilized on CM5 chips (Cytiva). V5 is 2-fold serially diluted (from 0.78 nM to 200 nM) and flowed through the chip in running buffer. V5 is injected for 120 seconds and dissociated for 240 seconds. The surface was regenerated for 30 seconds with 10 mmol / L glycine (pH 1.5). Data is analyzed using a 1:1 Langmuir binding model with BIAevaluation software. As for CD28 antigen, V5 is diluted with running buffer to 400 nM and then immobilized on Protein A chips (Cytiva) first. Human and mouse CD28 antigens are diluted in a 2-fold gradient (from 7.8 nM to 2000 nM) and flowed through the chip in running buffer. V5 is injected for 180 seconds, antigens are injected for 120 seconds and followed by a 400-second dissociation. The surface is regenerated for 30 seconds with 10 mmol / L glycine (pH 1.5). Data is analyzed using a steady stable affinity binding model to estimate kinetics and binding constants. Table 7 detail V5 binding affinities reached 10-8M for human PD-L1 and PD-L2, and 10-9M for mouse PD-L1 and PD-L2, which represents a significant improvement from V2. Table 7. Affinity measurements of V5 were determined by surface plasmon resonance (SPR). Antigens Apparent KD Antigens Apparent KD （M ） （M ） hPD-L12.04e-08mPD-L16.91e-09hPD-L21.67e-08mPD-L22.24e-09Atty. Ref.67176WO01 hCTLA-43.02e-08mCTLA-44.71e-09hCD281.43e-06mCD281.30e-07Example 8: Two-target bridging

[0128] V5 is shown to be a multifunctional protein that can potentially bind two cell surface targets to bridge cells. A dual-target ELISA method is used to study the simultaneous binding. hCD28 and hCTLA-4 antigens are pre-coated in ELISA plates at a concentration of 2 μg / mL individually via incubation overnight at 4℃. On the following day, PBST containing 3% milk is used for blocking. Different concentrations of V5 or an isotype antibody as negative control are added to the pre-coated plates and incubated at room temperature for 1 h. After washing away the unbound V5, biotin-PD-L1 (Cat# 10084-H08H-B, Sino biological) is added and incubated at room temperature for 1 h. Biotin-PD-L1nd to V5 is detected by Ultrasensitive Streptavidin- Peroxidase Polymer (Cat# S2438, Sigma), and the absorbance at 450 nm is read and the EC50is calculated. As shown in FIGS. 7A-7B, after binding to CTLA-4 or CD28, V5 can still bind PD- L1, suggesting its functional capacity and potential in bridging cells via surface receptor profile in vivo. Example 9. In vitro receptor blocking analysis

[0129] As noted above, V5 includes a mutant PD1 extracellular domain sequence having enhanced affinity to PD-L1 and PD-L2 (relative to native PD1), which can compete with PD1 for PD-L1 / PD-L2 binding. In the in vitro competition assay, V5 or an isotype antibody as negative control at different concentrations were mixed with 1 μg / mL human biotin-PD1(Cat# PD1- H82F1, ACROBiosystems) and added into ELISA plates (pre-coated with PD-L1 or PD-L2). After incubation at room temperature for 1 h, biotin-PD1 bound to PD-L1 or PD-L2 is detected with Ultrasensitive Streptavidin-Peroxidase Polymer (Cat# S2438, Sigma) at 450 nm. EC50 is calculated with GraphPad Prism. FIGS. 8A-8B and Table 9 illustrates, multifunctional fusion protein V5 is capable of blocking the interaction between PD1 and PD-L1 or PD-L2. Table 9: Summary of V5 Receptor Blocking AssayAtty. Ref.67176WO01 Proteins R squareEC50（nM）PD-L1 and biotin-PD1 Isotype 0.8691 20.46 V5 0.9929 0.1804 PD-L2 and biotin-PD1 Isotype 0.8832 N / A V5 0.9876 0.6752 Example 10: Activation of T Cell and Antitumor Activity in vitro

[0130] Besides PD1, the V5 construct also contains CD80 that is a natural T cell costimulator. V5 has the potential function for promoting T lymphocyte proliferation, cytokine release, and anti-tumor activity. T cell activation function in several in vitro models are examined, including T cell proliferation assay, superantigen stimulatory assay, mixed lymphocyte reaction test, CMV recall assay, and tumor specific lymphocyte cytotoxic assay.

[0131] In the T cell proliferation assay, anti-human CD3 antibody (Cat# 317325, Biolegend, 1 μg / mL) are precoated in a 24-well plate at 4℃ overnight. Fresh PBMCs stained with cell trace CFSE (Cat# C34570, Thermo Fisher Scientific) are added into the CD3-coated wells at a density of 2.5×105cells per well. At the same time, V5 and control proteins are added with a final concentration of 150 nM. Anti-human CD28 antibody (Cat# 302934, Biolegend, 4 μg / mL) is used as a positive control. No CD3 antibody wells are set up for background subtraction. Cell cultures are incubated at 37℃ with 5% CO2for 96 hours, then PBMC cells are collected, and the cell proliferation is observed by flow cytometry. FIGS. 9A-9B illustrates V5 had a weak activity of promoting T cell proliferation in the absence of the signal I (CD3). With the signal I from OKT3 antibody, V5 significantly promoted T cell proliferation. The proliferation ratio from the V5 / CD3 group reached 51.07%, higher than the 38.58% from the CD28 / CD3 group and the 12.86% from the combo / CD3 group.

[0132] In Superantigen stimulatory assay, fresh PBMCs are added to a 96-well plate (1.5 × 105per well) with RPMI-1640 medium containing 10% FBS and 2.77 ng / mL staphylococcal enterotoxin B (SEB, Cat# BT202, Toxin Technology). V5 and control proteins are added in an 8- fold dose titration from 0.097 nM to 50 nM. Cell cultures are incubated at 37℃ with 5% CO2. IL-2 and IFN-γ secretion levels in the cell supernatant are determined by ELISA after incubationAtty. Ref.67176WO01 for 72 h and 120 h respectively. FIGS. 9C-9D illustrates under the premise of superantigen stimulation, V5 group showed more IL-2 and IFNγ release compared to the combo group (CD80-Fc + Fc-PD1).

[0133] In Mixed lymphocyte reaction test assay, CD4+T cells are isolated from peripheral blood mononuclear cells (PBMC) using a Human CD4+T Cell Enrichment Kit (Cat# 17952, Stemcell). Monocytes from another donor are induced to dendritic cells by IL-4 and GM-CSF for 6 days, followed by 1 μg / mL lipopolysaccharide (LPS, Cat# L2630, Sigma) stimulation for 24 h. In a 96- well plate, 10,000 dendritic cells and 100,000 CD4+T cells are added per well and incubated with V5 or other control proteins at final concentrations of 3, 30, and 300 nM. The cell supernatant is collected to determine the IL-2 levels (after 72 h) and IFN-γ levels (after 120 h), following the instructions of the ELISA kit (R&D Systems). FIGS.9E-9F illustrates V5 promoted significantly higher antigen presentation of allogeneic dendritic cells and IL2 and IFN- γ cytokine secretion than the combo group or V2.

[0134] CMV recall assay is used to evaluate the potential effect of V5 on memory T cell recalling in the presence of homologous dendritic cells. The PBMCs used in this test were from CMV-seropositive healthy donors. PBMC cells were resuspended in AIM-V medium (Cat# 12055091, Gibco) and a total of 2.5× 105PBMCs per well were added to a 96-well plate, followed by the addition of V5 and control proteins (0.5 nM, 5 nM and 50 nM). After incubation at 37℃ and 5% CO2for 96h in medium with or without 0.3 μg / mL PepTivator CMV pp65 (Cat# 130-098-235, Miltenyi biotec), IFN-γ in the cell culture supernatant is detected. FIG. 9G illustrates in the presence of pp65 peptide, only V5 can efficiently recall memory T cells into activation state. In the absence of pp65 peptide (signal I), V5 did not recall memory T cells into activation state, indicating that V5 had ideal safety profile.

[0135] Tumor-specific T cells are the main effector cells enhanced by V5. To obtained tumor- specific T cells in vitro, fresh PBMCs are precultured with target cells for 36 h. NCI-H292-Luc and MDA-MB-231-Luc were chosen as target tumor cells. Cytotoxic activity of tumor specific lymphocyte is determined by co-culturing lymphocytes, and the target tumor cells for 24 h at an E / T ratio of 10, with V5 or control proteins, range from 0.002 to 600 nM. Target tumor cells can express luciferase and the luciferase activity of surviving target cells is measured using Steady- Glo kit (Cat# E2520, Promega). Percentage of target cells killing is defined as follows: target cell killing (%) = (C / B – A / B) × 100 where A is the value of the well containing the target cells andAtty. Ref.67176WO01 the tumor specific lymphocyte with V5 or other control proteins, B is the value of the control well of the target cells, and C is the value of the well containing the target cells and the tumor specific lymphocyte. As shown in FIGS.9H-9I and Table 10a-10b, V5 efficiently mobilized tumor-specific T cells to kill tumor cells efficiently, when single Fc fusion protein and combo groups did not kill tumor efficiently. Table 10a. The EC50 summary of MDA-MB-231-LUC cytotoxity. V5 CD80-Fc Fc-mPD1 Combo IC501.082 16.55 0.4535 1.977 R squared 0.9840 0.7974 0.6737 0.9443 Table 10b. The EC50 summary of NCI-H292-LUC cytotoxity. V5 CD80-Fc Fc-mPD1 Combo IC50 8.208 6.041 0.3384 0.3408 Rsquared0.9336 0.7340 0.9769 0.9854Example 11. Multifunctional fusion protein suppresses tumors in vivo.

[0136] In vivo anti-tumor activity of V5 is studied using tumor xenograft models in C57BL / 6 mice. A total of 3×105MC38, 3×105MC38-PD-L1 KO, 5×106Hepa1-6 or 1×106Pan02 cells suspended in 100 μl phosphate buffered saline (PBS) are inoculated subcutaneously into the right flanks of female C57BL / 6 mice. Mice are randomly divided into different treatment groups when tumors grew to about 80-120 mm3. Mice tumor size and weight are measured every 3 days. Tumor volumes are measured using length (l) and width (w) and calculated as tumor volume=lww / 2. For MC38 tumor, mice are divided into eight groups (n=6 per group). Vehicle control (PBS), Keytruda group (5 mg / kg), V5 (1.66, 5 or 15 mg / kg) or CD80-Fc / Fc-mPD1 group (1.66, 5 or 15 mg / kg) is administered intraperitoneally twice a week from day 0 until the tumor volumes are over 2000 mm3. For MC38-PD-L1 KO, Hepa1-6 and Pan02 tumor model, mice are divided into two groups, vehicle control (PBS) and V5 (15 mg / kg) are administered intraperitoneally twice a week from day 0 until the tumor volumes are over 2000 mm3. FIGS. 10A-10B illustrate V5 at 1.66mg / mL dose level significantly inhibited tumor growth. And, significantly, V5 showed better tumor growth inhibition activity than the combination ofAtty. Ref.67176WO01 equivalent concentration of CD80-Fc and Fc-PD1 (FIG. 10A). No significant weight loss was observed in the V5 group even at the high concentration of 15 mg / ml (FIG. 10B). In addition to the data for colon cancer cell line MC38, V5 was also shown to be very effective in inhibiting the growth of the mouse liver cancer cell line Hepa 1-6, and partially inhibiting the pancreatic cancer Pan02 (FIGS. 10C-10D). Interestingly, V5 could effectively inhibit the growth of MC38 PDL1 knock out tumors (FIG. 10E), which indicates that V5 may also regulate immune responses through mechanisms other than via recruiting T cells to kill tumors.

[0137] MC38 tumor re-challenge experiments are conducted to assess the immunological memory induced by V5. Mice with complete tumor disappearance are re-challenged with 3×105MC38 tumor cells on the left flanks on day 13 after the last administration. At the same time, control group (naive) mice are inoculated with 3×105MC38 tumor cells. The tumor growth is monitored every 3 days. FIG. 10F illustrates mice previously dosed with V5 exhibited persistent resistance to tumor relapse. The results suggest that mice cured by V5 developed immunological memory against the tumor recurrence.

Claims

Atty. Ref.67176WO01 We claim:

1. A fusion protein comprising a first binding domain that binds to PDL1 and / or PDL2, a first linker region, an IgG Fc region, a second linker region, and a second binding domain that binds to CD28 and / or CTLA4.

2. The fusion protein of claim 1, wherein first binding domain comprises an extracellular domain of PD1 or a variant thereof.

3. The fusion protein of claim 1, wherein the first linker region comprises SEQ ID NO.: 2, SEQ ID NO.: 4, or SEQ ID NO.:

13.

4. The fusion protein of claim 1, wherein the IgG Fc region is the human IgG Fc region and functionally modified Fc variants.

5. The fusion protein of claim 1, wherein the second binding domain comprises an extracellular domain of CD80 or a variant thereof.

6. The fusion protein of claim 1, wherein the second linker region comprises SEQ ID NO.: 2, SEQ ID NO.: 4, or SEQ ID NO.:

13.

7. A fusion protein comprising a PD1 sequence, a linker region, and an IgG Fc region.

8. A fusion protein comprising a CD80 sequence, a linker region, and an IgG Fc region.

9. A fusion protein comprising a PD1 sequence, a first linker region, an IgG Fc region, a second linker region, and CD80 sequence.

10. The fusion protein of any one of claims 7-9, wherein the PD1 sequence is attached to the Fc region at the N-terminal region.Atty. Ref.67176WO01 11. The fusion protein of any one of claims 7-9, wherein the PD1 sequence is attached to the Fc region at the C-terminal region.

12. The fusion protein of any one of claims 7-9, wherein the CD80 sequence is attached to the Fc region at the N-terminal region.

13. The fusion protein of any one of claims 7-9, wherein the CD80 sequence is attached to the Fc region at the C-terminal region.

14. The fusion protein of any one of claims 7-14, wherein the fusion protein comprises two PD1 sequences.

15. The fusion protein of any one of claims 7-14, wherein the fusion protein comprises two CD80 sequences.

16. The fusion protein of any one of claims 14-15, wherein the PD1 sequences are attached to the Fc region at the N-terminal region, and the CD80 sequences are attached to the Fc region at the C-terminal region.

17. The fusion protein of any one of claims 14-15, wherein the CD80 sequences are attached to the Fc region at the N-terminal region, and the PD1 sequences are attached to the Fc region at the C-terminal region.

18. The fusion protein of any one of claims 7-18, comprising SEQ ID NO.: 1, SEQ ID NO.: 3, SEQ ID NO.: 5, SEQ ID NO.: 6, SEQ ID NO.: 7, SEQ ID NO.: 8, SEQ ID NO.: 9, SEQ ID NO.: 10, SEQ ID NO.: 11, SEQ ID NO.: 12, SEQ ID NO.: 14, or SEQ ID NO.:

15.

19. The fusion protein of any one of claims 7-18, wherein the PD1 sequence comprises SEQ ID NO.:1 or SEQ ID NO.: 12.Atty. Ref.67176WO01 20. The fusion protein of any one of claims 7-18, wherein the CD80 sequence comprises SEQ ID NO.:

5.

21. A pharmaceutical composition comprising the fusion protein according to any one of claims 1-20, and one or more pharmaceutically acceptable excipients, diluents, or carriers.

22. A method of treating cancer comprising administering to a human patient in need thereof an effective amount of the protein of any one of claims 1-20, or the pharmaceutical composition of claim 21.

23. A method of enhancing or inducing an immune response comprising administering to a human patient in need thereof an effective amount of the fusion protein of any one of claims 1- 20, or the pharmaceutical composition of claim 21.

24. The fusion protein according to any of the preceding claims wherein the PD1 sequence comprises a variant sequence of the structure: [PD-1 N-terminal sequence]-Xaa1Xaa2Xaa3Xaa4Xaa5-[PD-1 C-terminal sequence], wherein Xaa1Xaa2Xaa3Xaa4Xaa5comprise five variable amino acid residues that comprise at least one amino acid residue that is non-native to a PD-1 loop-1 sequence; the PD-1 N-terminal sequence and the PD-1 C-terminal sequence comprise at least 80% homology to a wild type PD-1 sequence.

25. The fusion protein according to claim 24, wherein in the PD-1 protein variant sequence: Xaa1comprises M, V, I, L, or E; Xaa2comprises S, L, H, Y, L, G, D, T, or H; Xaa3 comprises P, E, S, T, D, K, Q, R, V, or A; Xaa4 comprises S, T, G, N, D, F, R, H, R, or E; Xaa5comprises N, or G; Xaa6comprises an amino acid residue that is not cysteine; andAtty. Ref.67176WO01 Xaa7comprises a non-polar amino acid residue.

26. The fusion protein according to any one of claims 24-25, wherein the N-terminal sequence comprises an amino acid sequence that is N-terminal to the loop 1 region of a native PD-1 sequence; and the C-terminal sequence comprises an amino acid sequence that is C-terminal to the loop 1 region of a native PD-1 sequence; or one or both of the N-terminal sequence and / or the C-terminal sequence comprises an amino acid sequence that is at least 80% homologous to a native N-terminal sequence and / or a C-terminal PD-1 sequence.

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