Compositions and methods for inhibiting IL-23 signaling

Abstract

The present disclosure provides recombinant molecules, compositions, and methods for modulating the IL-23 axis. In certain embodiments, these recombinant molecules are used in methods for the treatment and / or prophylactic treatment of cancer, autoimmune disease, and inflammatory disorders associated with the IL-23 axis and signal transduction. In one aspect, a recombinant molecule is provided herein that includes: (a) an interleukin-23 (IL-23) inhibitory polypeptide (IIP), the IIP including an IL-23 binding polypeptide or an IL-23R binding polypeptide; and (b) a target-binding polypeptide portion that binds to one or more immune checkpoint proteins or immunostimulatory receptors.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 254,387, filed October 11, 2021, which is incorporated by reference in its entirety.

[0002] Sequence Listing This application contains a Sequence Listing, which has been submitted via the Patent Center and is incorporated by reference in its entirety herein. The XML copy, created on XXX XX, 2022, is named 50048WO_sequencelisting.xml and is XX,XXX bytes in size. [Background technology]

[0003] background Interleukin 23 (IL-23) is known to be a vital pro-inflammatory cytokine in the development of chronic inflammatory diseases such as psoriasis, inflammatory bowel disease, multiple sclerosis, or rheumatoid arthritis. Excessive IL-23 signaling is associated with pathological consequences that can promote the production of inflammatory mediators (such as IL-17, IL-22, granulocyte-macrophage colony-stimulating factor (GM-CSF), or tumor necrosis factor (TNFα)) by target populations, mainly Th17 cells and IL-17-secreting TCRγδ cells (Tγδ17). IL-23 is also a key determinant of tumor-promoting pro-inflammatory signaling and antitumor adaptive immune deficiency.

[0004] Thus, there is a need for molecules that can modulate IL-23 signaling. Summary of the Invention [Means for solving the problem]

[0005] overview In one aspect, provided herein is a recombinant molecule comprising: (a) an interleukin-23 (IL-23) inhibitory polypeptide (IIP), where the IIP comprises an IL-23 binding polypeptide or an IL-23R binding polypeptide; and (b) a target binding polypeptide portion that binds to one or more immune checkpoint proteins or immunostimulatory receptors.

[0006] In various embodiments, the target binding polypeptide binds to an immune checkpoint protein as an antagonist or an agonist, hi various embodiments, the immune checkpoint protein is a T cell co-inhibitory receptor or ligand or a natural inhibitory receptor or ligand.

[0007] In some embodiments, the immune checkpoint protein is selected from programmed death-1 (PD1; CD279), programmed death ligand 1 (PDL1), programmed death ligand 2 (PDL2), cytotoxic T-lymphocyte antigen-4 (CTLA4; CD152), attenuator of B and T lymphocytes (BTLA), V-domain immunoglobulin suppressor of T-cell activation (VISTA), T cell immunoglobulin and ITIM domain (TIGIT), lymphocyte activation gene 3 (LAG-3; CD223), T cell immunoglobulin and mucin domain 3 (Tim-3; HAVCR2), carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), CD47, signal regulatory protein alpha (SIRPa), major histocompatibility complex class I,G (HLA-G), Ig-like transcript 2 (ILT2; LILRB1), or Ig-like transcript 4 (ILT4, LILRB2).

[0008] In some embodiments, the immunostimulatory receptor is selected from 4-1BB (CD137), inducible T cell costimulatory factor (ICOS; CD278), OX-40 (CD134), glucocorticoid-induced TNFR-related protein (GITR; CD357), CD40, herpes virus entry mediator (HVEM), CD28, or CD27.

[0009] In some embodiments, IIPs bind to and inhibit IL-23. In some embodiments, IIPs bind to and inhibit IL-23p19 subunit. In some embodiments, IIPs bind to and inhibit IL-23R.

[0010] In various embodiments, the IIP comprises an antibody or an antigen-binding fragment thereof. In some embodiments, the IIP is an antibody or an antigen-binding fragment thereof, wherein the antigen-binding fragment comprises a fragment crystallizable (Fc) region, a fragment antigen-binding (Fab) region, a single chain variable fragment (scFv), a light chain or a functional portion thereof, a variable region of the light chain (VL), a constant region of the light chain (CL), a heavy chain or a functional portion thereof, a variable region of the heavy chain (VH), a constant region of the heavy chain (CH), at least one complementarity determining region (CDR) or a portion thereof, or any combination thereof.

[0011] In some embodiments, the antibody or antigen-binding fragment thereof comprises a monoclonal antibody targeting the human IL-23p19 subunit. In some embodiments, the antibody or antigen-binding fragment thereof comprises one or more of the six CDRs or antigen-binding portions thereof selected from any one of risankizumab (VH: SEQ ID NO: 79; VL: SEQ ID NO: 80), guselkumab (VH: SEQ ID NO: 81; VL: SEQ ID NO: 82), tildrakizumab (VH: SEQ ID NO: 83; VL: SEQ ID NO: 84), brazikumab (VH: SEQ ID NO: 85; VL: SEQ ID NO: 86), and mirikizumab (VH: SEQ ID NO: 87; VL: SEQ ID NO: 88). In some embodiments, the antibody or antigen-binding fragment thereof is guselkumab.

[0012] In some embodiments, the target binding polypeptide comprises an antibody or an antigen-binding fragment thereof. In some embodiments, the target binding polypeptide is an antibody or an antigen-binding fragment thereof, the antigen-binding fragment of which comprises a fragment crystallizable (Fc) region, a fragment antigen-binding (Fab) region, a single chain variable fragment (scFv), a light chain or a functional portion thereof, a variable region of the light chain (VL), a constant region of the light chain (CL), a heavy chain or a functional portion thereof, a variable region of the heavy chain (VH), a constant region of the heavy chain (CH), at least one complementarity determining region (CDR) or a portion thereof, or any combination thereof.

[0013] In some embodiments, the target-binding polypeptide binds to an immune checkpoint protein as an antagonist, and the target-binding polypeptide comprises a ligand-binding sequence of the extracellular domain (ECD) of the immune checkpoint protein. In some embodiments, the ECD of the immune checkpoint protein can specifically bind to one or more of its cognate ligands expressed or presented on a tumor cell or an immune cell. In some embodiments, the immune cell is an antigen-presenting cell (APC), a myeloid-derived suppressor cell (MDSC), a CD4 T cell, or a T H There are 17 cells.

[0014] In some embodiments, the ECD is capable of specifically binding to programmed death-1 ligand 1 (PDL1; CD274; B7-H1) and / or programmed death-1 ligand 2 (PDL2). In some embodiments, the target binding polypeptide comprises the PD1 (CD279) extracellular domain (PD1-ECD) or a ligand-binding fragment thereof.

[0015] In some embodiments, the target binding polypeptide comprises the amino acid sequence of SEQ ID NO:56, or an amino acid sequence having at least 80% identity to SEQ ID NO:56, or a ligand-binding fragment thereof.

[0016] In some embodiments, the target binding polypeptide comprises one or more modifications of the amino acid sequence of SEQ ID NO:56 or a ligand-binding fragment thereof, wherein the target binding polypeptide comprises a substitution, deletion, insertion, or inversion of 1-10 amino acid residues. In some embodiments, the one or more modifications increase the affinity of the target binding polypeptide for PDL1 or PDL2 or both, compared to the affinity of wild-type PD1-ECD for its ligand. In some embodiments, the one or more modifications are selected from A132I, S87G, P89L, N116S, G124S, S127V, A140V. In some embodiments, the modification is A132I. In some embodiments, the target binding polypeptide has the amino acid sequence of SEQ ID NO:57.

[0017] In various embodiments, the IIP is linked to the target binding polypeptide moiety via a linker. In some embodiments, the target binding polypeptide moiety is linked to the C-terminus of the IIP. In some embodiments, the linker is selected from a non-cleavable linker, a peptide linker, a flexible linker, a rigid linker, a helical linker, and a non-helical linker. In some embodiments, the linker is a peptide linker having an amino acid sequence comprising (GGGGS)n, where n is 1, 2, 3, 4, 5, 6, 7, or 8. In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO:55. In some embodiments, the recombinant molecule comprises a first polypeptide having an amino acid sequence of SEQ ID NO:53 and a second polypeptide having an amino acid sequence of SEQ ID NO:54.

[0018] In some aspects, provided herein are hosts comprising the recombinant molecules described herein.

[0019] In some aspects, provided herein is a polynucleotide sequence encoding a recombinant molecule described herein, hi some aspects, provided herein is a vector comprising the polynucleotide.

[0020] In some aspects, provided herein are polypeptides comprising a recombinant molecule described herein.

[0021] In some aspects, provided herein is a pharmaceutical composition comprising a recombinant molecule described herein or a vector comprising a polynucleotide sequence encoding a recombinant molecule described herein. In some embodiments, the pharmaceutical composition further comprises a pharma- ceutically acceptable excipient.

[0022] In some aspects, provided herein are methods of treating a neoplastic disease, cancer, or immune disorder in a subject, comprising administering to the subject an effective amount of a pharmaceutical composition comprising a recombinant molecule described herein, or a vector comprising a polynucleotide sequence encoding a recombinant molecule described herein.

[0023] In some embodiments, the subject has cancer. In some embodiments, the recombinant molecule comprises a first polypeptide having the amino acid sequence of SEQ ID NO:53 and a second polypeptide having the amino acid sequence of SEQ ID NO:54.

[0024] In some embodiments, the cancer is selected from prostate cancer, pancreatic cancer, biliary tract cancer, colon cancer, rectal cancer, liver cancer, kidney cancer, lung cancer, testicular cancer, breast cancer, ovarian cancer, brain cancer, skin cancer, bladder cancer, and head and neck cancer, melanoma, sarcoma, multiple myeloma, leukemia, and / or lymphoma.

[0025] In some embodiments, the method inhibits tumor growth for at least 10, 15, or 20 days. In some embodiments, the method reduces tumor growth by at least 5%, 10%, 15%, or 20%.

[0026] In some embodiments, the method further comprises administering to the subject a therapeutic agent comprising an anti-CTLA4-TGFβRII molecule, hi some embodiments, the method further comprises administering one or more anti-cancer agents.

[0027] In some embodiments, the one or more anti-cancer agents comprise an immunotherapeutic agent, a chemotherapeutic molecule, an antibody, an antibody-drug conjugate, a small molecule kinase inhibitor, a hormonal agent, an androgen synthesis inhibitor, an androgen receptor antagonist, an anti-angiogenic agent, a cell therapy, a CAR-T cell therapy, a CAR-NK cell therapy, a radionuclide therapy, an ionizing radiation, an ultraviolet radiation, a cryoablation, a thermal ablation, a selective estrogen receptor modulator (SERM), a selective estrogen receptor degrader (SERD), or a radiofrequency ablation. In some embodiments, the immunotherapeutic agent is selected from an immune checkpoint inhibitor, an immunostimulatory receptor agonist, an immunostimulatory cytokine / cytokine receptor agonist, an immunoinhibitory cytokine / cytokine receptor antagonist, a tumor vaccine, an immunomodulatory imido drug, a CAR-T cell, a CAR-NK cell, or an oncolytic virus.

[0028] In some embodiments, administration of an anti-IL-23 agent in combination with one or more anti-cancer agents more effectively reduces or prevents severe immune-related adverse events or toxicity compared to administration of one or more anti-cancer agents alone. In some embodiments, administration of an anti-IL-23 agent in combination with one or more anti-cancer agents more effectively enhances tumor growth reduction or inhibits tumor growth compared to administration of one or more anti-cancer agents alone.

[0029] In some embodiments, the subject is a mammal, hi some embodiments, the subject is a human.

[0030] In one embodiment, a method of treating a neoplastic disease or cancer in a subject comprises administering to the subject one or more therapeutic agents, wherein the one or more therapeutic agents comprise: (a) a first therapeutic agent comprising an inhibitor of IL-23 signaling; and (b) a second therapeutic agent comprising: (i) one or more modulatory agents, each of which is an antagonist of one or more immune checkpoint proteins; (ii) one or more modulatory agents, each of which is an agonist of one or more immunostimulatory receptors; (iii) one or more modulatory agents, each of which is an agonist of one or more immunostimulatory receptors. (iv) one or more modulators, each of which is an agonist of one or more cytokine receptors; (v) one or more modulators, each of which modulates one or more cell surface molecules expressed or displayed on the cell surface of a tumor cell or immune cell; (vi) one or more immune cells, including CAR-T cells, CAR-NK cells, or hematopoietic stem cells; (vii) one or more immunogenic chemotherapeutic agents; and / or (viii) one or more modulators, each of which is an antagonist of one or more immune inhibitory enzymes.

[0031] In some embodiments, the inhibitor of IL-23 signaling comprises an IL-23 binding moiety that is a recombinant protein that binds to IL-23. In some embodiments, the inhibitor of IL-23 signaling comprises an IL-23R binding moiety that is a recombinant protein that binds to IL-23R. In some embodiments, the inhibitor of IL-23 signaling comprises a recombinant molecule described herein.

[0032] In various embodiments, the inhibitor of IL-23 signaling is an antibody or antigen-binding fragment thereof, wherein the antigen-binding fragment comprises a fragment crystallizable (Fc) region, a fragment antigen-binding (Fab) region, a single chain variable fragment (scFv), a light chain or a functional portion thereof, a variable region of the light chain (VL), a constant region of the light chain (CL), a heavy chain or a functional portion thereof, a variable region of the heavy chain (VH), a constant region of the heavy chain (CH), at least one complementarity determining region (CDR) or an antigen-binding portion thereof, or a combination thereof.

[0033] In some embodiments, the antibody or antigen-binding fragment thereof comprises a monoclonal antibody that targets an IL-23 subunit. In some embodiments, the monoclonal antibody targets an IL-23p19 subunit. In some embodiments, the antibody or antigen-binding fragment thereof comprises one or more of the six CDRs or antigen-binding portions thereof selected from any one of risankizumab, guselkumab, tildrakizumab, brazikumab, and mirikizumab.

[0034] In some embodiments, the IL-23R binding portion comprises an antibody or antigen-binding fragment thereof. In some embodiments, the antibody or antigen-binding fragment thereof comprises one or more of the six CDRs or antigen-binding portions thereof of AS2762900-00.

[0035] In various embodiments, the second therapeutic agent comprises a modulator that is an antagonist of one or more immune checkpoint proteins. In some embodiments, the immune checkpoint protein is selected from programmed death-1 (PD1; CD279), programmed death ligand 1 (PDL1), programmed death ligand 2 (PDL2), cytotoxic T-lymphocyte antigen-4 (CTLA4; CD152), attenuator of B and T lymphocytes (BTLA), V-domain immunoglobulin suppressor of T-cell activation (VISTA), T cell immunoglobulin and ITIM domain (TIGIT), lymphocyte activation gene 3 (LAG-3; CD223), T cell immunoglobulin and mucin domain 3 (Tim-3; HAVCR2), carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), CD47, signal regulatory protein alpha (SIRPa), major histocompatibility complex class I,G (HLA-G), Ig-like transcript 2 (ILT2; LILRB1), or Ig-like transcript 4 (ILT4, LILRB2).

[0036] In some embodiments, the second therapeutic agent comprises a modulator that is an antagonist of PD1 signaling. In some embodiments, the antagonist of PD1 signaling is a polypeptide that targets PD1. In some embodiments, the modulator is an inhibitor that comprises a monoclonal antibody or an antigen-binding fragment thereof that targets PD1 (CD279). In some embodiments, the antibody or an antigen-binding fragment thereof comprises one or more of six CDRs or antigen-binding portions thereof selected from pembrolizumab, nivolumab, cemiplimab, dostallimab, spartalizumab, camrelizumab, sintilimab, sasanlimab, ticelizumab, or toripalimab.

[0037] In some embodiments, the modulator is an inhibitor of a checkpoint protein selected from programmed death-1 ligand 1 (PDL1; CD274; B7-H1), programmed death-1 ligand 2 (PDL2), or both. In some embodiments, the modulator is a polypeptide that targets PDL1, PDL2, or both. In some embodiments, the polypeptide is an antibody or antigen-binding fragment thereof that targets PDL1, PDL2, or both. In some embodiments, the antibody or antigen-binding fragment thereof comprises one or more of the six CDRs or antigen-binding portions thereof selected from any one of durvalumab, avelumab, or atezolizumab.

[0038] In some embodiments, the modulator is a polypeptide inhibitor of the checkpoint protein CTLA-4. In some embodiments, the polypeptide is an antibody or antigen-binding fragment thereof that targets CTLA-4. In some embodiments, the antibody or antigen-binding fragment thereof comprises one or more of the six CDRs or antigen-binding portions thereof selected from any one of ipilimumab or tremelimumab.

[0039] In some embodiments, the modulator is a polypeptide inhibitor of the checkpoint protein LAG-3. In some embodiments, the polypeptide is an antibody or antigen-binding fragment thereof that targets LAG-3. In some embodiments, the antibody or antigen-binding fragment thereof comprises one or more of the six CDRs or antigen-binding portions thereof selected from any one of relatolimab, fianlimab, Sym022, GSK2831781, TSR-033, ielumilimab, favezelimab, tebotelimab, FS118, or pubunalimab.

[0040] In some embodiments, the regulator is a polypeptide inhibitor of the checkpoint protein TIGIT. In some embodiments, the polypeptide is an antibody or antigen-binding fragment thereof that targets TIGIT. In some embodiments, the antibody or antigen-binding fragment thereof comprises one or more of six CDRs or antigen-binding portions thereof selected from any one of tiragolumab, vibostolimab, BMS-986207, osipellimab, etigilimab, domvanalimab, EOS-448, SEA-TGT, ASP8374, COM902, or IBI939.

[0041] In various embodiments, the second therapeutic agent comprises a modulatory agent that is an agonist of one or more immunostimulatory receptors. In some embodiments, the immunostimulatory receptor is selected from 4-1BB (CD137), inducible T cell costimulator (ICOS; CD278), OX-40 (CD134), glucocorticoid-induced TNFR-related protein (GITR; CD357), CD40, herpes virus entry mediator (HVEM), CD28, or CD27. In some embodiments, the second therapeutic agent is a polypeptide comprising CD40L or a CD40-binding fragment thereof. In some embodiments, the second therapeutic agent is a polypeptide comprising CD80 or CD86; or a CD28-binding fragment thereof.

[0042] In various embodiments, the second therapeutic agent comprises a modulator of a cell surface molecule expressed or presented on tumor cells or tumor-associated stromal cells. In various embodiments, the cell surface molecule is selected from a growth factor receptor, a transforming growth factor-beta receptor (TGFβR), a tumor necrosis factor receptor (TNFR) superfamily receptor, an Ig superfamily receptor, a vascular endothelial growth factor receptor (VEGFR), an epidermal growth factor receptor (EGFR), a platelet-derived growth factor receptor (PDGFR), a tumor cell surface molecule, a cytokine receptor, or a chemokine receptor.

[0043] In some embodiments, the second therapeutic agent comprises a modulator of a cell surface molecule expressed or presented on an immune cell. In some embodiments, the immune cell is a T cell, a NK cell, or a myeloid cell.

[0044] In some embodiments, the cell surface molecule is a tumor necrosis factor receptor (TNFR) superfamily receptor, an Ig superfamily receptor, a cytokine receptor, a chemokine receptor, a T cell co-stimulatory molecule receptor, a T cell co-inhibitory molecule receptor, or a natural killer (NK) cell receptor. In some embodiments, the cell surface molecule is a myeloid cell inhibitory receptor, or a myeloid cell stimulatory receptor.

[0045] In various embodiments, the cell surface receptor is SIRPa or CD47. In some embodiments, the second therapeutic agent inhibits binding of SIRPa to CD47. In some embodiments, the second therapeutic agent is a polypeptide that binds to CD47. In some embodiments, the polypeptide is an antibody or antigen-binding fragment thereof that targets CD47. In some embodiments, the antibody or antigen-binding fragment thereof comprises one or more of six CDRs or antigen-binding portions thereof selected from magrolimab, ZL-1201, TJ011133, STI-6643, SRF231, SHR-1603, IMC-002, IBI188, CC-90002, AO-176, or AK117. In some embodiments, the polypeptide comprises a SIRPa extracellular domain or a CD47-binding fragment thereof. In some embodiments, the polypeptide is selected from evolupercept, TTI-621, or TTI-622. In some embodiments, the second therapeutic agent is a polypeptide that binds to SIRPa.

[0046] In various embodiments, the second therapeutic agent is an antagonist of one or more cytokine signaling. In some embodiments, the cytokine is transforming growth factor-beta (TGFb). In some embodiments, the modulator is an inhibitor of TGFb signaling selected from a small molecule kinase inhibitor, a polypeptide comprising TGFbRII ECD or a TGFb-binding fragment thereof, or an antibody or antigen-binding fragment thereof selected from an anti-TGFβ antibody, an anti-TGFβR antibody, an anti-GARP antibody, or an anti-LAP antibody.

[0047] In some embodiments, the small molecule kinase inhibitor is a TGFβR small molecule kinase inhibitor comprising galunisertib. In some embodiments, the anti-TGFβ antibody or antigen-binding fragment thereof comprises one or more of the six CDRs or antigen-binding portions thereof of fresolimumab. In some embodiments, the polypeptide comprising a TGFbRII ECD or a TGFb-binding fragment thereof is selected from AVID200, vintrafusp alfa (M7824), anti-CTLA4-TGFbRII, SIRPa ECD-TGFbRII, anti-CEA-TGFbRII, anti-PSMA-TGFbRII, anti-IL6R-TGFbRII, anti-PD1-TGFbRII, anti-EGFR-TGFbRII, or anti-HER2-TGFbRII.

[0048] In various embodiments, the cytokine is selected from one or more of the following: IL-4, IL-13, IL-10, IL-6, IL-1b, IL-17, IL-22, or VEGF. In some embodiments, the cytokine is IL-4 or IL-13.

[0049] In some embodiments, the modulator is a polypeptide that targets IL4 receptor alpha (IL4Ra). In some embodiments, the polypeptide is an antibody or antigen-binding fragment that includes one or more of the six CDRs of dupilumab, or an antigen-binding portion thereof.

[0050] In some embodiments, the cytokine is IL1b. In some embodiments, the second therapeutic agent comprises anakinra. In some embodiments, the second therapeutic agent comprises one or more of the six CDRs of canakinumab or antigen-binding portions thereof.

[0051] In some embodiments, the cytokine is IL10. In some embodiments, the second therapeutic agent comprises an IL10-binding sequence of the extracellular domain of IL10R, or an antibody or antigen-binding fragment thereof that targets IL10 or IL10R.

[0052] In various embodiments, the second therapeutic agent is an agonist of one or more cytokine receptors. In some embodiments, the cytokine receptors are selected from IL12R, IL15R, and IL18R. In some embodiments, the second therapeutic agent is a polypeptide comprising IL-12.

[0053] In various embodiments, the cell surface molecule is CA125, CA19-9, CD30, carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), CEACAM5 or cluster of differentiation, 66e (CD66e), CEACAM6, DLL3, DLL4, DPEP3, EGFR The tumor cell surface molecule is selected from EGFRvIII, GD2, HER2, HER3, HGF, IGF1R, IL13Ra2, LIV-1, LRRC15, MUC1, PRLR, PSCA, PSMA, PTK7, SEZ6, SLAMF7, TF, cMet, claudin, mesothelin, nectin4, uPAR, GPNMB, CD79b, CD22, NaPi2b, SLTRK6, STEAP1, MUC16, CD37, GCC, AGC-16, 5T4, CD70, TROP2, CD74, CD27L, Fra, CD138, CA6, CD38, SLAMF7, BCMA, CD20, CD19, CD33, or CD30.

[0054] In some embodiments, the regulator is an inhibitor comprising a monoclonal antibody or an antigen-binding fragment thereof that targets a tumor cell surface molecule. In some embodiments, the antibody or an antigen-binding fragment thereof comprises one or more of six CDRs or antigen-binding portions thereof selected from any one of labetuzumab, sergituzumab, cetuximab, necitumumab, panitumumab, depatuxizumab, trastuzumab, pertuzumab, enfortumab, or sacituzumab.

[0055] In various embodiments, the immune cells include antigen-presenting cells (APCs), myeloid-derived suppressor cells (MDSCs), dendritic cells, natural killer cells, or macrophages. H 17 cells, CD4 T cells, CD8 T cells, Treg cells, gamma delta T cells, NK cells, innate lymphoid cells (ILCs), or gamma delta T17 cells.

[0056] In some embodiments, treatment with a combination of a first and second agent reduces or inhibits tumor growth, prevents or reduces severe immune-related adverse events, increases overall survival or progression-free survival, reduces or prevents adverse events or toxicity, or reduces or prevents bone metastases or skeletal-related adverse events more effectively than treatment with the second agent alone.

[0057] In some embodiments, the cancer is selected from prostate cancer, pancreatic cancer, biliary tract cancer, colon cancer, rectal cancer, liver cancer, kidney cancer, lung cancer, testicular cancer, breast cancer, ovarian cancer, brain cancer, skin cancer, bladder cancer, and head and neck cancer, melanoma, sarcoma, multiple myeloma, leukemia, and / or lymphoma.

[0058] In some embodiments, the method further comprises administering to the subject a therapeutic agent comprising an anti-CTLA4-TGFβRII molecule.

[0059] In some embodiments, the method further comprises administering one or more anti-cancer therapies. In some embodiments, the one or more anti-cancer therapies comprise immunotherapeutic agents, chemotherapeutic molecules, antibodies, antibody-drug conjugates, small molecule kinase inhibitors, hormonal agents, androgen synthesis inhibitors, androgen receptor antagonists, anti-angiogenic agents, cell therapy, CAR-T cell therapy, CAR-NK cell therapy, radionuclide therapy, ionizing radiation, ultraviolet radiation, cryoablation, thermal ablation, selective estrogen receptor modulators (SERMs), selective estrogen receptor degraders (SERDs), or radiofrequency ablation. In some embodiments, the immunotherapeutic agents are selected from immune checkpoint inhibitors, immunostimulatory receptor agonists, immunostimulatory cytokines / cytokine receptor agonists, immunoinhibitory cytokines / cytokine receptor antagonists, tumor vaccines, immunomodulatory imid drugs, CAR-T cells, CAR-NK cells, or oncolytic viruses.

[0060] In some embodiments, the subject is a mammal, hi some embodiments, the subject is a human. [Brief description of the drawings]

[0061] These and other features, aspects, and advantages of the present invention will become better understood with reference to the following description and accompanying drawings.

[0062] [Figure 1A-D] 1A-1D show schematic diagrams corresponding to several exemplary designs of multispecific polypeptides described herein, where IIP comprises an antibody or antibody fragment and 2P represents a target-binding polypeptide.

[0063] [Figure 2A-C]2A-2G show schematic diagrams corresponding to several exemplary designs of the multispecific polypeptides described herein, where IIP comprises the ligand-binding fragment of the IL-23R ECD. D1, D2 and D3 represent IL-23R binding domains 1, 2 and 3, respectively. In these figures, dark grey corresponds to IIP and light grey corresponds to 2P of the multispecific polypeptide. 2P represents the target-binding polypeptide. [Fig. 2D-G] 2A-2G show schematic diagrams corresponding to several exemplary designs of the multispecific polypeptides described herein, where IIP comprises the ligand-binding fragment of the IL-23R ECD. D1, D2 and D3 represent IL-23R binding domains 1, 2 and 3, respectively. In these figures, dark grey corresponds to IIP and light grey corresponds to 2P of the multispecific polypeptide. 2P represents the target-binding polypeptide.

[0064] [Figure 3A] Figure 3A shows a schematic diagram corresponding to an exemplary design of a multispecific polypeptide having at least one amino acid modification in the PD1 extracellular domain (PD1 ECD A123I or GV-2). When compared to wild-type PD1, Figures 3B-3C show that PD1 ECD A123I or PD1 ECD GV-2 exhibit superior binding affinity to PD1, and Figures 3D-3E show superior binding affinity to PDL2. [Figure 3B-C] Figure 3A shows a schematic diagram corresponding to an exemplary design of a multispecific polypeptide having at least one amino acid modification in the PD1 extracellular domain (PD1 ECD A123I or GV-2). When compared to wild-type PD1, Figures 3B-3C show that PD1 ECD A123I or PD1 ECD GV-2 exhibit superior binding affinity to PD1, and Figures 3D-3E show superior binding affinity to PDL2. [Fig. 3D-E]Figure 3A shows a schematic diagram corresponding to an exemplary design of a multispecific polypeptide having at least one amino acid modification in the PD1 extracellular domain (PD1 ECD A123I or GV-2). When compared to wild-type PD1, Figures 3B-3C show that PD1 ECD A123I or PD1 ECD GV-2 exhibit superior binding affinity to PD1, and Figures 3D-3E show superior binding affinity to PDL2.

[0065] [Figure 4A] Figures 4A-4C demonstrate that IL-23 antibody enhances anti-tumor immune responses induced by PDL1 / PD1 blockade. Combined treatment with anti-IL-23 (anti-IL-23p19) and anti-PD-L1 antibodies (a-IL-23 / PDL1 blockade) resulted in superior survival and tumor shrinkage compared to mice treated with anti-PDL1 alone (a-PDL1) or anti-IL-23 antibodies alone (a-IL-23). ​​Figure 4A shows that combined treatment (a-IL-23 / PDL1 blockade) resulted in superior survival. ***p<0.005 by pairwise log-rank test. Y-axis: survival probability. X-axis: survival days. Figure 4B shows measurements of tumor volume (Y-axis) after tumor inoculation in mice implanted with B16F10 tumor cells. Combined treatment (right panel) resulted in superior tumor volume shrinkage when compared to treatment with anti-PD-L1 antibody alone (left panel). Figure 4C shows reduced lung tumor metastasis in mice treated with the combination treatment compared to mice treated with anti-PD-L1 antibody alone. White spots represent tumor metastases. [Figure 4B]Figures 4A-4C demonstrate that IL-23 antibody enhances anti-tumor immune responses induced by PDL1 / PD1 blockade. Combined treatment with anti-IL-23 (anti-IL-23p19) and anti-PD-L1 antibodies (a-IL-23 / PDL1 blockade) resulted in superior survival and tumor shrinkage compared to mice treated with anti-PDL1 alone (a-PDL1) or anti-IL-23 antibodies alone (a-IL-23). ​​Figure 4A shows that combined treatment (a-IL-23 / PDL1 blockade) resulted in superior survival. ***p<0.005 by pairwise log-rank test. Y-axis: survival probability. X-axis: survival days. Figure 4B shows measurements of tumor volume (Y-axis) after tumor inoculation in mice implanted with B16F10 tumor cells. Combined treatment (right panel) resulted in superior tumor volume shrinkage when compared to treatment with anti-PD-L1 antibody alone (left panel). Figure 4C shows reduced lung tumor metastasis in mice treated with the combination treatment compared to mice treated with anti-PD-L1 antibody alone. White spots represent tumor metastases. [Figure 4C] Figures 4A-4C demonstrate that IL-23 antibody enhances anti-tumor immune responses induced by PDL1 / PD1 blockade. Combined treatment with anti-IL-23 (anti-IL-23p19) and anti-PD-L1 antibodies (a-IL-23 / PDL1 blockade) resulted in superior survival and tumor shrinkage compared to mice treated with anti-PDL1 alone (a-PDL1) or anti-IL-23 antibodies alone (a-IL-23). ​​Figure 4A shows that combined treatment (a-IL-23 / PDL1 blockade) resulted in superior survival. ***p<0.005 by pairwise log-rank test. Y-axis: survival probability. X-axis: survival days. Figure 4B shows measurements of tumor volume (Y-axis) after tumor inoculation in mice implanted with B16F10 tumor cells. Combined treatment (right panel) resulted in superior tumor volume shrinkage when compared to treatment with anti-PD-L1 antibody alone (left panel). Figure 4C shows reduced lung tumor metastasis in mice treated with the combination treatment compared to mice treated with anti-PD-L1 antibody alone. White spots represent tumor metastases.

[0066] [Diagram 5] Figure 5 demonstrates that exemplary multispecific polypeptides described herein reduce tumor growth and limit toxicity in a humanized mouse model. Treatment with anti-IL-23-PD1ECD reduces tumor volume in NSG mice with hu-PBMC. *p=0.05 by one-tailed t-test. Control: vehicle only; a-IL-23: anti-IL-23 antibody; a-PDL1: anti-PDL1 antibody; a-IL-23-PD1: anti-IL-23-PD1ECD.

[0067] [Figure 6] Figure 6 demonstrates that the exemplary multispecific polypeptides described herein are more effective at inhibiting tumor growth compared to treatment with immune checkpoint inhibitor anti-PD1 antibody (a-PD1) (p=0.03) or a combination of checkpoint inhibitors anti-PD1 antibody and anti-CTLA4 antibody (a-PDL1+a-CTLA4). Combination treatment with anti-IL-23-PD1ECD and anti-PD1 antibody and anti-CTLA4 antibody (a-IL-23-PD1+a-CTLA4-TGFbRII) polypeptides inhibits tumor growth and shows a statistically synergistic anti-tumor efficacy (p<0.001) when compared to combination treatment with anti-PD1 antibody and anti-CTLA4 antibody (a-PD1+a-CTLA4).

[0068] [Figure 7A] 7A-7B illustrate an exemplary multispecific polypeptide described herein (anti-IL-23-PD1ECD) that suppresses the Th17 / MDSC axis by blocking PDL1 and PDL2 on tumor cells and antigen presenting cells (APCs) and sequestering IL-23. MDSC: myeloid-derived suppressor cells. [Figure 7B] 7A-7B illustrate an exemplary multispecific polypeptide described herein (anti-IL-23-PD1ECD) that suppresses the Th17 / MDSC axis by blocking PDL1 and PDL2 on tumor cells and antigen presenting cells (APCs) and sequestering IL-23. MDSC: myeloid-derived suppressor cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0069] Detailed Description Provided herein are novel polypeptides and methods for treating cancer, autoimmune diseases, and inflammatory disorders associated with the IL-23 axis.

[0070] In the case of cancer, activation of the immune system by immunotherapeutic agents (e.g., immune checkpoint inhibitors) or other anti-cancer treatments (e.g., conventional anti-cancer treatments) can lead to the adverse activation and expansion of IL-23-dependent immune cells such as Th17, gamma delta T17, and ILC3 cells. These cell types contribute to suppression of anti-tumor immunity and / or tumor-promoting inflammation in the presence of IL-23 signaling that maintains their phenotype. Furthermore, these cell types contribute to immune-related adverse events and treatment-related toxicity. As such, the multispecific polypeptides and methods of treatment described herein are designed to block IL-23 signaling while simultaneously activating anti-tumor immune responses. This strategy may allow for both better anti-tumor efficiency and lower toxicity (i.e., a broader therapeutic window of immune activation) than approaches that focus solely on activating the immune response.

[0071] In some cases, the multispecific polypeptides and combination treatment regimens described herein include one moiety that blocks IL-23 signaling and an additional moiety that promotes immune cell activation. In other cases, the additional moiety further serves to bias the phenotype of immune cells to an anti-tumor state. Although breaking tumor-induced immune tolerance by itself can lead to the adverse activation of IL-23-dependent immune cells, the present inventors disclose polypeptides and methods that direct the breaking of tumor-induced immune tolerance in situations where IL-23 signaling is inhibited.

[0072] The effect of increasing Th17 cells in the context of cancer treatment is unclear.In fact, some studies suggest that the infiltration of Th17 cells in TME is associated with better clinical prognosis; alternatively, the role of Th17 cells in cancer has been characterized.In light of such findings in the field, the multispecific polypeptides and various combinations described herein demonstrate the unexpected beneficial effect of IL-23 blockade in conjunction with immune activation (e.g., using immune checkpoint inhibitors) for both increasing efficacy and reducing undesirable immune-related adverse events.

[0073] In one embodiment, a recombinant molecule is provided herein, comprising: (a) an interleukin-23 (IL-23) inhibitory polypeptide (IIP), said IIP comprising an IL-23 binding polypeptide or an IL-23R binding polypeptide; and (b) a target-binding polypeptide portion that binds to one or more immune checkpoint proteins or immunostimulatory receptors. The IIP can be an IL-23 inhibitor or an IL-23R inhibitor. The target-binding polypeptide can specifically bind to an inhibitor of one or more cell surface molecules or their ligands, such as immune checkpoint protein receptors (e.g., PD-1, PD-L1, PD-L2, CTLA4). Thus, the recombinant molecule described herein is capable of specifically inhibiting IL-23 / checkpoint protein (e.g., PD-1) signaling in the tumor microenvironment (TME) and enhancing immune responses and reducing and / or suppressing tumor growth.

[0074] definition Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs.

[0075] As used herein, the terms "patient" and "subject" are used interchangeably and can be taken to mean any living organism that can be treated with the compounds of the invention. As such, the terms "patient" and "subject" include, but are not limited to, any non-human mammal, primate, and human.

[0076] In the context of this disclosure insofar as any of the conditions listed herein are concerned, the terms "treat" and "treatment" and the like refer to alleviating or relieving at least one symptom associated with such condition, or slowing or reversing the progression of such condition. Within the meaning of this disclosure, the term "treat" also refers to arresting, slowing the onset of disease (i.e., the period prior to clinical manifestation of disease) and / or reducing the risk of developing or worsening disease. Additionally, the terms "treat" and "treatment" and the like with respect to a state, disorder, or condition can include (1) preventing or delaying the appearance of at least one clinical or subclinical symptom of the state, disorder, or condition that develops in a subject who may be afflicted with or predisposed to the state, disorder, or condition, but who has not yet experienced or exhibited any clinical or subclinical symptoms of the state, disorder, or condition; or (2) inhibiting the state, disorder, or condition (i.e., arresting, reducing, or slowing the onset of the disease or its recurrence (in the case of maintenance treatment) or at least one clinical or subclinical symptom thereof); or (3) alleviating the disease (i.e., reversing the state, disorder, or condition or at least one of its clinical or subclinical symptoms).

[0077] As used herein, "prevention" of a disease refers to inhibiting the development of the disease altogether.

[0078] The term "biological sample" refers to any tissue, cell, fluid, or other material derived from an organism (e.g., a human subject). In certain embodiments, the biological sample is serum or blood.

[0079] The term "immunoglobulin" generally refers to a class of structurally related proteins that contain two pairs of polypeptide chains: one pair of light (L) chains and one pair of heavy (H) chains. In "intact immunoglobulins," all four of these chains are interconnected by disulfide bonds. The structure of immunoglobulins is well characterized. See, for example, Paul, Fundamental Immunology 7th ed., Ch. 5 (2013) Lippincott Williams & Wilkins, Philadelphia, PA. Briefly, each heavy chain typically contains a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region typically contains three domains, abbreviated as CH1, CH2, and CH3. Each light chain typically contains a light chain variable region (VL) and a light chain constant region. The light chain constant region typically contains one domain, abbreviated as CL.

[0080] The term "antibody" is used herein in its broadest sense and includes certain types of immunoglobulin molecules that contain one or more antigen-binding domains that specifically bind to an antigen or epitope. Antibodies specifically include intact antibodies (e.g., intact immunoglobulins), antibody fragments, and multispecific antibodies. An example of an antigen-binding domain is the antigen-binding domain formed by a VH-VL dimer. As used herein, "antibody" encompasses polyclonal and monoclonal antibodies and refers to immunoglobulin molecules of the IgA (e.g., IgA1 or IgA2), IgD, IgE, IgG (e.g., IgG1, IgG2, IgG3, and IgG4), or IgM class, or fragments or derivatives thereof, including, but not limited to, Fab, F(ab')2, Fd, single-chain antibodies, diabodies, bispecific antibodies, bifunctional antibodies, humanized antibodies, and various derivatives thereof.

[0081] The term "antigen-binding fragment" refers to a portion of an intact antibody and / or to the antigen-determining variable region of the intact antibody. It is known that fragments of a full-length antibody can exert the antigen-binding function of the antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, linear antibodies, single-chain antibodies, diabodies, and multispecific antibodies formed from antibody fragments.

[0082] The term "Fc region" refers to the C-terminal region of the immunoglobulin heavy chain that interacts with the Fc receptor and certain proteins of the complement system in naturally occurring antibodies. The structures of the Fc region of various immunoglobulins and the glycosylation sites contained therein are known in the art. See Schroeder and Cavacini, J. Allergy Clin. Immunol., 2010, 125:S41-52, which is incorporated by reference in its entirety. The Fc region may be a naturally occurring Fc region or may be an altered Fc region as described elsewhere in this disclosure.

[0083] The VH and VL regions can be further subdivided into regions of hypervariability ("hypervariable regions" (HVRs); also called "complementarity determining regions" (CDRs)) interspersed with more conserved regions. The more conserved regions are called framework regions (FRs). Each VH and VL generally contains three CDRs and four FRs arranged in the following order (N-terminus to C-terminus): FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The CDRs are involved in antigen binding and affect the antigen specificity and binding affinity of the antibody. See Kabat et al., Sequences of Proteins of Immunological Interest 5th ed. (1991) Public Health Service, National Institutes of Health, Bethesda, MD (incorporated by reference in its entirety).

[0084] Light chains from any vertebrate species can be assigned to one of two types, called kappa (κ) and lambda (λ), based on their constant domain sequence.

[0085] Heavy chains from any vertebrate species can be assigned to one of five different classes (or isotypes): IgA, IgD, IgE, IgG, and IgM. These classes are also designated α, δ, ε, γ, and μ, respectively. The IgG and IgA classes are further divided into subclasses based on differences in sequence and function. Humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

[0086] One of skill in the art can determine the amino acid sequence boundaries of the CDRs using any of several known numbering schemes, including those described in Kabat et al., supra (the "Kabat" numbering scheme); Al-Lazikani et al., 1997, J. Mol. Biol., 273:927-948 (the "Chothia" numbering scheme); MacCallum et al., 1996, J. Mol. Biol. 262:732-745 (the "Contact" numbering scheme); Lefranc et al., Dev. Comp. Immunol., 2003, 27:55-77 (the "IMGT" numbering scheme); and Honegge and Plueckthun, J. Mol. Biol., 2001, 309:657-70 (the "AHo" numbering scheme), each of which is incorporated by reference in its entirety.

[0087] Table 1 provides the positions of CDR1-L (CDR1 of VL), CDR2-L (CDR2 of VL), CDR3-L (CDR3 of VL), CDR1-H (CDR1 of VH), CDR2-H (CDR2 of VH), and CDR3-H (CDR3 of VH) as identified by the Kabat and Chothia scheme. For CDR1-H, residue numbering is provided using both the Kabat and Chothia numbering schemes.

[0088] CDRs can be assigned using, for example, antibody numbering software (such as Abnum, available at www.bioinf.org.uk / abs / abnum / and described in Abhinandan and Martin, Immunology, 2008, 45:3832-3839, which is incorporated by reference in its entirety). [Table 1]

[0089] An "Fv" fragment comprises a dimer of one heavy- and one light-chain variable domain in a non-covalent association.

[0090] A "Fab" fragment contains the variable domains of the heavy and light chains as well as the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab fragments can be produced, for example, by recombinant methods or by papain digestion of a full-length antibody.

[0091] "F(ab')2" fragments contain two Fab' fragments linked near the hinge region by disulfide bonds. F(ab')2 fragments can be produced, for example, by recombinant methods or by pepsin digestion of an intact antibody. For example, F(ab') fragments can be separated by treatment with β-mercaptoethanol.

[0092] "Single-chain Fv" or "sFv" or "scFv" antibody fragments comprise a VH domain and a VL domain in a single polypeptide chain. The VH and VL are generally linked by a peptide linker. See Plueckthun A. (1994). In some embodiments, the linker is (GGGGS)n (SEQ ID NO:55). In some embodiments, n=1, 2, 3, 4, 5, or 6. See Antibodies from Escherichia coli. In Rosenberg M. & Moore GP (Eds.), The Pharmacology of Monoclonal Antibodies vol.113 (pp.269-315). Springer-Verlag, New York (incorporated by reference in its entirety).

[0093] An "scFv-Fc" fragment comprises an scFv linked to an Fc domain. For example, the Fc domain may be linked to the C-terminus of the scFv. The Fc domain may follow the VH or VL depending on the orientation of the variable domains in the scFv (i.e., VH-VL or VL-VH). Any suitable Fc domain known in the art or described herein may be used. In some cases, the Fc domain comprises an IgG4 Fc domain.

[0094] "Humanized" forms of non-human antibodies are chimeric antibodies that contain minimal sequences derived from the non-human antibody. Humanized antibodies are generally human antibodies (recipient antibodies) in which residues from one or more CDRs have been replaced by residues from one or more CDRs of a non-human antibody (donor antibody). The donor antibody can be any suitable non-human antibody (such as a mouse, rat, rabbit, chicken, or non-human primate antibody with the desired specificity, affinity, or biological effect). In some instances, selected framework region residues of the recipient antibody are replaced by the corresponding framework region residues from the donor antibody. Humanized antibodies may also contain residues that are not found in either the recipient antibody or the donor antibody. Such modifications may be made to further improve antibody function. For further details, see Jones et al., Nature, 1986, 321:522-525; Riechmann et al., Nature, 1988, 332:323-329; and Presta, Curr. Op. Struct. Biol., 1992, 2:593-596 (each of which is incorporated by reference in its entirety).

[0095] A "human antibody" is an antibody that possesses an amino acid sequence that corresponds to that of an antibody produced by a human or human cell, or derived from a human antibody repertoire or a non-human source that utilizes human antibody coding sequences (e.g., obtained from a human source or designed de novo). Human antibodies specifically exclude humanized antibodies. In some embodiments, rodents are genetically engineered to replace the rodent antibody sequences with human antibody sequences.

[0096] The term "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues. As used herein, "cancer" refers to a disease caused by the uncontrolled division of abnormal cells. The terms "cancer," "neoplastic disease," and "tumor," as referred to herein, are not mutually exclusive. The terms "cell proliferative disorder" and "proliferative disorder" refer to disorders associated with some degree of abnormal cell proliferation. In some embodiments, the cell proliferative disorder is cancer. Non-limiting examples of cancer include prostate cancer, pancreatic cancer, biliary tract cancer, colon cancer, rectal cancer, liver cancer, kidney cancer, lung cancer, testicular cancer, breast cancer, ovarian cancer, brain cancer, skin cancer, bladder cancer, and head and neck cancer, melanoma, sarcoma, multiple myeloma, leukemia, and / or lymphoma.

[0097] The term "immune response" refers to the response of immune system cells (e.g., B cells, T cells, macrophages, or polymorphonuclear cells) to a stimulus such as an antigen (e.g., a viral antigen). An active immune response can involve the differentiation and proliferation of immunocompetent cells leading to the synthesis of antibodies or the development of cell-mediated reactivity, or both. An active immune response can be initiated by a host after exposure to an antigen (e.g., by infection or vaccination). An active immune response can be contrasted with passive immunity, which can be obtained by the transfer of substances such as, for example, antibodies, transfer factors, thymic grafts, and / or cytokines from an actively immunized host to a non-immunized host.

[0098] "Homology" or "identity" or "similarity" can refer to the sequence similarity between two peptides or two nucleic acid molecules. Homology can be determined by comparing positions in each sequence that can be aligned for comparison. If a position in the compared sequences can be occupied by the same base or amino acid, the molecules can be homologous at that position. The degree of homology between sequences can be a function of the number of matching or homologous positions shared by the aforementioned sequences. An "unrelated" or "non-homologous" sequence shares less than 40% identity, or alternatively less than 25% identity, with one of the sequences of the present disclosure. Sequence homology can refer to the % identity of a sequence to a reference sequence. In practice, whether any particular sequence may be at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to any sequence described herein (which may correspond to a particular nucleic acid sequence described herein) can be routinely determined by using known computer programs such as the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, Wis. 53711). When using Bestfit or any other sequence alignment program to determine whether a particular sequence is, for example, 95% identical to a reference sequence, the percent identity can be calculated over the entire length of the reference sequence, and parameters can be set such that gaps in sequence homology can be allowed up to 5% of the entire reference sequence. The terms "percent identity" or "homology" in the context of two or more nucleic acid or polypeptide sequences refer to two or more sequences or subsequences that have a specified percentage of nucleotide or amino acid residues that are identical when compared and aligned for maximum correspondence, as measured using one of the sequence comparison algorithms described below (e.g., BLASTP and BLASTN or other algorithms available to those of skill in the art) or by visual inspection.Depending on the application, the percentage of "identity" can be over the region of sequences being compared (e.g., over a functional domain) or over the entire length of the two sequences being compared. For sequence comparison, typically one sequence serves as a reference sequence to which a test sequence is compared. When using a sequence comparison algorithm, the test sequence and the reference sequence are input into a computer, subsequence coordinates are designated if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percentage of sequence identity for the test sequence(s) relative to the reference sequence based on the designated program parameters. For purposes herein, percentage identity and sequence similarity are determined using the BLAST algorithm described in Altschul et al., J. Mol. Biol. 215:403-410 (1990). Software for performing BLAST analysis is publicly available from the National Center for Biotechnology Information (on the World Wide Web at: ncbi.nlm.nih.gov / ).

[0099] In some cases, the identity between a reference sequence (query sequence, e.g., a sequence of the present disclosure) and a subject sequence (also referred to as global sequence alignment) can be determined using a FASTDB computer program. In some embodiments, the parameters for a particular embodiment that can locally interpret the identity used in FASTDB amino acid alignment can include: scoring scheme=PAM (allowed mutation rate) 0, k-tuple=2, mismatch penalty=1, binding penalty=20, randomization group length=0, cutoff score=1, window size=sequence length, gap penalty=5, gap size penalty=0.05, window size=500 or subject sequence length (any of which can be shorter). According to this embodiment, if the subject sequence may be shorter than the query sequence due to N- or C-terminal deletions rather than internal deletions, the results can be manually corrected to account for the fact that the FASTDB program does not consider the N- and C-terminal truncations of the subject sequence when calculating the global identity percentage. In the case of a subject sequence that is truncated at the N-terminus or C-terminus compared to the query sequence, the identity can be corrected by calculating the number of residues of the query sequence that can be located at the N-terminus and C-terminus of the subject sequence (that cannot be matched / aligned with the corresponding subject sequence) as a percentage of the total bases of the query sequence. Whether a residue can be matched / aligned can be determined by the result of FASTDB sequence alignment. This percentage can then be subtracted from the identity calculated by the FASTDB program using the specified parameters to arrive at a final identity score. This final identity score can be used for the purposes of this embodiment. In some cases, only the residues to the N-terminus and C-terminus of the subject sequence that cannot be matched / aligned with the query sequence can be considered for manually adjusting the identity score. That is, only the query residue positions outside the residues at the farthest N-terminus and C-terminus of the subject sequence can be considered for this manual correction.For example, a 90-residue subject sequence can be aligned with a 100-residue query sequence to determine percent identity. If a deletion occurs at the N-terminus of the subject sequence, the result is that the FASTDB alignment does not show a match / alignment of the first 10 residues at the N-terminus. Since the 10 mismatched residues represent 10% of the sequence (number of residues at the N-terminus and C-terminus that do not match / total number of residues in the query sequence), 10% can be subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 residues are perfectly matched, the final percent identity can be 90%. In another example, a 90-residue subject sequence can be compared with a 100-residue query sequence. In this case, the deletion can be an internal deletion, so there can be no residues at the N-terminus or C-terminus of the subject sequence that cannot be matched / aligned with the query. In this case, the percent identity calculated by FASTDB cannot be manually corrected. Again, only residue positions outside the N- and C-termini of the subject sequence displayed in the FASTDB alignment that cannot be matched / aligned with the query sequence can be manually corrected.

[0100] The phrase "pharmaceutically acceptable," as used in reference to compositions described herein, refers to molecular entities and other components of such compositions that are physiologically tolerable and typically do not produce adverse reactions when administered to a subject (e.g., a human). Preferably, the term "pharmaceutically acceptable" means approved by a federal or state regulatory agency for use in mammals, more specifically humans, or listed in the United States Pharmacopeia or other generally recognized pharmacopoeias.

[0101] As used herein, "carrier" includes pharma- ceutically acceptable carriers, excipients, or stabilizers that are non-toxic to cells or mammals exposed to the dosages and concentrations used. Often, physiologically acceptable carriers are pH-buffered aqueous solutions. Examples of physiologically acceptable carriers include buffers (such as phosphate buffers, citrate buffers, and other organic acid buffers); antioxidants (including ascorbic acid and methionine); preservatives (such as octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens (such as methyl or propyl paraben); catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins (such as serum albumin, gelatin, or immunoglobulins). amino acids (such as glycine, glutamine, asparagine, arginine, or lysine); monosaccharides, disaccharides, and other carbohydrates (including glucose, mannose, or dextrin); chelating agents (such as EDTA); sugars (such as sucrose, mannitol, trehalose, or sorbitol); salt-forming counterions (such as sodium); metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants (such as TWEEN™, polyethylene glycol (PEG), and PLURONICS™ or polyethylene glycol (PEG)).

[0102] As used herein, a "diluent" is a diluent that is pharma- ceutically acceptable (safe and non-toxic for administration to humans) and useful for preparing liquid formulations (such as formulations reconstituted after lyophilization). Exemplary diluents include sterile water, bacteriostatic water for injection (BWFI), pH buffer (e.g., phosphate buffered saline), sterile saline, Ringer's solution, or dextrose solution. In another embodiment, the diluent may include aqueous salt and / or buffer solutions.

[0103] A "preservative" is a compound that can be added to the formulation herein to reduce bacterial activity. The addition of a preservative can, for example, facilitate the production of a repeat-use (repeated administration) formulation. Examples of potential preservatives include octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride (a mixture of alkylbenzyldimethylammonium chlorides, where the alkyl group is a long-chain compound), and benzethonium chloride. Other types of preservatives include aromatic alcohols, such as phenol, butyl and benzyl alcohol, alkylparabens, such as methyl or propylparaben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol. The most preferred preservative herein is benzyl alcohol.

[0104] The term "pharmaceutical formulation" refers to a preparation that is in such a form that the biological activity of the active ingredient is effective and does not contain additional ingredients that are unacceptably toxic to the subject to which the formulation is intended to be administered. Such a formulation is sterile. A "sterile" formulation is aseptic or free of all viable microorganisms and their spores.

[0105] A "stable" formulation is one in which the protein in the formulation essentially retains its physical and chemical stability and integrity upon storage. A variety of analytical techniques for measuring protein stability are available in the art and are reviewed in Peptide and Protein Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, NY, Pubs. (1991) and Jones, A. Adv. Drug Delivery Rev. 10:29-90 (1993). Stability can be measured at a selected temperature for a selected period of time. For rapid screening, the formulation may be kept at 40°C for 2 weeks to 1 month, during which time stability is measured. If the formulation is to be stored at 2-8°C, generally the formulation should be stable at 30°C or 40°C for at least 1 month and / or at 2-8°C for at least 2 years. If the formulation is to be stored at 30°C, generally the formulation should be stable at 30°C for at least 2 years and / or at least 6 months at 40°C. For example, the degree of aggregation during storage can be used as an indicator of protein stability. Thus, a "stable" formulation can be one in which less than about 10%, preferably less than about 5%, of the protein is present as aggregates in the formulation. In other embodiments, any increase in aggregate formation during storage of the formulation can be determined.

[0106] A "reconstituted" formulation is one that has been prepared by dissolving a lyophilized protein or antibody formulation in a diluent such that the protein is dispersed throughout the formulation. The reconstituted formulation is suitable for administration (e.g., subcutaneous administration) to a patient to be treated with the protein of interest, and in certain embodiments may be a formulation suitable for parenteral or intravenous administration.

[0107] An "isotonic" formulation is one that has essentially the same osmotic pressure as human blood. Isotonic formulations generally have an osmotic pressure of about 250-350 mOsm. The term "hypotonic" describes a formulation that has an osmotic pressure less than human blood. Correspondingly, the term "hypertonic" is used to describe a formulation that has an osmotic pressure greater than human blood. Isotonicity can be measured, for example, using a vapor pressure or ice-freezing osmometer. The formulations of the present application can be hypertonic as a result of the addition of salts and / or buffers.

[0108] As used herein, "immune cells," "immune effector cells," or "immune response cells" include T lymphocytes, B lymphocytes, natural killer (NK) cells, NKT cells, monocytes, macrophages, dendritic cells (DCs), and antigen-presenting cells (APCs).

[0109] The term "effector T cells" or "T cells" includes T helper (i.e., CD4+) cells and cytotoxic (i.e., CD8+) T cells. CD4+ effector T cells contribute to the development of several immunological processes, including maturation of B cells into plasma cells and memory B cells, and activation of cytotoxic T cells and macrophages. CD8+ effector T cells destroy virus-infected cells and tumor cells. For more information regarding effector T cells, see Seder and Ahmed, Nature Immunol., 2003, 4:835-842 (incorporated by reference in its entirety).

[0110] The term "regulatory T cells" includes, for example, cells that regulate immunological tolerance by suppressing effector T cells. In some embodiments, regulatory T cells have a CD4+CD25+Foxp3+ phenotype. In some embodiments, regulatory T cells have a CD8+CD25+ phenotype. For more information regarding regulatory T cells, see Nocentini et al., Br. J. Pharmacol., 2012, 165:2089-2099 (incorporated by reference in its entirety).

[0111] The term "Th17 cells" includes a subset of CD4+ T cells characterized by expression of the signature transcription factor ROR gamma and cytokines such as interleukin-17 (IL-17). The term "gamma delta T17 cells" includes a subset of gamma delta T cells also characterized by expression of IL-17. The term "ILC3 cells" includes a subset of innate immune cells.

[0112] The term "dendritic cell" refers to professional antigen-presenting cells that can activate naive T cells and stimulate the growth and differentiation of B cells.

[0113] The term "treating" (and its variations such as "treat" or "treatment") refers to a clinical intervention that attempts to change the natural course of a disease or condition in a subject in need of treatment. Treatment can be performed both prophylactically and during the course of a clinical pathological condition. Desirable effects of treatment include prevention of disease onset or recurrence, alleviation of symptoms, reduction of any direct or indirect pathological consequences of the disease, prevention of metastasis, slowing the rate of disease progression, amelioration or palliation of the disease, and remission or improvement of prognosis.

[0114] As used herein, the term "therapeutically effective amount" or "effective amount" refers to an amount of any of the recombinant molecules, polypeptides, or pharmaceutical compositions provided herein that is effective to treat a disease or disorder when administered to a subject.

[0115] As used herein, a "target-binding moiety" or a "target-binding polypeptide" refers to a molecule that has the ability to localize and bind to a specific molecule or cellular component. The targeting binding moiety or polypeptide can be an antibody, an antibody fragment, an scFv, an Fc-containing polypeptide, a fusion antibody, a polypeptide, a peptide, an aptamer, a ligand, a nucleic acid, or any combination thereof. As a non-limiting example, the targeting moiety or polypeptide can bind to a molecule present in a cell or tissue, a molecule in a diseased cell or tissue (e.g., a cancer cell or a tumor), a normal cell or tissue (e.g., an immune cell), a cellular or extracellular molecule that modulates the immune response (e.g., a cytokine (e.g., IL-23, or an immune checkpoint protein (e.g., PD-1, CTLA4, or the like)), a growth factor receptor (e.g., TGFbRII, VEGFR, TNFR, EGFR), a growth factor, a cytokine receptor, a cytokine, or a cell surface molecule. As another example, the targeting moiety or polypeptide is a tumor targeting moiety capable of binding to a component of or near a tumor cell (e.g., tumor vasculature or tumor microenvironment), tumor microenvironment, tumor vasculature, tumor-associated lymphocytes, tumor antigens, tumor-associated antigens, tumor cell surface molecules, tumor antigenic determinants, tumor antigen-containing fusion proteins, tumor-associated cells, tumor-associated immune cells, or tumor vaccines. Non-limiting examples of molecules or components to which a targeting moiety or polypeptide can specifically bind include epidermal growth factor receptor (EGFR, EGFR1, ErbB-1, HER1), ErbB-2 (HER2 / neu), ErbB-3 / HER3, ErbB-4 / HER4, EGFR ligand family; insulin-like growth factor receptor (IGFR) family, IGF binding proteins (IGFBPs), IGFR ligand family (IGF-1R); platelet-derived growth factor receptor (PDGFR) family, PDGFR ligand family; fibroblast growth factor receptor (FGFR) family, FGFR ligand family, vascular endothelial growth factor receptor (VEGFR) family,VEGF family;HGF receptor family;TRK receptor family;ephrin (EPH) receptor family;AXL receptor family;leukocyte tyrosine kinase (LTK) receptor family;TIE receptor family, angiopoietin 1, 2;receptor tyrosine kinase-like orphan receptor (ROR) receptor family;discoidin domain receptor (DDR) family;RET receptor family;KLG receptor family;RYK receptor family;MuSK receptor family;transforming growth factor alpha (TGF-α),TG F-alpha receptor; transforming growth factor-beta (TGF-β), TGF-β receptor; interleukin-beta receptor alpha 2 chain (IL13Ralpha2), interleukin-6 (IL-6), IL-6 receptor, interleukin-4, IL-4 receptor, cytokine receptor, class I (hematopoietin family) and class II (interferon / IL-10 family) receptors, tumor necrosis factor (TNF) family, TNF-α, tumor necrosis factor (TNF) receptor superfamily (TNFRSF), death receptor family, T RAIL-receptor; cancer testis (CT) antigen, lineage specific antigen, differentiation antigen, alpha-actinin-4, ARTC1, fibronectin (FN), GPNMB, HLA-A2, MLA-A11, MART2, melanoma ubiquitous mutations 1, 2, 3 (MUM-1, 2, 3), prostatic acid phosphatase (PAP), neo-PAP, myosin class 1, NFYC, OGT, OS-9, pml-RAR alpha fusion protein, PRDX5, PTPRK, IRT2, SNRPD1, SYT-SSX1 or -SSX2 fusion protein, BAGE, BAGE-1-5, GAGE-1-5, GAGE-2-5, GAGE-3-5, GAGE-4-5, GAGE-5-5, GAGE-5-5, GAGE-6-5, GAGE-7-5, GAGE-8-5, GAGE-9-5, GAGE-1-5, GAGE-1-5, GAGE-1-5, GAGE-1-5, GAGE-1-5, GAGE-2 ... AGE-1-8, MGAT5, LAGE, LAGE-1, CTL-recognized antigen on melanoma (CAMEL), melanoma-associated antigen (MAGE) family members, mucin 1 (MUC1), MART-1 / Melan-A (MLANA), gp100, gp100 / Pme117 (S1LV), tyrosinase (TYR), TRP-1, HAGE, NA-88, NY-ESO-1, NY-ESO-1 / LAGE-2, SAGE, Sp17, SSX-1-4, carcinoembryonic antigen (CEA), caliculine 4, mammaglobin-A, OA1, prostate-specific antigen (PSA),Prostate-specific membrane antigen, TRP-2, adipophilin, interferon-inducible protein 2 (AIM-2) absent in melanoma (nielanorna), BING-4, CPSF, cyclin D1, epithelial cell adhesion molecule (Ep-CAM), EpbA3, fibroblast growth factor-5 (FGF-5), alpha-fetoprotein (AFP), M-CSF, MUCI, PBF, FRAME, RAGE-1, RNF43, RU2AS, SOX10, STEAP1, XAGE, ADAM2, PAGE-5, LIP1, CTAGE-1, CSAGE, MMA1, CAGE, BORIS, HOM-TES-85, AF15q14, HCA66I, LDHC, MORC, SGY-1, SPO11, TPX1, NY-SAR-35, FTHLI7, , TDRD1, TEX 15, FATE, TPTE, estrogen receptor (ER), androgen receptor (AR), CD40, CD30, CD20, CD19, CD33, CD4, CD25, CD3, cancer antigen 72-4 (CA72-4), cancer antigen 15-3 (CA15-3), cancer antigen 27-29 (CA27-29), cancer antigen 125 (CA125), cancer antigen 19-9 (CA19-9), beta-human chorionic gonadotropin, 1-2 microglobulin, squamous cell carcinoma antigen, GM2, 707 alanine proline (707-AP), adenocarcinoma antigen recognized by T cells 4 (ART-4), carcinoembryonic antigen peptide-1 (CAP-1), calcium-activated chloride channel-2 (CLCA2), cyclophilin B (Cyp-B), human ring body tumor-2 (HST-2). The compositions of the invention may further comprise the above as peptides / polypeptides and / or encoding same.

[0116] As used herein, the term "subject" refers to a mammalian subject. Exemplary subjects include humans, monkeys, dogs, cats, mice, rats, cows, horses, camels, goats, rabbits, and sheep. In certain embodiments, the subject is a human. In some embodiments, the subject has a disease or condition that can be treated with the multispecific polypeptides provided herein. In some aspects, the disease or condition is cancer. In some aspects, the disease or condition is an immune disorder.

[0117] The term "immunostimulatory receptor" refers to a polypeptide expressed on the cell surface of an immune cell that activates, mutates, proliferates, or stimulates said cell. In some embodiments, an immunostimulatory receptor may signal through one or more intracellular immunoreceptor tyrosine-based activation motifs (ITAMs) or immunoreceptor tyrosine-based switch motifs (ITSMs). A subset of immunostimulatory receptors expressed on T cells may be referred to as "T cell costimulatory receptors." In some embodiments, when a T cell costimulatory receptor is ligated by its cognate ligand, intracellular signaling occurs that activates the T cell. In some embodiments, this "signal 2" acts in concert with "signal 1" resulting from TCR ligation to fully activate the T cell. Non-limiting examples of T cell costimulatory receptors include 4-1BB (CD137), inducible T cell costimulatory factor (ICOS; CD278), OX-40 (CD134), glucocorticoid-induced TNFR-related protein (GITR; CD357), herpes virus entry mediator (HVEM), CD28, or CD27. A subset of immune stimulatory receptors expressed on innate immune cells may be referred to as "innate immune stimulatory receptors." Non-limiting examples of innate immune stimulatory receptors expressed on NK cells include TRAIL, CD16, NKp30, NKp44, NKp46, NKp80, NKG2C, NKG2D, 2B4 (CD244), DNAM-1 (CD226), CD137, OX40, and CD27. Non-limiting examples of innate immune stimulatory receptors expressed on myeloid cells include DAP12 and Fc receptor gamma, and receptors linked to ITAM-containing adaptors like DAP12 and Fc receptor gamma (such as TREM-2).

[0118] The term "immune checkpoint protein" refers to a polypeptide that attenuates the activation of immune cells. In some embodiments, immune checkpoint proteins include receptors that transmit inhibitory signals in immune cells (e.g., PD-1) and ligands that activate such receptors (e.g., PD-L1, PD-L2). In other embodiments, immune checkpoint proteins include receptors that sequester ligands of immunostimulatory receptors (e.g., CTLA-4 sequester the immunostimulatory ligand CD80, which prevents CD86 from interacting with the immunostimulatory receptor CD28). In some embodiments, immune checkpoint proteins may signal through one or more intracellular immunoreceptor tyrosine-based inhibitory motifs (ITIMs) or immunoreceptor tyrosine-based switch motifs (ITSMs). A subset of immune checkpoint receptors expressed on T cells may be referred to as "T cell co-inhibitory receptors" and their cognate ligands as "T cell co-inhibitory ligands." A subset of immune checkpoint receptors expressed on innate immune cells may be referred to as "natural inhibitory receptors" and their cognate ligands as "natural inhibitory ligands."

[0119] The term "immunogenic chemotherapeutic agent" refers to a chemotherapeutic agent that induces cancer cells to undergo immunogenic cell death. Immunogenic cell death refers to any mechanism by which cell death can drive an antigen-specific immune response. Many anti-cancer treatments, including chemotherapy, irradiation, and targeted therapy, can induce immunogenic cell death. In some embodiments, the immunogenic chemotherapeutic agent exhibits genotoxicity. Non-limiting examples of chemotherapeutic agent classes that can cause immunogenic cell death include alkylating agents (e.g., cyclophosphamide, ifosfamide), topoisomerase inhibitors (e.g., doxorubicin), platinum derivatives (e.g., cisplatin, carboplatin, oxaliplatin, nedaplatin), taxanes (e.g., paclitaxel, docetaxel), or anthracyclines (e.g., doxorubicin). In some embodiments, the immunogenic chemotherapeutic agent or its derivatives can be conjugated to an antibody or other polypeptide for delivery as an antibody-drug conjugate.

[0120] The term "immune inhibitory enzyme" refers to an enzyme whose metabolic activity has an immunosuppressive effect. In some embodiments, the immunoinhibitory enzyme is an ectonucleotidase (e.g., CD39, CD73) or indoleamine 2,3-dioxygenase. Non-limiting examples of suitable immunoinhibitory enzymes include quiescent sulfhydryl oxidase 1 (QSOX1), carbonic anhydrase 12 (CA12), and carbonic anhydrase IX (CAIX).

[0121] As used herein, a "modulator" of a particular target refers, without limitation, to an agent that, in certain embodiments, binds to said target and can inhibit the activity of said target (i.e., can act as an antagonist), or, in another embodiment, the modulator can promote the activity of said target (i.e., can act as an agonist). A modulator can directly or indirectly inhibit or promote the activity of a given target (e.g., by binding to its cognate binding partner, its upstream signaling molecule, or its downstream signaling molecule).

[0122] The term "tumor stromal cells" refers to non-malignant cells in the tumor microenvironment. In one embodiment, tumor stromal cells are components of structural or connective tissue in tumors. In another embodiment, tumor stromal cells form or participate in the formation of blood vessels. Non-limiting examples of tumor stromal cells include fibroblasts, cancer-associated fibroblasts (CAFs), vascular endothelial cells, pericytes, adippocytes, mesenchymal stromal cells, and myofibroblasts.

[0123] The term "immune-related adverse events" (irAEs) refers to any undesirable side effects caused by immune activation. irAEs may include gastrointestinal toxicity, endocrine toxicity, cardiac toxicity, pulmonary toxicity, hepatic toxicity, rheumatologic toxicity, renal toxicity, neurological or dermatological / skin toxicity. irAEs may be caused by or associated with treatment with immune checkpoint inhibitors or other anti-cancer therapies caused by or associated with immune cell activation. irAEs are further defined and reviewed in Martins et al., "Adverse effects of immune-checkpoint inhibitors: epidemiology, management and surveillance." Nat Rev Clin Oncol 2019;16:563, incorporated herein in its entirety.

[0124] The symbol "+" in the sequence listing denotes a fusion of the indicated polypeptide sequences, e.g., "A+B" denotes the fusion of A to B in the order indicated (i.e., N-terminus to ABC-terminus).

[0125] Multispecific polypeptides that block IL-23 / IL-23R IL-23 is a component of the tumor microenvironment (TME) that is involved in the development, progression, and metastasis of malignant cells. IL-23 can manipulate host immune responses, modulate cells in the TME, and directly affect a variety of pre-malignant and malignant tumors.

[0126] Treatment of cancer with agents that promote immune cell activation can result in the activation of T cell subsets that inhibit antitumor immunity and / or contribute to tumorigenic inflammatory signaling. For example, Th17 cells are a subset of CD4 T cells that are characterized by the expression of the signature transcription factor RORg and the expression of inflammatory cytokines such as IL-17, IL-21, IL-22, IL-1, and TNFa. Without being bound by any theory, Th17 cells can directly and indirectly promote tumor progression by activating fibroblasts leading to fibrosis, producing cytokines that contribute to epithelial cell survival / proliferation, and promoting angiogenesis via endothelial cell activation and ECM remodeling. Th17 cells can recruit and activate myeloid cells that exert an independent immunosuppressive program that inhibits antitumor immunity, such as myeloid-derived suppressor cells (MDSCs). Thus, treatment of cancer with agents that promote immune cell activation may result in the opposite outcome of activation / expansion of inflammatory T cells such as Th17 cells, gamma delta T17 cells, and ILC3 cells that inhibit antitumor immunity and / or contribute to tumorigenic inflammatory signaling. Furthermore, IL17+ T cells such as Th17 cells and gamma delta T17 cells are associated with induction of immune-related adverse events (irAEs) and toxicity in response to immunotherapy. This may limit the therapeutic scope of immunotherapeutic agents, and more generally any immunogenic anticancer agent. IL-23 is a STAT3-activating cytokine that plays a pivotal role in the differentiation and maintenance of these inflammatory cell phenotypes (such as Th17 cells, gamma delta T17 cells, and ILC3 cells). Furthermore, IL-23 signaling and the resulting STAT3 signaling use multiple mechanisms to inhibit IL-12 signaling and the resulting STAT4 signaling, thereby limiting the differentiation and maintenance of the antitumor Th1 T cell phenotype. Therefore, IL-23 blockade may enhance the efficacy and safety of therapeutic strategies aimed at enhancing immune cell activation, proliferation, and / or function.Non-limiting examples of such therapeutic strategies include antagonism of immune checkpoint proteins (including T cell co-inhibitory receptors and innate inhibitory receptors), agonism of immune stimulatory receptors (including T cell co-stimulatory receptors and innate stimulatory receptors), antagonism of specific cytokines / cytokine receptors, agonism of specific cytokine receptors, immunogenic chemotherapy, antagonism of immune inhibitory enzymes, and administration of cell therapy including CAR-T, CAR-NK, or hematopoietic stem cells. Such therapeutic strategies for cancer treatment are sometimes described as "breaking tolerance" or attempting to do so. As such, in some embodiments, the molecules and methods of the present invention provide a strategy to break tolerance while suppressing IL-23 (a major determinant of immune cell phenotype biased toward a tumor-promoting state). This can ensure that while breaking tolerance, the immune cell phenotype in the tumor microenvironment is not biased toward such an adverse outcome, thus improving both the safety and efficacy of therapies aimed at breaking tolerance.

[0127] Provided herein is a multispecific polypeptide capable of specifically blocking IL-23 / IL-23 receptor (IL-23 / IL-23R) signaling. The multispecific polypeptide comprises at least two moieties: (a) an IL-23 inhibitory polypeptide (IIP), and (b) a secondary polypeptide (2P), such as a target-binding polypeptide. In a preferred embodiment, the multispecific polypeptide disclosed herein effectively inhibits tumor growth and / or reduces tumor volume compared to treatment with an immune checkpoint inhibitor alone. In another preferred embodiment, the multispecific polypeptide disclosed herein prevents or reduces immune disorders, such as graft-versus-host disease.

[0128] In some embodiments, the IL-23 inhibitory polypeptide inhibits IL-23 / IL-23R signaling by blocking or disrupting the interaction of the IL-23 ligand with the IL-23 receptor. In some embodiments, the IL-23 inhibitory polypeptide can specifically bind to the IL-23 ligand and deplete the binding of IL-23 to the IL-23 receptor present on the surface of a cell (e.g., T cells, natural killer cells, natural killer T cells, dentritic cells, macrophages, tumor cells). In some embodiments, the IL-23 inhibitory polypeptide can specifically bind to the IL-23 receptor present on the cell surface. In some embodiments, the IL-23 inhibitory polypeptide is an anti-IL-23 antibody or an antigen-binding fragment thereof. In some embodiments, the IL-23 inhibitory polypeptide is an anti-IL-23R antibody or an antigen-binding fragment thereof. In some embodiments, the IL-23 inhibitory polypeptide comprises the extracellular domain of the IL-23 receptor (IL-23 ECD) and is capable of binding to an IL-23 ligand, thereby preventing IL-23 from binding to endogenous IL-23R and inhibiting IL-23 signaling.

[0129] In some embodiments, the second polypeptide (2P) is a target-binding polypeptide that binds to one or more immune checkpoint proteins expressed or present on the cell surface of an immune cell (e.g., antigen-presenting cell, CD4+ T cell, Th17 cell) or tumor cell, thereby blocking or disrupting immune checkpoint protein interactions. In some embodiments, the target-binding polypeptide is programmed death-1 (PD1; CD279), programmed death ligand 1 (PDL1; CD274; B7-H1), programmed death ligand 2 (PDL2), cytotoxic T-lymphocyte antigen-4 (CTLA4; CD152), attenuator of B and T lymphocytes (BTLA), V domain immunoglobulin suppressor of T cell activation (VISTA), T cell immunoglobulin and ITIM domain (TIGIT), lymphocyte activation gene 3 (LAG-3; CD223), The target-binding polypeptide binds to an immune checkpoint protein, a receptor or ligand-binding fragment thereof, or a ligand or receptor-binding fragment thereof selected from T-cell immunoglobulin and mucin domain 3 (Tim-3; HAVCR2), carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), CD47, signal regulatory protein alpha (SIRPa), major histocompatibility complex class I, G (HLA-G), Ig-like transcript 2 (ILT2; LILRB1), Ig-like transcript 4 (ILT4, LILRB2), or a combination thereof. In some embodiments, the target-binding polypeptide binds to PD1 (CD279). In some embodiments, the target-binding polypeptide is an anti-PD1 antibody or an antigen-binding fragment thereof. In some embodiments, the target-binding polypeptide binds to PDL1 (CD274; B7-H1). In some embodiments, the target-binding polypeptide is an anti-PDL1 antibody or an antigen-binding fragment thereof. In some embodiments, the target-binding polypeptide binds to PDL2. In some embodiments, the target-binding polypeptide is an anti-PDL2 antibody or an antigen-binding fragment thereof. In some embodiments, the target binding polypeptide comprises the extracellular domain of PD1 (PD1 ECD), which can bind to a ligand or receptor binding fragment of, for example, PDL1, PDL2.In some embodiments, the PD1 ECD has one or more mutations to increase its binding affinity to PDL1 and / or PDL2 compared to wild-type human PD1 ECD (SEQ ID NO: 56). The PD1 ECD may include a mutation at residue A132 (residue numbering defined by the complete human PD1 sequence as in UniProt Q15116). The PD1 ECD may include a conservative substitution at residue A132. In a preferred embodiment, the PD1 ECD includes the mutation A132I (SEQ ID NO: 57). In some embodiments, the PD1 ECD variant binds to PDL1 with an affinity of greater than 100 nM, 10 nM, 1 nM, or 0.1 nM. In some embodiments, the PD1 ECD variant binds to PDL2 with an affinity of greater than 100 nM, 10 nM, 1 nM, or 0.1 nM.

[0130] Tumor cells and myeloid-derived suppressor cells (MDSCs) can express PDL1 and / or PDL2. Tumor cells and MDSCs can also express IL-23. As such, a multispecific polypeptide that binds PDL1 and / or PDL2; and IL-23 can localize the blockade of IL-23 to the cell surface of PDL1+ and / or PDL2+ cells that also express IL-23 (e.g., cells in which IL-23, and PDL1 or PDL2, or both, are present or expressed).

[0131] IL-23 Inhibitory Polypeptides Thus, in some embodiments, an IL-23 inhibitory polypeptide (IIP) inhibits IL-23 / IL-23R signaling in one of the following ways: (a) inhibiting the interaction of IL-23 with IL-23R by binding to IL-23 (an IIP is an "IL-23 binder"), or (b) inhibiting the interaction of IL-23 with IL-23R by binding to IL-23R (an IIP is an "IL-23R binder").

[0132] IL-23 binders In some embodiments, the IL-23 inhibitory polypeptide binds to IL-23. In some embodiments, the IL-23 inhibitory polypeptide is an anti-IL-23 antibody. In some embodiments, the anti-IL-23 antibody is a humanized monoclonal antibody or antigen-binding fragment thereof that can specifically bind to an IL-23 subunit (e.g., IL-23p19, IL-23p40). In some embodiments, the IL-23 antibody or antigen-binding fragment thereof can specifically bind to the IL-23p19 subunit. As non-limiting examples, an IL-23 antibody or antigen-binding fragment thereof comprises one or more of six complementarity determining regions (CDRs) selected from any one of risankizumab (VH: SEQ ID NO:79; VL: SEQ ID NO:80), guselkumab (VH: SEQ ID NO:81; VL: SEQ ID NO:82), tildrakizumab (VH: SEQ ID NO:83; VL: SEQ ID NO:84), brazikumab (VH: SEQ ID NO:85; VL: SEQ ID NO:86), and mirikizumab (VH: SEQ ID NO:87; VL: SEQ ID NO:88).

[0133] IL-23R binder In some embodiments, the IL-23 inhibitory polypeptide binds to IL-23R. In some embodiments, the IL-23 inhibitory polypeptide is an anti-IL-23R antibody. In some embodiments, the anti-IL-23R antibody is a humanized monoclonal antibody or antigen-binding fragment thereof that can specifically bind to an IL-23 ligand or its receptor-binding fragment. As a non-limiting example, the IL-23R antibody or antigen-binding fragment thereof comprises one or more of the six CDRs of AS2762900-00. In some embodiments, the IL-23R antibody or antigen-binding fragment thereof is selected from the antibodies disclosed in U.S. Pat. No. 9,371,391 (incorporated herein in its entirety).

[0134] IL-23R ECD In some embodiments, the IL-23 inhibitory polypeptide comprises an IL-23 receptor, its ligand binding domain or fragment, or its extracellular domain (IL-23R-ECD) that can specifically bind to an endogenous IL-23 ligand. For example, the IL-23R-ECD can be the D1 subunit of IL-23R, the D1 and D2 subunits of IL-23R, or the D1, D2, and D3 subunits of IL-23R. The sequence of human IL-23R and its extracellular domain has been reported. As used herein, the ECD sequence has the amino acid sequence of SEQ ID NO: 1 (UniProt accession Q5VWK5).

[0135] The IL-23 ligand is a heterodimeric cytokine comprising a p19 subunit and a p40 subunit. The IL-12 ligand is a heterodimeric cytokine comprising a p35 subunit and a p40 subunit. In one aspect, the IL-23R-ECD of the fusion protein of the present invention exhibits a higher affinity for IL-23 than for IL-12. In one embodiment, the IL-23R-ECD preferentially binds to the p19 subunit compared to the p35 subunit. In some embodiments, the IL-23R-ECD comprises residues that interact with both the p19 and p40 subunits of IL-23. In other embodiments, the IL-23R-ECD comprises residues that interact only with the p19 subunit.

[0136] In some embodiments, the IL-23R-ECD domain comprises the GITNIN hexapeptide upstream of D1. In other embodiments, the IL-23R-ECD domain begins with the first amino acid sequence of D1 (CSGHI).

[0137] In some embodiments, the IL-23R-ECD used herein may be modified in one or more of the following ways with reference to the native human IL-23-R extracellular domain (e.g., wild-type IL-23R-ECD) sequence (SEQ ID NO:1). The IL-23R-ECD may have one or more substitutions or deletions of residues not required for ligand binding, one or more substitutions of residues to remove N-linked glycosylation sites, one or more substitutions, additions, or deletions of residues to increase affinity for IL-23, one or more substitutions, additions, or deletions of residues to improve expression of the fusion protein, one or more substitutions, additions, or deletions of residues to allow site-specific conjugation of drug conjugates, one or more substitutions, additions, or deletions of residues to decrease specificity of ligand capture for IL-12, while maintaining or increasing its specificity for IL-23, fusion of one or more discontinuous domains of IL-23R-ECD, or fusion of domains from different isoforms of IL-23R-ECD.

[0138] In some embodiments, the IL-23R-ECD has an amino acid sequence having at least 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or more sequence identity to the ligand binding sequence of wild-type human IL-23R-ECD (SEQ ID NO:1).

[0139] Secondary Polypeptide (2P) In some embodiments, the secondary polypeptide is a target-binding polypeptide that serves one or more of the following functions: (a) localization of the multispecific polypeptide to a specific tissue, cell type, or tumor cell; (b) antagonism of inhibitory immune checkpoint signaling; (c) agonism of immunostimulatory signaling; (d) antagonism of another cytokine or cytokine receptor, (e) agonism of a cytokine receptor, and / or (f) antagonism of a chemokine or chemokine receptor, etc.

[0140] In various embodiments, the second polypeptide (2P) is a target-binding polypeptide that is an immunoglobulin, an antibody, a bispecific or multispecific antibody, a nanobody, an antibody fragment, a single-chain variable fragment (scFv), a bivalent or multivalent scFv, an Affimer, a ligand-binding sequence derived from the extracellular domain (ECD) of a receptor, a receptor-binding sequence derived from the ECD of a ligand, or an antigen-binding domain of an Fc-containing polypeptide.

[0141] In some embodiments, 2P is a target binding polypeptide that is an antibody or an antigen-binding fragment thereof, such as a fragment crystallizable (Fc) region, a fragment antigen-binding (Fab) region, a single chain variable fragment (scFv), a light chain or a functional portion thereof, a variable region of a light chain (VL), a constant region of a light chain (CL), a heavy chain or a functional portion thereof, a variable region of a heavy chain (VH), a constant region of a heavy chain (CH), at least one complementarity determining region (CDR) or antigen-binding portion thereof, or a combination thereof.

[0142] In some embodiments, 2P is a target binding polypeptide having a ligand binding sequence of an extracellular domain of a receptor. In some embodiments, 2P is a target binding polypeptide having a receptor binding sequence of an extracellular domain of a ligand. In some embodiments, the ECD has one or more of the following modifications with reference to the wild-type ECD. In various embodiments, the ECD has one or more substitutions, additions, or deletions of residues to improve expression of the fusion protein, one or more substitutions or deletions of residues that are not required for ligand binding, one or more substitutions of residues to remove N-linked glycosylation sites, or one or more substitutions, additions, or deletions of residues to increase the affinity of the ECD for a natural binding partner.

[0143] Examples of 2P moieties for treating cancer or immune disorders In various embodiments, the second polypeptide (2P) is a target-binding polypeptide that can specifically bind to one or more cytokines or cytokine receptors, or one or more cell surface molecules. In some embodiments, the 2P is a target-binding polypeptide that can exchange the fusion protein through the blood-brain barrier. In some embodiments, the 2P is a target-binding polypeptide that includes an Fc domain, a CDR, or an antigen-binding fragment of an immunoglobulin.

[0144] In some embodiments, 2P is T H2P binds to a cytokine or cytokine receptor that promotes differentiation, maturation, or function of IL-17 cells. In some embodiments, 2P binds to IL-17 or IL-17R. In some embodiments, 2P is an antibody or antigen-binding fragment thereof that binds to and disables IL-17 or IL-17R. In some embodiments, 2P antibody or antigen-binding fragment thereof comprises one or more of six CDRs or antigen-binding portions thereof selected from any one of afasevicumab, bimekizumab, ixekizumab, netakimab, perakizumab, secukinumab, bunakizumab, or brodalumab. In some embodiments, 2P is a ligand-binding sequence of the extracellular domain of IL-17R or a fragment thereof. In some embodiments, 2P binds to IL-1a, IL-1b, or IL-1R. In some embodiments, 2P is an antibody or antigen-binding fragment thereof that binds to and disables IL-1a, IL-1b, and / or IL-1R. In some embodiments, 2P is a ligand binding sequence of the extracellular domain of IL-1R, IL-1 receptor antagonist (IL-1RA) (SEQ ID NO: 77), or a fragment thereof. In some embodiments, 2P comprises the amino acid sequence of anakinra. In some embodiments, 2P binds to IL-6 or IL-6R. In some embodiments, 2P is an antibody or antigen-binding fragment thereof that binds to and disables IL-6 or IL-6R. In some embodiments, 2P antibody or antigen-binding fragment thereof comprises one or more of six CDRs or antigen-binding portions thereof selected from any one of clazakizumab, olokizumab, siltuximab, sirukumab, diltibekimab, revilimab, sapelizumab, sarilumab, satralizumab, or tocilizumab. In some embodiments, 2P prevents the interaction of RANK with RANKL. In some embodiments, 2P is a RANKL binding sequence of the extracellular domain of RANK (SEQ ID NO: 76).

[0145] In some embodiments, 2P binds to a TNFR superfamily receptor or a ligand that binds to a TNFR superfamily receptor. In other embodiments, 2P binds to a type I cytokine receptor or a cytokine that binds to a type I cytokine receptor. In other embodiments, 2P binds to a type II cytokine receptor or a cytokine that binds to a type II cytokine receptor. In other embodiments, 2P binds to an Ig superfamily receptor or a cytokine that binds to an Ig superfamily receptor. In other embodiments, 2P binds to a chemokine receptor or a chemokine that binds to a chemokine receptor.

[0146] In some embodiments, 2P binds to a cell surface molecule of cells responsible for the production of IL-23, thereby sequestering IL-23 when it is expressed. In other embodiments, 2P binds to a cell surface molecule of cells that express IL-23R and that normally respond to IL-23, thereby sequestering IL-23 on cells that would otherwise initiate IL-23R signaling.

[0147] In some embodiments, 2P binds to a T cell surface molecule. H 17 cells. As such, in some embodiments, 2P can be designed to suppress inflammation mediated by T H 17 cells. In other embodiments, 2P binds to a T cell surface molecule expressed by CD4 T cells.

[0148] In some embodiments, 2P binds to the transferrin receptor (TfR). Without being bound by any theory, binding of 2P to TfR allows exchange of the fusion protein through the blood-brain barrier. In some embodiments, 2P is an antibody or antigen-binding fragment thereof that binds to TfR. In other embodiments, 2P is an antibody or antigen-binding fragment thereof with an engineered Fc region mutated to bind to TfR. In other embodiments, 2P comprises a synthetic peptide sequence engineered to bind to TfR. In some embodiments, the engineered Fc region comprises SEQ ID NO: 116. In some embodiments, the engineered Fc region comprises one or more mutations disclosed in Kariolis et al, ''Brain delivery of therapeutic proteins using an Fc fragment blood-brain barrier transport vehicle in mice and monkeys'' Sci Trans Med 2020,12:545, which is incorporated by reference in its entirety.

[0149] In some embodiments, 2P comprises an immunoglobulin Fc domain. In some embodiments, the Fc domain is a wild-type IgG. In some embodiments, the Fc domain carries one or more mutations designed to enhance or abolish its binding to various Fc receptors. In some embodiments, the Fc domain is an IgG1 Fc that contains a L234A and / or a L235A ("LALA") mutation. In some embodiments, the Fc domain is an IgG4 Fc that contains a S228P mutation.

[0150] Examples of 2P moieties for treating cancer In various embodiments, 2P has a target-binding polypeptide capable of binding to one or more target molecules for cancer treatment. In some embodiments, 2P binds to a tumor cell surface molecule. Without being bound by any theory, 2P may be "decorated" with an IL-23 binder to sequester any IL-23 in the tumor cell microenvironment and serve to localize the fusion protein to the tumor cell surface. Binding of 2P to its target may further serve to neutralize receptor / ligand interactions that exacerbate immune tolerance or tumor-promoting inflammation, or to neutralize growth factors, growth factor receptors, or other molecules that promote tumor cell survival, growth, or metastasis. In various embodiments, the tumor cell surface molecule is a T cell co-inhibitory ligand, tumor growth factor receptor, cytokine receptor, chemokine receptor, or tumor antigen. In some embodiments, 2P is an antibody or antigen-binding fragment thereof that binds to a specific tumor cell surface molecule. As non-limiting examples, 2P may be a tumor cell surface molecule selected from the following list (CA125, CA19-9, CD30, carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) or CD66e (e.g., labetuzumab, sergituzumab), CEACAM1, CEACAM6, DLL3, DLL4, DPEP3, EGFR (e.g., cetuximab, necitumumab, panitumumab), EGFRvIII (e.g., depatuxizumab), GD2, HER2 (e.g., trastuzumab, pertuzumab), HER3, HGF, IGF 1R, IL13Ra2, LIV-1, LRRC15, MUC1, PRLR, PSCA, PSMA, PTK7, SEZ6, SLAMF7, TF, cMet, claudins, mesothelin, nectin4 (e.g., enfortumab), uPAR, GPNMB, CD79b, CD22, NaPi2b, SLTRK6, STEAP1, MUC16, CD37, GCC, AGC-16, 5T4, CD70, TROP2 (e.g., sacituzumab), CD74, CD27L, Fra, CD138, and CA6). In various embodiments, 2P binds to a cell surface molecule of a tumor stromal cell.

[0151] In some embodiments, 2P binds to CEA (CEACAM5). In some embodiments, 2P is labetuzumab. In another embodiment, 2P is sergituzumab or CH1A1A-2F1. In one embodiment, 2P binds preferentially to membrane-bound CEA over soluble CEA. In some embodiments, this binding is by binding to a CEA epitope near the GPI-anchored site at the C-terminus of the CEA extracellular domain. In some embodiments, this binding is by binding to a CEA epitope that overlaps with the B3 domain of CEA.

[0152] In some embodiments, 2P binds to an antigen overexpressed by a hematological malignancy. In some embodiments, 2P binds to an antigen overexpressed by multiple myeloma. In some embodiments, 2P binds to CD38, SLAMF7, or BCMA. In some embodiments, 2P is an antibody selected from the following list: MEDI2228; CC-99712; belantamab; gemtuzumab (anti-CD33 mAb). In some embodiments, the antibody binds to CD20. In some embodiments, 2P binds to rituximab (chimeric mouse / human anti-CD20 mAb); obinutuzumab (anti-CD20 mAb); ofatumumab (anti-CD20 mAb). In some embodiments, 2P binds to CD19. In some embodiments, the antibody binds to CD30, or CD22. In some embodiments, 2P binds to an antigen overexpressed by leukemia. In some embodiments, 2P binds to CD33.

[0153] In some embodiments, 2P is an antagonist of immune checkpoint proteins. In some embodiments, 2P is an antagonist of innate immune checkpoint proteins. In some embodiments, 2P binds to T cell co-inhibitory molecules as an antagonist. In some embodiments, 2P has a ligand-binding sequence of the extracellular domain of a T cell co-inhibitory receptor. Such 2P has the effect of sequestering T cell co-inhibitory ligands and reducing ligand-induced signaling of native T cell co-inhibitory receptors expressed on the surface of T cells.

[0154] In some embodiments, 2P has the ligand-binding sequence of PD1 ECD. The PD1 ECD may contain one or more mutations to increase its binding affinity to PDL1 and / or PDL2 compared to wild-type human PD1 ECD. In some embodiments, the PD1 ECD has a mutation at residue A132 (residue numbering defined by the full-length human PD1 sequence as in UniProt Q15116). The A132 residue may be mutated (e.g., substituted, deleted, inserted, or inverted) at I (A132I), V (A132V), or L (A132L). In a preferred embodiment, the PD1 ECD has a mutation at A132I. In some embodiments, the PD1 ECD is mutated with multiple amino acid mutations (S87G, P89L, N116S, G124S, S127V, A140V, A125I, A125V, L122V, K78T, N74G, M70E, Y68H, N66V, N66I, L65I, L65V, V64H, or Miao et al., ``Neutralization of PD-L2 is Essential for Overcoming Immune Checkpoint Blockade Resistance in Ovarian Cancer.'' Clin Cancer Res. 2021.27(15):4435-4448, Maute et al., ``Engineering high-affinity PD-1 variants for optimized immunotherapy and immuno-PET imaging.'' Proceedings of the National Academy of Sciences. 2015.112(47), E6506-E6514, each of which is incorporated herein by reference in its entirety.

[0155] In other embodiments, 2P comprises the ligand-binding sequence of the TIM3 ECD (SEQ ID NO: 63). In other embodiments, 2P comprises the CTLA4-binding sequence of the CD80 ECD (SEQ ID NO: 66) or CD86 ECD (SEQ ID NO: 67).

[0156] In some embodiments, 2P binds to an immunostimulatory receptor as an agonist. In some embodiments, 2P binds to a T cell costimulatory molecule as an agonist. In some embodiments, the immunostimulatory receptor may be selected from 4-1BB (CD137), inducible T cell costimulatory factor (ICOS; CD278), OX-40 (CD134), glucocorticoid-induced TNFR-related protein (GITR; CD357), CD40, herpes virus entry mediator (HVEM), CD28, or CD27. In some embodiments, 2P comprises a receptor binding sequence of a T cell costimulatory ligand, or a receptor binding fragment thereof. In some embodiments, 2P comprises a CD40 binding sequence of CD40L (SEQ ID NO: 74). In some embodiments, 2P comprises multiple CD40L moieties that assemble into a trimer or hexameric configuration. In some embodiments, 2P comprises the receptor binding sequence of ICOS-L (SEQ ID NO: 73), 4-1BBL (SEQ ID NO: 70), OX40L (SEQ ID NO: 71), or GITRL (SEQ ID NO: 72). In some embodiments, 2P comprises the HVEM binding sequence of BTLA ECD (SEQ ID NO: 62) or LIGHT ECD (SEQ ID NO: 65). In some embodiments, 2P comprises the CD28 binding sequence of CD80 ECD or CD86 ECD.

[0157] In some embodiments, 2P binds to an innate immune stimulatory receptor as an agonist. In some embodiments, 2P binds to an innate immune stimulatory receptor expressed on NK cells as an agonist. In some embodiments, 2P binds to NKG2D as an agonist. In some embodiments, 2P comprises an NKG2D binding sequence of an NKG2D ligand (NKG2DL).

[0158] In some embodiments, 2P binds to a growth factor or growth factor receptor. In some embodiments, 2P inhibits TGFb signaling. In some embodiments, 2P binds to TGFb and prevents TGFb from binding to TGFbRII. In some embodiments, 2P comprises the ligand binding sequence of TGFbRII ECD (SEQ ID NO:58). In some embodiments, 2P is an anti-TGFb antibody or antigen-binding fragment thereof (e.g., fresolimumab, SRK-181, SAR439459, NIS793). In other embodiments, 2P binds to TGFbRII. In some embodiments, 2P is an anti-TGFbRII antibody or antigen-binding fragment thereof. In some embodiments, the multispecific polypeptide is an anti-IL-23-TGFbRII comprising the amino acid sequences of SEQ ID NO:90 and SEQ ID NO:54.

[0159] In some embodiments, 2P inhibits the interaction of VEGF and VEGFR. In some embodiments, 2P binds to VEGF. In some embodiments, 2P is a chimeric ECD comprising the ligand binding sequence of the extracellular domain of VEGFR1 (SEQ ID NO: 59) or VEGFR2 (SEQ ID NO: 60); or domains from VEGFR1 and VEGFR2. In some embodiments, the chimeric ECD comprises VEGFR1 domain 2 and VEGFR2 domain 3 (SEQ ID NO: 61). In some embodiments, 2P is aflibercept. In some embodiments, 2P is an antibody or antigen-binding fragment thereof that binds to VEGF (e.g., bevacizumab). In some embodiments, 2P binds to VEGFR. In some embodiments, 2P is an antibody or antigen-binding fragment thereof that binds to VEGF (e.g., ramucirumab). In some embodiments, the multispecific polypeptide is an anti-IL-23-VEGFR comprising the amino acid sequences of SEQ ID NOs: 91 and 54.

[0160] In some embodiments, 2P binds to and neutralizes a molecule expressed on the cell surface of a dendritic cell or macrophage. In some embodiments, 2P binds to and neutralizes a molecule expressed on the cell surface of a dendritic cell or macrophage that is SIRPa. In other embodiments, 2P binds to and neutralizes a ligand that binds to an inhibitory receptor on a dendritic cell or macrophage that is CD47. In some embodiments, 2P is a CD47 binding sequence of SIRPa ECD (SEQ ID NO: 68). In some embodiments, the multispecific polypeptide is an anti-IL-23-SIRPa ECD comprising the amino acid sequences of SEQ ID NOs: 68 and 54. In other embodiments, 2P binds to and neutralizes a ligand that inhibits the maturation or function of a dendritic cell or macrophage. In one embodiment, 2P is a ligand binding sequence of the extracellular domain of SIGLEC10.

[0161] In some embodiments, 2P is a cytokine / cytokine receptor antagonist. In some embodiments, 2P is an immunoinhibitory cytokine / cytokine receptor signaling antagonist. In some embodiments, 2P inhibits IL-8 signaling. In some embodiments, 2P binds to IL-8.

[0162] In some embodiments, 2P binds to a ligand or cytokine that inhibits the activation, maturation, or function of NK cells. In some embodiments, 2P binds to a ligand or cytokine that inhibits the activation, maturation, or function of T cells. In some embodiments, 2P is the ligand-binding domain of the extracellular domain of a receptor that binds to such a ligand or cytokine. In one embodiment, 2P is the ligand-binding domain of the extracellular domain of IL-10R.

[0163] In some embodiments, 2P binds to a cytokine receptor that promotes NK cell activation, maturation, or function. In some embodiments, 2P is a cytokine or a receptor-binding fragment thereof that promotes NK cell activation, maturation, or function. In some embodiments, 2P binds to a cytokine receptor that promotes T cell activation, maturation, or function. In some embodiments, 2P is a cytokine or a receptor-binding fragment thereof that promotes T cell activation, maturation, or function. In some embodiments, 2P is IL-15, IL-12, IL-18, or a receptor-binding fragment thereof. In some embodiments, 2P comprises a fusion of a receptor-binding fragment of IL-15 and a ligand-binding fragment of the IL-15R sushi domain.

[0164] In some embodiments, 2P binds to a cytokine receptor that acts as an agonist to promote T cell activation, maturation, or function, hi some embodiments, 2P comprises IL-2.

[0165] In some embodiments, 2P binds to a NK cell surface molecule. In some embodiments, 2P binds to a NK cell surface molecule. dim CD16 + 2P binds to NK cell surface molecules that are preferentially expressed by NK cells. In some embodiments, 2P binds to NK cell surface activation receptor as an agonist. In some embodiments, 2P is the NKG2D-binding fragment of the NKG2DL extracellular domain. NKG2DL can be selected from MICA, MICB, or ULBP1-6.

[0166] In some embodiments, 2P binds to FGF-2 or FGFR. In other embodiments, 2P binds to PDGF or PDGFR. In other embodiments, 2P binds to angiopoietin (1, 2, 3, or 4) or angiopoietin receptor (TIE-1 or TIE-2).

[0167] In some embodiments, 2P inhibits the activation, differentiation, maturation, or function of TH2 cells. In some embodiments, 2P binds to IL-4, IL-13, IL4RA, or IL13R. In some embodiments, 2P is an antibody or antigen-binding fragment thereof that binds to IL4RA (e.g., dupilumab).

[0168] Examples of 2P moieties for autoimmune conditions Multispecific polypeptides of the invention intended for the treatment of immune disorders such as autoimmune conditions do not attempt to "break tolerance". Instead, effective treatment of autoimmune disorders may involve inducing tolerance or suppressing one or more inflammatory mechanisms in addition to blocking IL-23. As such, in some embodiments, multispecific polypeptides of the invention comprise 2P as described below.

[0169] In some embodiments, 2P localizes the fusion protein to a specific tissue. In general, to reduce NK / macrophage-mediated aggravation of autoimmune conditions, 2P comprises the Fc domain of human immunoglobulin. In some embodiments, the Fc domain has one or more mutations to reduce or eliminate its binding to activating FcR. In some embodiments, the Fc domain has one or more mutations to increase its binding to inhibitory FcR.

[0170] In some embodiments, 2P binds to and neutralizes proinflammatory cytokines. For the treatment of certain autoimmune disorders, it may be further advantageous for the fusion protein to neutralize additional proinflammatory cytokines in addition to IL-23. The proinflammatory cytokines may be selected from IFNg, TNFa, IL-1a, IL-1b, IL-6, IL-17, IL-12, IL-18, RANKL, and GM-CSF. Exemplary such 2P include TNFR2-ECD (SEQ ID NO: 75) (e.g., etanercept), anti-IL17 mAb (e.g., secukinumab), RANK-ECD (SEQ ID NO: 76), or anti-GMCSF mAb (e.g., lenzilumab).

[0171] In some embodiments, 2P binds to and neutralizes a proinflammatory cytokine receptor, which may be selected from: IFNgR, TNFR, IL-1R, IL-6R, IL-17R, IL-12R, IL-18R, RANK, and GM-CSFR.

[0172] In some embodiments, 2P binds to a T cell costimulatory ligand and negates its effect. In some embodiments, 2P binds to one of the following costimulatory ligands: CD40L, 41BBL, OX40L, ICOSL, or GITRL. In some embodiments, 2P comprises a ligand binding sequence of the extracellular domain of one of the following costimulatory receptors: CD40-ECD, 41BB-ECD, OX40-ECD, ICOS-ECD, GITR-ECD. In other embodiments, 2P binds to a T cell costimulatory receptor as an antagonist. In some embodiments, 2P binds to CD40, 41BB, OX40, ICOS, or GITR as an antagonist.

[0173] In some embodiments, 2P binds to a T cell co-inhibitory receptor as an agonist. In some embodiments, 2P binds to one of the following co-inhibitory receptors: PD1, BTLA, VISTA, TIGIT, LAG-3. In some embodiments, 2P comprises a receptor binding sequence of the extracellular domain of one of the following co-inhibitory ligands: PDL1, PDL2, HVEM.

[0174] In some embodiments, 2P comprises the sequence of the extracellular domain of CTLA-4 (SEQ ID NO: 115) (eg, CTLA4-Fc; abatacept).

[0175] In some embodiments, 2P binds to an inhibitory receptor on macrophages and / or dendritic cells as an agonist. In some embodiments, 2P binds to SIRPa as an agonist. In some embodiments, 2P comprises the receptor binding sequence of the extracellular domain of CD47.

[0176] In some embodiments, 2P binds to the receptor of an anti-inflammatory cytokine as an agonist. In some embodiments, 2P can be the anti-inflammatory cytokine itself, or a receptor-binding fragment thereof. In other embodiments, 2P can be an agonist antibody that binds to a cytokine receptor to inhibit inflammation. In some embodiments, the anti-inflammatory cytokine receptor is selected from IL-4R, IL-10R, and TGFbR. In some embodiments, the anti-inflammatory cytokine is selected from IL-4, IL-10, and TGF-b, or a receptor-binding fragment thereof.

[0177] Design of multispecific polypeptides In some embodiments, the multispecific polypeptides of the invention are constructed as fusion proteins. In some embodiments, the components of the fusion proteins of the invention are fused via a flexible linker. In some embodiments, the flexible linker comprises a polypeptide sequence (GGGGS)n, where n is between 1 and 10. In some embodiments, a linker is used to link 2P to the C-terminus of the IIP. In some embodiments, the linker is selected from a non-cleavable linker, a peptide linker, a flexible linker, a rigid linker, a helical linker, and / or a non-helical linker. Non-limiting examples of possible linkers are disclosed in the art, for example, Chen et al. "Fusion Protein Linkers: Property, Design, and Functionality" Adv Drug Deliv Rev. 2014, 65(10):1357, which is incorporated herein by reference in its entirety. In some embodiments, the components of the fusion proteins of the invention are fused without a linker between them.

[0178] Exemplary designs of multispecific polypeptides for use herein are shown, for example, in Figures 1A-1D, 2A-2G.

[0179] In the following examples, N represents the N-terminus of the protein and C represents the C-terminus of the protein. In some embodiments, the IIP is an antibody or an antigen-binding fragment thereof. In such cases, the fusion protein (e.g., recombinant molecule) may have one of the following structures, where HC refers to the heavy chain of the antibody and LC refers to the light chain of the antibody: N-HC-linker-2P-C; N-LC-C (e.g., Figure 1A) N-2P-Linker-HC-C; N-LC-C N-HC-C; N-LC-Linker-2P-C (e.g., Figure 1B) N-HC-C; N-2P-Linker-LC-C

[0180] In some embodiments, the molecule is a bispecific antibody, where one Fab of the bispecific antibody is an IIP and the other Fab of the bispecific antibody is 2P (e.g., FIG. 1C). In other embodiments, the molecule is a fusion of two scFv antibody fragments, where one scFv is an IIP and the other scFv is 2P (e.g., FIG. 1D).

[0181] In other embodiments, 2P is an antibody. In such cases, the fusion protein may have one of the following structures, where HC refers to the heavy chain of the antibody and LC refers to the light chain of the antibody: N-HC-Linker-IIP-Fc; N-LC-C (e.g., Figures 2A-C) N-IIP-Linker-HC-Fc; N-LC-C N-HC-C; N-LC-Linker-IIP-C N-HC-C; N-IIP-Linker-LC-C

[0182] In some embodiments, the IIP portion and the 2P portion are fused in one of the following ways: N-IIP-Fc-2P-C (e.g., Figure 2D) N-IIP-2P-C (e.g., Figure 2F) N-IIP-Fc-Linker-2P-C N-IIP-Linker-2P-C N-2P-Fc-IIP-C (e.g., Figure 2E) N-2P-IIP-C (e.g., Figure 2G) N-2P-Fc-Linker-IIP-C N-2P-Linker-IIP-C

[0183] In some embodiments, the multispecific polypeptides of the invention are bispecific antibodies (bsAbs). In some embodiments, the bSab is an absolute or non-absolute bsAb.

[0184] In some embodiments, the bsAb is bivalent in a 1+1 format (i.e., one binding site for each target). In further embodiments, the bispecific antibody can be a tandem VHH nanobody fusion, tandem scFvs (e.g., BiTE), DART, diabody, F(ab)2, or scFv-Fab fusion. In another embodiment, the bispecific antibody can comprise two or more of the following asymmetric chains: for example, a heteroheavy chain with forced knob-and-hole HL pairing, a heteroheavy chain with CrossMab VH / VL swap domains, a heteroheavy chain with CrossMAB CH1 / CL swap domains, a DART-Fc, a LP-DART, or a half-life extended BiTE.

[0185] In other embodiments, the bsAb is trivalent in a 1+2 format (i.e., one binding site for one target and two binding sites for the other target). In a further embodiment, the bsAb is a CrossMab, having three F(ab) regions.

[0186] In other embodiments, the bsAb is tetravalent in a 2+2 format (i.e., two binding sites for each target). In further embodiments, the bsAb is a fusion of regular IgG with two scFv domains, a Bs4Ab, a DVD-Ig, a tetravalent DART-Fc, four scFv domains fused to Fc, a CODV-Ig, a pair of tandem VHH nanobodies fused to Fc, or a CrossMab with four F(ab) regions.

[0187] In some embodiments, the bsAb comprises the VH and VL of any one of risankizumab, guselkumab, tildrakizumab, brazikumab, mirikizumab, In some embodiments, the bsAb further comprises the VH and VL of another antibody or antigen-binding fragment thereof. In some embodiments, the bsAb comprises one or more of the six complementarity determining regions (CDRs) of any one of risankizumab, guselkumab, tildrakizumab, brazikumab, mirikizumab, In some embodiments, the bsAb further comprises an additional CDR of another antibody or antigen-binding fragment thereof.

[0188] Exemplary Multispecific Polypeptides As shown diagrammatically in Figures 1A and 3A, anti-IL-23-PD1ecd is an exemplary molecule having an anti-IL-23 antibody (IIP) fused or linked to the ligand binding sequence of PD1 ECD (2P) at the C-terminus of its heavy chain via a flexible linker (GGGGS)3. The PD1 ECD contains an A132I mutation. The sequence of this exemplary molecule is shown in SEQ ID NO:53 and SEQ ID NO:54.

[0189] In certain embodiments, the recombinant molecules described herein can include "conservative sequence modifications" of any of the sequences set forth in SEQ ID NOs: 1-116 (i.e., modifications of the nucleotide and amino acid sequences that do not abrogate antigen binding of the VH and VL sequences encoded by the nucleotide sequences or comprising the amino acid sequences). Such conservative sequence modifications include conservative nucleotide and amino acid substitutions, as well as nucleotide and amino acid additions and deletions. For example, SEQ ID NOs: 1-116 can be modified by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include those in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a predicted non-essential amino acid residue in any of the moieties described herein can be replaced with another amino acid residue from the same side chain family. Methods for identifying conservative nucleotide and amino acid substitutions that do not eliminate antigen binding are well known in the art (see, e.g., Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10):879-884 (1999); and Burks et al. Proc. Natl. Acad. Sci. USA 94:412-417 (1997)).

[0190] In certain embodiments, conservative amino acid sequence modifications refer to up to 1, 2, 3, 4, or 5 conservative amino acid substitutions relative to the CDR sequences described herein. For example, each such CDR may contain up to 5 conservative amino acid substitutions, such as up to 4 (i.e., no more than) conservative amino acid substitutions, such as up to 3 (i.e., no more than) conservative amino acid substitutions, such as up to 2 (i.e., no more than) conservative amino acid substitutions, or as few as 1 conservative amino acid substitution.

[0191] In some embodiments, the multispecific polypeptide of the invention comprises an antibody, IIP, and a polypeptide, 2P, fused to the C-terminus of the heavy chain of the IIP antibody. An exemplary fusion protein (using the anti-IL-23 antibody guselkumab as the IIP) is provided as follows: a heavy chain corresponding to any one of SEQ ID NOs: 53 or 97-109; and a light chain corresponding to SEQ ID NO: 54.

[0192] In some embodiments, the IIP is an antibody comprising one of the following VH / VL pairs: SEQ ID NO:79+80, SEQ ID NO:81+82, SEQ ID NO:83+84, SEQ ID NO:85+86, or SEQ ID NO:87+88. The IIP antibody may further comprise an IgG1 constant region fused to the C-terminus of the VH, which may be selected from SEQ ID NOs:111-112. The IIP antibody may further comprise a light chain constant region fused to the C-terminus of the VL. The IIP antibody may be fused to a 2P moiety, either with or without a linker, which may be SEQ ID NO:55. The 2P may be selected from SEQ ID NOs:56-78 or 89.

[0193] Design of recombinant IL-23R-ECD In various embodiments, an exemplary design of IL-23R-ECD is as follows: IL-23R D1D2D3 (SEQ ID NO: 6), IL-23R D1D2 (SEQ ID NO:5), or IL-23R D1 (Sequence number 2).

[0194] In some embodiments, the IIP of a multispecific polypeptide of the invention may comprise the ligand binding domain of the extracellular domain of human IL-23R and the ligand binding domain of the extracellular domain of human IL-12Rb (IL-23R / IL12R-ECD).

[0195] To signal, native IL-23R expressed on cell surface binds to IL-23 heterodimer (p19, p40). This heterotrimer generally binds to IL-12Rb to activate IL-23R signaling. Similarly, native IL12Ra expressed on cell surface binds to IL-12 heterodimer (p35, p40). This heterotrimer then binds to the same IL-12Rb to activate IL12R signaling.

[0196] Without being bound by any theory, it is possible that the IL-23R-ECD of the fusion protein binds to the complete IL-23 heterodimer (p19, p40), and this heterotrimer of IL-23R / p19 / p40 can bind to native IL-12Rb. This reduces the number of IL12Rb subunits available for IL12R signaling, and as such, can lead to reduced IL12R signaling. In the case of cancer treatment, this may be undesirable. Thus, the design of a chimeric IL-23R-ECD-IL12Rb-ECD fusion (IL-23R / IL12R-ECD) is described herein to prevent this undesirable consequence of sequestration of native IL12Rb.

[0197] The IL-23R / IL12R-ECD contains the ligand-binding sequence of IL-23R, which binds p19, and the ligand-binding sequence of IL-12Rb, which binds p40.

[0198] IL-23R is composed of one N-terminal Ig-like domain (D1), two fibronectin type III domains (D2 and D3) followed by a stalk region, a transmembrane domain, and a cytoplasmic domain. IL12Rb starts with two N-terminal fibronectin type III domains (D1, D2) followed by three fibronectin type III-like domains (D3, D4, D5) followed by a transmembrane domain and a cytoplasmic domain.

[0199] In some embodiments, the IL-23R / IL12R-ECD comprises one or more domains of IL-23R selected from D1, D2, D3. In some embodiments, the IL-23R / IL12R-ECD comprises one or more domains of IL12Rb selected from D1, D2. In some embodiments, the IL-23R / IL12R-ECD comprises one or more domains of IL12Rb selected from IL-23R. D1 , IL-23R D1D2 , or IL-23R D1D2D3 In some embodiments, the IL-23R / IL12R-ECD may comprise IL12Rb. D1 , IL12Rb D2 , or IL12Rb D1D2 may include.

[0200] In some embodiments, the IL12Rb domain and the IL-23R domain are fused or linked via a flexible linker. In some embodiments, the IL-23R / IL12R-ECD has the form N-IL-23R domain(s)-linker-IL12R domain(s)-C. In other embodiments, the IL-23R / IL12R-ECD has the form N-IL12R domain(s)-linker-IL-23R domain(s)-C. In a further aspect, the flexible linker comprises the polypeptide sequence (GGGGS)n, where n is between 1 and 10.

[0201] Exemplary embodiments of IL-23R / IL12R-ECD are as follows: IL12Rb D1 -Linker-IL-23R D1(SEQ ID NO: 29), IL12Rb D1 -Linker-IL-23R D1D2 (SEQ ID NO: 30), IL12Rb D1 -Linker-IL-23R D1D2D3 (SEQ ID NO: 31), IL12Rb D1D2 -Linker-IL-23R D1 (SEQ ID NO: 32), IL12Rb D1D2 -Linker-IL-23R D1D2 (SEQ ID NO: 33), IL-23R D1 -Linker-IL12Rb D1 (SEQ ID NO: 34), IL-23R D1D2 -Linker-IL12Rb D1 (SEQ ID NO: 35), IL-23R D1D2D3 -Linker-IL12Rb D1 (SEQ ID NO: 36), IL-23R D1 -Linker-IL12Rb D1D2 (SEQ ID NO: 37), or IL-23R D1D2 -Linker-IL12Rb D1D2 (Sequence number 38).

[0202] Exemplary embodiments of fusion proteins include: IL-23R fused to the C-terminus of HC D1 an anti-CEA antibody having the formula (SEQ ID NO: 12, 13), IL-23R fused to the C-terminus of LC; D1 an anti-CEA antibody having the formula (SEQ ID NO: 11, 14), IL-23R fused to the C-terminus of HC; D1D2 an anti-CEA antibody having the formula (SEQ ID NO: 12, 15), IL-23R fused to the C-terminus of LC; D1D2 an anti-CEA antibody having the formula (SEQ ID NO: 11, 16), IL-23R fused to the C-terminus of HC; D1D2D3 an anti-CEA antibody having the formula (SEQ ID NO: 12, 17), IL-23R fused to the C-terminus of LC; D1D2D3 an anti-CEA antibody having the formula (SEQ ID NO: 11, 18), IL-23R fused to the C-terminus of LC; D1 and anti-CEA antibody with TGFbRII-ECD fused to the C-terminus of HC (SEQ ID NOs: 14, 19), TNFR-ECD-Fc-IL-23R D1(SEQ ID NO: 20), TNFR-ECD-Fc-IL-23R D2 (SEQ ID NO: 21), TNFR-ECD-Fc-IL-23R D1D2D3 (SEQ ID NO: 22), Fc-IL-23R D1 (SEQ ID NO: 23), Fc-IL-23R D1D2 (SEQ ID NO: 24), Fc-IL-23R D1D2D3 (SEQ ID NO: 25), IL-23R D1 -Fc (SEQ ID NO: 26), IL-23R D1D2 -Fc (SEQ ID NO: 27), IL-23R D1D2D3 - Fc (SEQ ID NO: 28), IL12Rb fused to the C-terminus of HC D1 -Linker-IL-23R D1 an anti-CEA antibody having the formula (SEQ ID NO: 39), IL12Rb fused to the C-terminus of LC; D1 -Linker-IL-23R D1 Anti-CEA antibody (SEQ ID NO: 40) having the formula: VEGFR-Fc-IL-23R D1 (SEQ ID NO: 41), IL-23R D1 -Fc-VEGFR (SEQ ID NO: 42), TGFbRII-Fc-IL-23R D1 (SEQ ID NO: 43), IL-23R D1 -Fc-TGFbRII (SEQ ID NO: 44), Fc and IL-23R D1 HC fused to the C-terminus of IL-23R. D1 an anti-PSMA antibody having the formula (SEQ ID NO: 48, 47), IL-23R fused to the C-terminus of LC; D1 an anti-PSMA antibody having the formula (SEQ ID NO: 46, 49), IL-23R fused to the C-terminus of LC; D1 and an anti-PSMA antibody having a TGFbRII-ECD fused to the C-terminus of the HC (SEQ ID NOs: 50, 49), or an anti-IL-23 antibody having a TGFbRII-ECD fused to the C-terminus of the HC and a PSMA-binding peptide fused to the C-terminus of the LC (SEQ ID NOs: 51, 52).

[0203] Combination Therapies for the Treatment of Cancer Also provided herein is a method of treating a neoplastic disease or cancer in a subject, comprising administering to said subject an effective amount of a pharmaceutical composition(s) comprising one or more therapeutic agents, wherein said one or more therapeutic agents comprises at least a first therapeutic agent comprising an inhibitor of IL-23 / IL-23R signaling ("a-IL-23 agent"); and a second therapeutic agent ("combination agent"). The second therapeutic agent may comprise one or more antagonists of immune checkpoint proteins; one or more agonists of immune stimulatory receptors; one or more antagonists of cytokine signaling; one or more agonists of cytokine receptors; a modulator of one or more cell surface molecules expressed or displayed on the cell surface of tumor cells or immune cells; immune cells, including CAR-T cells, CAR-NK cells, or hematopoietic stem cells; an immunogenic chemotherapeutic agent; and / or one or more antagonists of immune inhibitory enzymes.

[0204] In some embodiments, the a-IL-23 agent comprises an antibody that binds to IL-23p19 (e.g., risankizumab, guselkumab, tildrakizumab, brazikumab, mirikizumab). In other embodiments, the a-IL-23 agent is an antibody that binds to IL-23R (e.g., AS2762900-00). In other embodiments, the a-IL-23 agent comprises a fusion protein comprising an antibody that binds to IL-23p19 or IL-23R. In other embodiments, the a-IL-23 agent is a multispecific polypeptide / fusion protein of the invention. In some aspects, the a-IL-23 agent is an antibody-ligand trap fusion protein comprising an IL-23R-ECD.

[0205] In some embodiments, the combination inhibits TGFb / TGFbR. In some embodiments, the TGFb / TGFbR inhibitor is selected from the following: a-TGFb antibody (e.g., fresolimumab); a-TGFbR antibody; TGFbRII ECD-containing fusion protein (e.g., TGFbRIIecd-Fc, AVID200); TGFbR TKI (e.g., galunisertib); anti-GARP antibody; anti-LAP antibody; fusion protein comprising an antibody and TGFbRII ECD (e.g., a-PDL1-TGFbRIIecd; vintrafusp alfa, SIRPa ECD-TGFbRII, anti-CEA-TGFbRII, anti-PSMA-TGFbRII, anti-IL6R-TGFbRII, anti-PD1-TGFbRII, anti-EGFR-TGFbRII, or anti-HER2-TGFbRII). In a specific embodiment, the combination is anti-EGFR-TGFbRII. In a specific embodiment, the co-agent is BCA101.

[0206] In some embodiments, the combination inhibits VEGF / VEGFR. In some embodiments, the VEGF / VEGFR inhibitor can be selected from the following: anti-VEGF antibody (e.g., bevacizumab), anti-VEGFR antibody (e.g., ramucirumab), VEGFR kinase inhibitor (e.g., sunitinib, sorafenib, axitinib, cabozantinib, regorafenib, pazopanib, vandetanib, lenvatenib), VEGFR ECD-Fc fusion protein (e.g., aflibercept), or fusion protein comprising antibody and VEGFR ECD.

[0207] In some embodiments, the combination inhibits the interaction of CD47 and SIRPa. In some aspects, the CD47 / SIRPPa inhibitor can be selected from the following: a-CD47 mAb (e.g., magrolimab, ZL-1201, TJ011133, STI-6643, SRF231, SHR-1603, IMC-002, IBI188, CC-90002, AO-176, or AK117, retaplimab, urabrelimab), a-SIRPa mAb, SIRPa-ECD-containing fusion protein (e.g., SIRPa-Fc, evolupercept, TTI-621, TTI-622).

[0208] In some embodiments, the combination inhibits the interaction of SIGLEC10 with CD24.

[0209] In some embodiments, the combination is an immune checkpoint inhibitor. In some embodiments, the combination is an antagonist of an innate immune checkpoint receptor or ligand. In some embodiments, the combination is an antagonist of a T cell co-inhibitory molecule. In some embodiments, the combination inhibits the interaction of PD-1 with PD-L1 or PD-L2. In some embodiments, the combination is an antibody that binds to PD-1 (e.g., nivolumab, pembrolizumab, cemiplimab, dostallimab, spartalizumab, camrelizumab, sintilimab, sasanlimab, ticelizumab, or toripalimab) or an antibody that binds to PDL1 (e.g., durvalumab, avelumab, atezolizumab). In other embodiments, the combination inhibits the interaction of BTLA and HVEM. In other embodiments, the combination inhibits the interaction of TIGIT and PVR. In some embodiments, the combination that inhibits TIGIT is selected from tiragolumab, vibostolimab, BMS-986207, osipellimab, etigilimab, domvanalimab, EOS-448, SEA-TGT, ASP8374, COM902, or IBI939. In other embodiments, the combination inhibits the interaction of TIM-3 with CEACAM. In some embodiments, the combination inhibits LAG-3. In some embodiments, the combination that inhibits LAG-3 is selected from leratolimab, fianlimab, Sym022, GSK2831781, TSR-033, yerumirimab, favezelimab, tebotelimab, FS118, or pubunalimab.

[0210] In some embodiments, the combination agent is an agonist of an immunostimulatory receptor. In some embodiments, the combination agent is an agonist of a T cell costimulatory molecule. In some embodiments, the combination agent is a polypeptide comprising a corresponding costimulatory ligand or a receptor-binding fragment thereof. In other embodiments, the combination agent is an agonist antibody that binds to a T cell costimulatory receptor. In some embodiments, the combination agent binds to 4-1BB (CD137), inducible T cell costimulatory factor (ICOS), OX-40 (CD134), herpes virus entry mediator (HVEM), glucocorticoid-induced TNFR-related protein (GITR), CD40, CD30, DNAM, or CD27. In some embodiments, the combination agent is a fusion protein comprising a receptor-binding sequence of the extracellular domain of CD30L, 4-1BBL, BTLA, LIGHT, OX-40L, ICOS-L, GITRL, CD80, CD86, or CD40L. In some embodiments, the combination agent is FPT-155. In some embodiments, the combination comprises an antibody or antigen-binding fragment thereof that binds to 4-1BB as an agonist (e.g., urelumab, utomirumab). In some embodiments, the combination comprises an antibody or antigen-binding fragment thereof that binds to OX40 as an agonist (e.g., tavolimab, PF-04518600, BMS-986178, MOXR-0916, GSK-3174998, INCAGN01949). In some embodiments, the combination comprises an antibody or antigen-binding fragment thereof that binds to ICOS as an agonist (e.g., GSK-3359609, JTX-2011). In some embodiments, the combination agent comprises an antibody or antigen-binding fragment thereof that binds GITR as an agonist (e.g., TRX-518, MK-4166, MK-1248, GWN-323, INCAGN01876, BMS-986156, AMG-228). In some embodiments, the combination agent comprises an antibody or antigen-binding fragment thereof that binds CD40 as an agonist (e.g., CDX-1140, SEA-CD40, RO7009789, JNJ-64457107, APX-005M, Chi Lob 7 / 4).In some embodiments, the combination comprises an antibody or antigen-binding fragment thereof (e.g., varlilumab) that binds to CD27 as an agonist. In some embodiments, the combination binds to a TNFR superfamily member receptor as an agonist.

[0211] In some embodiments, the combination agent is an agonist of the immunostimulatory receptor expressed on natural immune cells. In some embodiments, the combination agent is an agonist of the immunostimulatory receptor expressed on NK cells. In some embodiments, the immunostimulatory receptor of NK cells is NKG2D. In some embodiments, the combination agent is a polypeptide that comprises an NKG2D-binding fragment of an NKG2D ligand (NKG2DL).

[0212] In some embodiments, the combination agent is a tumor-targeting antibody. In some embodiments, the combination agent binds to a tumor cell surface molecule, a tumor antigen, or a tumor-associated antigen. In some embodiments, the tumor-targeting antibody has an Fc domain (e.g., FcgRI, FcgRIII) that binds to an activating receptor on NK cells and / or macrophages. In some embodiments, the Fc domain of the tumor-targeting antibody has a mutation designed to increase its binding to one or more Fc receptors.

[0213] In some embodiments, the combination agent is a cytokine that activates NK cells, or a fusion protein that includes a cytokine that activates NK cells. In some embodiments, the cytokine can be IL-15, IL-12, or IL-18. In some embodiments, the combination agent can be ST-067, nogapendekin alfa, SHR1501, BJ-001, SO-C101, or NHS-IL12. In some embodiments, the combination agent is a virus or plasmid that encodes a cytokine.

[0214] In some embodiments, the co-agent is a hormonal treatment. In some embodiments, the hormonal agent inhibits androgen synthesis or inhibits androgen receptor signaling. In some embodiments, the hormonal agent is an LHRH agonist (e.g., goserelin, histrelin, leuprolide, or triptorelin); an LHRH antagonist (e.g., degarelix), a first-generation antiandrogen (e.g., nilutamide, flutamide, or bicalutamide), a second-generation antiandrogen (e.g., apalutamide, enzalutamide, or darolutamide), or an androgen synthesis inhibitor (e.g., abiraterone acetate).

[0215] In some embodiments, the co-agent is a cytotoxic agent, hi some embodiments, the co-agent is a chemotherapeutic agent, radiation, or a tumor-targeting antibody.

[0216] In some embodiments, the co-agent is an antibody-drug conjugate. In some embodiments, the co-agent is selected from the list of: gemtuzumab ozogamicin, brentuximab vedotin, trastuzumab emtansine, inotuzumab ozogamicin, polatuzumab vedotin, enfortumab vedotin, trastuzumab deruxtecan, belantamab mafodotin, or sacituzumab govitecan.

[0217] In some embodiments, the combination agent is a small molecule kinase inhibitor. In some embodiments, the combination agent is a PARP inhibitor. In some embodiments, the combination agent is a tumor vaccine or a viral lytic agent. In some embodiments, the combination agent is an inhibitor of TH17 differentiation, maintenance, or function. In some embodiments, the combination agent inhibits IL-17 / IL-17R, IL-6 / IL-6R, or IL-1 / IL-1R. In some embodiments, the combination agent is an anti-IL6 antibody or an anti-IL6R antibody.

[0218] In some embodiments, the combination agent is an antagonist of signaling of one or more cytokines. In some embodiments, the cytokine is an immunoinhibitory cytokine. In some embodiments, the cytokine is selected from the following: IL-4, IL-13, IL-10, IL-6, IL-1b, IL-17, IL-22. In some embodiments, the combination agent is a polypeptide that binds to the cytokine. In other embodiments, the combination agent is a polypeptide that binds to the cytokine's cognate cytokine receptor. In some embodiments, the combination agent binds to and inhibits IL1b or IL1R. In some embodiments, the combination agent is selected from anakinra or canakinumab. In some embodiments, the combination agent binds to and inhibits IL-10 or IL-10R. In some embodiments, the combination agent is an antibody or antigen-binding fragment thereof that binds to IL-10 or IL-10R; or a polypeptide comprising an IL10-binding fragment of IL-10R. In some embodiments, the combination agent is an antibody that binds to IL-17 or IL-17R. In some embodiments, the combination agent is selected from afasevicumab, bimekizumab, ixekizumab, netakimab, perakizumab, secukinumab, bunakizumab, or brodalumab. In some embodiments, the combination agent is an antibody that binds to IL-6 or IL-6R. In some embodiments, the combination agent is selected from clazakizumab, olokizumab, siltuximab, sirukumab, diltibekimab, revilimab, sapelizumab, sarilumab, satralizumab, or tocilizumab. In some embodiments, the combination agent is an antibody that binds to IL-4, IL-13, IL4RA, or IL13R. In some embodiments, the combination agent is dupilumab.

[0219] In some embodiments, the combination agent is an antagonist of RANK / RANKL signaling. In some embodiments, the combination agent is an antibody that binds to RANKL or RANK. In some embodiments, the combination agent is denosumab. In other embodiments, the combination agent is a polypeptide that comprises a RANKL-binding fragment of RANK ECD.

[0220] In some embodiments, the combination comprises one or more agents selected from the following: immunotherapeutic agents, chemotherapeutic molecules, antibodies, antibody-drug conjugates, small molecule kinase inhibitors, hormonal agents, androgen synthesis inhibitors, androgen receptor antagonists, antiangiogenic agents, cell therapy, CAR-T cell therapy, CAR-NK cell therapy, radionuclide therapy, ionizing radiation, ultraviolet radiation, cryoablation, thermal ablation, selective estrogen receptor modulators (SERMs), selective estrogen receptor degraders (SERDs), or radiofrequency ablation. In some embodiments, the immunotherapeutic agent is selected from the following: immune checkpoint inhibitors, immunostimulatory receptor agonists, immunostimulatory cytokines / cytokine receptor agonists, immunoinhibitory cytokines / cytokine receptor antagonists, tumor vaccines, immunomodulatory imide drugs, CAR-T cells, CAR-NK cells, oncolytic viruses.

[0221] In some embodiments, the combination agent is an immunogenic chemotherapeutic agent.The mechanism of action of immunogenic chemotherapeutic agent may involve immune activation, and therefore may be hindered by the expansion / activation of IL-23-dependent inflammatory cells.In some embodiments, the immunogenic chemotherapeutic agent is an alkylating agent, a topoisomerase inhibitor, a platinum derivative, a taxane, or an anthracycline.

[0222] In some embodiments, the co-agent is an antagonist of an immuno-inhibitory enzyme. In some embodiments, the immuno-inhibitory enzyme is an ectonucleotidase (e.g., CD39, CD73) or indoleamine 2,3-dioxygenase.

[0223] In an IL-23-rich tumor immune microenvironment, engineered T cells or NK cells (CAR-T, CAR-NK cells, respectively) adoptively transferred into a patient may adopt an undesirable tumor-promoting phenotype. In some embodiments, the combination is a composition comprising CAR-T cells or CAR-NK cells.

[0224] In some embodiments, the cancer is a blood system or hematogenous cancer selected from the group consisting of acute leukemia, acute myelocytic leukemia, acute myelogenous leukemia, myeloblastic leukemia, promyelocytic leukemia, myelomonocytic leukemia, monocytic leukemia, erythroleukemia, chronic leukemia, chronic myelocytic (or granulocytic) leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (asymptomatic and aggressive forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia, and myelodysplasia, and any combination thereof.

[0225] In some embodiments, the cancer is selected from the group consisting of fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, and other sarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, lymphoid malignancies, pancreatic cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytoma sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, cholangiocarcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumor, seminoma, bladder cancer, melanoma, and CNS tumors, such as gliomas (such as brain stem glioma and mixed glioma), glioblastoma (also known as glioblastoma multiforme), astrocytoma, CNS lymphoma, germinoma, medulloblastoma, Schwannoma. The solid tumor is selected from the group consisting of craniopharyogioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, retinoblastoma, and brain metastasis.

[0226] In some embodiments, the method of treatment comprises administration of a multispecific polypeptide of the invention comprising 2P comprising a polypeptide sequence that binds to TfR, thereby crossing the blood-brain barrier. In some such embodiments, the cancer is a CNS tumor (e.g., glioma (such as brain stem glioma and mixed glioma), glioblastoma (also known as glioblastoma multiforme), astrocytoma, CNS lymphoma, germinoma, medulloblastoma, schwannoma craniopharyogioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, retinoblastoma, and brain metastasis.

[0227] In some embodiments, the method includes treatment with an a-IL-23 agent, either in conjunction with or sequentially with a combination agent. For example, treatment with an a-IL-23 agent can be administered simultaneously (e.g., in the same pharmaceutical composition or within a time frame of between about 0.1 hours and about 24 hours) with administration of the combination agent; treatment with an a-IL-23 agent can be administered 1-28 days after administration of the combination agent; or treatment with an a-IL-23 agent can be administered 1-28 days prior to administration of the combination agent.

[0228] In some embodiments, treatment with either the single agent or the combination agent is repeated periodically as maintenance treatment in a time frame of once per month to once every two months, once per three months, once per four months, once per five months, once per six months, or once per seven months, or once per eight months, or once per nine months, or once per ten months, or once per eleven months, or once per year, for as long as the patient's disease improves or becomes stable / non-progressive.

[0229] In some embodiments, the method of treatment with a combination of an anti-IL-23 agent and other anti-cancer agent(s) reduces the incidence and / or severity of immune-related adverse events (irAEs) compared to treatment with the other anti-cancer agent(s) alone. In some embodiments, the reduction in the proportion of patients who discontinue treatment due to toxicity is at least 10%, 30%, 50%, 70%, or 90%. In some embodiments, the reduction in the incidence of grade 3, grade 4, grade 3+4, or all grades of irAEs is at least 10%, 30%, 50%, 70%, or 90%. In some embodiments, the reduction in grade 3, grade 4, grade 3+4, or all grades of a particular class of irAEs is at least 10%, 30%, 50%, 70%, or 90%; where the class may be selected from, but is not limited to, gastrointestinal toxicity, endocrine toxicity, cardiac toxicity, pulmonary toxicity, hepatic toxicity, rheumatologic toxicity, renal toxicity, neurological or dermatological / skin toxicity. In some embodiments, the reduction in grade 3, grade 4, grade 3+4, or total grade of a specific irAE is at least 10%, 30%, 50%, 70%, or 90%; where the specific irAE is, but is not limited to, uveitis, Sjogren's syndrome, conjunctivitis, blepharitis, episcleritis, scleritis, retinitis, pneumonitis, pleuritis, sarcoid granulamatosis, hepatitis, pancreatitis, autoimmune diabetes, skin rash, pruritus. The adverse events may be selected from the group consisting of inflammatory bowel disease, vitiligo, DRESS, psoriasis, Stevens-Johnson syndrome, arthralgia, arthritis, myositis, dermatomyositis, encephalitis, meningitis, polyneuropathy, fatigue, Guillain-Barré syndrome, hypophysitis, thyroiditis, adrenalitis, myocarditis, pericarditis, interstitial nephritis, glomerulonephritis, colitis, enteritis, gastritis, anemia, neutropenia, thrombocytopenia, thrombotic microangiopathy, acquired hemophilia, vasculitis, or any Common Terminology Criteria for Adverse Events (CTCAE) adverse event.

[0230] In some embodiments, the method of treatment with a combination of an anti-IL-23 agent and other anti-cancer agent(s) extends overall survival or progression-free survival more effectively than treatment with the other anti-cancer agent(s) alone. In some embodiments, the method of treatment with a combination of an anti-IL-23 agent and other anti-cancer agent(s) results in a statistically significant improvement in any RECIST v1.1 criteria as fully described in the art, e.g., Eisenhauer et al., "New response evaluation criteria in solid tumours: Revised RECIST guideline (version 1.1)" European Journal of Cancer 2009;45:228, which is incorporated herein in its entirety.

[0231] In some embodiments, the method of treatment with a combination of an anti-IL-23 agent and another anti-cancer agent(s) reduces or prevents bone metastasis or skeletal-related events more effectively than treatment with the other anti-cancer agent(s) alone. In some embodiments, the reduction in bone metastasis complies with RECIST v1.1 criteria. In some embodiments, the reduction in skeletal-related events is at least 10%, 30%, 50%, 70%, or 90%.

[0232] In some embodiments, methods of treatment with a combination of an anti-IL-23 agent and other anti-cancer agent(s) improve both efficacy and toxicity as described above compared to treatment with the other anti-cancer agent(s) alone.

[0233] In some embodiments, the treatment is repeated periodically as a maintenance treatment in a time frame of once every 2 weeks to once every 3 weeks, once every month, once every 2 months, once every 3 months, once every 4 months, once every 5 months, once every 6 months, or once every 7 months, or once every 8 months, or once every 9 months, or once every 10 months, or once every 11 months, or once every year, for as long as the patient's disease improves or becomes stable / non-progressive.

[0234] In some embodiments, treatment prevents metastasis, inhibits tumor growth, and / or reduces tumor growth.

[0235] Therapies for Treating Immune Disorders or Autoimmune Conditions Provided herein are methods of treating an immune disorder in a subject, comprising administering to said subject an effective amount of a pharmaceutical composition comprising one or more therapeutic agents, wherein said one or more therapeutic agents comprise an anti-IL-23 agent, or a multispecific polypeptide of the invention.

[0236] In some embodiments, the method further comprises a second agent. In some embodiments, the second agent is an agonist of IL10 signaling. In some embodiments, the second agent comprises an agonist IL10R antibody or an IL10R binding sequence of IL10.

[0237] In some embodiments, the method of treatment comprises administration of a multispecific polypeptide of the invention comprising 2P, which comprises a polypeptide sequence that binds to TfR and thereby crosses the blood-brain barrier, hi some such embodiments, the immune disorder is multiple sclerosis or causes neuroinflammation.

[0238] In some embodiments, the immune disorder is an autoimmune disorder.As mentioned herein, non-limiting examples of immune disorder include Addison's disease, celiac disease, dermatomyositis, Graves' disease, Hashimoto's thyroiditis, multiple sclerosis, myasthenia gravis, pernicious anemia, reactive arthritis, rheumatoid arthritis, Sjogren's syndrome, scleroderma, systemic sclerosis, systemic lupus erythematosus, or type I diabetes, chronic inflammatory disease, psoriasis, ulcerative colitis, Crohn's disease, and inflammatory bowel disease.In some embodiments, the immune disorder is graft-versus-host disease (GVHD).

[0239] In one embodiment, the present invention discloses a method of treatment or prevention of acute or chronic graft-versus-host disease comprising an anti-IL-23 agent or a multispecific polypeptide of the present invention.

[0240] Kits and Products Further provided are kits, unit doses, and articles of manufacture comprising any of the recombinant molecules described herein. In some embodiments, kits are provided comprising any of the pharmaceutical compositions described herein, and the kits preferably provide instructions for use.

[0241] The kit of the present application is suitably packaged. Suitable packaging includes, but is not limited to, vials, bottles, jars, and flexible packaging (e.g., sealed Mylar or plastic bags). The kit may provide additional components, such as buffers and interpretative information, as needed. Thus, the present application also provides articles of manufacture, including vials (such as sealed vials), bottles, jars, and flexible packaging.

[0242] The article of manufacture may include a container and a label or package insert on or attached to the container. Suitable containers include, for example, bottles, vials, syringes, and the like. The containers may be formed from a variety of materials, such as glass or plastic. In general, the container holds a composition effective for treating a disease or disorder described herein and may have a sterile access port (e.g., the container may be an intravenous solution bag or vial having a stopper that can be pierced with a hypodermic needle). The label or package insert indicates that the composition is used to treat a particular condition in an individual. The label or package insert further includes instructions for administering the composition to an individual. The label may indicate instructions for reconstitution and / or use. The container holding the pharmaceutical composition may be a multi-use vial that allows for repeated administration (e.g., 2-6 administrations) of the reconstituted formulation. Package insert refers to instructions customarily included in commercial packages of therapeutic products that include information about indications, usage, dosage, administration, contraindications, and / or warnings regarding the use of such therapeutic products. Additionally, the article of manufacture may further comprise a second container comprising a pharma- ceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. The article of manufacture may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0243] The kit or article of manufacture can include multiple unit doses of the pharmaceutical composition and instructions for use, and can be packaged in an amount sufficient for storage and use in a pharmacy (eg, a hospital pharmacy and a compounding pharmacy). EXAMPLES

[0244] Example 1: Design of a multifunctional construct A multifunctional fusion protein may comprise an IIP that is an antibody or an antigen-binding fragment thereof. Examples of such constructs are shown in Figures 1A-1D.

[0245] Figures 1A-1D show schematic diagrams corresponding to several exemplary designs of multispecific polypeptides described herein, where IIP (dark grey) comprises an antibody or antibody fragment and 2P (light grey) represents a target binding polypeptide. Figure 1A shows an antibody IIP with 2P fused to the C-terminus of the heavy chain. Figure 1B shows an antibody IIP with 2P fused to the C-terminus of the light chain. Figure 1C shows a bispecific antibody where one Fab is an IIP and the other Fab is 2P. Figure 1D shows a fusion of two scFvs where one scFv is an IIP and the other scFv is 2P.

[0246] 2A-2G show diagrams corresponding to several exemplary designs of multispecific polypeptides described herein, in which IIP comprises a ligand-binding fragment of IL-23R ECD. D1, D2 and D3 represent IL-23R binding domains 1, 2 and 3, respectively. In these figures, dark grey corresponds to IIP and light grey. Multifunctional fusion proteins can include IIPs that comprise a ligand-binding fragment of IL-23 ECD.

[0247] The amino acid sequences of the exemplary fusion proteins of the invention were codon-optimized using GeneOptimizer®. Antibody heavy chain cDNA and antibody light chain cDNA were synthesized and subsequently cloned into separate plasmids (pEvi3; evitria AG, Switzerland) under the control of a mammalian promoter and polyadenylation signal. Plasmid DNA was amplified in E. coli and DNA was purified using an anion exchange kit for low-endotoxin plasmid DNA preparation. DNA concentration was determined by measuring absorbance at 260 nm wavelength. Sequence accuracy was verified by Sanger sequencing (up to two sequencing reactions per plasmid depending on the size of the cDNA). Heavy and light chain plasmid DNA were subsequently co-transfected into CHO K1 cells for suspension culture (originally obtained from ATCC and adapted for serum-free growth in suspension culture at evitria). Seeds were grown in eviGrow medium (a serum-free medium with defined animal components). Cells were transfected with eviFect (evitria AG, Switzerland) and CHO cells were cultured in eviMake2 (evitria AG, Switzerland), a serum-free and animal component-free medium. Production was stopped when viability reached 75% (8 days after transfection). Supernatants were harvested by centrifugation and subsequent filtration (0.2 um filter). Antibodies were purified using MabSelect™ Sure™ (Protein A affinity chromatography on a Bio-Rad BioLogic FuoFlow FPLC machine followed by gel filtration as a polishing and rebuffering step). In some cases, antibodies were further purified using SEC purification.

[0248] Fusion proteins of the invention can also be produced by stable transfection of mammalian cell lines (e.g., CHO K1 cells) with plasmid DNA encoding the chains of the fusion protein, selection of stably transfected cell clones or cell pools expressing the fusion protein, development of a master cell bank for production of the fusion protein, purification of the fusion protein by Protein A affinity chromatography and / or SEC, and formulation using methods well described in the art.

[0249] Example 2: Anti-IL-23-PD1ecd constructs with single amino acid mutations in PD1ecd show superior binding affinity to PDL1. As shown in Figures 3A-3D, the data demonstrate that the anti-IL-23-PD1ecd construct with a single amino acid mutation in PD1ecd exhibits superior binding affinity to PDL1 (Figures 3B-3C) and PDL2 (Figures 3D-3E) compared to wild-type PD1. Furthermore, it was demonstrated that this single mutation possesses essentially equivalent binding properties compared to the more extensively engineered PD1 variants reported in the literature. This means that the test construct, anti-IL-23-PD1ecd, has acquired favorable binding properties with lower immunogenicity risk since only a single amino acid mutation was required. (See Figures 3A-3D).

[0250] A fixed concentration of each construct was coated onto plates (1 μg / mL), followed by varying concentrations of biotinylated hu-PDL1 (detected by streptavidin-HRP). The binding EC50 of the test construct (a-IL-23-PD1(A123I)) was 57 nM, which is superior to the wild-type EC50 of 471 nM, but essentially equivalent to the more extensively mutated a-IL-23-PD1(G-V2) reported in the literature. A control construct with the A123I PD1ecd mutation (but a different targeting antibody) showed identical binding characteristics to anti-IL-23-PD1(A123I), confirming that the Fab does not contribute to the differential binding activity observed. (See Figure 3A)

[0251] Various concentrations of each construct were coated onto plates followed by a fixed concentration of biotinylated hu-PDL1 (100 ng / mL). The binding EC50 of a-IL-23-PD1(A123I)) is 1 μM, which is superior to the wild-type EC50 of 3.3 μM. a-IL-23-PD1(G-V2) is slightly better (0.6 μM). (See FIG. 3B).

[0252] A fixed concentration of each construct was coated onto plates (1 μg / mL), followed by various concentrations of biotinylated hu-PDL2 (detected by streptavidin-HRP). The binding EC50 of a-IL-23-PD1(A123I) was 54 nM, which is superior to the wild-type EC50 of 131 nM; essentially equivalent to anti-IL-23-PD1(G-V2) (62 nM). (See FIG. 3C).

[0253] Various concentrations of each construct were coated onto plates followed by a fixed concentration of biotinylated hu-PDL2 (500 ng / mL). The binding EC50 of a-IL-23-PD1(A123I) was 0.78 μM, which is superior to the wild-type EC50 of 6 μM; essentially equivalent to anti-IL-23-PD1(G-V2) (0.6 μM). (See FIG. 3D).

[0254] Example 3: IL-23p19 antibody enhances anti-tumor immune responses induced by PDL1 / PD1 blockade As shown in Figures 4A-4C, IL-23p19 antibody enhances anti-tumor immune responses induced by PDL1 / PD1 blockade. To evaluate the hypothesis that resistance to anti-PDL1 / PD1 therapy may be mediated by TH17b cells and that such resistance may be suppressed by simultaneous blockade of PD1 / PDL1 and IL-23, the following studies were performed. B16 tumor cells (4x10e5 cells, sc) were implanted in C57 / BL6 mice. Tumors were approximately 50 mm in size. 3 At that time, mice were randomized and treated with either anti-PDL1 Ab (5mg / kg ip weekly x 4) and / or anti-IL-23p19 Ab (anti-mouse p19 Ab=G23-8) (5mg / kg ip twice weekly x 4). (See Figures 4A-4C). Mice treated with the combination of a-PD-L1 Ab and a-IL-23p19 had superior survival and smaller tumors compared to mice treated with a-PDL1 alone (p<0.005). (See Figures 4A-4B).

[0255] The improved survival in mice treated with the combination of a-PD-L1 Ab and a-IL-23p19 compared to mice treated with a-PDL1 Ab alone was reflected in the significant inhibition of lung tumor metastasis (see Figure 4C).

[0256] Example 4: Anti-IL-23-PD1 Polypeptides Reduce Tumor Growth and Limit Toxicity in a Humanized Mouse Model Human tumor xenografts were established in NSG mice (humanized with human PBMCs). Tumor-bearing mice were randomized and treated with the following single agents or combinations: (i) vehicle only (control), (ii) anti-IL-23 antibody, (iii) anti-PDL1 antibody, (iv) anti-IL-23-PD1ecd. (See FIG. 5).

[0257] In the treatment group that received a-PDL1, 4 / 5 mice experienced significant GVHD during treatment, compared to mice in the other treatment groups. Anti-IL-23-PD1 significantly reduced tumor growth compared to anti-IL-23 alone, meaning that anti-IL-23-PD1 mitigated the undesirable immune-related toxicity associated with anti-PDL1 treatment while simultaneously limiting tumor growth.

[0258] Example 5: Anti-IL-23-PD1 Polypeptides are Effective in Inhibiting Tumor Growth Compared to Treatment with Immune Checkpoint Inhibitors Human tumor xenografts were established in NSG mice (humanized with human PBMCs). Tumor-bearing mice were randomized and treated with the following single agents or combinations: (i) vehicle only (control); (ii) anti-PD1 antibody (pembrolizumab); (iii) anti-PD1 antibody (pembrolizumab) + anti-CTLA4 antibody (ipilimumab); (iv) anti-IL-23-PD1; (v) anti-CTLA4-TGFbRII; (vi) anti-IL-23-PD1 + CTLA4-TGFbRII (see Figure 6). As described in U.S. Pat. No. 8,993,524; Ravi et al., ``Bifunctional immune checkpoint-targeted antibody-ligand traps that simultaneously disable TGFb enhance the efficacy of cancer immunotherapy.'' Nat. Commun. 2018;9:741 (each of which is incorporated herein by reference in its entirety), anti-CTLA4-TGFbRII has been reported to be more effective at reducing tumor-infiltrating Tregs and inhibiting tumor progression compared to CTLA-4 antibody (ipilimumab) alone.

[0259] Tumor-bearing mice failed to respond to treatment with either anti-PD1 (pembrolizumab) alone or even the combination of anti-PD1 and anti-CTLA4 (pembrolizumab + ipilimumab). In contrast, treatment with a-IL-23-PD1ecd alone was significantly more effective at inhibiting tumor growth compared to anti-PD1 antibody (p<0.03). Furthermore, treatment with the combination of anti-IL-23-PD1 and CTLA4-TGFbRII was able to completely halt tumor growth, and the synergistic antitumor efficiency of this combination was markedly superior to the current combination treatment with ICI (anti-PD1 + anti-CTLA4) (p<0.001). The data are consistent with reports in the literature showing that the majority of cancers fail to respond to immunotherapy with antibodies targeting immune checkpoints, such as cytotoxic T-lymphocyte antigen-4 (CTLA-4) or programmed death-1 (PD-1) / PD-1 ligand (PD-L1)). Ravi et al., ``Bifunctional immune checkpoint-targeted antibody-ligand traps that simultaneously disable TGFb enhance the efficacy of cancer immunotherapy.'' Nat. Commun. 2018;9:741; U.S. Patent No. 8,993,524 (each of which is incorporated herein by reference in its entirety). The synergy of IL-23 blockade with TGFb blockade demonstrates the efficacy of combining IL-23 blockade with immune inhibitory cytokine blockade.

[0260] These results demonstrate that tumor resistance to current ICIs (either anti-PD1 alone or anti-PD1 in combination with anti-CTLA4) can be effectively suppressed by treatment with a bifunctional fusion protein (anti-IL-23-PD1 and CTLA4-TGFbRII) that simultaneously disables IL-23 / TGFb signaling in the tumor microenvironment (TME).

[0261] Example 6: Mechanism of action of anti-IL-23-PD1 Without being bound by any theory, a possible mechanism of action for anti-IL-23-PD1 is shown in Figures 7A-7B. First, an intact tumor microenvironment (TME) is shown in Figure 7A. In this context, PDL1 and PDL2 expressed on tumor cells and myeloid-derived suppressor cells (MDSCs) inhibit T cell activation by binding to PD1 on T cells. In the context of IL-23 (expressed by tumor cells and MDSCs), CD4 T cells in this TME are biased toward a Th17 phenotype. Blockade of PD1 or PDL1 / PDL2 leads to activation and proliferation of Th17 cells. Factors from Th17 cells (e.g., IL-8, G-CSF, IL-17) feed back onto MDSCs to maintain their phenotype. Thus, in this context, blocking PD1 signaling alone may produce the opposite results in the presence of IL-23. Figure 7B shows the results of treatment with anti-IL-23-PD1: PD1 ECD binds to PDL1 and / or PDL2, neutralizing PD1 signaling. At the same time, PDL1+ and / or PDL2+ cells are decorated with anti-IL-23 antibodies, thereby sequestering IL-23 and suppressing the Th17 phenotype. This allows for the activation of these Th1 cells while simultaneously inducing an IL-12-mediated antitumor Th1 phenotype. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6] [Table 2-7] [Table 2-8] [Table 2-9]

[0262] Equivalents and Incorporation by Reference All references herein are incorporated by reference to the same extent as if each individual publication, database entry (e.g., Genbank sequence or GeneID entry), patent application, or patent was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. Applicant's incorporation-by-reference statement is intended to be consistent with 37 CFR §1.57(b)(1) with respect to any and all individual publications, database entries (e.g., Genbank sequence or GeneID entry), patent application, or patent, each of which is clearly identical in accordance with 37 CFR §1.57(b)(2), even if such citation is not immediately adjacent to the dedicated incorporation-by-reference statement. The inclusion of an incorporation-by-reference statement specifically in the specification does not in any way weaken this general incorporation-by-reference statement. The citation of references herein is not intended as an admission that the references are relevant prior art, nor does it constitute any admission as to the contents or dates of these publications or documents.

[0263] While the present invention has been shown and described in detail with reference to preferred and various alternative embodiments, it will be understood by those skilled in the relevant art that various changes in form and detail may be made therein without departing from the spirit and scope of the invention.

Claims

1. 1. A recombinant molecule comprising: a) an interleukin-23 (IL-23) inhibitory polypeptide (IIP), wherein the IIP comprises an IL-23 binding polypeptide or an IL-23R binding polypeptide; and b) a target-binding polypeptide portion that binds to one or more immune checkpoint proteins or immunostimulatory receptors. A recombinant molecule comprising: (i) the target-binding polypeptide binds to an immune checkpoint protein as an antagonist or agonist; (ii) the immune checkpoint protein is a T cell co-inhibitory receptor or ligand or a natural inhibitory receptor or ligand; (iii) the immune checkpoint protein is programmed death-1 (PD1; CD279), programmed death-ligand 1 (PDL1), programmed death-ligand 2 (PDL2), cytotoxic T-lymphocyte antigen-4 (CTLA4; CD152), attenuator of B- and T-lymphocytes (BTLA), V-domain immunoglobulin suppressor of T-cell activation (VISTA), T-cell immunoglobulin and ITIM domain (TIG IT), lymphocyte activation gene 3 (LAG-3; CD223), T-cell immunoglobulin and mucin domain 3 (Tim-3; HAVCR2), carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), CD47, signal regulatory protein alpha (SIRPa), major histocompatibility complex class I, G (HLA-G), Ig-like transcript 2 (ILT2; LILRB1), or Ig-like transcript 4 (ILT4, LILRB2); (iv) the immunostimulatory receptor is selected from 4-1BB (CD137), inducible T-cell costimulatory factor (ICOS; CD278), OX-40 (CD134), glucocorticoid-induced TNFR-related protein (GITR; CD357), CD40, herpes virus entry mediator (HVEM), CD28, or CD27; (v) the IIP binds to and inhibits IL-23, and optionally the IIP binds to and inhibits the IL-23p19 subunit or the IIP binds to and inhibits IL-23R; and / or (vi) the IIP comprises an antibody or antigen-binding fragment thereof, optionally wherein the IIP is an antibody or antigen-binding fragment thereof, wherein the antigen-binding fragment thereof is selected from the group consisting of a fragment crystallizable (Fc) region, a fragment antigen-binding (Fab) region, a single-chain variable fragment (scFv), a light chain or a functional portion thereof, a variable region of the light chain (VL), a constant region of the light chain (CL), a heavy chain or a functional portion thereof, a variable region of the heavy chain (VH), a constant region of the heavy chain (CH), at least one complementarity-determining region (CDR) or a portion thereof, or a combination thereof; 10. The recombinant molecule of claim 1, wherein the antibody or antigen-binding fragment thereof comprises a monoclonal antibody that targets the human IL-23p19 subunit, and optionally the antibody or antigen-binding fragment thereof comprises one or more of six CDRs or antigen-binding portions thereof selected from any one of risankizumab, guselkumab, tildrakizumab, brazikumab, and mirikizumab, and optionally the antibody or antigen-binding fragment thereof is guselkumab.

3. the target-binding polypeptide (i) comprises an antibody or antigen-binding fragment thereof, optionally wherein the target-binding polypeptide is an antibody or antigen-binding fragment thereof, and the antigen-binding fragment thereof comprises a fragment crystallizable (Fc) region, a fragment antigen-binding (Fab) region, a single-chain variable fragment (scFv), a light chain or a functional portion thereof, a variable region of the light chain (VL), a constant region of the light chain (CL), a heavy chain or a functional portion thereof, a variable region of the heavy chain (VH), a constant region of the heavy chain (CH), at least one complementarity-determining region (CDR) or a portion thereof, or any combination thereof; and / or (ii) binds to an immune checkpoint protein as an antagonist, wherein the target-binding polypeptide comprises a ligand-binding sequence of the extracellular domain (ECD) of the immune checkpoint protein, and optionally (a) the ECD of the immune checkpoint protein is capable of specifically binding to one or more of its cognate ligands expressed or presented on a tumor cell or an immune cell, optionally wherein the immune cell is an antigen-presenting cell (APC), a myeloid-derived suppressor cell (MDSC), a CD4 T cell, or a T H 17 cell; and / or 2. The recombinant molecule of claim 1, wherein (b) the ECD is capable of specifically binding to Programmed Death-1 Ligand 1 (PDL1; CD274; B7-H1) and / or Programmed Death-1 Ligand 2 (PDL2), and optionally the target-binding polypeptide comprises the PD1 (CD279) extracellular domain (PD1-ECD) or a ligand-binding fragment thereof. (i) the target-binding polypeptide is (a) the amino acid sequence of SEQ ID NO: 56, or an amino acid sequence having at least 80% identity to SEQ ID NO: 56, or a ligand-binding fragment thereof; or (b) one or more modifications of the amino acid sequence of SEQ ID NO: 56, or a ligand-binding fragment thereof. wherein the target-binding polypeptide comprises a substitution, deletion, insertion, or inversion of 1 to 10 amino acid residues, optionally wherein the one or more modifications increase the affinity of the target-binding polypeptide for PDL1 or PDL2, or both, compared to the affinity of wild-type PD1-ECD for its ligand, and / or is selected from A132I, S87G, P89L, N116S, G124S, S127V, A140V, optionally wherein the modification is A132I, and optionally wherein the target-binding polypeptide has the amino acid sequence of SEQ ID NO:57; (ii) the IIP is linked to the target-binding polypeptide moiety via a linker, and optionally (a) the target-binding polypeptide portion is linked to the C-terminus of the IIP; (b) the linker is selected from a non-cleavable linker, a peptide linker, a flexible linker, a rigid linker, a helical linker, and a non-helical linker; and / or (c) the linker is a peptide linker having an amino acid sequence comprising (GGGGS)n, where n is 1, 2, 3, 4, 5, 6, 7, or 8, and optionally the linker comprises the amino acid sequence of SEQ ID NO: 55; and / or (iii) The recombinant molecule of claim 1, wherein the recombinant molecule comprises a first polypeptide having the amino acid sequence of SEQ ID NO:53 and a second polypeptide having the amino acid sequence of SEQ ID NO:

54.

5. A host comprising a recombinant molecule according to any one of claims 1 to 4.

6. A polynucleotide sequence encoding a recombinant molecule according to any one of claims 1 to 4.

7. A vector comprising the polynucleotide of claim 6.

8. A polypeptide comprising a recombinant molecule according to any one of claims 1 to 4.

9. 5. A pharmaceutical composition comprising a recombinant molecule according to any one of claims 1 to 4 or a vector comprising a polynucleotide sequence encoding said recombinant molecule, optionally further comprising a pharmaceutically acceptable excipient.

10. 10. The pharmaceutical composition of claim 9 for use in a method for treating a neoplastic disease, cancer, or immune disorder in a subject, the method comprising administering an effective amount of the pharmaceutical composition to the subject.

11. (i) the subject has cancer, and optionally the recombinant molecule comprises a first polypeptide having the amino acid sequence of SEQ ID NO: 53 and a second polypeptide having the amino acid sequence of SEQ ID NO: 54, and / or the cancer is selected from prostate cancer, pancreatic cancer, biliary tract cancer, colon cancer, rectal cancer, liver cancer, kidney cancer, lung cancer, testicular cancer, breast cancer, ovarian cancer, brain cancer, skin cancer, bladder cancer, and head and neck cancer, melanoma, sarcoma, multiple myeloma, leukemia, and / or lymphoma; (ii) the method inhibits tumor growth for at least 10, 15, or 20 days; (iii) the method reduces tumor growth by at least 5%, 10%, 15%, or 20%; (iv) The method further comprises administering to the subject a therapeutic agent comprising an anti-CTLA4-TGFβRII molecule or one or more anti-cancer agents, optionally wherein the one or more anti-cancer agents are selected from the group consisting of an immunotherapeutic agent, a chemotherapeutic molecule, an antibody, an antibody-drug conjugate, a small molecule kinase inhibitor, a hormonal agent, an androgen synthesis inhibitor, an androgen receptor antagonist, an anti-angiogenic agent, a cell therapy, a CAR-T cell therapy, a CAR-NK cell therapy, a radionuclide therapy, ionizing radiation, ultraviolet radiation, cryoablation, thermal ablation, a selected and / or said administration of said one or more anti-cancer agents comprises a selective estrogen receptor modulator (SERM), a selective estrogen receptor degrader (SERD), or radiofrequency ablation, and optionally said immunotherapeutic agent is selected from an immune checkpoint inhibitor, an immunostimulatory receptor agonist, an immunostimulatory cytokine / cytokine receptor agonist, an immunoinhibitory cytokine / cytokine receptor antagonist, a tumor vaccine, an immunomodulatory imidazole, CAR-T cells, CAR-NK cells, or an oncolytic virus; and / or said administration of said one or more anti-cancer agents comprises (a) more effectively reduces or prevents severe immune-related adverse events or toxicity compared to administration of said one or more anti-cancer agents alone; or (b) more effectively enhances tumor growth reduction or inhibits tumor growth compared to administration of the one or more anti-cancer agents alone; and / or (v) The pharmaceutical composition for use according to claim 10, wherein the subject is a mammal, optionally a human.

12. 1. A composition for use in a method of treating a neoplastic disease or cancer in a subject, said composition comprising one or more therapeutic agents, said method comprising administering said composition to said subject, wherein said one or more therapeutic agents are: a. a first therapeutic agent comprising an inhibitor of IL-23 signaling; and b. A second therapeutic agent comprising any one or more of the following: (i) one or more modulators, each of which is an antagonist of one or more immune checkpoint proteins; (ii) one or more modulators, each of which is an agonist of one or more immunostimulatory receptors; (iii) one or more modulators, each of which is an antagonist of signaling of one or more cytokines; (iv) one or more modulators, each of which is an agonist of one or more cytokine receptors; (v) one or more modulators, each of which modulates one or more cell surface molecules expressed or displayed on the cell surface of tumor cells or immune cells; (vi) one or more immune cells comprising CAR-T cells, CAR-NK cells, or hematopoietic stem cells; (vii) one or more immunogenic chemotherapeutic agents; and / or (viii) one or more modulators, each of which is an antagonist of one or more immunoinhibitory enzymes; A composition comprising:

13. The inhibitor of IL-23 signaling, (i) an IL-23 binding portion that is a recombinant protein that binds to IL-23, and optionally, the inhibitor of IL-23 signaling is an antibody or an antigen-binding fragment thereof, the antigen-binding fragment thereof comprising a crystallizable fragment (Fc) region, an antigen-binding fragment (Fab) region, a single-chain variable fragment (scFv), a light chain or a functional portion thereof, a variable region of the light chain (VL), a constant region of the light chain (CL), a heavy chain or a functional portion thereof, a variable region of the heavy chain (VH), a constant region of the heavy chain (CH), at least one phase complementarity determining regions (CDRs) or antigen-binding portions thereof, or a combination thereof, optionally wherein the antibody or antigen-binding fragment thereof comprises a monoclonal antibody that targets an IL-23 subunit or the IL-23p19 subunit, optionally wherein the antibody or antigen-binding fragment thereof comprises one or more of six CDRs or antigen-binding portions thereof selected from any one of risankizumab, guselkumab, tildrakizumab, brazikumab, and mirikizumab; (ii) an IL-23R binding moiety that is a recombinant protein that binds to IL-23R, and optionally (a) the inhibitor of IL-23 signaling is an antibody or antigen-binding fragment thereof, wherein the antigen-binding fragment thereof comprises a fragment crystallizable (Fc) region, a fragment antigen-binding (Fab) region, a single-chain variable fragment (scFv), a light chain or a functional portion thereof, a variable region of the light chain (VL), a constant region of the light chain (CL), a heavy chain or a functional portion thereof, a variable region of the heavy chain (VH), a constant region of the heavy chain (CH), at least one complementarity-determining region (CDR) or antigen-binding portion thereof, or a combination thereof; optionally, the antibody or antigen-binding fragment thereof comprises a monoclonal antibody that targets an IL-23 subunit or the IL-23p19 subunit; optionally, the antibody or antigen-binding fragment thereof comprises one or more of six CDRs or antigen-binding portions thereof selected from any one of risankizumab, guselkumab, tildrakizumab, brazikumab, and mirikizumab; or (b) the IL-23R binding portion comprises an antibody or antigen-binding fragment thereof, optionally wherein the antibody or antigen-binding fragment thereof comprises one or more of the six CDRs or antigen-binding portions thereof of AS2762900-00; or (iii) A composition according to claim 12, comprising a recombinant molecule according to any one of claims 1 to 4.

14. the second therapeutic agent comprises a modulatory agent that is an antagonist of one or more immune checkpoint proteins, and optionally (i) the immune checkpoint protein is programmed death-1 (PD1; CD279), programmed death-ligand 1 (PDL1), programmed death-ligand 2 (PDL2), cytotoxic T-lymphocyte antigen-4 (CTLA4; CD152), attenuator of B and T lymphocytes (BTLA), V-domain immunoglobulin suppressor of T-cell activation (VISTA), T-cell immunoglobulin and ITIM domain (TIGIT), lymphocyte activation gene 3 (LAG-3; CD223), T-cell immunoglobulin and mucin domain 3 (Tim-3; HAVCR2), carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), CD47, signal regulatory protein alpha (SIRPa), major histocompatibility complex class I, G (HLA-G), Ig-like transformant and / or the second therapeutic agent comprises a modulator that is an antagonist of PD1 signaling, optionally wherein the antagonist of PD1 signaling is a polypeptide that targets PD1, and / or the modulator is an inhibitor comprising a monoclonal antibody or antigen-binding fragment thereof that targets PD1 (CD279), optionally wherein the antibody or antigen-binding fragment thereof comprises one or more of six CDRs or antigen-binding portions thereof selected from pembrolizumab, nivolumab, cemiplimab, dostallimab, spartalizumab, camrelizumab, sintilimab, sasanlimab, tiselizumab, or toripalimab; (ii) the modulator is an inhibitor of the checkpoint protein selected from programmed death-1 ligand 1 (PDL1; CD274; B7-H1), programmed death-1 ligand 2 (PDL2), or both, and optionally the modulator is a polypeptide that targets PDL1, PDL2, or both, and / or the polypeptide is an antibody or antigen-binding fragment thereof that targets PDL1, PDL2, or both, and optionally the antibody or antigen-binding fragment thereof comprises one or more of six CDRs or antigen-binding portions thereof selected from any one of durvalumab, avelumab, or atezolizumab; (iii) the modulator is a polypeptide inhibitor of the checkpoint protein CTLA-4, and optionally the polypeptide is an antibody or antigen-binding fragment thereof that targets CTLA-4, and / or the antibody or antigen-binding fragment thereof comprises one or more of the six CDRs or antigen-binding portions thereof selected from any one of ipilimumab or tremelimumab; (iv) the modulator is a polypeptide inhibitor of the checkpoint protein LAG-3, and optionally the polypeptide is an antibody or antigen-binding fragment thereof that targets LAG-3, and / or the antibody or antigen-binding fragment thereof comprises one or more of the six CDRs or antigen-binding portions thereof selected from any one of leratolimab, fianlimab, Sym022, GSK2831781, TSR-033, ielumilimab, favezelimab, tebotelimab, FS118, or pubunalimab; or (v) The composition of claim 12, wherein said modulator is a polypeptide inhibitor of the checkpoint protein TIGIT, and optionally said polypeptide is an antibody or antigen-binding fragment thereof that targets TIGIT, and / or said antibody or antigen-binding fragment thereof comprises one or more of six CDRs or antigen-binding portions thereof selected from any one of tiragolumab, vibostolimab, BMS-986207, osipellimab, etigilimab, domvanalimab, EOS-448, SEA-TGT, ASP8374, COM902, or IBI939. (i) the second therapeutic agent comprises a modulator that is an agonist of one or more immunostimulatory receptors, optionally wherein the immunostimulatory receptors are selected from 4-1BB (CD137), inducible T-cell costimulatory factor (ICOS; CD278), OX-40 (CD134), glucocorticoid-induced TNFR-related protein (GITR; CD357), CD40, herpes virus entry mediator (HVEM), CD28, or CD27, and optionally wherein the second therapeutic agent is a polypeptide comprising CD40L or a CD40-binding fragment thereof and / or CD80 or CD86; or a CD28-binding fragment thereof; (ii) the second therapeutic agent comprises a modulator of a cell surface molecule expressed or presented on tumor cells or tumor-associated stromal cells, optionally wherein the cell surface molecule is selected from (a) a growth factor receptor, a transforming growth factor-beta receptor (TGFβR), a tumor necrosis factor receptor (TNFR) superfamily receptor, an Ig superfamily receptor, a vascular endothelial growth factor receptor (VEGFR), an epidermal growth factor receptor (EGFR), a platelet-derived growth factor receptor (PDGFR), a tumor cell surface molecule, a cytokine receptor, or a chemokine receptor, or (b) SIRPa or CD47, optionally wherein the second therapeutic agent inhibits binding of SIRPa to CD47, optionally wherein the second therapeutic agent is a polypeptide that binds to SIRPa or CD47, optionally wherein the polypeptide is 1) an antibody or antigen-binding fragment thereof that targets CD47, optionally wherein the antibody or antigen-binding fragment thereof comprises one or more of six CDRs or antigen-binding portions thereof selected from magrolimab, ZL-1201, TJ011133, STI-6643, SRF231, SHR-1603, IMC-002, IBI188, CC-90002, AO-176, or AK117; or 2) comprising the SIRPa extracellular domain or a CD47-binding fragment thereof, optionally wherein the polypeptide is selected from evoluptor, TTI-621, or TTI-622; (iii) the second therapeutic agent comprises a modulator of a cell surface molecule expressed or presented on an immune cell, and optionally: (a) the immune cell is a T cell, an NK cell, or a myeloid cell, and / or the cell surface molecule is a tumor necrosis factor receptor (TNFR) superfamily receptor, an Ig superfamily receptor, a cytokine receptor, a chemokine receptor, a T cell costimulatory molecule receptor, a T cell costimulatory molecule receptor, or a natural killer (NK) cell receptor, and / or the cell surface molecule is a myeloid cell inhibitory receptor or a myeloid cell stimulatory receptor; (b) the cell surface molecule is CA125, CA19-9, CD30, carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), CEACAM5 or cluster of differentiation 66e (CD66e), CEACAM6, DLL3, DLL4, DPEP3, or EGFR. EGFRvIII, GD2, HER2, HER3, HGF, IGF1R, IL13Ra2, LIV-1, LRRC15, MUC1, PRLR, PSCA, PSMA, PTK7, SEZ6, SLAMF7, TF, cMet, claudins, mesothelin, nectin-4, uPAR, GPNMB, CD79b, CD22, NaPi2b, SLTRK6, STEAP1, MUC16, CD37, GCC, AGC-16, 5T4, CD70, TROP2, CD74, CD27L, Fra, CD138, CA6, CD38, SLAMF7, BCMA, CD20 , CD19, CD33 or CD30, and optionally said modulator is an inhibitor comprising a monoclonal antibody or antigen-binding fragment thereof that targets said tumor cell surface molecule, and optionally said antibody or antigen-binding fragment thereof comprises one or more of six CDRs or antigen-binding portions thereof selected from any one of labetuzumab, sergituzumab, cetuximab, necitumumab, panitumumab, depatuxizumab, trastuzumab, pertuzumab, enfortumab, or sacituzumab; (c) the immune cells comprise antigen-presenting cells (APCs), myeloid-derived suppressor cells (MDSCs), dendritic cells, natural killer cells, or macrophages; and / or the immune cells comprise T H 17 cells, CD4 T cells, CD8 T cells, Treg cells, gamma delta T cells, NK cells, innate lymphoid cells (ILCs), or gamma delta T17 cells; or (d) the cell surface receptor is SIRPa or CD47, and optionally the second therapeutic agent inhibits binding of SIRPa to CD47, and optionally the second therapeutic agent is a polypeptide that binds to SIRPa or CD47, and optionally the polypeptide is 1) an antibody or antigen-binding fragment thereof that targets CD47, optionally wherein the antibody or antigen-binding fragment thereof comprises one or more of six CDRs or antigen-binding portions thereof selected from magrolimab, ZL-1201, TJ011133, STI-6643, SRF231, SHR-1603, IMC-002, IBI188, CC-90002, AO-176, or AK117; or 2) comprising the SIRPa extracellular domain or a CD47-binding fragment thereof, optionally wherein the polypeptide is selected from evoluptor, TTI-621, or TTI-622; (iv) the second therapeutic agent is an antagonist of signaling of one or more cytokines, optionally wherein the cytokine is transforming growth factor-beta (TGFb), and optionally wherein the modulator is an inhibitor of TGFb signaling selected from a small molecule kinase inhibitor, a polypeptide comprising TGFbRII ECD or a TGFb-binding fragment thereof, or an antibody or antigen-binding fragment thereof selected from an anti-TGFβ antibody, an anti-TGFβR antibody, an anti-GARP antibody, or an anti-LAP antibody, and optionally (a) the small molecule kinase inhibitor is a TGFβR small molecule kinase inhibitor comprising galunisertib; (b) the anti-TGFβ antibody or antigen-binding fragment thereof comprises one or more of the six CDRs or antigen-binding portions thereof of fresolimumab; or (c) the polypeptide comprising the TGFbRII ECD or TGFb-binding fragment thereof is selected from AVID200, vintrafusp alfa (M7824), anti-CTLA4-TGFbRII, SIRPa ECD-TGFbRII, anti-CEA-TGFbRII, anti-PSMA-TGFbRII, anti-IL6R-TGFbRII, anti-PD1-TGFbRII, anti-EGFR-TGFbRII, or anti-HER2-TGFbRII, and / or optionally, the cytokine is selected from one or more of the following: IL-4, IL-13, IL-10, IL-6, IL-1b, IL-17, IL-22, or VEGF, optionally; (1) the cytokine is IL-4 or IL-13, and / or the modulator is a polypeptide that targets IL4 receptor alpha (IL4Ra), and optionally the polypeptide is an antibody or antigen-binding fragment comprising one or more of the six CDRs of dupilumab or antigen-binding portions thereof; (2) the cytokine is IL1b, and optionally the second therapeutic agent comprises one or more of the six CDRs or antigen-binding portions thereof of anakinra or canakinumab; or (3) the cytokine is IL10, and optionally the second therapeutic agent comprises an IL10-binding sequence of the extracellular domain of IL10R, or an antibody or antigen-binding fragment thereof that targets IL10 or IL10R; or (v) The composition of claim 12, wherein the second therapeutic agent is an agonist of one or more cytokine receptors, optionally wherein the cytokine receptors are selected from IL12R, IL15R, and IL18R, and / or wherein the second therapeutic agent is a polypeptide comprising IL-12. (i) treatment with the combination of the first and second agents reduces or inhibits tumor growth, prevents or reduces severe immune-related adverse events, increases overall survival or progression-free survival, reduces or prevents adverse events or reduces toxicity, or reduces or prevents bone metastases or skeletal-related adverse events more effectively than treatment with the second agent alone; (ii) the cancer is selected from prostate cancer, pancreatic cancer, biliary tract cancer, colon cancer, rectal cancer, liver cancer, kidney cancer, lung cancer, testicular cancer, breast cancer, ovarian cancer, brain cancer, skin cancer, bladder cancer, and head and neck cancer, melanoma, sarcoma, multiple myeloma, leukemia, and / or lymphoma; (iii) the method comprises: (a) a therapeutic agent comprising an anti-CTLA4-TGFβRII molecule; or (b) one or more anti-cancer therapies to the subject, optionally wherein the one or more anti-cancer therapies comprise an immunotherapeutic agent, a chemotherapeutic molecule, an antibody, an antibody-drug conjugate, a small molecule kinase inhibitor, a hormonal agent, an androgen synthesis inhibitor, an androgen receptor antagonist, an anti-angiogenic agent, a cell therapy, a CAR-T cell therapy, a CAR-NK cell therapy, a radionuclide therapy, ionizing radiation, ultraviolet radiation, cryoablation, thermal ablation, a selective estrogen receptor modulator (SERM), a selective estrogen receptor degrading drug (SERD), or radiofrequency ablation, optionally wherein the immunotherapeutic agent is chosen from an immune checkpoint inhibitor, an immunostimulatory receptor agonist, an immunostimulatory cytokine / cytokine receptor agonist, an immunoinhibitory cytokine / cytokine receptor antagonist, a tumor vaccine, an immunomodulatory imid drug, a CAR-T cell, a CAR-NK cell, or an oncolytic virus; and / or (iv) The composition of claim 12, wherein the subject is a mammal, optionally a human.

Images