Novel IL10 agonist and its usage

Abstract

To provide an IL10 agonist with improved antitumor therapeutic efficacy. [Solution] An IL10 agonist is provided, comprising (a) an IgG Fc domain; (b) a linker moiety; and (c) an IL10 moiety, arranged from the amino terminus to the carboxyl terminus. The IgG Fc domain may contain CH2 and CH3 domains derived from IgG1, IgG2, IgG3, IgG4, or combinations thereof, and the effector function of the Fc domain may be reduced.

Description

[Technical Field]

[0001] The present invention relates to a novel IL10 agonist and a method for using the same. [Background technology]

[0002] Interleukin-10 (IL-10 or IL10), a cytokine also known as a human cytokine synthesis inhibitor (CSIF), is a multifaceted cytokine that modulates multiple immune responses through its actions on T cells, B cells, macrophages, and antigen-presenting cells (APCs). IL10 is primarily expressed in macrophages, but its 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 antigen-presentation suppression (by reducing the expression of major histocompatibility complex type II (MHC-II) and the costimulatory ligand CD80 / CD86), suppression of inflammatory cytokine release by myeloid cells, and inhibition of T cell priming (via suppression of CD28 signaling). However, more recent studies have also described the role of IL10 in immunostimulation through B cell costimulation, enhancement of NK cell cytolytic activity, and proliferation, cytokine release, and enhanced cytolytic activity of cytolytic T cells (see Non-Patent Literature 1 for a review).

[0003] Human IL-10 is a non-covalently linked homodimer, and its receptor is a heterotetrameric complex composed of two IL10Rα (also called IL10R1) molecules and two IL10Rβ (also called IL10R2) molecules. IL10Rα is expressed on all IL10-responsive cells, while IL10Rβ is constitutively expressed in most cell types. When IL10Rα binds to IL10, it induces a conformational change in IL10Rβ, enabling IL10Rβ to also bind to IL10. Once the IL10 / IL10Rα / IL10Rβ complex is formed, tyrosine kinases Jak1 and Tyk2 are activated, phosphorylating specific tyrosine residues in the intracellular domain of IL10Rα, leading to the recruitment of signaling transcription factor 3 (STAT3), which mediates downstream signaling of IL10. Unlike IL10Rα, which is specific to IL10, the IL10Rβ subunit is shared with receptors for other type II cytokines, including IL22, IL26, and INFλ (see Non-Patent Literature 2 for a review).

[0004] Due to its multifaceted activity, IL-10 has been associated with a wide range of diseases, disorders, and conditions, including inflammatory states, immune-related disorders, fibrotic disorders, and cancer. One drawback of using IL-10, especially any form of recombinant IL-10, in therapeutic applications is its short serum half-life. The 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 [Overview of the project] [Problems that the invention aims to solve]

[0006] Having an IL10 agonist that can withstand systemic exposure during therapy by enhancing the circulating lifetime of IL10 (delaying clearance) and its solubility and stability is considered advantageous. This disclosure addresses this and other related needs in the art. [Means for solving the problem]

[0007] This disclosure stems from the discovery of an IL10 agonist that exhibits remarkably improved in vivo therapeutic efficacy, particularly in its antitumor activity. The IL10 portions that can be used in the IL10 agonists of this disclosure are described in Section 6.3.

[0008] The Fc domains that may be used as IL10 agonists in this disclosure are described in Section 6.4. The targeting portions that may be used in the IL10 agonists of this disclosure are described in Section 6.5.

[0009] Stabilization portions that may be used in the IL10 agonists of this disclosure are described in Section 6.6. Various exemplary configurations of the IL10 agonist of this disclosure are described in the following specific embodiments 60 to 77.

[0010] Linkers that can be used to connect multiple different components of the IL10 agonist of this disclosure are described in Section 6.7. This disclosure further provides nucleic acids encoding the IL10 agonists of this 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). This disclosure further provides host cells and cell lines engineered to express the nucleic acids and IL10 agonists of this disclosure. This disclosure further provides methods for producing the IL10 agonists of this disclosure. Exemplary nucleic acids, host cells, cell lines, and methods for producing IL10 agonists are described below in Section 6.8 and Specific Embodiments 199-204.

[0011] This disclosure further provides pharmaceutical compositions comprising the IL10 agonist of this disclosure. Exemplary pharmaceutical compositions are described in Section 6.8.3 and Specific Embodiments 211-223 below.

[0012] The present specification further provides methods for using the IL10 agonists and pharmaceutical compositions of the present disclosure to treat, for example, cancer and immunodeficiencies. Exemplary methods are described in Section 6.10. The IL10 agonists of the present disclosure are useful in combination therapy, for example, as adjuncts to CART therapy. Exemplary combination therapy methods are disclosed in 6.11. Specific embodiments of the therapeutic methods of the present disclosure are described in the following specific embodiments 224 to 304. [Brief explanation of the drawing]

[0013] [Figure 1A] A diagram showing the differences in the effects of IL10 on the priming and effector functions of CD8 T cells. [Figure 1B] Same as above. [Figure 2]Figures illustrating the IL10 agonists of this disclosure. Figure 2A shows a general form of the IL10 agonist, and Figure 2B shows a specific embodiment of the IL10 agonist of this disclosure. These explanatory figures are intended to depict the N-terminus to C-terminus sequence of the domains contained in the IL10 agonist and are not intended to convey scale or three-dimensional configuration. IL10M11 (not shown) is similar to IL10M3, but has a mouse IL10 portion instead of the human IL10 portion of IL10M3. [Figure 3-1] This figure shows the activity of IL10 mutein against STAT3-mediated luciferase reporter activity. Recombinant IL10 and IL10 mutein increase STAT3-responsive luciferase activity in engineered Ramos / STAT3-Luc (Figures 3A-3C) and TF-1 / STAT3-Luc (Figures 3D-3F) reporter cells. The graph is separated by the IL10 agonist used, with black circles representing IL10M1 (Figures 3A, 3D), IL10M2 (Figures 3B, 3E), or IL10M3 (Figures 3C, 3F), white squares representing isotype controls (human IgG4 stealth for Figures 3A, 3B, 3D, and 3E; mouse IgG1 for Figures 3C and 3F), and triangles representing commercially available human IL10 (purchased from Peptrotech). [Figure 3-2] Same as above. [Figure 4-1]Figures showing that IL10 mutain suppresses cytokine release from primary human T cells derived from donor 5500Y to a level comparable to that of recombinant human IL10. Release of IL2 (Figures 4A, 4D, 4G), TNFα (Figures 4B, 4E, 4H), and IFNγ (Figures 4C, 4F, 4I) from donor 5500Y T cells co-cultured with allogeneic PBMCs treated with mitomycin C and IL10M1 (Figures 4A-4C), IL10M2 (Figures 4D-4F), or IL10M3 (Figures 4G-4I). The graphs are separated by the IL10 mutain used. Black circles represent IL10 mutain, white squares represent isotype controls (hIgG4s for IL10M1 and IL10M2, and mIgG1 for IL10M3), and gray triangles represent recombinant human IL10. [Figure 4-2] Same as above. [Figure 4-3] Same as above. [Figure 5-1] Figures showing that IL10 mutain suppresses cytokine release from primary human T cells derived from donor 6900M to a level comparable to that of recombinant human IL10. Release of IL2 (Figures 5A, 5D, 5G), TNFα (Figures 5B, 5E, 5H), and IFNγ (Figures 5C, 5F, 5I) from donor 6900M-derived T cells co-cultured with allogeneic PBMCs treated with mitomycin C and IL10M1 (Figures 5A-5C), IL10M2 (Figures 5D-5F), or IL10M3 (Figures 5G-5I). The graphs are separated by the IL10 mutain used. Black circles represent IL10 mutain, white squares represent isotype controls (hIgG4s for IL10M1 and IL10M2, and mIgG1 for IL10M3), and gray triangles represent recombinant human IL10. [Figure 5-2] Same as above. [Figure 5-3] Same as above. [Figure 6] This figure shows the antitumor activity of IL10 mutein compared to isotype controls in a syngeneic mouse model. Tumor volume is shown on day 11 (Figure 6A) and day 32 (Figure 6B). [Figure 7-1]A figure showing the activity of various IL10 muteins in STAT3 luciferase analysis. [Figure 7-2] Same as above. [Figure 7-3] Same as above. [Figure 8A] Figures illustrating the antitumor activity of IL10M11. IL10M11 exhibits antitumor activity against various tumor cell lines after prophylactic or therapeutic administration (Figures 8A-8E). Administration of IL10M11 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] The figure shows the results of administering IL10M11 in combination with a PD-1 antagonist and a CD40 agonist. These results indicate that modulating these pathways enhances the sustained primary and memory antitumor responses induced by IL10M11. [Figure 9-2] Same as above. [Figure 10-1]Figures showing the density of overall CD45+ immune cells and analyzed immune cell subsets in A20, Colon25, MC38, and B16F10 tumors. These results indicate that the overall density of 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] This figure shows the expression of PD-1 and IL10R1 on various myeloid cells in the spleen, influx region lymph nodes (dLNs), and tumors. IL10R1 expression on T cells, particularly CD8 T cells, was very limited to tumor-infiltrating CD8 T cells, and was hardly observed on T cells in secondary lymphoid tissues, such as the spleen and influx region LNs of MC38-cOVA and Colon 26. Almost all IL10R1+CD8 T cells expressed the PD1 molecule. [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] This study shows PD-L1 expression in four different tumor types (A20, Colon26, MC38, and B16F10). These results indicate 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]Figures 13A and 13C show the experimental design of the A20 tumor model study and the results of treatment with controls, IL10M11, or PD-1 antagonist antibodies. A 150 mm3 A20 B-cell lymphoma responded to PD-1Ab treatment, showing a significant delay in tumor growth and approximately 50% tumor-free survival. IL10M11 induced a more advanced and widespread immune response against A20, resulting in approximately 85% tumor-free survival. [Figure 13-2] Same as above. [Figure 14-1] Figure 14A shows the experimental design of the Colon26 tumor model study and the results of treatment with controls, IL10M11, or PD-1 antagonist antibodies (Figures 14B and 14C). Tumors of 100 mm3 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] Figure 15A shows the experimental design of the B16F10 tumor model study and the results of treatment with control, IL10M11, or PD-1 antagonist antibodies (Figures 15B and 145). A 100 mm3 B16F10 tumor showed little response to PD1Ab. IL10M11 significantly delayed tumor growth and made the majority of the tumor responsive. [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 a combination of IL10M11 and PD-1 antagonist antibody (Figures 16B and 16C). Both PD1 Ab and IL10-Fc had antitumor effects comparable to those of monotherapy. The combination of PD1 Ab and IL10-Fc dramatically increased the antitumor response by delaying tumor growth and increasing the frequency of tumor-free survival. [Figure 16-2] Same as above. [Figure 17A-1]Figure showing the results of immunoprofiling of the tumor microenvironment after treatment. As shown, IL10 treatment significantly increased CD8 T cell density in both immunogenic MC38 tumor models (Figure 17A) and non-immunogenic 4T1 tumor models (Figure 17B), and this increase 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] Figure 18B shows the density of PD1+CD8 T cells in MC38 tumors (Figure 18A) and 4T1 tumors (Figure 18B). [Figure 18B] Same as above. [Figure 19-1] A diagram showing serum levels of various cytokines after treatment with IL10M11 in naive, non-tumor-carrying mice. [Figure 19-2] Same as above. [Figure 20A] This figure shows serum levels of various cytokines after treatment with IL10M11, IL12, IL1b, IL2, IL4, and IL5 in B16F10 tumor-bearing mice. Serum levels were upregulated by IL10 but not by the PD1 antibody. [Figure 20B] Same as above. [Figure 21] Figure 21A shows serum levels of IL12 and IL4 (Figure 21B) normalized to the mean in isotype control treated mice. Upregulation of IL12 and IL4 is shown across the A20, MC38, MC38-cOVA, B16F10, and Colon26 tumor models. [Figure 22]Figures 22B and 22C show the experimental design of the MC38 tumor model study (Figure 22A) 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 a PD-1 binding domain (mPD1-mIL10). Both PD1 Ab and IL10-Fc showed antitumor effects as monotherapy (slight effect for PD1 Ab, moderate effect for IL10-Fc). The combination of IL10-Fc and PD-1 Ab significantly improved efficacy. PD1-IL10-Fc did not show any difference in efficacy compared to the control Ab-IL10 and IL10-Fc. [Modes for carrying out the invention]

[0014] 6.1.Definition Related: In the context of IL10 agonists or their components (e.g., targeted moieties such as antibodies), the term “related” refers to a functional relationship between two or more polypeptide chains. In particular, the term “related” means that two or more polypeptides are related to each other, for example, non-covalently via molecular interactions, or covalently via one or more disulfide crosslinks or chemical crosslinks, in order to produce a functional IL10 agonist. Examples of associations that may exist in the IL10 agonists of this disclosure include, but are not limited to, associations between homodimeric or heterodimeric Fc domains in the Fc region, associations between the VH and VL regions in Fab or scFv, associations between CH1 and CL in Fab, and associations between CH3 and CH3 in domain-substituted Fab.

[0015] Cancer: The term "cancer" refers to a disease characterized by the uncontrolled (often rapid) proliferation of abnormal cells. Cancer cells may spread locally or spread to other parts of the body via the bloodstream and lymphatic system. Examples of various cancers described in this specification include, but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, adrenal cancer, autonomic ganglion cancer, biliary tract cancer, bone cancer, endometrial cancer, eye cancer, fallopian tube cancer, genital cancer, colorectal 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, thymic cancer, thyroid cancer, upper respiratory tract and digestive cancer, urinary tract cancer, vaginal cancer, vulvar cancer, lymphoma, leukemia, lung cancer, and others.

[0016] Complementarity-Determining Regions or CDRs: As used in this specification, the term “complementarity-determining region” or “CDR” refers to the sequence of amino acids within the antibody variable region that confer 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). Examples of exemplary conventions that may be used to identify the boundaries of CDRs include, for example, the definitions of Kabat, Chothia, ABM, and IMGT. For example, see Kabat, 1991, "Sequences of Proteins of Immunological Interest," National Institutes of Health [Bethesda, Maryland, USA] (Kabat's numbering scheme); Al-Lazikani et al., 1997, J.Mol.Biol.273:927-948 (Chothia's numbering scheme); Martin et al., 1989, Proc.Natl.Acad.Sci.USA 86:9268-9272 (ABM's numbering scheme); and Lefranc et al., 2003, Dev.Comp.Immunol.27:55-77 (IMGT's numbering scheme). Public databases can also be used to identify CDR sequences within antibodies.

[0017] EC50: The term "EC50" refers to the maximum half-maximal effective concentration of a molecule (e.g., an IL10 agonist) that induces an intermediate response between baseline and maximum after a specified exposure time. Essentially, EC50 represents the concentration of an antibody or IL10 agonist at which 50% of its maximum 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 the assay, as described in Section 7.1.2.

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

[0019] Fab: In the context of the targeted portion of this disclosure, the term “Fab” refers to a pair of polypeptide chains, wherein the first polypeptide chain includes a heavy chain variable (VH) domain located on the antibody N-terminal side of the first constant domain (referred to as C1 in this specification), and the second polypeptide chain includes a light chain variable (VL) domain located on the antibody N-terminal side of the second constant domain (referred to as C2 in this specification), which may pair with the first constant domain. In the native antibody, the VH is located on the N-terminal side of the first constant domain (CH1) of the heavy chain, and the VL is located on the N-terminal side of the constant domain (CL) of the light chain. The Fab of this disclosure may be positioned according to the native orientation, or may include domain substitutions or swaps to facilitate correct VH and VL pairing. For example, replacing the CH1 domain and CL domain pair in the Fab with a CH3 domain pair can facilitate correct pairing of the modified Fab chain of the heterodimer molecule. Furthermore, it is possible to reverse CH1 and CL, linking CH1 to VL and CL to VH; this configuration is generally known as a crossmab.

[0020] Fc domains and Fc regions: The term "Fc domain" refers to a portion of a heavy chain that pairs with a corresponding portion of another heavy chain. The term "Fc region" refers to a 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 may be identical or different from one another. In native antibodies, the Fc domains are typically identical, but one or both Fc domains may be modified to favor heterodimerization, for example, via knob-in-hole interactions. Furthermore, the Fc domains may contain chimeric sequences derived from multiple immunoglobulin isotypes.

[0021] Host Cells: As used in this specification, the term “host cells” refers to cells into which the nucleic acids of this disclosure have been introduced. The terms “host cells” and “recombinant host cells” are used interchangeably in this specification. Such terms are understood to refer to specific target cells and their offspring or potential offspring. Such offspring may not be identical to the parent cells in practice because certain changes may occur in subsequent generations due to either mutation or environmental influences, but they are still included within the scope of this term as used in this specification. 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 mutain: A variant IL10 molecule possessing IL10 activity. This variant may be an IL10 fusion protein (e.g., IL10 fused to IL-2Rα) and / or a mutant IL10, e.g., having one or more amino acid substitutions compared to wild-type IL10. IL10 mutain may have altered functions (e.g., receptor binding, affinity, cytokine activity) and / or pharmacokinetics compared to wild-type IL10. In the context of IL10 agonists as described herein, the term "IL10 mutain" may refer to the untargetable component of the IL10 molecule (and associated linker moiety), and unless otherwise indicated in the context, the term "IL10 mutain" should be understood to encompass the IL10 molecule, with or without a targeting moiety and with or without a multimerizing moiety.

[0023] Major Histocompatibility Complex and MHC: These terms refer to naturally occurring MHC molecules, individual chains of MHC molecules (e.g., MHC class Iα (heavy chain), β2 microglobulin, MHC class IIα chain, and MHC class IIβ chain), individual subunits of such chains of MHC molecules (e.g., α1, α2, and / or α3 subunits of MHC class Iα chain, α1-α2 subunits of MHC class IIα chain, β1-β2 subunits of MHC class IIβ chain), as well as their portions (e.g., peptide bonding portions, e.g., peptide bonding grooves), variants, and various derivatives (including fusion proteins) in which such portions, variants, and derivatives retain the ability to present antigenic peptides for recognition by T cell receptors (TCRs), e.g., antigen-specific TCRs. MHC class I molecules contain peptide bonding grooves formed by the α1 and α2 domains of the heavy chain, which can accommodate peptides of approximately 8-10 amino acids. Despite the fact that any class of MHC binds to a core of approximately 9 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, but shorter or longer lengths are not uncommon. As a result, peptides can move within the MHC class II peptide binding groove, and the position of which 12-mer directly occupies the groove can change at any given time. This specification uses conventional identification of specific MHC variants. These terms include "human leukocyte antigen" or "HLA."

[0024] Operafully linked: As used in this specification, the term “operably linked” refers to a functional relationship between two or more regions of a polypeptide chain in which two or more regions are linked to produce a functional polypeptide, or a functional relationship in which two or more nucleic acid sequences are linked to produce, for example, an infraframe fusion of two polypeptide components, or a regulatory sequence is linked to a coding sequence.

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

[0026] Specifically (or selectively) bind: As used herein, the term "specifically (or selectively) bind" means that a targeting moiety, such as an antibody, or its antigen-binding domain ("ABD"), forms a complex with a target molecule that is relatively stable under physiological conditions. Specific binding can be characterized by a K , -9 , -4 , -8 , -7 , , -6 , -5 , -5 , -10 , -9 ,

[0027] , -8 , -7 , -6 of about 5×10 -2 M or less (e.g., less than 5×10 -2 M, less than 10 -2 M, less than 5×10 -3 M, less than 10 -3 M, less than 5×10 -4 M, less than 10 -4 M, less than 5×10 -5 M, less than 10 -5 M, less than 5×10 -6 M, less than 10 -6 M, less than 5×l0 -7 M, less than 10 -7 M, less than 5×10 -8 M, less than 10 -8 M, less than 5×10 -9 M, less than 10 -9 M, or less than 10 -10 M). Methods for determining the binding affinity of an antibody or antibody fragment, such as an IL10 agonist or a component targeting moiety, to a target molecule are known in the art and include, for example, equilibrium dialysis, surface plasmon resonance (e.g., Biacore™ assay), fluorescence-activated cell sorting (FACS) binding assays, and the like. However, an IL10 agonist of the disclosure that contains a targeting moiety or its ABD that specifically binds to a target molecule from one species may have cross-reactivity with target molecules from one or more other species.

[0027] Subjects: "Subjects" include humans and non-human animals. Non-human animals include all vertebrates, e.g., mammals and non-mammals, e.g., non-human primates, sheep, dogs, cattle, chickens, amphibians, and reptiles. Unless otherwise noted, the terms "patient" and "subjects" are used interchangeably in this specification.

[0028] Target Molecules: As used in this specification, the term “target molecule” means any biomolecule (e.g., protein, carbohydrate, lipid, or combination thereof) expressed on the cell surface or in the extracellular matrix that can be specifically bound by the targeting moiety in the IL10 agonists of this disclosure.

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

[0030] To treat, to treat, to treat: As used in this specification, “to treat,” “to treat,” and “to treat” mean a reduction or improvement in the progression, severity, and / or duration of a proliferative disorder, or improvement in one or more symptoms (preferably one or more identifiable symptoms) of a proliferative disorder, which occurs by the administration of one or more IL10 agonists of this disclosure. In certain embodiments, the terms “to treat,” “to treat,” and “to treat” mean at least one measurable physical parameter of a proliferative disorder, e.g., tumor growth, which is not necessarily identifiable by the patient. In other embodiments, the terms “to treat,” “to treat,” and “to treat” mean inhibiting the progression of a proliferative disorder, physically, e.g., by stabilizing an identifiable symptom; physiologically, e.g., by stabilizing a physical parameter; or both. In other embodiments, the terms “to treat,” “to treat,” and “to treat” mean a reduction or stabilization of tumor size or cancer cell number.

[0031] Tumor: The term “tumor” is used interchangeably with the term “cancer” in this specification, and both terms, for example, encompass solid and liquid tumors, such as diffuse or circulating tumors. As used in this specification, the terms “cancer” or “tumor” include not only malignant cancers and tumors but also precancerous ones.

[0032] Tumor-associated antigens: The term “tumor-associated antigens” or “TAA” refers to molecules (typically proteins, carbohydrates, lipids, or any combination thereof) that are expressed on the surface of cancer cells, either whole or as fragments (e.g., MHC / peptides), and are useful for the preferential targeting of pharmacological agents to cancer cells. In some embodiments, TAAs are markers expressed by both normal and cancer cells, e.g., lineage markers, e.g., CD19 on B cells. In some embodiments, TAAs are cell surface molecules that are overexpressed in cancer cells compared to normal cells, e.g., 1-fold overexpression, 2-fold overexpression, 3-fold overexpression, or more compared to normal cells. In some embodiments, TAAs are cell surface molecules that are improperly synthesized in cancer cells, e.g., molecules containing deletions, additions, or mutations compared to molecules expressed on normal cells. In some embodiments, TAAs are expressed only on the cell surface of cancer cells, either whole or as fragments (e.g., MHC / peptides), and are not synthesized or expressed on the surface of normal cells. Therefore, the term "TAA" encompasses antigens specific to cancer cells, which are sometimes known in the art as tumor-specific antigens ("TSAs").

[0033] Universal Light Chain: As used in this specification in the context of the targeted moiety, the term “universal light chain” refers to a light chain polypeptide that can pair with the heavy chain region of the targeted moiety and with other heavy chain regions. Universal light chains are also known as “common light chains.”

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

[0035] 6.2. IL10 Agonist This disclosure provides an IL10 agonist comprising an IL10 moiety, an optional polymerizing moiety, and an optional targeting moiety.

[0036] IL-10, also known as a human cytokine synthesis inhibitor (CSIF), is classified as a type (class)-2 cytokine, and is part of a set of cytokines that include IL-19, IL-20, IL-22, IL-24 (Mda-7), and IL-26, interferons (e.g., IFNγ, IFNβ, IFNγ), and interferon-like molecules (e.g., limitin, IL-28A, IL-28B).

[0037] IL-10 is a cytokine with multifaceted effects in immunomodulation and inflammation. It is produced by mast cells and counteracts the inflammatory effects these cells exert at the site of allergic reactions. IL-10 can inhibit the synthesis of pro-inflammatory cytokines such as IFNγ, IL2, IL3, TNFα, and GM-CSF, while also stimulating certain T cells and mast cells, promoting B cell maturation, proliferation, and antibody production. IL-10 can block NFκB activity and is involved in regulating the JAK-STAT signaling pathway. This also induces cytotoxic activity in CD8+ T cells and antibody production by B cells, suppressing macrophage activity and pro-tumor inflammation. The regulation of CD8+ T cells is dose-dependent, with higher doses inducing a stronger cytotoxic response.

[0038] Human IL-10 is homodimer. Each monomer is produced as an immature molecule of 178 amino acids, the first 18 of which are a signal peptide, which is cleaved upon secretion to produce mature IL-10 containing 160 amino acids. The IL-10 monomers within the dimer are non-covalently associated, but each subunit contains two intrachain disulfide bonds between residues 12 and 108, and between residues 62 and 114. The monomers non-covalently dimerize to form a V-shaped structure, with each half consisting of six α-helices, four of which originate from one subunit and two from the other. The IL-10 agonists of this disclosure may be in monomeric or polymeric form, e.g., dimers (homodimers or heterodimers) or higher-order complexes. For convenience, IL10 agonists, which are homodimers (or higher-order multimers of the same polypeptide), are described by their constituent monomers; however, recombinant expression of the constituent monomers in a suitable cell line can produce homodimer (or higher-order multimer) molecules.

[0039] Examples of IL10 portions suitable for use in the IL10 agonists of this disclosure are described in Section 6.2. IL10 agonists may be monomers or homodimers of two polypeptide chains, each containing an IL10 moiety, an Fc moiety (e.g., an Fc domain consisting of a CH2 domain and a CH3 domain), an optional hinge moiety, an optional linker moiety, and an optional targeting moiety.

[0040] Therefore, the IL10 agonist may be a monomer, or a homodimer or heterodimer of two polypeptide monomers. Each monomer in the monomer or dimer may contain the following, from the amino terminus to the carboxyl terminus: i) Selective targeting portion ii) Optional hinge domains iii) Fc domains, for example, Fc domains consisting of CH2 domains and CH3 domains; iv) Linker portion; and v) IL10 part.

[0041] Dimerization of IL10 agonists can occur via 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 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 in Section 6.4 for the Fc domain.

[0042] An exemplary Fc portion is described in Section 6.4, which includes an Fc domain that confers dimerization ability to IL10 agonists. Active IL10 is a dimeric molecule, and its pharmacokinetics in vivo are very poor, partly due to monomerization in the bloodstream. While not constrained by theory, the inclusion of an Fc domain and an optional hinge domain is thought to improve the serum stability and pharmacokinetic profile of IL10 agonists, particularly by stabilizing the dimeric structure of IL10.

[0043] For convenience, the IL10 moiety and the Fc domain moiety, as well as any optional linker between them, may be referred to as IL10 mutain in this specification, although the term “mutein” also includes molecules having a targeting moiety. 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] It was found that when the IL10 moiety is located at the N-terminus of the Fc domain, the resulting recombinant IL10 agonist is shortened at both the N-terminus and / or C-terminus. For example, in the case of the N-terminal IL10 moiety, the resulting recombinant IL10 agonist either lacks the C-terminal lysine or is shortened at both the N-terminus and C-terminus, possessing only residues 3-402 of the 403-amino acid full-length construct. This was not observed in IL10 agonists with the IL10 moiety at the C-terminus of the Fc domain. Therefore, in some embodiments, the IL10 moiety is located at the C-terminus of the Fc domain.

[0045] In various embodiments, an 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 to splice-type isoforms of fibronectin and tenascin-C; or the variable region of an anti-CD86 antibody); (c) a non-targeting antibody variable region; (d) an unbound 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 located on the C-terminal side of the IL10 moiety; or (j) any combination of two, three, four, five, six, seven, eight, or all of the above. An IL10 agonist may include, for example, one or more linker sequences connecting various components of the molecule, e.g., multiple different domains present in the fusion protein. Examples of exemplary linkers are described in Section 6.7.

[0046] In other embodiments, the IL10 agonist may include (a) a stabilizing moiety, e.g., 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), a polysialic acid, and / or a fatty acid; and (b) an antibody variable region (e.g., L19, F16, G11, or F8 directed towards splice-type isoforms of fibronectin and tenascin-C). (c) Variable region; variable region of anti-CD86 antibody; or variable region of anti-PD-1 antibody; (d) non-targeting antibody variable region; (e) antibody CDR; (f) antibody CH1 domain; (g) antibody CL domain; (h) another cytokine (e.g., IL4); (i) Fc domain at the C-terminus of the IL10 portion; (j) human serum albumin binder or binding domain; or (k) not including any combination of two, three, four, five, six, seven, eight, nine, or all of the above.

[0047] In certain embodiments, the IL10 agonists of this disclosure increase the ratio of CD8+ T cells to Treg cells in a tumor after administration to a subject (e.g., a cancer patient or a tumor-bearing mouse). In some embodiments, treatment with the IL10 agonists of this disclosure causes an increase in CD8 T cell density in the tumor. This increase may be, for example, at least a twofold, at least a threefold, or at least a fourfold increase in CD8 T cell density in the tumor.

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

[0049] In some embodiments, treatment with the IL-10 agonist of the Disclosure upregulates serum IL-12 levels compared to a preferred control that has not been treated with the IL-10 agonist or a pharmaceutical composition containing the IL-10 agonist, or compared to the subject's own serum IL-12 levels before treatment with the IL-10 agonist or pharmaceutical composition. Compared to a preferred control or the subject's own serum IL-12 levels before treatment, the IL-10 agonist of the Disclosure can upregulate serum IL-12 levels by, for example, at least 2 times, at least 5 times, at least 10 times, at least 15 times, at least 20 times, at least 25 times, or at least 30 times.

[0050] In some embodiments, treatment with the IL10 agonist of this disclosure upregulates serum IL12 levels compared to a preferred control that has not been treated with the IL10 agonist or a pharmaceutical composition containing the IL10 agonist, or compared to the subject's own serum IL4 level before treatment with the IL10 agonist or pharmaceutical composition. Compared to a preferred control or the subject's own serum IL4 level before treatment, the IL10 agonist of this disclosure can upregulate serum IL4 levels by, for example, at least 2 times, at least 5 times, at least 10 times, at least 15 times, at least 20 times, at least 25 times, or at least 30 times.

[0051] In the IL10 agonists of this disclosure, if the targeting portion is the antigen-binding domain ("ABD") of an antibody, one or both monomers of the dimeric IL10 agonist can carry the ABD, for example, in the form of Fab or scFv.

[0052] The Fc domain may be, for example, the Fc domain of IgG1 or IgG4, and may or may not have 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 mutain 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, and having an optional targeting moiety.

[0054] Further details of the components of the IL10 agonist in this disclosure are presented below. 6.3.IL10 part The IL10 portion of the IL10 antagonists in this disclosure includes a wild-type or variant IL10 domain.

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

[0056] In eukaryotic cells, human IL-10 is synthesized as a 178-amino acid precursor polypeptide, from which 18 amino acids are removed to produce mature secreted IL-10. Therefore, in some embodiments, the IL10 portion of the present disclosure includes mature human IL10 corresponding to positions S19-N178 of a 178-amino acid precursor sequence, for example, mature human IL10 having the following amino acid sequence or the amino acid sequence shown in Section 7.1.1.

[0057] [ka]

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

[0059] Therefore, the “IL10 moiety” includes, more preferably, proteins having a sequence substantially similar to that of mature wild-type human, mouse, pig, or rat IL10. In various embodiments, the IL10 moiety includes 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 with human, mouse, pig, or rat IL10, for example, a mature human IIL10 sequence comprising amino acid residues S19-N178 of a human IIL10 precursor.

[0060] In certain embodiments, the IL10 agonists of this disclosure have one or more amino acid modifications, e.g., substitutions, deletions, or insertions, in the IL10 moiety compared to wild-type IL10. The one or more amino acid modifications may be introduced to alter one or more properties of IL10, e.g., its stability. A specific modified human IL10 molecule with improved monomeric stability, in which a linker sequence functioning as a hinge is inserted between N116 and K117 (i.e., between the helical domains D and E of IL10), is described by 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 six amino acids and / or includes the sequence GGGSGG (SEQ ID NO: 2). In one embodiment, the IL10 moiety does not have an insertion of a linker sequence between N116 and K117 (i.e., between the helical domains D and E of IL10). In some embodiments, the IL10 portion does not have any amino acid modifications, such as substitutions, deletions, or insertions, compared to the wild-type IL10.

[0061] Human IL10 contains a potential N-linked glycosylation site at N116 on the surface of the molecule, which is conserved in non-human species. Zdanov et al., 1995, Structure 3(6):591-601. Accordingly, this disclosure includes IL10 molecules having or not having an N-linked glycan at N116 or an equivalent position in IL10 of other species. This potential N-linked glycosylation site is conserved in rat, mouse, and pig 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). This disclosure includes mouse or rat IL10 portions having and not having this additional N-linked glycan.

[0062] 6.4. FC Domains The IL10 agonists of this disclosure may include Fc regions derived from any preferred 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 IgG4Fc 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 IgG Fc.

[0064] One embodiment of the present disclosure relates to a dimer comprising two Fc-fusion polypeptides created by fusing an IL10 domain with the Fc region of an antibody. This dimer can be prepared, for example, by inserting a gene fusion encoding the fusion protein into a suitable expression vector, expressing the gene fusion in host cells transformed by a recombinant expression vector, and assembling the expressed fusion protein in the same way as an antibody molecule, thereby forming an interchain bond between the Fc portions and producing a dimer.

[0065] The Fc domain may be derived from any preferred class of antibody, including IgA (including subclasses IgA1 and IgA2), IgD, IgE, IgG (including subclasses IgG1, IgG2, IgG3, and IgG4), and IgM. In one embodiment, the Fc domain is derived from IgG. In one embodiment, the Fc domain is derived from IgG1, IgG2, IgG3, or IgG4. In one embodiment, the Fc domain is derived from IgG1. In one embodiment, the Fc domain is derived from IgG4.

[0066] The two Fc domains within the Fc region may be identical or different. In native antibodies, the Fc domains are typically identical, but for the purpose of producing multispecific binding molecules, such as the IL10 agonist of this disclosure, it may be advantageous for the Fc domains to be different to enable heterodimerization, as described in Section 6.4.1 below.

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

[0068] In the IL10 agonists of this disclosure, the Fc region and / or the Fc domains within it may be chimeric, combining sequences derived from multiple immunoglobulin isotypes. Thus, the Fc region and / or the Fc domains within it may contain one or more different classes of antibodies, e.g., one, two, or three different classes of heavy chain constant domains.

[0069] In one embodiment, the Fc region includes CH2 and CH3 domains derived from lgG1. In one embodiment, the Fc region includes CH2 and CH3 domains derived from lgG2.

[0070] In one embodiment, the Fc region includes CH2 and CH3 domains derived from lgG3. In one embodiment, the Fc region includes CH2 and CH3 domains derived from lgG4.

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

[0072] In a further embodiment, the chimeric Fc domain may include 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, the chimeric Fc domain may also include a chimeric hinge region. For example, the chimeric hinge may include an “upper hinge” sequence derived from the hinge region of human IgG1, human IgG2, or human IgG4 combined with a “lower hinge” sequence derived from the hinge region of human IgG1, human IgG2, or human IgG4. A specific example of a chimeric Fc domain that can be included in any of the IL10 mutains shown in this specification includes, 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 shown in this specification is, from N-terminus to C-terminus, comprising: [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 and their variants having these common structural configurations may have alterations in Fc receptor binding, which in turn affects Fc effector function.

[0073] It will be understood that the heavy chain constant domains used to construct the Fc region for the IL10 agonist of this disclosure may include variants of naturally occurring constant domains. Such variants may include one or more amino acid changes (variations) compared to the wild-type constant domain. In one example, the Fc region of this disclosure includes at least one constant domain that is sequence-different 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 to the wild-type constant domain. In another example, the variant constant domain has at least 70% identity or similarity. In yet another example, the variant constant domain has at least 80% identity or similarity. In yet another example, the variant constant domain has at least 90% identity or similarity. In yet another example, the variant constant domain has at least 95% identity or similarity.

[0074] IgM and IgA exist naturally in humans as covalent polymers of the common H2L2 antibody unit. IgM exists as a pentamer if it incorporates a J chain, and as a hexamer if it lacks a J chain. IgA exists in monomeric and dimeric forms. The heavy chains of IgM and IgA have an 18-amino acid extension to a 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 a crucial role in polymerization. The tailpiece also contains glycosylation sites. In certain embodiments, the IL10 agonist of this disclosure does not contain a tailpiece.

[0075] The Fc domain incorporated into the IL10 agonist of this disclosure may include one or more modifications that alter the functional properties of the protein, such as binding to Fc receptors such as FcRn or leukocyte receptors, binding to complement, modification of the disulfide bond structure, or alteration of the glycosylation pattern. Exemplary Fc modifications that alter effector function are described in Section 6.4.1.

[0076] Furthermore, the Fc domains may be modified to include modifications that improve the manufacturability of asymmetric IL10 agonists, for example, by enabling heterodimerization, which is the preferential pairing of non-identical Fc domains with an identical Fc domain. Heterodimerization enables the production of IL10 agonists in which different polypeptide components are connected to each other by Fc regions containing Fc domains with 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 self-associating ability. 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. IL10 agonists can further be dimerized via the IL10 moiety. One example of a soluble monomeric Fc domain involves 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 include additional substitutions that degrade the effector function, as described in Section 6.4.1.

[0078] As used in this specification, 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 containing a hinge domain, positions 233–236 within the hinge domain may be G, G, G and unoccupied; G, G, unoccupied and unoccupied; G, unoccupied, unoccupied and unoccupied; or all unoccupied, with the positions numbered by EU numbering. Optionally, the heavy chain constant region includes a hinge domain, a CH2 domain and a CH3 domain from N-terminus to C-terminus. Optionally, the heavy chain constant region includes a CH1 domain, a hinge domain, a CH2 domain and a CH3 domain from N-terminus to C-terminus. Optionally, the CH1 region, if present, the remainder of the hinge region, 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, the CH2 region and the CH3 region are human IgG1. Optionally, the CH1 region (if present), the remainder of the hinge region (if present), the CH2 region, and the CH3 region are human IgG2. Optionally, the CH1 region (if present), the remainder of the hinge region (if present), the CH2 region, and the CH3 region are human IgG4.

[0079] Optionally, the constant region may have a modified CH3 domain that reduces binding to protein A. Examples of these and other Fc regions that may be included in any of the IL10 variants of this disclosure are described in International Publication WO2016 / 161010. Exemplary hinge arrangements are shown in Section 6.7.1 and its subsections.

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

[0081] 6.4.1. Fc domain with changes in effector function In some embodiments, the Fc domain includes one or more amino acid substitutions that reduce binding to and / or effector function of the Fc receptor.

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

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

[0084] Typically, one or more identical amino acid substitutions are present in each of the two Fc domains of the Fc region. Thus, in a particular embodiment, each Fc domain of the Fc region contains amino acid substitutions L234A, L235A, and P329G (Kabat EU index numbering), namely, 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 IgG1Fc domain, and more specifically, human IgG1Fc. In another embodiment, the Fc domain is an IgG4Fc domain with reduced binding affinity to the Fc receptor. An exemplary IgG4Fc domain with reduced binding affinity to the Fc receptor may include an amino acid sequence selected from Table 1 below. In some embodiments, the Fc domain includes 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 particular embodiment, the IgG4 with reduced effector function comprises the bolded portion of the amino acid sequence of SEQ ID NO: 31 (amino acids 99 to 326 of SEQ ID NO: 6, or SEQ ID NO: 31; the bolded sequence may also be referred to as IgG4 in this specification) of International Publication No. WO2014 / 121087 reproduced above, 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 another embodiment, 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 with SEQ ID NO: 31 (SEQ ID NO: 6) of International Publication No. WO2014 / 121087.

[0091] For the heterodimer IL10 agonists of this disclosure, in each case of International Publication No. WO2014 / 121087, a combination of the variant IgG4 Fc sequences shown above may be incorporated, for example, an Fc region containing the combination of SEQ ID NO: 30 (or its bolded portion) and SEQ ID NO: 37 (or its bolded portion), or an Fc region containing the combination of SEQ ID NO: 31 (or its bolded portion) and SEQ ID NO: 38 (or its bolded portion).

[0092] In a particular embodiment, the Fc domain includes an amino acid sequence designated as hIgG4s (SEQ ID NO: 31) 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.

[0093] In another specific embodiment, the Fc domain comprises an 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 an Fc sequence based on variant IgG1, including the D265A, N297A mutation (EU numbering) for reducing effector function.

[0094] 6.4.1.1. Fc Heterodimerized Variants Unlike native immunoglobulins, certain IL10 agonists require dimerization between two Fc domains operably linked to non-identical N-terminal regions, for example, one Fc domain being attached to Fab and the other Fc domain to the IL10 portion. Improper heterodimerization of the two Fc domains to form the Fc domain can hinder the yield of the desired heterodimer molecule and poses a challenge for purification. Various approaches available in the art can be used, for example, to enhance the dimerization of Fc domains that may be present in the IL10 agonists of this disclosure, as disclosed in European Patent Publication No. 1870459A1; U.S. Patent No. 5,582,996; U.S. Patent No. 5,731,168; U.S. Patent No. 5,910,573; U.S. Patent No. 5,932,448; U.S. Patent No. 6,833,441; U.S. Patent No. 7,183,076; U.S. Patent Publication No. 2006204493A1; and International Application No. WO2009 / 089004A1.

[0095] This disclosure provides IL10 agonists comprising Fc heterodimers, i.e., Fc regions containing heterogeneous and non-identical Fc domains. Typically, each Fc domain in the Fc heterodimer contains a CH3 domain of an antibody. The CH3 domain is derived from the constant region of an antibody of any isotype, class, or subclass, preferably an IgG (IgG1, IgG2, IgG3, and IgG4) class, as described in the previous section.

[0096] Heterodimerization of two different heavy chains at the CH3 domain yields the desired IL10 agonist, while homodimerization of the same heavy chain reduces the yield of the desired IL10 agonist. Therefore, in a preferred embodiment, the polypeptide that associates to form the IL10 agonist of this disclosure is considered to contain a CH3 domain with modifications that are favorable for heterodimerization compared to the unmodified Fc domain.

[0097] In a particular embodiment, the modification that promotes the formation of an Fc heterodimer is a so-called "knob-into-hole" or "knob-in-hole" modification, which includes a "knob" modification in one of the Fc domains and a "hole" modification in the other Fc domain. Knob-in-hole techniques are described, for example, in U.S. Patent No. 5,731,168 and U.S. Patent No. 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 cavity corresponding to the interface of a second polypeptide to the interface of a protrusion ("knob") so that the protrusion can be positioned in a cavity ("hole") to promote heterodimer formation and inhibit homodimer formation. The protrusion is constructed by replacing a small amino acid side chain (e.g., tyrosine or tryptophan) originating from the interface of the first polypeptide with a larger side chain. By replacing the larger amino acid side chain with a smaller amino acid side chain (e.g., alanine or threonine), a compensatory cavity of the same or similar size as the protrusion is created at the interface of the second polypeptide.

[0098] Therefore, in some embodiments, an amino acid residue in the CH3 domain of the first subunit of the Fc domain is replaced with an amino acid residue having a larger side chain volume, thereby creating a protrusion within the CH3 domain of the first subunit that can be positioned in a cavity within the CH3 domain of the second subunit, and an amino acid residue in the CH3 domain of the second subunit of the Fc domain is replaced with an amino acid residue having a smaller side chain volume, thereby creating a cavity within the CH3 domain of the second subunit that can accommodate the protrusion within 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 modifying the nucleic acid encoding the polypeptide, for example, by site-directed mutagenesis or by peptide synthesis. An exemplary substitution is Y470T.

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

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

[0101] Instead of using, 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, a polypeptide comprises a modified Fc domain that inhibits its binding to protein A, thereby enabling a purification method that produces a heterodimeric protein. See, for example, U.S. Patent No. 8,586,713. Thus, an IL10 agonist comprises a first CH3 domain and a second Ig CH3 domain, where the first and second Ig CH3 domains differ from each other by at least one amino acid, and this difference of at least one amino acid reduces the binding affinity of the IL10 agonist to protein A compared to a corresponding IL10 agonist with no amino acid difference. In one embodiment, the first CH3 domain binds to protein A, and the second CH3 domain includes a mutation / modification such as the H95R modification (by IMGT exon numbering; H435R by EU numbering) that reduces or inhibits protein A binding. The second CH3 may further include the Y96F variant (according to IMGT; Y436F according to the EU). Variations in this class are referred to in this specification as “star” variants.

[0102] 6.5. Targeting part By incorporating the targeting portion of the IL10 agonist of this disclosure, it becomes possible to deliver high concentrations of IL10 to the tumor microenvironment or to tumor-reactive lymphocytes, particularly CD8+ T lymphocytes, thereby exerting a local effect on them.

[0103] A preferred form of the targeting moiety is described in Section 6.5.2. The targeting moiety is preferably an antigen-binding moiety, for example, an antibody or an antigen-binding moiety of an antibody, for example, the scFv described in Section 6.5.2.1 or the Fab described in Section 6.5.2.2.

[0104] The antibody and antigen-binding moieties can generally bind to specific antigenic determinants, allowing the IL10 agonist to be directed to a target site, such as tumor cells or tumor stroma carrying that antigenic determinant. Exemplary target molecules recognized by the targeting moieties of this disclosure are described in Section 6.5.1.

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

[0106] Examples of target molecules include fibroblast-activating protein (FAP), the A1 domain of tenascin-C (TNC A1), and the A2 domain of tenascin-C (TNC A2) Fibronectin extradomain B (EDB), melanoma-associated chondroitin sulfate proteoglycan (MCSP), MART-1 / Melan A, gp100, dipeptidyl peptidase IV (DPPIV), adenosine deaminase-binding protein (ADAbp), cyclophyllin 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 / CD3ζ 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-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), GAGE ​​family tumor antigens (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 adenomatous polyposis protein (APC), fodrin, connexin 37, Ig-idiotype, p15, gp75, GM2 and GD2 gangliosides, viral products, e.g., human papillomavirus protein, tumor antigens of the Smad family, Imp-1, P1A, nuclear antigen encoded by EBV (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 (TCR-related heteromultimer), CD22 (B cell receptor), CD23 (low affinity IgE receptor), CD30 (cytokine receptor), CD33 (bone marrow 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, prostain, PSMA, survival and telomerase, prostate cancer tumor antigen-1 (PCTA-1), ELF2M, neutrophil elastase, ephrin B2, insulin growth factor (IGF-1)-I, IGF-II, IGFI receptor, 5T4, ROR1, Nkp30, NKG2D, tumor stromal antigen, extra domain A (EDA) and extra domain B (EDB) of fibronectin, and the A1 domain (TnC A1) of tenascin-C.

[0107] In some embodiments, the targeting moiety binds to an MHC peptide complex or a peptide within the MHC peptide complex, such as a tumor neoantigen. Some tumor neoantigens are viral antigens.

[0108] Non-specific examples of viral antigens include EBV antigen (e.g., Epstein-Barr virus LMP-1), hepatitis C virus antigen (e.g., hepatitis C virus E2 glycoprotein), HIV antigen (e.g., HIV gp160, HIV gp120); CMV antigen; HPV-specific antigen; or influenza virus antigen (e.g., influenza virus hemagglutinin). Specific embodiments of tumor neoantigens that can be conjugated by the targeting moieties of this disclosure include 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 These include 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), and WT1 (126-134).

[0109] Non-exclusive examples of ECM antigens include syndecan, heparanase, integrin, osteopontin, link, cadherin, laminin, laminin EGF type, lectin, fibronectin, notch, tenascin, collagen, and matrixin.

[0110] Other target molecules include cell surface molecules of tumor or viral lymphocytes, such as T cell costimulatory proteins like 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 molecules are checkpoint inhibitors, such as CTLA-4, PD1, PDL1, PDL2, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK1, and CHK2.

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

[0113] In various embodiments, the IL10 agonist does not contain a PD1-binding targeting domain. 6.5.2. Form of the target site In certain embodiments, the targeting moiety may 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 specifically an IgG1 or IgG4 immunoglobulin molecule. The antibody fragment may contain VH (or V H ) Fragment, VL (or V L This includes, but is not limited to, ) fragments, Fab fragments, F(ab')2 fragments, scFv fragments, Fv fragments, minibodies, diabodies, triabodies, and tetrabodies. In the IL10 agonists of this disclosure, it may be advantageous for the antibody fragments to be incorporated into the N-terminal side of the Fc domain.

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

[0115] Unless otherwise specified, when used in this specification, scFv may have the VL and VH variable regions in either order, for example, relative to the N-terminus and C-terminus of the polypeptide, and scFv may include VL-linker-VH or VH-linker-VL.

[0116] scFv may include VH and VL sequences derived from any suitable species, such as mouse, human, or humanized VH and VL sequences. To create the nucleic acid encoding scFv, the DNA fragments encoding VH and VL are operably ligated to another fragment encoding a linker, for example, one of the linkers described in Section 6.7 (typically a sequence containing amino acids glycine and serine, e.g., a repeat of amino acid sequence (Gly4~Ser)3 (SEQ ID NO: 53), such that the VH and VL sequences are expressed as a continuous single-chain protein and the VL and VH regions are joined by a mobile linker) (see, e.g., Bird et al., 1988, Science, 242:423-426; Houston 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 this disclosure, the Fab domain is typically recombinantly expressed as part of the IL10 agonist.

[0118] The Fab domain may contain constant domains and variable region sequences derived from any preferred species, and therefore may be mouse, chimeric, human, or humanized.

[0119] The Fab domain typically contains a CH1 domain bound to the VH domain, which pairs with a CL domain bound to the VL domain. In wild-type immunoglobulin, 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 this 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 the correct association of Fab domains belonging to the same Fab and to minimize abnormal pairing of Fab domains belonging to different Fab domains. For example, the Fab heterodimerization strategies shown in Table 2 below can be used.

[0121] [Table 2]

[0122] Therefore, in a particular embodiment, the correct association between two polypeptides of Fab is facilitated by, for example, exchanging the VL domain and VH domain of Fab with each other, or by exchanging the CH1 domain and CL domain with each other, as described in International Publication No. WO2009 / 080251.

[0123] Furthermore, correct Fab pairing can also be promoted by introducing one or more amino acid modifications into the CH1 domain of Fab, one or more amino acid modifications into the CL domain, and / or introducing one or more amino acid modifications into the VH domain and one or more amino acid modifications into the VL domain. The amino acids to be modified are typically parts of the VH:VL and CH1:CL interfaces so that components of Fab preferentially pair with each other more than components of other Fab.

[0124] In one embodiment, one or more amino acid modifications are limited to conserved framework residues of variable (VH, VL) and constant (CH1, CL) domains, as indicated by Kabat numbering of the 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, modifications introduced to the VH and CH1 domains 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 contact, electrostatic / charge interactions, or various combinations of interactions. Complementarity between protein surfaces has been widely described in the literature in terms of lock-and-key fit, knob-into-hole, protrusion and cavity, donor and acceptor, etc., all of which refer to the nature of structural and chemical compatibility between two interacting surfaces.

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

[0127] In some embodiments, the Fab domain includes a 192E substitution in the CH1 domain and 114A and 137K substitutions in the CL domain, which introduces 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 includes substitutions of 143Q and 188V in the CH1 domain and substitutions of 113T and 176V in the CL domain, which serve to exchange contact between hydrophobic and polar regions 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 may include modifications in part or all of the VH, CH1, VL, and CL domains to introduce an orthogonal Fab interface that facilitates the correct assembly of the Fab domain (Lewis et al., 2014, Nature Biotechnology, 32:191-198). In one embodiment, the 39K, 62E modification is introduced into the VH domain, the H172A, F174G modification is introduced into the CH1 domain, the 1R, 38D, (36F) modification is introduced into the VL domain, and the L135Y, S176W modification is introduced into the CL domain. In another embodiment, the 39Y modification is introduced into the VH domain and the 38R modification is introduced into the VL domain.

[0130] Furthermore, the Fab domain can be modified to improve the efficiency of Fab component pair formation by replacing the native CH1:CL disulfide bond with an engineered disulfide bond. For example, an engineered disulfide bond 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] Furthermore, the Fab domain can 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 the constant domain of the T cell receptor and the CL domain with the b domain of the T cell receptor, pairing these domain substitutions with additional charge-charge interactions between the VL and VH domains by introducing the 38D modification into the VL domain and the 39K modification into the VH domain.

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

[0133] 6.6. Stabilization part The IL-10 agonists of this disclosure may include a stabilizing moiety that can extend the serum half-life of the molecule in vivo. The serum half-life is often divided into α-phase and 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 the corresponding IL-10 agonist that does not contain the stabilizing moiety. For the purposes of this disclosure, serum half-life refers to the half-life in humans or other mammals (e.g., mice or non-human primates). Furthermore, it is recognized that including an Fc domain in an IL10 agonist extends the half-life of the IL10 portion; in the context of this disclosure, the term “stabilized portion” refers to the portion other than the Fc domain / Fc region.

[0134] The serum half-life of wild-type IL-10 is less than 30 minutes. The IL-10 agonists of this disclosure preferably have 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 IL-10 agonists of this disclosure have 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] The stabilizing portion includes a polyoxyalkylene moiety (e.g., polyethylene glycol), a sugar (e.g., sialic acid), and a highly tolerable protein moiety (e.g., Fc and its fragments and variants, transferrin, or serum albumin).

[0136] Other stabilizing components that may be used with the IL10 agonist of this disclosure include those described by Kontermann et al., 2011, Current Opinion in Biotechnology, 22:868-76. Such stabilizing components include, but are not limited to, human serum albumin fusions, human serum albumin conjugates, human serum albumin binders (e.g., adnectin PKE, AlbudAb, ABD), XTEN fusions, PAS fusions (i.e., recombinant PEG mimetic 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] Therefore, in some embodiments, the present disclosure provides an IL10 agonist comprising a stabilizing moiety that is a high molecular weight sugar. Furthermore, serum albumin can also be involved in half-life extension via a module having the ability to interact with albumin non-covalently. Therefore, the IL10 agonists of this disclosure may include albumin-binding proteins as stabilizing portions. Albumin-binding proteins can be conjugated to or genetically fused to one or more other components of the IL10 agonists of this disclosure. Proteins with albumin-binding activity are known to originate 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, e.g., in U.S. Patent Application Publications 2007 / 0178082 and 2007 / 0269422. Fusion of albumin-binding domains with proteins significantly extends the half-life (see Kontermann et al., 2011, Current Opinion in Biotechnology, 22:868-76).

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

[0139] The stabilization portion may be connected to one or more other components of the IL10 agonist of this disclosure via a linker, for example, as described in Section 6.7 below.

[0140] In certain embodiments, the IL10 agonist is not conjugated with 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 a particular embodiment, the IL10 agonist lacks a stabilizing moiety.

[0141] 6.6.1. Polyethylene glycol In some embodiments, the IL10 agonist includes polyethylene glycol (PEG) or another hydrophilic polymer as a stabilizing portion, 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, polyamine acid (either homopolymer or random copolymer), dextran or poly(n-vinylpyrrolidone) polyethylene glycol, propylene glycol (propropylene) homopolymer, propropylene oxide / ethylene oxide copolymer, polyoxyethylated polyol (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. The polymer may have 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 modification of the PEG ends, with the formula: XO(CH2CH2O) n It can be represented by -1CH2CH2OH, where n is between 20 and 2300, and X is H or terminal modification, e.g., C 1-4 It is alkyl. PEG may further contain chemical groups necessary for the bonding reaction, which may result from the chemical synthesis of the molecule; or they may function as spacers for the optimal distance between parts of the molecule. Furthermore, such PEG may consist of one or more PEG side chains linked together. PEG having multiple PEG chains is called multi-armed PEG or branched PEG. Branched PEG is 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 linked to different positions on an IL10 agonist, and such linkages are achieved by reaction with amines, thiols, or other suitable reactive groups. The amine moiety may be, for example, a primary amine found at the N-terminus of the IL10 agonist (or its components), or an amine group present in an amino acid, such as lysine or arginine.

[0144] PEGylation can be achieved by site-directed PEGylation, which involves introducing a suitable reactive group into a protein to create a site where PEGylation preferentially occurs. In some embodiments, IL10 agonists are modified to introduce a cysteine ​​residue at a desired position, enabling site-directed PEGylation at cysteine. Mutations can be introduced into the coding sequence of the IL10 agonists of this disclosure to produce 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. The residues to be mutated to cysteine ​​are preferably surface-exposed residues. Algorithms for predicting the surface accessibility of residues based on primary sequence or three-dimensional structure are well known in the art. The three-dimensional structure of IL10 is described, for example, by Wang et al., 2005, Science, 310(5751):1159-63, and can be used to identify surface-exposed residues that can be mutated to cysteine. Mutations can be selected so as not to interfere with the interaction between IL10 and one or more receptors. PEGylation of cysteine ​​residues can be performed using, for example, PEG-maleimide, PEG-vinyl sulfone, PEG-iodoacetamide, or PEG-orthopyridyl disulfide.

[0145] PEGs are typically activated by activating groups suitable for coupling to a desired site on a 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," J.M. Harris, Plenus Press, New York (1992), and Zalipsky, 1995, Advanced Drug Reviews, 16:157-182.

[0146] The PEG portion exhibits significant molecular weight variations and can be branched or linear. Typically, the weight-average molecular weight of PEG ranges from approximately 100 daltons to approximately 150,000 daltons. Exemplary weight-average molecular weights of PEG include approximately 20,000 daltons, 40,000 daltons, 60,000 daltons, and 80,000 daltons. In certain embodiments, the molecular weight of PEG is 40,000 daltons. Branched PEG with any of the above total molecular weights can also be used. In some embodiments, PEG has two branches. In other embodiments, PEG has four branches. In yet another embodiment, PEG is bis-PEG (NOF Corporation, DE-200MA) conjugated with two IL10-containing polypeptide chains.

[0147] PEGylated IL10 agonists can be purified using conventional separation and purification methods known in the art, such as size exclusion (e.g., gel filtration) and ion exchange chromatography. The products can also be separated using SDS-PAGE. The 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 a broader fraction before and after the elution peak to increase the proportion of mono-PEG in the composition. A mono-PEG conjugate of approximately 90% offers a good balance of yield and activity.

[0148] In some embodiments, it is preferable that the PEGylated IL10 agonist retains at least about 25%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 100% of the biological activity associated with the unmodified IL10 agonist. In some embodiments, the biological activity is K D , k on , or k off This refers to its ability to bind to IL10R1, as evaluated by [a specific method / tool].

[0149] 6.7. Linker In certain embodiments, the disclosure provides an IL10 agonist in which two or more components of the IL10 agonist are linked together by a peptide linker. For example, but not limited to, the linker can be used to link (a) the IL10 portion and the Fc domain; (b) the IL10 portion and the targeting portion; (c) the Fc domain and the targeting portion (e.g., the Fab domain or scFv); or (d) multiple different domains within the targeting portion (e.g., the VH domain and VL domain within scFv).

[0150] The peptide linker may range from 2 amino acids to 60 amino acids or more, and in certain embodiments, the peptide linker may be in the range of lengths 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.

[0151] Charged (e.g., charged hydrophilic linkers) and / or movable linkers are particularly preferred. Examples of movable linkers that may be used with the IL10 agonists of this 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 movable linkers are glycine and serine repeats, e.g., G n S (Sequence ID 9) or SG n A monomer or polymer of (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 It is a monomer or polymer of the repeat of (Sequence ID 11).

[0152] 6.7.1. Hinge Arrangement In other embodiments, the IL10 agonists of this disclosure include a linker, which is a hinge region. In particular, if the IL10 agonist contains an immunoglobulin-based targeting moiety, the hinge can be used to connect the targeting moiety, e.g., the Fab domain, to a multimerizing domain, e.g., the 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 can be native or a modified hinge region. The hinge region is typically found at the N-terminus of an Fc region. The native hinge region is the hinge region that is typically found between the Fab domain and the Fc domain in naturally occurring antibodies. A modified hinge region is any hinge that differs in length and / or composition from the native hinge region. Such hinges may include those derived from other species, e.g., humans, mice, rats, rabbits, sharks, pigs, hamsters, camels, llamas, and goats. Other modified hinge regions may include complete hinge regions derived from antibodies of a different class or subclass than those in the heavy chain Fc region. Alternatively, a modified hinge region may include a portion of the native hinge, or repeat units in which each unit in the repeat is derived from the native hinge region. As a further alternative, the native hinge region can be modified by converting one or more cysteine ​​or other residues to neutral residues such as serine or alanine, or by converting suitably positioned residues to cysteine ​​residues. Such means can increase or decrease the number of cysteine ​​residues within the hinge region. Other modified hinge regions may be fully synthetic and may be designed to have desired properties, such as length, cysteine ​​composition, and mobility.

[0154] Several modified hinge regions have already been described, for example, in U.S. Patent No. 5,677,425, International Publication No. WO99 / 15549, International Publication No. WO2005 / 003170, International Publication No. WO2005 / 003169, International Publication No. WO2005 / 003170, International Publication No. WO98 / 25971 and International Publication No. WO2005 / 003171, which are incorporated herein by reference.

[0155] In various embodiments, positions 233-236 within the hinge domain may be G, G, G and unoccupied; G, G, unoccupied and unoccupied; G, unoccupied, unoccupied and unoccupied; or all unoccupied, and the positions are numbered by EU numbering.

[0156] In some embodiments, the IL10 muteins of this disclosure include a modified hinge domain that reduces binding affinity to the Fcγ receptor compared to the 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 the dimer IL10 agonist of the present disclosure has an intact hinge region at its N-terminus. In one embodiment, the Fc region and hinge region of one or both chains of the dimer IL10 agonist of this disclosure are derived from lgG4, and the hinge region contains the modified sequence CPPC. The core hinge region of human lgG4 contains the sequence CPSC, whereas lgG1 contains the sequence CPPC. The serine residues present in the lgG4 sequence lead to increased mobility of this region, and therefore a certain percentage of the molecules form disulfide bonds (intrachain disulfides) within the same protein chain rather than crosslinking with other heavy chains within the IgG molecule to form interchain disulfides (Angel et al., 1993, Mol Immunol, 30(1):105-108). By changing the serine residues to proline to obtain the same core sequence as lgG1, complete formation of interchain disulfides within the lgG4 hinge region becomes possible, and heterogeneity of the purified product is reduced. This modified isotype is called lgG4P.

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

[0159] In certain embodiments, the chimeric hinge region includes the amino acid sequence EPKSCDKTHTCPPCPPAPPVA (SEQ ID NO: 12) (SEQ ID NO: 8 in International Publication WO2014 / 121087, which is incorporated in its entirety in this specification) or ESKYGPPCPCPCPPAPPVA (SEQ ID NO: 13) (SEQ ID NO: 9 in International Publication WO2014 / 121087). Such a chimeric hinge sequence can be suitably linked to the IgG4 CH2 region (for example, by incorporating a human or mouse Fc domain into the IgG4 Fc domain, and further modifying it in the CH2 and / or CH3 domains, as described in Section 6.4.1, to reduce effector function).

[0160] 6.7.1.2. Hinge arrangement with reduced effector functionality In further embodiments, the hinge region can be modified to reduce the effector function, for example, as described in International Publication WO2016161010A2 (which is incorporated in its entirety in this specification). In various embodiments, the modified hinge region positions 233–236 are G, G, G, and unoccupied; G, G, unoccupied and unoccupied; G, unoccupied, unoccupied and unoccupied; or all unoccupied, with the positions numbered by EU numbering (as shown in Figure 1 of International Publication WO2016161010A2). These segments can be represented as GGG-, GG--, G---, or ----, where "-" indicates an unoccupied position.

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

[0162] Hinge modifications within positions 233-236 can be combined with position 228, which is 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 for 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] The exemplary hinge region comprises residues 226–236, which are sometimes referred to as the intermediate (or core) hinge and the lower hinge, and is occupied by modified hinge sequences designated as GGG-(233–236), GG--(233–236), G---(233–236), and G-less(233–236). Optionally, the amino acid sequence of the hinge domain may include CPPPAPGGG-GPSVF (Sequence ID 14) (Sequence ID 1 in International Publication No. WO2016161010A2), CPPPAAPGG--GPSVF (Sequence ID 15) (Sequence ID 2 in International Publication No. WO2016161010A2), CPPPAPG---GPSVF (Sequence ID 16) (Sequence ID 3 in International Publication No. WO2016161010A2), or CPPPAAP----GPSVF (Sequence ID 17) (Sequence ID 4 in International Publication No. 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., an upper hinge) adjacent to the specified region. Such additional constant region segments present are typically of the same isotype, preferably a human isotype, but may be hybrids of multiple different isotypes. The isotype of such additional human constant region segments is preferably human IgG4, but may 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 detailed embodiments, the modified hinge sequence can be linked to the IgG4 CH2 region (for example, by incorporating a human or mouse Fc domain into the IgG4 Fc domain; for example, this can be further modified in the CH2 and / or CH3 domains to reduce effector function, as described in Section 6.4.1).

[0166] 6.8. Nucleic acids and host cells In another embodiment, the Disclosure provides nucleic acids encoding the IL10 agonist of the Disclosure. In some embodiments, the IL10 agonist is encoded by a single nucleic acid. In other embodiments, for example, in the case of a molecule containing a targeting moiety composed of a heterodimer molecule or multiple polypeptide chains, the IL10 agonist may be encoded by multiple (e.g., two, three, four, or more) nucleic acids.

[0167] A single nucleic acid can encode an IL10 agonist containing a single polypeptide chain, an IL10 agonist containing two or more polypeptide chains, or a portion of an IL10 agonist containing more than two polypeptide chains (for example, a single nucleic acid can encode two polypeptide chains of an IL10 agonist containing three, four or more polypeptide chains, or three polypeptide chains of an IL10 agonist containing four or more polypeptide chains). To control expression separately, 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 intra-sequence ribosome entry site (IRES) sequence, allowing translation into separate polypeptides.

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

[0169] The nucleic acids in this disclosure may be DNA or RNA (e.g., mRNA). In another aspect, the Disclosure provides host cells and vectors containing the nucleic acids of the Disclosure. These nucleic acids may be present in a single vector or in separate vectors present in the same host cell or in separate host cells, as described in further detail below.

[0170] 6.8.1. Vectors This disclosure provides a vector comprising one or two nucleotide sequences encoding an IL10 agonist or IL10 agonist component as described in this specification, for example, a polypeptide chain of a dimeric IL10 agonist. The vector may include, but is not limited to, a virus, plasmid, cosmid, lambda phage, or yeast artificial chromosome (YAC).

[0171] A wide variety of vector systems can be employed. For example, one class of vectors utilizes DNA elements derived from animal viruses, such as bovine papillomavirus, polyomavirus, 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 Semlik Forest virus, Eastern equine encephalitis virus, or flavivirus.

[0172] Furthermore, by introducing one or more markers that enable the selection of transfected host cells, cells in which the DNA is stably integrated into the chromosome can be selected. The markers can confer, for example, prototropy to a trophic-dependent host, biocide resistance (e.g., antibiotics), or resistance to heavy metals such as copper. The selection marker gene may be directly ligated to the DNA sequence to be expressed, or it may be introduced into the same cell by co-transformation. Additional elements may be required for optimal mRNA synthesis. These elements may include splice signals, as well as transcription promoters, enhancers, and termination signals.

[0173] Once an expression vector or DNA sequence containing a construct is prepared for expression, the expression vector can be transfected or introduced into a suitable host cell. To achieve this, various methods can be employed, such as protoplast fusion, calcium phosphate precipitation, electroporation, retroviral introduction, viral transfection, gene guns, lipid-based transfection, or other conventional methods. 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 according to the specific expression vector and mammalian host cell employed, based on this description.

[0174] 6.8.2.Cells This disclosure also provides host cells containing the nucleic acids of this disclosure. In one embodiment, a host cell is genetically engineered to contain one or more nucleic acids as described in this specification.

[0175] In one embodiment, host cells are genetically engineered using an expression cassette. The term “expression cassette” refers to a nucleotide sequence that can influence the expression of a gene in a host that conforms to such a sequence. Such a cassette may include a promoter, an open reading frame with or without introns, and a termination signal. Additional factors necessary or helpful in influencing expression, such as an inducible promoter, may also be used.

[0176] This disclosure also provides host cells containing the vector described in this specification. In addition, IL10 agonists, which are products of recombinant cell expression in mammalian cells, are also provided.

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

[0178] 6.8.3. Production Method This disclosure also provides a method for producing the IL10 agonist of this disclosure. In some embodiments, the IL10 agonist is produced by culturing host cells as described in Section 6.8.2 and recovering the IL10 expressed by the host cells. In certain embodiments, this 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 may be any affinity column configured to capture an IL10 agonist. For example, the affinity column may include 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 comprising five regions that bind to the Fc region of IgG. These regions are often coupled with Sepharose® and can freely bind to IgG Fc, so that one molecule of coupled protein A can bind to at least two molecules of IgG. In another embodiment, the affinity column includes 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 the elution buffer to produce an eluate containing the IL10 agonist. Many elution buffers are known in the art, and a suitable buffer can be selected by those skilled in the art. In some embodiments, the elution buffer is Pierce® Gentle elution buffer (pH 6.6).

[0181] Next, the eluate containing the IL10 agonist is subjected to size exclusion chromatography to isolate and purify the IL10 agonist that does not form aggregates. 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 ultrahigh performance liquid chromatography is used. In one particular embodiment, a Superdex® 200, 26 / 600 pg column (MilliporeSigma, Inc., St. Louis, Missouri, USA) is used.

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

[0183] By benefiting from this disclosure, those skilled in the art can realize a desired IL10 agonist by selecting an IL10 agonist construct having a certain positioning (i.e., whether the IL10 moiety is N-terminus or C-terminus), linker length, and purification method. In some embodiments, the desired IL10 agonist exhibits minimal aggregation and is not shortened 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 which 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 this disclosure can be formulated as pharmaceutical compositions, for example, as described in Section 6.9. 6.9. Pharmaceutical Compositions 6.9.1. Pharmaceutical compositions containing IL10 agonist polypeptides The IL10 agonists of this disclosure, for example, IL10 agonists produced by the methods described in Section 6.8.3, may be provided in the form of compositions comprising the IL10 agonist and one or more carriers, excipients, and / or diluents. These compositions may be formulated for specific uses, e.g., veterinary or human pharmaceutical applications. The form of the composition (e.g., dry powder, liquid formulation, etc.) and the excipients, diluents, and / or carriers used are considered to depend on the intended use of the IL10 agonist and, in the case of therapeutic applications, 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 preferred form (depending on the desired method of administration to the patient). The pharmaceutical composition can be administered to the patient via various routes, including orally, transdermally, subcutaneously, intranasally, intravenously, intramuscularly, intratumorally, intrathecally, topically, or locally. In any case, the most preferred route of administration is considered to depend on the specific antibody, the target, the nature and severity of the disease, and the physical condition of the target. Typically, the pharmaceutical composition is considered to be administered intravenously or subcutaneously.

[0186] Pharmaceutical compositions may be conveniently presented in unit dosage forms containing a predetermined amount of the IL10 agonist of this disclosure per single dose. The amount of IL10 agonist contained in a unit dose is thought to depend on the disease being treated as well as other factors known in the art. Such unit doses may be in the form of a lyophilized powder containing an amount of IL10 agonist suitable for a single dose, or in liquid form. The lyophilized 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. Unit doses in liquid form can be conveniently supplied in the form of a syringe pre-filled with an amount of IL10 agonist suitable for a single dose.

[0187] In some embodiments, the pharmaceutical compositions of the present disclosure include 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 this disclosure comprise only full-length IL10 agonists or nearly full-length IL10 agonists. For example, in some embodiments, the pharmaceutical compositions do not contain a detectable amount of C-terminal shortened variants and / or N-terminal shortened variants of the IL10 agonist. Placing the IL10 portion at the N-terminus of the Fc domain, as specified in this specification, may result in shortening of the N-terminus and / or C-terminus of the recombinantly expressed IL10 agonist.

[0188] In some embodiments, the pharmaceutical composition of the present disclosure minimally or does not contain any IL10 agonist aggregates. As defined herein, size exclusion chromatography can be used to produce non-aggregated IL10 agonists. In some embodiments, the pharmaceutical composition contains 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 aggregate form, as determined by size exclusion ultra-performance liquid chromatography (SE-UPLC). In some embodiments, the pharmaceutical composition does not contain a detectable amount of IL10 agonist aggregates as determined by size exclusion ultra-performance liquid chromatography (SE-UPLC).

[0189] Also, it is possible to supply in a batch a pharmaceutical composition containing an amount of IL10 agonist suitable for multiple administrations. The pharmaceutical composition can be prepared for storage as a lyophilized formulation or an aqueous solution by mixing an IL10 agonist having the desired purity with any optional pharmaceutically acceptable carrier, excipient or stabilizer (all of which are referred to herein as "carriers"), i.e., buffers, stabilizers, preservatives, isotonic agents, non-ionic surfactants, antioxidants, and various other additives. See Remington’s Pharmaceutical Sciences, 16th Edition (ed. Osol, 1980). Such additives should be non-toxic to the recipient at the dosages and concentrations used.

[0190] Buffers help maintain pH within a range close to physiological conditions. They can exist in a wide variety of concentrations, but are typically found in concentrations ranging from about 2 mM to about 50 mM. Suitable buffers for use with this disclosure include both organic and inorganic acids and their salts, e.g., citrate buffers (e.g., monosodium citrate-disodium citrate mixture, citrate-trisodium citrate mixture, citrate-monosodium citrate mixture, etc.), succinate buffers (e.g., succinate-monosodium succinate mixture, succinate-sodium hydroxide mixture, succinate-disodium succinate mixture, etc.), tartaric acid buffers (e.g., tartaric acid-sodium tartrate mixture, tartaric acid-potassium tartrate mixture, tartaric acid-sodium hydroxide mixture, etc.), fumarate buffers (e.g., fumarate-monosodium fumarate mixture, fumarate These include oxalic acid-disodium fumarate mixtures, monosodium fumarate-disodium fumarate mixtures, etc., gluconate buffers (e.g., gluconate-sodium glycoside mixtures, gluconate-sodium hydroxide mixtures, gluconate-potassium gluconate mixtures, etc.), oxalate buffers (e.g., oxalic acid-sodium oxalate mixtures, oxalic acid-sodium hydroxide mixtures, oxalic acid-potassium oxalate mixtures, etc.), lactate buffers (e.g., lactate-sodium lactate mixtures, lactate-sodium hydroxide mixtures, lactate-potassium lactate mixtures, etc.), and acetate buffers (e.g., acetate-sodium acetate mixtures, acetate-sodium hydroxide mixtures, etc.). Furthermore, phosphate buffers, histidine buffers, and trimethylamine salts, such as Tris, may also be used.

[0191] Preservatives may be added to slow microbial growth, and can be added in amounts ranging from approximately 0.2% to 1% (w / v). Suitable preservatives for use with this 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. Isotonic agents, sometimes known as “stabilizers,” may be added to ensure the isotonicity of the liquid compositions of this disclosure, and these 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, which can vary in function from volume extenders to additives that solubilize therapeutic agents, or additives that help prevent denaturation or adhesion to container walls. Typical stabilizers include polyhydric sugar alcohols (listed above); amino acids, e.g., arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, ornithine, L-leucine, 2-phenylalanine, glutamic acid, threonine, etc.; and organic sugars or sugar alcohols, e.g., lactose, trehalose, stachyose, mannitol, sorbitol, xylitol, ribitol, myo-inititol, galactitol, glycerol, etc.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, and monosaccharides such as xylose, mannose, fructose, glucose, disaccharides such as lactose, maltose, sucrose and trehalose, 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 IL1 agonist.

[0192] Adding a nonionic surfactant or surfactant (also known as a "wetting agent") can help solubilize the therapeutic agent and protect the glycoprotein from aggregation induced by agitation, thereby exposing the formulation to a stressed shear surface without causing protein denaturation. Suitable nonionic surfactants include polysorbates (such as 20, 80, etc.), poloxamers (such as 184, 188, etc.), and Pluronic® polyols. The nonionic surfactant can be present in the range of about 0.05 mg / mL to about 1.0 mg / mL, or in the range of about 0.07 mg / mL to about 0.2 mg / mL.

[0193] Other various excipients include bulking agents (such as starch), chelating agents (such as EDTA), antioxidants (such as ascorbic acid, methionine, vitamin E), and co-solvents.

[0194] 6.9.2. Pharmaceutical composition for delivery of nucleic acid encoding an IL10 agonist The IL10 agonists of this 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] Examples of gene therapy vectors include therapeutic drugs based on adenoviruses or AAVs. Non-limiting examples of adenovirus-based or AAV-based therapeutic agents for use in the methods, uses, or compositions described herein include, for example: rAd-p53, a recombinant adenovirus vector encoding the wild-type human tumor suppressor protein p53, used, for example, in the treatment of cancer (also known as Gendicine® or Genkaxin®; see, for example, Russell et al., 2006, Modern Oncology, 14:1295-1297); Ad5_d11520, an adenovirus lacking the E1B gene that inactivates host p53 (also known as H101 or ONYX-015; see, for example, Russell et al., 2012, Nature Biotechnology, 30:658-670); and AD5-D24-GM-CSF, an adenovirus containing the cytokine GM-CSF, used in the treatment of cancer (see, for example, Cerullo et al., 2010, Cancer). Res., 70:4297); rAd-HSVtk, this is a replication-deficient adenovirus possessing the HSV thymidine kinase gene, and is used, for example, for the treatment of cancer (developed as Cerepro® by Ark Therapeutics).See, for example, U.S. Patent No. 6,579,855; developed by Advantagene as ProstAtak®; International Publication No. WO2005 / 049094); rAd-TNFα, which is a replication-deficient adenovirus vector that expresses human tumor necrosis factor α (TNFα) under the control of a chemoradiation-inducible EGR-1 promoter, for example, cancer treatment (TNFerade®, GenVec); Rasmussen et al., 2002, Cancer Gene Ther., 9:951-7; Ad-IFNβ is an adenovirus serotype 5 vector lacking E1 and E3 genes that expresses the human interferon-β gene under the direction of the cytomegalovirus (CMV) pre-early promoter, and is used, for example, for cancer treatment (BG00001 and H5.110CMVhIFN-β, Biogen; Sterman et al., 2010, Mol. Ther., 18:852-860).

[0196] Nucleic acid molecules (e.g., mRNA) or viruses may be formulated as the sole pharmaceutically active ingredient in a pharmaceutical composition, or in combination with other active agents for a specific disorder to be treated. Optionally, other agents, pharmaceuticals, carriers, adjuvants, and diluents may be included in the compositions provided herein. For example, wetting agents, emulsifiers, and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as one or more of the following may be included in the composition: colorants, release agents, coating agents, sweeteners, flavorings, and fragrances, preservatives, antioxidants, chelating agents, and inert gases. Other exemplary agents and excipients that may be included in the composition include, for example, water-soluble antioxidants such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfite, sodium metabisulfite, and sodium sulfite; oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, and α-tocopherol; and metal chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, and phosphoric acid.

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

[0198] 6.10. Indications and methods of treatment The IL10 agonists of this disclosure are useful in treating conditions treatable by IL10, such as inflammation-related and immune-related disorders, fibrotic disorders, cancer and cancer-related disorders, or cardiovascular disorders (e.g., atherosclerosis).

[0199] In certain embodiments, the conditions treated by the IL10 agonists of this disclosure are autoimmune conditions, transplant rejection, post-traumatic immune responses, infections, or graft-versus-host diseases. In certain embodiments, these conditions are autoimmune diseases, organ or bone marrow transplant rejection, graft-versus-host diseases, parasitic infections, granulomas, Crohn's disease, colitis, pancreatitis, inflammatory lung disease, allergic conditions, asthma, atopic dermatitis, or rhinitis.

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

[0201] In some embodiments, the disease to be treated is resistant to treatment with anti-PD1 antibodies. In some embodiments, the disease to be treated is resistant to treatment with anti-PD1 antibody monotherapy. Blocking the PD1 / PD-L1 checkpoint has proven to be an effective treatment for some malignancies, but it is ineffective in a significant proportion of patients, and resistance develops in some initial responders, leading to disease recurrence (Nowicki et al., 2018, Cancer J., 24(1):47-53). Resistance to treatment with anti-PD1 antibodies can be primary or acquired. The mechanisms leading to primary and acquired resistance to PD1 inhibition are both known in the art, as are the methods for identifying malignancies resistant to PD1 inhibition. For example, see Nowicki et al., 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 this disclosure are generally considered to be used in an effective amount to achieve the intended purpose. When used to treat or prevent a disease condition, the IL10 agonists of this disclosure, or their pharmaceutical compositions, are administered or applied in a therapeutically effective dose. Determining the therapeutically effective dose is well within the capabilities of those skilled in the art, particularly 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 is not considered to result in a cure, but rather to provide only partial benefit. In some embodiments, any physiological change that provides some benefit is also considered therapeutically beneficial. Accordingly, the terms “effective dose” or “therapeutically effective dose” encompass doses or drug regimens that provide partial benefit.

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

[0204] Suitable control samples for determining reference or baseline values ​​for IL12 and / or IL4 may be derived from individuals having the same disease condition as the individuals being treated with the IL10 agonist or its pharmaceutical composition according to this disclosure. The control samples may be of an age consistent with the subjects being treated with the IL10 agonist or its pharmaceutical composition according to this disclosure. Reference or baseline values ​​may be obtained from suitable individuals or populations of suitable individuals and used as general reference values ​​for multiple analyses. Alternatively, the control samples may be based on the subjects' own values ​​prior to the initiation of IL10 agonist therapy. Identifying and selecting suitable controls is within the ordinary skill of those skilled in the art.

[0205] The subjects, patients, or individuals requiring treatment are typically mammals, more specifically humans. The appropriate dose of the IL10 agonist of this disclosure (used alone or in combination with one or more other additional therapeutic agents) for the prevention or treatment of disease is considered to 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. In any case, the physician administering the drug is considered to determine the concentration of the active ingredient in the composition and the appropriate dose for the individual subject. Various dosing schedules, including but not limited to single doses or multiple doses at various time points, bolus doses, and pulse infusions, are envisioned in this specification.

[0206] A single dose of non-conjugated IL-10 can range from approximately 50,000 IU / kg to approximately 1,000,000 IU / kg or more, more typically up to approximately 600,000 IU / kg. This may be repeated several times a day (e.g., 2-3 times) for several days (e.g., approximately 3-5 consecutive days), followed by a rest period (e.g., approximately 7-14 days), and then one or more repetitions. Therefore, a therapeutically effective dose may consist of a single dose or a number of doses over a period of time (e.g., approximately 20-30 individual doses of approximately 600,000 IU / kg of IL-10 over a period of approximately 10-20 days).

[0207] Similarly, IL10 agonists are preferably administered to patients as a single dose or over a series of treatments. Depending on the type and severity of the disease, for example, whether by one or more separate doses or by continuous infusion, an initial candidate dose of IL10 agonist of approximately 1 μg / kg to 15 mg / kg (e.g., 0.1 mg / kg to 10 mg / kg) may be administered to a patient. A typical daily dose may range from approximately 1 μg / kg to 100 mg / kg or more, depending on the factors mentioned above. For repeated administrations over several days or more, treatment is generally considered to be continued until the desired suppression of disease symptoms is achieved, depending on the patient's condition. One exemplary dose of IL10 agonist is considered to be in the range of approximately 0.005 mg / kg to approximately 10 mg / kg. In other non-limiting examples, a single dose may be thought to consist of approximately 1 μg / kg / body weight, 5 μg / kg / body weight, 10 μg / kg / body weight, 50 μg / kg / body weight, 100 μg / kg / body weight, 200 μg / kg / body weight, 350 μg / kg / body weight, 500 μg / kg / body weight, 1 mg / kg / body weight, 5 mg / kg / body weight, 10 mg / kg / body weight, 50 mg / kg / body weight, 100 mg / kg / body weight, 200 mg / kg / body weight, 350 mg / kg / body weight, 500 mg / kg / body weight, up to approximately 1000 mg / kg / body weight or more, and any range that can be derived within that range. In non-limiting examples derived from the values ​​specified in this specification, doses can be administered in ranges such as approximately 5 mg / kg / body weight to approximately 100 mg / kg / body weight, or approximately 5 μg / kg / body weight to approximately 500 mg / kg / body weight. Therefore, one or more doses of approximately 0.5 mg / kg, 2.0 mg / kg, 5.0 mg / kg, or 10 mg / kg (or any combination thereof) can be administered to the patient. Such doses can be administered intermittently, for example, every week or every three weeks (e.g., the patient receives approximately 2 to 20 doses of the IL-10 agonist, or, for example, approximately 6 doses). One or more lower doses may be administered after an initial high-load dose. However, other drug regimens may also be useful. The progression of this treatment is readily monitored by prior art and assays.

[0208] In the case of systemic administration, the therapeutically effective dose can first be estimated from in vitro assays such as cell culture assays. Subsequently, in animal models, the EC determined in cell cultures is used. 50 A dose can be set to achieve a circulating concentration range that includes [specific concentration range]. Using such information, an effective dose in humans can be determined more accurately.

[0209] The initial dose can also be estimated from in vivo data, such as animal models, using techniques known in the art. Those skilled in the art will likely find it easy to optimize the dose to humans based on animal data.

[0210] Dosage and dosing intervals may be individually adjusted to obtain plasma concentrations of the IL10 agonist sufficient to maintain therapeutic effect. Typical patient doses for injectable administration range from approximately 0.1 to 50 mg / kg / day, typically from approximately 0.5 to 1 mg / kg / day. Therapeutically effective plasma levels may be achieved by administering multiple doses daily. Plasma levels can be measured, for example, by ELISA HPLC.

[0211] In the case of local administration or selective uptake, the effective local concentration of the IL10 agonist may not be related to the plasma concentration. Those skilled in the art can likely optimize the therapeutically effective local dose without excessive experimentation.

[0212] The therapeutically effective doses of IL10 agonists described in this specification are generally considered to provide therapeutic benefits 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, for example, 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 50(The dose that is therapeutically effective in 50% of the population) can be determined. The dose ratio between the toxic effect and the therapeutic effect is the therapeutic index, which can be expressed as the ratio LD 50 / ED 50 . IL10 agonists showing a high therapeutic index are preferred. In one embodiment, the IL10 agonist according to the present disclosure shows a high therapeutic index. Data obtained from cell culture assays and animal tests can be used to set the range of dosages suitable for use in humans. The dosage preferably falls within the range of circulating concentrations that contain little or no toxicity, the ED 50 . The dosage 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 selected by the individual physician considering the condition of the patient. (See, for example, Fingl et al., 1975, "The Pharmacological Basis of Therapeutics", Chapter 1, p. 1, which is hereby incorporated by reference in its entirety).

[0213] The attending physician of a patient being treated with the IL10 agonist of the present disclosure will know how and when to terminate, interrupt, or adjust the administration in cases of toxicity, organ dysfunction, etc. Conversely, the attending physician will also know to adjust the treatment to a higher level if the clinical response is insufficient (excluding toxicity). The degree of dosage in the management of the target disorder is thought to vary depending on the severity of the condition to be treated and the route of administration, etc. The severity of the condition can be evaluated in part, for example, by standard prognostic assessment methods. Furthermore, the dosage and perhaps the frequency of administration are also thought to vary depending on the age, weight, and response of the individual patient.

[0214] 6.11. Combination Therapy The IL10 agonists according to this disclosure may be administered in combination with one or more additional agents in a therapeutic setting. For example, the IL10 agonists according to this disclosure may be administered concurrently with at least one additional therapeutic agent. The term “therapeutic agent” encompasses any agent administered to treat a target symptom or disease requiring such treatment. Such additional therapeutic agents may include any active ingredients suitable for the specific indication being treated, preferably those having complementary activities that do not adversely affect each other.

[0215] In the treatment of cancer, the IL10 agonists of this disclosure may be administered in combination with one or more anti-cancer drugs (e.g., chemotherapeutic agents) or modalities of anti-cancer treatment (e.g., radiation therapy). The entity of the additional drug depends largely on the nature of the underlying condition being treated (e.g., for the treatment of bladder cancer, the addition of an alkylating agent such as cisplatin may be appropriate). One or more additional drugs (e.g., chemotherapeutic agents) administered in combination with the IL10 agonist are administered in amounts effective for the intended purpose. The effective amount of such additional drugs depends on the amount of IL10 agonist used, the type of disorder or treatment, and other factors considered above.

[0216] This IL10 agonist is generally used in the same dosage and route of administration as described in this specification, or in approximately 1 to 99% of the dosage described in this specification, or in any dosage and route of administration that is deemed empirically / clinically appropriate.

[0217] Examples of chemotherapeutic agents used in combination with the IL10 agonists of this disclosure include alkylating agents, e.g., thiotepa and cyclophosphamide; alkyl sulfonates, e.g., busulfan, improsulfan and pigosulfan; aziridines, e.g., benzodopa, carbocone, meturedopa, and uredopa; ethyleneimines and methylamelanines, e.g., altoretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiofo Suphoramide and trimethylolomelamime; nitrogen mustards, e.g., chlorambucil, chlornafadin, chlorophosphamide, estramustine, ifosfamide, mechloretamine, mechloretamine oxide hydrochloride, melphalan, nobenbicin, phenesterine, prednimustine, trophosphamide, uracil mustard; nitrosoureas, e.g., carmustine, chlorozoto Syn, fotemustine, lomustine, nimustine and ranimustine; antibiotics, e.g., acrasinomycin, actinomycin, anthramycin, azaserin, bleomycin, kactinomycin, calicheamicin, carabicin, caminomycin, cardinophilin, chromomycin, dactinomycin, daunorubicin, detrubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcelomycin, ma Itomycin, mycophenolic acid, nogaramycin, olibomycin, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidine, ubenimex, dinostatin, zolubicin; antimetabolites, e.g., methotrexate and 5-fluorouracil (5-FU); folate analogs, e.g., denopterin, methotrexate, pteropterin, trimethrexate;Purine analogs, e.g., fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs, e.g., ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; androgens, e.g., carsterone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone; anti-adrenal agents, e.g., aminoglutethimide, mitotane, trilostane; folic acid supplements / replenispers, e.g., frolinic acid acid); acegraton; aldofamide glycoside; aminolevulinic acid; amsacrin; bestlabsil; bisanthren; edatraxate; defofamine; demecoltin; diazicon; elformithine; eriptinium acetate; etogluside; gallium nitrate; hydroxyurea; lentinan; ronidamin; mitogluzone; mitoxantrone; mopidamol; nitracrin; pentostatin; fenamet; pirarubicin; podophyllic acid; 2-ethylhydrazide; procarbazine; razoxane; schizophyllan; spirogermanium; tenuazonic acid; triadicone; 2,2',2"-trichlorotriethylamine; urethane; vindesine; dacarbazine; mannomustine; mitobronito Lu; Mitractol; Pipobroman; Gacytosine; Arabinoside ("Ara-C"); Cyclophosphamide; Thiotepa; Taxoids, e.g., Paclitaxel and Docetaxel; Chlorambucil; Gemcitabine; 6-Thiogunine; Mercaptopurine; Methotrexate; Platinum and platinum-coordinated complexes, e.g., 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;This also includes, but is not limited to, any pharmaceutically acceptable salts, acids, or derivatives of any of the above. Furthermore, the term “chemotherapeutic agent” can also refer to anti-hormonal agents that modulate or inhibit the hormonal effects on tumors, such as anti-estrogen agents including, for example, tamoxifen, raloxifen, 4(5)-imidazole-inhibiting aromatase, 4-hydroxytamoxifen, trioxyfen, keoxyfen, onapristone, and toremifene; and anti-androgen agents, such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; as well as any pharmaceutically acceptable salts, acids, or derivatives of any of the above. In some embodiments, the additional agents may include, but are not limited to, one or more chemical or biological agents identified in the art as useful for treating neoplastic diseases, including, but not limited to, cytokines or cytokine antagonists such as IL12 and INFα, or anti-epidermal growth factor receptors, radiotherapy, antibodies against tumor antigens, monoclonal antibody-toxin complexes, T cell adjuvants, bone marrow transplants, or antigen-presenting cells (e.g., dendritic cell therapy), antitumor vaccines, replicable viruses, and CART cells as described in Section 6.11.1.

[0218] For the treatment of immune and inflammatory conditions, the IL10 agonists of this disclosure can be used in combination with immunosuppressive or immunomodulatory therapies. Non-limiting examples of immunosuppressive therapies include immunosuppressive compounds such as cyclosporine A, cyclophosphamide, FK506, tacrolimus, corticosteroids, azathioprine, mycophenolate mofetil, sirolimus, rapamycin, rapamycin analogs, deoxyspagarine, and prednisone.

[0219] In some embodiments, the IL10 agonists of this disclosure can be used in combination with anti-PD-1 antibodies. Examples of anti-PD-1 antibodies for use in combination with the IL10 agonists of this 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 therapies described above include combined administration (where two or more therapeutic agents are contained in the same or separate compositions) and separate administration, in the latter case, administration of the IL10 agonist of this disclosure may be performed before, concurrently with, and / or after administration of additional therapeutic agents and / or adjuvants.

[0221] 6.11.1. Combination therapy using IL10 agonist therapy and immunotherapy The IL10 agonists of this disclosure can be advantageously used in combination with chimeric antigen receptor ("CAR") expressing cells, such as CAR-expressing T ("CAR-T") cells, such as CAR-T cells, in the treatment of cancer or autoimmune diseases.

[0222] Conditioning or lymphodepletion therapy, such as regimens of cyclophosphamide and fludarabine, can also be administered to subjects receiving CAR and IL10 agonist therapy. Such treatment is usually performed several days before administration of CAR-expressing cells to the subject. For example, cyclophosphamide can be administered for two days prior to the infusion of CAR-expressing cells, e.g., on day -8 and day -7 (with the infusion day being zero), and fludarabine can be administered for five consecutive days from day -6 to day -2. In one embodiment, 60 mg / kg of cyclophosphamide is administered to the subject. In another embodiment, 25 mg / m² 2 Fludarabine is administered to the subject. In one embodiment, there is a no-treatment day on day (-1), which is the day immediately preceding the injection of CAR-expressing cells into the subject.

[0223] CAR-expressing cells are 10 4 from 10 9 pieces / kg body weight, preferably 10 5 from 10 6 The T cell composition can be administered in amounts encompassing all integer values ​​within the range of cells / kg body weight. Multiple doses of the T cell composition may also be administered at these doses. In some embodiments, CAR-expressing cells are 1 × 10⁶ 6 From 1 x 10 11 individual or 1 x 10 7 From 1 x 10 8 It is administered in individual doses.

[0224] Prior to administration to human subjects, CAR-expressing cells can be activated with anti-CD3 and / or anti-CD28 antibodies in conjunction with an increase in IL10. CAR-expressing cells, such as T cells, are preferably of autologous origin to the target, but may also be of homogeneous origin.

[0225] In one embodiment, the IL10 agonist is administered to human subjects by bolus injection for four consecutive days starting from the administration date of a population of CAR-expressing cells. In one embodiment, the IL10 agonist is administered to human subjects by bolus for at least 5 consecutive days starting from the administration date of a population of CAR-expressing cells.

[0226] IL10 agonists can be administered for longer periods, such as one week, two weeks, one month, or longer. The frequency of administration can be reduced, for example, after the depletion of CAR-expressing cells. For example, IL10 agonists can be initially administered daily, and then the frequency can be reduced to once a week.

[0227] IL10 therapy can be initiated on the same day as the administration of CAR-expressing cells, or it can be initiated 1, 2, 3, 4, 5, 6 days, or 1 week after administration. In one embodiment, the cell population includes T cells obtained from subjects engineered to recombinantly express CARs.

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

[0229] 6.11.1.1. Components of CAR A typical CAR includes an extracellular region containing an antigen-binding domain, e.g., the antigen-binding domain of an antibody, which is 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] Antigen-binding domains are typically linkers (e.g., linkers as described in Section 6.7), optional spacers (e.g., as described in Section 6.11.1.1.1), optional hinges (e.g., as described in Section 6.11.1.1.2), transmembrane domains (e.g., as described in Section 6.11.1.1.3), and intracellular signaling blocks (e.g., as described in Section 6.11.1.1.4).

[0231] 6.11.1.1.1. 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, enabling proper cell / cell contact, antigen binding, and activation (Patel et al., 1999, Gene Therapy 6:412-419). The spacer domains may be derived from natural, synthetic, semi-synthetic, or recombinant sources. In certain embodiments, the spacer domains are parts of an immunoglobulin, including, but not limited to, one or more heavy chain constant regions, e.g., CH2 and CH3. The spacer domains may include amino acid sequences of naturally occurring or modified immunoglobulin hinge regions.

[0232] In one embodiment, the spacer domain includes the CH2 and CH3 domains of IgG1 or IgG4. 6.11.1.1.2. Hinged 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 play a role in positioning the antigen-binding domain away from the effector cell surface, enabling proper cell / cell contact, antigen binding, and activation. CARs generally contain one or more hinge domains between the binding domain and the transmembrane domain (TM). The hinge domains may originate from natural, synthetic, semi-synthetic, or recombinant sources. The hinge domains may contain amino acid sequences from naturally occurring immunoglobulin hinge regions or modified immunoglobulin hinge regions.

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

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

[0235] 6.11.1.1.3. Transmembrane (TM) domain The “transmembrane domain” is the portion of the CAR that fuses the extracellular binding portion with the intracellular signaling domain, thereby fixing the CAR to the plasma membrane of an immunoeffector cell. As used in this specification, 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 a natural, synthetic, semi-synthetic, or recombinant source. The TM domain may be derived from 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 (for example, including at least these transmembrane regions). In a particular embodiment, the TM domain is synthetic and consists mainly of hydrophobic residues such as leucine and valine.

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

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

[0239] 6.11.1.1.4. Intracellular signal transduction domains CARs typically contain an intracellular signaling domain. The “intracellular signaling domain” refers to the portion of the CAR that transmits effective antigen-binding messages within immune effector cells, thereby inducing effector cell function, such as activation including the release of cytotoxic factors to the CAR-bound target cell, cytokine production, proliferation, and cytotoxic activity, or other cellular responses induced by antigen binding to the extracellular CAR domain.

[0240] The term "effector function" refers to the specialized function of immune effector cells. The effector function of T cells is, for example, assisting or activating cytolytic activity or cytokine secretion. Therefore, the term "intracellular signaling domain" refers to the portion of a protein that transmits effector function signals and directs cells to perform their specialized functions. 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 shortened portion of an intracellular signaling domain is used, such a shortened portion may be used in place of the entire domain, as long as it transmits effector function signals. The term "intracellular signaling domain" means that it includes any shortened portion of an intracellular signaling domain that is sufficient to transmit effector function signals.

[0241] It is known that signals generated solely via the TCR are insufficient for complete T cell activation, and that secondary or co-stimulatory signals are also required. Therefore, T cell activation can be said to be mediated by two distinct classes of intracellular signaling domains: primary signaling domains that induce antigen-dependent primary activation via the TCR (e.g., the TCR / CD3 complex), and co-stimulatory signaling domains that act antigen-independently to provide secondary or co-stimulatory signals. In preferred embodiments, the CAR envisioned in this specification comprises an intracellular signaling domain including one or more "co-stimulatory signaling domains" and "primary signaling domains."

[0242] The primary signaling domain modulates the primary activation of the TCR complex to either stimulate or inhibit it. Primary signaling domains that act in a stimulating manner may contain a signaling motif known as an immunoreceptor tyrosine system activating motif or ITAM.

[0243] Specific examples of ITAMs containing primary signaling domains particularly used in the methods of this 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 co-stimulatory signaling domains. The intracellular primary signaling domains and co-stimulatory signaling domains may be ligated in a line to the carboxyl terminus of the transmembrane domain in any order.

[0244] The CARs envisioned in this specification include one or more costimulatory signaling domains for enhancing the efficacy and growth of T cells expressing CAR receptors. As used in this specification, the terms “costimulatory signaling domain” or “costimulatory domain” refer to the intracellular signaling domain of a costimulatory molecule. A costimulatory molecule is a cell surface molecule other than an antigen receptor or Fc receptor that provides a second signal necessary for the efficient activation and function of T lymphocytes when bound to an antigen. Specific examples of such co-stimulatory 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 a co-stimulatory endodomain of CD28, 4-1BB (also known as CD137), CD70, or OX40 (also known as CD134), or a combination thereof, or in a line. These endodomains enable robust T cell activation upon TCR recognition by antigen-presenting cells (APCs), improving cytokine production and proliferation of CAR-T cells.

[0246] In another embodiment, the CAR includes CD28 and CD137 co-stimulatory signaling domains, as well as a CD3ζ primary signaling domain. In yet another embodiment, the CAR includes CD28 and CD134 co-stimulatory signaling domains, as well as a CD3ζ primary signaling domain.

[0247] In one embodiment, the CAR includes CD137 and CD134 co-stimulatory signaling domains, as well as a CD3ζ primary signaling domain. An exemplary CD3ζ signaling region may include any of the following amino acid sequences:

[0248] [ka]

[0249] An exemplary CD28 signaling region may include any of the following amino acid sequences:

[0250] [ka]

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

[0252] [ka]

[0253] 6.11.1.1.5. Tags In some embodiments, the CAR includes a tag used for CAR identification; for example, the V5 epitope tag is derived from a small epitope (Pk) present on the P and V proteins of the paramyxovirus Simianvirus 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] 6.11.1.1.6. Signal Peptides In some embodiments, the CAR includes a signal peptide. The signal peptide promotes 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 positioned at the N-terminus of the antigen-binding moiety of the CAR. This is typically not present in the mature molecule, as the signal peptide is cleaved when the CAR is expressed and processed within cells such as T cells.

[0255] 6.11.1.2. Preparation of CART cells To produce CART cells ex vivo, for example, PBMCs, peripheral blood lymphocytes, or T cells enriched therefrom can be expanded before and / or after introducing CAR-encoding nucleic acids into the cells, such as by viral introduction.

[0256] T cells useful for producing CART cells can be isolated from peripheral blood lymphocytes by lysing erythrocytes and depleting monocytes, for example, by centrifugation via a PERCOLL® gradient or by countercurrent centrifugation. Specific subpopulations of T cells, e.g., 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 incubation period is approximately 30 minutes. In a further embodiment, the incubation period ranges from 30 minutes to 36 hours or more, and all integer values ​​in between. In a further embodiment, the incubation period is at least 1, 2, 3, 4, 5, or 6 hours. In yet another preferred embodiment, the period is 10 to 24 hours. In one preferred embodiment, the incubation period is 24 hours. For the isolation of T cells from leukemia patients, the cell yield can be increased by using a relatively long incubation time, for example, 24 hours. In any situation where T cells are few in number compared to other cell types, such as when isolating tumor-infiltrating lymphocytes (TILs) from tumor tissue or immunodeficient individuals, a longer incubation time can be used to isolate T cells. Furthermore, by using a longer incubation time, the capture efficiency of CD8+ T cells can be increased. Thus, a subset of T cells can be selectively selected or excluded at the start of culture or at any other point in the process by simply shortening or lengthening the time that T cells are bound to CD3 / CD28 beads, and / or by increasing or decreasing the ratio of beads to T cells.In addition, by increasing or decreasing the ratio of anti-CD3 and / or anti-CD28 antibodies to beads or other surfaces, a subset of T cells can be selectively selected or excluded at the start of culture or at any other desired point in time. Those skilled in the art will recognize that multiple rounds of selection may be used in the context of this disclosure. In certain embodiments, it may be desirable to perform a selection procedure and use "unselected" cells in the process of activation and growth. "Unselected" cells may also be subjected to further rounds of selection.

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

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

[0259] Before or after genetic modification of T cells to express a desired CAR, T cells can be activated and enlarged using methods known in the art, for example, as described in U.S. Patent No. 7,144,575; No. 7,067,318; No. 7,172,869; No. 7,232,566; or No. 7,175,843.

[0260] Generally, T cells useful in the methods of this disclosure are increased by contact between a surface to which a drug that stimulates CD3 / TCR complex-related signaling is attached and a ligand that stimulates co-stimulatory molecules on the surface of the T cells. 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 the surface, or by contacting them with a protein kinase C activator (e.g., bryostatin) together with a calcium ionophore. For co-stimulation of accessory molecules on the T cell surface, ligands that bind to the accessory molecules are used. For example, under conditions suitable for stimulating T cell proliferation, 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.

[0261] Prior to administration to human subjects, CAR-expressing cells can also be pre-treated with IL12 (see, for example, Emtage et al., 2003, J.Immunother., 16(2):97-106, as incorporated herein).

[0262] 6.11.1.3. Cancer Immunotherapy Accordingly, the present disclosure provides a method for treating cancer in a human subject requiring cancer treatment, comprising the steps of administering an effective dose of the IL10 agonist of the present disclosure to the subject, and administering CAR-expressing cells, for example, CAR-expressing T cells (or "CART cells"). A T cell subtype particularly useful for cancer treatment is T cells with robust CAR-mediated cytotoxicity, for example, CD3+CD8+ T cells, which can be prepared as described in 6.11.1.2 above.

[0263] For the treatment of cancer, the extracellular domain of a CAR can be targeted to tumor-associated antigens, for example, as described in Section 6.5.1. In certain embodiments, the tumor-associated antigens are CD20, EGFR, FITC, CD19, CD22, CD33, PSMA, GD2, EGFR variants, 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, IgGκ chain, folate receptor α, PSCA, or EpCAM. In certain embodiments: • CARs are designed to target CD22 to treat diffuse large B-cell lymphoma.

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

[0265] CAR is designed to target c-Met to treat triple-negative breast cancer, non-small cell lung cancer, and other cancers. CARs are designed to target PSMA to treat conditions such as prostate cancer.

[0266] CARs are designed to target the glycolipid F77 to treat conditions such as prostate cancer. CARs are designed to target EGFRvIII to treat conditions such as glioblastoma.

[0267] CARs are designed to target GD-2 to treat neuroblastoma, melanoma, and other conditions. CARs are designed to target the NY-ESO-1 TCR to treat myeloma, sarcoma, melanoma, and other conditions.

[0268] CARs are designed to target the MAGE A3 TCR to treat myeloma, sarcoma, melanoma, and other conditions. Particularly useful for CAR-IL10 agonist combination therapy are IL10 agonists that contain a targeting moiety that recognizes cell surface antigens present on the surface of CAR-expressing lymphocytes.

[0269] 6.11.1.4. Immunotherapy for Autoimmune Diseases Chimeric antigen receptor (CAR) T cells have become a powerful treatment option for blood cancers. Using the same idea of ​​modifying T cells to efficiently target affected cells, scientists have efficiently created T cells with predetermined antigen specificity through transfection with viral vectors encoding chimeric antigen receptors (CARs). CAR-modified T cells, manipulated in an MHC-independent manner, have the advantage of having broad applications, particularly in transplantation and autoimmunity.

[0270] When used in the treatment of autoimmune diseases, the extracellular domain of CARs is preferably specific to target antigens or ligands associated with the autoimmune response. Such modifications induce activation of redirected Tregs at the inflammatory site, suppressing the inflammatory effector immune response. regExamples of autoimmune diseases that can be targeted by this therapy include multiple sclerosis, inflammatory bowel disease (IBD), rheumatoid arthritis, systemic lupus erythematosus, Crohn's disease, psoriasis, type 1 diabetes, Sjögren's disease, myasthenia gravis (MG), Hashimoto's thyroiditis; Graves' disease; and uveitis.

[0271] In certain embodiments, CART cells are engineered to express CARs that target antigens or ligands specific to the following: • 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 antigen present in the epitope of collagen or in the joints; • Type 1 diabetes or autoimmune islet inflammation, where the antigen or ligand is the pancreatic β-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 neurogenic antigen; • Autoimmune thyroiditis, where the antigen or ligand is the thyroid antigen; • Autoimmune gastritis, where the antigen or ligand is the gastric antigen; • Autoimmune uveitis or uveoretinitis, where the antigen or ligand is the S-antigen or another uveal antigen or retinal antigen; • Autoimmune orchitis, where the antigen or ligand is the testicular antigen; • Autoimmune oophoritis, where the antigen or ligand is the 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 melanocyte antigen such as melanin or tyrosinase; • Autoimmune prostatitis, where the antigen or ligand is the prostate antigen; • Any undesirable immune response, where the antigen or ligand is an activating antigen or other antigen expressed on T effector cells present at the site of the undesirable 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 inflammation.

[0272] In one embodiment, T cells can be modified to express chimeric autoantigen receptor (CAAR) T cells in order to specifically eliminate B cells that cause autoimmune diseases. Therefore, unless otherwise indicated by the context, references to CAR-expressing T cells include references to CAAR expression. [Examples]

[0273] 7. Examples 7.1. Materials and Methods 7.1.1. Production of IL10 Agonists Constructs encoding IL10 and IL10 mutain (identified by IL10M_), as listed in Table 3 below, as well as Fc controls, were constructed. The IL10 mutain constructs included mouse or human IL10 of various configurations, 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 then 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 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), we developed a STAT3-driven luciferase-based reporter assay to evaluate the ability of IL10 and IL10 mutein to activate transcription via STAT3.

[0288] 7.1.2.1. Manipulation of Reporter TF-1 Cells TF-1 cells (ATCC, #CRL-2003) were transduced using lentiviral particles containing a STAT3 luciferase reporter construct (Cignal STAT3-Luc lentireporter, SA Biosciences, CLS-6028L-8) in the presence of 5 μg / mL polybren. 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 using lentiviral particles containing a STAT3 luciferase reporter construct (Cignal STAT3-Luc lentireporter, SA Biosciences, CLS-6028L-8) in the presence of 5 μg / mL polybren. 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 via either recombinant IL10 or IL10 mutain. This cytokine recruits the β subunit (IL10Rb) via binding to the IL10 receptor subunit α (IL10Ra), enabling the assembly of the 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 assay medium for preparing cell suspensions and antibody dilutions.

[0292] One day before screening, the manipulated reporter cells were diluted to a 1:3 ratio. On the day of the assay, the cells were centrifuged and precipitated, and 1 × 10⁶ cells were added to the assay medium. 6 The solutions were resuspended at 1 / mL. IL10, IL10 mutaine, and the control were diluted 1:5, and then diluted in 11 steps from 100 nM to 10 fM, with step 12 being the one that did not contain recombinant protein. 5 × 10 4 Reporter cells were added to a 96-well white flat-bottom plate and incubated with serially diluted IL10, IL10 mutaine, or a control protein. After incubation at 37°C / 5% CO2 for 5 hours and 30 minutes, cells were lysed with 100 μL of ONE-Glo® (Promega) reagent, and luciferase activity was detected. Synchrotron radiation was acquired as relative light units (RLU) using a multi-label plate reader Envision® (PerkinElmer). All serial dilutions were tested twice.

[0293] In determining the EC50 value, we excluded points that would introduce a hook effect, where the signal decreases in a dose-dependent manner when the protein concentration is higher than the maximum signal. The antibody's EC50 value was 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 mutein in cell-based assays using primary human immune cells To evaluate the effects of IL10 stimulation on the release of IL2, TNFα, and IFNγ during T cell activation, we developed a functional assay for primary mixed-type T cells / allogeneic PBMCs.

[0296] 7.1.3.1. Isolation of the first human PBMC Human peripheral blood mononuclear cells (PBMCs) were isolated from healthy donor LeucoPak (Donor 123). PBMC isolation was achieved by density gradient centrifugation using 50 mL SepMate® tubes, following the manufacturer's recommended protocol. Briefly, LeucoPak was diluted 1:2 with D-PBS, and a 30 mL layer of this was placed on top of 15 mL of Ficoll® added to a 50 mL SepMate® tube. Subsequent steps were followed 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 two healthy donor LeucoPac cells (donors 5500Y and 6900M). T cell isolation was achieved using the RosetteSep® Human T cell enrichment cocktail (StemCell) in accordance with the manufacturer's protocol. T cells were isolated by density gradient centrifugation using 50 mL SepMate® tubes according to the manufacturer's recommended protocol. The 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 co-culture with allogeneic PBMCs treated with mitomycin C to halt PBMC proliferation. During co-culture, IL10 or IL10 mutein titration was added, and its effect on T cell activity was determined by measuring the release of IL2, TNFα, and IFNγ in the cell culture supernatant using a homogeneous no-wash AlphaLISA® kit (Perkin Elmer, AL208F AL217S AL221F).

[0299] Human T cells previously isolated and frozen from donors 5500Y and 6900M were thawed on the day of analysis in a stimulating medium containing 50 U / mL of benzonase nuclease (X-VIVO® 15 cell medium supplemented with 10% FBS, HEPES, NaPyr, NEAA, and 0.01 mM BME). The cells were centrifuged at 1200 rpm for 10 minutes and resuspended in the stimulating medium, resulting in 1 × 10⁶ cells. 5 Cells were plated at a concentration of cells / well into 96-well round-bottom plates. Human PBMCs previously isolated and frozen from donor 123 were thawed on the day of analysis in stimulating medium containing 50 U / mL benzonase nuclease. Cells were centrifuged at 1200 rpm for 10 minutes and 1 × 10⁶ cells were placed in stimulating medium containing 50 μg / mL mitomycin C.7 The cells were resuspended at a concentration of cells / mL. After incubation at 37°C for 1 hour, the PBMCs were washed three times with D-PBS containing 2% FBS, resuspended in stimulation medium, and 1.5 × 10⁶ T cells were added per well. 5 The final concentrations were added to the wells. Subsequently, IL10, IL10 mutain, and control were diluted 1:5 and then diluted in nine steps, ranging from 500 nM to 1.28 pM for IL10, and from 100 nM to 0.256 pM for IL10 mutain or control antibody, with the 10th dilution being free of recombinant protein. The serially diluted proteins were added to the wells, and the plates were incubated at 37°C / 5% CO2 for 72 hours. The plates were then centrifuged to pellet the cells, and 50 μL of the supernatant was collected. 5 μL of the supernatant was taken from there, and tested using the AlphaLISA® assay for human IL-2, TNFα, and IFNγ according to the manufacturer's protocol. Measurements were obtained using the Envision® multi-label plate reader. Standard curves for known concentrations of IL-2, TNFα, and IFNγ were created to extrapolate the pg / mL of each cytokine released into the assay wells. All serial dilutions were tested twice.

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

[0301]

number

[0302] 7.1.4. Activity of IL10 mutein in syngeneic tumor allograft models 7.1.4.1. Tumor transplantation and treatment group allocation Tumor cells were transplanted subcutaneously into female BALB / c mice (Jackson Laboratory, located in Bar Harbor, Maine, USA). 1 × 10⁶ female BALB / c mice (Jackson Laboratory, located in Bar Harbor, Maine, USA) between 6 and 8 weeks of age were transplanted into the right flank. 6 Individual tumor cells were transplanted subcutaneously.

[0303] Therapeutic intervention or administration of an isotype control was performed intraperitoneally for 3 weeks. The test substance was injected intraperitoneally into mice twice a week for 3 weeks, and tumor volume and body weight were monitored twice a week throughout the study period.

[0304] 7.1.4.2. Calculation of Tumor Size and Growth Inhibition For each group, the mean tumor size, median tumor size, and tumor growth inhibition rate compared to the control group were calculated. Tumor length and width were measured twice a week with calipers, and tumor volume was calculated using the formula (length × width). 2 The calculation was performed using ) / 2. The measurement was performed when the mean tumor size of the control group was 4000 mm. 3 This was continued until the tumor growth inhibition was reached, or until mice in either group required euthanasia due to ulceration or weight loss exceeding 20%. Tumor growth inhibition was calculated according to the following formula: [1-(T final -T initial ) / (C final -C initial )]*100, in the formula, T (treatment 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 dose) to determine the frequency of tumor-free mice.

[0305] 7.2. Example 1: Activity of IL10 mutein in STAT3 reporter assay The ability of recombinant IL10 mutein to stimulate the IL10 receptor was evaluated using a bioassay based on STAT3 reporter cells as described in Section 7.1.2.

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

[0307] No increase in luciferase activity was detected when reporter cells were treated with the control protein. 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). Luciferase activity was also induced by incubation of Ramos / STAT3-Luc and TF-1 / STAT3-Luc with IL10 mutein (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 EC50 values ​​below nanomolar, as shown in Table 4 and Figure 3.

[0308] [Table 4]

[0309] 7.3. Example 2: Activity of IL10 mutein on cytokine release in primary human T cells The ability of IL10 to inhibit T cell stimulation was evaluated using a functional primary T cell assay that measures the production of IL2, TNFα, and IFNγ cytokines.

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

[0311] Co-incubation of T cells derived from two donors (Figure 4: Donor 5500Y and Figure 5: Donor 6900M) and allogeneic PBMCs treated with mitomycin C resulted in measurable IL2 (A), TNFα (B), and IFNγ release (C). Addition of human IL10 or IL10 Fc mutein titration during T cell / PBMC co-incubation 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 mutein The activity of IL10 mutein was evaluated in a syngeneic tumor allogeneic transplantation model. Colon 26 tumor cells were transplanted into BALB-C mice as described in Section 7.1.4. On day 11, the tumors had an average volume of 110 mm². 3 (80mm 3 From 140mm 3When the mice reached the specified range, they were randomized into groups (n=7 mice / group) and administered either the hIgG4s isotype control, IL10M1, or IL10M2 in equimolar amounts of the isotype control (0.167 mg / kg) or IL10 mutain (0.1 mg / kg).

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

[0316] [Table 7]

[0317] At day 32, when all mice were still alive, six doses of IL10M1 resulted in approximately 90% inhibition of tumor growth, while IL10M2 administration had only a slight effect (approximately 7% inhibition of tumor growth) compared to the isotype control group. Upon extended observation, 4 out of 7 mice in the IL10M1 group became tumor-free, compared to only 2 out of 7 mice in the IL10M2 group, and there were no tumor-free mice in the control group. These data support the conclusion that IL10 fused to the C-terminus of hIgG4s-Fc (IL10M1) exhibits superior efficacy compared to 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 mutaine activity The activity of IL10 mutaine with different linker lengths (including two different lots each of IL10M1 and IL10M2, referred to as Lot 1 and Lot 2 or L1 and L2, respectively) was evaluated using Ramos / STAT3-Luc and TF-1 / STAT3-Luc assays as described in Section 7.1.2. Cells were collected, centrifuged, resuspended in assay medium (Jurkat medium), and then stored in 50 μl volumes of 5 × 10⁶ cells in a flat-bottomed culture dish with a white bottom / sides. 5Each cell / well was plated, and then 50 μl of medium containing titration of IL10 or IL10 mutein was added.

[0319] Cells were incubated for 5.5 hours, after which 100 μL of ONE-Glo® luciferase (Promega Corporation, Madison, Wisconsin, USA) was added. This plate was incubated in the presence of ONE-Glo® for 3 minutes, and the activity was read using an Envision® fluorescence plate reader (Perkin Elmer, Waltham, Massachusetts, USA).

[0320] The induction factor and EC50 were determined as described in Section 7.1.2.4. The results are summarized in Tables 8-1 and 8-2 and shown in Figures 7A to 7F.

[0321] [Table 8]

[0322] These results show that IL10 muteins with the IL10 moiety at the C-terminus of the Fc moiety are more active than IL10 muteins with the IL10 moiety at the N-terminus of the Fc moiety, and EC 50 This indicates that the value is low. Furthermore, the G4S (SEQ ID NO: 51) linker length is low in terms of the induction multiplier and EC. 50 Although the effect is minimal, a complete loss of the linker from the IL10 mutaine located at the C-terminus of the Fc moiety in the IL10 region results in a greater decrease in activity.

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

[0324] In short, female mice (Jackson Laboratory, located in Bar Harbor, Maine, USA) were subcutaneously transplanted with tumor cells from a matching strain (Tissue Culture Core, Regeneron Pharmaceuticals, Inc.): C57BL / 6 syngeneic tumor cells MC38-cOVA (MC38 colon cancer cells engineered to overexpress chicken ovalbumin); BALB / c syngeneic tumor cells Colon26 (colon cancer), A20 (B-cell lymphoma), 4T1 (breast cancer), or RENCA (renal cell carcinoma).

[0325] In the prophylactic setting, treatment was initiated two days before MC38-cOVA tumor transplantation and continued twice weekly. In the therapeutic setting, the mean tumor volume (typically 60–120 mm) 3 Mice were randomly assigned to treatment groups (n=7-9) based on the following criteria. Treatment was initiated after tumor randomization and continued twice weekly.

[0326] IL10M11 or isotype controls were administered intraperitoneally at the same molar dose (6-9 doses per mouse). In IL10M11 dose-finding studies, the dose of the isotype control was equivalent to the maximum molar dose of IL10M11.

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

[0328] 7.6.1. Materials and Methods Tumor transplantation and treatment group assignment: For MC38-cOVA, Colon26, and RENCA syngeneic tumor models, 1 × 10¹⁶ female C57BL / 6 or BALB / c mice (Jackson Laboratory, Bar Harbor, Maine, USA) were transplanted into the right flank. 6 Each tumor cell (Tissue Culture Core, Regeneron Pharmaceuticals, Inc.) was subcutaneously transplanted. For the A20 and 4T1 syngeneic tumor models, 1 × 10⁶ cells were transplanted. 7 individual or 1 x 10 5 Nucleic tumor cells (Tissue Culture Core, Regeneron Pharmaceuticals, Inc.) were subcutaneously transplanted into the right flank of 6-8 week old female BALB / c mice (Jackson Laboratory, Bar Harbor, Maine, USA). The tumors averaged 60-120 mm in volume. 3 At the point when (according to each unique tumor model) mice reached a certain stage, they were randomized into groups (n=7-9 mice / group) and administered IL10M11, CD40, PD-1, a control reagent, or, in some cases, a combination thereof. The test substances were administered intraperitoneally to the mice twice a week for 6-9 sessions, and tumor volume and body weight were monitored twice a week throughout the study period. When the antitumor memory response was tested, mice that rejected the primary Colon26 tumor load showed a response of 5 × 10⁶. 6 While the mice received a dose of 1 x 10¹¹ Colon26 tumor cells, the naive mice received 1 x 10¹¹ 6 They were only able to receive a single dose of Colon26 tumor cells.

[0329] Tumor size and growth inhibition calculation: For each group, the mean and median tumor size, as well as the tumor growth inhibition rate compared to the control treatment group, were calculated. Tumor length and width were measured twice weekly with calipers, and tumor volume was calculated using the formula (length × width). 2The calculation was performed using ) / 2. The measurement was performed when the mean tumor size of the control group was 4000 mm. 3 The procedure was continued until the tumor growth inhibition rate was reached, or until mice in either group required euthanasia due to ulcers or weight loss exceeding 20%. The tumor growth inhibition rate was calculated using the following formula: [1-(T final -T initial ) / (C final -C initial )*100% is used for calculation, where T (treatment group) and C (control group) represent the mean tumor mass on the days when all mice were alive.

[0330] 7.6.2.Results Prophylactic treatment of MC38-cOVA tumors with the maximum test dose of IL10M11 (0.1 mg / kg) showed a slight antitumor effect, with tumor growth inhibition (TGI) of approximately 30% and tumor-free mice (1 out of 8 mice) (Figure 8A). Low doses (0.005 or 0.0003 mg / kg) showed 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 tumor-free mice (4 out of 7 mice) (Figure 8B).

[0331] The therapeutic efficacy 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 the Colon26 and A20 models (Figures 8C and 8D). Significant TGI was also observed in the 4T1 tumor model, including inhibition of growth in large established tumors exceeding 100 mm3; however, tumor-free survival was not achieved (Figure 8E).

[0332] The 4T1 tumors collected at the end of the study were dissociated and subjected to flow cytometry analysis of the composition and function of immune cells. 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 (Figure 8F and Figure 8G).

[0333] Mice that survived primary Colon26 or A20 tumor loading underwent reloading tests with bilateral transplantation of either Colon26 and RENCA (Figure 8H) or A20 and Colon26 (Figure 8I) tumors. Both the positive control bilateral tumors of Colon26 and RENCA, or A20 and Colon26, proliferated rapidly when transplanted into naive mice. However, mice that completely rejected the primary tumor with IL10M11 therapy induced a long-term antitumor memory response and rejected secondary loading with the same tumor, but did not reject unrelated tumor types (Figures 8H and 8I).

[0334] Finally, the antitumor effects of IL10M11 in combination with either a CD40 agonist or a PD-1 antagonist were determined. IL10M11, CD40Ab, and PD-1Ab each showed an antitumor response as monotherapy (Figure 9A). Combinations of any two of these therapies 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 Using multiple syngeneic tumor allograft models, we evaluated immune infiltration in the tumor microenvironment pretreatment as a baseline, and used this to compare the in vivo antitumor efficacy of mouse IL-10 fused to mouse IgG1 (IL10M11) and the anti-PD-1 antagonist Ab (mouse IgG1) in a therapeutic setting.

[0336] In short, female mice (Jackson Laboratory, located in Bar Harbor, Maine, USA) were subcutaneously transplanted with strain-matched tumor cells: C57BL / 6 mice carrying MC38 colon cancer cells and B16F10 melanoma cells, or BALB / c mice carrying Colon26 colon cancer cells and A20 B-cell lymphoma cells (all cells were from Regeneron Pharmaceuticals, Inc.'s Tissue Culture Core). The tumor size was approximately 100 mm. 3 Upon reaching a certain stage, the tumor was removed and subjected to standard protocols for immunoprofiling of CD45+ immune cells and listed subsets within the CD45 gate. In some cases, along with the tumor, the spleen and inflow region lymph nodes were subjected to analysis of IL10R1, PD1, or PD-L1 surface expression on immune cells / tumor cells. In therapeutic settings, treatment was administered a few days after tumor transplantation, typically when the tumor size was an average volume of 75–150 mm. 3 The procedure was performed when the tumor size reached a certain stage (depending on the type of tumor), and mice were randomly assigned to one of several different treatment groups (n=7-8 mice / group). Mice were administered either an mIgG1 isotype control, IL10M11, or an anti-mouse PD1 antibody. Treatment, tumor size, and body weight measurements were performed twice a week.

[0337] 7.7.1. Materials and Methods Tumor transplantation and treatment group allocation: For syngeneic tumor models of B16F10, MC38, MC38-cOVA, Colon26, and A20, 0.5–10 × 10 6Individual tumor cells (Tissue Culture Core of Regeneron Pharmaceuticals, Inc.) were each subcutaneously implanted into the right flank of 6- to 8-week-old female C57BL / 6 or BALB / c mice (from the Jackson Laboratory, located in Bar Harbor, Maine, USA). When the tumors reached an average volume of 75-150 mm 3 (depending on each specific tumor model), the mice were randomized into each group (n = 7-8 mice / group), and IL10M11, PD1 Ab, PD1-IL10, control reagents, or in some cases combinations thereof were administered. The test substance was intraperitoneally injected into the mice twice a week for 6-9 times, and tumor volume and body weight were monitored twice a week throughout the test period.

[0338] Calculation of tumor size and growth inhibition: For each group, the mean and median of tumor size, and the tumor growth inhibition rate compared to the control treatment group were calculated. The length and width of the tumor were measured with calipers twice a week, and the tumor volume was calculated using the formula (length × width 2 ) / 2. The measurements were performed until the average tumor size of the control group reached 4000 mm 3 , or until the mice in any group required euthanasia due to ulceration or weight loss exceeding 20%. Tumor growth inhibition was calculated according to the following formula: [1 - (T final - T initial ) / (C final - C[[ID=1十七]] initial )] * 100 (where T (treatment group) and C (control group) represent the average tumor amount on days when all mice were alive). Observation was extended until day 54 (3 weeks after the final treatment), and the frequency of tumor-free mice was determined.

[0339] MSD multiplexing of cytokines in serum was performed according to the standard protocol of MSD (Meso Scale Diagnostics, located in Rockville, Maryland, USA).

[0340] 7.7.2.Results As shown in Figures 10A–10E, the density of CD45+ immune cells as a whole, as well as all immune cell subsets analyzed in A20, Colon26, and MC38 tumors, was significantly higher than that of their counterparts in B16F10 tumors. Further analysis also showed that B16F10 tumors had fewer Ki67-proliferating CD4 and CD8 T cells and exhibited a memory phenotype.

[0341] As expected, IL10R1 was constitutively and extensively 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 very limited to tumor-infiltrating CD8 T cells and was hardly expressed on T cells in secondary lymphoid tissues, for example, in the spleen and influx region LN of MC38-cOVA and Colon 26 (see Figures 11A-11I). This study further revealed that almost all IL10R1+ CD8 T cells express the PD1 molecule. This data suggests 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 explain the biological reasons behind the favorable safety profile of IL10M11 in mice; and 3) the combination of IL10-Fc and the PD-1 antagonist Ab is a possible strategy to enhance the antitumor response.

[0342] Differences in PD-L1 expression were observed among the four tumor types tested (Figures 12A-12G). Next, the response of each tumor type to treatment was determined by comparing the PD1 antagonist Ab with IL10M11. As shown in Figures 13A-13C, 150 mm 3A20 B-cell lymphoma responded very well to PD-1Ab treatment, showing a significant delay in tumor growth and approximately 50% tumor-free survival. However, IL10M11 induced an even more advanced and extensive immune response against A20, resulting in approximately 85% tumor-free survival. In the Colon26 tumor model (Figures 14A-14C), 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 have low immunogenicity and far less immune cell infiltration, which was confirmed in our study (Figures 10A-10E). 100mm 3 The B16F10 tumor showed little response to PD1 Ab. However, IL10M11 significantly delayed tumor growth and made the majority of the tumor responsive (Figures 15A-15C). 90-100 mm 3 Based on observations that the MC38 tumor showed little response to PD1 Ab (see Figures 22A-22B), the 75mm tumor was found to be... 3 The study was conducted on MC38 colon cancer. In this study, both PD1 Ab and IL10-Fc showed similar antitumor effects when used as monotherapy, but when used in combination, the antitumor response dramatically increased, as tumor growth was delayed and tumor-free survival rates (Figures 16A-16C) were improved.

[0343] To elucidate the relevant mechanisms of action of IL10-Fc (e.g., IL10M11), we performed immunoprofiling of the tumor microenvironment after treatment. IL10 treatment significantly increased CD8 T cell density in both immunogenic MC38 tumor models (Figure 17A) and less immunogenic 4T1 tumor models (Figure 17B), and in both cases, correlated with a better prognosis. 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 that had been activated (Figures 18A-18B). IL10M11 tends to increase overall CD45 immune infiltration, including CD4 T cells and myeloid cells, in addition to CD8 T cells, in the 4T1 tumor model (Figures 17A-17B and 18A-18B), which provides a reasonable explanation for why IL10 functioned in less immunogenic tumor models such as 4T1 and B16F10.

[0344] To identify potential pharmacodynamic (PD) markers of IL10-Fc, serum levels of various cytokines were analyzed using a commercially available multiplex MSD platform. In both naive non-tumor-bearing mice (Figure 19) and B16F10 tumor-bearing mice (Figure 20), the signature panel of cytokines (IL12, IL1b, IL2, IL4, and IL5) were upregulated in a manner specific to IL10 but not to PD1 Ab. When normalized to the mean of isotype-controlled 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–21B). Some samples were collected from the same tumor model at different time points (MC38 and Colon26), but still showed a consistent trend.

[0345] Based on previous findings regarding the co-expression of PD1 and IL10R1, and the enhancement of antitumor efficacy by combining IL10-Fc with the PD-1 antagonist Ab, we investigated the potential of PD1-targeted IL10 as a therapeutic agent. Clinical trials have reported some toxicity with high doses of pagirodecaquine (PEGylated IL10). It is appropriate to normalize the molar amount of IL10, rather than the molar amount of PD1, across different therapeutic modes (IL10-Fc, control Ab-IL10, PD1-IL10, and PD1 Ab). As shown in Figures 22A and 22B, PD1 Ab and IL10-Fc each demonstrated the expected antitumor effect as monotherapy (a very slight effect with PD1 Ab and a moderate effect with IL10-Fc). The combination of IL10-Fc and PD-1Ab significantly improved efficacy, but PD1-IL10-Fc did not show any difference in efficacy compared to the control Ab-IL10 or IL10-Fc.

[0346] 7.8. Example 8: Purification of IL10 agonist Multiple iterations of IL10 agonists (i.e., IL10M1 to IL10M11) were evaluated to determine their ability to be recombinetically expressed and recovered. IL10 agonists with the IL10 moiety located at the N-terminus or C-terminus of the Fc domain were recoverable, but all configurations resulted in a significant amount of aggregated IL10 agonist. A purification method was developed to produce non-aggregated IL10 agonists.

[0347] 7.8.1. Materials and Methods As described in Section 7.1, constructs encoding IL10 mutain were prepared, recombinantly expressed in mammalian cell lines, and purified. The cells were lysed, and the cell lysate in PBS buffer was passed through 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 at pH 6.6. The buffer was then replaced by dialysis with 1× TBS + 5% glycerol, followed by 2× PBS + 5% glycerol. The replaced buffer eluate was then subjected to size exclusion chromatography using a Superdex® 200, 26 / 600 pg column (MilliporeSigma, Inc., St. Louis, Missouri, USA). Fractions containing non-aggregating IL10 agonists were collected.

[0348] 7.8.2.Results Since IL10 agonists have the IL10 moiety at the C-terminus of the Fc domain, this purification protocol produced intact, non-aggregating IL10 agonists that did not exhibit the abnormal peaks observed in size exclusion ultrahigh-performance liquid chromatography. IL10 agonists with the IL10 moiety at the N-terminus of the Fc domain were also recovered, but these were not intact, lacking the C-terminal lysine or being recovered as fragments containing amino acids 3-402, compared to the full-length construct which contains residues 1-403.

[0349] Purified, non-aggregating IL10 agonists can be formulated as pharmaceutical compositions with excipients, and these formulations contain minimal IL10 agonist aggregates.

[0350] 8. Specific embodiments and citation of references While various specific embodiments have been described and explained, it will be understood that various modifications can be made without departing from the spirit and scope of this disclosure. This disclosure is illustrated by the numbered embodiments shown below.

[0351] In preferred embodiments of the numbered embodiments and subsequent claims described below, the IL10 domain, Fc domain, and variants thereof preferably include the amino acid sequences of human IL10, human Fc domain, and variants thereof, for example, 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 with such human sequences.

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

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

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

[0355] 6. An IL10 agonist according to any one of Embodiments 1 to 5, wherein the Fc domain is IgG1. 7. The IL10 agonist according to any one of Embodiments 1 to 6, wherein the IgG1 is human IgG1.

[0356] 8. An IL10 agonist according to any one of Embodiments 1 to 6, wherein the IgG1 is mouse IgG1. 9. An IL10 agonist according to any one of Embodiments 1 to 5, wherein the Fc domain is IgG4.

[0357] 10. The IL10 agonist according to Embodiment 9, wherein the IgG1 is human IgG4. 11. The IL10 agonist according to Embodiment 9, wherein the IgG1 is mouse IgG4.

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

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

[0360] 14. An IL10 agonist according to 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 with SEQ ID NO: 31.

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

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

[0363] 17. An IL10 agonist according to 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. An IL10 agonist according to any one of Embodiments 1 to 7, wherein the Fc domain comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 33.

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

[0365] 20. An IL10 agonist according to 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 with SEQ ID NO: 33.

[0366] 21. An IL10 agonist according to 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 with SEQ ID NO: 33.

[0367] 22. An IL10 agonist according to 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 with SEQ ID NO: 33.

[0368] 23. An IL10 agonist according to 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. An IL10 agonist according to any one of embodiments 1 to 17, wherein the effector function of the Fc domain is reduced.

[0369] 25. An IL10 agonist according to 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. An IL10 agonist according to any one of Embodiments 5 to 24, wherein the hinge domain is derived from an IgG different from the CH2 and / or CH3 domain.

[0370] 27. An IL10 agonist according to any one of Embodiments 2 to 26, wherein the hinge domain includes a chimeric hinge array. 28. The IL10 agonist according to 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 according to 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 the linker is 10 to 60 amino acid residues in length.

[0372] 31. The IL10 agonist according to any one of Embodiments 1 to 18, wherein the 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 The IL10 agonist according to any one of Embodiments 1 to 32, comprising a monomer or multimer thereof, and optionally, n is an integer from 1 to 7.

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

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

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

[0377] 40. The IL10 agonist according to Embodiment 35, wherein the linker includes (G4S)5. 41. The IL10 agonist according to Embodiment 35, wherein the linker includes (G4S)6.

[0378] 42. An IL10 agonist according to any one of embodiments 1 to 41, wherein the linker connects the Fc domain and the IL10 portion. 43. An IL10 agonist according to any one of Embodiments 1 to 41, wherein the IL10 portion is located on the C-terminal side of the Fc domain.

[0379] 44. An IL10 agonist according to any one of Embodiments 1 to 41, wherein the IL10 portion is located on the N-terminal side of the Fc domain. 45. An IL10 agonist according to any one of Embodiments 1 to 41, wherein the IL10 portion includes an amino acid sequence having at least 95% sequence identity with the sequence of mature human IL10 (for example, IL10 having the amino acid sequence of SEQ ID NO: 1).

[0380] 46. ​​An IL10 agonist according to any one of Embodiments 1 to 42, wherein the IL10 portion includes an amino acid sequence having at least 96% sequence identity with the sequence of mature human IL10 (for example, IL10 having the amino acid sequence of SEQ ID NO: 1).

[0381] 47. An IL10 agonist according to any one of Embodiments 1 to 43, wherein the IL10 portion includes an amino acid sequence having at least 97% sequence identity with the sequence of mature human IL10 (for example, IL10 having the amino acid sequence of SEQ ID NO: 1).

[0382] 48. An IL10 agonist according to any one of Embodiments 1 to 44, wherein the IL10 portion includes an amino acid sequence having at least 98% sequence identity with the sequence of mature human IL10 (for example, IL10 having the amino acid sequence of SEQ ID NO: 1).

[0383] 49. An IL10 agonist according to any one of Embodiments 1 to 45, wherein the IL10 portion comprises an amino acid sequence having at least 99% of the sequence of mature human IL10 (for example, IL10 having the amino acid sequence of SEQ ID NO: 1).

[0384] 50. An IL10 agonist according to any one of Embodiments 1 to 49, wherein the IL10 portion comprises the amino acid sequence of mature human IL10 (for example, IL10 having the amino acid sequence of SEQ ID NO: 1).

[0385] 51. An IL10 agonist according to 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 (for example, IL10 having the amino acid sequence of SEQ ID NO: 30).

[0386] 52. An IL10 agonist according to 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 (for example, IL10 having the amino acid sequence of SEQ ID NO: 30).

[0387] 53. An IL10 agonist according to 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 (for example, IL10 having the amino acid sequence of SEQ ID NO: 30).

[0388] 54. An IL10 agonist according to 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 (for example, IL10 having the amino acid sequence of SEQ ID NO: 30).

[0389] 55. An IL10 agonist according to 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 (for example, IL10 having the amino acid sequence of SEQ ID NO: 30).

[0390] 56. An IL10 agonist according to any one of embodiments 1 to 41 and 51 to 55, wherein the IL10 portion comprises the amino acid sequence of mature mouse IL10 (for example, IL10 having the amino acid sequence of SEQ ID NO: 30).

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

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

[0393] 60. An IL10 agonist according to any one of embodiments 1 to 56, which is a homodimer. 61. An IL10 agonist according to Embodiment 58, comprising two of the aforementioned Fc domains.

[0394] 62. The IL10 agonist according to Embodiment 61, which is dimerized via the Fc domain. 63. The IL10 agonist according to Embodiment 62, wherein the Fc domain includes an amino acid sequence named hIgG4-Fc (for example, Fc having the amino acid sequence of amino acids 18-228 of SEQ ID NO: 31).

[0395] 64. The IL10 agonist according to Embodiment 62, wherein the Fc domain comprises an amino acid sequence named hIgG4s-Fc (for example, Fc having the amino acid sequence of SEQ ID NO: 31).

[0396] 65. The IL10 agonist according to Embodiment 62, wherein the Fc domain comprises an amino acid sequence named mIgG1-Fc (for example, Fc having the amino acid sequence of SEQ ID NO: 33).

[0397] 66. The IL10 agonist according to Embodiment 61, wherein the Fc domain is non-dimerizable. 67. An IL10 agonist according to any one of embodiments 58 to 67, comprising two IL10 parts.

[0398] 68. The IL10 agonist according to embodiment 67, which is dimerized via the IL10 portion. 69. The IL10 agonist according to embodiment 67, wherein the IL10 portion is non-dimerizable.

[0399] 70. An IL10 agonist according to any one of embodiments 1 to 56, which is a heterodimer. 71. An IL10 agonist according to Embodiment 70, comprising two of the aforementioned Fc domains.

[0400] 72. The IL10 agonist according to Embodiment 71, which is dimerized via the Fc domain. 73. The IL10 agonist according to Embodiment 71, wherein the Fc domain is non-dimerizable.

[0401] 74. An IL10 agonist according to any one of embodiments 70 to 73, comprising two IL10 parts. 75. The IL10 agonist according to embodiment 74, which is dimerized via the IL10 portion.

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

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

[0404] 79. An 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. An IL10 agonist according to Embodiment 78 or Embodiment 79, comprising an amino acid sequence having at least 97% sequence identity with the amino acid sequence of IL10M1.

[0405] 81. An IL10 agonist according to any one of embodiments 78 to 80, comprising an amino acid sequence having at least 98% sequence identity with the amino acid sequence of IL10M1. 82. An IL10 agonist according to any one of embodiments 78 to 81, comprising 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. An 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. An IL10 agonist according to Embodiment 84 or Embodiment 85, comprising an amino acid sequence having at least 97% sequence identity with the amino acid sequence of IL10M2.

[0408] 87. An 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. An 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. An 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. An IL10 agonist according to Embodiment 90 or Embodiment 91, comprising an amino acid sequence having at least 97% sequence identity with the amino acid sequence of IL10M3.

[0411] 93. An 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. An 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. An 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. An IL10 agonist according to Embodiment 96 or Embodiment 97, comprising an amino acid sequence having at least 97% sequence identity with the amino acid sequence of IL10M4.

[0414] 99. An 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 (i.e., an IL10 agonist having the amino acid sequence of SEQ ID NO: 38).

[0416] 103. An 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. An IL10 agonist according to Embodiment 102 or Embodiment 103, comprising an amino acid sequence having at least 97% sequence identity with the amino acid sequence of IL10M5.

[0417] 105. An IL10 agonist according to any one of embodiments 102 to 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 (i.e., an IL10 agonist having the amino acid sequence of SEQ ID NO: 39).

[0419] 109. An 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. An IL10 agonist according to Embodiment 108 or Embodiment 109, comprising an amino acid sequence having at least 97% sequence identity with the amino acid sequence of IL10M6.

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

[0421] 113. An IL10 agonist according to any one of embodiments 108 to 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 (i.e., an IL10 agonist having the amino acid sequence of SEQ ID NO: 40).

[0422] 115. An 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. An IL10 agonist according to Embodiment 114 or Embodiment 115, comprising an amino acid sequence having at least 97% sequence identity with the amino acid sequence of IL10M7.

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

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

[0425] 121. An 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. An IL10 agonist according to Embodiment 120 or Embodiment 121, comprising an amino acid sequence having at least 97% sequence identity with the amino acid sequence of IL10M8.

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

[0427] 125. An 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 (i.e., an IL10 agonist having the amino acid sequence of SEQ ID NO: 42).

[0428] 127. An 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. An IL10 agonist according to Embodiment 126 or Embodiment 127, comprising an amino acid sequence having at least 97% sequence identity with the amino acid sequence of IL10M9.

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

[0430] 131. An 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 (i.e., an IL10 agonist having the amino acid sequence of SEQ ID NO: 43).

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

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

[0433] 137. An IL10 agonist according to any one of embodiments 132 to 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 (i.e., an IL10 agonist having the amino acid sequence of SEQ ID NO: 44).

[0434] 139. An 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. An IL10 agonist according to Embodiment 138 or Embodiment 139, comprising an amino acid sequence having at least 97% sequence identity with the amino acid sequence of IL10M11.

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

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

[0437] 145. The IL10 agonist according to Embodiment 144, wherein the targeting portion is located on the N-terminal side of the Fc domain, and if a hinge domain is present, the targeting portion is located on the N-terminal side of the hinge domain.

[0438] 146. The targeting portion is (a) Binds to tumor-associated antigens; (b) Binds to tumor microenvironment antigens; (c) Binding to cell surface molecules of tumor-reactive lymphocytes; or (d) Binds to checkpoint inhibitors, An IL10 agonist according to Embodiment 144 or Embodiment 145.

[0439] 147. The IL10 agonist according to embodiment 146, wherein the targeting portion binds to a tumor-associated antigen. 148. The tumor-associated antigen is fibroblast-activating protein (FAP), the A1 domain of tenascin-C (TNC A1), and the A2 domain of tenascin-C (TNC A2) Fibronectin extradomain B (EDB), melanoma-associated chondroitin sulfate proteoglycan (MCSP), MART-1 / Melan A, gp100, dipeptidyl peptidase IV (DPPIV), adenosine deaminase-binding protein (ADAbp), cyclophyllin 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 / CD3ζ 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-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), GAGE ​​family tumor antigens (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 protein (APC), fodrin, connexin 37, Ig idiotype, p15, gp75, GM2 and GD2 gangliosides, viral products, e.g., human papillomavirus protein, tumor antigens of the Smad family, Imp-1, P1A, nuclear antigen encoded by EBV (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-related heteromultimers), 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 (IL-6 receptor), CD20, MCSP, PDGFβR (β-platelet-derived growth factor receptor), ErbB2 epithelial cell adhesion molecule (EpCAM), EGFR variant III (EGFRvIII), CD19, disiaroganglioside GD2, ductal epithelial mucin, gp36, TAG-72, glioma-associated antigens, β-human chorionic gonadotropin, α-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, MN-CA An IL10 agonist according to Embodiment 147, wherein the agonist is 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, survivorbin 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 A1 domain (TnC A1) of tenascin-C.

[0440] 149. The IL10 agonist according to Embodiment 147, wherein the tumor-associated antigen is a viral antigen. 150. The IL10 agonist according to 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 portion 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 according to Embodiment 152, wherein the extracellular matrix protein is 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 portion binds to the cell surface molecules of tumor-reactive lymphocytes. 155. The IL10 agonist according to 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 portion binds to a checkpoint inhibitor. 157. The IL10 agonist according to 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. An IL10 agonist according to Embodiment 144 or Embodiment 145, wherein the targeting portion binds to an MHC-peptide complex.

[0446] 160. The IL10 agonist according to Embodiment 159, wherein the peptide in the peptide-MHC complex contains a tumor neoantigen. 161. The tumor neoantigens are 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. An IL10 agonist according to 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. An IL10 agonist according to any one of embodiments 144 to 161, wherein the targeting portion is an antibody or an antigen-binding fragment thereof. 163. The IL10 agonist according to embodiment 162, wherein the targeting portion is Fab.

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

[0449] 166. An IL10 agonist according to any one of embodiments 1 to 164, lacking a targeting domain that binds to a checkpoint inhibitor, wherein the checkpoint inhibitor is optionally PD1.

[0450] 167. An IL10 agonist according to any one of embodiments 1 to 139, lacking a targeting portion. 168. Targeted portion, for example, not limited to, (a) Binds to tumor-associated antigens; (b) Binds to tumor microenvironment antigens; (c) Binding to cell surface molecules of tumor-reactive lymphocytes; or (d) Binds to checkpoint inhibitors An IL10 agonist according to any one of embodiments 1 to 99 and 167, lacking a targeting portion.

[0451] 169. An IL agonist according to embodiment 168, lacking a targeting portion that binds to PD1. An IL10 agonist according to any one of embodiments 1 to 139 and 165, lacking the 170.CH1 domain.

[0452] 171. An IL10 agonist according to any one of embodiments 1 to 139, 165, and 168, lacking a CL domain. 172. An IL10 agonist according to any one of embodiments 1 to 139 and 165 to 171, lacking an antibody variable region.

[0453] 173. An IL10 agonist according to any one of Embodiments 1 to 172, further comprising a hydrophilic polymer. 174. The IL10 agonist according to 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 in the range of about 7.5 kDa to about 80 kDa. 176. The IL10 agonist according to Embodiment 175, wherein the PEG has a molecular weight in the range of about 30 kDa to about 60 kDa, and optionally the molecular weight is about 50 kDa.

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

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

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

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

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

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

[0461] 189. An IL10 agonist according to any one of embodiments 1 to 172 and 179 to 187, which does not contain O-linked glycans. 190. An IL10 agonist according to any one of embodiments 1 to 172 and 179 to 189, lacking a stabilizing portion.

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

[0463] 193. An IL10 agonist according to any one of Embodiments 1 to 192, wherein the IL10 portion does not contain an Fc domain at the C-terminal end. 194. An IL10 agonist according to any one of Embodiments 1 to 193, which is a product of recombinant cell expression in mammalian cells.

[0464] 195. The IL10 agonist according to Embodiment 194, wherein the mammalian cells are from a mouse cell line. 196. The IL10 agonist according to Embodiment 194, wherein the mammalian cells are from a rat cell line.

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

[0466] 199. One or more nucleic acids encoding an IL10 agonist as described in any one of Embodiments 1 to 198. 200. Host cells manipulated 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 according to Embodiment 200, wherein the host cell is a mouse cell line. 202. The IL10 agonist according to Embodiment 200, wherein the mammalian cells are from a rat cell line.

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

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

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

[0471] 207. The method according to Embodiment 206, wherein the affinity column is a protein A affinity column. 208.(a) A step of culturing host cells according to any one of embodiments 200 to 204; and (b) A step of recovering the IL10 agonist expressed thereby. IL10 agonists produced by methods including the following.

[0472] 209. The above method, (a) The step of bringing the recovered IL10 agonist into contact with an affinity column configured to capture the IL10 agonist; (b) A step of eluting from the affinity column to produce an eluate; and (c) A step of performing size exclusion chromatography on the eluate. The IL10 agonist according to embodiment 208 further includes the following:

[0473] 210. A method for preparing a pharmaceutical composition, comprising the step of formulating an IL10 agonist prepared by any one of the methods of Embodiments 205 to 207 together with an excipient. 211. A pharmaceutical composition comprising one IL10 agonist from any one of embodiments 1 to 198 and an excipient.

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

[0475] 214. A pharmaceutical composition according to Embodiment 210 or Embodiment 211, comprising at least 20 mg of the IL10 agonist. 215. A pharmaceutical composition according to Embodiment 210 or Embodiment 211, comprising at least 50 mg of the IL10 agonist.

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

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

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

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

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

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

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

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

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

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

[0486] 230. The method according to Embodiment 225, wherein the solid tumor is a tumor of the pancreas. 231. The method according to Embodiment 225, wherein the solid tumor is a tumor of the lung. 232. The method according to any one of Embodiments 224 to 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 according to any one of embodiments 224 to 234, wherein the solid tumor is resistant to treatment with an anti-PD1 antibody. 236. The method according to any one of embodiments 224 to 235, wherein the solid tumor is resistant to treatment with anti-PD1 antibody monotherapy.

[0489] 237. The method according to any one of embodiments 224 to 236, further comprising the step of administering an anti-PD1 antibody to a target. 238. The method according to any one of Embodiments 235 to 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 according to any one of embodiments 225 to 238, which results in an increase in CD8 T cell density in the solid tumor. 240. The method according to Embodiment 239, wherein the increase in CD8 T cell density in the solid tumor is at least a twofold increase.

[0491] 241. The method according to Embodiment 239, wherein the increase in CD8 T cell density in the solid tumor is at least a threefold increase. 242. The method according to Embodiment 239, wherein the increase in CD8 T cell density in the solid tumor is at least a fourfold increase.

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

[0493] 245. CD45 in the solid tumor + The method according to Embodiment 243, wherein the aforementioned increase in immune cell infiltration is a 10% increase. 246. CD45 in the aforementioned solid tumor + The method according to Embodiment 243, wherein the aforementioned increase in immune cell infiltration is a 15% increase.

[0494] 247. CD45 in the solid tumor + The method according to Embodiment 243, wherein the aforementioned increase in immune cell infiltration is a 20% increase. 248. The aforementioned CD45 + The method according to any one of embodiments 243 to 247, wherein the immune cells include CD4 T cells, myeloid cells, or both CD4 T cells and myeloid cells.

[0495] 249. The method according to any one of Embodiments 225 to 248, wherein the method upregulates serum IL12 compared to a preferred control that has not been treated with the IL10 agonist or pharmaceutical composition, or compared to the serum IL12 level of the subject itself before treatment with the IL10 agonist or pharmaceutical composition.

[0496] 250. The method according to Embodiment 249, wherein the method upregulates the serum IL12 level by at least twofold compared to the control or compared to the serum IL12 level of the subject itself before treatment with the IL10 agonist or pharmaceutical composition.

[0497] 251. The method according to Embodiment 249, wherein the method upregulates the serum IL12 level by at least five times compared to the control or compared to the serum IL12 level of the subject itself before treatment with the IL10 agonist or pharmaceutical composition.

[0498] 252. The method according to Embodiment 249, wherein the method upregulates the serum IL12 level by at least 10 times compared to the control or compared to the serum IL12 level of the subject itself before treatment with the IL10 agonist or pharmaceutical composition.

[0499] 253. The method according to Embodiment 249, wherein the method upregulates the serum IL12 level by at least 15 times compared to the control or compared to the serum IL12 level of the subject itself before treatment with the IL10 agonist or pharmaceutical composition.

[0500] 254. The method according to Embodiment 249, wherein the method upregulates the serum IL12 level by at least 20 times compared to the control or compared to the serum IL12 level of the subject itself before treatment with the IL10 agonist or pharmaceutical composition.

[0501] 255. The method according to Embodiment 249, wherein the method upregulates the serum IL12 level by at least 25 times compared to the control or compared to the serum IL12 level of the subject itself before treatment with the IL10 agonist or pharmaceutical composition.

[0502] 256. The method according to Embodiment 249, wherein the method upregulates the serum IL12 level by at least 30 times compared to the control or compared to the serum IL12 level of the subject itself before treatment with the IL10 agonist or pharmaceutical composition.

[0503] 257. The method according to any one of Embodiments 225 to 256, wherein the method upregulates serum IL4 levels compared to a preferred control that has not been treated with the IL10 agonist or pharmaceutical composition, or compared to the serum IL4 levels of the subject itself before treatment with the IL10 agonist or pharmaceutical composition.

[0504] 258. The method according to Embodiment 257, wherein the method upregulates the serum IL4 level by at least twofold compared to the control or compared to the subject's own serum IL4 level before treatment with the IL10 agonist or pharmaceutical composition.

[0505] 259. The method according to Embodiment 257, wherein the method upregulates the serum IL4 level by at least 5 times compared to the control or compared to the subject's own serum IL4 level before treatment with the IL10 agonist or pharmaceutical composition.

[0506] 260. The method according to Embodiment 257, wherein the method upregulates the serum IL4 level by at least 10 times compared to the control or compared to the subject's own serum IL4 level before treatment with the IL10 agonist or pharmaceutical composition.

[0507] 261. The method according to Embodiment 257, wherein the method upregulates the serum IL4 level by at least 15 times compared to the control or compared to the subject's own serum IL4 level before treatment with the IL10 agonist or pharmaceutical composition.

[0508] 262. The method according to Embodiment 257, wherein the method upregulates the serum IL4 level by at least 20 times compared to the control or compared to the subject's own serum IL4 level before treatment with the IL10 agonist or pharmaceutical composition.

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

[0510] 264. The method according to Embodiment 263, wherein the IL10 agonist is administered to the subject within one week of the administration of the CART cells. 265. The method according to 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 according to any one of embodiments 263 to 265, comprising the step of administering the IL10 agonist to the subject over a period of at least two weeks. 267. The method according to embodiment 266, wherein the IL10 agonist is administered by continuous infusion.

[0512] 268. The method according to Embodiment 266, wherein the IL10 agonist is administered by daily doses for at least a portion of the period of at least two weeks. 269. The IL10 agonist, (a) administering the IL10 agonist at a first dosing frequency for the first part of the period of at least two weeks; and (b) The step of administering the IL10 agonist at a second dosing frequency for the subsequent portion of the period of at least two weeks. The method according to embodiment 266, administered according to a divided dosing regimen including the following.

[0513] 270. The method according to Embodiment 269, wherein the first drug administration frequency is daily. 271. The method according to Embodiment 269 or Embodiment 270, wherein the second drug administration frequency is lower than the first drug administration frequency.

[0514] 272. The method according to embodiment 271, wherein the second drug administration frequency is weekly. 273. The method according to any one of embodiments 269 to 272, wherein the subject transitions from a first drug administration frequency to a second drug administration frequency simultaneously with or after the depletion of the CART cells.

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

[0516] 276. The method according to any one of embodiments 263 to 275, further comprising the step of administering an anti-PD1 antibody to a target. 277. The method according to 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 aforementioned CARs include 5T4, α-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, and HER2. The method according to 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, κ chain, λ 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 according to any one of embodiments 263 to 278, wherein the CAR is configured according to Section 6.11.1 and its subsections. 280. The method according to any one of Embodiments 263 to 279, wherein the IL10 agonist is in the form of the pharmaceutical composition described in any one of Embodiments 211 to 223.

[0519] 281. The method according to any one of embodiments 263 to 280, which results in an increase in CD8 T cell density in the solid tumor. 282. The method according to Embodiment 281, wherein the increase in CD8 T cell density in the solid tumor is at least a twofold increase.

[0520] 283. The method according to Embodiment 281, wherein the increase in CD8 T cell density in the solid tumor is at least a threefold increase. 284. The method according to Embodiment 281, wherein the increase in CD8 T cell density in the solid tumor is at least a fourfold increase.

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

[0522] 286. CD45 in the aforementioned solid tumor + The method according to Embodiment 285, wherein the aforementioned increase in immune cell infiltration is a 5% increase. 287. CD45 in the aforementioned solid tumor + The method according to Embodiment 285, wherein the aforementioned increase in immune cell infiltration is a 10% increase.

[0523] 288. The method according to Embodiment 285, wherein the increase in CD45+ immune cell infiltration in the solid tumor is a 15% increase. 289. CD45 in the aforementioned solid tumor + The method according to Embodiment 285, wherein the aforementioned increase in immune cell infiltration is a 20% increase.

[0524] 290. The aforementioned CD45 + The method according to any one of embodiments 285 to 289, wherein the immune cells include CD4 T cells and myeloid cells. 291. The method according to any one of Embodiments 263 to 286, wherein the method upregulates serum IL12 levels compared to a preferred control that has not been treated with the IL10 or pharmaceutical composition.

[0525] 292. The method according to Embodiment 291, wherein the method upregulates the serum IL12 level by at least twofold compared to the control, or compared to the serum IL12 level of the subject itself before treatment with the IL10 agonist or pharmaceutical composition.

[0526] 293. The method according to Embodiment 291, wherein the method upregulates the serum IL12 level by at least 5 times compared to the control or compared to the serum IL12 level of the subject itself before treatment with the IL10 agonist or pharmaceutical composition.

[0527] 294. The method according to Embodiment 291, wherein the method upregulates the serum IL12 level by at least 10 times compared to the control or compared to the serum IL12 level of the subject itself before treatment with the IL10 agonist or pharmaceutical composition.

[0528] 295. The method according to Embodiment 291, wherein the method upregulates the serum IL12 level by at least 15 times compared to the control or compared to the serum IL12 level of the subject itself before treatment with the IL10 agonist or pharmaceutical composition.

[0529] 296. The method according to Embodiment 291, wherein the method upregulates the serum IL12 level by at least 20 times compared to the control or compared to the serum IL12 level of the subject itself before treatment with the IL10 agonist or pharmaceutical composition.

[0530] 297. The method according to Embodiment 291, wherein the method upregulates the serum IL12 level by at least 25 times compared to the control or compared to the serum IL12 level of the subject itself before treatment with the IL10 agonist or pharmaceutical composition.

[0531] 298. The method according to Embodiment 291, wherein the method upregulates the serum IL12 level by at least 30 times compared to the control or compared to the serum IL12 level of the subject itself before treatment with the IL10 agonist or pharmaceutical composition.

[0532] 299. The method according to any one of Embodiments 263 to 298, wherein the method upregulates serum IL4 levels compared to a preferred control that has not been treated with the IL10 or pharmaceutical composition.

[0533] 300. The method according to Embodiment 299, wherein the method upregulates the serum IL4 level by at least twofold compared to the control or compared to the subject's own serum IL4 level before treatment with the IL10 agonist or pharmaceutical composition.

[0534] 301. The method according to Embodiment 299, wherein the method upregulates the serum IL4 level by at least five times compared to the control or compared to the subject's own serum IL4 level before treatment with the IL10 agonist or pharmaceutical composition.

[0535] 302. The method according to Embodiment 299, wherein the method upregulates the serum IL4 level by at least 10 times compared to the control or compared to the subject's own serum IL4 level before treatment with the IL10 agonist or pharmaceutical composition.

[0536] 303. The method according to Embodiment 299, wherein the method upregulates the serum IL4 level by at least 15 times compared to the control or compared to the subject's own serum IL4 level before treatment with the IL10 agonist or pharmaceutical composition.

[0537] 304. The method according to Embodiment 299, wherein the method upregulates the serum IL4 level by at least 20 times compared to the control or compared to the subject's own serum IL4 level before treatment with the IL10 agonist or pharmaceutical composition.

[0538] All publications, patents, patent applications, and other documents referenced in this application are incorporated by reference in whole for all purposes to the same extent that each individual publication, patent, patent application, or other document is individually directed to be incorporated by reference for all purposes. In the event of any conflict between the teachings of one or more references incorporated in this specification and the disclosure, the teachings of this specification shall prevail.

Claims

1. An IL10 agonist comprising a first polypeptide chain and a second polypeptide chain, wherein each polypeptide chain is arranged from an amino terminus to a carboxyl terminus. (a) IgG Fc domain; (b) Linker portion; and (c) IL10 part Includes IL10 agonists.

2. The IL10 agonist according to claim 1, wherein the first polypeptide chain and the second polypeptide chain each contain an amino acid sequence having at least 95% sequence identity with the amino acid sequence of IL10M1 (SEQ ID NO: 34).

3. The IL10 agonist according to claim 1, wherein the first polypeptide chain and the second polypeptide chain each contain an amino acid sequence having at least 96% sequence identity with the amino acid sequence of IL10M1 (SEQ ID NO: 34).

4. The IL10 agonist according to claim 1, wherein the first polypeptide chain and the second polypeptide chain each contain an amino acid sequence having at least 97% sequence identity with the amino acid sequence of IL10M1 (SEQ ID NO: 34).

5. The IL10 agonist according to claim 1, wherein the first polypeptide chain and the second polypeptide chain each contain an amino acid sequence having at least 98% sequence identity with the amino acid sequence of IL10M1 (SEQ ID NO: 34).

6. The IL10 agonist according to claim 1, wherein the first polypeptide chain and the second polypeptide chain each contain an amino acid sequence having at least 99% sequence identity with the amino acid sequence of IL10M1 (SEQ ID NO: 34).

7. The IL10 agonist according to claim 1, wherein the first polypeptide chain and the second polypeptide chain each contain the amino acid sequence of IL10M1 (SEQ ID NO: 34).

8. The IL10 agonist according to claim 1, wherein the first polypeptide chain and the second polypeptide chain each contain an amino acid sequence having at least 95% sequence identity with the amino acid sequence of IL10M4 (SEQ ID NO: 37).

9. The IL10 agonist according to claim 1, wherein the first polypeptide chain and the second polypeptide chain each contain an amino acid sequence having at least 96% sequence identity with the amino acid sequence of IL10M4 (SEQ ID NO: 37).

10. The IL10 agonist according to claim 1, wherein the first polypeptide chain and the second polypeptide chain each contain an amino acid sequence having at least 97% sequence identity with the amino acid sequence of IL10M4 (SEQ ID NO: 37).

11. The IL10 agonist according to claim 1, wherein the first polypeptide chain and the second polypeptide chain each contain an amino acid sequence having at least 98% sequence identity with the amino acid sequence of IL10M4 (SEQ ID NO: 37).

12. The IL10 agonist according to claim 1, wherein the first polypeptide chain and the second polypeptide chain each contain an amino acid sequence having at least 99% sequence identity with the amino acid sequence of IL10M4 (SEQ ID NO: 37).

13. The IL10 agonist according to claim 1, wherein the first polypeptide chain and the second polypeptide chain each contain the amino acid sequence of IL10M4 (SEQ ID NO: 37).

14. The IL10 agonist according to claim 1, wherein the first polypeptide chain and the second polypeptide chain each contain an amino acid sequence having at least 95% sequence identity with the amino acid sequence of IL10M5 (SEQ ID NO: 38).

15. The IL10 agonist according to claim 1, wherein the first polypeptide chain and the second polypeptide chain each contain an amino acid sequence having at least 96% sequence identity with the amino acid sequence of IL10M5 (SEQ ID NO: 38).

16. The IL10 agonist according to claim 1, wherein the first polypeptide chain and the second polypeptide chain each contain an amino acid sequence having at least 97% sequence identity with the amino acid sequence of IL10M5 (SEQ ID NO: 38).

17. The IL10 agonist according to claim 1, wherein the first polypeptide chain and the second polypeptide chain each contain an amino acid sequence having at least 98% sequence identity with the amino acid sequence of IL10M5 (SEQ ID NO: 38).

18. The IL10 agonist according to claim 1, wherein the first polypeptide chain and the second polypeptide chain each contain an amino acid sequence having at least 99% sequence identity with the amino acid sequence of IL10M5 (SEQ ID NO: 38).

19. The IL10 agonist according to claim 1, wherein the first polypeptide chain and the second polypeptide chain each contain the amino acid sequence of IL10M5 (SEQ ID NO: 38).

20. The IL10 agonist according to claim 1, wherein the linker portion includes or consists of a monomer or polymer of G n S or SG n (wherein n is an integer from 1 to 7).

21. The IL10 agonist according to claim 20, wherein the linker portion comprises a monomer or polymer of amino acid sequence G4S (SEQ ID NO: 51).

22. The IL10 agonist according to claim 21, wherein the linker portion comprises one, two, or three repeats of the amino acid sequence G4S (SEQ ID NO: 51).

23. The IL10 agonist according to any one of claims 1 to 22, wherein the IL10 portion comprises or consists of an amino acid sequence having at least 98% sequence identity with mature human IL10 (SEQ ID NO: 1).

24. The IL10 agonist according to any one of claims 1 to 22, wherein the IL10 portion comprises or consists of an amino acid sequence having at least 99% sequence identity with mature human IL10 (SEQ ID NO: 1).

25. The IL10 agonist according to any one of claims 1 to 22, wherein the IL10 portion comprises the amino acid sequence of mature human IL10 (SEQ ID NO: 1).

26. The IL10 agonist according to any one of claims 1 to 22, which is a homodimer.

27. ​​The IL10 agonist according to any one of claims 1 to 22, lacking a targeting moiety, wherein the IL10 agonist (a) binds to a tumor-associated antigen; (b) binds to a tumor microenvironment antigen; (c) binds to a cell surface molecule of a tumor-reactive lymphocyte; or (d) binds to a checkpoint inhibitor.

28. The IL10 agonist according to claim 27, wherein the IL10 agonist lacks a targeting portion that binds to PD-1.

29. The IL10 agonist according to any one of claims 1 to 22, wherein the IL10 agonist lacks an antibody variable region.

30. The IL10 agonist according to any one of claims 1 to 22, wherein the IL10 agonist lacks a CH1 domain.

31. The IL10 agonist according to any one of claims 1 to 22, wherein the IL10 agonist lacks a CL domain.

32. An IL10 agonist according to any one of claims 1 to 22, which contains no cytokines other than IL10.

33. One or more nucleic acids encoding the IL10 agonist according to any one of claims 1 to 22.

34. A host cell manipulated to express an IL10 agonist according to any one of claims 1 to 22, or one or more nucleic acids according to claim 33.

35. The host cell according to claim 34, which is a mammalian cell.

36. A method for producing an IL10 agonist according to any one of claims 1 to 22, comprising culturing the host cells described in claim 34 and recovering the IL10 agonist expressed thereby.

37. (a) Contacting the recovered IL10 agonist with an affinity column configured to capture the IL10 agonist, optionally the affinity column being a protein A affinity column; (b) Eluting the affinity column to generate eluents; and (c) Perform size exclusion chromatography on the eluted substance. The method according to claim 36, further comprising:

38. A pharmaceutical composition comprising an IL10 agonist and an excipient according to any one of claims 1 to 22.

39. The pharmaceutical composition according to claim 38, comprising at least 10 mg, at least 20 mg, or at least 50 mg of the IL10 agonist.

40. The pharmaceutical composition according to claim 38, 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-high performance liquid chromatography (SE-UPLC).

41. The pharmaceutical composition according to claim 38, wherein it does not contain a detectable amount of aggregates of the IL10 agonist as determined by size exclusion ultra-high performance liquid chromatography (SE-UPLC).

42. The pharmaceutical composition according to claim 38, wherein (a) it does not contain a detectable amount of the C-terminal shortened variant of the IL10 agonist, and / or (b) it does not contain a detectable amount of the N-terminal shortened variant of the IL10 agonist.

43. A therapeutic agent for treating cancer, wherein the therapeutic agent comprises an IL10 agonist according to any one of claims 1 to 22 or a pharmaceutical composition according to any one of claims 38 to 42, and the therapeutic agent is administered to a subject in need thereof.

44. The therapeutic agent according to claim 43, wherein the cancer is a solid tumor.

45. The therapeutic agent according to claim 44, wherein the solid tumor is colon cancer, melanoma, squamous cell carcinoma, lymphoma, pancreatic tumor, or lung tumor.

46. The therapeutic agent according to claim 44, wherein the solid tumor is resistant to treatment with an anti-PD1 antibody.

47. The therapeutic agent according to claim 44, wherein the solid tumor is resistant to treatment with anti-PD1 antibody monotherapy.

48. The therapeutic agent according to claim 44, wherein the therapeutic agent is administered to the subject together with an anti-PD1 antibody.

49. The therapeutic agent according to any one of claims 46 to 48, wherein the anti-PD1 antibody is MDX-1106 (nivolumab), MK-3475 (pembrolizumab), MEDI-0680 (AMP-514), PDR001, REGN2810, or BGB-108.

50. The therapeutic agent according to any one of claims 44 to 48, which results in an increase in CD8 T cell density in a solid tumor, wherein the increase is optionally at least twofold, at least threefold, or at least fourfold.

51. The therapeutic agent according to any one of claims 44 to 48, which results in an increase in CD45+ immune cell infiltration in solid tumors, wherein the increase is optionally at least 10%.

52. The therapeutic agent according to claim 51, wherein the CD45+ immune cells include CD4 T cells, myeloid cells, or both CD4 T cells and myeloid cells.

53. The therapeutic agent according to any one of claims 44 to 48, wherein the administration of the therapeutic agent upregulates serum IL-12 compared to a preferred control not treated with the therapeutic agent, or compared to the subject's own serum IL-12 level before treatment with the therapeutic agent, and optionally, the administration of the therapeutic agent upregulates serum IL-12 by at least 2 times, at least 5 times, at least 10 times, at least 15 times, at least 20 times, at least 25 times, or at least 30 times compared to the preferred control, or compared to the subject's own serum IL-12 level before treatment with the therapeutic agent.

54. The therapeutic agent according to any one of claims 44 to 48, wherein administration of the therapeutic agent upregulates serum IL-4 compared to a preferred control not treated with the therapeutic agent, or compared to the subject's own serum IL-4 level before treatment with the therapeutic agent, and optionally, administration of the therapeutic agent upregulates serum IL-4 by at least 2 times, at least 5 times, at least 10 times, at least 15 times, or at least 20 times compared to the preferred control, or compared to the subject's own serum IL-4 level before treatment with the therapeutic agent.

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