Therapeutic agent containing a novel IL10 agonist

Abstract

The present disclosure relates to IL10 agonists with improved anti-tumor therapeutic efficacy.

Description

[Technical Field]

[0001] The present invention relates to novel IL10 agonists and methods of using them. [Background technology]

[0002] The cytokine interleukin-10 (IL-10 or IL10), also known as human cytokine synthesis inhibitory factor (CSIF), is a pleiotropic cytokine that regulates multiple immune responses through its effects on T cells, B cells, macrophages, and antigen-presenting cells (APCs). IL10 is primarily expressed in macrophages, but expression has also been detected in activated T cells, B cells, mast cells, and monocytes. IL10 was initially reported to inhibit immune responses due to its suppression of antigen presentation (by downregulating the expression of major histocompatibility complex type II (MHC-II) and the costimulatory ligands CD80 / CD86), suppression of inflammatory cytokine release by myeloid cells, and inhibition of T cell priming (through suppression of CD28 signaling). However, more recent studies have also described immunostimulatory roles for IL10 through costimulation of B cells, enhancement of the cytolytic activity of NK cells, and enhancement of the proliferation, cytokine release, and cytolytic activity of cytolytic T cells (reviewed by [1]).

[0003] Human IL10 is a noncovalently linked homodimer, and its receptor is a heterotetrameric complex composed of two IL10Rα (also called IL10R1) and two IL10Rβ (also called IL10R2) molecules. IL10Rα is expressed on all IL10-responsive cells, whereas IL10Rβ is constitutively expressed on most cell types. Upon binding to IL10, IL10Rα induces a conformational change in IL10Rβ, allowing IL10Rβ to also bind to IL10. Once the IL10 / IL10Rα / IL10Rβ complex is assembled, the tyrosine kinases Jak1 and Tyk2 are activated, phosphorylating specific tyrosine residues in the intracellular domain of IL10Rα, leading to the recruitment of signal transducer and activator of transcription 3 (STAT3), which mediates downstream signaling of IL10. Unlike IL10Rα, which is unique to IL10, the IL10Rβ subunit is shared by receptors for other type II cytokines, including IL22, IL26, and INFλ (reviewed by Non-Patent Document 2).

[0004] As a result of its pleiotropic activities, IL10 has been implicated in a wide range of diseases, disorders and conditions, including inflammatory conditions, immune-related disorders, fibrotic disorders and cancer. One drawback to the therapeutic use of IL-10, especially recombinant IL-10 in any form, is its short serum half-life. Loss of IL-10 activity in vivo is thought to be due to several factors, including renal clearance and proteolysis. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Mosser and Zhang, 2008, Immunol Rev. 226:205-18 [Non-patent document 2] Shouval et al., 2014, Adv. Immunol. 122:177-210 Summary of the Invention [Problem to be solved by the invention]

[0006] It would be advantageous to have an IL10 agonist that can withstand systemic exposure during therapy by enhancing the circulating lifetime of IL10 (delayed clearance), its solubility, and stability. The present disclosure addresses this and other related needs in the art. [Means for solving the problem]

[0007] The present disclosure stems from the discovery of IL10 agonists that have surprisingly improved therapeutic efficacy in vivo, particularly anti-tumor activity. IL10 moieties that may be used in the IL10 agonists of the present disclosure are described in Section 6.3.

[0008] Fc domains that may be used in the IL10 agonists of the present disclosure are described in Section 6.4. Targeting moieties that may be used in the IL10 agonists of the present disclosure are described in Section 6.5.

[0009] Stabilizing moieties that may be used in the IL10 agonists of the present disclosure are described in Section 6.6. Various exemplary configurations of the IL10 agonists of the present disclosure are described in specific embodiments 60-77 below.

[0010] Linkers that can be used to connect different components of the IL10 agonists of the present disclosure are described in Section 6.7. The present disclosure further provides nucleic acids encoding the IL10 agonists of the present disclosure. The nucleic acids encoding the IL10 agonists may be a single nucleic acid (e.g., a vector encoding all polypeptide chains of the IL10 agonist) or multiple nucleic acids (e.g., two or more vectors encoding multiple different polypeptide chains of the IL10 agonist). The present disclosure further provides host cells and cell lines engineered to express the nucleic acids and IL10 agonists of the present disclosure. The present disclosure further provides methods of producing the IL10 agonists of the present disclosure. Exemplary nucleic acids, host cells, cell lines, and methods of producing the IL10 agonists of the present disclosure are described in Section 6.8 below and in specific embodiments 199-204.

[0011] The present disclosure further provides pharmaceutical compositions comprising the IL10 agonists of the present disclosure. Exemplary pharmaceutical compositions are described in Section 6.8.3 below and in specific embodiments 211-223.

[0012] Further provided herein are methods of using the IL10 agonists and pharmaceutical compositions of the present disclosure, for example, to treat cancer and immune disorders. Exemplary methods are described in Section 6.10. The IL10 agonists of the present disclosure are useful in combination therapy, for example, as an adjunct to CART therapy. Exemplary combination therapy methods are disclosed in Section 6.11. Specific embodiments of the therapeutic methods of the present disclosure are described below in specific embodiments 224-304. [Brief explanation of the drawings]

[0013] [Figure 1A] Diagram showing the differential effects of IL10 on the priming and effector functions of CD8 T cells. [Figure 1B] Same as above. [Figure 2]

[0023] Figures 2A and 2B show the general format of an IL10 agonist, and Figure 2B shows a specific embodiment of an IL10 agonist of the present disclosure. These illustrations are intended to depict the N- to C-terminal order of the domains contained in the IL10 agonist and are not intended to convey scale or three-dimensional organization. IL10M11 (not shown) is similar to IL10M3, but has a murine IL10 portion instead of the human IL10 portion of IL10M3. [Figure 3-1] Figure 3 shows the activity of IL10 muteins on STAT3-mediated luciferase reporter activity. Recombinant IL10 and IL10 muteins increase STAT3-responsive element-driven luciferase activity in engineered Ramos / STAT3-Luc (Figures 3A-C) and TF-1 / STAT3-Luc (Figures 3D-F) reporter cells. Graphs are divided by IL10 agonist used: IL10M1 (Figures 3A-D), IL10M2 (Figures 3B-E), or IL10M3 (Figures 3C-F). Open squares represent isotype controls (human IgG4 stealth for Figures 3A-E, human IgG4 stealth for Figures 3B-E, mouse IgG1 for Figures 3C-F). Triangles represent commercially available human IL10 (purchased from Peptrotech). [Figure 3-2] Same as above. [Figure 4-1]Figure 4 shows that IL10 muteins suppress cytokine release from primary human T cells from donor 5500Y to levels comparable to recombinant human IL10. Figure 4 shows release of IL2 (Figures 4A, 4D, 4G), TNFα (Figures 4B, 4E, 4H), and IFNγ (Figures 4C, 4F, 4I) from T cells from donor 5500Y cocultured with mitomycin C-treated allogeneic PBMCs and IL10M1 (Figures 4A-C), IL10M2 (Figures 4D-F), or IL10M3 (Figures 4G-I). The graphs are separated by the IL10 mutein used: black circles represent IL10 muteins, open squares represent isotype controls (hIgG4s for IL10M1 and IL10M2, mIgG1 for IL10M3), and grey triangles represent recombinant human IL10. [Figure 4-2] Same as above. [Figure 4-3] Same as above. [Figure 5-1] Figure 5 shows that IL10 muteins suppress cytokine release from primary human T cells from donor 6900M to levels comparable to recombinant human IL10. Figure 5 shows release of IL2 (Figures 5A, 5D, 5G), TNFα (Figures 5B, 5E, 5H), and IFNγ (Figures 5C, 5F, 5I) from T cells from donor 6900M cocultured with mitomycin C-treated allogeneic PBMCs and IL10M1 (Figures 5A-C), IL10M2 (Figures 5D-F), or IL10M3 (Figures 5G-I). The graphs are separated by the IL10 mutein used: black circles represent IL10 muteins, open squares represent isotype controls (hIgG4s for IL10M1 and IL10M2, mIgG1 for IL10M3), and grey triangles represent recombinant human IL10. [Figure 5-2] Same as above. [Figure 5-3] Same as above. [Figure 6] Figure 6 shows the antitumor activity of IL10 muteins compared to isotype controls in a syngeneic mouse model. Tumor volumes are shown on days 11 (Figure 6A) and 32 (Figure 6B). [Figure 7-1]FIG. 1 shows the activity of various IL10 muteins in a STAT3 luciferase assay. [Figure 7-2] Same as above. [Figure 7-3] Same as above. [Figure 8A] Figure 8 shows the antitumor activity of IL10M11. IL10M11 has antitumor activity against various tumor cell lines after prophylactic or therapeutic administration (Figures 8A-8E). IL10M11 administration increases the frequency of CD4 and CD8 T cells producing TNFα and IFNγ (Figures 8F and 8G, respectively). IL10M11 induces long-term memory and rejection of secondary tumor burden (Figures 8H and 8I, respectively). [Figure 8B] Same as above. [Figure 8C] Same as above. [Figure 8D] Same as above. [Figure 8E-1] Same as above. [Figure 8E-2] Same as above. [Figure 8E-3] Same as above. [Figure 8F] Same as above. [Figure 8G] Same as above. [Figure 8H] Same as above. [Figure 8I] Same as above. [Figure 9-1] Figure 1 shows the results of administering IL10M11 in combination with a PD-1 antagonist and a CD40 agonist. These results demonstrate that modulation of these pathways enhances the sustained primary and memory antitumor responses induced by IL10M11. [Figure 9-2] Same as above. [Figure 10-1]Figure 1 shows the density of total CD45+ immune cells and analyzed immune cell subsets in A20, Colon25, MC38, and B16F10 tumors. These results show that the density of total CD45+ immune cells was much higher in A20, Colon26, and MC38 cells than in B16F10 tumors, and that B16F10 tumors had fewer Ki67-proliferating CD4 and CD8 T cells and memory phenotypes. [Figure 10-2] Same as above. [Figure 10-3] Same as above. [Figure 11-1] Figure 1 shows the expression of PD-1 and IL10R1 on various myeloid cells in the spleen, draining lymph nodes (dLNs), and tumors. IL10R1 expression on T cells, particularly CD8+ T cells, was highly restricted to tumor-infiltrating CD8+ T cells, with almost no expression observed on T cells in secondary lymphoid tissues, such as the spleen and draining LNs of MC38-cOVA and Colon26. Nearly all IL10R1+ CD8+ T cells expressed PD1. [Figure 11-2] Same as above. [Figure 11-3] Same as above. [Figure 11-4] Same as above. [Figure 11-5] Same as above. [Figure 11-6] Same as above. [Figure 12-1] Figure 1 shows the expression of PD-L1 in four different tumor types (A20, Colon26, MC38, and B16F10). These results indicate that there were differences in PD-L1 expression depending on the tumor type. [Figure 12-2] Same as above. [Figure 12-3] Same as above. [Figure 13-1]Experimental design of the A20 tumor model study (Figure 13A) and results of treatment with control, IL10M11, or PD-1 antagonist antibody (Figures 13B and 13C). 150 mm3 A20 B-cell lymphomas responded to PD-1 Ab treatment, showing significant tumor growth delay and approximately 50% tumor-free survival. IL10M11 induced an even higher and broader immune response against A20, resulting in approximately 85% tumor-free survival. [Figure 13-2] Same as above. [Figure 14-1] Diagram showing the experimental design of the Colon26 tumor model study (FIG. 14A) and the results of treatment with control, IL10M11, or PD-1 antagonist antibody (FIGS. 14B and 14C). 100 mm tumors were completely resistant to PD1Ab treatment. Treatment with IL10M11 resulted in approximately 85% tumor-free survival. [Figure 14-2] Same as above. [Figure 15-1] Experimental design of the B16F10 tumor model study (FIG. 15A) and results of treatment with control, IL10M11, or PD-1 antagonist antibody (FIG. 15B and FIG. 14). 100 mm3 B16F10 tumors were largely unresponsive to PD1Ab. IL10M11 significantly delayed tumor growth and rendered the majority of tumors reactive. [Figure 15-2] Same as above. [Figure 16-1] Figure 16A shows the experimental design of the MC38 tumor model study and the results of treatment with control, IL10M11, PD-1 antagonist antibody, or the combination of IL10M11 and PD-1 antagonist antibody (Figures 16B and 16C). PD1 Ab and IL10-Fc both had antitumor effects comparable to those of the single agents. The combination of PD1 Ab and IL10-Fc dramatically increased the antitumor response by slowing tumor growth and increasing the frequency of tumor-free survival. [Figure 16-2] Same as above. [Figure 17A-1]Figure 17 shows results from immune profiling of the tumor microenvironment following treatment. As shown, IL10 treatment significantly increased CD8 T cell density in both the immunogenic MC38 tumor model (Figure 17A) and the less immunogenic 4T1 tumor model (Figure 17B), which correlated with improved prognosis in both cases. [Figure 17A-2] Same as above. [Figure 17B-1] Same as above. [Figure 17B-2] Same as above. [Figure 18A] Diagram showing the density of PD1+CD8 T cells in MC38 tumors (FIG. 18A) and 4T1 tumors (FIG. 18B). [Figure 18B] Same as above. [Figure 19-1] FIG. 1 shows serum levels of various cytokines following treatment with IL10M11 in naive, non-tumor-bearing mice. [Figure 19-2] Same as above. [Figure 20A] Diagram showing serum levels of various cytokines after treatment of B16F10 tumor-bearing mice with IL10M11 IL12, IL1b, IL2, IL4, and IL5. Serum levels were upregulated by IL10 but not by PD1 antibody. [Figure 20B] Same as above. [Figure 21] Serum levels of IL12 (FIG. 21A) and IL4 (FIG. 21B) normalized to the mean in isotype control-treated mice. Upregulation of IL12 and IL4 is shown across A20, MC38, MC38-cOVA, B16F10, and Colon26 tumor models. [Figure 22]Figure 22A shows the experimental design of the MC38 tumor model study and the results of treatment with control, IL10M11, PD-1 antagonist antibody, a combination of IL10M11 and PD-1 antagonist antibody, or IL10 linked to the PD-1 binding domain (mPD1-mIL10) (Figures 22B and 22C). Both PD1 Ab and IL10-Fc showed antitumor effects as single agents (a small effect for PD1 Ab and a moderate effect for IL10-Fc). Combination of IL10-Fc with PD-1 Ab significantly improved efficacy. PD1-IL10-Fc showed no difference in efficacy from control Ab-IL10 and IL10-Fc. DETAILED DESCRIPTION OF THE INVENTION

[0014] 6.1.Definition Related: The term "related" in the context of an IL10 agonist or a component thereof (e.g., a targeting moiety such as an antibody) refers to a functional relationship between two or more polypeptide chains. In particular, the term "associated" means that two or more polypeptides are associated with each other, for example, non-covalently via molecular interactions or covalently via one or more disulfide or chemical crosslinks, to produce a functional IL10 agonist. Examples of relationships that may exist in the IL10 agonists of the present disclosure include, but are not limited to, the relationship between homodimeric or heterodimeric Fc domains in the Fc region, the relationship between the VH and VL regions in a Fab or scFv, the relationship between CH1 and CL in a Fab, and the relationship between CH3 and CH3 in a domain-substituted Fab.

[0015] Cancer: The term "cancer" refers to a disease characterized by the uncontrolled (often rapid) growth of abnormal cells. Cancer cells can spread locally or to other parts of the body via the bloodstream and lymphatic system. Examples of various cancers are described herein, including, but not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain malignancies, adrenal cancer, autonomic ganglion cancer, biliary tract cancer, bone cancer, endometrial cancer, eye cancer, fallopian tube cancer, reproductive cancer, colon cancer, meningeal cancer, esophageal cancer, peritoneal cancer, pituitary cancer, penile cancer, placental cancer, pleural cancer, salivary gland cancer, small intestine cancer, stomach cancer, testicular cancer, thymus cancer, thyroid cancer, upper aerodigestive tract cancer, urinary tract cancer, vaginal cancer, vulvar cancer, lymphoma, leukemia, lung cancer, and the like.

[0016] Complementarity-Determining Region or CDR: The term "complementarity-determining region" or "CDR," as used herein, refers to the sequence of amino acids in an antibody variable region that confers antigen specificity and binding affinity. Generally, each heavy chain variable region has three CDRs (CDR-H1, CDR-H2, HCDR-H3), and each light chain variable region has three CDRs (CDR1-L1, CDR-L2, CDR-L3). Exemplary conventions that can be used to identify CDR boundaries include, for example, the Kabat definition, the Chothia definition, the ABM definition, and the IMGT definition. See, e.g., Kabat, 1991, "Sequences of Proteins of Immunological Interest," National Institutes of Health, Bethesda, MD (Kabat numbering scheme); Al-Lazikani et al., 1997, J. Mol. Biol. 273:927-948 (Chothia numbering scheme); Martin et al., 1989, Proc. Natl. Acad. Sci. USA 86:9268-9272 (ABM numbering scheme); and Lefranc et al., 2003, Dev. Comp. Immunol. 27:55-77 (IMGT numbering scheme). Public databases are also available to identify CDR sequences within antibodies.

[0017] EC50: The term "EC50" refers to the half-maximal effective concentration of a molecule (e.g., an IL10 agonist) that induces a response halfway between baseline and maximum after a specified exposure time. EC50 essentially represents the concentration of an antibody or IL10 agonist at which 50% of its maximal effect is observed. In certain embodiments, the EC50 value is equal to the concentration of an IL10 agonist that results in half-maximal activation of STAT3 in an assay, as described in Section 7.1.2.

[0018] Epitope: An epitope or antigenic determinant is the part of an antigen (e.g., a target molecule) that is recognized by an antibody or other antigen-binding moiety as described herein. Epitopes can be linear or conformational.

[0019] Fab: The term "Fab" in the context of targeting moieties of the present disclosure refers to a pair of polypeptide chains in which the first polypeptide chain comprises a heavy chain variable (VH) domain N-terminal to a first constant domain (referred to herein as C1) and the second polypeptide chain comprises a light chain variable (VL) domain N-terminal to a second constant domain (referred to herein as C2) that can pair with the first constant domain. In native antibodies, the VH is N-terminal to the first constant domain (CH1) of the heavy chain and the VL is N-terminal to the constant domain (CL) of the light chain. Fabs of the present disclosure can be oriented according to their native orientation or can include domain substitutions or swaps that facilitate correct VH and VL pairing. For example, replacing the pair of CH1 and CL domains in a Fab with a pair of CH3 domains can facilitate correct pairing of engineered Fab chains in a heterodimeric molecule. It is also possible to reverse CH1 and CL, linking CH1 to VL and CL to VH; this configuration is commonly known as a Crossmab.

[0020] Fc domain and Fc region: The term "Fc domain" refers to the portion of a heavy chain that pairs with the corresponding portion of another heavy chain. The term "Fc region" refers to the region of an antibody-based binding molecule formed by the association of two heavy chain Fc domains. The two Fc domains within an Fc region can be the same or different from each other. In native antibodies, the Fc domains are typically identical, although one or both Fc domains may be advantageously modified to enable heterodimerization, for example, via knob-in-hole interactions. Furthermore, Fc domains can also contain chimeric sequences derived from multiple immunoglobulin isotypes.

[0021] Host cell: As used herein, the term "host cell" refers to a cell into which a nucleic acid of the present disclosure has been introduced. The terms "host cell" and "recombinant host cell" are used interchangeably herein. Such terms are understood to refer to the particular subject cell and the progeny or potential progeny of such a cell. Because certain changes may occur in subsequent generations, either due to mutation or environmental influences, such progeny may not actually be identical to the parent cell, but are still included within the scope of the term as used herein. Typical host cells are eukaryotic host cells, e.g., mammalian host cells. Exemplary eukaryotic host cells include yeast cells and mammalian cells, e.g., vertebrate cells such as mouse, rat, monkey, or human cell lines, e.g., HKB11 cells, PER.C6 cells, HEK cells, or CHO cells.

[0022] IL10 mutein: A variant IL10 molecule having IL10 activity. The variant may be an IL10 fusion protein (e.g., IL10 fused to IL-2Rα) and / or a mutant IL10, e.g., one having one or more amino acid substitutions compared to wild-type IL10. IL10 muteins may have altered function (e.g., receptor binding, affinity, cytokine activity) and / or altered pharmacokinetics compared to wild-type IL10. In the context of the IL10 agonists of the present disclosure, the term "IL10 mutein" may refer to the non-targeting component of the IL10 molecule (and any associated linker moieties), and unless the context dictates otherwise, the term "IL10 mutein" should be understood to encompass IL10 molecules with or without a targeting moiety and with or without a multimerization moiety.

[0023] Major histocompatibility complex and MHC: These terms refer to naturally occurring MHC molecules, individual chains of MHC molecules (e.g., MHC class I α (heavy chain), β2 microglobulin, MHC class II α chain, and MHC class II β chain), individual subunits of such chains of MHC molecules (e.g., α1, α2, and / or α3 subunits of the MHC class I α chain, α1-α2 subunits of the MHC class II α chain, and β1-β2 subunits of the MHC class II β chain), as well as portions (e.g., peptide-binding portions, e.g., peptide-binding grooves), variants, and various derivatives (including fusion proteins) thereof, which retain the ability to present antigenic peptides for recognition by T cell receptors (TCRs), e.g., antigen-specific TCRs. MHC class I molecules contain a peptide-binding groove formed by the α1 and α2 domains of the heavy chain that can accommodate peptides of approximately 8-10 amino acids. Despite the fact that MHC of any class binds to a core of approximately nine amino acids (e.g., 5-17 amino acids) within a peptide, the expandable nature of the MHC class II peptide-binding groove (the α1 domain of a class II MHC polypeptide associated with the β1 domain of a class II MHC β polypeptide) allows for a wider range of peptide lengths. Peptides that bind to MHC class II are typically between 13 and 17 amino acids in length, although shorter or longer lengths are not uncommon. As a result, peptides may shift within the MHC class II peptide-binding groove, potentially changing which nonamer is directly positioned within the groove at any given time. Conventional identification of specific MHC variants is used herein. These terms encompass "human leukocyte antigen" or "HLA."

[0024] Operably linked: As used herein, the term "operably linked" refers to a functional relationship between two or more regions of a polypeptide chain, where two or more regions are joined to produce a functional polypeptide, or where two or more nucleic acid sequences are joined, for example, to produce an in-frame fusion of two polypeptide components, or where a regulatory sequence is linked to a coding sequence.

[0025] Single-chain Fv or scFv: As used herein, the term "single-chain Fv" or "scFv" refers to a polypeptide chain comprising the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain.

[0026] Specific (or selective) binding: As used herein, the term "specifically (or selectively) binding" means that a targeting moiety, e.g., an antibody, or antigen-binding domain ("ABD") thereof, forms a complex with a target molecule that is relatively stable under physiological conditions. Specific binding is defined as a binding activity that is greater than or equal to the K D is about 5 × 10 -2 M or less (e.g., 5 × 10 -2 Under M, 10 -2 Less than M, 5 x 10 -3 Under M, 10 -3 Less than M, 5 x 10 -4 Under M, 10 -4 Less than M, 5 x 10 -5 Under M, 10 -5 Less than M, 5 x 10 -6 Under M, 10 -6 Less than M, 5 x 10 -7 Under M, 10 -7 Less than M, 5 x 10 -8 Under M, 10 -8 Less than M, 5 x 10 -9 Under M, 10 -9 Less than M or 10 -10 M or less). Methods for determining the binding affinity of an antibody or antibody fragment, e.g., an IL10 agonist or component targeting moiety, to a target molecule are known in the art and include, for example, equilibrium dialysis, surface plasmon resonance (e.g., a Biacore™ assay), fluorescence-activated cell sorting (FACS) binding assays, etc. However, an IL10 agonist of the present disclosure that includes a targeting moiety or its ABD that specifically binds to a target molecule from one species may have cross-reactivity to target molecules from one or more other species.

[0027] Subject: A "subject" includes human and non-human animals. Non-human animals include all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cows, chickens, amphibians, and reptiles. Unless otherwise noted, the terms "patient" and "subject" are used interchangeably herein.

[0028] Target molecule: As used herein, the term "target molecule" refers to any biomolecule (e.g., a protein, carbohydrate, lipid, or combination thereof) that is expressed on a cell surface or in the extracellular matrix and can be specifically bound by a targeting moiety in an IL10 agonist of the present disclosure.

[0029] Targeting moiety: As used herein, the term "targeting moiety" refers to any molecule or binding portion thereof (e.g., an immunoglobulin or antigen-binding fragment) that can bind to a cell surface or extracellular matrix molecule at a site where an IL10 agonist of the present disclosure is to be localized, e.g., on a tumor cell or on a lymphocyte within the tumor microenvironment. In addition to localizing an IL10 agonist to a specific site, a targeting moiety can also have functional activity. For example, a targeting moiety that is an anti-PD1 antibody or antigen-binding portion thereof can exhibit anti-tumor activity or enhance the anti-tumor activity of an IL10 mutein by inhibiting PD1 signaling.

[0030] Treat, Treatment, Treating: As used herein, "treat," "treatment," and "treating" refer to the reduction or amelioration of the progression, severity, and / or duration of a proliferative disorder, or the amelioration of one or more symptoms (preferably one or more discernible symptoms) of a proliferative disorder, resulting from the administration of one or more IL10 agonists of the present disclosure. In certain embodiments, the terms "treat," "treat," and "treating" refer to at least one measurable physical parameter of a proliferative disorder, not necessarily discernible by the patient, e.g., tumor growth. In other embodiments, the terms "treat," "treatment," and "treating" refer to inhibiting the progression of a proliferative disorder physically, e.g., by stabilization of a discernible symptom, physiologically, e.g., by stabilization of a physical parameter, or both. In other embodiments, the terms "treat," "treatment," and "treating" refer to the reduction or stabilization of tumor size or cancer cell number.

[0031] Tumor: The term "tumor" is used interchangeably herein with the term "cancer," e.g., both terms encompass solid and liquid tumors, e.g., diffuse or circulating. As used herein, the terms "cancer" or "tumor" include malignant cancers and tumors as well as precancerous forms.

[0032] Tumor-associated antigen: The term "tumor-associated antigen" or "TAA" refers to a molecule (typically a protein, carbohydrate, lipid, or some combination thereof) that is expressed on the surface of cancer cells, either whole or as a fragment (e.g., MHC / peptide), and that is useful for preferential targeting of pharmacological agents to cancer cells. In some embodiments, the TAA is a marker expressed by both normal and cancer cells, e.g., a lineage marker, e.g., CD19 on B cells. In some embodiments, the TAA is a cell surface molecule that is overexpressed in cancer cells compared to normal cells, e.g., greater than 1-fold overexpression, greater than 2-fold overexpression, greater than 3-fold overexpression, or more, compared to normal cells. In some embodiments, the TAA is a cell surface molecule that is inappropriately synthesized in cancer cells, e.g., a molecule that contains a deletion, addition, or mutation compared to the molecule expressed on normal cells. In some embodiments, the TAA is expressed only on the cell surface of cancer cells, either whole or as a fragment (e.g., MHC / peptide), and is not synthesized or expressed on the surface of normal cells. Thus, the term "TAA" encompasses antigens specific to cancer cells, sometimes known in the art as tumor-specific antigens ("TSAs").

[0033] Universal light chain: As used herein in the context of a targeting moiety, the term "universal light chain" refers to a light chain polypeptide that is capable of pairing with a heavy chain region of a targeting moiety and also with other heavy chain regions. A universal light chain is also known as a "common light chain."

[0034] VH: The term "VH" refers to the variable region of an immunoglobulin heavy chain of an antibody, including the heavy chain of an scFv or Fab. VL: The term "VL" refers to the variable region of an immunoglobulin light chain, including the light chain of an scFv or Fab.

[0035] IL-10 agonists The present disclosure provides an IL10 agonist comprising an IL10 moiety, an optional multimerization moiety, and an optional targeting moiety.

[0036] IL10, also known as human cytokine synthesis inhibitory factor (CSIF), is classified as a type (class)-2 cytokine, a set of cytokines that includes IL19, IL20, IL22, IL24 (Mda-7), and IL26, interferons (e.g., IFNγ, IFNβ, IFNγ), and interferon-like molecules (e.g., limitin, IL-28A, IL-28B).

[0037] IL-10 is a cytokine with pleiotropic effects in immunoregulation and inflammation. It is produced by mast cells and counteracts the inflammatory effects of these cells at the site of an allergic reaction. IL-10 can inhibit the synthesis of proinflammatory cytokines such as IFNγ, IL2, IL3, TNFα, and GM-CSF, while also being stimulatory to certain T cells and mast cells and stimulating B cell maturation, proliferation, and antibody production. IL10 can block NFκB activity and is involved in regulating the JAK-STAT signaling pathway. It also induces the cytotoxic activity of CD8+ T cells and antibody production by B cells, and suppresses macrophage activity and tumor-promoting inflammation. The modulation of CD8+ T cells is dose-dependent, with higher doses inducing stronger cytotoxic responses.

[0038] Human IL10 is a homodimer. Each monomer is produced as an immature molecule of 178 amino acids, the first 18 of which are a signal peptide that is cleaved during secretion to generate mature IL10 containing 160 amino acids. The IL10 monomers within the dimer are noncovalently associated, with each subunit containing two intrachain disulfide bonds between residues 12 and 108 and between residues 62 and 114. The monomers noncovalently dimerize to form a V-shaped structure, with each half of each consisting of six α-helices, four of which originate from one subunit and two from the other. The IL10 agonists of the present disclosure can be in the form of monomers or multimers, e.g., dimers (homodimers or heterodimers) or higher-order complexes. For convenience, IL10 agonists that are homodimers (or higher order multimers of the same polypeptide) will be described in terms of the constituent monomers; however, recombinant expression of the constituent monomers in suitable cell lines can produce homodimeric (or higher order multimeric) molecules.

[0039] Exemplary IL10 moieties suitable for use in the IL10 agonists of the present disclosure are described in Section 6.2. The IL10 agonist may be a monomer or a homodimer of two polypeptide chains, each comprising an IL10 portion, an Fc portion (e.g., an Fc domain consisting of a CH2 domain and a CH3 domain), an optional hinge portion, an optional linker portion, and an optional targeting portion.

[0040] Thus, the IL10 agonist may be a monomer, or a homodimer or heterodimer of two polypeptide monomers. The monomer, or each monomer in the dimer, may comprise, from amino terminus to carboxy terminus: i) Optional Targeting Moiety ii) an optional hinge domain iii) an Fc domain, e.g., an Fc domain consisting of a CH2 domain and a CH3 domain; iv) a linker moiety; and v) IL10 part.

[0041] Dimerization of the IL10 agonist can occur through disulfide bonds between the hinge domains of two monomers, disulfide bonds between the Fc domains of two monomers, non-covalent bonds between the IL10 moieties of two monomers, or a combination of two or all three of the above. If a monomeric form of the IL10 agonist is desired, the ability of the monomers to dimerize is reduced by modifying the IL10 moiety and the Fc domain, as described in Section 6.3 for the IL10 moiety and Section 6.4 for the Fc domain.

[0042] Exemplary Fc moieties are described in Section 6.4 and include an Fc domain that confers dimerization ability to the IL10 agonist. Active IL10 is a dimeric molecule, and the pharmacokinetics of IL10 in vivo are very poor, in part due to monomerization in the bloodstream. Without being bound by theory, it is believed that the inclusion of the Fc domain and optional hinge domain improves the serum stability and pharmacokinetic profile of the IL10 agonist by, among other things, stabilizing the dimeric structure of IL10.

[0043] For convenience, the IL10 portion and the Fc domain portion and the optional linker therebetween are sometimes referred to herein as IL10 muteins, although the term "mutein" also encompasses molecules having targeting moieties. Exemplary targeting moieties are described in Section 6.5 and include antigen-binding domains (e.g., scFv or Fab) that bind to tumor-associated antigens, tumor microenvironment antigens, tumor lymphocytes, or MHC-peptide complexes.

[0044] When the IL10 moiety is located at the N-terminus of the Fc domain, the resulting recombinant IL10 agonist was found to be truncated at the N-terminus and / or C-terminus. For example, in the case of an N-terminal IL10 moiety, the resulting recombinant IL10 agonist either lacked the C-terminal lysine or was truncated at both the N-terminus and C-terminus, possessing only residues 3-402 of the full-length 403 amino acid construct. This was not observed with IL10 agonists having an IL10 moiety at the C-terminus of the Fc domain. Thus, in some embodiments, the IL10 moiety is located at the C-terminus of the Fc domain.

[0045] In various embodiments, the IL10 agonist does not include (a) a stabilizing moiety, e.g., polyethylene glycol and / or albumin; (b) an antibody variable region (e.g., the variable regions of L19, F16, G11, or F8 directed against splice isoforms of fibronectin and tenascin-C; or the variable region of an anti-CD86 antibody); (c) a non-targeting antibody variable region; (d) a non-binding antibody variable region; (e) an antibody CDR; (f) an antibody CH1 domain; (g) an antibody CL domain; (h) another cytokine (e.g., IL4); (i) an Fc domain C-terminal to the IL10 moiety; or (j) a combination of two, three, four, five, six, seven, eight, or all of the above. The IL10 agonist may include, for example, one or more linker sequences connecting various components of the molecule, e.g., multiple different domains present in a fusion protein. Exemplary linker examples are described in Section 6.7.

[0046] In other embodiments, the IL10 agonist comprises (a) a stabilizing moiety, such as a hydrophilic polymer (e.g., polyethylene glycol), albumin, XTEN, PAS, a carbohydrate conjugate (e.g., hydroxyethyl starch), a glycan (e.g., N- and O-linked glycans), polysialic acid, and / or a fatty acid; (b) an antibody variable region (e.g., L19, F16, G11, or F8 directed against splice isoforms of fibronectin and tenascin-C); (c) a non-targeting antibody variable region; (d) a non-binding antibody variable region; (e) an antibody CDR; (f) an antibody CH1 domain; (g) an antibody CL domain; (h) another cytokine (e.g., IL4); (i) an Fc domain C-terminal to the IL10 portion; (j) a human serum albumin binder or binding domain; or (k) a combination of two, three, four, five, six, seven, eight, nine, or all of the above.

[0047] In certain aspects, the IL10 agonists of the present disclosure increase the ratio of CD8+ T cells to Treg cells in the tumor after administration to a subject (e.g., a cancer patient or a tumor-bearing mouse). In some embodiments, treatment with an IL10 agonist of the present disclosure causes an increase in the density of CD8 T cells in the tumor. This increase can be, for example, at least a two-fold increase, at least a three-fold increase, or at least a four-fold increase in the density of CD8 T cells in the tumor.

[0048] In certain embodiments, treatment with the IL10 agonist of the present disclosure causes an increase in CD45+ immune cell infiltration in tumors.The increase in CD45+ immune cell infiltration in tumors can be, for example, at least 10% increase.Examples of CD45+ immune cells include, for example, CD4 T cells and myeloid cells (e.g., CD45+ leukocytes).

[0049] In some embodiments, treatment with an IL10 agonist of the present disclosure upregulates serum IL12 levels relative to a suitable control not treated with the IL10 agonist or pharmaceutical composition comprising an IL10 agonist, or relative to the subject's own serum IL12 levels prior to treatment with the IL10 agonist or pharmaceutical composition. Compared to a suitable control or the subject's own serum IL-12 levels prior to treatment, an IL10 agonist of the present disclosure can upregulate serum IL-12 levels, for example, by at least 2-fold, at least 5-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, or at least 30-fold.

[0050] In some embodiments, treatment with an IL10 agonist of the present disclosure upregulates serum IL12 levels relative to a suitable control not treated with an IL10 agonist or a pharmaceutical composition comprising an IL10 agonist, or relative to the subject's own serum IL4 levels prior to treatment with the IL10 agonist or pharmaceutical composition. Compared to a suitable control or the subject's own serum IL4 levels prior to treatment, an IL10 agonist of the present disclosure can upregulate serum IL4 levels, for example, by at least 2-fold, at least 5-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, or at least 30-fold.

[0051] In the IL10 agonists of the present disclosure, when the targeting moiety is an antibody antigen binding domain (“ABD”), one or both monomers of the dimeric IL10 agonist can carry the ABD, for example, in the form of a Fab or scFv.

[0052] The Fc domain may be, for example, an IgG1 or IgG4 Fc domain, with or without substitutions that reduce glycosylation and / or effector function, as described in Section 6.4.1 and its subsections.

[0053] In certain embodiments, the IL10 agonist includes an IL10 mutein comprising the amino acid sequence of IL10M1, IL10M2, IL10M3, IL10M4, IL10M5, IL10M6, IL10M7, IL10M8, IL10M9, or IL10M10, or an amino acid sequence having at least 90% or at least 95% sequence identity thereto, optionally with a targeting moiety.

[0054] Further details of the components of the IL10 agonists of the present disclosure are provided below. 6.3.IL10 part The IL10 portion of the IL10 antagonist of the present disclosure comprises a wild-type or variant IL10 domain.

[0055] The IL10 moiety encompasses mature human and non-human (e.g., mouse, rat, pig, non-human primate) IL10 polypeptides, including homologues, variants and fragments thereof, as well as IL10 polypeptides having, for example, a leader sequence (e.g., a signal peptide), and modified versions of the above.

[0056] In eukaryotic cells, human IL10 is synthesized as a 178 amino acid precursor polypeptide, from which 18 amino acids are removed to generate the mature secreted form of IL10. Thus, in some embodiments, the IL10 portion of the present disclosure includes mature human IL10 corresponding to positions S19 to N178 of the 178 amino acid precursor sequence, for example, mature human IL10 having the amino acid sequence below or the amino acid sequence set forth in Section 7.1.1.

[0057] [ka]

[0058] The sequences of mature mouse, porcine, and rat IL10 are disclosed in Figure 5 of Zdanov et al., 1995, Structure 3(6):591-601, which is incorporated herein by reference in its entirety.

[0059] Thus, an "IL10 portion" encompasses proteins having a sequence substantially similar to mature, wild-type human, mouse, porcine, or rat IL10, and more preferably, proteins having a sequence substantially similar to mature, wild-type human IL10. In various embodiments, the IL10 portion comprises an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to human, mouse, porcine, or rat IL10, e.g., the mature human IL10 sequence comprising amino acid residues S19 to N178 of the human IL10 precursor.

[0060] In certain embodiments, the IL10 agonist of the present disclosure has one or more amino acid modifications, e.g., substitutions, deletions, or insertions, in the IL10 portion compared to wild-type IL10. The one or more amino acid modifications can be introduced to alter one or more properties of IL10, such as its stability. A specific modified human IL10 molecule with improved stability in the monomeric form, which has a linker sequence inserted between N116 and K117 (i.e., between helical domains D and E of IL10) that functions as a hinge, is described in Josephson et al., 2000, J Biol Chem, 275:13552-13557. In one embodiment, the sequence inserted between N116 and K117 (or the equivalent position in IL10 of a non-human species) is 6 amino acids long and / or includes the sequence GGGSGG (SEQ ID NO: 2). In one embodiment, the IL10 portion does not have a linker sequence inserted between N116 and K117 (i.e., between helical domains D and E of IL10). In some embodiments, the IL10 moiety does not comprise any amino acid modifications, eg, substitutions, deletions, or insertions, in the IL10 moiety compared to wild-type IL10.

[0061] Human IL10 contains a potential N-linked glycosylation site at N116, located on the surface of the molecule, which is conserved in non-human species. Zdanov et al., 1995, Structure 3(6):591-601. Thus, the present disclosure includes IL10 molecules with or without an N-linked glycan at N116 or the equivalent position in IL10 of other species. This potential N-linked glycosylation site is conserved in rat, mouse, and porcine IL10. Mouse and rat IL10 contain a second potential N-linked glycosylation site near the N-terminus (N8 in mIL10 and N1 in rat IL10). The present disclosure encompasses mouse or rat IL10 moieties with and without this additional N-linked glycan.

[0062] Fc Domain The IL10 agonist of the present disclosure can comprise an Fc region derived from any suitable species. In one embodiment, the Fc region is derived from a human Fc domain. In a preferred embodiment, the IL10 domain is fused to an IgG Fc region (e.g., an IgG1 or IgG4 Fc region).

[0063] The IL10 domain can be fused to the N-terminus or C-terminus of the IgG Fc region. As shown in the Examples, fusion to the C-terminus of the IgG Fc region maintains the activity of the IL10 domain to a greater extent than fusion to the N-terminus of the IgG Fc region.

[0064] One embodiment of the present disclosure is directed to a dimer comprising two Fc fusion polypeptides created by fusing an IL10 domain with an Fc region of an antibody. The dimer can be produced, for example, by inserting a gene fusion encoding the fusion protein into an appropriate expression vector, expressing the gene fusion in a host cell transformed with the recombinant expression vector, and allowing the expressed fusion protein to assemble in the same manner as antibody molecules, resulting in the formation of interchain bonds between the Fc portions to form a dimer.

[0065] The Fc domain may be derived from any suitable class of antibody, including IgA (including subclasses IgA1 and IgA2), IgD, IgE, IgG (including subclasses lgG1, lgG2, lgG3, and lgG4), and IgM. In one embodiment, the Fc domain is derived from IgG. In one embodiment, the Fc domain is derived from lgG1, lgG2, lgG3, or lgG4. In one embodiment, the Fc domain is derived from lgG1. In one embodiment, the Fc domain is derived from lgG4.

[0066] The two Fc domains within the Fc region can be the same or different from one another. In native antibodies, the Fc domains are typically identical, but for purposes of producing multispecific binding molecules, such as the IL10 agonists of the present disclosure, it can be advantageous for the Fc domains to be different to allow for heterodimerization, as described in Section 6.4.1 below.

[0067] In native antibodies, the heavy chain Fc domain of IgA, IgD, and IgG consists of two heavy chain constant domains (CH2 and CH3), while the heavy chain Fc domain of IgE and IgM consists of three heavy chain constant domains (CH2, CH3, and CH4), which dimerize to create the Fc region.

[0068] In the IL10 agonists of the present disclosure, the Fc region and / or Fc domains therein may be chimeric, combining sequences from multiple immunoglobulin isotypes, and thus the Fc region and / or Fc domains therein may include heavy chain constant domains from one or more different classes of antibodies, for example, one, two, or three different classes.

[0069] In one embodiment, the Fc region comprises CH2 and CH3 domains derived from IgG1. In one embodiment, the Fc region comprises CH2 and CH3 domains derived from IgG2.

[0070] In one embodiment, the Fc region comprises CH2 and CH3 domains derived from IgG3. In one embodiment, the Fc region comprises CH2 and CH3 domains derived from IgG4.

[0071] In one embodiment, the Fc region comprises a CH4 domain from IgM. The IgM CH4 domain is typically located C-terminal to the CH3 domain. In one embodiment, the Fc region is composed of the CH2 and CH3 domains derived from an IgG and the CH4 domain derived from an IgM.

[0072] In a further embodiment, a chimeric Fc domain can comprise part or all of a CH2 sequence derived from the CH2 region of human IgG1, human IgG2, or human IgG4, and part or all of a CH3 sequence derived from human IgG1, human IgG2, or human IgG4. As described in Section 6.7.1.1, a chimeric Fc domain can also contain a chimeric hinge region. For example, a chimeric hinge can comprise an "upper hinge" sequence derived from a human IgG1, human IgG2, or human IgG4 hinge region combined with a "lower hinge" sequence derived from a human IgG1, human IgG2, or human IgG4 hinge region. A specific example of a chimeric Fc domain that can be included in any of the IL10 muteins described herein comprises, from N-terminus to C-terminus: [IgG4 CH1]-[IgG4 upper hinge]-[IgG2 lower hinge]-[IgG4 CH2]-[IgG4 CH3]. Another example of a chimeric Fc domain that can be included in any of the antigen-binding molecules described herein comprises, from N-terminus to C-terminus: [IgG1 CH1]-[IgG1 upper hinge]-[IgG2 lower hinge]-[IgG4 CH2]-[IgG1 CH3]. These and other examples of chimeric Fc domains that can be included in any of the antigen-binding molecules of the present invention are described in WO 2014 / 121087. Chimeric Fc regions having these general structural arrangements, and variants thereof, can have altered Fc receptor binding, which in turn affects Fc effector function.

[0073] It will be understood that heavy chain constant domains for use in generating Fc regions for the IL10 agonists of the present disclosure can include variants of naturally occurring constant domains. Such variants may contain one or more amino acid variations compared to the wild-type constant domain. In one example, the Fc region of the present disclosure includes at least one constant domain that differs in sequence from the wild-type constant domain. It will be understood that the variant constant domain may be longer or shorter than the wild-type constant domain. Preferably, the variant constant domain has at least 60% identity or similarity with the wild-type constant domain. In another example, the variant constant domain has at least 70% identity or similarity. In another example, the variant constant domain has at least 80% identity or similarity. In another example, the variant constant domain has at least 90% identity or similarity. In another example, the variant constant domain has at least 95% identity or similarity.

[0074] IgM and IgA naturally exist in humans as covalently linked multimers of the common H2L2 antibody unit. IgM exists as a pentamer when incorporating a J chain and as a hexamer when lacking a J chain. IgA exists in both monomeric and dimeric forms. The heavy chains of IgM and IgA have an 18-amino acid extension to the C-terminal constant domain known as the tailpiece. This tailpiece contains cysteine ​​residues that form disulfide bonds between heavy chains in the polymer and is thought to play an important role in polymerization. The tailpiece also contains glycosylation sites. In certain embodiments, the IL10 agonist of the present disclosure does not contain a tailpiece.

[0075] The Fc domain incorporated into the IL10 agonists of the present disclosure can include one or more modifications that alter the functional properties of the protein, for example, binding to an Fc receptor such as FcRn or a leukocyte receptor, binding to complement, altering the disulfide bond structure, or changing the glycosylation pattern. Exemplary Fc modifications that alter effector function are described in Section 6.4.1.

[0076] The Fc domain can also be modified to include modifications that improve the manufacturability of asymmetric IL10 agonists, for example, by allowing heterodimerization, the preferential pairing of non-identical Fc domains over identical Fc domains. Heterodimerization allows the production of IL10 agonists in which different polypeptide components are connected to each other by Fc regions that contain Fc domains of different sequences. Examples of heterodimerization strategies are illustrated in Section 6.4.1.1.

[0077] Alternatively, the Fc domain may be a soluble monomeric Fc domain with reduced ability to self-associate. See, for example, Helm et al., 1996, J. Biol. Chem., 271:7494-7500 and Ying et al., 2012, J. Biol. Chem., 287(23):19399-19408. The IL10 agonist can further dimerize via the IL10 moiety. One example of a soluble monomeric Fc domain includes amino acid substitutions at positions corresponding to T366 and / or Y407 in CH3, as described in U.S. Patent Application Publication No. 2019 / 0367611. In some embodiments, the IL10 agonist is not substituted at positions corresponding to T366 and / or Y407 in CH3. The monomeric Fc domain may be of any Ig subtype and may contain additional substitutions that reduce effector function, as described in Section 6.4.1.

[0078] As used herein, the term "Fc region" can include an Fc domain with or without a hinge sequence. In various embodiments in which the Fc region includes a heavy chain constant region that includes a hinge domain, positions 233-236 within the hinge domain can be G, G, G, and unoccupied; G, G, unoccupied, and unoccupied; G, unoccupied, unoccupied, and unoccupied; or all unoccupied, with positions numbered according to EU numbering. Optionally, the heavy chain constant region includes, from N-terminus to C-terminus, a hinge domain, a CH2 domain, and a CH3 domain. Optionally, the heavy chain constant region includes, from N-terminus to C-terminus, a CH1 domain, a hinge domain, a CH2 domain, and a CH3 domain. Optionally, the CH1 region, if present, the remainder of the hinge region, if any, the CH2 region, and the CH3 region are of the same human isotype. Optionally, the CH1 region, if present, the remainder of the hinge region, if any, the CH2 region, and the CH3 region are human IgG1. Optionally, the CH1 region, if present, the remainder of the hinge region, if any, the CH2 region, and the CH3 region are human IgG2. Optionally, the CH1 region, if present, the remainder of the hinge region, if any, the CH2 region, and the CH3 region are human IgG4.

[0079] Optionally, the constant region has a CH3 domain that is modified to reduce binding to Protein A. These and other examples of Fc regions that can be included in any of the IL10 variants of the present disclosure are described in International Publication No. WO2016 / 161010. Exemplary hinge sequences are provided in Section 6.7.1 and its subsections.

[0080] It will be appreciated that any of the above modifications can be combined in any suitable manner to achieve the desired functional properties and / or can be combined with other modifications to alter the properties of the IL10 agonist.

[0081] 6.4.1. Fc Domains with Altered Effector Functions In some embodiments, the Fc domain comprises one or more amino acid substitutions that reduce binding to Fc receptors and / or effector function.

[0082] In one specific embodiment, the Fc receptor is an Fcγ receptor. In one embodiment, the Fc receptor is a human Fc receptor. In one embodiment, the Fc receptor is an activating Fc receptor. In one specific embodiment, the Fc receptor is an activating human Fcγ receptor, more particularly human FcγRIIIa, FcγRI, or FcγR11a, most particularly human FcγR111a. In one embodiment, the effector function is one or more selected from the group consisting of complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and cytokine secretion. In one specific embodiment, the effector function is ADCC.

[0083] In one embodiment, the Fc region comprises an amino acid substitution at a position selected from the group consisting of E233, L234, L235, N297, P331, and P329 (numbering according to Kabat EU index). In a more particular embodiment, the Fc region comprises an amino acid substitution at a position selected from the group consisting of L234, L235, and P329 (numbering according to Kabat EU index). In some embodiments, the Fc region comprises amino acid substitutions L234A and L235A (numbering according to Kabat EU index). In one such embodiment, the Fc region is an Igd Fc region, particularly a human Igd Fc region. In one embodiment, the Fc region comprises an amino acid substitution at position P329. In a more particular embodiment, the amino acid substitution is P329 or P329G, particularly P329G (numbering according to Kabat EU index). In one embodiment, the Fc region comprises an amino acid substitution at position P329 and an additional amino acid substitution at a position selected from E233, L234, L235, N297, and P331 (numbering according to the Kabat EU index). In a more particular embodiment, the additional amino acid substitution is E233P, L234A, L235A, L235E, N297A, N297D, or P331S. In a particular embodiment, the Fc region comprises amino acid substitutions at positions P329, L234, and L235 (numbering according to the Kabat EU index). In a more particular embodiment, the Fc region comprises amino acid mutations L234A, L235A, and P329G ("P329G LALA," "PGLALA," or "LALAPG").

[0084] Typically, the same one or more amino acid substitutions are present in each of the two Fc domains of the Fc region. Thus, in one specific embodiment, each Fc domain of the Fc region comprises amino acid substitutions L234A, L235A, and P329G (Kabat EU index numbering), i.e., in each of the first and second Fc domains of the Fc region, the leucine residue at position 234 is replaced with an alanine residue (L234A), the leucine residue at position 235 is replaced with an alanine residue (L235A), and the proline residue at position 329 is replaced with a glycine residue (P329G) (Kabat EU index numbering).

[0085] In one embodiment, the Fc domain is an IgG1 Fc domain, in particular a human IgG1 Fc. In another embodiment, the Fc domain is an IgG4 Fc domain with reduced binding to Fc receptors. Exemplary IgG4 Fc domains with reduced binding to Fc receptors can comprise an amino acid sequence selected from Table 1 below. In some embodiments, the Fc domain comprises only the bolded portion of the sequence shown below.

[0086] [Table 1-1]

[0087] [Table 1-2]

[0088] [Table 1-3]

[0089] [Table 1-4]

[0090] In one specific embodiment, the IgG4 with reduced effector function comprises the bolded portion of the amino acid sequence of SEQ ID NO: 31 of WO 2014 / 121087, reproduced above (amino acids 99 to 326 of SEQ ID NO: 6, or SEQ ID NO: 31; said bolded sequence may also be referred to herein as IgG4), or an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity thereto. In other embodiments, the IgG4 with reduced effector function comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 31 of WO 2014 / 121087 (SEQ ID NO: 6).

[0091] For IL10 agonists of the present disclosure that are heterodimers, in each case of WO2014 / 121087, one may incorporate a combination of the variant IgG4 Fc sequences set out above, for example an Fc region comprising a combination of SEQ ID NO: 30 (or a bolded portion thereof) and SEQ ID NO: 37 (or a bolded portion thereof), or an Fc region comprising a combination of SEQ ID NO: 31 (or a bolded portion thereof) and SEQ ID NO: 38 (or a bolded portion thereof).

[0092] In one specific embodiment, the Fc domain comprises the amino acid sequence designated in Section 7.1.1 as hIgG4s (SEQ ID NO: 31), or an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity thereto.

[0093] In another specific embodiment, the Fc domain comprises the amino acid sequence designated as hIgG1 (SEQ ID NO: 32) in Section 7.1.1, or an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity thereto. hIgG1 (SEQ ID NO: 32) is a variant IgG1-based Fc sequence that includes D265A, N297A mutations (EU numbering) to reduce effector function.

[0094] 6.4.1.1. Fc Heterodimerization Variants Certain IL10 agonists, unlike native immunoglobulins, require dimerization between two Fc domains operably linked at non-identical N-terminal regions, e.g., one Fc domain connected to a Fab and the other Fc domain connected to an IL10 moiety. Incorrect heterodimerization of two Fc regions to form an Fc domain can be an obstacle to increasing the yield of the desired heterodimeric molecule and presents a challenge for purification. Various approaches available in the art can be used to enhance dimerization of Fc domains that may be present in the IL10 agonists of the present disclosure, as disclosed, for example, in EP 1870459 A1; U.S. Pat. No. 5,582,996; U.S. Pat. No. 5,731,168; U.S. Pat. No. 5,910,573; U.S. Pat. No. 5,932,448; U.S. Pat. No. 6,833,441; U.S. Pat. No. 7,183,076; U.S. Pat. Appl. Publ. No. 2006204493 A1; International Application No. WO2009 / 089004 A1.

[0095] The present disclosure provides IL10 agonists comprising Fc heterodimers, i.e., Fc regions comprising heterologous, non-identical Fc domains. Typically, each Fc domain in the Fc heterodimer comprises an antibody CH3 domain. The CH3 domain is derived from the constant region of an antibody of any isotype, class, or subclass, preferably the IgG (lgG1, lgG2, lgG3, and lgG4) class, as described in the previous section.

[0096] Heterodimerization of two different heavy chains at the CH3 domains results in the desired IL10 agonist, whereas homodimerization of the same heavy chain reduces the yield of the desired IL10 agonist. Thus, in a preferred embodiment, polypeptides that associate to form the IL10 agonists of the present disclosure will contain a CH3 domain with modifications that favor heterodimeric association relative to an unmodified Fc domain.

[0097] In a specific embodiment, the modification that promotes Fc heterodimer formation is a so-called "knob-into-hole" or "knob-in-hole" modification, comprising a "knob" modification in one of the Fc domains and a "hole" modification in the other Fc domain. Knob-in-hole technology is described, for example, in U.S. Pat. Nos. 5,731,168 and 7,695,936; Ridgway et al., 1996, Prot Eng, 9:617-621; and Carter, 2001, Immunol Meth, 248:7-15. Generally, this method involves introducing a protrusion ("knob") at the interface of a first polypeptide and a corresponding cavity ("hole") at the interface of a second polypeptide, such that the protrusion ("knob") can be positioned within the cavity ("hole") to promote heterodimer formation and prevent homodimer formation. The protrusions are constructed by replacing small amino acid side chains from the interface of the first polypeptide with larger side chains (e.g., tyrosine or tryptophan). Compensatory cavities of identical or similar size to the protrusions are created in the interface of the second polypeptide by replacing the large amino acid side chains with smaller amino acid side chains (e.g., alanine or threonine).

[0098] Thus, in some embodiments, an amino acid residue in the CH3 domain of a first subunit of an Fc domain is replaced with an amino acid residue having a larger side chain volume, thereby creating a protrusion in the CH3 domain of the first subunit that can be placed in a cavity in the CH3 domain of a second subunit, and an amino acid residue in the CH3 domain of a second subunit of an Fc domain is replaced with an amino acid residue having a smaller side chain volume, thereby creating a cavity in the CH3 domain of the second subunit that can accommodate the protrusion in the CH3 domain of the first subunit. Preferably, the amino acid residue having a larger side chain volume is selected from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W). Preferably, the amino acid residue having a smaller side chain volume is selected from the group consisting of alanine (A), serine (S), threonine (T), and valine (V). The protrusion and cavity can be created by altering a nucleic acid encoding the polypeptide, for example, by site-directed mutagenesis or by peptide synthesis. An exemplary substitution is Y470T.

[0099] In one particular such embodiment, in the first Fc domain, the threonine residue at position 366 is replaced with a tryptophan residue (T366W), the tyrosine residue at position 407 is replaced with a valine residue (Y407V) in the Fc domain, and optionally, the threonine residue at position 366 is replaced with a serine residue (T366S) and the leucine residue at position 368 is replaced with an alanine residue (L368A) (numbering according to the Kabat EU index). In a further embodiment, the first Fc domain further comprises a replacement of the serine residue at position 354 with a cysteine ​​residue (S354C), a replacement of the glutamic acid residue at position 356 with a cysteine ​​residue (E356C) (particularly the replacement of the serine residue at position 354 with a cysteine ​​residue), and the second Fc domain further comprises a replacement of the tyrosine residue at position 349 with a cysteine ​​residue (Y349C) (numbering according to the Kabat EU index). In a particular embodiment, the first Fc domain comprises the amino acid substitutions 354C and T366W, and the second Fc domain comprises the amino acid substitutions Y349C, T366S, L368A, and Y407V (numbering according to the Kabat EU index).

[0100] In some embodiments, electrostatic steering (e.g., as described in Gunasekaran et al., 2010, J Biol Chem, 285(25):19637-46) can be used to promote association of the first and second Fc domains of the Fc region.

[0101] Alternatively, or in addition to, using an Fc domain modified to promote heterodimerization, the Fc domain can be modified to enable a purification strategy that allows for the selection of Fc heterodimers. In one such embodiment, one polypeptide contains a modified Fc domain that abrogates its binding to Protein A, thereby enabling a purification method that yields a heterodimeric protein. See, e.g., U.S. Pat. No. 8,586,713. Thus, the IL10 agonist comprises a first CH3 domain and a second Ig CH3 domain, wherein the first and second Ig CH3 domains differ from each other by at least one amino acid, such that the at least one amino acid difference reduces the binding of the IL10 agonist to Protein A compared to a corresponding IL10 agonist without the amino acid difference. In one embodiment, the first CH3 domain binds to Protein A, and the second CH3 domain contains a mutation / modification, such as an H95R modification (according to IMGT exon numbering; H435R according to EU numbering), that reduces or abrogates Protein A binding. The second CH3 can further comprise a Y96F modification (according to IMGT; Y436F according to EU). This class of modification is referred to herein as a "star" mutation.

[0102] 6.5. Targeting part Incorporation of a targeting moiety into the IL10 agonists of the present disclosure allows for the delivery of high concentrations of IL10 within the tumor microenvironment or to tumor-reactive lymphocytes, particularly CD8+ T lymphocytes, where they can exert a localized effect.

[0103] Suitable targeting moiety formats are described in Section 6.5.2. The targeting moiety is preferably an antigen-binding portion, e.g., an antibody or an antigen-binding portion of an antibody, e.g., an scFv, as described in Section 6.5.2.1, or a Fab, as described in Section 6.5.2.2.

[0104] Antibodies and antigen-binding moieties generally bind to a specific antigenic determinant and can direct the IL10 agonist to a target site, e.g., a tumor cell type or tumor stroma that bears that antigenic determinant. Exemplary target molecules recognized by targeting moieties of the present disclosure are described in Section 6.5.1.

[0105] 6.5.1.Target molecules The target molecules recognized by the targeting moieties of the IL10 agonists of the present disclosure are generally found, for example, on the surface of activated T cells, tumor cells, virus-infected cells, other diseased cells, free in serum, in the extracellular matrix (ECM), or on immune cells present on the targeting moiety, such as tumor-reactive lymphocytes, or peptides in peptide-MHC complexes. When immune cells (e.g., T cells expressing a chimeric antigen receptor ("CAR")) are administered exogenously, the targeting moiety can recognize the chimeric antigen receptor or another molecule found on the surface of the CAR T cells.

[0106] Exemplary target molecules include fibroblast activation protein (FAP), the A1 domain of tenascin-C (TNC A1), the A2 domain of tenascin-C (TNC A2), fibronectin extra domain B (EDB), melanoma-associated chondroitin sulfate proteoglycan (MCSP), MART-1 / MelanA, gp100, dipeptidyl peptidase IV (DPPIV), adenosine deaminase-binding protein (ADAbp), cyclophilin b, colorectal-associated antigen (CRC)-C017-1A / GA733, carcinoembryonic antigen (CEA) and its immunogenic epitopes CAP-1 and CAP-2, etv6, aml1, prostate-specific antigen (PSA) and its immunogenic epitopes PSA-1, PSA-2, and PSA-3, prostate-specific membrane antigen (PSMA), T-cell receptor / CD3 ζ chain, tumor antigens of the MAGE family (e.g., MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-B10, MAGE-B20, MAGE-B3, MAGE-B4, MAGE-B5, MAGE-B6, MAGE-B7, MAGE-B8, MAGE-B9, MAGE-B10, MAGE-B11, MAGE-B12, MAGE-B13, MAGE-B14, MAGE-B15, MAGE-B16, MAGE-B17, MAGE-B18, MAGE-B19, MAGE-B20, MAGE-B21, MAGE-B22, MAGE-B23, MAGE-B24, MAGE-B25, MAGE-B26, MAGE-B30, MAGE-B41, MAGE-B42, MAGE-B53, MAGE-B64, MAGE-B75, MAGE-B86, MAGE-B97, MAGE-B118 A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (MAGE-B4), MAGE-C1, MAGE-C2, MAGE-C3, MAGE-C4, MAGE-C5), tumor antigens of the GAGE ​​family (e.g., GAGE-1, GAGE-2, GAGE- 3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, GAGE-9), BAGE, RAGE, LAGE-1, NAG, GnT-V, MUM-1, CDK4, tyrosinase, p53, MUC family, HER2 / neu, p21ras, RCAS1, α-fetoprotein, E-cadherin, α-catenin, β-catenin and γ-catenin, p120ctn, gp100Pmel117, PRAME, NY-ESO-1, cdc27, adenomatous polyposis coli protein (APC), fodrin, connexin 37, Ig-idiotype, p15, gp75, GM2 and GD2 gangliosides, viral products such as human papillomavirus proteins, tumor antigens of the Smad family, Imp-1, P1A, EBV-encoded nuclear antigen (EBNA)-1, brain glycogen phosphorylase, SSX-1, SSX-2, SSX-4, SSX-5, HOMOGEN-MEL-40, SSX-1, SSX-2, SSX-3, SSX-4, SSX-5, HOMOGEN-MEL-40 ... SCP-1 and CT-7, c-erbB-2, Her2, EGFR, IGF-1R, CD2 (T cell surface antigen), CD3 (TCR-associated heteromultimer), CD22 (B cell receptor), CD23 (low affinity IgE receptor), CD30 (cytokine receptor), CD33 (myeloid cell surface antigen), CD40 (tumor necrosis factor receptor), IL-6R- (IL-6 receptor), CD20, MCSP, PDGFβR (β platelet-derived growth factor receptor), ErbB2 epithelial cell adhesion molecule (EpCAM), EGFR variant III (EGFR vIII), CD19, disialoganglioside GD2, ductal epithelial mucin, gp36, TAG-72, glioma-associated antigen, β-human chorionic gonadotropin, α-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxylesterase, mut These include hsp70-2, M-CSF, prostase, prostase-specific antigen (PSA), PAP, LAGA-1, p53, prostein, PSMA, surviving and telomerase, prostate cancer tumor antigen-1 (PCTA-1), ELF2M, neutrophil elastase, ephrin B2, insulin growth factor (IGF-1)-I, IGF-II, IGF-1 receptor, 5T4, ROR1, Nkp30, NKG2D, tumor stromal antigen, fibronectin extra domain A (EDA) and extra domain B (EDB), and tenascin-C A1 domain (TnC A1).

[0107] In some embodiments, the targeting moiety binds to an MHC-peptide complex or a peptide in an MHC-peptide complex, such as a tumor neo-antigen. Some tumor neo-antigens are viral antigens.

[0108] Non-limiting examples of viral antigens include EBV antigens (e.g., Epstein-Barr virus LMP-1), hepatitis C virus antigens (e.g., hepatitis C virus E2 glycoprotein), HIV antigens (e.g., HIV gp160, HIV gp120); CMV antigens; HPV-specific antigens, or influenza virus antigens (e.g., influenza virus hemagglutinin). Specific embodiments of tumor neo-antigens that can be bound by the targeting moieties of the present disclosure include the LCMV-derived peptide gp33-41, APF (126-134), BALF (276-284), CEA (571-579), CMV pp65 (495-503), FLU-M1 (58-66), gp100 (154-162), gp100 (209-217), HBV core (18-27), Her2 / neu (369-377; V2v9); HPV E7(11-20), KLK4(11-19), LMP1(125-133), MAG-A3(112-120), NYESO1(157-165, C165A), NYESO1(157-165, C165V), p54WT(264-272), PAP-3(136-143), PSMA(4-12), PSMA(135-145), survivin(96-014), tyrosinase(369-377, 371D), WT1(126-134).

[0109] Non-limiting examples of ECM antigens include syndecans, heparanase, integrins, osteopontin, link, cadherins, laminins, laminin EGF type, lectins, fibronectin, notch, tenascin, collagens, and matrixins.

[0110] Other target molecules include cell surface molecules of tumor or viral lymphocytes, for example, T cell costimulatory proteins such as CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, and B7-H3.

[0111] In certain embodiments, the target molecule is a checkpoint inhibitor, e.g., CTLA-4, PD1, PDL1, PDL2, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK1, CHK2.

[0112] In some embodiments, the IL10 agonist does not comprise a targeting moiety, for example, but not limited to, a targeting moiety that binds to a tumor-associated antigen, binds to a tumor microenvironment antigen, binds to a cell surface molecule of a tumor-reactive lymphocyte, or binds to a checkpoint inhibitor.

[0113] In various embodiments, the IL10 agonist does not comprise a PD1-binding targeting domain. 6.5.2. Targeting Moiety Format In certain aspects, the targeting moiety can be any type of antibody or fragment thereof that retains specific binding to an antigenic determinant. In one embodiment, the antigen-binding moiety is a full-length antibody. In one embodiment, the antigen-binding moiety is an immunoglobulin molecule, particularly an IgG class immunoglobulin molecule, more particularly an IgG1 or IgG4 immunoglobulin molecule. Antibody fragments include VH (or V H ) fragment, VL (or V L Antibody fragments include, but are not limited to, F(ab')2 fragments, Fab fragments, F(ab')2 fragments, scFv fragments, Fv fragments, minibodies, diabodies, triabodies, and tetrabodies. Antibody fragments may be advantageously incorporated N-terminal to the Fc domain in the IL10 agonists of the present disclosure.

[0114] 6.5.2.1.scFv Single-chain Fv or "scFv" antibody fragments comprise the VH and VL domains of an antibody in a single polypeptide chain, can be expressed as single-chain polypeptides, and retain the specificity of the intact antibody from which they are derived. Generally, scFv polypeptides further comprise a polypeptide linker between the VH and VL domains, which enables the scFv to form the desired structure for target binding. Examples of linkers suitable for connecting the VH and VL chains of scFvs are the linkers identified in Section 6.7.

[0115] Unless otherwise specified, as used herein, an scFv may have the VL and VH variable regions in either order, e.g., relative to the N-terminus and C-terminus of the polypeptide, and may comprise a VL-linker-VH or a VH-linker-VL.

[0116] The scFv can comprise VH and VL sequences derived from any suitable species, eg, murine, human or humanized VH and VL sequences. To generate a nucleic acid encoding an scFv, the VH- and VL-encoding DNA fragments are operably linked to another fragment encoding a linker, e.g., any of the linkers described in Section 6.7 (typically a sequence containing the amino acids glycine and serine, e.g., repeats of the amino acid sequence (Gly4 to Ser)3 (SEQ ID NO: 53), such that the VH and VL sequences are expressed as a contiguous single-chain protein, with the VL and VH regions joined by a flexible linker (see, e.g., Bird et al., 1988, Science, 242:423-426; Huston et al., 1988, Proc. Natl. Acad. Sci. USA, 85:5879-5883; McCafferty et al., 1990, Nature, 348:552-554).

[0117] 6.5.2.2.Fab Fab domains have traditionally been produced by proteolytic cleavage of immunoglobulin molecules using enzymes such as papain. In the IL10 agonists of the present disclosure, the Fab domain is typically recombinantly expressed as part of the IL10 agonist.

[0118] The Fab domain can comprise constant domain and variable region sequences derived from any suitable species, and therefore can be murine, chimeric, human, or humanized.

[0119] The Fab domain typically comprises a CH1 domain connected to a VH domain, which is paired with a CL domain connected to a VL domain. In wild-type immunoglobulins, the VH domain pairs with the VL domain to form the Fv region, and the CH1 domain pairs with the CL domain to further stabilize the binding module. Disulfide bonds between the two constant domains can further stabilize the Fab domain.

[0120] For the IL10 agonists of the present disclosure, particularly when IL10 contains two different Fab domains and the light chain is not a common or universal light chain, it is advantageous to use a Fab heterodimerization strategy to enable correct association of Fab domains belonging to the same Fab and minimize aberrant pairing of Fab domains belonging to different Fabs. For example, the Fab heterodimerization strategies shown in Table 2 below can be used.

[0121] [Table 2]

[0122] Thus, in a specific embodiment, correct association between the two polypeptides of a Fab is facilitated by swapping the VL and VH domains of the Fab with one another, or swapping the CH1 and CL domains with one another, as described, for example, in International Publication No. WO2009 / 080251.

[0123] Correct Fab pairing can also be promoted by introducing one or more amino acid modifications into the CH1 domain of the Fab, one or more amino acid modifications into the CL domain, and / or one or more amino acid modifications into the VH domain and one or more amino acid modifications into the VL domain. The modified amino acids are typically part of the VH:VL and CH1:CL interfaces, such that the Fab members pair preferentially with each other over other Fab members.

[0124] In one embodiment, the one or more amino acid modifications are limited to conserved framework residues of the variable (VH, VL) and constant (CH1, CL) domains, as indicated by the Kabat numbering of residues. Almagro, 2008, Frontiers In Bioscience, 13:1619-1633, provides definitions of framework residues based on the Kabat, Chothia, and IMGT numbering schemes.

[0125] In one embodiment, the modifications introduced into the VH and CH1 and / or VL and CL domains are complementary to each other. Complementarity at the heavy-light chain interface can be achieved based on steric and hydrophobic contacts, electrostatic / charge interactions, or a combination of various interactions. Complementarity between protein surfaces has been widely described in the literature in terms of lock and key fit, knobs-into-holes, protrusions and cavities, donors and acceptors, etc., all of which refer to the nature of the structural and chemical compatibility between two interacting surfaces.

[0126] In one embodiment, one or more of the modifications introduced introduce new hydrogen bonds across the interface of the Fab component. In one embodiment, one or more of the modifications introduced introduce new salt bridges across the interface of the Fab component. Exemplary substitutions are described in International Publication Nos. WO2014 / 150973 and WO2014 / 082179, the contents of which are incorporated herein by reference.

[0127] In some embodiments, the Fab domain comprises a 192E substitution in the CH1 domain and 114A and 137K substitutions in the CL domain, which introduce a salt bridge between the CH1 and CL domains (see, e.g., Golay et al., 2016, J Immunol, 196:3199-211).

[0128] In some embodiments, the Fab domain comprises substitutions 143Q and 188V in the CH1 domain and 113T and 176V in the CL domain, which serve to exchange hydrophobic and polar region contacts between the CH1 and CL domains (see, e.g., Golay et al., 2016, J Immunol, 196:3199-211).

[0129] In some embodiments, the Fab domain can contain modifications in part or all of the VH, CH1, VL, and CL domains to introduce an orthogonal Fab interface that promotes correct assembly of the Fab domain (Lewis et al., 2014, Nature Biotechnology, 32:191-198). In one embodiment, a 39K, 62E modification is introduced in the VH domain, an H172A, F174G modification is introduced in the CH1 domain, a 1R, 38D, (36F) modification is introduced in the VL domain, and an L135Y, S176W modification is introduced in the CL domain. In another embodiment, a 39Y modification is introduced in the VH domain and a 38R modification is introduced in the VL domain.

[0130] Fab domains can also be modified to replace the native CH1:CL disulfide bond with an engineered disulfide bond, thereby increasing the efficiency of pairing of the Fab components. For example, engineered disulfide bonds can be introduced by introducing 126C into the CH1 domain and 121C into the CL domain (see, e.g., Mazor et al., 2015, MAbs, 7:377-89).

[0131] Fab domains can also be modified by replacing the CH1 and CL domains with alternative domains that promote correct assembly. For example, Wu et al., 2015, MAbs, 7:364-76, describe replacing the CH1 domain with a constant domain of a T cell receptor and the CL domain with a b domain of a T cell receptor, pairing these domain replacements with additional charge-charge interactions between the VL and VH domains by introducing a 38D modification into the VL domain and a 39K modification into the VH domain.

[0132] Instead of, or in addition to, using a Fab heterodimerization strategy to promote correct VH-VL pairing, a VL of a common light chain (also referred to as a universal light chain) can be used for each Fab VL region of the IL10 agonist of the present disclosure. In various embodiments, employing a common light chain as described herein reduces the number of inappropriate species of the IL10 agonist compared to employing the original cognate VL. In various embodiments, the VL domain of the IL10 agonist is identified from a monospecific antibody that comprises a common light chain. In various embodiments, the VH region of the IL10 agonist comprises human heavy chain variable gene segments rearranged in vivo in mouse B cells previously engineered to express a limited human light chain repertoire or a single human light chain, cognate with the human heavy chain, and in response to exposure to an antigen of interest, generates an antibody repertoire containing multiple human VHs that are cognate with one or one of two possible human VLs, and this antibody repertoire is specific for the antigen of interest. Typical light chains are derived from rearranged human Vκ1-39Jκ5 or rearranged human Vκ3-20Jκ1 sequences, including somatically mutated (e.g., affinity matured) versions. See, e.g., U.S. Patent No. 10,412,940.

[0133] 6.6. Stabilization part The IL10 agonists of the present disclosure may contain a stabilizing moiety that can extend the serum half-life of the molecule in vivo. Serum half-life is often divided into an α-phase and a β-phase. Either or both phases may be significantly improved by the addition of an appropriate stabilizing moiety. For example, a stabilizing moiety can extend the serum half-life of an IL-10 agonist by 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 150, 200, 400, 600, 800, 1000% or more compared to a corresponding IL10 agonist that does not contain a stabilizing moiety. For purposes of this disclosure, serum half-life refers to half-life in humans or other mammals (e.g., mice or non-human primates). Furthermore, it is recognized that the inclusion of an Fc domain in an IL10 agonist increases the half-life of the IL10 moiety; in the context of this disclosure, the term "stabilizing moiety" refers to a moiety other than the Fc domain / Fc region.

[0134] The serum half-life of wild-type IL10 is less than 30 minutes. The IL10 agonist of the present disclosure preferably has a serum half-life of at least about 2 hours, at least about 4 hours, at least about 6 hours, or at least about 8 hours in humans and / or mice. In some embodiments, the IL10 agonist of the present disclosure has a serum half-life of at least 10 hours, at least 12 hours, at least 15 hours, at least 18 hours, at least 24 hours, at least 36 hours, at least 48 hours, at least 60 hours, or at least 72 hours.

[0135] Stabilizing moieties include polyoxyalkylene moieties (e.g., polyethylene glycol), sugars (e.g., sialic acid), and well-tolerated protein moieties (e.g., Fc and fragments and variants thereof, transferrin, or serum albumin).

[0136] Other stabilizing moieties that may be used in the IL10 agonists of the present disclosure include those described in Kontermann et al., 2011, Current Opinion in Biotechnology, 22:868-76. Such stabilizing moieties include, but are not limited to, human serum albumin fusions, human serum albumin conjugates, human serum albumin binders (e.g., Adnectins PKE, AlbudAb, ABD), XTEN fusions, PAS fusions (i.e., recombinant PEG mimetics based on the three amino acids proline, alanine, and serine), carbohydrate conjugates (e.g., hydroxyethyl starch (HES)), glycosylation, polysialic acid conjugates, and fatty acid conjugates.

[0137] Thus, in some embodiments, the present disclosure provides an IL10 agonist that includes a stabilizing moiety that is a polymeric sugar. Serum albumin can also be involved in half-life extension via a module capable of non-covalently interacting with albumin. Thus, the IL10 agonist of the present disclosure can include an albumin-binding protein as a stabilizing moiety. The albumin-binding protein can be conjugated or genetically fused to one or more other components of the IL10 agonist of the present disclosure. Proteins with albumin-binding activity are known from certain bacteria. For example, streptococcal protein G contains several small albumin-binding domains consisting of approximately 50 amino acid residues (6 kDa). Other examples of serum albumin-binding proteins are described, for example, in U.S. Patent Application Publication Nos. 2007 / 0178082 and 2007 / 0269422. Fusing an albumin-binding domain to a protein significantly extends half-life (see Kontermann et al., 2011, Current Opinion in Biotechnology, 22:868-76).

[0138] In other embodiments, the stabilizing moiety is human serum albumin. In other embodiments, the stabilizing moiety is transferrin. In still other embodiments, the stabilizing moiety is a polyethylene glycol moiety or another polymer, as described in Section 6.6.1 below.

[0139] The stabilizing moiety can be connected to one or more other components of the IL10 agonist of the present disclosure via a linker, for example, as described in Section 6.7 below.

[0140] In certain embodiments, the IL10 agonist is not conjugated to polyethylene glycol, other hydrophilic polymers, albumin, human serum albumin binders, XTEN, PAS, polysialic acid, and / or hydroxyethyl starch. In some embodiments, the IL10 agonist does not contain N-linked glycans and / or O-linked glycans. In one particular embodiment, the IL10 agonist lacks a stabilizing moiety.

[0141] Polyethylene glycol In some embodiments, the IL10 agonist comprises polyethylene glycol (PEG) or another hydrophilic polymer as a stabilizing moiety, such as ethylene glycol / propylene glycol copolymer, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymer, polyamic acid (either homopolymer or random copolymer), dextran or poly(n-vinylpyrrolidone) polyethylene glycol, propylene glycol (propropylene) homopolymer, propylene oxide / ethylene oxide copolymer, polyoxyethylated polyol (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. The polymer may be of any molecular weight and may be branched or unbranched.

[0142] PEG is a well-known water-soluble polymer that is commercially available or can be prepared by ring-opening polymerization of ethylene glycol according to methods known in the art (Sandler and Karo, Polymer Synthesis, Academic Press, New York, Vol. 3, 138-161). The term "PEG" is used broadly to encompass any polyethylene glycol molecule, regardless of size or PEG terminus modification, and has the formula: XO(CHCHO). n -1CH2CH2OH, where n is 20 to 2300, and X is H or a terminal modification, e.g., C 1-4 The PEG is an alkyl. PEG can further contain chemical groups necessary for conjugation reactions, which are generated by chemical synthesis of the molecule; or they serve as spacers for optimal distance between molecular parts. Furthermore, such PEG can consist of one or more PEG side chains linked together. PEGs with multiple PEG chains are called multi-arm PEGs or branched PEGs. Branched PEGs are described, for example, in European Patent Application Publication No. 473084A and U.S. Patent No. 5,932,462.

[0143] One or more PEG molecules can be attached to different positions on the IL10 agonist, and such attachment is achieved by reaction with an amine, thiol, or other suitable reactive group. The amine moiety may be, for example, a primary amine found at the N-terminus of the IL10 agonist (or a component thereof), or may be an amine group present in an amino acid, such as lysine or arginine.

[0144] PEGylation can be achieved by site-specific PEGylation, in which a suitable reactive group is introduced into a protein to create a site where PEGylation preferentially occurs. In some embodiments, an IL10 agonist is modified to introduce a cysteine ​​residue at a desired position, allowing site-specific PEGylation at the cysteine. Mutations can be introduced into the coding sequence of an IL10 agonist of the present disclosure to generate cysteine ​​residues. This can be achieved, for example, by mutating one or more amino acid residues to cysteine. Preferred amino acids for mutating to cysteine ​​residues include serine, threonine, alanine, and other hydrophilic residues. Residues mutated to cysteine ​​are preferably surface-exposed residues. Algorithms for predicting surface accessibility of residues based on primary sequence or three-dimensional structure are well known in the art. The three-dimensional structure of IL10, described, for example, in Wang et al., 2005, Science, 310(5751):1159-63, can be used to identify surface-exposed residues that can be mutated to cysteine. Mutations can be selected so as not to disrupt the interaction of IL10 with one or more receptors. PEGylation of cysteine ​​residues can be accomplished using, for example, PEG-maleimide, PEG-vinylsulfone, PEG-iodoacetamide, or PEG-orthopyridyl disulfide.

[0145] PEG is typically activated with a suitable activating group for coupling to the desired site on the polypeptide. Methods of PEGylation are known in the art and are further described in Zalipsky et al., "Use of Functionalized Poly(Ethylene Glycols) for Modification of Polypeptides," "Polyethylene Glycol Chemistry: Biotechnical and Biomedical Applications," JM Harris, Plenus Press, New York (1992), and Zalipsky, 1995, Advanced Drug Reviews, 16:157-182.

[0146] The PEG moiety can vary widely in molecular weight and can be branched or linear. Typically, the weight-average molecular weight of PEG is about 100 daltons to about 150,000 daltons. Exemplary weight-average molecular weights of PEG include about 20,000 daltons, about 40,000 daltons, about 60,000 daltons, and about 80,000 daltons. In certain embodiments, the molecular weight of PEG is 40,000 daltons. Branched PEGs having any of the above total molecular weights can also be used. In some embodiments, the PEG has two branches. In other embodiments, the PEG has four branches. In another embodiment, the PEG is a bis-PEG (NOF Corporation, DE-200MA) to which two IL10-containing polypeptide chains are conjugated.

[0147] The PEGylated IL10 agonist can be purified using conventional separation and purification techniques known in the art, such as size exclusion (e.g., gel filtration) and ion exchange chromatography. Products can also be separated using SDS-PAGE. Separable products include free PEG as well as mono-, di-, tri-, poly-, and non-PEGylated IL10 agonists. The proportion of mono-PEG conjugates can be controlled by pooling broader fractions around the elution peak to increase the proportion of mono-PEG in the composition. Approximately 90% mono-PEG conjugates provide a good balance between yield and activity.

[0148] In some embodiments, PEGylated IL10 agonists preferably retain at least about 25%, 50%, 60%, 70%, 80%, 85%, 90%, 95% or 100% of the biological activity associated with an unmodified IL10 agonist. D , k on , or k off It refers to its ability to bind to IL10R1, as assessed by

[0149] Linker In certain aspects, the present disclosure provides IL10 agonists in which two or more components of the IL10 agonist are connected to each other by a peptide linker. By way of example and not limitation, a linker can be used to link (a) an IL10 moiety and an Fc domain; (b) an IL10 moiety and a targeting moiety; (c) an Fc domain and a targeting moiety (e.g., a Fab domain or an scFv); or (d) multiple different domains within a targeting moiety (e.g., a VH domain and a VL domain within an scFv).

[0150] Peptide linkers can range from 2 amino acids to 60 or more amino acids, and in certain embodiments, peptide linkers range from 3 to 50 amino acids, 4 to 30 amino acids, 5 to 25 amino acids, 10 to 25 amino acids, 10 to 60 amino acids, 12 to 20 amino acids, 20 to 50 amino acids, or 25 to 35 amino acids in length.

[0151] Charged (eg, charged hydrophilic linkers) and / or flexible linkers are particularly preferred. Examples of flexible linkers that may be used in the IL10 agonists of the present disclosure include those disclosed by Chen et al., 2013, Adv Drug Deliv. Rev. 65(10):1357-1369 and Klein et al., 2014, Protein Engineering, Design & Selection, 27(10):325-330. Particularly useful flexible linkers are repeats of glycine and serine, e.g., G n S (SEQ ID NO: 9) or SG n (SEQ ID NO: 10), where n is an integer from 1 to 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In one embodiment, the linker is G4S (SEQ ID NO: 51), e.g., (GGGGS) n (SEQ ID NO: 11).

[0152] 6.7.1. Hinge arrangement In other embodiments, the IL10 agonist of the present disclosure comprises a linker that is a hinge region. In particular, when the IL10 agonist contains an immunoglobulin-based targeting moiety, the hinge can be used to connect the targeting moiety, for example, a Fab domain, to a multimerization domain, for example, an Fc domain. Even in the absence of a targeting moiety, the hinge sequence can be used to stabilize the IL10 agonist dimer.

[0153] The hinge region may be a native or modified hinge region. Hinge regions are typically found at the N-terminus of the Fc region. A native hinge region is the hinge region that would normally be found between the Fab and Fc domains in a naturally occurring antibody. A modified hinge region is any hinge that differs in length and / or composition from the native hinge region. Such hinges can include hinge regions from other species, such as humans, mice, rats, rabbits, sharks, pigs, hamsters, camels, llamas, and goats. Other modified hinge regions can include complete hinge regions from antibodies of a different class or subclass than that of the heavy chain Fc region. Alternatively, the modified hinge region can comprise a portion of a native hinge or a repeating unit in which each unit in the repeat is derived from a native hinge region. As a further alternative, the native hinge region can be altered by converting one or more cysteine ​​or other residues to neutral residues such as serine or alanine, or by converting appropriately positioned residues to cysteine ​​residues. By such means, the number of cysteine ​​residues in the hinge region can be increased or decreased. Other modified hinge regions may be entirely synthetic and may be designed to have desired properties, such as length, cysteine ​​composition and flexibility.

[0154] Several modified hinge regions have been previously described, for example, in U.S. Pat. No. 5,677,425, WO 99 / 15549, WO 2005 / 003170, WO 2005 / 003169, WO 2005 / 003170, WO 98 / 25971 and WO 2005 / 003171, which are incorporated herein by reference.

[0155] In various embodiments, positions 233-236 in the hinge domain can be G, G, G, and unoccupied; G, G, unoccupied, and unoccupied; G, unoccupied, unoccupied, and unoccupied; or all unoccupied, and the positions are numbered according to EU numbering.

[0156] In some embodiments, an IL10 mutein of the present disclosure comprises a modified hinge domain that confers reduced binding affinity to an Fcγ receptor relative to a wild-type hinge domain of the same isotype (e.g., human IgG1 or human IgG4).

[0157] In one embodiment, the Fc region of one or both chains of a dimeric IL10 agonist of the present disclosure has an intact hinge region at its N-terminus. In one embodiment, the Fc and hinge regions of one or both chains of the dimeric IL10 agonist of the present disclosure are derived from IgG4, and the hinge region contains the modified sequence CPPC. The core hinge region of human IgG4 contains the sequence CPSC, whereas IgG1 contains the sequence CPPC. The serine residues present in the IgG4 sequence increase the flexibility of this region; therefore, a certain percentage of the molecules form disulfide bonds within the same protein chain (intrachain disulfides) rather than cross-linking to other heavy chains within an IgG molecule to form interchain disulfides (Angel et al., 1993, Mol Immunol, 30(1):105-108). Changing the serine residues to proline to obtain the same core sequence as IgG1 allows for the complete formation of interchain disulfides within the IgG4 hinge region, reducing heterogeneity of the purified product. This modified isotype is referred to as IgG4P.

[0158] 6.7.1.1. Chimeric Hinge Sequences The hinge region may be a chimeric hinge region. For example, a chimeric hinge can comprise an "upper hinge" sequence derived from a human IgG1, human IgG2, or human IgG4 hinge region combined with a "lower hinge" sequence derived from a human IgG1, human IgG2, or human IgG4 hinge region.

[0159] In certain embodiments, the chimeric hinge region comprises the amino acid sequence EPKSCDKTHTCPPCPPAPPVA (SEQ ID NO: 12) (SEQ ID NO: 8 of WO 2014 / 121087, which is incorporated herein by reference in its entirety) or ESKYGPPCPCPCPPAPPVA (SEQ ID NO: 13) (SEQ ID NO: 9 of WO 2014 / 121087). Such a chimeric hinge sequence may be suitably linked to an IgG4 CH2 region (which may be further modified in the CH2 and / or CH3 domains to reduce effector function, e.g., by incorporating a human or mouse Fc domain into the IgG4 Fc domain, e.g., as described in Section 6.4.1).

[0160] 6.7.1.2. Hinge sequences with reduced effector function In further embodiments, the hinge region can be modified to reduce effector function, for example, as described in International Publication No. WO2016161010A2 (incorporated herein in its entirety). In various embodiments, positions 233-236 of the modified hinge region are G, G, G, and unoccupied; G, G, unoccupied, and unoccupied; G, unoccupied, unoccupied, and unoccupied; or all unoccupied, with positions numbered according to EU numbering (as shown in Figure 1 of International Publication No. WO2016161010A2). These segments can be represented as GGG-, GG--, G---, or ----, where "-" represents an unoccupied position.

[0161] Position 236 is unoccupied in standard human IgG2 but occupied in other standard human IgG isotypes. Positions 233-235 are occupied by residues other than G in all four human isotypes (as shown in Figure 1 of WO2016161010A2).

[0162] A hinge modification within positions 233-236 can be combined with position 228 occupied by P. Position 228 is naturally occupied by P in human IgG1 and IgG2, but by S in human IgG4 and by R in human IgG3. The S228P mutation in IgG4 antibodies is advantageous for stabilizing IgG4 antibodies and reducing heavy-light chain pair exchange between exogenous and endogenous antibodies. Preferably, positions 226-229 are occupied by C, P, P, and C, respectively.

[0163] An exemplary hinge region has residues 226-236, which are sometimes referred to as the middle (or core) hinge and lower hinge, and are occupied by modified hinge sequences designated as GGG-(233-236), GG--(233-236), G---(233-236), and no G(233-236). Optionally, the amino acid sequence of the hinge domain comprises CPPCPAPGGG-GPSVF (SEQ ID NO: 14) (SEQ ID NO: 1 of WO2016161010A2), CPPCPAPGG--GPSVF (SEQ ID NO: 15) (SEQ ID NO: 2 of WO2016161010A2), CPPCPAPG---GPSVF (SEQ ID NO: 16) (SEQ ID NO: 3 of WO2016161010A2), or CPPCPAP----GPSVF (SEQ ID NO: 17) (SEQ ID NO: 4 of WO2016161010A2).

[0164] The modified hinge region described above can be incorporated into a heavy chain constant region, which typically includes CH2 and CH3 domains and may have additional hinge segments (e.g., upper hinges) adjacent to the designated regions. Such additional constant region segments are typically of the same isotype, preferably a human isotype, but may also be hybrids of multiple different isotypes. The isotype of such additional human constant region segments is preferably human IgG4, but can also be human IgG1, IgG2, or IgG3, or hybrids in which the domains are of different isotypes. Exemplary sequences of human IgG1, IgG2, and IgG4 are shown in Figures 2-4 of International Publication No. WO2016161010A2.

[0165] In particular embodiments, a modified hinge sequence can be linked to the IgG4 CH2 region (e.g., by incorporating a human or mouse Fc domain into the IgG4 Fc domain; this can be further modified in the CH2 and / or CH3 domains to reduce effector function, e.g., as described in Section 6.4.1).

[0166] 6.8. Nucleic Acids and Host Cells In another aspect, the present disclosure provides nucleic acids encoding the IL10 agonists of the present disclosure. In some embodiments, the IL10 agonist is encoded by a single nucleic acid. In other embodiments, for example, in the case of heterodimeric molecules or molecules containing a targeting moiety composed of multiple polypeptide chains, the IL10 agonist can be encoded by multiple (e.g., two, three, four, or more) nucleic acids.

[0167] A single nucleic acid can encode an IL10 agonist comprising a single polypeptide chain, an IL10 agonist comprising two or more polypeptide chains, or a portion of an IL10 agonist comprising more than two polypeptide chains (e.g., a single nucleic acid can encode two polypeptide chains of an IL10 agonist comprising three, four, or more polypeptide chains, or three polypeptide chains of an IL10 agonist comprising four or more polypeptide chains). To separately control expression, open reading frames encoding two or more polypeptide chains are placed under the control of separate transcriptional regulatory elements (e.g., promoters and / or enhancers). Alternatively, open reading frames encoding two or more polypeptides can be controlled by the same transcriptional regulatory element and separated by an internal ribosome entry site (IRES) sequence, allowing translation into separate polypeptides.

[0168] In some embodiments, an IL10 agonist comprising two or more polypeptide chains is encoded by two or more nucleic acids, and the number of nucleic acids encoding the IL10 agonist is equal to or less than the number of polypeptide chains in the IL10 agonist (e.g., when multiple polypeptide chains are encoded by a single nucleic acid).

[0169] The nucleic acids of the present disclosure can be DNA or RNA (eg, mRNA). In another aspect, the disclosure provides host cells and vectors containing the nucleic acids of the disclosure, which can be present in a single vector or in separate vectors present in the same host cell or in separate host cells, as described in more detail below.

[0170] Vectors The present disclosure provides vectors comprising nucleotide sequences encoding the IL10 agonists or IL10 agonist components described herein, e.g., one or two of the polypeptide chains of a dimeric IL10 agonist. Vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phage, or yeast artificial chromosomes (YACs).

[0171] Numerous vector systems can be employed. For example, one class of vectors utilizes DNA elements derived from animal viruses, such as bovine papilloma virus, polyoma virus, adenovirus, vaccinia virus, baculovirus, retrovirus (Rous sarcoma virus, MMTV, or MOMLV), or SV40 virus. Another class of vectors utilizes RNA elements derived from RNA viruses, such as Semliki Forest virus, eastern equine encephalitis virus, and flaviviruses.

[0172] Furthermore, cells in which the DNA has been stably integrated into the chromosome can be selected by introducing one or more markers that allow for the selection of transfected host cells. Markers can confer, for example, prototropy to auxotrophic hosts, biocide resistance (e.g., antibiotics), or resistance to heavy metals such as copper. The selectable marker gene can be directly linked to the DNA sequence to be expressed or introduced into the same cell by cotransformation. Additional elements may be required for optimal mRNA synthesis. These elements can include splice signals as well as transcription promoters, enhancers, and termination signals.

[0173] Once the expression vector or DNA sequence containing construct is prepared for expression, the expression vector can be transfected or introduced into suitable host cells.To achieve this, various methods can be adopted, such as protoplast fusion, calcium phosphate precipitation, electroporation, retrovirus introduction, viral transfection, gene gun, lipid-based transfection or other conventional methods.The methods and conditions for culturing the resulting transfected cells and recovering the expressed polypeptide are known to those skilled in the art, and can be modified or optimized based on the present description according to the specific expression vector and mammalian host cell used.

[0174] 6.8.2.Cells The present disclosure also provides a host cell comprising a nucleic acid of the present disclosure. In one embodiment, the host cell is genetically engineered to contain one or more of the nucleic acids described herein.

[0175] In one embodiment, the host cell is genetically engineered by using an expression cassette. The term "expression cassette" refers to a nucleotide sequence that can affect the expression of a gene in a host that is compatible with such a sequence. Such a cassette can include a promoter, an open reading frame with or without introns, and a termination signal. Additional factors that are necessary or useful for affecting expression, such as an inducible promoter, can also be used.

[0176] The present disclosure also provides host cells comprising the vectors described herein. Also provided are IL10 agonists that are the product of recombinant cell expression in mammalian cells.

[0177] The cells may be, but are not limited to, eukaryotic cells, bacterial cells, insect cells, or human cells. Suitable eukaryotic cells include, but are not limited to, Vero cells, HeLa cells, COS cells, CHO cells, HEK293 cells, BHK cells, and MDCKII cells. Suitable insect cells include, but are not limited to, Sf9 cells.

[0178] 6.8.3. Production Method The present disclosure also provides methods of producing the IL10 agonists of the present disclosure. In some embodiments, the IL10 agonist is produced by culturing the host cells described in Section 6.8.2 and recovering IL10 expressed by the host cells. In certain embodiments, the method also includes a) contacting the recovered IL10 agonist with an affinity column configured to capture the IL10 agonist, b) eluting from the affinity column to produce an eluate, and c) performing size exclusion chromatography on the eluate.

[0179] The affinity column can be any affinity column configured to capture an IL10 agonist. For example, the affinity column can contain a binding molecule (e.g., an antibody or affinity ligand) that specifically binds to the Fc domain or IL10 portion of the IL10 agonist. In one embodiment, the affinity column is a Protein A affinity column. Protein A is an affinity ligand that contains five regions that bind to the Fc region of IgG. These regions are often coupled to Sepharose® and are free to bind to IgG Fc, such that one molecule of coupled Protein A can bind to at least two molecules of IgG. In another embodiment, the affinity column contains an IL10 antibody that can specifically and reversibly bind to the IL10 portion of the IL10 agonist.

[0180] Once bound to the affinity column, the captured IL10 agonist is eluted with an elution buffer to produce an eluate containing the IL10 agonist. Many elution buffers are known in the art, and one skilled in the art can select an appropriate buffer. In some embodiments, the elution buffer is Pierce™ Gentle Elution Buffer (pH 6.6).

[0181] The eluate containing the IL10 agonist is then subjected to size exclusion chromatography to isolate and purify the aggregate-free IL10 agonist. Many size exclusion chromatography methods and columns are known in the art. Those skilled in the art can select an appropriate size exclusion chromatography method and / or column. In some embodiments, size exclusion ultra-high performance liquid chromatography is used. In one particular embodiment, a Superdex® 200, 26 / 600 pg column (MilliporeSigma, St. Louis, Missouri, USA) is used.

[0182] In some embodiments, the elution buffer containing IL10 after affinity chromatography is exchanged with another buffer before passing the eluted IL10 agonist through size exclusion chromatography. This can be achieved by methods known in the art, such as dialysis. If the size exclusion chromatography column requires the use of a specific buffer, buffer exchange may be necessary. In one embodiment, before performing size exclusion chromatography, the elution buffer (e.g., Pierce™ Gentle) is exchanged with PBS + 5% glycerol.

[0183] With the benefit of this disclosure, one of skill in the art will be able to achieve a desired IL10 agonist by selecting an IL10 agonist construct with a certain orientation (i.e., whether the IL10 moiety is at the N-terminus or C-terminus), linker length, and purification method. In some embodiments, the desired IL10 agonist has minimal aggregation and is not truncated at the N-terminus and / or C-terminus of the construct. In certain embodiments, the IL10 agonist comprises an IL10 moiety at the C-terminus of the Fc domain, a linker that is one, two, or three repeats of G4S (SEQ ID NO: 51), and is purified using a process including size exclusion chromatography.

[0184] After production, the IL10 agonists of the present disclosure can be formulated as pharmaceutical compositions, for example, as described in Section 6.9. Pharmaceutical Compositions 6.9.1. Pharmaceutical Compositions Comprising IL10 Agonist Polypeptides The IL10 agonists of the present disclosure, e.g., those produced by the methods described in Section 6.8.3, can be provided in the form of compositions comprising the IL10 agonist and one or more carriers, excipients, and / or diluents. These compositions can be formulated for a particular use, e.g., veterinary use or human pharmaceutical use. The form of the composition (e.g., dry powder, liquid formulation, etc.) and the excipients, diluents, and / or carriers used will depend on the intended use of the IL10 agonist and, in the case of therapeutic uses, the method of administration.

[0185] For therapeutic use, the composition may be supplied as part of a sterile pharmaceutical composition containing a pharmaceutically acceptable carrier. This composition may be in any suitable form (depending on the desired method of administration to a patient). The pharmaceutical composition may be administered to a patient by a variety of routes, including oral, transdermal, subcutaneous, intranasal, intravenous, intramuscular, intratumoral, intrathecal, topical, or local. The most suitable route of administration in any given case will depend on the particular antibody, the subject, the nature and severity of the disease, and the physical condition of the subject. Typically, the pharmaceutical composition will be administered intravenously or subcutaneously.

[0186] The pharmaceutical composition may be conveniently presented in a unit dosage form containing a predetermined amount of the IL10 agonist of the present disclosure per administration. The amount of IL10 agonist contained in a unit dose will depend on the disease being treated as well as other factors known in the art. Such a unit dose may be in the form of a lyophilized powder containing an amount of IL10 agonist suitable for a single administration, or may be in liquid form. The dry powder unit dosage form may be packaged in a kit with a syringe, an appropriate amount of diluent, and / or other components useful for administration. A unit dosage in liquid form may conveniently be supplied in the form of a syringe pre-filled with an amount of IL10 agonist suitable for a single administration.

[0187] In some embodiments, the pharmaceutical compositions of the present disclosure comprise at least 5 mg, at least 10 mg, at least 20 mg, or at least 50 mg of an IL10 agonist. In certain embodiments, the pharmaceutical compositions of the present disclosure comprise only or almost only a full-length IL10 agonist. For example, in some embodiments, the pharmaceutical compositions do not comprise detectable amounts of C-terminal truncated variants of the IL10 agonist and / or detectable amounts of N-terminal truncated variants of the IL10 agonist. As defined herein, placing the IL10 moiety N-terminally of the Fc domain may result in N- and / or C-terminal truncation of the recombinantly expressed IL10 agonist.

[0188] In some embodiments, the pharmaceutical compositions of the present disclosure contain minimal or no IL10 agonist aggregates. Size exclusion chromatography, as defined herein, can be used to produce non-aggregated IL10 agonists. In some embodiments, the pharmaceutical compositions contain less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, or less than 5% of the IL10 agonist present in the form of aggregates, as determined by size exclusion ultra-performance liquid chromatography (SE-UPLC). In some embodiments, the pharmaceutical compositions do not contain detectable amounts of IL10 agonist aggregates, as determined by size exclusion ultra-performance liquid chromatography (SE-UPLC).

[0189] Alternatively, a pharmaceutical composition containing an amount of an IL10 agonist suitable for multiple administrations can be supplied in bulk. Pharmaceutical compositions can be prepared for storage as lyophilized formulations or aqueous solutions by mixing an IL10 agonist having the desired purity with optional pharmaceutically acceptable carriers, excipients, or stabilizers (all of which are referred to herein as "carriers") typically used in the art, i.e., buffers, stabilizers, preservatives, tonicity agents, non-ionic surfactants, antioxidants, and various other additives. See Remington's Pharmaceutical Sciences, 16th Edition (Osol, ed., 1980). Such additives should be nontoxic to recipients at the dosages and concentrations used.

[0190] Buffering agents help maintain pH in a range close to physiological conditions. They can be present in a wide variety of concentrations, but are typically present at concentrations ranging from about 2 mM to about 50 mM. Suitable buffering agents for use with the present disclosure include both organic and inorganic acids and their salts, such as citrate buffers (e.g., monosodium citrate-disodium citrate mixtures, citric acid-trisodium citrate mixtures, citric acid-monosodium citrate mixtures, etc.), succinate buffers (e.g., succinic acid-monosodium succinate mixtures, succinic acid-sodium hydroxide mixtures, succinic acid-disodium succinate mixtures, etc.), tartaric acid buffers (e.g., tartaric acid-sodium tartrate mixtures, tartaric acid-potassium tartrate mixtures, tartaric acid-sodium hydroxide mixtures, etc.), fumaric acid buffers (e.g., fumaric acid-monosodium fumarate mixtures, fumaric acid-monosodium fumarate mixtures, etc.), and the like. Examples of buffers include oxalic acid-disodium fumarate mixtures, monosodium fumarate-disodium fumarate mixtures, etc.), gluconic acid buffers (e.g., gluconic acid-sodium gluconate mixtures, gluconic acid-sodium hydroxide mixtures, gluconic acid-potassium gluconate mixtures, etc.), oxalic acid buffers (e.g., oxalic acid-sodium oxalate mixtures, oxalic acid-sodium hydroxide mixtures, oxalic acid-potassium oxalate mixtures, etc.), lactate buffers (e.g., lactic acid-sodium lactate mixtures, lactic acid-sodium hydroxide mixtures, lactic acid-potassium lactate mixtures, etc.), acetate buffers (e.g., acetic acid-sodium acetate mixtures, acetic acid-sodium hydroxide mixtures, etc.). Phosphate buffers, histidine buffers, and trimethylamine salts, such as Tris, may also be used.

[0191] Preservatives may be added to retard microbial growth and can be added in the range of about 0.2% to 1% (w / v). Suitable preservatives for use with the present disclosure include phenol, benzyl alcohol, metacresol, methylparaben, propylparaben, octadecyldimethylbenzylammonium chloride, benzalkonium halides (e.g., chloride, bromide, iodide, etc.), hexamethonium chloride, and alkylparabens, such as methyl or propylparaben, catechol, resorcinol, cyclohexanol, and 3-pentanol. Tonicity adjusting agents, sometimes known as "stabilizers," can be added to ensure the isotonicity of the liquid compositions of the present disclosure and include polyhydric sugar alcohols, such as trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol, and mannitol. Stabilizers refer to a broad category of excipients that can vary in function, from bulking agents to additives that solubilize therapeutic agents or help prevent denaturation or adhesion to container walls. Typical stabilizers include polyhydric sugar alcohols (listed above); amino acids such as arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, ornithine, L-leucine, 2-phenylalanine, glutamic acid, threonine, and the like; organic sugars or sugar alcohols such as lactose, trehalose, stachyose, mannitol, sorbitol, xylitol, ribitol, myo-inititol, galactitol, glycerol, and the like.Also included are cyclitols such as inositol; polyethylene glycol; amino acid polymers; sulfur-containing reducing agents such as urea, glutathione, thioctic acid, sodium thioglycolate, thioglycerol, α-monothioglycerol, and sodium thiosulfate; low molecular weight polypeptides (e.g., peptides of 10 residues or less); proteins such as human serum albumin, bovine serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; monosaccharides such as xylose, mannose, fructose, and glucose; disaccharides such as lactose, maltose, sucrose, and trehalose; and trisaccharides such as raffinose; and polysaccharides such as dextran. The stabilizer may be present in an amount ranging from 0.5 to 10% by weight per weight of the IL10 agonist.

[0192] Non-ionic surfactants or detergents (also known as "wetting agents") can be added to aid in solubilizing the therapeutic agent and to protect glycoproteins from agitation-induced aggregation, allowing the formulation to be exposed to stressed shear surfaces without denaturing the protein. Suitable non-ionic surfactants include polysorbates (e.g., 20, 80), poloxamers (e.g., 184, 188), and Pluronic® polyols. The non-ionic surfactant can be present in a range of about 0.05 mg / mL to about 1.0 mg / mL, or in a range of about 0.07 mg / mL to about 0.2 mg / mL.

[0193] Other miscellaneous excipients include bulking agents (eg, starch), chelating agents (eg, EDTA), antioxidants (eg, ascorbic acid, methionine, vitamin E), and cosolvents.

[0194] 6.9.2. Pharmaceutical Compositions for Delivery of Nucleic Acids Encoding IL10 Agonists The IL10 agonists of the present disclosure can be delivered by any method useful for gene therapy, for example, as mRNA or via a viral vector encoding the IL10 agonist under the control of a suitable promoter.

[0195] Exemplary gene therapy vectors include adenovirus- or AAV-based therapeutics. Non-limiting examples of adenovirus-based or AAV-based therapeutics for use in the methods, uses, or compositions herein include, for example, rAd-p53, which is a recombinant adenovirus vector encoding wild-type human tumor suppressor protein p53, used, for example, to treat cancer (Gendicine®, also known as Genkaxin®. Qi et al., 2006, Modern Oncology, 14:1295-1297); Ad5_d11520, which is an adenovirus lacking the E1B gene to inactivate host p53 (also referred to as H101 or ONYX-015; see, e.g., Russell et al., 2012, Nature Biotechnology, 30:658-670); AD5-D24-GM-CSF, which is an adenovirus containing the cytokine GM-CSF, used to treat cancer (Cerullo et al., 2010, Cancer Res., 70:4297; rAd-HSVtk, which is a replication-deficient adenovirus carrying the HSV thymidine kinase gene, for example, for the treatment of cancer (developed as Cerepro®, Ark Therapeutics).See, e.g., U.S. Pat. No. 6,579,855; developed by Advantagene as ProstAtak™; International Publication No. WO 2005 / 049094; rAd-TNFα, which is a replication-deficient adenoviral vector expressing human tumor necrosis factor alpha (TNFα) under the control of the chemo-radiation-inducible EGR-1 promoter, for use in, e.g., cancer therapy (TNFerade™, GenVec; Rasmussen et al., 2002, Cancer Genetics). Ther., 9:951-7; Ad-IFNβ, which is an E1- and E3-gene deleted adenovirus serotype 5 vector expressing the human interferon β gene under the direction of the cytomegalovirus (CMV) immediate-early promoter, for example, for cancer therapy (BG00001 and H5.110CMVhIFN-β, Biogen; Sterman et al., 2010, Mol. Ther., 18:852-860).

[0196] The nucleic acid molecule (e.g., mRNA) or virus can be formulated as the sole pharmaceutically active ingredient of the pharmaceutical composition or can be combined with other active agents for the particular disorder being treated. Optionally, other medicinal agents, pharmaceutical agents, carriers, adjuvants, diluents can be included in the compositions provided herein. For example, any one or more of wetting agents, emulsifying agents, and lubricating agents, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweeteners, flavorings, and perfuming agents, preservatives, antioxidants, chelating agents, and inert gases can be included in the composition. Exemplary other agents and excipients that can be included in the compositions include, for example, water-soluble antioxidants such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite; oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol; metal chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid.

[0197] When used as an adjuvant therapy for adoptive cell transfer therapy, such as the CAR-expressing cell therapy described in Section 6.11.1, the cell therapy, e.g., CAR-expressing cells, can be engineered to express the IL10 agonist of the present disclosure. The IL10 agonist can be targeted to a specific genomic locus, for example, the endogenous IL10 locus, or another locus active in activated or dysfunctional lymphocytes, for example, the PD-1 locus, or another locus inserted into a non-specific genomic locus. Targeting to a specific genomic locus can be achieved, for example, by gene editing using zinc finger proteins, CRISPR / Cas9 systems, etc.

[0198] 6.10. Treatment Indications and Methods The IL10 agonists of the present disclosure are useful in treating conditions treatable by IL10, such as inflammatory and immune-related disorders, fibrotic disorders, cancer and cancer-related disorders, or cardiovascular disorders (eg, atherosclerosis).

[0199] In certain embodiments, the condition treated by the IL10 agonists of the present disclosure is autoimmunity, transplant rejection, post-traumatic immune response, infectious disease, or graft-versus-host disease, hi certain embodiments, these conditions are autoimmune diseases, organ or bone marrow transplant rejection, graft-versus-host disease, parasitic infections, granulomas, Crohn's disease, colitis, pancreatitis, pulmonary inflammation, allergic conditions, asthma, atopic dermatitis, or rhinitis.

[0200] In other embodiments, the disease to be treated is a proliferative disorder, preferably cancer, for example, a solid tumor. Non-limiting examples of cancer include bladder cancer, brain malignancies, head and neck cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, uterine cancer, cervical cancer, endometrial cancer, esophageal cancer, colon cancer, colorectal cancer, rectal cancer, gastric cancer, prostate cancer, blood cancer, skin cancer, squamous cell carcinoma, bone cancer, and kidney cancer. Other cell proliferative disorders that can be treated using the IL10 agonists of the present disclosure include, but are not limited to, tumors located in the abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal glands, parathyroid glands, pituitary gland, testes, ovaries, thymus, thyroid), eyes, head and neck, nervous system (central and peripheral), lymphatic system, pelvis, skin, soft tissue, spleen, thoracic region, and genitourinary system. Precancerous conditions or lesions and cancer metastases are also included. In certain embodiments, the cancer is selected from the group consisting of renal cell carcinoma, skin cancer, lung cancer, colon cancer, breast cancer, brain malignancies, and head and neck cancer. Similarly, other cell proliferative disorders can be treated with the IL10 agonists of the present disclosure. Examples of such cell proliferative disorders include, but are not limited to, hypergammaglobulinemia, lymphoproliferative disorders, paraproteinemia, purpura, sarcoidosis, Sézary syndrome, Waldenstrom's macroglobulinemia, Gaucher disease, histiocytosis, and any other cell proliferative disorder of a tumor located in the organ systems listed above.

[0201] In some embodiments, the disease to be treated is resistant to treatment with an anti-PD1 antibody. In some embodiments, the disease to be treated is resistant to treatment with an anti-PD1 antibody as a monotherapy. While blockade of the PD1 / PD-L1 checkpoint has proven to be an effective treatment for some malignancies, it is ineffective in a significant proportion of patients, and some initial responders develop resistance, accompanied by disease recurrence (Nowicki et al., 2018, Cancer J., 24(1):47-53). Resistance to treatment with an anti-PD1 antibody can be primary or acquired. Both mechanisms leading to primary and acquired resistance to PD1 inhibition are known in the art, as are methods for identifying malignancies that are resistant to PD1 inhibition. See, e.g., Nowicki et al., supra; Shergold et al., 2019, Pharmacological Research, 145:204258; and Fares et al., 2019, American Society of Clinical Oncology Educational Book, 39:147-164.

[0202] The IL10 agonists of the present disclosure are generally considered to be used in an amount effective to achieve their intended purpose. When used to treat or prevent a disease state, the IL10 agonists of the present disclosure, or pharmaceutical compositions thereof, are administered or applied in a therapeutically effective amount. Determining a therapeutically effective amount is well within the capabilities of those skilled in the art, especially in light of the detailed disclosure provided herein. Those skilled in the art will readily recognize that in many cases, treatment with an IL10 agonist will not result in a cure but will only provide a partial benefit. In some embodiments, physiological changes that have some benefit are also considered therapeutically beneficial. Thus, the terms "effective amount" or "therapeutically effective amount" encompass dosages or dosing regimens that provide a partial benefit.

[0203] In some embodiments, the IL10 agonist of the present disclosure, or a pharmaceutical composition thereof, is administered or applied in an amount sufficient to increase the density of CD8 T cells in solid tumors relative to a suitable control (see Section 7.7 and Figures 18A-18B). The IL10 agonist of the present disclosure, or a pharmaceutical composition thereof, may increase the density of CD45 T cells in solid tumors. + It can be administered or applied in an amount that increases immune cell infiltration. + Immune cells include, for example, CD4 T cells and myeloid cells. Furthermore, the IL10 agonist of the present disclosure, or a pharmaceutical composition thereof, can be administered or applied in an amount sufficient to upregulate serum IL12 and / or IL4 expression relative to a suitable control (see Section 7.7 and Figures 21A-21B). In certain embodiments, the IL10 agonist of the present disclosure, or a pharmaceutical composition thereof, can upregulate IL-12 serum levels by at least 5-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, or at least 30-fold relative to a control (Figure 21A). In further embodiments, the IL10 agonist of the present disclosure, or a pharmaceutical composition thereof, can upregulate IL-4 serum levels by at least 5-fold, at least 10-fold, at least 15-fold, or at least 20-fold relative to a control (Figure 21A).

[0204] A suitable control sample for determining the reference or baseline values ​​of IL12 and / or IL4 can be derived from an individual with the same disease condition as the individual being treated with the disclosed IL10 agonist or pharmaceutical composition thereof. The control sample can be age-matched to the subject being treated with the disclosed IL10 agonist or pharmaceutical composition thereof. Reference or baseline values ​​can be obtained from a suitable individual or a population of suitable individuals and used as a common reference value for multiple analyses. Alternatively, the control sample can be based on the subject's own values ​​before the initiation of IL10 agonist therapy. Identifying and selecting a suitable control is within the ordinary skill of one of ordinary skill in the art.

[0205] The subject, patient, or individual in need of treatment is typically a mammal, more particularly a human. The appropriate dose of the IL10 agonist of the present disclosure (when used alone or in combination with one or more other additional therapeutic agents) for the prevention or treatment of a disease will depend on the type of disease to be treated, the route of administration, the patient's weight, the specific IL10 agonist, the severity and course of the disease, whether the antibody is administered for prophylactic or therapeutic purposes, previous or concurrent therapeutic interventions, the patient's medical history and response to the IL10 agonist, and the discretion of the attending physician. The physician responsible for administration will, in any case, determine the concentration of active ingredient in the composition and the appropriate dose for each individual subject. Various dosing schedules are contemplated herein, including, but not limited to, single administration or multiple administrations over various time periods, bolus administration, and pulse infusion.

[0206] A single dose of unconjugated IL10 can range from about 50,000 IU / kg to about 1,000,000 IU / kg or more, more typically up to about 600,000 IU / kg of IL10. This may be repeated several times daily (e.g., 2-3 times) for several days (e.g., about 3-5 consecutive days), followed by one or more rest periods (e.g., about 7-14 days). Thus, a therapeutically effective amount may consist of only a single dose or multiple doses over a period of time (e.g., about 20-30 individual doses of about 600,000 IU / kg of IL10 over a period of about 10-20 days).

[0207] Similarly, the IL10 agonist is suitably administered to the patient at once or over a series of treatments. Depending on the type and severity of the disease, for example, about 1 μg / kg to 15 mg / kg (e.g., 0.1 mg / kg to 10 mg / kg) of the IL10 agonist may be an initial candidate dose for administration to the patient, whether by one or more separate administrations or by continuous infusion. A typical daily dosage may range from about 1 μg / kg to 100 mg / kg or more, depending on the factors mentioned above. For repeated administration over several days or longer, depending on the condition, treatment would generally be continued until a desired suppression of disease symptoms is achieved. One exemplary dosage of the IL10 agonist would be in the range of about 0.005 mg / kg to about 10 mg / kg. In other non-limiting examples, a single dose would comprise about 1 μg / kg / body weight, about 5 μg / kg / body weight, about 10 μg / kg / body weight, about 50 μg / kg / body weight, about 100 μg / kg / body weight, about 200 μg / kg / body weight, about 350 μg / kg / body weight, about 500 μg / kg / body weight, about 1 mg / kg / body weight, about 5 mg / kg / body weight, about 10 mg / kg / body weight, about 50 mg / kg / body weight, about 100 mg / kg / body weight, about 200 mg / kg / body weight, about 350 mg / kg / body weight, about 500 mg / kg / body weight, up to about 1000 mg / kg / body weight or more, and any range derivable therein. Non-limiting examples of ranges derivable from the numerical values ​​recited herein include ranges based on the above numerical values, such as about 5 mg / kg / body weight to about 100 mg / kg / body weight, about 5 μg / kg / body weight to about 500 mg / kg / body weight, etc. Thus, one or more doses of about 0.5 mg / kg, 2.0 mg / kg, 5.0 mg / kg, or 10 mg / kg (or any combination thereof) can be administered to a patient. Such doses can be administered intermittently, for example, weekly or every three weeks (e.g., a patient receives about two to about 20, or, for example, about six, doses of an IL-10 agonist). An initial high loading dose can be followed by one or more lower doses. However, other dosing regimens may also be useful. The progress of this therapy is easily monitored by conventional techniques and assays.

[0208] For systemic administration, the therapeutically effective dose can be estimated initially from in vitro assays, such as cell culture assays, or in animal models based on the EC determined in cell culture. 50 Doses can be formulated to achieve a circulating concentration range comprising: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,

[0209] Initial dosages can also be estimated from in vivo data, e.g., animal models, using techniques known in the art. One skilled in the art would be able to readily optimize human dosage based on animal data.

[0210] The dosage and administration interval can be individually adjusted to achieve a plasma concentration of the IL10 agonist sufficient to maintain therapeutic efficacy. Typical patient dosages for administration by injection range from about 0.1 to 50 mg / kg / day, typically about 0.5 to 1 mg / kg / day. Therapeutically effective plasma levels can be achieved by administering multiple doses daily. Plasma levels can be measured, for example, by ELISA or HPLC.

[0211] In cases of local administration or selective uptake, the effective local concentration of the IL10 agonist may not be related to plasma concentration. One skilled in the art would be able to optimize the therapeutically effective local dosage without undue experimentation.

[0212] The therapeutically effective dose of the IL10 agonists described herein is generally believed to provide therapeutic benefit without causing substantial toxicity. The toxicity and therapeutic efficacy of IL10 agonists can be determined by standard pharmaceutical procedures in cell culture or experimental animals (see, e.g., Examples 8 and 9). Using cell culture assays and animal studies, LD 50 (the dose that is lethal in 50% of the population) and ED 50The dose ratio between toxic and therapeutic effects is the therapeutic index, which is the ratio LD 50 / ED 50 IL10 agonists that exhibit a high therapeutic index are preferred. In one embodiment, the IL10 agonists of the present disclosure exhibit a high therapeutic index. Data obtained from cell culture assays and animal studies can be used to establish a range of dosages suitable for use in humans. The dosage is determined based on the ED that results in little or no toxicity. 50 It is preferred that the circulating concentration of the compound be within a range including the range of 0.1 to 1.5 mg / kg of the active ingredient. Dosages can vary within this range depending on various factors, such as the dosage form employed, the route of administration utilized, the condition of the subject, etc. The exact formulation, route of administration, and dosage can be chosen by the individual physician in view of the patient's condition. (See, e.g., Fingl et al., 1975, The Pharmacological Basis of Therapeutics, Chapter 1, p. 1, which is incorporated herein by reference in its entirety.)

[0213] The attending physician of a patient being treated with an IL10 agonist of the present disclosure will know how and when to terminate, interrupt, or adjust administration in the event of toxicity, organ dysfunction, etc. Conversely, the attending physician will also know to adjust treatment to higher levels if the clinical response is inadequate (excluding toxicity). The magnitude of the administered dose in the management of the disorder of interest will vary depending on, for example, the severity of the condition to be treated and the route of administration. The severity of the condition may be assessed, in part, by standard prognostic evaluation methods. Furthermore, the dose, and perhaps the frequency of administration, will also vary depending on the age, weight, and response of the individual patient.

[0214] Combination therapy The IL10 agonist of the present disclosure may be administered in combination with one or more other additional agents in a therapeutic regimen. For example, the IL10 agonist of the present disclosure may be co-administered with at least one additional therapeutic agent. The term "therapeutic agent" encompasses any agent administered to treat a symptom or disease of a subject in need of such treatment. Such additional therapeutic agents may include any active ingredients suitable for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other.

[0215] In the case of cancer treatment, the IL10 agonist of the present disclosure can be administered in combination with one or more anti-cancer drugs (e.g., chemotherapeutic agents) or anti-cancer treatment modalities (e.g., radiation). The identity of the additional agent will largely depend on the nature of the underlying condition being treated (e.g., the addition of an alkylating agent such as cisplatin may be appropriate for the treatment of bladder cancer). The one or more additional agents (e.g., chemotherapeutic agents) administered in conjunction with the IL10 agonist are administered in an amount effective for the intended purpose. The effective amount of such additional agent will depend on the amount of IL10 agonist used, the type of disorder or treatment, and other factors discussed above.

[0216] The present IL10 agonists are generally used at the same dosages and by the same routes of administration as described herein, or at about 1 to 99% of the dosages described herein, or at any dosage and by any route determined empirically / clinically appropriate.

[0217] Examples of chemotherapeutic agents that may be used in combination with the IL10 agonists of the present disclosure include alkylating agents, such as thiotepa and cyclophosphamide; alkyl sulfonates, such as busulfan, improsulfan, and piposulfan; aziridines, such as benzodopa, carboquone, meturedopa, and uredopa; ethyleneimines and methylameramines, such as altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphine, and methylameramine. sulforamide and trimethylolomelamime; nitrogen mustards, such as chlorambucil, chlornaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, nobembicine, phenesterine, prednimustine, trophosfamide, uracil mustard; nitrosureas, such as carmustine, chlorozoto antibiotics such as aclacinomycin, actinomycin, anthramycin, azaserine, bleomycin, cactinomycin, calicheamicin, carabicin, caminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, macromycin, Itomycin, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate;Purine analogues, such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogues, such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; androgens, such as calsterone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone; antiadrenal agents, such as aminoglutethimide, mitotane, trilostane; folic acid supplements, replenishers, such as florinic acid acid); aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestravcil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; etoglucide; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; fenamet; pirarubicin; podophyllic acid; 2-ethylhydrazide; procarbazine; razoxane; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; urethane; vindesine; dacarbazine; mannomustine; mitobronitol acetaminophen; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, such as paclitaxel and docetaxel; chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum and platinum coordination complexes, such as cisplatin and carboplatin; vinblastine; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT11; topoisomerase inhibitors; difluoromethylornithine (DMFO); retinoic acid; esperamicin; capecitabine;The term "chemotherapeutic agent" includes, but is not limited to, any of the above-mentioned pharmaceutically acceptable salts, acids or derivatives. The term "chemotherapeutic agent" also includes antihormonal agents that regulate or inhibit hormone action on tumors, such as antiestrogens, including, for example, tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazole, 4-hydroxytamoxifen, trioxifene, keoxifene, onapristone and toremifene; and antiandrogens, such as flutamide, nilutamide, bicalutamide, leuprolide and goserelin; and any of the above-mentioned pharmaceutically acceptable salts, acids or derivatives. In some embodiments, the additional agent may be one or more chemical or biological agents identified in the art as useful in the treatment of neoplastic diseases, including, but not limited to, cytokines or cytokine antagonists such as IL12, INFα, or anti-epidermal growth factor receptor, radiation therapy, antibodies against tumor antigens, monoclonal antibody-toxin conjugates, T-cell adjuvants, bone marrow transplants, or antigen-presenting cells (e.g., dendritic cell therapy), anti-tumor vaccines, replication-competent viruses, and CART cells, e.g., as described in Section 6.11.1;

[0218] For the treatment of immune and inflammatory conditions, the IL10 agonists of the present disclosure can be used in combination with immunosuppressive or immunomodulatory therapy, including, but not limited to, immunosuppressive compounds such as cyclosporin A, cyclophosphamide, FK506, tacrolimus, corticosteroids, azathioprine, mycophenolate mofetil, sirolimus, rapamycin, rapamycin analogs, deoxyspergualin, and prednisone.

[0219] In some embodiments, the IL10 agonists of the present disclosure can be used in combination with anti-PD1 antibodies. Examples of anti-PD-1 antibodies for use in combination with the IL10 agonists of the present disclosure include, but are not limited to, MDX-1106 (nivolumab), MK-3475 (pembrolizumab), MEDI-0680 (AMP-514), PDR001, REGN2810, or BGB-108.

[0220] Such combination therapy as described above encompasses both combined administration (where two or more therapeutic agents are contained in the same or separate compositions) and separate administration, in which case administration of an IL10 agonist of the present disclosure may occur before, simultaneously with, and / or after administration of the additional therapeutic agent(s) and / or adjuvant.

[0221] 6.11.1. Combination Therapy Using IL10 Agonist Therapy and Immunotherapy The IL10 agonists of the present disclosure can be advantageously used in combination with chimeric antigen receptor ("CAR")-expressing cells, e.g., CAR-expressing T ("CAR-T") cells, e.g., CAR-T, in the treatment of cancer or autoimmune disease.

[0222] Conditioning or lymphodepletion therapy, for example, a regimen of cyclophosphamide and fludarabine, can also be administered to subjects receiving CAR and IL10 agonist therapy. Such treatment is usually administered several days before the administration of CAR-expressing cells to the subject. For example, cyclophosphamide can be administered for two days, for example, on days -8 and -7 (day of infusion being zero), before the infusion of CAR-expressing cells, and fludarabine can be administered for five consecutive days from days -6 to -2. In one embodiment, 60 mg / kg of cyclophosphamide is administered to the subject. In one embodiment, 25 mg / m 2 of fludarabine is administered to the subject. In one embodiment, there is a treatment-free day on day -1, which is the day immediately before the CAR-expressing cells are infused into the subject.

[0223] CAR-expressing cells were cultured at 10 4 From 10 9 Pieces / kg body weight, preferably 10 5 From 10 6 The T cell compositions can be administered in amounts ranging from 1×10 cells / kg body weight, including all integer values ​​within such ranges. The T cell compositions can also be administered multiple times at these dosages. In some embodiments, the CAR-expressing cells are administered in amounts ranging from 1×10 cells / kg body weight to 1×10 cells / kg body weight, including all integer values ​​within such ranges. The T cell compositions can also be administered multiple times at these dosages. In some embodiments, the CAR-expressing cells are administered in amounts ranging from 6 From 1×10 11 pcs or 1 x 10 7 From 1×10 8 It is administered in doses.

[0224] Prior to administration to a human subject, the CAR-expressing cells can be activated with anti-CD3 and / or anti-CD28 antibodies in concert with the expansion of IL10. The CAR-expressing cells, e.g., T cells, are preferably autologous to the subject, but may also be allogeneic.

[0225] In one embodiment, the IL10 agonist is administered to the human subject by bolus infusion for four consecutive days starting from the day of administration of the population of CAR-expressing cells. In one embodiment, the IL10 agonist is administered to the human subject by bolus for at least five consecutive days from the day of administration of the population of CAR-expressing cells.

[0226] IL10 agonist can be administered for a longer period, for example, for one week, two weeks, one month or longer.The frequency of administration can be reduced, for example, after the CAR-expressing cell is exhausted.For example, IL10 agonist can be administered every day at first, and then the frequency of administration can be reduced to once a week.

[0227] Initiation of IL10 therapy can be on the same day as administration of the CAR-expressing cells, or can begin 1, 2, 3, 4, 5, 6 days, or 1 week after administration. In one embodiment, the population of cells comprises T cells obtained from a subject that have been engineered to recombinantly express a CAR.

[0228] In one embodiment, the plasma levels of the IL10 agonist are maintained for one to two weeks following administration of the cell population to the subject. In one embodiment, the plasma levels of the IL10 agonist are maintained for one month following administration of the cell population to the subject.

[0229] CAR Components A typical CAR comprises an extracellular region containing an antigen-binding domain, e.g., the antigen-binding domain of an antibody, linked to an intracellular signaling block containing a CD3 signaling domain (e.g., the CD3ζ signaling region of a T cell receptor) that induces T cell activation after antigen binding. The antigen-binding domain may be in the form of an scFv, as described in Section 6.5.2.1.

[0230] The antigen binding domain typically comprises a linker (e.g., a linker described in Section 6.7), an optional spacer (e.g., as described in Section 6.11.1.1.1), an optional hinge (e.g., as described in Section 6.11.1.1.2), a transmembrane domain (e.g., as described in Section 6.11.1.1.3), and an intracellular signaling block (e.g., as described in Section 6.11.1.1.4).

[0231] Spacer Domain In certain embodiments, the antigen-binding domain of the CAR (followed by an optional linker) is followed by one or more "spacer domains," which refer to regions that move the antigen-binding domain away from the effector cell surface to allow proper cell-cell contact, antigen binding, and activation (Patel et al., 1999, Gene Therapy 6:412-419). Spacer domains may be derived from either natural, synthetic, semi-synthetic, or recombinant sources. In certain embodiments, the spacer domain is a portion of an immunoglobulin, including, but not limited to, one or more heavy chain constant regions, e.g., CH2 and CH3. The spacer domain can comprise the amino acid sequence of a naturally occurring immunoglobulin hinge region or an altered immunoglobulin hinge region.

[0232] In one embodiment, the spacer domain comprises the CH2 and CH3 domains of IgG1 or IgG4. Hinge Domain The antigen-binding domain of a CAR is generally followed by one or more "hinge domains" (downstream of an optional linker and / or spacer), which serve to position the antigen-binding domain away from the effector cell surface to allow for proper cell-cell contact, antigen binding, and activation. CARs generally include one or more hinge domains between the binding domain and the transmembrane domain (TM). The hinge domain may be derived from either natural, synthetic, semi-synthetic, or recombinant sources. The hinge domain can comprise the amino acid sequence of a naturally occurring immunoglobulin hinge region or an altered immunoglobulin hinge region.

[0233] An "altered hinge region" refers to (a) a naturally occurring hinge region with up to 30% amino acid changes (e.g., up to 25%, 20%, 15%, 10%, or 5% amino acid substitutions or deletions), (b) a portion of a naturally occurring hinge region that is at least 10 amino acids in length (e.g., at least 12, 13, 14, or 15 amino acids) with up to 30% amino acid changes (e.g., up to 25%, 20%, 15%, 10%, or 5% amino acid substitutions or deletions), or (c) a portion of a naturally occurring hinge region that includes the core hinge region (which may be 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in length, or may be at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in length). In certain embodiments, one or more cysteine ​​residues in a naturally occurring immunoglobulin hinge region may be substituted with one or more other amino acid residues (e.g., one or more serine residues). The altered immunoglobulin hinge region may alternatively or additionally have a proline residue in the wild-type immunoglobulin hinge region substituted with another amino acid residue (e.g., a serine residue).

[0234] Other exemplary hinge domains suitable for use in the CARs described herein include hinge regions derived from the extracellular regions of type 1 membrane proteins, such as CD8α, CD4, CD28, and CD7, which may be the wild-type hinge region from these molecules or may be altered. In another embodiment, the hinge domain comprises a CD8α hinge region.

[0235] Transmembrane (TM) Domain The "transmembrane domain" is the portion of the CAR that fuses the extracellular binding moiety with the intracellular signaling domain and anchors the CAR to the plasma membrane of an immune effector cell. As used herein, the term "transmembrane domain" refers to any polypeptide structure that is thermodynamically stable in a cell membrane, preferably a eukaryotic cell membrane (e.g., a mammalian cell membrane).

[0236] The TM domain may be derived from either natural, synthetic, semi-synthetic, or recombinant sources. The TM domain may be derived from (e.g., including at least the transmembrane regions of) the α, β, or ζ chain of the T cell receptor, CD3ε, CD3ζ, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, or PD1. In a specific embodiment, the TM domain is synthetic and composed primarily of hydrophobic residues such as leucine and valine.

[0237] In certain embodiments, the CAR comprises a CD3ζ transmembrane domain (e.g., a transmembrane domain comprising the amino acid sequence of LCYLLDGILFIYGVILTALFL (SEQ ID NO: 18) or LDPKLCYLLDGILFIYGVILTALFLRVK (SEQ ID NO: 19)), a CD28 transmembrane domain (e.g., a transmembrane domain comprising the amino acid sequence FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 20)), or a CD8α transmembrane domain (e.g., a transmembrane domain comprising the amino acid sequence KPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFA (SEQ ID NO: 21)).

[0238] The TM can be followed by a short linker, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in length, that connects the TM domain and the intracellular signaling domain of the CAR. Glycine-serine based linkers (e.g., linkers according to Section 6.7) are particularly preferred linkers.

[0239] 6.11.1.1.4. Intracellular Signaling Domain CARs typically contain an intracellular signaling domain. "Intracellular signaling domain" refers to the portion of the CAR that is involved in transmitting the message of effective antigen binding inside the immune effector cell to induce effector cell function, such as activation, cytokine production, proliferation and cytotoxic activity, including the release of cytotoxic factors to the target cell to which the CAR is bound, or other cellular response elicited by antigen binding to the extracellular CAR domain.

[0240] The term "effector function" refers to the specialized function of an immune effector cell. Effector functions of T cells are, for example, cytolytic activity, or aid or activity including cytokine secretion. Accordingly, the term "intracellular signaling domain" refers to the portion of a protein that transmits an effector function signal and directs the cell to perform a specialized function. While the entire intracellular signaling domain can usually be employed, it is often not necessary to use the entire domain. To the extent that a truncated portion of the intracellular signaling domain is used, such a truncated portion may be used in place of the entire domain, so long as it transmits the effector function signal. The term intracellular signaling domain is meant to include any truncated portion of the intracellular signaling domain sufficient to transmit the effector function signal.

[0241] It is known that the signal generated through TCR alone is insufficient for the complete activation of T cells, and a secondary signal or costimulatory signal is also required. Therefore, it can be said that T cell activation is mediated by two different classes of intracellular signaling domains: a primary signaling domain that initiates antigen-dependent primary activation through TCR (e.g., TCR / CD3 complex), and a costimulatory signaling domain that acts antigen-independently to provide a secondary signal or costimulatory signal. In a preferred embodiment, the CAR envisioned herein comprises an intracellular signaling domain that includes one or more "costimulatory signaling domains" and "primary signaling domains."

[0242] The primary signaling domain regulates the primary activation of the TCR complex, either stimulatory or inhibitory. Primary signaling domains that act in a stimulatory manner may contain signaling motifs known as immunoreceptor tyrosine-based activation motifs, or ITAMs.

[0243] Specific examples of ITAMs containing primary signaling domains that are particularly useful in the methods of the present disclosure include those derived from TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d. In certain preferred embodiments, the CAR comprises a CD3ζ primary signaling domain and one or more costimulatory signaling domains. The intracellular primary signaling domain and costimulatory signaling domain may be linked in series to the carboxyl terminus of the transmembrane domain in any order.

[0244] The CARs envisioned herein comprise one or more costimulatory signaling domains to enhance the efficacy and expansion of T cells expressing the CAR receptor. As used herein, the term "costimulatory signaling domain" or "costimulatory domain" refers to the intracellular signaling domain of a costimulatory molecule. A costimulatory molecule is a cell surface molecule other than an antigen receptor or an Fc receptor that, upon binding to an antigen, provides a second signal necessary for efficient activation and function of T lymphocytes. Specific examples of such costimulatory molecules include CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD152 (CTLA4), CD223 (LAG3), CD270 (HVEM), CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), DAP10, LAT, NKD2C SLP76, TRIM, and ZAP70.

[0245] In some embodiments, the CD3 signaling region has two signaling domains of CD3ζ linked to or aligned with the costimulatory endodomains of CD28, 4-1BB (also known as CD137), CD70, or OX40 (also known as CD134), or combinations thereof. These endodomains enable robust T cell activation upon TCR recognition by antigen-presenting cells (APCs) and improve cytokine production and proliferation of CAR-T cells.

[0246] In another embodiment, the CAR comprises CD28 and CD137 costimulatory signaling domains and a CD3ζ primary signaling domain. In yet another embodiment, the CAR comprises CD28 and CD134 costimulatory signaling domains and a CD3ζ primary signaling domain.

[0247] In one embodiment, the CAR comprises CD137 and CD134 costimulatory signaling domains and a CD3ζ primary signaling domain. Exemplary CD3 zeta signaling regions can include any of the following amino acid sequences:

[0248] [ka]

[0249] Exemplary CD28 signaling regions can include any of the following amino acid sequences:

[0250] [ka]

[0251] An exemplary CD137(41BB) signaling region can include the following amino acid sequence:

[0252] [ka]

[0253] TAGS In some embodiments, the CAR comprises a tag used to identify the CAR, for example, the V5 epitope tag, which is derived from a small epitope (Pk) present on the P and V proteins of the paramyxovirus Simian Virus 5 (SV5). The V5 tag is typically used with all 14 amino acids (GKPIPNPLLGLDST (SEQ ID NO: 28)), but may also be used with a shorter 9 amino acid sequence (IPNPLLGLD (SEQ ID NO: 29)).

[0254] Signal Peptide In some embodiments, the CAR comprises a signal peptide. The signal peptide facilitates the expression of the CAR on the cell surface. Signal peptides, including naturally occurring protein signal peptides or synthetic non-natural signal peptides, suitable for use in the CARs described herein will be apparent to those skilled in the art. In some embodiments, the signal peptide is located at the N-terminus of the antigen-binding portion of the CAR. When the CAR is expressed and processed in cells such as T cells, the signal peptide is cleaved, and therefore is typically not present in the mature molecule.

[0255] 6.11.1.2. Preparation of CART Cells To generate CART cells ex vivo, PBMCs, peripheral blood lymphocytes, or T cells enriched therefrom can be expanded before and / or after introducing a nucleic acid encoding a CAR into the cells, e.g., by viral transduction.

[0256] T cells useful for generating CART cells can be isolated from peripheral blood lymphocytes by lysing red blood cells and depleting monocytes, for example, by centrifugation through a PERCOLL™ gradient or by counterflow centrifugal elutriation. Specific subpopulations of T cells, such as CD3+, CD28+, CD4+, CD8+, CD45RA+, and CD45RO+ T cells, can be further separated by positive or negative selection techniques. For example, in one embodiment, T cells are isolated by incubation with anti-CD3 / anti-CD28 (e.g., 3x28)-conjugated beads, e.g., DYNABEADS® M-450 CD3 / CD28 T, for a time sufficient for positive selection of the desired T cells. In one embodiment, the time period is approximately 30 minutes. In a further embodiment, the time period ranges from 30 minutes to 36 hours or more, and all integer values ​​therebetween. In a further embodiment, the time period is at least 1, 2, 3, 4, 5, or 6 hours. In yet another preferred embodiment, the incubation period is 10 to 24 hours. In a preferred embodiment, the incubation period is 24 hours. For the isolation of T cells from leukemia patients, a relatively long incubation period, e.g., 24 hours, can be used to increase cell yield. In any situation where there are few T cells compared to other cell types, such as when isolating tumor-infiltrating lymphocytes (TILs) from tumor tissue or immunocompromised individuals, a longer incubation period can be used to isolate T cells. Furthermore, using a longer incubation period can increase the efficiency of capturing CD8+ T cells. Therefore, by simply shortening or lengthening the time that T cells are bound to CD3 / CD28 beads and / or increasing or decreasing the ratio of beads to T cells, subpopulations of T cells can be preferentially selected or eliminated at the beginning of the culture or at other points during the process.Additionally, by increasing or decreasing the ratio of anti-CD3 and / or anti-CD28 antibodies to beads or other surfaces, subpopulations of T cells can be preferentially selected for or eliminated at the initiation of culture or at other desired time points. Those skilled in the art will recognize that multiple rounds of selection can be used in the context of the present disclosure. In certain embodiments, it may be desirable to perform a selection procedure and use "unselected" cells in the activation and expansion process. "Unselected" cells can also be subjected to additional rounds of selection.

[0257] Enrichment of T cell populations by negative selection can be achieved by combining antibodies directed against surface markers specific to negatively selected cells. One method is cell sorting and / or selection via negative magnetic immunoadhesion or flow cytometry using a cocktail of monoclonal antibodies directed against cell surface markers present on negatively selected cells. For example, to enrich CD4+ cells by negative selection, the monoclonal antibody cocktail typically contains antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8. Treg cells can also be depleted by anti-C25-conjugated beads or other similar selection methods.

[0258] In certain embodiments, for example, for applications related to the treatment of autoimmune diseases as described in Section 6.11.1.4, typically CD4+, CD25+, CD62L hi , G.I.T.R. + , and FoxP3 + It may be preferable to enrich for or positively select for regulatory T cells that express FoxP3. Tregs can also be induced by recombinant expression of FoxP3.

[0259] Before or after genetic modification of the T cells to express the desired CAR, the T cells can be activated and expanded using methods known in the art, for example, as described in U.S. Pat. Nos. 7,144,575; 7,067,318; 7,172,869; 7,232,566; or 7,175,843.

[0260] Generally, T cells useful in the methods of the present disclosure are expanded by contacting a surface to which an agent that stimulates CD3 / TCR complex-associated signals and a ligand that stimulates costimulatory molecules on the surface of T cells are attached. In particular, T cell populations can be stimulated by contacting them with an anti-CD3 antibody or its antigen-binding fragment or an anti-CD2 antibody immobilized on a surface, or by contacting them with a protein kinase C activator (e.g., bryostatin) together with a calcium ionophore. To costimulate accessory molecules on the surface of T cells, a ligand that binds to the accessory molecule is used. For example, a population of T cells can be contacted with an anti-CD3 antibody and optionally an anti-CD28 antibody, for example, on anti-CD3 and anti-CD28 beads, under conditions suitable for stimulating T cell proliferation.

[0261] Prior to administration to a human subject, CAR-expressing cells can also be pretreated with IL12 (see, e.g., Emtage et al., 2003, J. Immunother., 16(2):97-106, incorporated herein by reference).

[0262] Cancer immunotherapy Thus, the present disclosure provides a method of treating cancer in a human subject in need thereof, the method comprising administering to the subject an effective amount of an IL10 agonist of the present disclosure and administering CAR-expressing cells, e.g., CAR-expressing T cells (or "CART cells"). Particularly useful T cell subtypes for cancer treatment are T cells with robust CAR-mediated cytotoxicity, e.g., CD3+CD8+ T cells, which can be prepared as described above in 6.11.1.2.

[0263] For cancer treatment, the extracellular domain of the CAR can be targeted to a tumor-associated antigen, for example, as described in Section 6.5.1. In certain embodiments, the tumor-associated antigen is CD20, EGFR, FITC, CD19, CD22, CD33, PSMA, GD2, EGFR variant, ROR1, c-Met, HER2, CEA, mesothelin, GM2, CD7, CD10, CD30, CD34, CD38, CD41, CD44, CD74, CD123, CD133, CD171, MUC16, MUC1, CS1 (CD319), IL-13Ra2, BCMA, Lewis Y, IgG kappa chain, folate receptor alpha, PSCA, or EpCAM. In certain embodiments: CARs are designed to target CD22 to treat diffuse large B-cell lymphoma.

[0264] CARs will be engineered to target mesothelin to treat cancers such as mesothelioma, pancreatic cancer, and ovarian cancer. CARs are designed to target CD33 / IL3Ra to treat diseases such as acute myeloid leukemia.

[0265] CARs will be engineered to target c-Met to treat triple-negative breast cancer, non-small cell lung cancer, etc. CARs will be engineered to target PSMA to treat cancers such as prostate cancer.

[0266] CARs will be designed to target the glycolipid F77 to treat cancers such as prostate cancer. CARs will be designed to target EGFRvIII to treat tumors such as glioblastoma.

[0267] CARs will be engineered to target GD-2 to treat neuroblastoma, melanoma, etc. CARs will be engineered to target the NY-ESO-1 TCR to treat myeloma, sarcoma, melanoma, etc.

[0268] CARs will be engineered to target the MAGE A3 TCR to treat myeloma, sarcoma, melanoma, etc. Particularly useful for CAR-IL10 agonist combination therapy are IL10 agonists that include a targeting moiety that recognizes a cell surface antigen present on the surface of lymphocyte cells expressing the CAR.

[0269] Immunotherapy for autoimmune diseases Chimeric antigen receptor (CAR) T cells have become a powerful therapeutic option for hematological cancers. By using the same idea of ​​modifying T cells to efficiently target diseased cells, scientists have efficiently generated T cells with predetermined antigen specificity through transfection of viral vectors encoding chimeric antigen receptors (CARs). CAR-modified T cells engineered in an MHC-unrestricted manner have the advantage of being broadly applicable, especially in transplantation and autoimmunity.

[0270] When used to treat autoimmune diseases, the extracellular domain of the CAR is preferably specific for a target antigen or ligand associated with the autoimmune response. Such modifications result in the activation of redirected Tregs at the site of inflammation, suppressing the inflammatory effector-type immune response. regExamples of autoimmune diseases that may be targeted by therapy include multiple sclerosis, inflammatory bowel disease (IBD), rheumatoid arthritis, systemic lupus erythematosus, Crohn's disease, psoriasis, type 1 diabetes, Sjogren's disease, myasthenia gravis (MG), Hashimoto's thyroiditis; Graves' disease, and uveitis.

[0271] In certain embodiments, CART cells are engineered to express a CAR that targets an antigen or ligand specific for: Inflammatory bowel disease (IBD), where the antigen or ligand is expressed in the affected colon or ileum; rheumatoid arthritis, where the antigen or ligand is an epitope of collagen or an antigen present in the joint; Type 1 diabetes or autoimmune insulitis, where the antigen or ligand is a pancreatic beta cell antigen; multiple sclerosis, where the antigen or ligand is, for example, myelin basic protein (MBP) antigen, or MOG-1 or MOG2-2, or a neuroantigen; · autoimmune thyroiditis, where the antigen or ligand is a thyroid antigen; · autoimmune gastritis, where the antigen or ligand is a gastric antigen; · autoimmune uveitis or uveoretinitis, where the antigen or ligand is S-antigen or another uveal or retinal antigen; · Autoimmune orchitis, where the antigen or ligand is a testicular antigen; · autoimmune oophoritis, where the antigen or ligand is an ovarian antigen; · psoriasis, where the antigen or ligand is a keratinocyte antigen or another antigen present in the dermis or epidermis; · Vitiligo, where the antigen or ligand is a melanocyte antigen such as melanin or tyrosinase; · autoimmune prostatitis, where the antigen or ligand is a prostate antigen; any unwanted immune response, wherein the antigen or ligand is an activating antigen or other antigen expressed on T effector cells present at the site of the unwanted response; tissue rejection, where the antigen or ligand is MHC specific to the transplanted tissue; or Inflammatory conditions, where the antigen or ligand is expressed on non-lymphoid cells of the hematopoietic lineage involved in the inflammation.

[0272] In one embodiment, T cells can be engineered to express chimeric autoantigen receptor (CAAR) T cells to specifically eliminate B cells responsible for autoimmune disease. Thus, unless the context dictates otherwise, reference to CAR-expressing T cells includes reference to CAAR expression. [Example]

[0273] 7. Working Example 7.1. Materials and Methods 7.1.1. Production of IL10 agonists Constructs were made encoding IL10 and IL10 muteins (identified by IL10M_), as well as Fc controls, listed below in Table 3. The IL10 mutein constructs included various configurations of mouse or human IL10, IgG1 or IgG4 Fc domains, and linkers of various lengths with various repeats of G4S (SEQ ID NO: 51).

[0274] [ka]

[0275] [ka]

[0276] [ka]

[0277] [ka]

[0278] [ka]

[0279] [ka]

[0280] These constructs were recombinantly expressed in mammalian cell lines and purified.

[0281] [Table 3-1]

[0282] [Table 3-2]

[0283] [Table 3-3]

[0284] [Table 3-4]

[0285] [Table 3-5]

[0286] [Table 3-6]

[0287] 7.1.2.STAT3 Reporter Assay A STAT3-driven luciferase-based reporter assay was developed to assess the ability of IL10 and IL10 muteins to activate STAT3-mediated transcription in TF-1 and Ramos, two cell lines previously reported to express the IL10 receptor (Tan et al., 1993, J Biol Chem., 268(28):21053-9).

[0288] 7.1.2.1. Engineering Reporter TF-1 Cells TF-1 cells (ATCC, #CRL-2003) were transduced with lentiviral particles carrying a STAT3 luciferase reporter construct (Cignal STAT3-Luc Lenti Reporter, SA Biosciences, CLS-6028L-8) in the presence of 5 μg / mL polybrene. Puromycin-resistant cells (TF-1 / STAT3-Luc) were selected and maintained in RPMI + 10% FBS + P / S / G + 2 ng / mL GM-CSF + 1 μg / mL puromycin.

[0289] 7.1.2.2. Manipulation of Reporter Ramos Cells Ramos.2G6.4C10 cells (ATCC, # CRL-1923) were transduced with lentiviral particles carrying a STAT3 luciferase reporter construct (Cignal STAT3-Luc Lenti Reporter, SA Biosciences, CLS-6028L-8) in the presence of 5 μg / mL polybrene. Puromycin-resistant cells (Ramos / STAT3-Luc) were selected and maintained in RPMI + 10% FBS + P / S / G + 1 μg / mL puromycin.

[0290] 7.1.2.3. IL10 stimulation of reporter cells In this experiment, engineered reporter cells are stimulated with either recombinant IL10 or an IL10 mutein. This cytokine binds to the IL10 receptor subunit α (IL10Ra) and recruits the β subunit (IL10Rb), allowing the assembly of a signaling complex and inducing STAT3 phosphorylation (Yoon et al., 2006, J Biol Chem., 281(46):35088-96). STAT3 phosphorylation leads to enhanced transcriptional activity of the STAT3 response element, driving the production of the reporter gene luciferase.

[0291] 7.1.2.4. Luciferase assay setup RPMI1640 supplemented with 10% FBS and P / S / G was used as the assay medium for preparing cell suspensions and antibody dilutions.

[0292] One day before screening, engineered reporter cells were diluted 1:3. On the day of the assay, cells were spun down and diluted to 1 x 10 in assay medium. 6 IL10, IL10 muteins and controls were diluted 1:5 and then diluted in 11 steps ranging from 100 nM to 10 fM, with step 12 containing no recombinant protein. 4 Reporter cells were added to a 96-well white flat-bottom plate and incubated with serially diluted IL10, IL10 muteins, or control proteins. After incubating the plate at 37°C / 5% CO2 for 5 hours and 30 minutes, 100 μL of ONE-Glo™ (Promega) reagent was added to lyse the cells and detect luciferase activity. Light emission was measured as relative light units (RLU) using a multilabel plate reader Envision® (PerkinElmer). All serial dilutions were tested in duplicate.

[0293] For the determination of EC50 values, the hook effect, which causes a dose-dependent decrease in signal when the protein concentration is higher than the maximum signal, was excluded. Antibody EC50 values ​​were determined using GraphPad Prism™ software from a four-parameter logistic equation for a 12-step dose-response curve, where the 12th dilution step did not contain recombinant protein. Fold induction was calculated using the following formula:

[0294]

number

[0295] 7.1.3. Characterization of Human IL10 Muteins in Cell-Based Assays Using Primary Human Immune Cells To assess the effect of IL10 stimulation on the release of IL2, TNFα, and IFNγ during T cell activation, a functional assay of primary mixed T cell / allogeneic PBMCs was developed.

[0296] 7.1.3.1. Isolation of Human Primary PBMCs Human peripheral blood mononuclear cells (PBMCs) were isolated from leukopacks (donor 123) of a healthy donor. PBMC isolation was achieved by density gradient centrifugation using 50 mL SepMate™ tubes according to the manufacturer's recommended protocol. Briefly, the leukopacks were diluted 1:2 with D-PBS, and a 30 mL layer was layered on top of 15 mL of Ficoll® in a 50 mL SepMate™ tube. Subsequent procedures were performed according to the SepMate™ manufacturer's protocol. Isolated PBMCs were frozen in FBS supplemented with 10% DMSO.

[0297] 7.1.3.2. Isolation of Human Primary T Cells T cells were isolated from leukopacks of two healthy donors (donors 5500Y and 6900M). T cell isolation was achieved using RosetteSep® Human T cell enrichment cocktail (StemCell) according to the manufacturer's protocol. T cells were isolated by density gradient centrifugation using 50 mL SepMate™ tubes according to the manufacturer's recommended protocol. Isolated T cells were placed in FBS supplemented with 10% DMSO.

[0298] 7.1.3.3. Cytokine Release from Primary T Cells Co-cultured with IL10 and Allogeneic PBMCs In this experiment, primary T cells were stimulated via a 3-day coculture with allogeneic PBMCs treated with mitomycin C to arrest PBMC proliferation. Titrations of IL10 or IL10 muteins were added during the coculture, and their effects on T cell activity were determined by measuring the release of IL2, TNFα, and IFNγ in the cell culture supernatant using homogenous no-wash AlphaLISA® kits (PerkinElmer, AL208F AL217S AL221F).

[0299] Previously isolated and frozen human T cells from donors 5500Y and 6900M were thawed on the day of analysis in stimulation medium (X-VIVO™ 15 cell medium supplemented with 10% FBS, HEPES, NaPyr, NEAA, 0.01 mM BME) containing 50 U / mL of benzonase nuclease. Cells were centrifuged at 1200 rpm for 10 minutes, resuspended in stimulation medium, and diluted to 1 x 10 5 Cells were plated in 96-well round-bottom plates at a concentration of 1 x 10 cells / well. Human PBMCs previously isolated and frozen from donor 123 were thawed on the day of analysis in stimulation medium containing 50 U / mL of benzonase nuclease. Cells were centrifuged at 1200 rpm for 10 minutes and plated at 1 x 10 cells / well in stimulation medium containing 50 μg / mL of mitomycin C.7 After 1 hour of incubation at 37°C, PBMCs were washed three times with D-PBS containing 2% FBS and resuspended in stimulation medium to give 1.5 x 10 T cells per well. 5 IL10, IL10 muteins, and controls were then diluted 1:5 and then serially diluted in nine steps, ranging from 500 nM to 1.28 pM for IL10 and from 100 nM to 0.256 pM for IL10 muteins or control antibodies, with step 10 containing no recombinant protein. Serially diluted proteins were added to the wells, and the plates were incubated at 37°C / 5% CO for 72 hours. The plates were then centrifuged to pellet the cells, and 50 μL of supernatant was collected. Five μL of supernatant was then taken and tested using the AlphaLISA® assay for human IL-2, TNFα, and IFNγ according to the manufacturer's protocol. Measurements were taken using the Envision® multilabel plate reader. Standard curves of known concentrations of IL-2, TNFα, and IFNγ were generated to extrapolate pg / mL of each cytokine released into the assay wells. All serial dilutions were tested in duplicate.

[0300] Cytokine IC50 values ​​were determined using GraphPad Prism™ software from a four-parameter logistic equation for 10-step dose-response curves where the 10th dilution step did not contain recombinant protein. Percent inhibition was calculated using the following formula:

[0301]

number

[0302] 7.1.4. Activity of IL-10 Muteins in Syngeneic Tumor Allograft Models 7.1.4.1. Tumor Implantation and Treatment Group Allocation Tumor cells were implanted subcutaneously into the right flank of female BALB / c mice (The Jackson Laboratory, Bar Harbor, Maine, USA) at 1 × 10 cells per mouse. Six to eight weeks of age, female BALB / c mice (The Jackson Laboratory, Bar Harbor, Maine, USA) were implanted subcutaneously with tumor cells at 1 × 10 cells per mouse. 6 Tumor cells were implanted subcutaneously.

[0303] Therapeutic intervention or isotype control was administered intraperitoneally for 3 weeks. Mice were injected intraperitoneally with test substances twice a week for 3 weeks, and tumor volume and body weight were monitored twice a week throughout the study.

[0304] 7.1.4.2. Calculation of Tumor Size and Growth Inhibition For each group, the mean and median tumor size, as well as the percentage of tumor growth inhibition compared to the control-treated group, were calculated. The length and width of tumors were measured twice weekly with calipers, and tumor volume was calculated using the formula (length x width). 2 ) / 2. Measurements were taken at a mean tumor size of 4000 mm in the control group. 3 The tumor growth inhibition was calculated according to the following formula: [1-(T final -T initial ) / (C final -C initial )]*100, where T (treated group) and C (control group) represent the mean tumor mass on the day all mice were alive. Observation was extended to day 54 (3 weeks after the last administration) to determine the frequency of tumor-free mice.

[0305] 7.2. Example 1: Activity of IL10 muteins in a STAT3 reporter assay The ability of recombinant IL10 muteins to stimulate the IL10 receptor was assessed in a STAT3 reporter cell-based bioassay described in Section 7.1.2.

[0306] Activation curves are shown in Figure 3, and EC50 and fold induction values ​​are summarized in Table 4 for engineered reporter cells, Ramos / STAT3-Luc, or TF-1 / STAT3-Luc cells incubated with recombinant human IL10, IL10 muteins, human IgG4s isotype control, or mouse IgG1 isotype control.

[0307] No increase in luciferase activity was detected when reporter cells were treated with control proteins. In contrast, incubation of reporter cells Ramos / STAT3-Luc and TF-1 / STAT3-Luc with human IL10 induced luciferase activity (4.5-fold and 5.9-fold, respectively). Incubation of Ramos / STAT3-Luc and TF-1 / STAT3-Luc with IL10 muteins also induced luciferase activity (Ramos / STAT3: IL10M1: 3.7-fold, IL10M2: 3.6-fold, IL10M3: 3.2-fold; TF-1 / STAT3-Luc, IL10M1: 4.8-fold, IL10M2: 4.1-fold, IL10M3: 3.4-fold), with subnanomolar EC50 values, as shown in Table 4 and Figure 3.

[0308] [Table 4]

[0309] 7.3. Example 2: Activity of IL10 Muteins on Cytokine Release in Primary Human T Cells The ability of IL10 to inhibit T cell stimulation was assessed in functional primary T cell assays measuring IL2, TNFα and IFNγ cytokine production.

[0310] Data on cytokine release for T cells co-incubated with mitomycin C-treated allogeneic PBMCs along with titrations of H4sH10154P3 (human IgG4 stealth isotype control), mIgG1 (mouse IgG1 isotype control), human IL10, or an IL10 mutein are shown in Figures 4 and 5 (for donors 5500Y and 6900M, respectively), and IC50 and percent inhibition values ​​are summarized in Tables 5 and 6 (for donors 5500Y and 6900M, respectively).

[0311] Coincubation of T cells from two donors (Figure 4: donor 5500Y and Figure 5: donor 6900M) with mitomycin C-treated allogeneic PBMCs resulted in measurable IL2 (A), TNFα (B), and IFNγ release (C). Addition of titrations of human IL10 or IL10 Fc muteins during T cell / PBMC coincubation reduced IL2, TNFα, and IFNγ release in a dose-dependent manner (donor 5500Y: Table 5 and Figure 4; donor 6900M: Table 6 and Figure 5).

[0312] [Table 5]

[0313] [Table 6]

[0314] 7.4. Example 3: Antitumor Activity of IL10 Muteins The activity of IL10 muteins was evaluated in a syngeneic tumor allograft model. Colon 26 tumor cells were implanted into BALB-C mice as described in Section 7.1.4. On day 11, tumors grew to a mean volume of 110 mm. 3 (80mm 3 From 140mm 3Once the mice reached a normal serum concentration (range 0.167 mg / kg), they were randomized into groups (n=7 / group) and administered either hIgG4s isotype control, IL10M1, or IL10M2 at equimolar doses of isotype control (0.167 mg / kg) or IL10 mutein (0.1 mg / kg).

[0315] The results are summarized in Table 7 and shown in Figure 6.

[0316] [Table 7]

[0317] By day 32, when all mice were still alive, six doses of IL10M1 resulted in approximately 90% tumor growth inhibition, whereas IL10M2 administration was only marginally effective (approximately 7% tumor growth inhibition) compared with the isotype control-treated group. Extended observations revealed that 4 of 7 mice in the IL10M1-treated group were tumor-free, whereas only 2 of 7 mice in the IL10M2-treated group were tumor-free, with no mice in the control group tumor-free. These data support the conclusion that IL10 fused to the C-terminus of hIgG4s-Fc (IL10M1) exhibits superior efficacy compared with the N-terminal Fc-IL-10 fusion protein (IL10M2) in the Colon26 in vivo tumor model.

[0318] 7.5. Example 4: Effect of Linker Length on IL10 Mutein Activity The activity of IL10 muteins with different linker lengths (including two different lots each of IL10M1 and IL10M2, designated Lot 1 and Lot 2 or L1 and L2, respectively) was assessed in the Ramos / STAT3-Luc and TF-1 / STAT3-Luc assays as described in Section 7.1.2. Cells were harvested, centrifuged, resuspended in assay medium (Jurkat medium), and plated at 5 × 10 cells per well in a 50 μl volume onto flat-bottom culture dishes with white bottom / sides. 5Cells / well were plated and then 50 μl of medium containing a titration of IL10 or IL10 muteins was added.

[0319] Cells were incubated for 5.5 hours, after which 100 μL of ONE-Glo™ luciferase reagent (Promega Corporation, Madison, Wis.) was added. The plate was incubated in the presence of ONE-Glo™ for 3 minutes, and activity was read on an Envision™ fluorescent plate reader (Perkin Elmer, Waltham, Mass.).

[0320] Fold induction and EC50 were determined as described in section 7.1.2.4. The results are summarized in Table 8-1 and Table 8-2 and shown in Figures 7A-7F.

[0321] [Table 8]

[0322] These results suggest that IL10 muteins with the IL10 moiety at the C-terminal end of the Fc moiety are more active than IL10 muteins with the IL10 moiety at the N-terminal end of the Fc moiety, and that IL10 muteins have a higher EC 50 Furthermore, the G4S (SEQ ID NO: 51) linker length was found to have a low induction fold and EC 50 Although complete loss of the linker from IL10 muteins in which the IL10 portion is C-terminal to the Fc portion only slightly affects activity, a larger loss of activity results.

[0323] 7.6. Example 5: Mechanism of Action of IL10 Antitumor Activity Using a syngeneic tumor allograft model, the in vivo antitumor efficacy of murine IL10 fused to murine Fc (IL10M11) was evaluated in either a prophylactic or therapeutic setting.

[0324] Briefly, female mice (the Jackson Laboratory, Bar Harbor, ME, USA) were subcutaneously implanted with lineage-matched tumor cells (Tissue Culture Core, Regeneron Pharmaceuticals, Inc.): C57BL / 6 syngeneic tumor cells MC38-cOVA (MC38 colon carcinoma cells engineered to overexpress chicken ovalbumin); BALB / c syngeneic tumor cells Colon26 (colon carcinoma), A20 (B-cell lymphoma), 4T1 (breast carcinoma), or RENCA (renal cell carcinoma).

[0325] In the preventative setting, treatment was initiated 2 days before MC38-cOVA tumor implantation and continued twice weekly. In the therapeutic setting, treatment was initiated 2 days before MC38-cOVA tumor implantation and continued twice weekly until the mean tumor volume (typically 60-120 mm) was reached. 3 Mice were randomly assigned to treatment groups (n = 7-9) based on tumor size. Treatment began after tumor randomization and continued twice weekly.

[0326] IL10M11 or isotype control was administered intraperitoneally at the same molar dose (6–9 doses per mouse). In IL10M11 titration studies, the dose of isotype control was equal to the highest molar dose of IL10M11.

[0327] Tumor size and mouse weight were measured twice weekly. To investigate the mechanism of action of IL10's antitumor activity, IL10M11 was evaluated prophylactically and therapeutically in a tumor allograft model. As described in section 7.1.4, tumor cells were implanted into BALB-C or C57BL / 6 mice, and IL10M11 was administered prophylactically (before tumor implantation) or therapeutically (7 days after tumor implantation). The results are shown in Figure 8A-8D.

[0328] 7.6.1 Materials and Methods Tumor implantation and treatment group assignment: For the MC38-cOVA, Colon26, and RENCA syngeneic tumor models, 1 × 10 tumor cells were implanted into the right flank of 6- to 8-week-old female C57BL / 6 or BALB / c mice (the Jackson Laboratory, Bar Harbor, ME, USA). 6 1 × 10 tumor cells (Tissue Culture Core, Regeneron Pharmaceuticals, Inc.) were subcutaneously implanted into the tumor cells. For the A20 and 4T1 syngeneic tumor models, 1 × 10 7 pcs or 1 x 10 5 Tumor cells (Tissue Culture Core, Regeneron Pharmaceuticals, Inc.) were subcutaneously implanted into the right flank of 6- to 8-week-old female BALB / c mice (The Jackson Laboratory, Bar Harbor, Maine, USA). Tumors grew to an average volume of 60-120 mm. 3 Upon reaching a tumor size of 1000 mg / mL (depending on each specific tumor model), mice were randomized into groups (n=7-9 / group) and administered IL10M11, CD40, PD-1, control reagents, or in some cases a combination. Mice were intraperitoneally injected with test substances twice weekly for 6-9 doses, and tumor volume and body weight were monitored twice weekly throughout the study. Anti-tumor memory responses were tested in mice that rejected the primary Colon26 tumor challenge with a 5x10 6 Colon26 tumor cells were administered to naive mice, whereas naive mice received 1 × 10 6 Only one dose of Colon26 tumor cells was received.

[0329] Calculation of tumor size and growth inhibition: For each group, the mean and median tumor size, as well as the percentage of tumor growth inhibition compared to the control-treated group, were calculated. The length and width of the tumor were measured twice weekly with calipers, and tumor volume was calculated using the formula (length x width). 2) / 2. Measurements were taken at a mean tumor size of 4000 mm in the control group. 3 The tumor growth inhibition rate was calculated using the following formula: [1-(T final -T initial ) / (C final -C initial )]*100%, where T (treated group) and C (control group) represent the mean tumor mass on the day all mice were alive.

[0330] 7.6.2.Results Prophylactic treatment of MC38-cOVA tumors with the highest tested dose of IL10M11 (0.1 mg / kg) demonstrated a modest antitumor effect, with tumor growth inhibition (TGI) of approximately 30% and 1 of 8 tumor-free mice (Figure 8A). Lower doses (0.005 or 0.0003 mg / kg) had no antitumor effect. In contrast, treatment of established MC38-cOVA tumors with IL10M11 (0.1 mg / kg) resulted in a TGI of approximately 65% ​​and 4 of 7 tumor-free mice (Figure 8B).

[0331] The therapeutic effect of IL10M11 was further tested in Colon26, A20, and 4T1 tumor models. This treatment resulted in a 100% overall response rate (ORR), 100% TGI, and nearly 100% tumor-free survival in both Colon26 and A20 models (Figures 8C and 8D). Significant TGI was also observed in the 4T1 tumor model, including growth inhibition of large established tumors exceeding 100 mm3; however, tumor-free survival was not achieved (Figure 8E).

[0332] 4T1 tumors harvested at the end of the study were dissociated and subjected to flow cytometric analysis of immune cell composition and function. Analysis of tumor-infiltrating lymphocytes from IL10M11-treated mice revealed increased frequency, density, and mean fluorescence intensity (MFI) of IFNγ / TNFα-producing CD4 and CD8 T cells (Figures 8F and 8G).

[0333] Mice that survived primary Colon26 or A20 tumor challenge were rechallenged with bilateral implants of either paired Colon26 and RENCA tumors (Figure 8H) or paired A20 and Colon26 tumors (Figure 8I). Both positive control Colon26 and RENCA or A20 and Colon26 tumors grew aggressively when implanted in naive mice. However, mice that completely rejected their primary tumors with IL10M11 therapy induced long-term antitumor memory responses and rejected secondary challenges with the same tumors, but not unrelated tumor types (Figures 8H and 8I).

[0334] Finally, we determined the antitumor effects of combining IL10M11 with CD40 agonist Abs or PD-1 antagonist Abs. IL10M11, CD40 Abs, and PD-1 Abs each induced antitumor responses as single agents (Figure 9A). Combining any two therapeutic modalities further enhanced their antitumor efficacy (Figure 9A) and increased tumor-free survival (Figure 9B).

[0335] 7.7. Example 6: Antitumor Activity of IL10 in Anti-PD-1 Antibody-Sensitive and Responsive Tumors Multiple syngeneic tumor allograft models were used to assess baseline immune infiltration in the tumor microenvironment before treatment and were also used to compare the in vivo antitumor efficacy of murine IL-10 fused to murine IgG1 (IL10M11) and an anti-PD-1 antagonist Ab (murine IgG1) in a therapeutic setting.

[0336] Briefly, female mice (the Jackson Laboratory, Bar Harbor, ME, USA) were subcutaneously implanted with lineage-matched tumor cells: C57BL / 6 mice with MC38 colon carcinoma cells and B16F10 melanoma cells, or BALB / c mice with Colon26 colon carcinoma cells and A20 B-cell lymphoma cells (all from the Tissue Culture Core at Regeneron Pharmaceuticals, Inc.). When tumor size was approximately 100 mm 3 At the time of tumor size, tumors were removed and subjected to standard protocols for immune profiling of CD45+ immune cells and the subsets listed within the CD45 gate. In some cases, the spleen and draining lymph nodes, along with the tumor, were subjected to analysis of IL10R1, PD1, or PD-L1 surface expression on immune / tumor cells. In a therapeutic setting, treatment begins several days after tumor implantation, typically when tumor size reaches an average volume of 75–150 mm. 3 At the time of tumor arrival, mice were randomly assigned to different treatment groups (n=7-8 / group). Mice were administered mIgG1 isotype control, IL10M11, or anti-mouse PD1 antibody. Treatments, tumor size, and body weight were measured twice weekly.

[0337] 7.7.1 Materials and Methods Tumor implantation and treatment group assignment: For the B16F10, MC38, MC38-cOVA, Colon26, and A20 syngeneic tumor models, 0.5–10 × 10 6Tumor cells (Regeneron Pharmaceuticals, Inc., Tissue Culture Core) were subcutaneously implanted into the right flank of 6- to 8-week-old female C57BL / 6 or BALB / c mice (The Jackson Laboratory, Bar Harbor, Maine, USA). Tumors grew to an average volume of 75–150 mm. 3 At the time of reaching 1000 mg / kg (depending on each specific tumor model), mice were randomized into groups (n=7-8 / group) and administered IL10M11, PD1 Ab, PD1-IL10, control reagent, or in some cases a combination thereof. Mice received 6-9 intraperitoneal injections of test substances twice weekly, and tumor volume and body weight were monitored twice weekly throughout the study.

[0338] Calculation of tumor size and growth inhibition: For each group, the mean and median tumor size, as well as the percentage of tumor growth inhibition compared to the control-treated group, were calculated. The length and width of the tumor were measured twice weekly with calipers, and tumor volume was calculated using the formula (length x width). 2 ) / 2. Measurements were taken at a mean tumor size of 4000 mm in the control group. 3 The tumor growth inhibition was calculated using the following formula: [1-(T final -T initial ) / (C final -C initial )]*100, where T (treated group) and C (control group) represent the mean tumor burden on the day all mice were alive. Observation was extended until day 54 (3 weeks after the final treatment) to determine the frequency of tumor-free mice.

[0339] MSD multiplexing of serum cytokines was performed according to standard protocols from MSD (Meso Scale Diagnostics, Rockville, MD, USA).

[0340] 7.7.2.Results As shown in Figures 10A-10E, the densities of total CD45+ immune cells and all analyzed immune cell subsets in A20, Colon26, and MC38 tumors were much higher than their counterparts in B16F10 tumors. Further analysis also showed that B16F10 tumors contained fewer Ki67-positive CD4 and CD8 T cells, which were of a memory phenotype.

[0341] As expected, IL10R1 was constitutively and ubiquitously expressed by a wide variety of myeloid cells in both the spleen and tumors. Surprisingly, IL10R1 expression on T cells, particularly CD8 T cells, was highly restricted to tumor-infiltrating CD8 T cells, with almost no expression observed on T cells in secondary lymphoid tissues, e.g., the spleen and draining LNs of MC38-cOVA and Colon26 (see Figures 11A-11I). This study further revealed that nearly all IL10R1+ CD8 T cells expressed PD1. This data suggested the following: 1) these IL10R1+PD1+ CD8 T cells may be tumor antigen-specific; 2) the highly restricted expression pattern of IL10R1 on a subset of T cells in tumors may represent the biological reason behind the favorable safety profile of IL10M11 in mice; and 3) the combination of IL10-Fc with PD-1 antagonist Ab is a possible strategy to enhance antitumor responses.

[0342] PD-L1 expression varied among the four tumor types tested (Figures 12A-12G). Next, PD1 antagonist Ab was compared with IL10M11 to determine the response of each tumor type to treatment. As shown in Figures 13A-13C, 150 mm 3The A20 B-cell lymphoma responded very well to PD-1 Ab treatment, showing significant tumor growth delay and approximately 50% tumor-free survival. However, IL10M11 induced a more intense and broad immune response against A20, resulting in approximately 85% tumor-free survival. In the Colon26 tumor model (Figures 14A-C), 100 mm 3 The tumors were completely resistant to PD1 Ab treatment. In contrast, IL10M11 again showed a tumor-free survival rate of approximately 85%. B16F10 is known to be less immunogenic and has much less immune cell infiltration, which was confirmed in our study (Figures 10A-10E). 3 B16F10 tumors were largely unresponsive to PD1 Ab. However, IL10M11 significantly delayed tumor growth and rendered the majority of tumors responsive (Figures 15A-C). 3 Based on the observation that MC38 tumors in this group hardly respond to PD1 Ab (see Figures 22A-22B), 3 In this study, PD1 Ab and IL10-Fc showed similar antitumor efficacy as single agents, whereas their combination dramatically increased the antitumor response by slowing tumor growth and increasing tumor-free survival (Figures 16A-C).

[0343] To elucidate the relevant mechanism of action of IL10-Fc (e.g., IL10M11), we performed immune profiling of the tumor microenvironment after treatment. IL10 treatment significantly increased CD8 T cell density in both the immunogenic MC38 tumor model (Figure 17A) and the less immunogenic 4T1 tumor model (Figure 17B), correlating with a better prognosis in both cases. Further testing showed that IL10 increased the density of PD1+ CD8 T cells, suggesting that these CD8 T cells were likely tumor-specific CD8 T cells once activated (Figures 18A-B). IL10M11 tended to increase the overall CD45 immune infiltrate, including CD4 T cells and myeloid cells, in addition to CD8 T cells in the 4T1 tumor model (Figures 17A-B and 18A-B), which plausibly explains why IL10 worked in less immunogenic tumor models such as 4T1 and B16F10.

[0344] To identify potential pharmacodynamic (PD) markers for IL10-Fc, we analyzed serum levels of various cytokines using a commercially available multiplex MSD platform. In both naive, non-tumor-bearing mice (Figure 19) and B16F10 tumor-bearing mice (Figure 20), a signature panel of cytokines (IL12, IL1b, IL2, IL4, and IL5) was uniquely upregulated by IL10 but not by PD1 Ab. When normalized to the mean values ​​of isotype control-treated mice, a very clear trend of multiple-fold upregulation of IL12 and IL4 was observed across multiple tumor models, including A20, MC38, MC38-cOVA, B16F10, and Colon26 (Figures 21A-B). Some samples were collected from the same tumor model at different time points (MC38 and Colon26) but still showed consistent trends.

[0345] Based on previous findings of coexpression of PD1 and IL10R1 and the enhanced antitumor efficacy of IL10-Fc combined with a PD-1 antagonist Ab, we investigated the potential of PD1-targeted IL10 as a therapeutic agent. Clinical trials have reported that high doses of pagilodecaquin (PEGylated IL10) have shown some toxicity. It is appropriate to normalize the molar dose of IL10, rather than the molar dose of PD1, between the different treatment modalities (IL10-Fc, control Ab-IL10, PD1-IL10, and PD1 Ab). As shown in Figures 22A-B, PD1 Ab and IL10-Fc each exhibited the expected antitumor effects as single agents (PD1 Ab exhibited a very modest effect, while IL10-Fc exhibited a moderate effect). Combining IL10-Fc with PD-1 Ab significantly improved efficacy, whereas PD1-IL10-Fc showed no difference in efficacy from control Ab-IL10 or IL10-Fc.

[0346] 7.8. Example 8: Purification of IL10 Agonists Multiple iterations of the IL10 agonist (i.e., IL10M1-IL10M11) were evaluated to determine their ability to be recombinantly expressed and recovered. IL10 agonists with IL10 moieties located at either the N- or C-terminus of the Fc domain were recoverable, but both constructs yielded significant amounts of aggregated IL10 agonist. A purification method was developed to produce non-aggregated IL10 agonist.

[0347] 7.8.1 Materials and Methods Constructs encoding IL10 muteins were generated, recombinantly expressed in mammalian cell lines, and purified as described in Section 7.1. Cells were lysed, and the cell lysate in PBS buffer was applied to a PrismA™ Protein A affinity column at a flow rate of 0.4 ml / min. Elution from the affinity column was performed with Pierce™ Gentle elution buffer, pH 6.6. The buffer was then exchanged by dialysis against 1x TBS + 5% glycerol, followed by 2x PBS + 5% glycerol. The exchanged buffer eluate was then subjected to size-exclusion chromatography using a Superdex® 200, 26 / 600 pg column (MilliporeSigma, St. Louis, MO, USA). Fractions containing unaggregated IL10 agonist were collected.

[0348] 7.8.2.Results Because the IL10 agonist has an IL10 moiety at the C-terminus of the Fc domain, this purification protocol produced intact, non-aggregated IL10 agonist without any aberrant peaks observed by size-exclusion ultra-performance liquid chromatography. IL10 agonists with an IL10 moiety at the N-terminus of the Fc domain were also recovered, but these were not intact; they either lacked the C-terminal lysine or were recovered as fragments containing amino acids 3-402, whereas the full-length construct contains residues 1-403.

[0349] The purified, non-aggregated IL10 agonist can be formulated as a pharmaceutical composition together with excipients, the formulated pharmaceutical composition having minimal aggregates of the IL10 agonist.

[0350] 8. Specific embodiments, references While various specific embodiments have been illustrated and described, it will be understood that various changes can be made without departing from the spirit and scope of the present disclosure, which is exemplified by the numbered embodiments set forth below.

[0351] In preferred aspects of the numbered embodiments below and in the claims that follow, the IL10 domain, the Fc domain, and variants thereof preferably comprise the amino acid sequence of human IL10, the human Fc domain, and variants thereof, e.g., variants having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% sequence identity to such human sequences.

[0352] 1. (a) Fc domain; (b) a linker; and (c)IL10 part IL10 agonists, including

[0353] 2. The IL10 agonist of embodiment 1, further comprising a hinge domain N-terminal to the Fc domain. 3. The IL10 agonist of embodiment 1 or embodiment 2, wherein said Fc domain is an IgG Fc domain.

[0354] 4. The IL10 agonist of any one of embodiments 1 to 3, wherein said Fc domain is an IgG1, IgG2, IgG3 or IgG4 Fc domain. 5. The IL10 agonist of any one of embodiments 1 to 4, wherein said Fc domain comprises CH2 and Ch3 domains derived from IgG1, IgG2, IgG3, IgG4, or a combination thereof.

[0355] 6. The IL10 agonist of any one of embodiments 1 to 5, wherein said Fc domain is IgG1. 7. The IL10 agonist of any one of embodiments 1 to 6, wherein said IgG1 is human IgG1.

[0356] 8. The IL10 agonist of any one of embodiments 1 to 6, wherein said IgG1 is a murine IgG1. 9. The IL10 agonist of any one of embodiments 1 to 5, wherein said Fc domain is IgG4.

[0357] 10. The IL10 agonist of embodiment 9, wherein said IgG1 is human IgG4. 11. The IL10 agonist of embodiment 9, wherein said IgG1 is a murine IgG4.

[0358] 12. The IL10 agonist of any one of embodiments 1 to 4 and 9, wherein said Fc domain comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 31.

[0359] 13. The IL10 agonist of any one of embodiments 1 to 4, 9 and 12, wherein said Fc domain comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO: 31.

[0360] 14. The IL10 agonist of any one of embodiments 1 to 4, 9, 12 and 13, wherein the Fc domain comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 31.

[0361] 15. The IL10 agonist of any one of embodiments 1-4, 9 and 12-14, wherein the Fc domain comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 31.

[0362] 16. The IL10 agonist of any one of embodiments 1-4, 9 and 12-15, wherein the Fc domain comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 31.

[0363] 17. The IL10 agonist of any one of embodiments 1 to 4, 9, and 12 to 16, wherein the Fc domain comprises the amino acid sequence of SEQ ID NO: 31. 18. The IL10 agonist of any one of embodiments 1 to 7, wherein the Fc domain comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 33.

[0364] 19. The IL10 agonist of any one of embodiments 1 to 7 and 18, wherein the Fc domain comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO: 33.

[0365] 20. The IL10 agonist of any one of embodiments 1 to 7, 18 and 19, wherein the Fc domain comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 33.

[0366] 21. The IL10 agonist of any one of embodiments 1 to 7 and 18 to 20, wherein the Fc domain comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 33.

[0367] 22. The IL10 agonist of any one of embodiments 1 to 7 and 18 to 21, wherein the Fc domain comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 33.

[0368] 23. The IL10 agonist of any one of embodiments 1 to 7 and 18 to 22, wherein the Fc domain comprises the amino acid sequence of SEQ ID NO: 33. 24. The IL10 agonist according to any one of embodiments 1 to 17, wherein the effector function of said Fc domain is reduced.

[0369] 25. The IL10 agonist of any one of embodiments 5 to 24, wherein the hinge domain is derived from the same IgG as the CH2 and / or CH3 domain. 26. The IL10 agonist of any one of embodiments 5-24, wherein the hinge domain is derived from a different IgG than the CH2 and / or CH3 domain.

[0370] 27. The IL10 agonist of any one of embodiments 2 to 26, wherein said hinge domain comprises a chimeric hinge sequence. 28. The IL10 agonist of any one of embodiments 2 to 27, wherein the hinge domain comprises the amino acid sequence of SEQ ID NO: 12.

[0371] 29. The IL10 agonist of any one of embodiments 2 to 27, wherein the hinge domain comprises the amino acid sequence of SEQ ID NO: 13. 30. The IL10 agonist according to any one of embodiments 1 to 18, wherein said linker is between 10 and 60 amino acid residues in length.

[0372] 31. The IL10 agonist according to any one of embodiments 1 to 18, wherein said linker is 15 to 25 amino acid residues in length. 32. The IL10 agonist according to any one of embodiments 1 to 18, wherein the linker is 15 to 20 amino acid residues in length.

[0373] 33. The linker is G n S or SG n 33. The IL10 agonist according to any one of embodiments 1 to 32, comprising a monomer or multimer of:

[0374] 34. The IL10 agonist of embodiment 33, wherein the linker comprises a G4S monomer or multimer. 35. The IL10 agonist of embodiment 34, wherein the linker comprises 1 to 6 repeats of G4S.

[0375] 36. The IL10 agonist of embodiment 35, wherein the linker comprises (G4S)1. 37. The IL10 agonist of embodiment 35, wherein the linker comprises (G4S)2.

[0376] 38. The IL10 agonist of embodiment 35, wherein the linker comprises (G4S)3. 39. The IL10 agonist of embodiment 35, wherein the linker comprises (G4S)4.

[0377] 40. The IL10 agonist of embodiment 35, wherein the linker comprises (G4S)5. 41. The IL10 agonist of embodiment 35, wherein the linker comprises (G4S)6.

[0378] 42. The IL10 agonist of any one of embodiments 1-41, wherein said linker connects the Fc domain and said IL10 moiety. 43. The IL10 agonist of any one of embodiments 1-41, wherein the IL10 moiety is C-terminal to the Fc domain.

[0379] 44. The IL10 agonist of any one of embodiments 1-41, wherein the IL10 moiety is N-terminal to the Fc domain. 45. The IL10 agonist of any one of embodiments 1 to 41, wherein the IL10 portion comprises an amino acid sequence having at least 95% sequence identity with the sequence of mature human IL10 (e.g., IL10 having the amino acid sequence of SEQ ID NO: 1).

[0380] 46. ​​The IL10 agonist of any one of embodiments 1 to 42, wherein the IL10 portion comprises an amino acid sequence having at least 96% sequence identity with the sequence of mature human IL10 (e.g., IL10 having the amino acid sequence of SEQ ID NO: 1).

[0381] 47. The IL10 agonist of any one of embodiments 1 to 43, wherein the IL10 portion comprises an amino acid sequence having at least 97% sequence identity with the sequence of mature human IL10 (e.g., IL10 having the amino acid sequence of SEQ ID NO: 1).

[0382] 48. The IL10 agonist of any one of embodiments 1 to 44, wherein the IL10 portion comprises an amino acid sequence having at least 98% sequence identity with the sequence of mature human IL10 (e.g., IL10 having the amino acid sequence of SEQ ID NO: 1).

[0383] 49. The IL10 agonist of any one of embodiments 1 to 45, wherein the IL10 portion comprises an amino acid sequence that has at least 99% sequence identity with that of mature human IL10 (e.g., IL10 having the amino acid sequence of SEQ ID NO: 1).

[0384] 50. The IL10 agonist of any one of embodiments 1-49, wherein the IL10 portion comprises the amino acid sequence of mature human IL10 (eg, IL10 having the amino acid sequence of SEQ ID NO: 1).

[0385] 51. The IL10 agonist of any one of embodiments 1 to 41, wherein the IL10 portion comprises an amino acid sequence having at least 95% sequence identity with the sequence of mature mouse IL10 (e.g., IL10 having the amino acid sequence of SEQ ID NO: 30).

[0386] 52. The IL10 agonist of any one of embodiments 1 to 41 and 51, wherein the IL10 portion comprises an amino acid sequence having at least 96% sequence identity with the sequence of mature mouse IL10 (e.g., IL10 having the amino acid sequence of SEQ ID NO: 30).

[0387] 53. The IL10 agonist of any one of embodiments 1 to 41, 51 and 52, wherein the IL10 portion comprises an amino acid sequence having at least 97% sequence identity with the sequence of mature mouse IL10 (e.g., IL10 having the amino acid sequence of SEQ ID NO: 30).

[0388] 54. The IL10 agonist of any one of embodiments 1 to 41 and 51 to 53, wherein the IL10 portion comprises an amino acid sequence having at least 98% sequence identity with the sequence of mature mouse IL10 (e.g., IL10 having the amino acid sequence of SEQ ID NO: 30).

[0389] 55. The IL10 agonist of any one of embodiments 1 to 41 and 51 to 54, wherein the IL10 portion comprises an amino acid sequence having at least 99% sequence identity with the sequence of mature mouse IL10 (e.g., IL10 having the amino acid sequence of SEQ ID NO: 30).

[0390] 56. The IL10 agonist of any one of embodiments 1-41 and 51-55, wherein the IL10 portion comprises the amino acid sequence of mature mouse IL10 (e.g., IL10 having the amino acid sequence of SEQ ID NO: 30).

[0391] 57. The IL10 agonist according to any one of embodiments 1 to 56, which is a monomer, and optionally which monomer has the insertion of a linker sequence between N116 and K117 of said IL10 moiety, and / or wherein the ability of said Fc to self-associate is reduced due to, for example, one or more substitutions at positions corresponding to T366 and / or Y407 in CH3.

[0392] 58. The IL10 agonist according to any one of embodiments 1-56, which is a monomer, and which monomer lacks the insertion of a linker sequence between N116 and K117 of said IL10 moiety. 59. The IL10 agonist according to any one of embodiments 1 to 56 and 58, which is a monomer, and which monomer is not substituted at positions corresponding to T366 and / or Y407 in CH3.

[0393] 60. The IL10 agonist according to any one of embodiments 1-56, which is a homodimer. 61. The IL10 agonist according to embodiment 58, comprising two of said Fc domains.

[0394] 62. The IL10 agonist of embodiment 61, which dimerizes via the Fc domain. 63. The IL10 agonist of embodiment 62, wherein the Fc domain comprises an amino acid sequence designated hIgG4-Fc (e.g., an Fc having the amino acid sequence of amino acids 18 to 228 of SEQ ID NO: 31).

[0395] 64. The IL10 agonist of embodiment 62, wherein the Fc domain comprises an amino acid sequence designated hIgG4s-Fc (e.g., an Fc having the amino acid sequence of SEQ ID NO: 31).

[0396] 65. The IL10 agonist of embodiment 62, wherein the Fc domain comprises an amino acid sequence designated mIgG1-Fc (e.g., an Fc having the amino acid sequence of SEQ ID NO: 33).

[0397] 66. The IL10 agonist of embodiment 61, wherein the Fc domain is non-dimerizing. 67. The IL10 agonist according to any one of embodiments 58 to 67, which comprises two IL10 moieties.

[0398] 68. The IL10 agonist according to embodiment 67, which dimerizes via the IL10 moiety. 69. The IL10 agonist according to embodiment 67, wherein the IL10 moiety is non-dimerizing.

[0399] 70. The IL10 agonist according to any one of embodiments 1-56, which is a heterodimer. 71. The IL10 agonist according to embodiment 70, comprising two of said Fc domains.

[0400] 72. The IL10 agonist of embodiment 71, which dimerizes via the Fc domain. 73. The IL10 agonist of embodiment 71, wherein the Fc domain is non-dimerizing.

[0401] 74. An IL10 agonist according to any one of embodiments 70 to 73, comprising two IL10 moieties. 75. The IL10 agonist according to embodiment 74, which dimerizes via the IL10 moiety.

[0402] 76. The IL10 agonist of embodiment 74, wherein the IL10 moiety is non-dimerizing. 77. An IL10 agonist according to any one of embodiments 70-73, comprising a single IL10 moiety.

[0403] 78. An IL10 agonist comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of IL10M1 (ie, an IL10 agonist having the amino acid sequence of SEQ ID NO: 34).

[0404] 79. The IL10 agonist according to embodiment 78, comprising an amino acid sequence having at least 96% sequence identity with the amino acid sequence of IL10M1. 80. The IL10 agonist according to embodiment 78 or embodiment 79, which comprises an amino acid sequence having at least 97% sequence identity with the amino acid sequence of IL10M1.

[0405] 81. The IL10 agonist according to any one of embodiments 78 to 80, which comprises an amino acid sequence having at least 98% sequence identity with the amino acid sequence of IL10M1. 82. The IL10 agonist according to any one of embodiments 78 to 81, which comprises an amino acid sequence having at least 99% sequence identity with the amino acid sequence of IL10M1.

[0406] 83. An IL10 agonist according to any one of embodiments 78 to 82, comprising the amino acid sequence of IL10M1. 84. An IL10 agonist comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of IL10M2 (i.e., an IL10 agonist having the amino acid sequence of SEQ ID NO: 35).

[0407] 85. The IL10 agonist according to embodiment 84, comprising an amino acid sequence having at least 96% sequence identity with the amino acid sequence of IL10M2. 86. The IL10 agonist according to embodiment 84 or embodiment 85, which comprises an amino acid sequence having at least 97% sequence identity with the amino acid sequence of IL10M2.

[0408] 87. The IL10 agonist according to any one of embodiments 84 to 86, comprising an amino acid sequence having at least 98% sequence identity with the amino acid sequence of IL10M2. 88. The IL10 agonist according to any one of embodiments 84 to 87, comprising an amino acid sequence having at least 99% sequence identity with the amino acid sequence of IL10M2.

[0409] 89. An IL10 agonist according to any one of embodiments 84 to 88, comprising the amino acid sequence of IL10M2. 90. An IL10 agonist comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of IL10M3 (i.e., an IL10 agonist having the amino acid sequence of SEQ ID NO: 36).

[0410] 91. The IL10 agonist according to embodiment 90, comprising an amino acid sequence having at least 96% sequence identity with the amino acid sequence of IL10M3. 92. The IL10 agonist according to embodiment 90 or embodiment 91, which comprises an amino acid sequence having at least 97% sequence identity with the amino acid sequence of IL10M3.

[0411] 93. The IL10 agonist according to any one of embodiments 90 to 92, comprising an amino acid sequence having at least 98% sequence identity with the amino acid sequence of IL10M3. 94. The IL10 agonist according to any one of embodiments 90 to 93, comprising an amino acid sequence having at least 99% sequence identity with the amino acid sequence of IL10M3.

[0412] 95. An IL10 agonist according to any one of embodiments 90 to 94, comprising the amino acid sequence of IL10M3. 96. An IL10 agonist comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of IL10M4 (i.e., an IL10 agonist having the amino acid sequence of SEQ ID NO: 37).

[0413] 97. The IL10 agonist according to embodiment 96, comprising an amino acid sequence having at least 96% sequence identity with the amino acid sequence of IL10M4. 98. The IL10 agonist according to embodiment 96 or embodiment 97, which comprises an amino acid sequence having at least 97% sequence identity with the amino acid sequence of IL10M4.

[0414] 99. The IL10 agonist according to any one of embodiments 96 to 98, comprising an amino acid sequence having at least 98% sequence identity with the amino acid sequence of IL10M4. 100. An IL10 agonist according to any one of embodiments 96 to 99, comprising an amino acid sequence having at least 99% sequence identity with the amino acid sequence of IL10M4.

[0415] 101. An IL10 agonist according to any one of embodiments 96 to 100, comprising the amino acid sequence of IL10M4. 102. An IL10 agonist comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of IL10M5 (ie, an IL10 agonist having the amino acid sequence of SEQ ID NO: 38).

[0416] 103. The IL10 agonist according to embodiment 102, comprising an amino acid sequence having at least 96% sequence identity with the amino acid sequence of IL10M5. 104. The IL10 agonist according to embodiment 102 or embodiment 103, which comprises an amino acid sequence having at least 97% sequence identity with the amino acid sequence of IL10M5.

[0417] 105. The IL10 agonist according to any one of embodiments 102-104, comprising an amino acid sequence having at least 98% sequence identity with the amino acid sequence of IL10M5. 106. An IL10 agonist according to any one of embodiments 102 to 105, comprising an amino acid sequence having at least 99% sequence identity with the amino acid sequence of IL10M5.

[0418] 107. An IL10 agonist according to any one of embodiments 102 to 106, comprising the amino acid sequence of IL10M5. 108. An IL10 agonist comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of IL10M6 (ie, an IL10 agonist having the amino acid sequence of SEQ ID NO: 39).

[0419] 109. The IL10 agonist according to embodiment 108, comprising an amino acid sequence having at least 96% sequence identity with the amino acid sequence of IL10M6. 110. The IL10 agonist according to embodiment 108 or embodiment 109, which comprises an amino acid sequence having at least 97% sequence identity with the amino acid sequence of IL10M6.

[0420] 111. The IL10 agonist according to any one of embodiments 108-110, which comprises an amino acid sequence having at least 98% sequence identity with the amino acid sequence of IL10M6. 112. The IL10 agonist according to any one of embodiments 108-111, comprising an amino acid sequence having at least 99% sequence identity with the amino acid sequence of IL10M6.

[0421] 113. The IL10 agonist according to any one of embodiments 108-112, comprising the amino acid sequence of IL10M6. 114. An IL10 agonist comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of IL10M7 (ie an IL10 agonist having the amino acid sequence of SEQ ID NO: 40).

[0422] 115. The IL10 agonist according to embodiment 114, comprising an amino acid sequence having at least 96% sequence identity with the amino acid sequence of IL10M7. 116. The IL10 agonist according to embodiment 114 or embodiment 115, which comprises an amino acid sequence having at least 97% sequence identity with the amino acid sequence of IL10M7.

[0423] 117. The IL10 agonist according to any one of embodiments 114-116, comprising an amino acid sequence having at least 98% sequence identity with the amino acid sequence of IL10M7. 118. The IL10 agonist according to any one of embodiments 114-117, comprising an amino acid sequence having at least 99% sequence identity with the amino acid sequence of IL10M7.

[0424] 119. The IL10 agonist according to any one of embodiments 114 to 118, comprising the amino acid sequence of IL10M7. 120. An IL10 agonist comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of IL10M8 (ie, an IL10 agonist having the amino acid sequence of SEQ ID NO: 41).

[0425] 121. The IL10 agonist according to embodiment 120, comprising an amino acid sequence having at least 96% sequence identity with the amino acid sequence of IL10M8. 122. The IL10 agonist according to embodiment 120 or embodiment 121, which comprises an amino acid sequence having at least 97% sequence identity with the amino acid sequence of IL10M8.

[0426] 123. The IL10 agonist according to any one of embodiments 120-122, comprising an amino acid sequence having at least 98% sequence identity with the amino acid sequence of IL10M8. 124. The IL10 agonist according to any one of embodiments 120-123, comprising an amino acid sequence having at least 99% sequence identity with the amino acid sequence of IL10M8.

[0427] 125. The IL10 agonist according to any one of embodiments 120 to 124, comprising the amino acid sequence of IL10M8. 126. An IL10 agonist comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of IL10M9 (ie an IL10 agonist having the amino acid sequence of SEQ ID NO: 42).

[0428] 127. The IL10 agonist according to embodiment 126, comprising an amino acid sequence having at least 96% sequence identity with the amino acid sequence of IL10M9. 128. The IL10 agonist according to embodiment 126 or embodiment 127, which comprises an amino acid sequence having at least 97% sequence identity with the amino acid sequence of IL10M9.

[0429] 129. The IL10 agonist according to any one of embodiments 126 to 128, comprising an amino acid sequence having at least 98% sequence identity with the amino acid sequence of IL10M9. 130. The IL10 agonist according to any one of embodiments 126-129, comprising an amino acid sequence having at least 99% sequence identity with the amino acid sequence of IL10M9.

[0430] 131. The IL10 agonist according to any one of embodiments 126 to 130, comprising the amino acid sequence of IL10M9. 132. An IL10 agonist comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of IL10M10 (ie an IL10 agonist having the amino acid sequence of SEQ ID NO: 43).

[0431] 133. The IL10 agonist according to embodiment 132, which comprises an amino acid sequence having at least 96% sequence identity with the amino acid sequence of IL10M10. 134. The IL10 agonist according to embodiment 132 or embodiment 133, which comprises an amino acid sequence having at least 97% sequence identity with the amino acid sequence of IL10M10.

[0432] 135. The IL10 agonist according to any one of embodiments 132-134, which comprises an amino acid sequence having at least 98% sequence identity with the amino acid sequence of IL10M10. 136. The IL10 agonist according to any one of embodiments 132-135, comprising an amino acid sequence having at least 99% sequence identity with the amino acid sequence of IL10M10.

[0433] 137. The IL10 agonist according to any one of embodiments 132-136, comprising the amino acid sequence of IL10M10. 138. An IL10 agonist comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of IL10M11 (ie an IL10 agonist having the amino acid sequence of SEQ ID NO: 44).

[0434] 139. The IL10 agonist according to embodiment 138, comprising an amino acid sequence having at least 96% sequence identity with the amino acid sequence of IL10M11. 140. The IL10 agonist according to embodiment 138 or embodiment 139, which comprises an amino acid sequence having at least 97% sequence identity with the amino acid sequence of IL10M11.

[0435] 141. The IL10 agonist according to any one of embodiments 138-140, which comprises an amino acid sequence having at least 98% sequence identity with the amino acid sequence of IL10M11. 142. The IL10 agonist according to any one of embodiments 138-141, comprising an amino acid sequence having at least 99% sequence identity with the amino acid sequence of IL10M11.

[0436] 143. The IL10 agonist according to any one of embodiments 138-142, comprising the amino acid sequence of IL10M11. 144. The IL10 agonist of any one of embodiments 1-143, further comprising a targeting moiety.

[0437] 145. The IL10 agonist of embodiment 144, wherein the targeting moiety is N-terminal to the Fc domain and, if a hinge domain is present, N-terminal to the hinge domain.

[0438] 146. The targeting moiety is (a) binds to tumor-associated antigens; (b) binds to tumor microenvironment antigens; (c) binds to a cell surface molecule of a tumor-reactive lymphocyte; or (d) binds to a checkpoint inhibitor; An IL10 agonist according to embodiment 144 or embodiment 145.

[0439] 147. The IL10 agonist according to embodiment 146, wherein the targeting moiety binds to a tumor-associated antigen. 148. The tumor-associated antigen is fibroblast activation protein (FAP), the A1 domain of tenascin-C (TNC A1), the A2 domain of tenascin-C (TNC A2), fibronectin extra domain B (EDB), melanoma-associated chondroitin sulfate proteoglycan (MCSP), MART-1 / MelanA, gp100, dipeptidyl peptidase IV (DPPIV), adenosine deaminase-binding protein (ADAbp), cyclophilin b, colorectal-associated antigen (CRC)-C017-1A / GA733, carcinoembryonic antigen (CEA) and its immunogenic epitopes CAP-1 and CAP-2, etv6, aml1, prostate-specific antigen (PSA) or its immunogenic epitopes PSA-1, PSA-2, and PSA-3, prostate-specific membrane antigen (PSMA), T-cell receptor / CD3 ζ chain, tumor antigens of the MAGE family (e.g., MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7), A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (MAGE-B4), MAGE-C1, MAGE-C2, MAGE-C3, MAGE-C4, MAGE-C5), tumor antigens of the GAGE ​​family (e.g., GAGE-1, GAGE-2, GAGE- 3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, GAGE-9), BAGE, RAGE, LAGE-1, NAG, GnT-V, MUM-1, CDK4, tyrosinase, p53, MUC family, HER2 / neu, p21ras, RCAS1, α-fetoprotein, E-cadherin, α-catenin, β-catenin and γ-catenin, p120ctn, gp100Pmel117, PRAME, NY-ESO-1, cdc27, adenomatous polyposis coli protein (APC), fodrin, connexin 37, Ig idiotype, p15, gp75, GM2 and GD2 gangliosides, viral products such as human papillomavirus proteins, tumor antigens of the Smad family, Imp-1, P1A, EBV-encoded nuclear antigen (EBNA)-1, brain glycogen phosphorylase, SSX-1, SSX-2 (HOM-MEL-40), SSX-1, SSX-4, SSX-5, SCP-1 and CT-7, c-erbB-2, Her2, EGFR, IGF-1R, CD2 (T cell surface antigen), CD3 (T CR-associated heteromultimer), CD22 (B cell receptor), CD23 (low-binding affinity IgE receptor), CD30 (cytokine receptor), CD33 (myeloid cell surface antigen), CD40 (tumor necrosis factor receptor), IL-6R (IL6 receptor), CD20, MCSP, PDGFβR (β-platelet-derived growth factor receptor), ErbB2, epithelial cell adhesion molecule (EpCAM), EGFR variant III (EGFRvIII), CD19, disialoganglioside GD2, ductal epithelial mucin, gp36, TAG-72, glioma-associated antigen, β-human chorionic gonadotropin, α-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, prostase, prostase specific antigen (PSA), PAP, LAGA-1a, p53, prostein, PSMA, survivin and telomerase, prostate cancer tumor antigen-1 (PCTA-1), ELF2M, neutrophil elastase, ephrin B2, insulin growth factor (IGF1)-I, IGF-II, IGFI receptor, 5T4, ROR1, Nkp30, NKG2D, tumor stromal antigen, extra domain A (EDA) or extra domain B (EDB) of fibronectin, or the A1 domain of tenascin-C (TnC A1).

[0440] 149. The IL10 agonist according to embodiment 147, wherein the tumor-associated antigen is a viral antigen. 150. The IL10 agonist of embodiment 149, wherein the viral antigen is Epstein-Barr virus LMP-1, Hepatitis C virus E2 glycoprotein, HIV gp160 or HIV gp120, HPV E6m, HPV E7, CMV early membrane antigen (EMA) or CMV late membrane antigen (LMA).

[0441] 151. The IL10 agonist according to embodiment 146, wherein the targeting moiety binds to a tumor microenvironment antigen. 152. The IL10 agonist according to embodiment 151, wherein the tumor microenvironment antigen is an extracellular matrix protein.

[0442] 153. The IL10 agonist of embodiment 152, wherein the extracellular matrix protein is a syndecan, heparanase, integrin, osteopontin, link, cadherin, laminin, laminin EGF type, lectin, fibronectin, notch, tenascin, collagen, or matrixin.

[0443] 154. The IL10 agonist according to embodiment 146, wherein the targeting moiety binds to a cell surface molecule of a tumor-reactive lymphocyte. 155. The IL10 agonist of embodiment 154, wherein the cell surface molecule is CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, LAG3, TIM3, or B7-H3.

[0444] 156. The IL10 agonist according to embodiment 146, wherein the targeting moiety binds to a checkpoint inhibitor. 157. The IL10 agonist of embodiment 156, wherein the checkpoint inhibitor is CTLA-4, PD1, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK1, VISTA, PSGL1, or CHK2.

[0445] 158. The IL10 agonist according to embodiment 157, wherein the checkpoint inhibitor is PD1. 159. The IL10 agonist according to embodiment 144 or embodiment 145, wherein the targeting moiety binds to an MHC-peptide complex.

[0446] 160. The IL10 agonist of embodiment 159, wherein the peptide in the peptide-MHC complex comprises a tumor neoantigen. 161. The tumor neoantigen is selected from the group consisting of LCMV-derived peptides gp33-41, APF (126-134), BALF (276-284), CEA (571-579), CMV pp65 (495-503), FLU-M1 (58-66), gp100 (154-162), gp100 (209-217), HBV Core (18-27), Her2 / neu (369-377; V2v9); HPV E7 (11-20), KLK4 (11-19), LMP1 (125-133), MAG-A3 (112-120), NYESO1 (157-165, C165A), NYESO1 (157-165, C165V), and p54 161. The IL10 agonist of embodiment 160, which is WT(264-272), PAP-3(136-143), PSMA(4-12), PSMA(135-145), survivin(96-014), tyrosinase(369-377, 371D), or WT1(126-134).

[0447] 162. The IL10 agonist of any one of embodiments 144 to 161, wherein the targeting moiety is an antibody or an antigen-binding fragment thereof. 163. The IL10 agonist according to embodiment 162, wherein the targeting moiety is a Fab.

[0448] 164. The IL10 agonist according to embodiment 162, wherein the targeting moiety is an scFv. 165. The IL10 agonist according to any one of embodiments 144 to 164, wherein the targeting moiety does not bind to a checkpoint inhibitor, and optionally, the checkpoint inhibitor is PD1.

[0449] 166. The IL10 agonist of any one of embodiments 1-164, which lacks a targeting domain that binds to a checkpoint inhibitor, and optionally, the checkpoint inhibitor is PD1.

[0450] 167. The IL10 agonist of any one of embodiments 1-139, which lacks a targeting moiety. 168. Targeting moieties, including but not limited to: (a) binds to tumor-associated antigens; (b) binds to tumor microenvironment antigens; (c) binds to a cell surface molecule of a tumor-reactive lymphocyte; or (d) binds to checkpoint inhibitors The IL10 agonist of any one of embodiments 1 to 99 and 167, which lacks a targeting moiety.

[0451] 169. The IL agonist according to embodiment 168, which lacks a targeting moiety that binds to PD1. 170. The IL10 agonist of any one of embodiments 1-139 and 165, which lacks the CH1 domain.

[0452] 171. The IL10 agonist of any one of embodiments 1-139, 165 and 168, which lacks the CL domain. 172. The IL10 agonist of any one of embodiments 1-139 and 165-171, which lacks antibody variable regions.

[0453] 173. The IL10 agonist of any one of embodiments 1-172, further comprising a hydrophilic polymer. 174. The IL10 agonist of embodiment 173, wherein the hydrophilic polymer is polyethylene glycol ("PEG").

[0454] 175. The IL10 agonist according to embodiment 174, wherein the PEG has a molecular weight ranging from about 7.5 kDa to about 80 kDa. 176. The IL10 agonist of embodiment 175, wherein the PEG has a molecular weight ranging from about 30 kDa to about 60 kDa, optionally wherein the molecular weight is about 50 kDa.

[0455] 177. The IL10 agonist according to any one of embodiments 1-172, comprising a stabilizing moiety. 178. The IL10 agonist according to embodiment 177, wherein the stabilizing moiety is albumin, a human serum albumin binder, XTEN, PAS, a hydrophilic polymer, polysialic acid, or a fatty acid.

[0456] 179. The IL10 agonist according to any one of embodiments 1 to 172, which is not conjugated to polyethylene glycol. 180. The IL10 agonist according to any one of embodiments 1 to 172 and 179, which is not conjugated to albumin.

[0457] 181. The IL10 agonist according to any one of embodiments 1 to 172, 179 and 180, which is not conjugated to a human serum albumin binder. 182. The IL10 agonist according to any one of embodiments 1 to 172 and 179 to 181, which is not conjugated to XTEN.

[0458] 183. An IL10 agonist according to any one of embodiments 1 to 172 and 179 to 182, which is not conjugated to a PAS. 184. The IL10 agonist according to any one of embodiments 1 to 172 and 179 to 183, which is not conjugated to a hydrophilic polymer.

[0459] 185. The IL10 agonist according to any one of embodiments 1 to 172 and 179 to 184, which is not conjugated to polysialic acid. 186. The IL10 agonist according to any one of embodiments 1 to 172 and 179 to 185, which is not conjugated to hydroxyethyl starch.

[0460] 187. The IL10 agonist according to any one of embodiments 1-172 and 179-186, which is not conjugated to a fatty acid. 188. The IL10 agonist according to any one of embodiments 1-172 and 179-187, which does not comprise N-linked glycans.

[0461] 189. The IL10 agonist of any one of embodiments 1-172 and 179-187, which does not comprise O-linked glycans. 190. The IL10 agonist according to any one of embodiments 1-172 and 179-189, which lacks a stabilizing moiety.

[0462] 191. The IL10 agonist according to any one of embodiments 1 to 180, which does not contain IL4. 192. The IL10 agonist according to any one of embodiments 1-191, which does not contain any cytokine other than IL10.

[0463] 193. The IL10 agonist according to any one of embodiments 1-192, wherein the IL10 moiety does not contain an Fc domain C-terminally. 194. The IL10 agonist according to any one of embodiments 1 to 193, which is the product of recombinant cell expression in a mammalian cell.

[0464] 195. The IL10 agonist according to embodiment 194, wherein the mammalian cells are of a murine cell line. 196. The IL10 agonist according to embodiment 194, wherein the mammalian cells are of a rat cell line.

[0465] 197. The IL10 agonist according to embodiment 194, wherein the mammalian cells are of a monkey cell line. 198. The IL10 agonist according to embodiment 194, wherein the mammalian cells are of a human cell line.

[0466] 199. A nucleic acid or nucleic acids encoding an IL10 agonist according to any one of embodiments 1 to 198. 200. A host cell engineered to express an IL10 agonist according to any one of embodiments 1 to 198 or a nucleic acid according to embodiment 194.

[0467] 201. The host cell of embodiment 200, wherein the host cell is of a mouse cell line. 202. The IL10 agonist according to embodiment 200, wherein the mammalian cells are of a rat cell line.

[0468] 203. The IL10 agonist according to embodiment 200, wherein the mammalian cells are of a monkey cell line. 204. The IL10 agonist according to embodiment 200, wherein the mammalian cells are of a human cell line.

[0469] 205. A method for producing an IL10 agonist according to any one of embodiments 1 to 198, comprising culturing a host cell according to any one of embodiments 200 to 204 and recovering the IL10 agonist expressed thereby.

[0470] 206. (a) contacting the recovered IL10 agonist with an affinity column configured to capture the IL10 agonist; (b) eluting from the affinity column to produce an eluate; and (c) performing size exclusion chromatography on the eluate. 206. The method of embodiment 205, further comprising:

[0471] 207. The method of embodiment 206, wherein the affinity column is a Protein A affinity column. 208. (a) Culturing a host cell according to any one of embodiments 200 to 204; and (b) recovering the IL10 agonist expressed thereby. An IL10 agonist produced by a method comprising:

[0472] 209. The method comprises: (a) contacting the recovered IL10 agonist with an affinity column configured to capture the IL10 agonist; (b) eluting from the affinity column to produce an eluate; and (c) performing size exclusion chromatography on the eluate. 209. The IL10 agonist of embodiment 208, further comprising:

[0473] 210. A method for preparing a pharmaceutical composition, comprising formulating an IL10 agonist prepared by the method of any one of embodiments 205-207 with an excipient. 21. A pharmaceutical composition comprising an IL10 agonist according to any one of embodiments 1 to 198 and an excipient.

[0474] 212. A pharmaceutical composition obtained by the method described in embodiment 210. 213. The pharmaceutical composition according to embodiment 210 or embodiment 211, comprising at least 10 mg of said IL10 agonist.

[0475] 214. The pharmaceutical composition according to embodiment 210 or embodiment 211, comprising at least 20 mg of said IL10 agonist. 215. The pharmaceutical composition according to embodiment 210 or embodiment 211, comprising at least 50 mg of said IL10 agonist.

[0476] 216. The pharmaceutical composition according to any one of embodiments 211-215, wherein less than 30% of said IL10 agonist is present in aggregate form as determined by size exclusion ultra-performance liquid chromatography (SE-UPLC).

[0477] 217. The pharmaceutical composition according to any one of embodiments 211-216, wherein less than 25% of said IL10 agonist is present in aggregate form as determined by size exclusion ultra-performance liquid chromatography (SE-UPLC).

[0478] 218. The pharmaceutical composition according to any one of embodiments 211-217, wherein less than 20% of said IL10 agonist is present in aggregate form as determined by size exclusion ultra-performance liquid chromatography (SE-UPLC).

[0479] 219. The pharmaceutical composition according to any one of embodiments 211-218, wherein less than 15% of said IL10 agonist is present in aggregate form as determined by size exclusion ultra-performance liquid chromatography (SE-UPLC).

[0480] 220. The pharmaceutical composition according to any one of embodiments 211-219, wherein less than 10% of said IL10 agonist is present in aggregate form as determined by size exclusion ultra-performance liquid chromatography (SE-UPLC).

[0481] 221. The pharmaceutical composition according to any one of embodiments 211-220, wherein less than 5% of said IL10 agonist is present in aggregate form as determined by size exclusion ultra-performance liquid chromatography (SE-UPLC).

[0482] 222. The pharmaceutical composition according to any one of embodiments 211-215, which does not contain detectable amounts of aggregates of said IL10 agonist, as determined by size-exclusion ultra-performance liquid chromatography (SE-UPLC).

[0483] 223. The pharmaceutical composition according to any one of embodiments 211-222, which does not contain a detectable amount of a C-terminal truncated variant of said IL10 agonist. 224. A method for treating an inflammatory condition, an immune-related disorder, a fibrotic disorder or cancer, comprising administering to a subject in need thereof an IL10 agonist according to any one of embodiments 1-198 or a pharmaceutical composition according to any one of embodiments 211-223.

[0484] 225. The method according to embodiment 224, which is a method for treating cancer, wherein the cancer is a solid tumor. 226. The method of embodiment 225, wherein the solid tumor is colon cancer.

[0485] 227. The method of embodiment 225, wherein the solid tumor is melanoma. 228. The method of embodiment 225, wherein the solid tumor is squamous cell carcinoma. 229. The method of embodiment 225, wherein the solid tumor is a lymphoma.

[0486] 230. The method of embodiment 225, wherein the solid tumor is a pancreatic tumor. 231. The method of embodiment 225, wherein the solid tumor is a lung tumor. 232. The method of any one of embodiments 224-231, wherein the IL10 agonist or pharmaceutical composition is administered in combination with 5-fluorouracil, folinic acid, and a platinum coordination complex.

[0487] 233. The method according to any one of embodiments 224 to 231, wherein the IL10 agonist or pharmaceutical composition is administered subcutaneously. 234. The method according to any one of embodiments 224 to 231, wherein the IL10 agonist or pharmaceutical composition is administered intravenously.

[0488] 235. The method of any one of embodiments 224-234, wherein the solid tumor is resistant to treatment with an anti-PD1 antibody. 236. The method of any one of embodiments 224-235, wherein the solid tumor is resistant to treatment with anti-PD1 antibody monotherapy.

[0489] 237. The method of any one of embodiments 224-236, further comprising administering an anti-PD1 antibody to the subject. 238. The method of any one of embodiments 235-237, wherein the anti-PD1 antibody is MDX-1106 (nivolumab), MK-3475 (pembrolizumab), MEDI-0680 (AMP-514), PDR001, REGN2810, or BGB-108.

[0490] 239. The method of any one of embodiments 225 to 238, which results in an increase in CD8 T cell density in said solid tumor. 240. The method of embodiment 239, wherein said increase in CD8 T cell density in said solid tumor is at least a two-fold increase.

[0491] 241. The method of embodiment 239, wherein said increase in CD8 T cell density in said solid tumor is at least a three-fold increase. 242. The method of embodiment 239, wherein said increase in CD8 T cell density in said solid tumor is at least a 4-fold increase.

[0492] 243. CD45 in solid tumors + The method of any one of embodiments 225 to 242, which results in increased immune cell infiltration. 244. CD45 in the aforementioned solid tumors + 244. The method of embodiment 243, wherein said increase in immune cell infiltration is a 5% increase.

[0493] 245. CD45 in solid tumors + 244. The method of embodiment 243, wherein said increase in immune cell infiltration is a 10% increase. 246. CD45 in solid tumors + 244. The method of embodiment 243, wherein said increase in immune cell infiltration is a 15% increase.

[0494] 247. CD45 in solid tumors + 244. The method of embodiment 243, wherein said increase in immune cell infiltration is a 20% increase. 248. CD45 + 248. The method of any one of embodiments 243-247, wherein the immune cells comprise CD4 T cells, myeloid cells, or both CD4 T cells and myeloid cells.

[0495] 249. The method of any one of embodiments 225-248, wherein the method upregulates serum IL12 relative to a suitable control not treated with the IL10 agonist or pharmaceutical composition, or relative to the subject's own serum IL12 level before treatment with the IL10 agonist or pharmaceutical composition.

[0496] 250. The method of embodiment 249, wherein said method upregulates serum IL12 levels by at least 2-fold compared to the control or compared to the subject's own serum IL12 levels before treatment with said IL10 agonist or pharmaceutical composition.

[0497] 251. The method of embodiment 249, wherein the method upregulates serum IL12 levels by at least 5-fold compared to the control or compared to the subject's own serum IL12 levels before treatment with the IL10 agonist or pharmaceutical composition.

[0498] 252. The method of embodiment 249, wherein said method upregulates serum IL12 levels by at least 10-fold compared to the control or compared to the subject's own serum IL12 levels before treatment with the IL10 agonist or pharmaceutical composition.

[0499] 253. The method of embodiment 249, wherein said method upregulates serum IL12 levels by at least 15-fold compared to the control or compared to the subject's own serum IL12 levels before treatment with the IL10 agonist or pharmaceutical composition.

[0500] 254. The method of embodiment 249, wherein said method upregulates serum IL12 levels by at least 20-fold compared to the control or compared to the subject's own serum IL12 levels before treatment with the IL10 agonist or pharmaceutical composition.

[0501] 255. The method of embodiment 249, wherein the method upregulates serum IL12 levels by at least 25-fold compared to the control or compared to the subject's own serum IL12 levels before treatment with the IL10 agonist or pharmaceutical composition.

[0502] 256. The method of embodiment 249, wherein the method upregulates serum IL12 levels by at least 30-fold compared to the control or compared to the subject's own serum IL12 levels before treatment with the IL10 agonist or pharmaceutical composition.

[0503] 257. The method of any one of embodiments 225-256, wherein the method upregulates serum IL4 levels relative to a suitable control not treated with the IL10 agonist or pharmaceutical composition, or relative to the subject's own serum IL4 levels prior to treatment with the IL10 agonist or pharmaceutical composition.

[0504] 258. The method of embodiment 257, wherein said method upregulates serum IL4 levels by at least 2-fold compared to the control or compared to the subject's own serum IL4 levels before treatment with said IL10 agonist or pharmaceutical composition.

[0505] 259. The method of embodiment 257, wherein said method upregulates serum IL4 levels by at least 5-fold compared to the control or compared to the subject's own serum IL4 levels before treatment with said IL10 agonist or pharmaceutical composition.

[0506] 260. The method of embodiment 257, wherein said method upregulates serum IL4 levels by at least 10-fold compared to the control or compared to the subject's own serum IL4 levels before treatment with said IL10 agonist or pharmaceutical composition.

[0507] 261. The method of embodiment 257, wherein said method upregulates serum IL4 levels by at least 15-fold compared to the control or compared to the subject's own serum IL4 levels before treatment with said IL10 agonist or pharmaceutical composition.

[0508] 262. The method of embodiment 257, wherein said method upregulates serum IL4 levels by at least 20-fold compared to the control or compared to the subject's own serum IL4 levels before treatment with said IL10 agonist or pharmaceutical composition.

[0509] 263. For subjects requiring cancer treatment: (a) CD8+ T cells expressing chimeric antigen receptors ("CARs") ("CART cells"); and (b) an IL10 agonist according to any one of embodiments 1 to 198; A method for treating cancer, comprising administering

[0510] 264. The method of embodiment 263, wherein the IL10 agonist is administered to the subject within one week of administering the CART cells. 265. The method of embodiment 264, wherein the IL10 agonist is administered to the subject on the same day as the administration of the CART cells.

[0511] 266. The method of any one of embodiments 263-265, comprising medicating said subject with said IL10 agonist for a period of at least two weeks. 267. The method of embodiment 266, wherein the IL10 agonist is administered by continuous infusion.

[0512] 268. The method of embodiment 266, wherein said IL10 agonist is administered by daily administration for at least a portion of said at least two week period. 269. The IL10 agonist is (a) administering the IL10 agonist at a first dosing frequency during an initial portion of the at least two week period; and (b) administering said IL10 agonist at a second dosing frequency for a subsequent portion of said at least two week period. 267. The method of embodiment 266, wherein the patient is administered according to a split dosing regimen comprising:

[0513] 270. The method of embodiment 269, wherein the first dosing frequency is daily. 271. The method of embodiment 269 or embodiment 270, wherein the second dosing frequency is lower than the first dosing frequency.

[0514] 272. The method of embodiment 271, wherein the second dosing frequency is weekly. 273. The method of any one of embodiments 269-272, wherein the subject is transitioned from a first dosing frequency to a second dosing frequency concurrently with or after depletion of the CART cells.

[0515] 274. The method of any one of embodiments 263-273, wherein the cancer is resistant to treatment with an anti-PD1 antibody. 275. The method of any one of embodiments 263-274, wherein the cancer is resistant to treatment with anti-PD1 antibody monotherapy.

[0516] 276. The method of any one of embodiments 263-275, further comprising administering an anti-PD1 antibody to the subject. 277. The method of any one of embodiments 274 to 276, wherein the anti-PD1 antibody is MDX-1106 (nivolumab), MK-3475 (pembrolizumab), MEDI-0680 (AMP-514), PDR001, REGN2810, or BGB-108.

[0517] 278. The CAR is selected from the group consisting of 5T4, alpha-fetoprotein, B-cell maturation antigen (BCMA), CA-125, carcinoembryonic antigen, CD19, CD20, CD22, CD23, CD30, CD33, CD56, CD123, CD138, c-Met, CSPG4, C-type lectin-like molecule 1 (CLL-1), EGFRvIII, epithelial tumor antigen, ERBB2, FLT3, folate binding protein, GD2, GD3, HER1-HER2 combination, HER2 - The method of any one of embodiments 263 to 277, which is designed to target a combination of HER3, HER2 / Neu, HERV-K, HIV-1 envelope glycoprotein gp41, HIV-1 envelope glycoprotein gp120, IL-IIRα, kappa chain, lambda chain, melanoma-associated antigen, mesothelin, MUC-1, mutant p53, mutant ras, prostate-specific antigen, ROR1, or VEGFR2, or a combination thereof.

[0518] 279. The method of any one of embodiments 263 to 278, wherein the CAR is configured in accordance with Section 6.11.1 and its subsections. 280. The method according to any one of embodiments 263-279, wherein the IL10 agonist is in the form of a pharmaceutical composition according to any one of embodiments 211-223.

[0519] 281. The method of any one of embodiments 263 to 280, which results in an increase in CD8 T cell density in said solid tumor. 282. The method of embodiment 281, wherein said increase in CD8 T cell density in said solid tumor is at least a two-fold increase.

[0520] 283. The method of embodiment 281, wherein said increase in CD8 T cell density in said solid tumor is at least a three-fold increase. 284. The method of embodiment 281, wherein said increase in CD8 T cell density in said solid tumor is at least a four-fold increase.

[0521] 285. CD45 in solid tumors + The method of any one of embodiments 263 to 281, which results in an increase in immune cell infiltration, optionally wherein the increase is at least a 10% increase.

[0522] 286. CD45 in solid tumors + 286. The method of embodiment 285, wherein said increase in immune cell infiltration is a 5% increase. 287. CD45 in solid tumors + 286. The method of embodiment 285, wherein said increase in immune cell infiltration is a 10% increase.

[0523] 288. The method of embodiment 285, wherein said increase in CD45+ immune cell infiltration in said solid tumor is a 15% increase. 289. CD45 in solid tumors + 286. The method of embodiment 285, wherein said increase in immune cell infiltration is a 20% increase.

[0524] 290. CD45 + 289. The method of any one of embodiments 285-289, wherein the immune cells comprise CD4 T cells and myeloid cells. 291. The method of any one of embodiments 263-286, wherein said method upregulates serum IL12 levels relative to a suitable control not treated with said IL10 or pharmaceutical composition.

[0525] 292. The method of embodiment 291, wherein the method upregulates serum IL12 levels by at least 2-fold compared to the control or compared to the subject's own serum IL12 levels before treatment with the IL10 agonist or pharmaceutical composition.

[0526] 293. The method of embodiment 291, wherein the method upregulates serum IL12 levels by at least 5-fold compared to the control or compared to the subject's own serum IL12 levels before treatment with the IL10 agonist or pharmaceutical composition.

[0527] 294. The method of embodiment 291, wherein the method upregulates serum IL12 levels by at least 10-fold compared to the control or compared to the subject's own serum IL12 levels before treatment with the IL10 agonist or pharmaceutical composition.

[0528] 295. The method of embodiment 291, wherein the method upregulates serum IL12 levels by at least 15-fold compared to the control or compared to the subject's own serum IL12 levels before treatment with the IL10 agonist or pharmaceutical composition.

[0529] 296. The method of embodiment 291, wherein the method upregulates serum IL12 levels by at least 20-fold compared to the control or compared to the subject's own serum IL12 levels before treatment with the IL10 agonist or pharmaceutical composition.

[0530] 297. The method of embodiment 291, wherein the method upregulates serum IL12 levels by at least 25-fold compared to the control or compared to the subject's own serum IL12 levels before treatment with the IL10 agonist or pharmaceutical composition.

[0531] 298. The method of embodiment 291, wherein the method upregulates serum IL12 levels by at least 30-fold compared to the control or compared to the subject's own serum IL12 levels before treatment with the IL10 agonist or pharmaceutical composition.

[0532] 299. The method of any one of embodiments 263-298, wherein said method upregulates serum IL4 levels relative to a suitable control not treated with said IL10 or pharmaceutical composition.

[0533] 300. The method of embodiment 299, wherein said method upregulates serum IL4 levels by at least 2-fold compared to the control or compared to the subject's own serum IL4 levels before treatment with said IL10 agonist or pharmaceutical composition.

[0534] 301. The method of embodiment 299, wherein said method upregulates serum IL4 levels by at least 5-fold compared to the control or compared to the subject's own serum IL4 levels before treatment with said IL10 agonist or pharmaceutical composition.

[0535] 302. The method of embodiment 299, wherein said method upregulates serum IL4 levels by at least 10-fold compared to the control or compared to the subject's own serum IL4 levels before treatment with said IL10 agonist or pharmaceutical composition.

[0536] 303. The method of embodiment 299, wherein said method upregulates serum IL4 levels by at least 15-fold compared to the control or compared to the subject's own serum IL4 levels before treatment with said IL10 agonist or pharmaceutical composition.

[0537] 304. The method of embodiment 299, wherein said method upregulates serum IL4 levels by at least 20-fold compared to said control or compared to the subject's own serum IL4 levels before treatment with said IL10 agonist or pharmaceutical composition.

[0538] All publications, patents, patent applications, and other documents cited in this application are incorporated herein by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, or other document was individually indicated to be incorporated by reference for all purposes. In the event of a conflict between the teachings of one or more references incorporated herein and the present disclosure, the teachings of the present disclosure are intended to control.

Claims

1. A therapeutic agent for treating cancer, the therapeutic agent comprising: From the amino terminus to the carboxy terminus (a) an IgG Fc domain; (b) a linker moiety; and (c) IL10 part and an IL10 agonist comprising: The IL10 agonist comprises an amino acid sequence having at least 95% sequence identity with the sequence of IL10M1 (SEQ ID NO: 34), and the therapeutic agent is administered to a subject in need thereof.

2. The method of claim 1 , wherein the IL10 agonist comprises an amino acid sequence having at least 96% sequence identity with the sequence of IL10M1 (SEQ ID NO: 34).

3. The method of claim 1 , wherein the IL10 agonist comprises an amino acid sequence having at least 97% sequence identity with the sequence of IL10M1 (SEQ ID NO: 34).

4. The method of claim 1 , wherein the IL10 agonist comprises an amino acid sequence having at least 98% sequence identity with the sequence of IL10M1 (SEQ ID NO: 34).

5. The method of claim 1 , wherein the IL10 agonist comprises an amino acid sequence having at least 99% sequence identity with the sequence of IL10M1 (SEQ ID NO: 34).

6. 6. The therapeutic agent of any one of claims 1 to 5, wherein the Fc domain comprises or consists of an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:31, or to amino acids 18 to 228 of SEQ ID NO:

31.

7. The therapeutic agent according to any one of claims 1 to 6, wherein the effector function of the Fc domain is reduced.

8. The therapeutic agent according to any one of claims 1 to 7, comprising a hinge domain at the N-terminus of the Fc domain.

9. The therapeutic agent of claim 8, wherein the hinge domain is derived from the same IgG as the CH2 and / or CH3 domain.

10. The therapeutic agent of claim 8, wherein the hinge domain is derived from an IgG different from the CH2 and / or CH3 domain.

11. The therapeutic agent according to any one of claims 8 to 10, wherein the hinge domain comprises a chimeric hinge sequence, and the chimeric hinge sequence comprises or consists of the amino acid sequence of SEQ ID NO: 12 or SEQ ID NO:

13.

12. The linker moiety is G ｎ S or SG ｎ 12. The therapeutic agent according to any one of claims 1 to 11, comprising or consisting of a monomer or polymer of the formula:

13. The linker portion is an amino acid sequence G ４ The therapeutic agent according to any one of claims 1 to 12, comprising a monomer or multimer of S (SEQ ID NO: 51).

14. The linker portion is an amino acid sequence G ４ The therapeutic agent of any one of claims 1 to 13, comprising one, two or three repeats of S (SEQ ID NO: 51).

15. The therapeutic agent according to any one of claims 1 to 14, wherein the linker moiety connects the Fc domain and the IL10 moiety.

16. The therapeutic agent of any one of claims 1 to 15, wherein the IL10 portion comprises or consists of an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to mature human IL10 (SEQ ID NO: 1).

17. The therapeutic agent according to any one of claims 1 to 16, which is a homodimer.

18. The therapeutic agent according to any one of claims 1 to 16, which is a heterodimer.

19. The therapeutic agent according to claim 17 or 18, comprising two Fc domains, wherein the IL10 agonist dimerizes via the Fc domains.

20. The therapeutic agent according to any one of claims 1 to 19, wherein the IL10 portion lacks an amino acid spacer located between helix D and helix E of the IL10 portion interdomain region.

21. The therapeutic agent according to any one of claims 1 to 20, which is not conjugated with polyethylene glycol.

22. The therapeutic agent according to any one of claims 1 to 21, which is not conjugated to albumin.

23. The therapeutic agent according to any one of claims 1 to 22, which is not conjugated to a human serum albumin binder.

24. 24. The therapeutic agent of any one of claims 1 to 23, which is not conjugated to XTEN, PAS, a hydrophilic polymer, polysialic acid, a fatty acid, or hydroxyethyl starch.

25. The therapeutic agent of any one of claims 1 to 24, which is not conjugated to any stabilizing moiety.

26. The method of any one of claims 1 to 25, wherein the IL10 portion does not include an N-linked glycan, an O-linked glycan, or both an N-linked and an O-linked glycan.

27. The IL10 agonist is: (a) binds to a tumor-associated antigen; (b) binds to tumor microenvironment antigens; (c) binds to a cell surface molecule of a tumor-reactive lymphocyte; or (d) binds to a checkpoint inhibitor The therapeutic agent of any one of claims 1 to 26, which lacks a targeting moiety.

28. The therapeutic agent of any one of claims 1 to 27, wherein the IL10 agonist lacks a targeting moiety that binds to PD-1.

29. The therapeutic agent according to any one of claims 1 to 28, wherein the IL10 agonist lacks an antibody variable region.

30. The method of any one of claims 1 to 28, wherein the IL10 agonist lacks a CH1 domain.

31. The therapeutic agent according to any one of claims 1 to 28, wherein the IL10 agonist lacks a CL domain.

32. The therapeutic agent according to any one of claims 1 to 28, which does not contain any cytokine other than IL10.

33. The therapeutic agent described in any one of claims 1 to 32, further comprising an excipient.

34. 34. The therapeutic agent of any one of claims 1 to 33, comprising at least 10 mg, at least 20 mg, or at least 50 mg of the IL10 agonist.

35. 35. The therapeutic agent of any one of claims 1 to 34, wherein less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, or less than 5% of the IL10 agonist is present in aggregate form as determined by size exclusion ultra performance liquid chromatography (SE-UPLC).

36. 35. The therapeutic agent of any one of claims 1 to 34, which does not contain detectable amounts of aggregates of the IL10 agonist as determined by size exclusion ultra-performance liquid chromatography (SE-UPLC).

37. The therapeutic agent according to any one of claims 1 to 36, which does not contain (a) a detectable amount of a C-terminal truncated variant of the IL10 agonist and / or (b) a detectable amount of an N-terminal truncated variant of the IL10 agonist.

38. The therapeutic agent according to any one of claims 1 to 37, wherein the cancer is a solid tumor.

39. 39. The method of claim 38, wherein the solid tumor is colon cancer, melanoma, squamous cell carcinoma, lymphoma, pancreatic tumor, or lung tumor.

40. The therapeutic agent of claim 38 or claim 39, wherein the solid tumor is resistant to treatment with an anti-PD1 antibody.

41. The therapeutic agent according to any one of claims 38 to 40, wherein the solid tumor is resistant to treatment with anti-PD1 antibody monotherapy.

42. The therapeutic agent according to any one of claims 38 to 41, which is administered to the subject together with an anti-PD1 antibody.

43. The therapeutic agent of claim 42, wherein the anti-PD1 antibody is MDX-1106 (nivolumab), MK-3475 (pembrolizumab), MEDI-0680 (AMP-514), PDR001, REGN2810, or BGB-108.

44. 44. The method of any one of claims 38 to 43, which results in an increase in CD8 T cell density in the solid tumor, said increase being at least a two-fold increase, at least a three-fold increase, or at least a four-fold increase.

45. The therapeutic agent of any one of claims 38 to 44, which results in an increase in CD45+ immune cell infiltration in the solid tumor, wherein the increase is at least a 10% increase.

46. 46. ​​The method of claim 45, wherein the CD45+ immune cells comprise CD4 T cells, myeloid cells, or both CD4 T cells and myeloid cells.

47. The therapeutic agent of any one of claims 1 to 46, wherein administration of the therapeutic agent upregulates serum IL12 relative to a suitable control not treated with the therapeutic agent, or relative to the subject's own serum IL12 level before treatment with the therapeutic agent, and wherein administration of the therapeutic agent upregulates serum IL12 by at least 2-fold, at least 5-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, or at least 30-fold relative to the suitable control, or relative to the subject's own serum IL12 level before treatment with the therapeutic agent.

48. 48. The therapeutic agent of any one of claims 1 to 47, wherein administration of the therapeutic agent upregulates serum IL4 relative to a suitable control not treated with the therapeutic agent, or relative to the subject's own serum IL4 level prior to treatment with the therapeutic agent, and wherein administration of the therapeutic agent upregulates serum IL4 by at least 2-fold, at least 5-fold, at least 10-fold, at least 15-fold, or at least 20-fold relative to the suitable control, or relative to the subject's own serum IL4 level prior to treatment with the therapeutic agent.

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