The present disclosure provides fusion proteins comprising an IL-10 polypeptide and a second polypeptide, e.g., an Fc polypeptide. Certain aspects of the present disclosure are directed to methods of treating a subject comprising administering the IL-10 fusion protein. In certain aspects, the subject is afflicted with a cancer.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application Ser. No. 62/970,957, filed Feb. 6, 2020; the disclosure of which is incorporated herein by reference.

SEQUENCE LISTING

[0002] Incorporated herein by reference in its entirety is a Sequence Listing named “20210205_SEQL_13311WOPCT_ST25.txt,” comprising SEQ ID NO:1 through SEQ ID NO:45, which include nucleic acid and/or amino acid sequences disclosed herein. The Sequence Listing has been submitted herewith in ASCII text format via EFS-Web, and thus constitutes both the paper and computer readable form thereof. The Sequence Listing was created on Feb. 3, 2021, and is approximately 104 KB in size.

BACKGROUND OF THE DISCLOSURE

[0003] IL-10 is a pleiotropic immunomodulatory cytokine produced by M, B, NK, and T cells (CD4+, CD8+, and Tregs). IL-10 binds as a noncovalent homodimer with high affinity to the IL-10 receptor alpha (IL-10Ra) leading to recruitment of the IL-10 receptor beta (IL-10Rβ). Receptor binding activates a complex signaling cascade, including phosphorylation of STAT3 and STAT1. Signaling through this pathway can lead to both anti-inflammatory and pro-inflammatory effects on various immune cell subsets where the receptor is expressed. The pro-inflammatory effects include expansion, activation, and cytolytic potentiation of primed CD8.sup.+ T cells and NK cells. Comparatively, the anti-inflammatory effects include suppression of myeloid cytokine production and priming capacity. Treatment with an IL-10 molecule can induce the expansion and activation of tumor-specific CD8.sup.+ T and NK cells, driving IFNγ-dependent tumor killing mechanisms in solid tumors, and can have potential benefit in combination with IO agents or standard of care (Autio, et al., Current Oncology Reports, 2019; 2 1:19).

[0004] Despite its dual immunomodulatory roles, IL-10 has been identified as an anti-tumor agent. Early studies in the IL-10 knockout mouse revealed a strain-dependent prevalence for development of colon adenocarcinoma (Berg et al., J Clin Invest, 1996; 98:1010-1020) as well increased incidence of DMBA induced skin tumors with reduced T cells (Mumm, et al, Cancer cell, 2011; 20:781-796). Similarly, humans with deficiencies in IL-10 signaling through mutations in the IL-10 receptor develop lymphomas with a much lower frequency of infiltrating cytolytic T cells (Neven et al., Blood, 2013; 122:3713-3722). Beyond genetic evidence, therapeutic administration of recombinant IL-10 or pegylated IL-10 have also shown anti-tumor activity in several mouse models, where efficacy was shown to require CD8+ T cells and IFNγ-dependent upregulation of tumor MHC class I antigens (Mumm, et al, Cancer cell, 2011; 20:781-796). However, both in mouse and man, repeated daily dosing is often required for therapeutic activity due to the cytokine's short half-life. A pegylated human IL-10 (PEGIL-10), now in clinical trials, is showing encouraging clinical signals across several tumor indications, yet still requires daily dosing to maintain a pharmacokinetic (PK) profile needed for activity (Naing, et al., Cancer Cell, 2018; 34:775-791). Moreover, hematologic toxicity such as anemia and thrombocytopenia has been observed clinically with repeated daily dosing (Autio, et al., Current Oncology Reports, 2019; 2 1:19; Naing, et al., Journal of Clinical Oncology, 2016; 34, 3562-3569; Sosman, et al., British Journal of Haematology, 2000; 111(1), 104-111). Thus, a human IL-10 agonist that is efficacious with a less frequent dosage regimen is needed. Such less frequent dosing not only obviates the need for daily injection, but also facilitates recovery from hematologic toxicity between doses.

SUMMARY OF THE DISCLOSURE

[0005] Certain aspects of the present disclosure are directed to an IL-10 fusion protein comprising (i) an IL-10 polypeptide, comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1; and (ii) a second polypeptide, wherein the IL-10 fusion protein comprises an IL-10 activity. In some aspects, the second polypeptide comprises an albumin polypeptide. In some aspects, the second polypeptide comprises an Fc polypeptide. In some aspects, the Fc polypeptide comprises an amino acid sequence having at least about 95% sequence identity to an amino acid sequence selected from SEQ ID NOs: 4-12.

[0006] Certain aspects of the present disclosure are directed to an IL-10 fusion protein comprising (i) an IL-10 polypeptide, comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1; and (ii) a second polypeptide comprising an Fc polypeptide, wherein the IL-10 fusion protein is capable of treating cancer in a subject in need thereof when the IL-10 fusion protein is administered to the subject no more than about once a week. In some aspects, the IL-10 fusion protein is capable of treating cancer in a subject in need thereof when the IL-10 fusion protein is administered to the subject no more than once about every two weeks. In some aspects, the IL-10 fusion protein is capable of treating cancer in a subject in need thereof when the IL-10 fusion protein is administered to the subject no more than once about every four weeks.

[0007] In some aspects, the second polypeptide is fused to the N-terminus of the IL-10 polypeptide. In some aspects, the second polypeptide is fused to the C-terminus of the IL-10 polypeptide.

[0008] In some aspects, the IL-10 polypeptide is fused to the second polypeptide by a linker. In some aspects, the linker comprises at least about 4 amino acids, at least about 5 amino acids, at least about 6 amino acids, at least about 7 amino acids, at least about 8 amino acids, at least about 9 amino acids, at least about 10 amino acids, at least about 11 amino acids, at least about 12 amino acids, at least about 13 amino acids, at least about 14 amino acids, at least about 15 amino acids, at least about 16 amino acids, at least about 17 amino acids, at least about 18 amino acids, at least about 19 amino acids, at least about 20 amino acids, or at least about 21 amino acids. In some aspects, the linker comprises at least about 15 amino acids. In some aspects, the linker comprises at least about 20 amino acids. In some aspects, the linker comprises at least about 21 amino acids.

[0009] In some aspects, the linker comprises a Glycine and a Serine. In some aspects, the linker comprises a GGGGS (SEQ ID NO: 39) motif or a GGGS (SEQ ID NO: 38) motif. In some aspects, the linker comprises an amino acid sequence selected from SEQ ID NOs: 38-45.

[0010] In some aspects, the IL-10 polypeptide comprises an amino acid sequence having at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1. In some aspects, the IL-10 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 1.

[0011] In some aspects, the Fc polypeptide comprises an amino acid sequence having at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to an amino acid sequence selected from SEQ ID NOs: 4-12. In some aspects, the Fc polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 4-12.

[0012] In some aspects, the IL-10 fusion protein comprises an amino acid sequence having at least 95% sequence identity to an amino acid sequence selected from SEQ ID NOs: 14-32. In some aspects, the IL-10 fusion protein comprises an amino acid sequence having at least 98% sequence identity to an amino acid sequence selected from SEQ ID NOs: 14-32. In some aspects, the IL-10 fusion protein comprises an amino acid sequence having at least 99% sequence identity to an amino acid sequence selected from SEQ ID NOs: 14-32. In some aspects, the IL-10 fusion protein comprises an amino acid sequence selected from SEQ ID NOs: 14-32 with 3 or fewer substitutions, insertions, or deletions. In some aspects, the IL-10 fusion protein comprises an amino acid sequence selected from SEQ ID NOs: 14-32 with 2 or fewer substitutions, insertions, or deletions. In some aspects, the IL-10 fusion protein comprises an amino acid sequence selected from SEQ ID NOs: 14-32 with 1 substitution, insertion, or deletion. In some aspects, the IL-10 fusion protein comprises an amino acid sequence selected from SEQ ID NOs: 14-32.

[0013] In some aspects, the IL-10 fusion protein comprises an amino acid sequence having at least 95% sequence identity to an amino acid sequence selected from SEQ ID NOs: 33-36. In some aspects, the IL-10 fusion protein comprises an amino acid sequence having at least 98% sequence identity to an amino acid sequence selected from SEQ ID NOs: 33-36. In some aspects, the IL-10 fusion protein comprises an amino acid sequence having at least 99% sequence identity to an amino acid sequence selected from SEQ ID NOs: 33-36. In some aspects, the IL-10 fusion protein comprises an amino acid sequence selected from SEQ ID NOs: 33-36 with 3 or fewer substitutions, insertions, or deletions. In some aspects, the IL-10 fusion protein comprises an amino acid sequence selected from SEQ ID NOs: 33-36 with 2 or fewer substitutions, insertions, or deletions. In some aspects, the IL-10 fusion protein comprises an amino acid sequence selected from SEQ ID NOs: 33-36 with 1 substitution, insertion, or deletion. In some aspects, the IL-10 fusion protein comprises an amino acid sequence selected from SEQ ID NOs: 33-36.

[0014] In some aspects, the IL-10 fusion protein comprises an IL-10 dimer comprising, a first polypeptide and a second polypeptide, wherein the first polypeptide comprises an IL-10 fusion protein disclosed herein, and wherein the second polypeptide comprises a second Fc polypeptide. In some aspects, the second polypeptide comprises a second IL-10 polypeptide fused to the second Fc polypeptide.

[0015] In some aspects, the IL-10 dimer is a homodimer. In some aspects, the first polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 1 and the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 1. In some aspects, the first polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 14-36 and the second polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 14-36.

[0016] In some aspects, the IL-10 dimer is a heterodimer.

[0017] In some aspects, the first polypeptide and the second polypeptide are linked by a covalent bond. In some aspects, the first polypeptide and the second polypeptide are linked by a disulfide bond. In some aspects, the first polypeptide and the second polypeptide are linked by a peptide bond. In some aspects, the first polypeptide and the second polypeptide are linked by a peptide linker. In some aspects, the peptide linker is a cleavable linker.

[0018] Certain aspects of the present disclosure are directed to a polynucleotide or a set of polynucleotides encoding an IL-10 fusion protein disclosed herein.

[0019] Certain aspects of the present disclosure are directed to a vector or a set of vectors comprising a polynucleotide or a set of polynucleotides disclosed herein. In some aspects, the vector is a viral vector.

[0020] Certain aspects of the present disclosure are directed to a host cell comprising an IL-10 fusion protein disclosed herein, a polynucleotide or a set of polynucleotides disclosed herein, or a vector or a set of vectors disclosed herein. In some aspects, the host cell is a mammalian cell. In some aspects, the host cell is selected from a Chinese hamster ovary (CHO) cell, an HEK293 cell, a BHK cell, a murine myeloma cell (NS0 and Sp2/0), a monkey kidney (COS) cell, a VERO cell, a fibrosarcoma HT-1080 cell, and a HeLa cell.

[0021] Certain aspects of the present disclosure are directed to a pharmaceutical composition comprising an IL-10 fusion protein disclosed herein, a polynucleotide or a set of polynucleotides disclosed herein, or a vector or a set of vectors disclosed herein, and a pharmaceutically acceptable excipient.

[0022] Certain aspects of the present disclosure are directed to a method of treating a cancer in a subject in need thereof, comprising administering to the subject an effective amount of an IL-10 fusion protein disclosed herein, a polynucleotide or a set of polynucleotides disclosed herein, a vector or a set of vectors disclosed herein, or a pharmaceutical composition disclosed herein.

[0023] Certain aspects of the present disclosure are directed to a method of killing a cancer cell in a subject in need thereof, comprising administering to the subject an effective amount of an IL-10 fusion protein disclosed herein, a polynucleotide or a set of polynucleotides disclosed herein, a vector or a set of vectors disclosed herein, or a pharmaceutical composition disclosed herein.

[0024] Certain aspects of the present disclosure are directed a method of treating cancer in a subject in need thereof, comprising administering to the subject an effective amount of an IL-10 fusion protein at a dosing interval of at least about 7 days, wherein the IL-10 fusion protein comprises an IL-10 polypeptide and a second polypeptide, which comprises an albumin polypeptide or an Fc polypeptide.

[0025] Certain aspects of the present disclosure are directed a method of killing a cancer call in a subject in need thereof, comprising administering to the subject an effective amount of an IL-10 fusion protein at a dosing interval of at least about 7 days, wherein the IL-10 fusion protein comprises an IL-10 polypeptide and a second polypeptide, which comprises an albumin polypeptide or an Fc polypeptide.

[0026] In some aspects, the second polypeptide is an albumin polypeptide. In some aspects, the second polypeptide is an Fc polypeptide. In some aspects, the IL-10 fusion protein further comprises a linker. In some aspects, the linker comprises a linker disclosed herein.

[0027] In some aspects, the IL-10 fusion protein is administered at a dosing interval of at least about 7 days, at least about 10 days, at least about 14 days, at least about 17 days, at least about 21 days, at least about 24 days, or at least about 28 days. In some aspects, the IL-10 fusion protein is administered no more than once week. In some aspects, the IL-10 fusion protein is administered no more than once every 2 weeks. In some aspects, the IL-10 fusion protein is administered no more than once every 3 weeks. In some aspects, the IL-10 fusion protein is administered no more than once every 4 weeks. In some aspects, the IL-10 fusion protein is administered at a dosing interval of at least about 7 days to at least about 28 days. In some aspects, the IL-10 fusion protein is administered at a dosing interval of at least about 14 days. In some aspects, the IL-10 fusion protein is administered at a dosing interval of at least about 21 days. In some aspects, the IL-10 fusion protein is administered at a dosing interval of at least about 28 days. In some aspects, the IL-10 fusion protein is administered about once a week. In some aspects, the IL-10 fusion protein is administered once about every 2 weeks. In some aspects, the IL-10 fusion protein is administered once about every 3 weeks. In some aspects, the IL-10 fusion protein is administered once about every 4 weeks. In some aspects, the IL-10 fusion protein is administered once about every 6 weeks. In some aspects, the IL-10 fusion protein is administered once about every 2 months.

[0028] In some aspects, the IL-10 fusion protein is administered as a single dose. In some aspects, the effective amount of the IL-10 fusion protein consists essentially of or consists of a single dose.

[0029] In some aspects, the IL-10 fusion protein comprises an amino acid sequence having at least 99% sequence identity to an amino acid sequence selected from SEQ ID NOs: 14-32, and wherein the IL-10 fusion protein is administered at a dosing interval of at least about 2 weeks. In some aspects, the IL-10 fusion protein comprises an amino acid sequence selected from SEQ ID NOs: 14-32. In some aspects, the IL-10 fusion protein comprises of SEQ ID NO: 14. In some aspects, the IL-10 fusion protein is administered once every 2 weeks. In some aspects, the IL-10 fusion protein is administered about once every 3 weeks. In some aspects, the IL-10 fusion protein is administered once about every 4 weeks. In some aspects, the IL-10 fusion protein is administered once every 5 weeks. In some aspects, the IL-10 fusion protein is administered once about every 6 weeks.

[0030] In some aspects, the cancer comprises a tumor. In some aspects, the cancer is selected from the group consisting of small-cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), squamous NSCLC, nonsquamous NSCLC, glioma, gastrointestinal cancer, renal cancer, clear cell carcinoma, ovarian cancer, liver cancer, colorectal cancer, endometrial cancer, kidney cancer, renal cell carcinoma (RCC), prostate cancer, hormone refractory prostate adenocarcinoma, thyroid cancer, neuroblastoma, pancreatic cancer, glioblastoma (glioblastoma multiforme), cervical cancer, stomach cancer, bladder cancer, hepatoma (hepatocellular carcinoma, HCC), breast cancer, colon carcinoma, head and neck cancer (or carcinoma), head and neck squamous cell carcinoma (HNSCC), gastric cancer, germ cell tumor, pediatric sarcoma, sinonasal natural killer/T-cell lymphoma, melanoma, metastatic malignant melanoma, cutaneous or intraocular malignant melanoma, mesothelioma, bone cancer, skin cancer, uterine cancer, cancer of the anal region, testicular cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, solid tumors of childhood, cancer of the ureter, carcinoma of the renal pelvis, neoplasm of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain cancer, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, squamous cell cancer, environmentally-induced cancers including those induced by asbestos, virus-related cancers or cancers of viral origin, human papilloma virus (HPV)-related or -originating tumors, and combinations of said cancers.

[0031] In some aspects, the cancer is selected from acute leukemia (ALL), acute myelogenous leukemia (AML), chronic lymphocytic leukemia (CLL), and chronic myelogenous leukemia (CML), undifferentiated AML, myeloblastic leukemia, myeloblastic leukemia, promyelocytic leukemia, myelomonocytic leukemia, monocytic leukemia, erythroleukemia, megakaryoblastic leukemia, isolated granulocytic sarcoma, chloroma, Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma (NHL), B-cell lymphoma, T-cell lymphoma, lymphoplasmacytoid lymphoma, monocytoid B-cell lymphoma, mucosa-associated lymphoid tissue (MALT) lymphoma, anaplastic large-cell lymphoma, adult T-cell lymphoma/leukemia, mantle cell lymphoma, angio immunoblastic T-cell lymphoma, angiocentric lymphoma, intestinal T-cell lymphoma, primary mediastinal B-cell lymphoma, precursor T-lymphoblastic lymphoma, T-lymphoblastic peripheral T-cell lymphoma, lymphoblastic lymphoma, post-transplantation lymphoproliferative disorder, true histiocytic lymphoma, primary central nervous system lymphoma, primary effusion lymphoma, lymphoblastic lymphoma (LBL), hematopoietic tumors of lymphoid lineage, acute lymphoblastic leukemia, diffuse large B-cell lymphoma, Burkitt's lymphoma, follicular lymphoma, diffuse histiocytic lymphoma (DHL), immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, cutaneous T-cell lymphoma (CTLC), lymphoplasmacytoid lymphoma (LPL) with Waldenstrom's macroglobulinemia; myeloma, IgG myeloma, light chain myeloma, nonsecretory myeloma, smoldering myeloma (indolent myeloma), solitary plasmocytoma, multiple myeloma, chronic lymphocytic leukemia (CLL), hairy cell lymphoma; and any combinations of said cancers.

[0032] In some aspects, the cancer is selected from RCC, NSCLC, gastric cancer, HCC, Squamous cell carcinoma of the head and neck (SCCHN), and any combinations of said cancers. In some aspects, the cancer is selected from RCC, NSCLC, gastric cancer, SCCHN, and any combinations of said cancers. In some aspects, the cancer is selected from melanoma, bladder cancer, pancreatic cancer, colon cancer, SCLC, mesothelioma, hepatocellular carcinoma, prostate cancer, multiple myeloma, and combinations of said cancers.

[0033] In some aspects, the method further comprises administering to the subject a second anticancer therapy. In some aspects, the second anticancer therapy comprises a therapy selected from the group consisting of an immunotherapy, a chemotherapy, a radiation therapy, a surgery, an agent that activates innate immune cells, an agent that enhances survival of NK and/or CD8+ T-cells, an agent that inhibits Tregs (T regulatory cells), TAMs (tumor-associated macrophages), CAFs (cancer-associated fibroblasts), or MDSCs (myeloid-derived suppressor cells), and any combination thereof. In some aspects, the second anticancer therapy comprises an effective amount of an antibody or an antigen-binding fragment thereof that specifically binds a protein selected from Inducible T cell Co-Stimulator (ICOS), CD137 (4-1BB), CD134 (OX40), NKG2A, CD27, CD38, CD73, CD96, Glucocorticoid-Induced TNFR-Related protein (GITR), and Herpes Virus Entry Mediator (HVEM), Programmed Death-1 (PD-1), Programmed Death Ligand-1 (PD-L1), CTLA-4, B and T Lymphocyte Attenuator (BTLA), T cell Immunoglobulin and Mucin domain-3 (TIM-3), Lymphocyte Activation Gene-3 (LAG-3), adenosine A2a receptor (A2aR), Killer cell Lectin-like Receptor G1 (KLRG-1), Natural Killer Cell Receptor 2B4 (CD244), CD160, T cell Immunoreceptor with Ig and ITIM domains (TIGIT), and the receptor for V-domain Ig Suppressor of T cell Activation (VISTA), KIR, TGFβ, IL-10, IL-8, B7-H4, Fas ligand, CXCR4, mesothelin, CEACAM-1, CD52, HER2, SLAMF7, BCMA, MICA, MICB, CCR8, and any combination thereof.

[0034] In some aspects, the second anticancer therapy comprises an antibody or antigen-binding fragment thereof that specifically binds PD-1 (“an anti-PD-1 antibody”). In some aspects, the anti-PD-1 antibody comprises nivolumab or pembrolizumab.

[0035] In some aspects, the second anticancer therapy comprises an antibody or an antigen-binding fragment thereof that specifically binds PD-L1 (“an anti-PD-L1 antibody”). In some aspects, the anti-PD-L1 antibody is selected from atezolizumab, durvalumab, and avelumab.

[0036] In some aspects, the second anticancer therapy comprises an antibody or an antigen-binding fragment thereof that specifically binds CTLA-4 (“an anti-CTLA-4 antibody”). In some aspects, the anti-CTLA-4 antibody comprises tremelimumab or ipilimumab.

[0037] In some aspects, the second anticancer therapy comprises an antibody or an antigen-binding fragment thereof that specifically binds CTLA-4, e.g., tremelimumab or ipilimumab, and an antibody or antigen-binding fragment thereof that specifically binds PD-1, e.g., nivolumab or pembrolizumab. In some aspects, the second anticancer therapy comprises an antibody or an antigen-binding fragment thereof that specifically binds CTLA-4, e.g., tremelimumab or ipilimumab, and an antibody or antigen-binding fragment thereof that specifically binds PD-L1, e.g., atezolizumab, durvalumab, or avelumab.

[0038] In some aspects, the second anticancer therapy comprises a chemotherapy selected from a proteasome inhibitor, an IMiD, a Bet inhibitor, an IDO antagonist, a platinum-based chemotherapy, STING agonists, NLRP3 agonists, TLR7 agonists, and any combination thereof.

[0039] In some aspects, the second therapy comprises an agent elected from doxorubicin (ADRIAMYCIN®), cisplatin, carboplatin, bleomycin sulfate, carmustine, chlorambucil (LEUKERAN®), cyclophosphamide (CYTOXAN®; NEOSAR®), lenalidomide (REVLIMID®), bortezomib (VELCADE®), dexamethasone, mitoxantrone, etoposide, cytarabine, bendamustine (TREANDA®), rituximab (RITUXAN®), ifosfamide, Folinic acid (leucovorin), Fluorouracil (5-FU), Oxaliplatin (Eloxatin), FOLFOX, Paclitaxel, Docetaxel, vincristine (ONCOVIN®), fludarabine (FLUDARA®), thalidomide (THALOMID®), alemtuzumab (CAMPATH®, ofatumumab (ARZERRA®), everolimus (AFINITOR®, ZORTRESS®), and carfilzomib (KYPROLISTM).

[0040] In some aspects, the second anticancer therapy comprises an agent that enhances survival of NK and/or CD8+ T-cells selected from an agent comprising IL-2, such as pegylated IL-2, IL-18, and IL-15.

[0041] In some aspects, the second anticancer therapy comprises a CAR-T therapy, such as CD19-targeted CAR-T.

[0042] In some aspects, the second anticancer therapy comprises a bispecific antibody therapy, such as CD3-targeted biospecific antibody, e.g., anti-CD3/CD20, anti-CD3/BCMA biospecifics.

[0043] In some aspects, the second anticancer therapy comprises a standar-of-care therapy, such as an anti-angiogenic therapy (e.g., Bevacuzimab, Sorafinib etc), or radiation.

[0044] Certain aspects of the present disclosure are directed to a method of preparing an IL-10 fusion protein, comprising expressing a polynucleotide or a set of polynucleotides disclosed herein or a vector or a set of vectors disclosed herein in a host cell under suitable conditions. In some aspects, the method further comprises collecting the IL-10 fusion protein.